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Novel Pharmacological Modulators of P2X7
receptor
A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy
Kshitija Dhuna
MSc, Guru Nanak Dev University, Punjab, India.
School of Health and Biomedical Sciences
College of Science, Engineering and Health
RMIT University
March,2019
ii
Declaration
I certify that except where due acknowledgement has been made, the work is that of the author
alone; the work has not been submitted previously, in whole or in part, to qualify for any other
academic award; the content of the thesis/project is the result of work which has been carried
out since the official commencement date of the approved research program; any editorial
work, paid or unpaid, carried out by a third party is acknowledged; and, ethics procedures and
guidelines have been followed. I acknowledge the support I have received for my research
through the provision of an Australian Government Research Training Program Scholarship.
Kshitija Dhuna
Date: 18/03/19
iii
Acknowledgement
It is an extreme pleasure to express my sincere gratitude to my worthy guide, Dr Leanne Stokes,
for her guidance, encouragement and useful critiques of this research work. You enabled me
to undertake one of the most rewarding journeys of my life. You encouraged me to aspire to
achieve my goals. My thesis, and the achievements throughout my candidature, would not have
been possible without you. I feel blessed that I was given the opportunity to work under the
supervision of a person who is an excellent scientist, a great teacher and a beautiful human
being.
I feel short of words to express my thanks to my co-supervisor A/Prof Samantha Richardson,
whose unconditional support and encouragement made it possible for this work to see the light
of the day. You took me by my hand and led me to the finish line. Thank you.
I am indebted to Dr Martin Stebbing, my associate supervisor, for providing all the essential
amenities for this project and for continuous and active involvement at each step of research
work embodied in this thesis.
I wish to convey my sincere thanks to Dr Ray M Helliwell, for making me a part of the
ginsenosides story. Your in-depth knowledge of Pharmacology helped me thoroughly with all
aspects of this project. Thank you!
I would like to extend my gratitude to Dr Narin Osman for her constant words of
encouragement and generous support during my entire candidature. Your smiles and
reassurance helped me through some very difficult times.
I would like to extend my heartfelt thanks to Dr Brett Cromer for his extensive help in the
automated patch clamp experiments.
iv
The primary macrophage work in thesis would not have been possible without the generous
contribution of Dr Simone de Luca and A/Prof Sarah Spencer who supplied the rat tissue for
primary macrophage isolation.
I am sincerely grateful to Prof Emilio Badoer and Dr Trisha Jenkins of the Neurobiology
Research Group, thank you for your advice and support. I would also like to extend my
gratitude to Dr Celine Velery, whose kind words made it a bit easier to survive this long
journey.
I would like to convey my sincere gratitude to members of the HDR administration team, Helen
Cassidy and Mary Briganti. Thank you for answering the endless emails and also wiping away
the occasional tears. I am grateful to our HDR co-ordinator, Prof Aj Jaworowski, for his
support during my candidature.
I would like to thank my friends at RMIT University, Saher Ali, Sefaa Al-Aryahi, Merfat
Algathami and Durga Dharmadana for their friendship over the years. I would also like to
mention the staff of Refuel cafeteria, who served coffee with a pinch of love! Thank you for
making this journey less lonely.
Thank you to my family for their outstanding support. Vikram, my husband, you stood by me
through thick and thin like the Rock of Gibraltar. My beautiful daughter, Kainaat, your support
and love made this thesis possible. My mother and sister, and my father in his time, without
the constant support and help you graciously offered when I needed it; I could not have reached
my goals.
I acknowledge the support I have received for my research through the provision of an
Australian Government Research Training Program Scholarship.
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Academic Contribution
During the course of this thesis, the candidate’s aim was to contribute to the fields of
Purinergic signalling and to characterise certain pharmacological modulators so as to
attain a better insight into the P2X7 function and modulation. The candidate has
contributed to the following publications and presentations:
i. JOURNAL ARTICLES
1. Ginsenosides act as positive modulators of P2X4 receptors
Kshitija Dhuna, Matthew Felgate, Stefan Bidula, Samuel Walpole, Lucka Bibic, Brett A.
Cromer, Jesus Angulo , Julie Sanderson , Martin J. Stebbing, Leanne Stokes: Molecular
pharmacology. 2018:mol.118.113696.
2. Neonatal overfeeding by small-litter rearing sensitizes hippocampal microglial responses
to immune challenge: reversal with neonatal repeated injections of saline as well as
minocycline
Simone De Luca, Ilvana Ziko, Kshitija Dhuna, Luba Sominsky, Mary Tolcos, Leanne
Stokes, Sarah Spencer: Journal of Neuroendocrinology 29(11):e12540 · October 2017.
3. P2X4 Receptor Function in the Nervous System and Current Breakthroughs in
Pharmacology Leanne Stokes, Janice Layhadi, Lucka Bibic, Kshitija Dhuna, Samuel
Fountain: Frontiers in Pharmacology 8 May 2017.
4. Selected ginsenosides of the protopanaxdiol series are novel positive allosteric modulators
of P2X7 receptors.
Ray M Helliwell, Charlene ShioukHuey, Kshitija Dhuna, Juan C Molero, Jiming M Ye,
Charlene C Xue. British Journal of Pharmacology. 2015; 172(13):3326-40.
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ii. CONFERENCE PRESENTATIONS
1. Ginsenosides cause cell death by potentiating calcium influx via P2X7 channels in a murine
macrophage cell line.
Kshitija Dhuna, Martin J Stebbing, Ray M Helliwell, Leanne Stokes, MGPCRS-ASCEPT,
Melbourne, December 2016
2. Investigating the selectivity of ginsenoside positive modulators on P2X7
Kshitija Dhuna, Martin J Stebbing, Ray M Helliwell, Leanne Stokes, ASCEPT, Tasmania,
December 2015
3. Minocycline: A novel pharmacological modulator of P2X7
Kshitija Dhuna, Martin J Stebbing, Leanne Stokes, GAGE conference, Canberra, April
2015
4. Minocycline modulates purinergic receptors
Kshitija Dhuna, Martin J Stebbing, Leanne Stokes, HDR Conference, RMIT University,
Melbourne, October 2014
vii
TABLE OF CONTENT
DECLARATION ...................................................................................................................... II
ACKNOWLEDGEMENT ....................................................................................................... III
ACADEMIC CONTRIBUTION .............................................................................................. V
ABSTRACT ............................................................................................................................... 1
CHAPTER 1 .............................................................................................................................. 4
A REVIEW OF THE LITERATURE........................................................................................ 4
Introduction ................................................................................................................................ 4
1.1 Immune response: An overview ..................................................................................... 4
1.2 Initial recognition of danger: PAMPs and DAMPs ........................................................ 7
1.3 ATP: The endogenous “signal 0” ................................................................................. 10
1.4 ATP release from cells .................................................................................................. 11
1.5 Purinergic Signalling- An overview ............................................................................. 15
1.5.1 Discovery of Purinergic Signalling ................................................................................. 15
1.5.2 Classification and distribution of purinergic receptors ................................................... 15
1.5.3 Purinergic receptors in immune cells .............................................................................. 19
1.5.4 Role of ATP in short-term and long term (trophic) Purinergic Signalling ..................... 21
1.5.5 P2X receptors in macrophages and microglia ................................................................ 22
1.5.6 P2X7 receptor structure .................................................................................................. 24
1.5.7 Membrane pore formation by P2X7 receptors................................................................ 27
1.6 P2X7R in inflammation ..................................................................................................... 29
1.7 Pharmacological and therapeutic roles of P2X7 ................................................................ 31
1.8 Overall Aims ...................................................................................................................... 35
CHAPTER 2 ............................................................................................................................ 36
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MATERIALS AND METHODS ............................................................................................. 36
Materials .................................................................................................................................. 36
2.1 Drugs and Reagents ........................................................................................................... 36
2.2 Chemicals ........................................................................................................................... 37
Methods.................................................................................................................................... 37
2.3 Cell culture ......................................................................................................................... 37
2.4 Transfections ...................................................................................................................... 38
2.5 Dye uptake experiments ..................................................................................................... 39
2.6 Measurement of Ca2+ concentration using Fura-2 AM. ..................................................... 40
2.7 Immunostaining and flow cytometric analysis .................................................................. 40
2.8 Enzyme-Linked Immunosorbent Assay (ELISA) .............................................................. 41
2.9 MTS cell viability assay..................................................................................................... 43
2.10 Measurement of caspase 3/7 activity using ImageXpress ............................................... 45
2.11 Measurement of current amplitudes using IonFlux-16 system. ....................................... 45
2.12 Cellular reactive oxygen species (ROS) measurement using CM-H2DCFDA ................ 47
2.13 Mitochondrial superoxide measurement using MitoSOXTM Red .................................... 48
2.14 Rat macrophage isolation ................................................................................................. 49
2.15 Statistical analysis ............................................................................................................ 49
CHAPTER 3 ............................................................................................................................ 51
EFFECT OF TETRACYCLINE DERIVATIVES ON PURINERGIC RECEPTORS ........... 51
3.1 Introduction ........................................................................................................................ 51
Minocycline: an anti-inflammatory and neuroprotective tetracycline antibiotic ..................... 51
Possible molecular targets of MINO........................................................................................ 54
3.2 Rationale for the research .................................................................................................. 55
3.3 Research Question ............................................................................................................. 55
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3.4 Results ................................................................................................................................ 56
3.4.1 P2X7 responses in BV-2 microglia................................................................................. 56
3.4.2 P2X7-mediated pro-inflammatory responses ................................................................. 61
3.4.3 Investigating the selectivity of MINO ............................................................................ 64
3.4.4 Investigating internalisation of P2X7 receptors .............................................................. 71
3.4.5 Investigating the effect of MINO on YOPRO-1 fluorescence........................................ 71
3.4.6 Investigating the pharmacological properties of MINO towards P2X7 and P2X4. ....... 74
3.7 Investigating the effect of tetracycline and its structural analogues on P2X7 ................... 77
3.8 Effect of MINO and DOX on P2X7 responses using primary macrophages .................... 80
3.4.9 Investigating the effect of MINO and DOX on P2Y1 and P2Y2 receptors.................... 82
3.4.10 Investigating the effect of MINO and DOX on TRPV-1 ion channels......................... 84
3.5 Key Points .......................................................................................................................... 86
3.6 Discussion .......................................................................................................................... 86
Mechanism of action of MINO via P2X receptors .................................................................. 91
3.7 Conclusion ......................................................................................................................... 93
CHAPTER 4 ............................................................................................................................ 94
PURINERGIC RECEPTOR MODULATION BY GINSENOSIDES .................................... 94
4.1 Introduction ................................................................................................................... 94
4.2 Biodegradation and bioavailability of ginsenosides ...................................................... 99
4.3 Mechanism of ginsenosides action ............................................................................. 100
4.4 Research Question ........................................................................................................... 105
4.5 Results .............................................................................................................................. 106
4.4.1 Effect of ginseng extract G115 on human P2X7 .......................................................... 106
4.4.2 Potentiation of human P2X7 mediated dye uptake by the ginseng metabolite CK ...... 107
4.4.3 Potentiation of human P2X7 mediated Ca2+ influx by ginseng metabolite CK ........... 107
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4.4.4 Lack of difference in effect of CK among different species of P2X7 .......................... 108
4.4.5 Potentiation of P2X7 mediated cell death by ginseng metabolite CK .......................... 109
4.4.6 Investigating effect of ginsenosides on P2X4 ............................................................... 118
4.4.7 Effect of ginsenosides on P2X4 mediated dye uptake .................................................. 118
4.4.8 Ginseng metabolite CK increases Ca2+ influx via P2X4 activation.............................. 118
4.4.9 Effect of selected ginsenosides on ATP concentration response curve in HEK-P2X4
cells ........................................................................................................................................ 119
4.4.10 Effect of selected ginsenosides on Ca2+ responses in HEK-P2X4 cells ..................... 119
4.4.11 Effect of ginsenosides on P2X4 inward currents using high throughput IonFlux-16
automated patch clamp system. ............................................................................................. 120
4.4.12 Effect of P2X4 and ginsenosides on cell viability ...................................................... 120
4.4.13 Role of ginsenosides in modulation of P2X2 receptor function ................................. 130
4.4.14 Effect of ginsenosides in modulation of P2Y1 and P2Y2-mediated Ca2+ responses . 134
4.6 Key Points ........................................................................................................................ 137
4.7 Discussion ........................................................................................................................ 137
4.8 Conclusions ...................................................................................................................... 143
CHAPTER 5 .......................................................................................................................... 144
SELECTED GINSENOSIDES MODULATE P2X7 RECEPTOR RESPONSES IN
MACROPHAGES ................................................................................................................. 144
5.1 Introduction ...................................................................................................................... 144
5.2 Research Question ........................................................................................................... 146
5.3 Results .............................................................................................................................. 146
5.4.1 J774 macrophages express functional P2X7................................................................. 146
5.4.2 CK and Rd enhance P2X7 mediated Ca2+ influx in J774 macrophages ....................... 149
5.4.3 Potentiation of P2X7 mediated YOPRO-1 uptake and Ca2+ influx by CK increases in a
concentration dependent manner ........................................................................................... 149
5.4.4 LPS does not affect P2X7 channel activity in J774 macrophages ................................ 155
xi
5.4.5 CK and Rd increase the ATP induced cell death .......................................................... 159
5.4.6 CK and Rd increase P2X7 mediated cellular ROS production..................................... 162
5.4.7 CK and Rd increase P2X7 mediated mitochondrial superoxide (mtSOX) production 166
5.8 CK increases caspase 3/7 activity in ATP activated J774 macrophages ......................... 171
5.4.9 CK and Rd increase caspase 3/7 activity in the presence of LPS in ATP activated
macrophages .......................................................................................................................... 178
5.4.10 Effect of selected ginsenosides on P2X7 mediated responses in primary peritoneal
macrophages .......................................................................................................................... 181
5.4.11 CK and Rd increase Ca2+ influx in ATP activated primary peritoneal macrophages . 181
5.4.12 Effect of ginsenosides on cell viability in primary peritoneal macrophages .............. 182
5.4.13 Effect of ginsenosides on mitochondrial superoxide production in primary peritoneal
macrophages .......................................................................................................................... 182
5.4.14 Effect of CK on caspase 3/7 activity in primary peritoneal macrophages .................. 182
5.4.15 Effect of ginsenosides on cytokine secretion in J774 macrophages ........................... 189
5.4.16 Effect of ginsenosides on cytokine secretion in primary peritoneal macrophages ..... 194
5.5 Key results from this study are summarised in Figure 5.30 ............................................ 198
5.6 Discussion ........................................................................................................................ 200
5.7 Conclusion ....................................................................................................................... 205
General Conclusions .............................................................................................................. 215
Future Directions ................................................................................................................... 215
REFERENCE ......................................................................................................................... 218
APPENDICES ....................................................................................................................... 267
Cell Line Information ............................................................................................................ 267
A. HEK-293 cells ................................................................................................................ 267
B. J774A.1 macrophage cell line ........................................................................................ 268
C. BV-2 microglia cell line ................................................................................................. 269
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D. Buffers ............................................................................................................................ 270
Phosphate Buffer Saline (PBS, pH 7.3) ................................................................................. 270
Electrolyte Low Divalent Buffer (ELDV, pH 7.3) ................................................................... 270
Physiological buffer (Etotal, pH 7.3) ....................................................................................... 271
Krebs-HEPES buffer (pH 7.3) ............................................................................................... 271
HBSS-HEPES Buffer (pH 7.3) .............................................................................................. 272
Ammonium chloride lyse (10X concentration) ..................................................................... 272
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LIST OF FIGURES
Figure 1.1: Interaction of innate and adaptive immune responses (11) ..................................... 6
Figure 1.2 PAMPs and DAMPs : Critical mediators of immune system .................................. 9
Figure 1.3 Sources of ATP and its effects on immune system ................................................ 12
Figure 1.4 Extracellular ATP acts as a legend for Purinergic Signalling ................................ 14
Figure 1.5 Classification of purinergic receptors (71) ............................................................. 17
Figure 1.6: Extracellular Purinergic Signalling ....................................................................... 20
Figure 1.7: General structure of P2X7 ..................................................................................... 26
Figure 1.8 Domain structure of P2X7 ...................................................................................... 26
Figure 2.1 Sandwich ELISA protocol (243) ............................................................................ 43
Figure 2.2 Schematic representation of the MTS cell viability detection method ................... 44
Figure 2.3 The special FluxIon plates used for automated whole cell current measurement and
the pattern within each plate. ............................................................................................ 46
Figure 3.1 Chemical structure of MINO (C23H27N3O7) ........................................................... 51
Figure 3.2 A schematic diagram displaying P2X7 and associated signalling pathways. ........ 53
Figure 3.3 Effect of MINO on P2X7 mediated YOPRO-1 uptake in BV-2 microglial cells .. 57
Figure 3.4 Effect of MINO on P2X7 activated by various concentrations of ATP in BV-2
microglial cells ................................................................................................................. 58
Figure 3.5 Effect of MINO on P2X7 mediated Ca2+ influx in BV-2 microglial cells ............. 59
Figure 3.6 Dose dependent inhibitory effect of MINO on P2X7 mediated Ca2+ influx in BV-2
microglial cells ................................................................................................................. 60
Figure 3.7 Effect of MINO on IL-1β release in BV-2 microglial cells as measured by ELISA
.......................................................................................................................................... 62
Figure 3.8 Effect of MINO on cell proliferation in BV-2 microglial cells as measured by Cell
Titer 96® AQueous One Solution Assay ......................................................................... 63
Figure 3.9 Effect of MINO on YOPRO-1 uptake in HEK-293 cells expressing rat P2X7 ..... 66
Figure 3.10 Effect of MINO on YOPRO-1 uptake in HEK-293 cells expressing rat P2X4 ... 67
Figure 3.11 Effect of MINO on ATP-induced dye uptake in HEK-293 cells stably expressing
human P2X7 ..................................................................................................................... 68
Figure 3.12 Effect of MINO on ATP-induced dye uptake in HEK-293 cells stably expressing
human P2X4 ..................................................................................................................... 69
Figure 3.13 Comparison of the effect of MINO on ATP-induced dye uptake in HEK-293 cells
stably expressing human P2X7 or human P2X4. ............................................................. 70
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Figure 3.14 MINO does not quench fluorescence of YOPRO-1 dye and does not alter
receptor expression on the cell surface. ............................................................................ 72
Figure 3.15 MINO does not reduce the maximum YOPRO-1 uptake in lysed cells ............... 73
Figure 3.16 MINO acts as a non-competitive antagonist at human P2X7............................... 75
Figure 3.17 MINO acts as a non-competitive antagonist at human P2X4 receptors ............... 76
Figure 3.18 Effect of tetracycline class antibiotics on ATP-induced dye uptake in HEK-
P2X7. ................................................................................................................................ 78
Figure 3.19 TET analogue DOX also attenuates P2X7 mediated responses ........................... 79
Figure 3.20 MINO and DOX have an inhibitory effect on P2X7 mediated Ca2+ influx in
primary peritoneal macrophages ...................................................................................... 81
Figure 3.21: MINO inhibits P2Y1 and P2Y2 mediated intracellular Ca2+ influx in HEK- 293
cells ................................................................................................................................... 83
Figure 3.22: MINO inhibits dye uptake in T-Rex-HEK rat TRPV-1 cells .............................. 85
Figure 4.1 Chinese herb Panax ginseng has a number of reported medicinal applications .... 94
Figure 4.2 Possible biotransformation of ginsenosides by lactic acid bacteria in human
intestines (371) ................................................................................................................. 99
Figure 4.3 Standardised ginseng extract G115 increases P2X7 mediated YOPRO-1 uptake.
........................................................................................................................................ 110
Figure 4.4 Some protopanaxadiol ginseng compounds increase P2X7-mediated YOPRO-1
uptake.............................................................................................................................. 111
Figure 4.5 CK acts as a positive allosteric modulator of human P2X7 receptors ................. 112
Figure 4.6 CK acts as a positive allosteric modulator of human P2X7 receptors activated by
BzATP. ........................................................................................................................... 113
Figure 4.7 Rd enhances the sustained dye uptake associated with hP2X7 receptor activation.
........................................................................................................................................ 114
Figure 4.8 CK enhances the sustained Ca2+ response associated with hP2X7 receptor
activation. ....................................................................................................................... 115
Figure 4.9 There is no difference in the potentiating effect of selected ginsenosides on P2X7
mediated YOPRO-1 uptake in humans and rats. ............................................................ 116
Figure 4.10 CK enhanced the ability of ATP to cause cell death in HEK-P2X7 cells. ......... 117
Figure 4.11 Effect of ginseng extract G115 on ATP-induced dye uptake in HEK-293 cells
expressing human P2X4 ................................................................................................. 122
Figure 4.12 Effect of ginsenosides on ATP-induced dye uptake in HEK-293 cells expressing
human P2X4 ................................................................................................................... 123
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Figure 4.13 ATP-induced dye uptake in HEK-293 cells expressing human P2X4 in the
presence of varying doses of CK .................................................................................... 124
Figure 4.14 Concentration dependent effect of ginsenosides on ATP-induced dye uptake in
HEK- human P2X4 cells ................................................................................................ 125
Figure 4.15 CK enhances the sustained Ca2+ response associated with hP2X4 receptor
activation. ....................................................................................................................... 126
Figure 4.16 CK acts a positive modulator of P2X4 receptor ................................................. 127
Figure 4.17 Effect of ginsenosides on current amplitude in HEK-hP2X4 cells .................... 128
Figure 4.18 ATP does not induce cell death in HEK-hP2X4 cells ........................................ 129
Figure 4.19 Effect of ginsenosides on P2X2a dye uptakes in transiently transfected HEK-293
cells ................................................................................................................................. 131
Figure 4.20 Effect of ginsenosides on P2X2a Ca2+ influx in transiently transfected HEK-293
cells ................................................................................................................................. 132
Figure 4.21 Selected ginsenosides do not alter P2Y mediated Ca2+ influx ........................... 135
Figure 4.22 Comparative analyses of ginsenosides on P2X7, P2X4 and P2X2a mediated
YOPRO-1 uptake and Ca2+ responses ............................................................................ 136
Figure 5.1 Expression of functional P2X7 receptor in J774 macrophages ............................ 148
Figure 5.2 Selected ginsenosides enhance the sustained Ca2+ response associated with P2X7
receptor activation in J774 macrophages........................................................................ 151
Figure 5.3 CK increases P2X7 mediated dye uptake in J774 macrophages in a concentration
dependent manner ........................................................................................................... 152
Figure 5.4 Effect of CK on Ca2+ influx in ATP activated P2X7 responses in J774
macrophages ................................................................................................................... 153
Figure 5.5 Effect of CK on ATP-induced Ca2+ influx in P2X7 responses in J774 macrophages
........................................................................................................................................ 154
Figure 5.7 Effect of selected ginsenosides on Ca2+ influx in LPS primed J774 macrophages
........................................................................................................................................ 158
Figure 5.8 Effect of LPS priming on cell viability in ATP stimulated J774 macrophages in the
presence of ginsenosides ................................................................................................ 161
Figure 5.9 Optimisation of CM-H2DCFDA assay with H2O2 as positive control in the
presence and absence of LPS priming in J774 macrophages for measurement of cellular
ROS ................................................................................................................................ 163
Figure 5.10 Effect of selected ginsenosides on cellular ROS production in the presence and
absence of LPS priming in J774 macrophages in CM-H2DCFDA assay. ...................... 164
xvi
Figure 5.11 Effect of ginsenosides on cellular ROS production in the presence and absence of
LPS priming in J774 macrophages. ................................................................................ 165
Figure 5.12 Effect of ginsenosides on mitochondrial superoxide (mtSOX) productions in the
presence of FCCP and Actinomycin in ATP activated J774 macrophages. ................... 168
Figure 5.13 Effect of ginsenosides on mitochondrial superoxide productions in ATP activated
J774 macrophages........................................................................................................... 169
Figure 5.14 Effect of ginsenosides on mitochondrial superoxide production in presence of
100 ng/ml LPS in ATP activated J774 macrophages. .................................................... 170
Figure 5.15 Measurement of caspase 3/7 activity in the presence of apoptotic inducer
staurosporine for optimisation of assay .......................................................................... 174
Figure 5.16 NucView 488 3/7 caspase activity in presence of 0.01% DMSO, 0.5 µM STR and
20 µM CK ....................................................................................................................... 175
Figure 5.17 Measurement of caspase 3/7 activity in the presence of selected ginsenosides CK,
Rd, Rb1 and Rh2 ............................................................................................................ 176
Figure 5.18 NucView 488 3/7 caspase activity in presence of 500 µM ATP, ATP + CK and
ATP + CK + AZ10606120 ............................................................................................. 177
Figure 5.19 Measurement of caspase 3/7 activity in the presence of selected ginsenosides CK,
Rd, Rb1 and Rh2 in LPS primed J774 macrophages. .................................................... 179
5.10 Effect of selected ginsenosides on P2X7 mediated responses in primary peritoneal
macrophages ................................................................................................................... 181
Figure 5.21 Effect of ginsenosides on Ca2+ influx in primary peritoneal macrophages ........ 184
Figure 5.22 Effect of CK on ATP-induced Ca2+ influx in P2X7 responses in primary
peritoneal macrophages .................................................................................................. 185
Figure 5.24 Effect of ginsenosides on mtSOX production in primary peritoneal macrophages
........................................................................................................................................ 187
Figure 5.24 TNF-α production by J774 macrophages with and without LPS stimulation .... 191
Figure 5.25 IL-1β productions by J774 macrophages with and without LPS stimulation .... 192
Figure 5.26 IL-6 production by J774 macrophages with and without LPS stimulation ........ 193
Figure 5.27 TNF-α production by primary peritoneal macrophages ± LPS stimulation ....... 195
Figure 5.28 IL-1β production by primary peritoneal macrophages ± LPS stimulation ......... 196
Figure 5.29 IL-6 production by rat macrophages with and without LPS stimulation ........... 197
Figure 5.30 Diagrammatic representations of the experimental findings in the present study.
........................................................................................................................................ 198
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LIST OF TABLES
Table 4.1 Classification of ginsenosides based upon their backbone structures [adapted from
Chen et al. 2009 (368) ...................................................................................................... 97
Table 4.2: Substituent R groups of some of the common ginsenosides [adapted from Sergiy
Oliynk and Seikwan oh,2013 (369) .................................................................................. 98
Table 4.3 Recent studies correlating the physiological effects of ginsenosides have been
correlated with their capability to modulate various signalling pathways. .................... 103
Table 4.4 Comparative list of EC50 values of ATP for human and rat P2X7 in the presence of
selected ginsenosides ...................................................................................................... 109
xviii
LIST OF ABBREVIATIONS
Symbols Meanings
ADP Adenosine diphosphate
ALS Amyotrophic lateral sclerosis
AMP Adenosine monophosphate
ANOVA Analysis of variance
APC Antigen presenting cells
ATP Adenosine triphosphate
CK Compound K
COPD Chronic obstructive pulmonary disorder
CREB cAMP response element binding protein
DAMPs Danger associated molecular patterns
DC Dendritic cells
DMEM Dulbecco’s modified Eagle’s medium
DNA Deoxyribonucleic acid
DOX Doxycycline
D-PBS Dulbecco's phosphate-buffered saline
EDTA Ethylene diamine tetraacetate
FDA U.S Food and Drug Administration
FRT-site Flippase recognition target
GSK GlaxoSmithKline
HBSS Hank’s buffer saline solution
HEK-293 Human embryonic kidney-293 cells
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIV Human immunodeficiency virus
IFN-ɤ Interferon-ɤ
IL-18 Interleukin-18
xix
IL-1β Interleukin-1 β
IP3 Inositol triphosphate
LDH Lactate dehydrogenase
LPS Lipopolysaccharide
MAPK Mitogen activated protein kinases
MINO Minocycline
NAD+ Nicotinamide adenine dinucleotide
NADPH reduced Nicotinamide adenine dinucleotide phosphate
NANC Non-adrenergic Non-cholinergic
NF-κB Nuclear factor kappa-light-chain-enhancer of activated
B-cells
NLRP-3 Nod-like receptor protein-3
NMDG+ N-methyl D-glucosamine
NMMHC-IIA Non-muscle myosin heavy chain IIA
NOD Nucleotide-binding oligomerization domain
NSAID Nonsteroidal anti-inflammatory drugs
PAMPs Pathogen associated molecular patterns
PLC Phospholipase C
PRR Pattern recognition receptors
RBC Red blood cells
RNA Ribonucleic acid
ROS Reactive oxygen species
RPMI Roswell Park Memorial Institute medium
S.E.M Standard error of the mean
STAT-3 Signal transducer and activator of transcription 3
TET Tetracycline
TIGE Tigecycline
TNF-α Tumour necrosis factor alpha
xx
TNP-ATP Trinitophenol-adenosine triphosphate
TRPA-1 Transient receptor potential cation channel, subfamily A,
Member 1
TRPV Transient receptor potential cation channel subfamily V
UDP Uridine diphosphate
UTP Uridine triphosphate
YOPRO-1 YO-PRO™-1Iodide
ABSTRACT
Inflammation is a biological response invoked by the body when challenged by harmful stimuli
such as pathogens, injury or irritants. Inflammatory responses can be classified as being acute
or chronic. In cases where the acute inflammatory response is unable to eliminate the infection
or resolve the damage, the characteristics of the immune response are altered. The neutrophil
response is supplanted by monocyte-derived macrophages and if there is infection, T cells from
the adaptive immune system are employed as well. If the infection persists, a state of chronic
inflammation ensues. This is accompanied by continued production of pro-inflammatory
cytokines. The chronic inflammatory response may cause tissue destruction due to the
production of reactive oxygen species (ROS) and hydrolytic enzymes. The over-production of
pro-inflammatory cytokines contributes to the pathophysiology of many diseases such as heart
disorders, cancer, obesity, stroke, chronic respiratory diseases and diabetes. According to the
World Health Organisation (WHO), the chronic inflammatory diseases are the major cause of
deaths in the world. According to the Australian Institute of Health and Welfare, 50% of
Australians were reported having 1 of 8 chronic inflammatory diseases is high. The economic
impact of chronic inflammatory disease is high. For example, the direct healthcare costs
incurred by patients of chronic obstructive pulmonary disorders in the year 2010 were
calculated to be approximately $50 million. There is a growing need to identify molecular
targets (enzymes and receptors) which are key regulators of inflammatory pathways and to
develop approaches to modulate them in order to attenuate inflammatory damage. Previous
studies have indicated significant involvement of adenosine triphosphate in initiating and
regulating inflammatory pathways.
Extracellular ATP (eATP) is involved in activating inflammatory responses and is considered
as an acute ‘danger signal’ or DAMP (Danger Associated Molecular Pattern). eATP can bind
to and activate purinergic receptors. Purinergic receptors, also known as purinoceptors, are a
2
family of plasma membrane complexes that are found in almost all mammalian tissues and are
activated by purines. P2X7, a trimeric purinergic ion channel is commonly expressed on
immune cells such as microglia, monocytes, macrophages and dendritic cells. P2X7 is
characterised by the formation of a large non-selective membrane pore following prolonged
exposure to its ligand ATP, which allows the passage of small organic molecules (up to 900
Da in size) as well as high calcium flux into the cells. P2X7 regulates the maturation and
secretion of inflammatory cytokines such as IL-1β and IL-18. Alteration in function and
expression of P2X7 has been implicated in the development and progression of many disease
types such as neurodegenerative disorders, cancer, neuropathic and inflammatory pain and
pulmonary fibrosis. Inhibition of receptor or deletion of the P2X7 gene has shown a down-
regulation of inflammatory pathways. Thus, the P2X7 receptor is a potential drug target for
treating inflammation-based disorders. Identification and characterisation of small molecular
weight modulators for this receptor are important for establishing effective treatments for
inflammation-related disorders. Several pharmaceutical companies (GSK, AstraZeneca, and
Evotech) have been developing novel antagonists for the past decade and some of them have
entered clinical trials for treatment of rheumatoid arthritis.
The project described in this thesis explores existing compounds that have been demonstrated
to have pharmacological actions on P2X7-dependent signalling pathways and characterises the
effects of two unrelated groups of compounds found to have pharmacological action on P2X7,
namely, tetracycline antibiotics and ginsenosides derived from the Chinese herb ginseng.
Minocycline is a broad-spectrum antibiotic, which has been reported to have anti-inflammatory
properties independent of its anti-microbial properties. Minocycline is an endogenous
microglial inhibitor. In this study, it was observed that by inhibiting P2X7 responses,
minocycline inhibited the influx of Ca2+ ions, and hence decreased pro-inflammatory cytokines
3
secretion, ROS generation and subsequent cell death. These results provided evidence that the
anti-inflammatory action of minocycline is due, in part, to blocking the P2X7 receptor.
Panax ginseng is a Chinese herb which has been used traditionally for treatment of anaemia,
diabetes, inflammation of the stomach lining (gastritis), fever, chronic obstructive pulmonary
disease (COPD), and asthma, all of which are diseases where resolution of inflammation or
immune responses are compromised. The main bioactive components among these are ginseng
saponins, also known as ginseng glycosides or ginsenosides. Previous work in our laboratory
has indicated that specific ginsenosides potentiate P2X7 responses. In this study, selected
ginsenosides (CK, Rd, Rb1 and Rh2) were shown to act as positive allosteric regulators of
P2X7 receptors in HEK-293 cells overexpressing P2X7. Potentiation of channel activity by
these ginsenosides was found to translate to physiological responses mediated by P2X7 in both
J774 macrophages and primary rat macrophages. These results indicate that ginsenosides may
re-sensitize the immune system in individuals with sub-optimal immune responses by
potentiating P2X7 responses in macrophages.
Overall, this thesis describes experiments which comprehensively characterise two unrelated
classes of compounds; semi-synthetic tetracycline antibiotics and Panax ginseng derived
ginsenosides, as effective pharmacological modulators of P2X7. Due to the involvement of
P2X7 in numerous diseases, there is a mounting need to characterise physiological modulators
of P2X7, which can act as tools to unravel the exact mechanism by which this receptor regulates
inflammatory pathways. The information gathered with the help of these physiological tools
may assist in designing safe, specific and selective drugs to target the P2X7 receptor and
thereby develop novel treatments for inflammatory disorders.
4
CHAPTER 1
A REVIEW OF THE LITERATURE
Introduction
1.1 Immune response: An overview
All organisms possess well-developed defence mechanisms in order to survive and flourish in
the living world. This host-defence mechanism is the immune system, which comprises a
complex series of cells, tissues and organs that interact in order to fight off any invading
pathogens and keep the body healthy. The immune system becomes increasingly sophisticated
as the complexity of the organism grows (1). Humans are constantly exposed to organisms that
can be swallowed, inhaled or can inhabit our skin or mucosal membranes. This exposure can
manifest into disease or discomfort depending upon the level of virulence of the invading
pathogens and the strength of the host’s defence system (2). The indispensable role of the
immune systems is most realised when it fails. A sub-optimal immune system may lead to
severe infections, tumours and immunodeficiency while an overactive defence mechanism may
cause allergies and autoimmune disorders (3). In humans, the immune system can be broadly
classified into two sub-systems: relatively less specific (innate) and highly specific (adaptive)
immunity (4).
If any bacteria, viruses, fungi or protozoans are able to cross the physical barriers to enter the
body, then these are dealt with by the innate immune system (5). The characteristic feature of
the innate immune response is the recruitment of neutrophils to the site of infection or injury,
which is followed by a cascade of events, termed the inflammatory response, which is meant
to eliminate infectious agents and restore normality (6). If this process is imbalanced or
5
inappropriate it may lead to conditions such as vaculitis, inflammation of connective tissue and
systemic inflammatory response syndrome (7).
The adaptive immune response is a characteristic feature of higher organisms and is specific in
nature as it involves antigen recognition with the help of T and B lymphocytes (8). In
comparison to innate immune response, which are fast but not specific (and can cause damage
to self-cells in the process), the adaptive response is slower to develop but targets particular
antigens. The adaptive immune response also has memory, which makes it able to respond
quicker and stronger in cases where infection reoccurs (9).
When confronted by danger signals released at sites of infection, the body first employs the
innate immune response and then the adaptive immune response in order to counter this attack
(10). Most opportunistic microorganisms that are able to cross the physical barriers of the body
are dealt with by neutrophils and macrophages of the innate immune response. In case the
innate immune response is unable to eliminate the infection or if these initial responders do not
recognise the invading pathogen, then the adaptive immune response is brought into play. The
adaptive immune response engages its highly specific lymphocytes to recognise and eliminate
the invading pathogens. The initial adaptive response may take up to 4-7 days to develop;
therefore, the innate immune cells are vital in restricting the extent of infection until the full
adaptive immune response ensues (Figure 1.1) (11).
6
Figure 1.1: Interaction of innate and adaptive immune responses (11)
Components of innate and adaptive immunity interact with each other to combat the attacking
pathogens (bacteria/fungi/parasites and viruses) in the vertebrates. Dendritic cells (DCs) are
accessory cells that act as connecting links between the innate and the adaptive immune system.
Dendritic cells process the antigen and present it on their cell surface to the T cells of the
immune system. They are, therefore, also called the antigen presenting cells (APCs). The
antigen is presented to either Tcytotoxic or Thelper cells.
7
1.2 Initial recognition of danger: PAMPs and DAMPs
In case the danger signals are microorganisms, they are recognised by the cells of the innate
immune system via the conserved molecular motifs expressed by the attacking micro-organism
but not present in the host organism. These conserved molecular patterns are called pathogen
associated molecular patterns (PAMPs). For example, lipopolysaccharide (LPS) in bacterial
cell membranes (12), zymosan in fungal cell walls (13) and profilin in protozoan cell
membranes act as PAMPs (14). Thus, PAMPs are functional components of the attacking
pathogen and help the immune system in differentiating self from non-self (stranger
hypothesis) (6). The receptors on immune cell surfaces that recognise and bind to PAMPs are
called pattern recognition receptors (PRRs) such as Toll like receptors (TLRs), nucleotide-
binding oligomerization domain (NOD)-like receptors, and the mannose receptor. These are
expressed on innate immune cells (15).
In addition to molecular patterns displayed on the surface of invading pathogens, the host cells
can also release endogenous alarm signals in order to apprise the immune system of incoming
stress, unscheduled cell death or microbial infection. These endogenous signals are called
danger associated molecular patterns (DAMPs). These can be intracellular proteins such as
heat shock proteins, high-mobility group protein 1 (HMGB1/amphoterin) or non-protein
cellular products such as ATP (adenosine triphosphate), uric acid, heparin sulphate and DNA
(16). PAMP and DAMP-mediated signalling and induction of an innate immune response
usually result in resolution of infection but may also result in chronic inflammation or
autoimmunity by changing various cell death and survival mechanisms.
The concept that an initial signal (signal 0) is mandatory for instigation of the innate immune
response was first put forward by Charles Janeway in 1989 (17). He proposed that recognition
of PAMPs and DAMPs by the innate immune system was the first step for initiation of the
immune response, which was followed by a series of events involving components of both the
8
innate and the adaptive immune response, which work in collaboration in order to eliminate
the imposing threat by the incoming danger signal.
This concept was thoroughly reviewed in 2002 by Medzitov and Janeway (18), where they
explained that upon activation by invading pathogen (PAMPs), the cells of the innate immune
response undergo differentiation into effector cells. In case, these effector cells are able to get
rid of the infection by themselves, the adaptive immune response is not switched on. However,
if the elimination of the pathogen is not completed by the innate immune response, the adaptive
immune response is called into action. The information regarding the nature of the pathogenic
microbe is relayed to the cells of the adaptive immune response by components of the innate
immunity. This is done via the help of co-stimulatory molecules, such as CD80 and CD86
present on the surface of antigen presenting cells (APCs). Other theories to explain the immune
system activation have also been put forward. Most notable of which is Polly Matzinger’s idea
that the immune system becomes activated in response to danger signals sent out by dying or
injured cells rather than by self/non-self recognition or via Janeway’s pathogen-PRR binding.
According to her “danger model”, the immune system is consistently updated in response to
cellular damage (19). Even though Matzinger’s danger signal theory is not accepted in its
entirety by most immunologists, the term DAMP was first described by Seong and Matzinger
in 2004 and is still used to describe various immunological reactions (20). It is now considered
that in addition to the external pathogens attacking the body, the immune system can be
activated by endogenous danger signals as well. ATP is an omnipresent molecule, which is a
fundamental energy donor as well as an efficient cell-to-cell communicator (21). Besides these
roles, ATP is also an alarm signal or DAMP, which stimulates the host immune response to
protect against stress or injury. Figure 1.2 illustrates some of the signalling pathways that are
initiated by various molecules acting as PAMPs and DAMPs for the immune system (22).
9
Figure 1.2 PAMPs and DAMPs : Critical mediators of immune system (22)
Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular pattern
molecules (DAMPs) activate various signalling pathways. LPS activates both the TRIF-
dependent TLR4 and MyD88-dependent pathways. The MyD88-dependent pathway is
responsible for NF-κB and MAPK activation, which controls induction of pro-inflammatory
cytokines. The TRIF-dependent pathway activates IRF3 by TBK1/IKKε, which is required for
the induction of IFN-inducible genes. TLR1-TLR2 and TLR2-TLR6 recognize bacterial
triacylated lipopeptide or diacyl lipopeptide, respectively, and recruit TIRAP and MyD88 at
the plasma membrane to activate the MyD88-dependent pathway. TLR5 recognizes flagellin
and activates the MyD88-dependent pathway. TLR3, TLR7, TLR8, and TLR9 reside in the
endosome and recognize dsRNA, ssRNA, CpG DNA, or mitochondrial DNA. DAMPs such as
HMGB1, S100s, and HSPs recognize the RAGE end products, TLR4 or TREM-1 and activates
the MyD88-MAPK-NF-κB pathway. HMGB1 and RAGE activate the TLR9-MyD88
dependent pathway, which contributes to autoimmune pathogenesis. CD24 is a negative
receptor to inhibit the DAMP-induced TLR4 pathway. ATP binding of the P2X7 receptor
increases activation of caspase-1 by the NALP3 inflammasome and other nucleotide-binding
oligomerization domain (NOD)-like receptors (NLRs) to promote secretion of IL-1β and IL-
18.
*LPS-Lipopolysaccharide, TRIF- TIR-domain-containing adapter-inducing interferon-β,
TLR4- Toll-like receptor 4, MyD88- the myeloid differentiation factor 88, MAPK-mitogen-
activated protein kinase, TBK1- TANK-binding kinase 1, IFN- Interferon, TIRAP -TIR
adapter protein, HMGB1-High mobility box-1, S-100- S100 proteins, HSP- heat shock
proteins, RAGE- receptor for advanced glycation, TREM-1-triggering receptor expressed on
myeloid cells-1
10
1.3 ATP: The endogenous “signal 0”
ATP is an ancient and fundamentally important biological molecule involved in vital cellular
processes (23). It was discovered independently by Karl Lohmann in Germany and by Cyrus
Hartwell Fiske and Yellagaprada SubbaRow in the USA in the year 1929 (24). Due to its
ubiquitous presence and its role as an energy donor in most metabolic reactions, ATP is
considered as the most important intracellular biochemical in living systems. The fact that ATP
and its metabolite adenosine may have an extracellular role in the heart and coronary blood
vessels was first demonstrated by Drury and Szent-Gyӧrgyi in 1929 (25). A number of studies
then focused on the action of ATP on heart muscles and its subsequent effect on cardiovascular
functions (26, 27). The effect of ATP in the nervous system was also studied by injecting ATP
into various parts of the cat brain where a number of electrophysiological and biochemical
changes were observed (28).
One of the first pieces of evidence that ATP may have a neurotransmitter role came from
studies conducted by Holton et al. where it was reported that antidromic stimulation of the great
auricular nerve of a rabbit ear artery caused ATP release, indicating some role of ATP in
vasodilation (29). Buchthal and Folkow also observed that acetylcholine-mediated contraction
of skeletal muscle fibres in frogs was enhanced in the presence of ATP (30). Studies conducted
on the physiological and pharmacological role of adenosine and its derivatives indicated a
strong extracellular role of these purines. In 1963, Bernes proposed that adenosine was the
compound responsible for the causing vasodilation in myocardial hypoxia (31). This
hypothesis was later replaced by Geoffrey Burnstock’s hypothesis that stress and hypoxia lead
to release of ATP from endothelial cells which acts upon certain receptors present in these
cells, causing coronary vasodilation associated with myocardial hypoxia (32). The
accumulating evidence from these pioneering studies conducted on adenylyl compounds
quashed any initial resistance to the idea of ATP’s extracellular role.
11
It is known that the cellular concentration of ATP varies from 3-10 mM while the extracellular
concentration of ATP is very low (~10 nM) (33). Low extracellular concentrations of ATP are
maintained by ectoenzymes that convert any Replaced by ATP released from cells to adenosine
diphosphate (ADP). This ensures that in extracellular compartments the nucleotide signalling
is relatively short-lived (34).
1.4 ATP release from cells
ATP is released from many cell types such as endothelial cells, astrocytes, macrophages and
urothelial cells in response to mechanical disturbances such as sheer stress, hypoxia and
pathogen invasion and also during necrosis (a type of cell death) (35-37). The concentration of
extracellular ATP has been observed to increase by more than 3 fold in inflammatory diseases
(38) such as atherosclerosis (39) and preeclampsia (40). ATP is released into the extracellular
matrix by a variety of mechanisms including vesicles, secretory granules and membrane
channels such as pannexins and connexin hemi-channels (41). Pannexins are transmembrane
proteins that connect the intracellular matrix with the extracellular environment and have been
implicated in the release of ATP and other nucleotides from cells (42, 43). Connexins are gap
junction proteins which, in addition to connecting with other connexins in adjacent cells, can
also exist independently within an individual cell as a hemi-channel or conduit (44). It has been
shown that the connexins are involved in the release of ATP from the cell by over-expression
in connexin-deficient glioma cells (45, 46). ATP is released during synaptic transmissions and
platelet aggregation (47, 48). Moreover, ATP release has been reported from dying cancer cells
via the Golgi apparatus/endoplasmic reticulum related pathway (49). Figure 1.3 illustrates the
various stimuli which initiate the release of ATP in different cell type leading to unique cellular
responses in each cell type.
12
Figure 1.3 Sources of ATP and its effects on immune system (50)
ATP is released by platelets, neurons, epithelial and endothelial cells in response to various
stimuli such as soluble mediators, mechanical stress or membrane depolarization. T cells,
macrophages and microglia have also been suggested to release ATP following activation with
antigen or bacterial lipopolysaccharide. ATP in the extracellular environment activates P2
receptors and stimulates many important responses in immunity and inflammation. From this
information, it was suggested that ATP might be a short-range immune cell modulator.
13
The released ATP is converted to ADP and adenosine by the membrane bound enzymes called
ectonucleotidases. These can be classified into four main groups which include ectonucleoside
triphosphate diphosphohydrolases (CD39), ecto-5’-nucleotidases (CD73), ectonucleotide
pyrophosphatase/phosphodiesterases and alkaline phosphatases (51). The extracellular ATP
and its major breakdown product adenosine, can affect almost all types of immune cells by
acting on a family of cell surface proteins called the purinergic receptors (52).
The ATP/adenosine balance is oppositely regulated in inflammatory responses (53). An
increase in ATP release is essential for TLR mediated activation of monocytes/macrophages,
inflammasome activation, cytokine release and host immune response during infection (54).
The ATP concentration is increased during the initiation of inflammatory responses and the
ATP concentrations are maintained during inflammation by inhibition of ATP breakdown
enzymes (CD39 and CD72) by oxidative stress and TNF-α (55). The end of the inflammatory
response is marked with a decrease in concentration of ATP and an increase in adenosine
(Figure 1.4) (53). Lower ATP concentrations and high levels of adenosine inhibit the
inflammatory response (56). Purinergic receptors responding to ATP and adenosine play a
critical role in perceiving danger, triggering the immune responses and many
pathophysiological changes in the living system. Purinergic Signalling is the focal point of this
thesis and its detailed background is discussed in the following sections.
14
Figure 1.4 Extracellular ATP acts as a legend for purinergic signalling (34)
Inside a cell, mitochondria act as the powerhouse of the cell and generate ATP as the end
product of oxidative phosphorylation reaction. ATP then acts as a ubiquitous energy source
catalysing metabolic reactions and cellular processes. ATP as well as other nucleotides can be
release actively from stressed cells. ATP released is converted into AMP or adenosine by
ectonucleoside triphosphate diphosphohydrolase (CD39) and ecto-5′-nucleotidase
(CD73).This enzymatic conversion of ATP to adenosine helps to maintain the optimum levels
of extracellular ATP and also to abolish ATP signalling.
15
1.5 Purinergic Signalling- An overview
Purinergic receptors are defined as proteins on the surface of cells that can bind and respond to
purines. The discovery, characterisation and physiological roles of these receptors are
discussed in detail in the following sections.
1.5.1 Discovery of Purinergic Signalling
In the year 1972, after relentlessly working for over a decade on mechanical and electrical
activities of smooth muscle cells, Geoffery Burnstock put forward the then controversial
hypothesis that ATP, the universal energy donor, also acted a neurotransmitter (57). He coined
the term “Purinergic Signalling” for ATP mediated signalling as ATP is a purine molecule (58).
Further work established that ATP was released from sympathetic nerves supplying smooth
muscle (59). This discovery led Burnstock to challenge Dale’s principle of “one nerve - one
transmitter” hypothesis by suggesting that ATP acted as a co-transmitter along with
noradrenalin (60). The function of ATP as a co-transmitter was initially opposed (61) but later
gained universal acceptance with many studies indicating that ATP acted as a co-transmitter in
most peripheral nerves (62-66). The receptors specific for binding purines and initiating the
subsequent signalling were named “purinergic receptors”. Based on the literature showing the
neuromodulatory effects of purines in peripheral as well as central nervous systems, Burnstock
made the first attempt at classifying purinergic receptors.
1.5.2 Classification and distribution of purinergic receptors
In 1978, Burnstock proposed that there are two separate sets of receptors for adenosine and
ATP/ADP. He called the adenosine receptors P1 receptors and the ATP/ADP receptors P2
receptors (67). In 1985, the P2 receptors were further sub-classified into the P2X and P2Y
receptors by Burnstock and Kennedy (68). In the beginning, this classification was based on
the differences in affinities of the receptors for various agonists/ antagonists but later it was
16
established that there are significant structural and physiological differences between the two
classes of P2 receptors (67). Consequently, the term P2X was used for ligand gated ion
channels and P2Y for G-protein coupled receptors (Figure 1.5). The concept of Purinergic
Signalling was widely accepted after the cloning and characterisation of the receptors involved.
At present, International Union of Pharmacology Committee on Receptor Nomenclature and
Drug Classification recognises eight distinct P2Y receptor subtypes (P2Y1, P2Y2, P2Y4,
P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) receptors and seven P2X subunits. Of the seven
P2X receptors, six subtypes can exist in homomeric form (P2X1–P2X5 and P2X7) (69), and at
least six heteromeric channels (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6, and P2X4/6) (70).
Figure 1.5 illustrates the presently accepted classification of purinergic receptors.
17
Figure 1.5 Classification of purinergic receptors (71)
Purinergic receptors are classified into two major categories: P1 receptors that bind to
adenosine and P2 receptors that bind to ATP/ADP . P1 receptors have further four subtypes:
A1, A2A, A2B and A3. P2 receptors are either ligand gated ion channels called the P2X
receptors - P2X and metabotropic G-protein coupled receptors called P2Y receptors
18
The P1 and P2Y receptors were found to be G-protein coupled receptors with 7 transmembrane
domains connected by three extracellular (ELs) and three intracellular (IL) loops, the N
terminus in the extracellular part and the C terminus in the intracellular part of the receptors
(72). The P2Y receptors were further sub-classified on the basis of their sequence homology
and their phylogenetic relationship. Members of the first group (P2Y1, 2, 4, 6 and 11) were
found to share a sequence homology of 35-52% and contain a conserved Y-Q/K-X-X-R motif
on their seventh transmembrane α-helix. The members of the second group (P2Y12, 13 and
14) were characterised by a sequence homology of 47- 48% and a conserved K-E-X-X-L motif
on transmembrane α-helix 7 (69). Some studies suggested that there might be a link between
the structural homology and function in these groups (73-77). On the contrary, there are some
studies indicating a functional similarity between P2Y receptors with lesser structural
homology (78). Thus, the P2Y receptors are very diverse in the regulation of signalling
pathways despite similarity in the amino acid sequences.
The P2X receptors were found to be structurally distinct with two transmembrane domains and
a large extracellular hydrophilic loop. The first hydrophobic transmembrane region lies
between amino acid 30-50 and the second lies between residues 330-353. The extracellular
loop starts close to amino acid 52 and ends at position 329 (79). The loop largely extends out
of the plasma membrane and contains the ligand-binding site. The P2X receptors are functional
in their trimeric form i.e three polypeptide subunits come together to form an ion channel.
Three ATP molecules can bind to trimeric P2X7 and can activate the channel so as to allow
the influx of Na+ and Ca2+ ions and efflux of K+. Binding of ATP to P2X7 increases the
intracellular Ca2+ levels and causes membrane depolarisation (80). There are seven known
subunits of P2X receptors and P2X4 shares the maximum sequence homology with P2X7 in
all the species tested. The purinoceptors were first described and characterised in neuronal cells
19
(60) but now, with the help of immunohistochemistry, it has been confirmed that these proteins
are expressed in a number of non-neuronal cells of the body (71)
1.5.3 Purinergic receptors in immune cells
The interaction of nucleotides/nucleosides with their receptors can lead to either stimulation of
the immune system or can develop immune tolerance, depending upon the quantity and the
time of nucleotide exposure (16). Since the ectonucleotidases are involved in the hydrolysis of
ATP and are highly expressed in all cells, their function is thought to be related to termination
of P2X signalling (81). The extracellular purinergic signalling illustrating the role of
ectonucleotidases in breakdown of ATP is shown in Figure 1.6. P2X signalling leads to the
initiation of pro-inflammatory responses in the cells and the removal of ATP by its conversion
to adenosines prevents these inflammatory changes. Due to this reason, P1 receptors are
thought to be involved in preventing tissue injury and acute inflammation (82, 83). This
protective role of P1 receptor mediated signalling is also confirmed by studies where CD39
and CD73 knockout mice were more prone to inflammatory tissue damage in comparison to
control due to the lack of P1 receptors (84, 85). CD39 deletion or dysfunction has been
associated with the development of inflammatory diseases such as Crohn’s disease and
inflammatory bowel disease (86). The CD39/CD73 pathway also affects the CD4+ T regulatory
cells (Tregs), the suppression of which can attenuate HIV infection (87). Taken together, these
studies suggest that P1 signalling has an anti-inflammatory role as it prevents the ATP binding
to the P2X7 receptor and thereby preventing the activation of pro-inflammatory pathways
controlled by P2X7 receptors (88).
20
Figure 1.6: Extracellular Purinergic Signalling (89)
Simplified representation of Purinergic Signalling where ATP is released from the cells and
gets converted to adenosine by the action of various ectonucleotidases. ATP and its breakdown
products further act on purinergic receptors to modulate various downstream signals. E-
NTPDases (CD39) are ectonucleosidase triphosphate diphosphohydrolase family of enzymes
which covert ATP to ADP/AMP. E-NPPs are ectonucleotide pyrophosphate/phosphodiesterase
family (CD73) which catalyse the conversion of ATP and ADP to AMP. The E-5’
nucleotidases convert AMP to adenosine.
21
1.5.4 Role of ATP in short-term and long term (trophic) Purinergic Signalling
The influence of purinergic receptors on signalling pathways can be either short term or long
term (trophic) (90). The short-term Purinergic Signalling effects include neurotransmission,
neuromodulation, secretion, chemoattraction, and platelet aggregation (91). The long-term
Purinergic Signalling can affect processes such as embryonic development, cell proliferation
and differentiation, angiogenesis, restenosis after angioplasty, atherosclerosis and glial cell
activities. The effect of short-term signalling by purinergic receptors can be witnessed in blood
vessels supplied by parasympathetic nerves. ATP is released as a co-transmitter along with
noradrenaline by parasympathetic nerves and this ATP acts on P2X receptors to excite smooth
muscles in the blood vessels leading to vasoconstriction (32). ATP release from endothelial
cells during shear stress and hypoxia can activate P2Y receptors by autocrine signalling and
cause the release of endothelial derived relaxing factor (EDRF) or nitric oxide, which may lead
to vasodilation. Adenosine, formed by ATP breakdown, can also participate in short-term
Purinergic Signalling by acting on P1 receptors in the muscles and leading to vasodilation (52,
92).
Long-term trophic signals are induced by ATP and adenosine binding to purinergic receptors
coupled to signal-transducing effector molecules (Src, GTPases, phospholipase-A,
phospholipase-D and adenylyl cyclase). These effector molecules lead to the generation of
secondary messengers or protein kinases which can regulate transcriptional factors to control
the synthesis of proteins required in long term trophic factors such as those involved in cell
proliferation, migration, differentiation and apoptosis (90)
Purinergic Signalling plays a significant role in health and disease. Therapeutic approaches for
treatment of pathological disorders caused by alteration in purinergic receptor functions can be
achieved by development of selective agonists and antagonists for P1 and P2 receptors.
Development of small molecule drugs which can be orally administered to the patients and are
22
able to target purinergic receptors, ecto-nucleotidases or ATP transporters could be a crucial
step in discovering novel treatments for inflammatory disorders.
1.5.5 P2X receptors in macrophages and microglia
The various subtypes of purinergic receptors are expressed by a variety of immune cells like
B-lymphocytes, dendritic cells, mast cells, microglia and macrophages (71). Purinergic
receptors including P2X4, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6 are expressed in macrophages.
Macrophages are large phagocytic cells and components of the innate immune system that play
an important role in immunity. They defend the living system through to their ability to engulf
the invading microbes and pathogens (93). Macrophages can regulate activation and
proliferation of lymphocytes and are also responsible for T and B cell activation via antigens
or alleges (94). Macrophages exposed to antigens are said to be “activated” and display
increased bactericidal capabilities (95).
In 1994, it was reported by Hickman et al. that human mononuclear phagocytes expressed
P2Z/P2X7 like activity. In this study, an increase in P2X7 responses was observed as
monocytes matured into macrophages (96). The macrophage responses to extracellular ATP
were evaluated and it was observed that development of a P2X7 mediated macrospore
following 15 minutes exposure to ATP increased intracellular Ca2+. Results from this study
indicated that P2X7 mediated pro-inflammatory responses and polykaryon formation in
macrophages (97). The role of P2X7 in macrophage fusion was also confirmed in a later study
(98). P2X7 mediated spontaneous cell death in J774 macrophages was also observed (99).
DiVirgilio et al. in 1996 reported the presence of P2X7 in microglia, mast cells and
macrophages (100). The effect of P2X7/P2Z receptors on macrophage and lymphocyte
function had already been indicated (93). ATP release by LPS stimulation of RAW264.7
macrophages resulted in increased nitric oxide production (101). Initially, P2X7 receptor’s
ability to cause IL-1β release from macrophages and monocytes was thought to be a secondary
23
effect of P2X7 related cytotoxicity because it was well understood that factors causing
apoptosis are among the most effective stimuli for IL-1β release (102). It was later understood
that rather than being a side-effect of P2X7 cytotoxicity, IL-1β was a product of a complex
signalling cascade that was orchestrated by P2X7. Solle et al. evaluated the role of P2X7 in IL-
1β post translational processing and found that peritoneal macrophages from P2X7 knockout
animals had an impaired cytokine signalling pathway and confirmed that the receptor directly
affects the maturation and release of IL-1β (103). Cell death in LPS primed macrophages
observed to occur via the activation of caspase-1 mechanism, independent of cytokine release
(104). In addition to caspase-1, an essential role of Ca2+ concentration was found in regulating
IL-1β production from macrophages (105, 106). Macrophage cell death following transient
P2X7 activation was suggested to be independent of TLR-2 and 4, caspase-1 as well as Panx-
1 (107). Pelegrin et al. in 2008 compared the IL-1β release from different macrophage type and
suggested two distinct pathways of cytokine release: release of processed IL-1β via a caspase-
1 cascade that could be selectively inhibited by blocking caspase-1 or Panx1, and a second
mechanism of pro-IL-1β release (Ca2+/caspase-1/Panx-1 independent pathway) that could be
inhibited by glycine (108). Eventually, the P2X7-NLRP3 inflammasome was established,
placing the role of P2X7 in right context (21, 109).
The involvement of P2X7 signalling in inflammatory and cell death pathways in macrophages
has significance in development of various pathological conditions. Stimulation of
macrophages and microglia resulted in secretion of IL-1β via ATP activated P2X7 responses,
suggesting a role of P2X7 receptor in development of Alzheimer’s disease (110). P2X7
immunoreactivities were observed to be increased in activated microglia and macrophages of
the spinal cord in amyotrophic lateral sclerosis and multiple sclerosis (111). Gain-of-function
P2X7 mutations increased inflammatory responses in macrophages and polymorphism of the
P2X7 gene was implicated in mood disorders and depression (112, 113). Activation of P2X7
24
responses in macrophages have also been cited to be beneficial in limiting Toxoplasma gondii
infection by inflammasome activation and ROS production (114).
P2X7 mediated cytokine release and cell death have been observed in microglia (115-117).
Microglia are the resident immune cells of central nervous system that undergo activation and
proliferation in response to inflammatory stimuli (118). Pharmacological characterisation of
the P2X7 receptors was also performed in the NTW8 microglial cell line and it was confirmed
that microglia expressed cytosolic pore forming P2X7 receptor which could be both negatively
and positively modulated (119, 120). Brough et al. in 2002 determined that the presence of IL-
1β was not necessary for the P2X7 mediated cell death, as the absence of the IL-1β gene did
not affect the P2X7 related cytotoxicity whereas the absence of the receptor lead to the
complete absence of cytokine release or cell death in LPS/ATP activated microglia (121).
Association of P2X7 with inflammatory pathways has now been well established (122).
Modulation of these pathways by targeting P2X7 in macrophages and microglia can have
significant therapeutic potential.
1.5.6 P2X7 receptor structure
P2X7 receptor is a ligand gated ion channel which becomes activated on binding ATP. The
human P2X7 gene is located on the long arm of chromosome 12 (12q24.31) and the resulting
protein is made up of 595 amino acids (79). P2X7 has a molecular mass of 69 kDa but
glycosylation in the extracellular loop increases this to 75-85kDa (123). Amongst all the
members of the P2X family, P2X7 is the most divergent and bears the closest resemblance to
P2X4 (79). The gene for the P2X4 receptor in humans is also located on 12q24.31 and radiation
hybrid mapping indicates that these two genes are may be located less than 130 bp apart and
these might have arisen by gene duplication (124). The general ectodomain structure of P2X7
is similar to that of P2X4 elucidated by Gouaux and colleagues in 2009. The crystal structure
of P2X is described as resembling a dolphin rising from water (80).
25
P2X7 opening on binding with appropriate ligand, allows the passage of positively charged
cations in a non-selective manner. P2X7 differs from the other P2X in many structural, function
and pharmacological features. It is functional only in its homotrimeric form, gets activated at
higher concentrations of ATP (EC50 > 100 μM), it is fully activated by 2’, 3’-(benzoyl-4-
benzoyl)-ATP which is 10-30 times more potent than ATP (EC50 = 7 μM) and it is the largest
P2X receptor (595 amino acids) bearing a long carboxyl terminal tail (242 amino acids). The
activation of P2X7 by ATP/BzATP is enhanced if extracellular Ca2+ and Mg2+ are removed
(125). Repeated exposure to the agonists results inward current through P2X7 receptor (126).
These properties make P2X7 unique and its effect on downstream signalling pathways very
significant in health and disease.
26
Figure 1.7: General structure of P2X7
P2X7 receptor is a trimeric receptor, with each of the three subunits made up of two transmembrane
domains. In the above Figure, 2 transmembrane domains of one of the trimeric subunits are shown.
Each subunit has an extracellular domain, which has an ATP binding site. The short N terminal and
long C-terminal tail is intracellular.
Figure 1.8 Domain structure of P2X7
The figure shows different components of P2X7 and the regions encoded by them. (127).
27
1.5.7 Membrane pore formation by P2X7 receptors
One of the properties that set the P2X7 receptor apart from the rest of its family members is its
ability to consistently form large conductance pores after prolonged exposure to its agonist
ATP. For most channels, the ion selectivity remains stable after channel activation, but for a
few channels including TRPV-1 (128, 129), TRPV-2 (130) , TRPA-1 (131), acid-sensing ion
channels (ASICs) (132) and purinergic receptors (primarily P2X7, sometimes P2X4 and P2X2)
there is a dilation of the ion conductance pore on prolonged exposure to ligand (133, 134).
It is thought that binding of the ATP to P2X7 receptor for a prolonged period of time triggers
the movement of TM2 that opens up a secondary permeability pathway also called the
membrane pore (macropore), which allows the passage of molecules as large as 900 Da into
the cell (133). The ability of P2X7 to allow otherwise impermeable organic dyes, to enter the
intracellular environment, after lengthy exposure to its ligand is often employed as reliable tool
for the measurement of pore formation. Fluorescent dyes such as lucifer yellow, ethidium and
YOPRO-1 iodide are used to observe the opening of the P2X7 macropore (135).
Overexpression of P2X7 allows the uptake of large cationic dyes following activation by ATP
(135, 136). Macrophages derived from P2X7 knockout mice are unable to exhibit this dye
uptake, response indicating a lack of membrane pore formation in the absence of P2X7 (103).
The mechanism of formation of this macropore is highly debated (137-139). It has been
suggested that the conversion from the channel to the pore state in P2X7 may be due to either
a change in conformation within the receptor's internal selectivity filter or the sequential
oligomerization of additional trimeric P2X7 subunits (140). It is understood that the TM2
domain of P2X7 is essential for the surface expression of this receptor and for pore formation.
Specific amino acids in the TM2 domain necessary for pore formation have been identified
(141). Some investigators argue that it is the inherent property of the ion channel while others
suggest the involvement of at least one other protein. Initial studies indicated the involvement
28
of pannexin-1 (Panx-1), a membrane hemichannel protein, in the formation of a membrane
pore on interaction with ligand bound P2X7 receptor. These studies suggest that P2X7 is
physically associated with Panx-1 and regulates the formation of membrane pore which can let
in large sized molecules such as organic dyes ethidium and YOPRO-1 iodide as well as the
concomitant release of IL-1β from activated macrophages (108, 142, 143). Thymocytes lacking
Panx-1 but containing functional P2X7 were found to show defective dye uptake during early
apoptosis (144). However, later studies provided evidence against the Panx-1 being the
obligatory protein involved in P2X7 pore pathway. For example, mouse macrophages deficient
in the Panx-1 gene demonstrated normal ATP-mediated YOPRO-1 iodide uptake (145); siRNA
knockdown of Panx-1 gene did not alter the dye uptake by mouse macrophages (146) and
blockade of this hemichannel by antagonists had no effect on the pore forming activity of the
ligand bound receptor (147). A study indicated that P2X7 initiated autocrine release of ATP
increased the fast motility of dendritic cells and this was further enhanced by Panx-1 channels.
Functional Panx-1 channels were required for migration of dendritic cells to lymph nodes even
though the overall maturity of these cells was independent of Panx-1 hemichannels (148).
Therefore, it is inconclusive whether Panx-1 is essential for membrane pore formation
following P2X7 activation, but this protein does play a role in inflammation and immune
responses in collaboration with P2X7.
There have also been some studies indicating the contribution of long C-terminal tail of P2X7
in pore formation as truncation of this tail and the presence of SNPs in this region have been
observed to cause loss of pore formation by the receptor (141, 149, 150). 95 % of the C-terminal
end of P2X7 has been found to be required for dye uptake/ membrane pore formation (151).
P2X7 was reported to interact with non-muscle myosin (NMMHC-IIA and myosin Va) and
extracellular ATP caused the dissociation of the P2X7-myosin complex. The dissociation of
P2X7 from non-muscle myosin was suggested to be essential for the formation of a membrane
29
pore (152). The transition to the secondary permeability pore following P2X7 activation is also
studied using electrophysiology to measure the permeability of cells towards the large cation
NMDG+. It is suggested that the characteristic shift in equilibrium potential observed after
prolonged activation of P2X receptors is not due to pore dilation but an outcome of time-
dependent change in ion concentration in the cells (153) . The authors of this study suggested
due to the electrophysiology techniques applied to evaluate the phenomenon and the pore
dilation was only “apparent” (153). In 2016, Karasawa and Kawate generated a x-ray crystal
structure of panda (Ailuropoda melanoleuca) (154). It has been suggested that membrane pore
formation depends upon the lipid composition of the plasma membrane (155). Information
regarding P2X7 mediated membrane pore formation may not be conclusive so far, but there is
a definite need for a unifying central dogma to gain better understanding of the working and
associated effects of this receptor.
The physiological relevance of this secondary permeation pathway in normal cell is often
speculated (156, 157). The formation of this secondary pore in a normal cell may aid in uptake
of ATP or other molecules required for meeting an enhanced metabolic demand during stress
or injury, or it may be a mechanism for ATP release to drive further Purinergic Signalling. It
could also be pathway for secreting chemical transmitter molecules such as glutamate (158).
1.6 P2X7R in inflammation
ATP binding to P2X7 leads to the activation of a number of downstream signalling events that
may play crucial roles in the development of many disease phenotypes such as multiple
sclerosis (159 , 160, 161), ALS (162), Alzheimer’s disease (163), Huntington’s disease (164),
cancer (165-168), neuropathic and inflammatory pain (122, 169), pulmonary fibrosis (170),
ischemia (171-173) and Sjogren's syndrome (174, 175). Single nucleotide polymorphisms
(SNPs) in the P2X7 gene have been associated with alteration of receptor function which makes
the individuals carrying these SNPs potentially more susceptible to certain disorders such as
30
systemic lupus erythematosus (176), osteoporosis (177-179), rheumatoid arthritis (180),
tuberculosis (181) and inflammatory disorders (113, 182).
The association of P2X7 receptors with diseases may be due to the initiation of many signalling
pathways by P2X7 activation. These include the activation of NLRP-3 inflammasome
pathways (122, 183), cytokine secretion (165, 184-186), reactive oxygen species formation
(187) and formation of phagolysosomes (158). The NLRP3 inflammasome is the name given
(Nod like receptor family members) regulate the maturation of IL-1β (188). Activation of the
NLRP3 inflammasome is associated with many pathological conditions such as brain injury,
encephalitis, skin allograft rejections (21). Activation of inflammasome occurs in two steps;
the priming step involves the increase in expression of inflammasome constituents such as pro-
IL-1β and NLRP3 (189) and the second step involves the assembly of inflammasome
components and secretion of IL-1β and IL-18 (190). The activation of caspase-1 has been
linked to the efflux of K+ through P2X7 (191). Due to the association of NLRP3 inflammasome
with cytokine secretion, P2X7 is considered a major physiological regulator of pro-
inflammatory responses in vivo.
The significance of P2X7 in inflammatory responses is evident from the studies where absence
or inhibition of the receptor has led to the downregulation of pro-inflammatory responses in
the host organism. For example, genetic ablation of P2X7 decreased pathophysiological
parameters in mouse model of Duchenne muscular dystrophy (192, 193). P2X7 affects graft-
versus host disease and allograft rejection (194, 195). A decrease in the clinical and
histopathalogical symptoms related to experimental autoimmune uveitis was seen P2X7
deficient mice (196). Similarly, P2X7 reduced the expansion of Treg cells, thereby decreasing
the renal injury associated with ischemia-reperfusion injury. P2X7 was implicated in the spread
of HIV infection as autocrine ATP release causes activation of macrophage P2X7 causing
release of microvesicles containing the HIV-1 virus (197). These studies provide strong
31
evidence that P2X7 is a key player in the immune responses in the body and modulation of this
receptor may have significant physiological implications.
The ATP mediated activation of the P2X7 receptor is also involved in generation of reactive
oxygen species in a variety of cells including microglia, macrophages, submandibular cells and
erythroid cells (198). This was found to activate the NADPH oxidase 2 enzyme in these cell
types (114, 199) . Other signalling pathways affected by P2X7 activation include protease
activation and release, prostaglandin secretion, glutamate release, cell proliferation and
phagocytosis (158). All these signalling pathways are involved in inflammation. Different cell
types respond differentially due to uniqueness of the effector molecules in each of these cells.
Thus, there is a mounting need to characterise physiological modulators of P2X7, which can
act as tools to unravel the exact mechanism by which this receptor regulates inflammatory
pathways. The information gathered with the help of these physiological tools may be helpful
in future for designing safe, specific and selective drugs to target the P2X7 receptor and thereby
develop cures for inflammatory disorders.
1.7 Pharmacological and therapeutic roles of P2X7
The role of P2X7 in inflammation is well defined and almost all immune cells express this
receptor. Therefore, the idea of regulating the activity of P2X7 by suitable drugs and
modulators holds a great therapeutic potential. Inhibition of this receptor can lead to
suppression of inflammatory responses which may be beneficial in ameliorating neuropathic
pain and cancer to name a few. Several attempts have been made to identify, characterise or
synthesise specific antagonists for P2X7. The antagonists for this receptor may be either
orthosteric (binding within the ATP binding site) or allosteric (binding at a site other than the
ATP binding site).
32
Orthosteric or competitive antagonists characterised for P2X7 include suramin or suramin-like
compounds, ATP derivatives (TNP-ATP), cynoguinidine derivatives (A740003 and A804598),
tetrazole derivatives (A839977, A438079) (200, 201). Of the entire orthosteric antagonist
characterised for P2X7, the cyonguinidine derivatives are the most potent and selective (202)
. Honore et al characterised A740003 as a competitive, selective P2X7 antagonist and observed
a dose dependent decreasein neuropathic pain in rats (203). Pharmacological inhibition of
P2X7 by A804598 resulted in inhibition of autophagic death in muscle cells in a mouse model
of Duchenne muscular dystrophy (204). Both cynoguanidine and tetrazole derivatives have
antagonistic affects at a very low micromolar IC50 value (205). ATP derivatives (oATP and
TNP) on the other hand are irreversible, less selective and antagonistic at high concentrations
(206).
Allosteric and non-competitive inhibitors for P2X7 responses include Brilliant Blue G (BBG)
(207), KN-62 (208), AZD9056 (209), GW791343 (210, 211), GSK314181A (212),
GSK1482160 (213), CE-224535(214), AFC-5128 (215), AZ-11645373 (216), AZ-10606120
(217) and JNJ-479655 (215, 218). Recently, 1, 4-Naphthoquinones compounds (A01 and A04)
were tested for their inhibitory activity for P2X7 (219). BBG has been found to diminish HIV-
gp120 related neuropathic pain by blocking P2X7 (220). P2X7 blockade by BBG also prevents
intrauterine inflammation related pre-term birth and perinatal brain injury in mice (221). BBG
prevents neuroinflammation, endocrinal dysregulation and other inflammatory disorders in
which P2X7 is implicated (220, 222-225). Stokes et al. characterised the thiazolidinedione
derivative AZ-11645373 in 2006 and found that while BBG was more potent in inhibiting rat
P2X7 responses, human P2X7 was better blocked by AZ11645373 (226). Physiological studies
conducted using this inhibitor indicate that AZ11645373 is a potent and selective blocker of
P2X7 mediated inflammatory responses (227-229). Studies involving characterisation of the
benzamide compound GSK314181A for its role in inhibiting P2X7 responses have shown that
33
this compound can ameliorate the P2X7 mediated neuropathic pain and inflammation (212).
More recently, a first in-human study was conducted for evaluating the pharmacokinetics,
pharmacodynamics, safety and tolerability of JNJ-479655, a brain permeable P2X7 antagonist
and it was found that this antagonist was able to downregulate LPS-induced, P2X7-mediated
cytokine secretion in peripheral blood via passive penetration into the brain (230).
Biotechnology company Evotec has confirmed a good safety profile of the compound EVT-
401, an oral antagonist of P2X7 after successful completion of the Phase-1 study (231). Many
other pharmaceutical groups including Affectis Pharmaceuticals AG, AstraZeneca, Merck,
Abbott and Pfizer have characterised and patented other P2X7 inhibitors but the functional
properties of these compounds are unpublished yet (232). Even though a great number of these
antagonists are potent and non-competitive, still many of them can also affect other purinergic
receptors such as P2X4, P2X2 and P2Ys (233). It is evident that pharmacological inhibition
of P2X7 is effective in decreasing the inflammatory responses elicited by the activation of
P2X7. Therefore, the pursuit to develop potent, selective and safer antagonists for P2X7 is still
on-going.
In contrast, there are not many specific agonists of P2X7. Even ATP, the endogenous activator
of P2X7 can stimulate the receptor only at high concentrations (EC50 ≥ 100µM) (134, 234).
ATP is also easily hydrolysed by ectonucleotidases, therefore the concentration of extracellular
ATP is reduced down to nanomolar or low micromolar concentrations, which are more suitable
concentrations for activating other purinergic receptors such as P2Y(235). Due to this reason,
non-hydrolysable ATP analogues such as ATPɤS are better suited for experimental evaluation
of P2X7 pharmacology, although ATPɤS is not a very potent activator of P2X7 (236). BzATP,
the benzoyl benzoyl derivative of ATP, is 10-30 times more potent than ATP and also offers
better selectivity for human P2X7 in comparison to other purinergic receptors (136). BzATP
does get metabolised to other adenine derivatives and also has an effect on other purinergic
34
receptors including P2X1, P2X2, P2X3 and P2X4. There are other non-nucleotide compounds
that can act as positive allosteric modulators of P2X7 (237). Studies have shown that
cathelicidin or LL-37 (the antimicrobial polypeptide found in macrophages), is an activator of
P2X7 receptors and has been found to induce IL-1β maturation and release (238). P2X7
potentiation by LL-37 inhibits macrophage pyroptosis and improves the survival of
polybacterial septic mice (239).It has been found that P2X7 receptors are critical in mediating
Aβ-mediated chemokine release, in particular CCL3, which is associated with recruitment of
CD8+ T cells (163, 240). Cytoplasmic factors such as Alu-RNA accumulation in the cytoplasm
have also been suggested to activate P2X7 activation independent of ATP release (241). A
number of studies have provided evidence that activation of P2X7 by nucleotide as well as
non-nucleotide agonists can have anti-viral, anti-parasitic and anti-inflammatory effects.
From the above analysis of literature, it can be concluded that P2X7 and its related purinergic
receptors play an important role in inflammatory responses. Therefore, modulation of P2X7
receptors via appropriate pharmacological modulators may provide an effective therapeutic
mechanism for modifying downstream inflammatory responses. The current thesis has
therefore investigated unrelated compounds; minocycline and protopanaxadiol ginsenosides,
for their potential to target and modulate P2X7 receptors.
35
1.8 Overall Aims
1 To characterise the inhibitory action of tetracycline antibiotics, specifically minocycline
on P2X7.
2 To investigate selectivity of tetracyclines on other purinergic receptors (P2X4 and P2Ys).
3 To investigate the effect of ginsenoside formulation G115 and purified ginsenoside
compounds Rb1, Rh2, Rd and compound K on purinergic receptor responses.
4 To investigate the effect of selected ginsenosides on P2X7 mediated physiological
responses in the J774 macrophage cell line and primary peritoneal macrophages.
Aims 1 and 2: Investigation and characterisation of P2X7 as a molecular target of minocycline
have been addressed in Chapter 3.
Aim 3: The effect of ginsenosides on P2X receptors has been discussed in Chapter 4.
Aim 4: The effect of selected ginsenosides on physiological responses mediated by P2X7
receptor in macrophages has been addressed in Chapter 5.
36
CHAPTER 2
MATERIALS AND METHODS
Materials
2.1 Drugs and Reagents
Dulbecco’s Modified Eagle’s Medium (DMEM), Hank’s Buffer Saline Solution (1X), HEPES
(1M), Trypsin-EDTA (0.25%, phenol red) were from Gibco™. Lipofectamine® 2000, Opti-
MEM, Penicillin/Streptomycin, Geneticin® (G4118) were obtained from Life Technologies
(Thermo Fisher scientific), Australia.
DMEM: F12, RPMI 1640 and Foetal Bovine Serum were obtained from Bovogen, Australia.
ATP (A7699), ADP (A2754), UTP (U6875), suramin (S2671), MINO (M9511), tetracycline
(T7660), DOX (D9891) and tigecycline (PZ0021) were purchased from Sigma.
AZ438079, AZ10606120 and 5-BDBD were purchased from Tocris Biosciences, UK.
YO-PRO®-1 Iodide (Y3603), Fluo-4 AM, Fura-2 AM, cell permeant (F-1201), NucBlue® Live
ReadyProbes® Reagent CellEvent™ (C37605), NucView 488 Caspase-3/7 Green Detection
Reagent (C10423), CM-H2DCFDA and MitoSOX™ Red were obtained from Molecular
Probes™ Thermo Fisher Scientific, Australia. The CellTiter 96® AQueous Non-Radioactive
Cell Proliferation Assay kit was obtained from Promega, Australia. Ginsenosides (certified as
98 % pure) Rd, Rb1, Rh2, Compound K were obtained from Chengdu Biopurify Chemicals
Ltd, China. PPD1 and Rf were obtained from Sichuan Weikeqi Biological Technology Co.
Ltd., China.
37
2.2 Chemicals
NaCl, KCl, Na2HPO4, KH2HPO4, CaCl2.2H2O, MgCl2.6 H2O, Na2HPO4, KH2PO4, NaOH,
NH4Cl, NaHCO3 (analytical grade) were obtained from Merck, VIC, Australia. HCl and
glucose were from Sigma-Aldrich, Australia. HBSS (Hank’s Buffered saline solution) and
HEPES were from Gibco™ (Fisher Scientifics) Australia.
Methods
2.3 Cell culture
Mouse microglial BV-2 cells and HEK-293 cells were maintained in DMEM: F12 media (Life
Technologies) supplemented with 10% foetal bovine serum (Bovogen, Australia) and 100 U/ml
penicillin plus 100 µg/ml streptomycin (Life Technologies). J774 macrophages and primary
peritoneal macrophages were maintained in RPMI 1640 media supplemented with 10% foetal
bovine serum (Bovogen, Australia) and 100 U/ml penicillin plus 100 µg/ml streptomycin (Life
Technologies). Flp-In™ 293 T-REx cell lines were used for generation of stable rat TRPV-1
cell line that ensured homogenous expression of TRPV-1 from Flp-In™ expression vector.
These cells contained a single stably integrated FRT site at a transcriptionally active genomic
locus. Targeted integration of Flp-In™ expression vector ensured high-level expression of the
gene of interest. The Flp-In™ T-REx™-293 cell line contained pFRT ⁄ lacZeo and pcDNA™6
⁄ TR (from the T-REx™ System) stably integrated (https://www.thermofisher.com). Co-
transfection of the Flp-In™ Cell Lines with the Flp-In™ expression vector and the Flp
recombinase vector, pOG44, resulted in targeted integration of the expression vector to the
same locus in every cell, ensuring homogeneous levels of gene expression. Transient
transfections of HEK-293 cells were performed in 35 mm petri dishes using 0.1-1 μg of plasmid
DNA and Lipofectamine 2000 as per the manufacturer’s instructions. Stable cell lines
38
expressing human P2X7 and human P2X4 were established by clonal dilution and kept under
selection using 400 μg/ml geneticin (Life Technologies). Cells were plated into poly-D-lysine
coated 96 well plates (Thermo Fisher Scientifics) at optimised specified cell densities and
incubated overnight before experiments.
2.4 Transfections
HEK-237 cells have been used to overexpress P2X receptors. HEK-237 cells do not express
P2X receptors endogenously (242). Thus, overexpressing the P2X receptors in HEK-237 cells
allows to study the characteristics and functions. The study of structure and function of P2X
would not be as effective if the cell line used for transfection already expressed P2X receptors
as the it would be difficult to isolate the responses from P2X receptors transfected from those
endogenously expressed. The plasmids for P2X7, P2X4 and P2X2a were originally obtained
from Prof Annmarie Surprenant. The stable cell lines were generated in the lab following the
described method. Cells were seeded in 35 mm dishes and allowed to grow overnight. A
cocktail of 1 μg DNA and 3 μl Lipofectamine 2000 (Invitrogen) was prepared in Opti-MEM
and incubated at room temperature for 10 minutes. This mixture was added to the petri dish
containing cells. The cells were left in a CO2 incubator for 37 oC and media was changed after
24 hours. The cells were returned to the incubator for another 24 hours. For transient
transfections the transfected and mock cells were plated in 96 well plates and left to grow
overnight in a CO2 incubator (5% CO2 levels) and used to run the assay the next day. In order
to select stably transfected colonies of cells, 800 μg/ml of G418 (Geneticin®) was added to the
cells. When grown in medium containing Geneticin® selection agent, stable colonies of cells
expressing resistance markers were generated in 2-3 weeks. The selection of transfected cells
was based upon 100 % death of mock transfected cells. To generate single cell colonies, the
cells were plated in 96 well plates using limiting dilution. This method allowed the isolation of
each individual cell that carried selection marker by plating them at very low cell densities (~1
39
well per well in 96 well plates) and expanding colonies from those single cells in separate
wells. The cells transfected with P2X receptor were tested for their functional responses by
stimulating them with ATP and measuring their dye uptake (macropore formation measured
by YOPRO-1 uptake, details in section 2.5) on Flexstation III. The clones that had good
functional responses were grown in T 25-flasks and cryopreserved for future use.
2.5 Dye uptake experiments
For experiments with stably or transiently transfected HEK-293 cells, cells were plated at a
density of 2-2.5 x 104 cells per well in DMEM: F12 media. BV-2 microglia were plated at
density of 5 x 103 cells/ well in DMEM: F12 media and the J774 macrophages were plated at
a density of 2 x 104 cells/well in RPMI media in 96-well plates (Thermo Fisher Scientific,
Scoresby, Victoria, Australia). For testing the effect of MINO on dye uptake, media was
replaced with low divalent cation buffer (145 mM NaCl, 5 mM KCl, 13 mM D-glucose, 10
mM HEPES and 0.2 mM CaCl2, pH 7.3) containing 2 µM YOPRO-1 iodide (±MINO) for 30
minutes at 37 oC before the dye uptake was measured. For experiments with ginsenosides, the
cells were either pre-incubated with drugs (made up in YOPRO-1 containing low divalent
cation buffer) or were co-injected with the agonist automatically using a Flexstation III
microplate reader (Molecular Devices, Sunnyvale, CA, USA). The dye uptake over time was
recorded with excitation at 488 nm and an emission filter at 520 nm on the Flexstation III.
Basal fluorescence measurements were acquired for 40 seconds followed by automatic
injection of ATP (1 mM final concentration) and the kinetic measurement of fluorescence
intensity was performed for 300 seconds using Softmax Pro software. Dye uptake responses
were calculated as area under the curve from 50-300 seconds (or 50-180 seconds) using
normalised data.
40
2.6 Measurement of Ca2+ concentration using Fura-2 AM.
Cells were loaded with a 2.25 µM Fura-2 acetoxymethyl (AM), 0.001 % Pluronic solution in
HBSS buffer containing zero Ca2+. Fura-2 AM was loaded on the cells for 40 minutes at 37 °C.
Loading buffer was removed and replaced with standard extracellular assay buffer (145 mM
NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 13 mM glucose, 10 mM HEPES, pH 7.3). Cells
were pre-incubated with drugs for 30 minutes at 37° C before measurements started and ATP
was injected automatically after 15 seconds in case of MINO experiments. In assays involving
ginsenosides, the pre-incubation time was 10 minutes and in most cases the drugs were co-
injected with agonist automatically using Flexstation 3. Fluorescence was measured using a
485 nm excitation.
2.7 Immunostaining and flow cytometric analysis
The cells were detached from the cell culture flask by using either a sterile scraper or Trypsin-
EDTA. A cell pellet was obtained by centrifuging the cell suspension at 10,000 x g for 5
minutes. The cell count and cell viability were checked using Muse® Cell Analyzer or
haemocytometer. The cell suspension (final cell count-5 x 105cells/tube) was added to 12 x 75
mm round bottom test tubes for staining purposes. For staining proteins on the cell surface, the
cells were washed with 0.5 % BSA in PBS. The cell pellet obtained after centrifugation at
10,000 x g for 5 minutes was re-suspended in pre-determined optimal concentration of a
required antibody or appropriate negative control. If the primary antibody was labelled with a
fluorochrome as in case of P2X7 antibody L4-FITC, then incubation on ice for 1 hour followed
by washing with BSA / PBS made the cells ready for flow cytometry. The washing step
involved removing the solution contacting antibody and replacing it with 300 µl of BSA/PBS
solution. The base of each test tube was flicked gently so as resuspend the cell pellet. The test
41
tubes were centrifuged at 10,000 x g for 5 minutes. The supernatant was removed and replaced
by 300 µl of PBS solution. If the primary antibody was not pre-labelled, then the cells were
washed with PBS / BSA twice and then appropriate secondary antibody was added. Cells were
incubated on ice for 30 minutes, washed with PBS / BSA and then re-suspended in 300 μl of
PBS for flow cytometry analysis. For staining of intracellular proteins, the BD Cytofix /
Cytoperm™ kit was used. The cells were washed with 1 x BD Perm/Wash buffer to obtain a
pellet. The cells were fixed by incubating on ice with 250 μl of BD Cytofix / Cytoperm™
solution. Permeabilization of fixed cells was one by washing 2 times in 1 x BD Perm/Wash
buffer (e.g.1ml/wash/tube). Cells were pelleted. The fixed/permeabilized cells were thoroughly
re-suspended in 50 µL of BD Perm/Wash buffer containing a pre-determined optimal
concentration of a required antibody or appropriate negative control. The cells were incubated
for1 hour on ice. The cells were washed 2 times with 1 x BD Perm/Wash buffer (1
ml/wash/tubes). Next, the secondary antibody was added and incubated on ice for 30 minutes.
The cells were washed twice with 1 x BD Perm / Wash buffer as previously and then
resuspended in staining buffer prior to flow cytometric analysis.
2.8 Enzyme-Linked Immunosorbent Assay (ELISA)
Cells were grown in 24 wells plates at densities of 3 x 105 cells/ml. Drugs were added to the
media at appropriate pre-calculated concentrations. The cells were incubated with the
appropriate drugs (LPS/MINO /ginsenosides) for 4 hours at 37 oC in a CO2 incubator. After 4
hours, the plates were removed, and ATP was added to “test” wells. The plates were returned
to the incubator for 30 minutes. The plates were again taken out after ATP treatment and the
media was collected from each well in labelled Eppendorf tubes. The tubes are centrifuged at
252 G for 5 minutes to obtain a pellet of cells. The supernatants were collected in fresh
Eppendorf tubes and stored at -80 oC till the ELISA was to be performed. The BD OptEIA was
used for detection of IL-1β secretion by microglial / macrophage cells. The kit contained
42
coating buffer, assay diluent, wash buffer and stop solution. The ELISA plates were coated
with capture antibody (1:250) at 4 oC overnight. The plate was washed three times with wash
buffer. The washing of the plates was done by adding 200 µl of washing buffer in each well
using a multichannel pipette. The plate was then kept on a rocker (BioNova lab rocker) at 20
G for 5 minutes. The washing solution was removed by tipping the solution out of the plate
gently in a waste container. The step was repeated 3 times. After the final wash, blocking buffer
(200 μl) was added to each well of the 96 well ELISA plate and incubated for 1 hour at room
temperature. The plates were washed twice with washing buffer and samples and standards
were added for 2 hours at room temperature. The samples and standards were aspirated, and
plates were washed 5 times with washing buffer before adding 100 μl detection antibodies
(1:1000) to each well. After an incubation period of one hour, the detection antibody was
aspirated out and wells were washed 5 times. Diluted streptavidin-HRP (100 μl) was added to
each well and incubated for 30 minutes. Washing steps were repeated 7 times before adding
100 μl substrate solutions to each well. The plates were incubated in dark at room temperature.
The reaction was stopped after 30 minutes by adding stop solution. Absorbance was read at
450 nm in a Clariostar plate reader. This method is called sandwich ELISA (Figure 2.1).
43
Figure 2.1 Sandwich ELISA protocol (243)
A known quantity of capture antibody is bound to the surface of the ELISA maxisorp plate.
The sample with the antigen is added to the plate and is captured by the antibody on the surface
of the plate. A specific antibody (called detection antibody) is added which binds to antigen-
antibody complex. Due to this reason, this method is called the 'sandwich' as the antigen is
sandwiched between two antibodies. The detection antibodies are generally enzyme-linked
secondary antibodies. The enzymes can catalyse a fluorescent or electrochemical reaction. The
absorbance or fluorescence or electrochemical signal of the plate wells determines the quantity
of antigen.
2.9 MTS cell viability assay
The cell viability and cell numbers were determined by using the CellTiter 96® AQueous Non-
Radioactive Cell Proliferation Assay. The CellTiter 96® AQueous Non-Radioactive Cell
Proliferation Assay is a colorimetric method for determining the number of viable cells in
proliferation assays. It is composed of solutions of a tetrazolium compound [3-(4, 5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner
salt; MTS] and an electron coupling reagent (phenazinemethosulfate; PMS). The tetrazolium
was converted into soluble formazan by mitochondrial dehydrogenases of viable cells (Figure
2.2). Thus, the absorbance measured at 490 nm was a direct measure of viability as non-viable
cells do not bio-reduced this dye.
44
For determining the cell viability following treatment with drugs, the cell suspension
containing 1 x 105 cells/ml of BV-2 microglia and 1 x 106 cells/ml of HEK-293-
transfected/J774 macrophages were prepared and 50 µl of this suspension was added per well
so that the final seeding density was halved. The cells were allowed to grow in a CO2 incubator
for 24 hours. MTS solution (20 µl) was added to media in each well 4 hours before the
stipulated end point time. The plate was kept in the dark in a CO2 incubator for these 4 hours.
At the end of the incubation time, the absorbance was read at 490 nm in a Clariostar plate
reader.
Figure 2.2 Schematic representation of the MTS cell viability detection method
The CellTiter 96® AQueous One Solution Cell Proliferation Assay is a colorimetric method
for determining the number of viable cells in proliferation or cytotoxicity assays. The MTS
tetrazolium compound is reduced by cells into a coloured formazan product which is soluble
in tissue culture medium. This conversion is likely caused by NADPH or NADH produced by
dehydrogenase enzymes in metabolically active cells.
45
2.10 Measurement of caspase 3/7 activity using ImageXpress
NucView™ 488 Caspase-3 substrate detects caspase-3 activity within individual intact cells
without inhibiting caspase-3 activity. The substrate consists of a fluorogenic DNA dye and a
DEVD substrate moiety specific for caspase 3. The substrate, which is both non-fluorescent
and non-functional as a DNA dye, rapidly crosses cell membranes to enter the cytoplasm,
where it is cleaved by caspase-3 to form a high-affinity DNA dye that stains the nucleus bright
green. Thus, the NucView™ 488 caspase-3 substrate is bi-functional, allowing detection of
intracellular caspase-3 activity and visualization of changes in nuclear morphology during
apoptosis. NucBlue® Live ReadyProbes® Reagent is a temperature stable cell permeant nuclear
counter stain that emits blue fluorescence when bound to DNA. When used in conjunction with
NucView™ 488, the total number of live cells and their corresponding caspase 3/7 activity can
be measured.
J774 cells in 96 well glass bottomed plates (1.5-2 x 104cells/well) were loaded with NucBlue
(nuclear dye) and 1µM NucView 488 (caspase 3/7) for 40-45 minutes at room temperature.
Compounds were then added, and images were taken following excitation at the relevant
wavelengths (x10 magnification) using the ImageExpress Micro imaging system.Two sites per
well were used for the analysis (n = 500-700 cells). Experimental solution used in these assays
was HBSS saline containing 1mM MgCl2 and 2 mM CaCl2 supplemented with 20mM HEPES,
pH 7.3 with 5M NaOH.
2.11 Measurement of current amplitudes using IonFlux-16 system.
For whole cell patching, a fully integrated automated patch clamp system called the IonFlux
system was used. The 96-well IonFlux plates which record currents in a 20-cell ensemble
format were used. Each pattern in an IonFlux plate has 8 compound wells, 2 trap recordings
filled with intracellular fluid, one inlet for cells and intracellular solution and one outlet for
46
waste.IonFlux-16 plates were emptied, filled with reagents and compounds and primed before
preparing the cells. The HEK-293 cells stably expressing P2X7 or P2X4 were used at a cell
density of 4-10 x 106 cells/2 ml. The cells were grown in T-175 cm2 flask and on the day of the
experiment, complete media was removed, and the cells are washed with 10 ml of warm Ca2+
and Mg2+ free D-PBS. The D-PBS was then replaced by 5 ml Tryp-LE. The cells were
incubated at 37 oC for 5-6 minutes. The dislodged cells were then pelleted and re-suspended in
extracellular solution (4 mM KCl, 138 mM NaCl, 1mM MgCl2, 1.8mM CaCl2, 5.6 mM glucose,
10 mM HEPES, pH 7.45, Osm - 285-290 mOsm), so that the final cell density is 2.5 x 106 cells/
ml. Cell suspension (250 µl) was added to each “IN” well of the Ion-flux 16 plate. The plate
was loaded on to the IonFlux Automated Patch Clamp System. The drug solutions were
prepared in 2 x concentrations. ATP was applied first and the responses (current amplitude)
were measured. Then the cells were washed out with running buffer for 2 minutes. ATP was
then reapplied in the presence of various ginsenosides. Current amplitudes were then measured
for each condition using IonFlux software. Data was expressed as fold change in current
amplitude.
Figure 2.3 The special FluxIon plates used for automated whole cell current
measurement and the pattern within each plate.
Each pattern in an IonFlux plate has 8 compound wells, 2 trap recordings filled with
intracellular fluid, one inlet for cells and intracellular solution and one outlet for waste.
(https://ionflux.fluxionbio.com/library-docs)
47
2.12 Cellular reactive oxygen species (ROS) measurement using CM-H2DCFDA
CM-H2DCFDA is a chloromethyl derivative of H2DCFDA (2’, 7’ –dichlorodihydrofluorescin
diacetate) which is a chemically reduced form of fluorescein used as an indicator for reactive
oxygen species (ROS) in cells, such as detection of reactive oxygen intermediates in
neutrophils and macrophages. CM-H2DCFDA passively diffuses into cells, where its acetate
groups are cleaved by intracellular esterases and its thiol-reactive chloromethyl group reacts
with intracellular glutathione and other thiols. Subsequent oxidation yields a fluorescent adduct
(DCF) that is trapped inside the cell, thus facilitating long-term studies. DCF is a highly
fluorescent compound, which can be detected by fluorescence spectroscopy with maximum
excitation and emission spectra of 492–495 nm and 517–527 nm respectively.
For determining cellular ROS production by macrophages in the presence of ginsenosides, the
cells were plated in black 96 well clear bottom plate (Costar®) at a seeding density of 5 x 105
cells per ml and were allowed to grow in a CO2 incubator overnight. ROS indicator CM-
H2DCFDA (10 mM stock in DMSO) was diluted to 10 µM (2 x concentrations) using Krebs-
HEPES buffer. The dye (50 µl of 2 x concentration) was added to each well. Ginsenosides (CK,
Rd, Rb1, Rh2 and PPD1), ATP and positive control H2O2 were all prepared in 2 x
concentrations and added on the dye. This diluted the dye as well as the drugs to the appropriate
working concentration. The fluorescence was read from time point zero (on addition of drugs)
and the readings were taken every 15 minutes for 2 hours. The plate was placed at 37 oC in
between reads.
48
2.13 Mitochondrial superoxide measurement using MitoSOXTM Red
MitoSOX™ Red is a mitochondrial superoxide indicator (MW = 759). The production of
superoxide by mitochondria can be measured using the MitoSOX™ Red reagent as it can
permeate live cells and selectively target mitochondria. It is rapidly oxidized by superoxide but
not by other reactive oxygen species (ROS) and reactive nitrogen species (RNS). The oxidized
product is highly fluorescent upon binding to nucleic acid. For measuring mitochondrial
superoxides, 50 μg of MitoSOX™ were dissolved in 13 μl of dimethylsulfoxide (DMSO) to
make a 5 mM MitoSOX™ reagent stock solution. MitoSOX™ reagent working solution was
prepared by diluting the 5 mM MitoSOX™ reagent stock solution in Krebs-HEPES to make a
5 μM concentration. For measuring superoxide production by macrophage mitochondria in the
presence of ginsenosides, the cells were plated in black 96 well clear bottom plates (Costar®)
at a seeding density of 5 x 105cells per ml and were allowed to grow in CO2 incubator overnight.
Mitochondrial ROS indicator MitoSOX was diluted to 10 µM (2 x concentrations) using Krebs-
HEPES buffer. 2 x dye (50 µl) was added to each well. Ginsenosides (CK, Rd, Rb1, Rh2 and
PPD1), ATP and positive controls (Antimycin-A, FCCP) were all prepared in 2 x
concentrations and added on to the dye. This diluted the dye and the drugs to the appropriate
working concentration. The fluorescence was read from time point zero (on addition of drugs)
and the readings taken every 15 minutes for 2 hours. The plate was placed at 37oC in between
reads. The mitochondrial superoxide release was quantified as fold change in ROS release from
time point zero to the last measured time point.
49
2.14 Rat macrophage isolation
Wistar rats (control rats from experiments conducted in A/Prof. Sarah Spencer’s laboratory)
were used to collect peritoneal macrophages. The dead animal was transferred in to a sterile
fume hood and the abdomen area cleaned with 70% ethanol. A lateral incision was made in the
abdominal wall with sterile equipment and conditions. The abdominal cavity was washed with
10 ml warm sterile PBS and the cell suspension was collected in a 15 ml sterile falcon tube.
The cell suspension was centrifuged at 10,000 x g for 10 minutes. The cell pellet was then re-
suspended in 1 ml RPMI-1640 media. Cells were counted using a haemocytometer (only the
large cells were counted). The cells were plated out in a specific cell number as required by
each experimental set up (For example- 2.5 x 105 cells/well were seeded for Fura-2 AM assay).
Cells from each animal were considered as a separate biological sample.
Note: If there was red blood cells (RBC) contamination, 2 ml of RBC lysis buffer was added
to the cell pellet after the centrifugation step and incubated at room temperature for 10 minutes
on a rocker to lyse the red blood cells. Centrifugation and washing with PBS was done to
remove RBCs completely.
2.15 Statistical analysis
All graphs were plotted using GraphPad Prism versions 6 or 7 (La Jolla, USA). For
concentration-response graphs, concentrations were first transformed using the in-built
function X = log[X] and plotted against response (either raw data or normalised data). Curve
fitting was performed using the built-in dose – response – stimulation function using a four-
parameter fit with variable slope. The equation used to fit raw data is
Y = Bottom + (Top-Bottom) / (1 + 10^ (LogEC50 – X) * hillslope) )
The equation used to fit normalised data is
Y = 100 / (1 + 10^ ( (LogEC50 – X ) )
50
EC50 values were defined as the concentration of agonist that gives a response halfway between
top and bottom of the curve and were calculated as part of the curve fitting. Data were analysed
for statistical significance using one way or two-way ANOVA with post hoc test as appropriate.
Significance was taken as P˂0.05.
51
CHAPTER 3
EFFECT OF TETRACYCLINE DERIVATIVES ON PURINERGIC
RECEPTORS
3.1 Introduction
Minocycline: an anti-inflammatory and neuroprotective tetracycline antibiotic
Minocycline (MINO) is a second generation, semi-synthetic, broad-spectrum bacteriostatic
antibiotic derived from four carbon rings containing polyketide, tetracycline, a naturally
occurring antibiotic (Figure 3.1) (244, 245). The antibiotic properties of MINO can be
attributed to its ability to inhibit protein synthesis in bacteria by binding to the 30S ribosomal
subunit, thereby preventing the tRNA from binding to the ribosomal complex (246). MINO
exhibits a good pharmacokinetic profile with rapid and complete absorption, almost 100%
bioavailability and a long half-life (247-249). It is used clinically for the treatment of acne
vulgaris, some sexually transmitted diseases and rheumatoid arthritis (250-253). It is well
tolerated by individuals of all ages and has a good safety profile, which makes it a
recommended drug by US FDA (Food and Drug Association, United States) (254).
Figure 3.1 Chemical structure of MINO (C23H27N3O7)
More recently MINO has attracted interest because of its anti-inflammatory and anti-apoptotic
actions independent of its antibiotic properties. It is highly lipophilic allowing it to cross the
52
blood-brain barrier and inhibit microglial activation (255-258) , amyotrophic lateral sclerosis
(259-262), depression (263, 264), Parkinson’s disease (265-268), schizophrenia (269), stroke,
autism and fragile X-syndrome (254, 270-272). Furthermore, MINO is under clinical
assessment as an adjuvant treatment for bipolar depression (271, 273), multiple sclerosis (274),
bipolar disorders (275) and schizophrenia (276). It is in early clinical trials for schizophrenia,
ischaemic stroke and pain (277-279). Meta-analysis of randomised clinical trials before
September 2017 has indicated that MINO has a significant anti-depressant effect and good
tolerance in comparison to a placebo in humans (271). A randomised, double-blind placebo-
controlled trial of MINO on at-risk mental state (ARMS) people has indicated the beneficial
effects of this anti-inflammatory drug in delaying the manifestation of psychosis (280).
The mammalian CNS consists of two main cell types: neurons and glia. Glial cells are sub-
divided into microglia, Bergmann cells (cerebellum) and Müller cells (only in the retina) (281).
Microglia are the immunomodulatory cells of central nervous system (282). In case of injury
or insult to the CNS, microglia become activated and change into motile, phagocytic and
cytotoxic cells, which infiltrate the site of injury (283). The levels of ATP around glial cells
can increase due to either neuronal release from synapses and along axons, from astrocytes via
P2 purinergic receptors or through cellular damage (284). The elevated levels of ATP activate
the purinergic receptors in microglia leading to increases in intracellular Ca2+ (285). The Ca2+
influx further affects a number of downstream pathways leading to inflammation or apoptosis
(286). In vitro studies have shown that MINO can block LPS-induced inflammatory cytokine
release (287-289). In one study, IFN-α was used to induce depression-like behaviour in mice
and it was observed that MINO was able to downregulate these pro-inflammatory signals,
thereby alleviating symptoms of depression (290). Microglial activation and glutamate
receptor-1 phosphorylation has been observed in the brains of chronically stressed mice and
the cognitive disturbances were seen to be ameliorated by MINO treatment (291). Microglial
53
activation has also been observed in schizophrenia and MINO administration attenuated
microglial proliferation and behavioural symptoms in a mouse model of schizophrenia (292).
The basic hypothesis tested by many of the studies mentioned above is that microglial
activation and proliferation occur in the brain during stress, injury or ischemia. The activated
microglia produce inflammatory cytokines and these inflammatory signals can ultimately cause
irreparable neuronal cell injury and MINO can prevent this by inhibiting the microglial
activation (Figure 3.2) (254).
Figure 3.2 A schematic diagram displaying P2X7 and associated signalling pathways.
MINO may affect the P2X7 channel activity directly or the associated membrane pores in order
to inhibit the downstream signalling pathways, thereby preventing pro-inflammatory responses
and apoptosis.
54
Possible molecular targets of MINO
Many of the reported cellular actions of MINO include inhibition of protein kinase C (289,
293), p38 and other MAP kinases (294-296), cytochrome c (297) along with cytokine release,
nitric oxide and ROS production, as reviewed in (279, 298). There is some evidence of MINO
directly targeting membrane metalloproteinases (MMP) thereby preventing the T- lymphocytes
from migrating through the fibronectin matrix, making MINO efficacious in treating diseases
such as multiple sclerosis, cerebral haemorrhage, encephalitis and Fragile X-syndrome (299).
Nanomolar concentrations of MINO have been seen to successfully inhibit poly(ADP-ribose)
polymerase-1 (PARP-1), the enzyme involved in DNA damage-induced cell death and
inflammation, providing some explanation to the anti-inflammatory and neuroprotective
activities of MINO (300, 301). However, it is currently unclear whether direct inhibition of
PARP-1 underlies the clinical effects of MINO. Besides PARP-1, MINO has not yet been
shown to act directly on most of the reported targets and therefore inhibition of such signalling
pathways may be indirect. Indeed, MINO may act on multiple targets to evoke its
neuroprotective action (254).
As mentioned earlier, microglia are known to endogenously express a heterogeneous collection
of purinergic receptors including ionotropic P2X7, P2X4 as well as metabotropic P2Y
receptors (117-119, 302). The expression of purinergic receptor P2X7 is known to increase in
activated microglia (303). These ATP-activated ion channels are coupled to many of the
intracellular signalling events that MINO is reported to block including microglial activation
and proliferation (116), ROS production (158), apoptosis (304) and cytokine secretion (304).
Furthermore, P2X7 and P2X4 receptors have been implicated in the pathology of many of the
diseases for which MINO has reported efficacy (305, 306). In a study conducted by Itoh et al.,
activation of P2X7 receptors in vivo was involved in the development of central sensitization
in an acute inflammatory pain model. In this study, the activation of P2X7 by specific agonists
55
led to an increase in central sensitization and blocking by P2X7 inhibitors and MINO reduced
this inflammatory pain (307). From this study, it was concluded that the P2X7 receptors are
mainly expressed on the microglia in the medullary dorsal horn and that they play a central role
in nociceptive responses. In another study investigating the rat model of chronic prostatitis, rats
induced with the disease had higher levels of inflammatory cytokines and blocking of microglia
by MINO or a P2X7 antagonist downregulated this cytokine production. The results from this
study suggested that microglia and P2X7 activation are involved in pain associated with
chronic prostatitis (308)
3.2 Rationale for the research
A large array of research indicates that MINO acts on microglia and downregulates
inflammatory cytokine production. P2X7 receptors are located on glia and are involved in
chronic inflammatory signalling pathways. Therefore, this project investigated whether
MINO’s inhibitory action on microglial activation works through direct blocking of the
purinergic P2X7 ion channels or the associated signalling pathways.
In order to attain a better insight into the selectivity and structure-function relationship of the
drugs towards these ion channels, the effects of tetracycline and two of its other derivatives
(doxycycline and tigecycline) on P2X7 and its close relative, P2X4 were also been tested.
The data from this study may help elucidate the underlying mechanism of action of MINO in
a variety of neurological diseases and conditions.
3.3 Research Question
The present study tested the hypothesis that MINO may be eliciting its inhibitory effect on
microglial activation and proliferation via purinergic ion channels, P2X7 and P2X4.
56
3.4 Results
3.4.1 P2X7 responses in BV-2 microglia
Minocycline (MINO) is known to inhibit microglial activation in vivo and in vitro (309, 310).
In the present study, the BV-2 mouse microglial cell line was used to investigate ATP-induced
responses and the effect of MINO. First, an ATP-mediated dye uptake assay was utilised to
measure P2X responses in BV-2 cells and the increase in YO-PRO™-1 Iodide (YOPRO-1)
fluorescence over time was inhibited with AZ10606120 (Figure 3.3 A), a selective P2X7
inhibitor (211). It was found that MINO effectively reduced ATP-induced responses in BV-2
cells at concentrations >50 µM (Figure 3.3 B and Figure 3.4). Intracellular Ca2+ responses were
measured to ATP in Fura-2 AM loaded BV-2 cells using a plate-reader based assay.
Intracellular Ca2+ increased with time after the application of ATP (Figure 3.5). This ATP-
induced Ca2+ response was a P2X7-mediated response as the sustained phase of Fura-2
fluorescence was abolished in the presence of the P2X7 antagonist AZ10606120 (10 μM). It
was observed that MINO significantly reduced the sustained Ca2+ responses in BV-2 microglia.
57
Figure 3.3 Effect of MINO on P2X7 mediated YOPRO-1 uptake in BV-2 microglial cells
(A) YOPRO-1 uptake was measured in BV-2 cells as an increase in fluorescence after agonist
application at 40 seconds on a Flexstation 3. Cells were pre-incubated with MINO for 30
minutes at 37 oC before ATP (1 mM) injection. (B) The dye uptake was quantified as area
under the dye uptake curve and plotted as bar graph. Control YOPRO-1 uptake to ATP (100
µM and 1 mM) in the absence of drugs is shown in black bar, 10 μM AZ10606120 is shown
in white open bar, in presence of 50 μM is shown in light grey bar and in the presence of 100
μM MINO is shown in dark grey bar. Bars represent mean data from 18-20 wells from 2–3
separate experiments. Data was calculated as area under the normalised curve (50-300
seconds). Error bars represent S.E.M, * P<0.05 calculated by one-way ANOVA (Dunnett’s
post-test).
58
Figure 3.4 Effect of MINO on P2X7 activated by various concentrations of ATP in BV-2
microglial cells
Dye uptake was induced by stimulating cells with different concentrations of ATP (1mM, 500
μM, 250 μM, 100 μM, 50 μM, 25 μM, 10 μM and 1 μM) in low divalent cation solution on a
Flexstation 3. The fluorescence was measured for 300 seconds and non-linear fit of transformed
responses were fitted in GraphPad Prism. MINO at 100 µM concentration increases the EC50
of ATP for P2X7 activation from 184.1 µM to 243.6 µM (95% confidence interval- 300.9 to
701.2). The data was collated from 3 different experiments and the dose response curves were
generated using non-linear fit of transformed data on GraphPad prism software. Error bars
represent S.E.M.
59
Figure 3.5 Effect of MINO on P2X7 mediated Ca2+ influx in BV-2 microglial cells
(A) Intracellular Ca2+ responses were measured in Fura-2 AM loaded BV-2 cells using a
Flexstation 3 plate reader. Fura-2 ratio (340nm/380nm) was normalised to zero baseline. ATP
(1mM) was injected after 30 seconds as indicated by the arrow. (B) represents the effect of
different drugs on the sustained phase of the response to 1mM ATP. Bar charts represent mean
data from 24-32 wells from 4 separate experiments. Ca2+ in the presence of P2X7 agonist ATP
is shown here in black bar, in the presence of 100 µM, 50 µM and 10 µM MINO are
represented by grey bars. The ATP-mediated Ca2+ response was inhibited by AZ10606120.
Data was calculated as mean of sustained responses (100 -300 seconds); baseline corrected.
Error bars represent S.E.M, * denotes P<0.05 calculated by one-way ANOVA (Dunnett’s post-
test). The concentration of ATP added to all treatments is 1 mM.
60
Figure 3.6 Dose dependent inhibitory effect of MINO on P2X7 mediated Ca2+ influx in
BV-2 microglial cells
The inhibitory effect of MINO on the sustained phase of the Ca2+ response to 1 mM ATP in
the presence of various concentrations of MINO was measured using Ca2+ sensitive ratiometric
dye Fura-2 AM. BV-2 microglia pre-treated with various concentrations of MINO (0.1-100
µM) were activated with 1 mM ATP. Fura-2 fluorescence was measured on Flexstation 3. The
IC50 was calculated from the non-linear fit of transformed data. IC50 of MINO was found to be
6.5 µM (95% confidence interval - 0.1 to 1.8). Data collated from 3 independent experiments.
Error bars represent S.E.M.
61
3.4.2 P2X7-mediated pro-inflammatory responses
P2X7 mediates a number of pro-inflammatory events such as release of IL-1 β from ATP
activated immune cells (311). In Figure 3.7, the effect of MINO was evaluated on P2X7-
mediated IL-1β release in LPS-primed BV-2 microglia and it was found that 100 µM MINO
pre-treatment was able to reduce ATP-induced IL-1β release in LPS-primed microglial cells.
An increase in IL-1β levels was observed in ATP treated cells in comparison to control cells.
No significant difference in IL-1β levels was observed between LPS primed and non-primed
cells. Multiple comparisons using one-way ANOVA showed ~ 30% decrease in IL-1β release
between the MINO treated LPS primed ATP activated cells and the non-MINO treated cells.
These results indicate that MINO has an inhibitory effect on pro-inflammatory responses
mediated by P2X7. MINO is known to inhibit microglial activation and proliferation (312). In
Figure 3.8, the effect of varying concentrations of MINO on proliferation rates in BV-2 cells
was measured using the CellTiter 96® AQueous One Solution assay. BV-2 microglia in the
presence of MINO (100 µM and 50 µM; 24 hours) showed a significant decrease in cell number
in comparison to untreated controls. This effect could be P2X7-mediated as A438079 (a P2X7
specific antagonist) also reduced BV-2 cell number in comparison to untreated controls (Figure
3.8). The reduction in cell number was also observed in the presence of the P2X4 antagonist
5-BDBD (Figure 3.8) but the cells showed some morbid changes in morphology suggesting
the involvement of toxicity.
62
Figure 3.7 Effect of MINO on IL-1β release in BV-2 microglial cells as measured by
ELISA
IL-1β levels in each treatment group were measured using a Thermo Fisher Mouse-IL-1β kit
using manufacturer’s instructions. The IL-1β levels in untreated controls are shown as pale
pink while the orange bar represents IL-1β secretions LPS treated cell. IL-1β production in
LPS primed cells activated by 500 µM ATP is shown in black while IL-1β production by cells
activated with LPS + 500 µM ATP is shown in orange with black dots. The IL-1β production
is LPS + ATP treated cells is reduced by treatment with 100 µM MINO (dark grey bar). The
P2X7 antagonist AZ438079 (light grey bar), however, does not significantly reduce IL-1β
release under these conditions. The bar graph was generated from collated data from 3 separate
experiments (n=3). The IL-1 β release was quantified using linear regression equation from a
standard curve. Error bars represent S.E.M, * P<0.05 calculated by one-way ANOVA
(Dunnett’s multiple comparison test).
63
Figure 3.8 Effect of MINO on cell proliferation in BV-2 microglial cells as measured by
Cell Titer 96® AQueous One Solution Assay
CellTiter 96® AQueous One Solution Reagent was used to measure cell viability after 24 h
treatment period. The absorbance was read at 490 nm on a Clariostar microplate reader. Bar
charts represent mean data from 9-12 wells from 3 separate experiments. Data was calculated
by normalising absorbance of media to 100% and the other conditions are plotted as percentage
of media control. The black bar represents the absorbance of untreated control cells (normalised
to 100%). 0.01 % DMSO has no significant effect on proliferation rates (black chequered bar).
The effect could be P2X7 mediated as a reduction in cell number is seen in the presence of
AZ438790 (P2X7 antagonist), shown in white open bar. Reduction in cell number is also seen
with P2X4 antagonist 5-BDBD (light grey bar) but the cells exhibited some abnormal
morphological changes suggesting mechanisms other that inhibition in cell proliferation. The
grey bars represent effect of MINO (different concentrations) and it was seen that MINO
decreases cell proliferation rates in a concentration dependent manner. Error bars represent
S.E.M, * denotes P<0.05 calculated by one-way ANOVA (Dunnett’s multiple comparison
test).
64
3.4.3 Investigating the selectivity of MINO
To investigate these channels individually, recombinant hP2X7 or hP2X4 were expressed in
HEK-293 cells and functional responses were measured using the YOPRO-1 dye uptake assay
(147, 313). The use of such dye uptake assays to measure P2X4 responses is less well defined
in the literature, however, ATP-induced activation of P2X4 does lead to a secondary
permeability state (314) and Bernier et al have demonstrated YOPRO-1 iodide uptake in P2X4-
expressing HEK-293 cells (315). The effect of MINO was first investigated on rat P2X7
(Figure 3.9) and rat P2X4 (Figure 3.10) expressed in HEK-293 cells as preliminary data
obtained in our laboratory on rat primary microglial cells demonstrated an effect of MINO on
ATP-induced dye uptake both in the absence or presence of AZ10606120 (a P2X7 antagonist)
or MINO (Jack Watson, Martin Stebbing, personal communication). Different concentrations
of ATP were used to stimulate the two P2X channels; 1 mM ATP for rat P2X7 and 10 µM ATP
for rat P2X4. AZ10606120 (10 µM) completely abolished the response to ATP only in cells
expressing rat P2X7 (Figure 3.9). AZ10606120 had no effect on rat P2X4 responses,
demonstrating that it is not a P2X7 response (Figure 3.10). MINO (100 µM) significantly
reduced responses at both rat P2X7 and rat P2X4. One-way ANOVA analysis for rat P2X7
responses revealed a 69.5 % decrease between dye uptake in presence of ATP (1 mM) and ATP
+ 100 µM MINO. This was a P2X7 mediated response as P2X7 specific antagonist
AZ10606120 decreased the ATP responses by 96.8 %. Similarly, 100 µM MINO inhibited
ATP (10 µM) responses in HEK- rat P2X4 by 68.8 %.
The effect of MINO was further investigated on human P2X7 (hP2X7) and human P2X4
(hP2X4) as it is well known that many compounds can have substantial species-selective
effects on P2X7 (211) and very little is known about human P2X4 pharmacology. AZ10606120
effectively inhibits rat, mouse and human P2X7 (211, 316) and clearly abolished hP2X7
responses (Figure 3.10). ATP-induced dye uptake via human P2X7 was substantially reduced
65
(~ 70 % decrease) by 100 µM MINO (Figure 3.11). To test the its selectivity for P2X7, MINO
was tested for its effect on closely related purinergic receptor, P2X4. HEK-293 cells expressing
hP2X4 displayed ATP-induced dye uptake that was not blocked by AZ10606120 (10 µM).
MINO (100 µM) showed a significant inhibition of hP2X4 responses (Figure 3.12). Following
the establishment that MINO inhibited both P2X7 and P2X4 mediated YOPRO-1 uptake, a
comparison between the magnitudes of inhibitory effects of MINO for each of these receptors
was done by comparing the area under the dye uptake curves from the above experiments
(Figure 3.11). Multiple comparisons using one-way ANOVA indicated a decrease of 76.11 %
between dye uptake in ATP treated and ATP + 100 µM MINO treated human P2X7 expressing
HEK-293 cells. Even though, MINO was found to inhibit dye uptake in both HEK-P2X7 and
HEK-P2X4, the amount of inhibition was more for P2X7 receptor as compared to P2X4
responses. This was found out by comparing the dye uptake data from the two types of
receptors tested (Figure 3.12). For comparing the level of inhibition caused by MINO for P2X7
and P2X4 mediated dye uptake, the area under the dye uptake curve for controls was
normalised to 100 % and dye uptake by MINO treated cells was plotted as percentage of
control. It was found that 100 µM MINO was able to inhibit ~ 70 % of the dye uptake caused
by ATP in HEK-P2X7 cells while the % inhibition caused by 100 µM MINO in HEK-P2X4
cells was ~ 45 %. Thus, MINO had a larger effect on P2X7 in comparison to P2X4 (Figure
3.13).
66
Figure 3.9 Effect of MINO on YOPRO-1 uptake in HEK-293 cells expressing rat P2X7
YOPRO-1 uptake was measured in HEK-rP2X7 expressing cells as an increase in fluorescence
after agonist application at 40 seconds on a Flexstation 3. Cells were pre-incubated with MINO
for 30 minutes at 37 oC before 1 mM ATP injection. (A) The raw traces of dye uptake from
Flexstation 3 are shown here. The fluorescence increased immediately following injection of 1
mM ATP on to HEK-293 cells expressing rat P2X7 at 40 seconds (black circles) and is
inhibited by MINO (charcoal grey circles) and P2X7 antagonist AZ10606120 (light grey
circles). (B) The dye uptake was quantified as the area under the dye uptake curve and plotted
as a bar graph. Control YOPRO-1 uptake in response to ATP in the absence of drugs is shown
in black, and P2X7 antagonist 10 μM AZ10606120 is shown in grey bar, in the presence of 10
μM MINO is shown in light grey bar and the 100 μM MINO is shown in white. Bar charts
represent mean data from 18-20 wells from 2–3 separate experiments. Data was calculated as
area under the normalised curve (50-300 seconds). Error bars represent S.E.M, *represents
P<0.05 calculated by one-way ANOVA (Dunnett’s post-test).
67
Figure 3.10 Effect of MINO on YOPRO-1 uptake in HEK-293 cells expressing rat P2X4
YOPRO-1 uptake was measured in HEK-rP2X4 expressing cells as an increase in the
fluorescence after agonist application at 40 seconds on a Flexstation 3. Cells were pre-
incubated with MINO for 30 minutes at 37 oC before 10 µM ATP for P2X4 injection. (A)
represents the raw traces from a Flexstation 3. The fluorescence increases immediately
following the injection of 10 µM ATP on to HEK-P2X4 cells at 40 seconds (black circles) and
was inhibited by MINO (grey circles). (B) Quantification of dye uptake was done as the area
under the dye uptake curve and plotted as bar graph. Bar charts represent the mean of data from
18-20 wells from 2–3 separate experiments. Dye uptake in the presence of 10 µM ATP is shown
as a black bar, in the presence of ATP + 10 µM MINO is shown in light grey and in the dark
grey. Data was calculated as the area under the normalised curve (50-300 seconds). Error bars
represent S.E.M, * P<0.05 calculated by one-way ANOVA (Dunnett’s post-test).
68
Figure 3.11 Effect of MINO on ATP-induced dye uptake in HEK-293 cells stably
expressing human P2X7
YOPRO-1 uptake was measured in HEK-hP2X7 pre-incubated with MINO for 30 minutes at
37 oC before 1 mM ATP injection. The raw traces of dye uptake from a Flexstation 3 are shown
here. The fluorescence increased immediately following the injection of 1mM ATP on to HEK-
P2X7 cells at 40 seconds (black circles) and was inhibited by MINO (grey circles) and P2X7
antagonist AZ10606120 (white circles). (B) The dye uptake was quantified as the area under
the dye uptake curve and plotted as a bar graph. Bar charts represent the mean of data from 18-
20 wells from 3 separate experiments. Black bar represents dye uptake in the presence of 1 mM
ATP, white (+ grey error bar) represents AZ10606120, ATP + 10 µM MINO is shown in light
grey and ATP + 100 µM MINO is shown in dark grey bar. Data was calculated as the area
under the normalised curve (50-300 seconds). Error bars represent S.E.M, * P<0.05 calculated
by one-way ANOVA (Dunnett’s post-test).
69
Figure 3.12 Effect of MINO on ATP-induced dye uptake in HEK-293 cells stably
expressing human P2X4
YOPRO-1 uptake was measured in HEK-hP2X4 expressing cells as an increase in fluorescence
after agonist application at 40 seconds on a Flexstation 3. Cells were pre-incubated with MINO
for 30 minutes at 37 oC before addition of 10 µM ATP . (A) The raw traces of dye uptake from
a Flexstation 3 are shown here. The fluorescence increased immediately following injection of
1 mM ATP on to HEK-P2X4 cells at 40 seconds (black circles) and was inhibited by MINO
(charcoal grey circles). (B) The dye uptake was quantified as the area under the dye uptake
curve and plotted as a bar graph. Bar charts represent the mean of data from 18-20 wells from
2–3 separate experiments. Data was calculated as the area under the normalised curve (50-300
seconds). Error bars represent S.E.M, * P<0.05 calculated by one-way ANOVA (Dunnett’s
post-test).
70
Figure 3.13 Comparison of the effect of MINO on ATP-induced dye uptake in HEK-293
cells stably expressing human P2X7 or human P2X4.
MINO attenuates the ATP induced dye uptake or macropore formation mediated by both
human P2X7 and P2X4, but the magnitude of this reduction is greater in HEK-293 cells
expressing P2X7 in comparison to cells HEK-293 expressing P2X4 receptors. This graph
represents the dye uptake by ATP activated HEK-P2X7 and HEK-P2X4. The black bars
represent the dye uptake by control cells in the presence of ATP only (1 mM for P2X7 and 10
µM for P2X4). The open black bars represent dye uptake in cells pre-treated with 100 µM
MINO for 30 minutes and then activated by ATP. For comparison, the area under the dye
uptake curve for controls was normalised to 100 % and dye uptake by MINO treated cells was
plotted as percentage of control. Error bars represent S.E.M, * P<0.05 calculated by one-way
ANOVA (Dunnett’s post-test).
71
3.4.4 Investigating internalisation of P2X7 receptors
It is known that P2X4 channel responses can desensitize rapidly via agonist-induced
internalisation of receptors (317), therefore, the possibility of MINO stimulating internalisation
of P2X7 receptors was investigated. When the surface expression of P2X7 was quantified using
an extracellular domain-binding antibody (clone L4) and live cell flow cytometry, there was
no effect of 100 µM MINO on P2X7 expression (Figure 3.14 B). This suggested that
stimulation of internalisation was not the mechanism by which MINO was affecting dye uptake
responses mediated by P2X7 receptors.
3.4.5 Investigating the effect of MINO on YOPRO-1 fluorescence
To ensure that the inhibition of fluorescence exhibited by MINO was an actual effect on the
channel and not quenching of fluorescence by the drugs, HEK-hP2X7 cells were incubated
with ATP (1 mM or 100 μM) for 5 - 10 minutes at 37 oC to allow YOPRO-1 to be taken up by
the cells. Fluorescence was recorded using the Flexstation and MINO was injected to a final
concentration of 100 μM. If MINO was having a quenching action on fluorescence, it was
expected that the fluorescent signal would decrease. The data show that there was no decrease
in fluorescence signal following MINO injection (Figure 3.14 A). To further ensure that MINO
was not directly acting on the YOPRO-1 fluorescence, dye uptake was measured for HEK-
293 cells pre-incubated with 100 µM and then lysed with a low concentration of detergent,
0.01 % Triton X-100. The rationale for this assay was that if P2X receptors were not required
for MINO action, then MINO would be able to act on YOPRO-1 bound to DNA of lysed cells,
thereby decreasing the YOPRO-1 fluorescence. The results from these experiments (Figure
3.15) show that MINO is unable to cause any decrease in maximum YOPRO-1 fluorescence
of lysed cells, thus indicating that the presence of a specific receptor is required for MINO
action.
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Figure 3.14 MINO does not quench fluorescence of YOPRO-1 dye and does not alter
receptor expression on the cell surface.
(A) The effect of MINO on fluorescence of the YOPRO-1 dye was performed by incubating
HEK-hP2X7 cells with ATP (1 mM or 100 μM) for 5-10 minutes at 37 oC and then injecting
MINO to a final concentration of 100 μM. The treatment of cells with MINO does not show
any reduction in fluorescent signal. The bar charts represent mean data from 6 wells from 2
separate experiments. The black bars represent fluorescence in presence of ATP alone and grey
bars represent fluorescence in presence of ATP + MINO. Data was calculated at 41-60 second
time point as baseline corrected endpoint data. (B) The effect of MINO on expression of P2X7
was measured by flow cytometer with HEK-hP2X7 stable cells. Cells were treated with ATP
+ 100 μM MINO (red trace) or ATP alone (black trace) for 30 minutes and then detached with
0.05 % Trypsin EDTA. The cells were washed with 0.5 % BSA in 0.1 mM PBS and then
incubated on ice with an anti-P2X7-FITC (1:100) antibody or isotype control for 1 hour.
Staining was measured using a BD FACS Canto flow cytometer.
73
Figure 3.15 MINO does not reduce the maximum YOPRO-1 uptake in lysed cells
Triton X-100 (0.01 %) was used to lyse the cell membrane in order to obtain maximum dye
uptake. HEK-P2X7 cells pre-incubated with 100 µM MINO did not show inhibition in dye
uptake providing evidence that MINO acts via P2X mediated responses and not on fluorescence
of the dye itself. Bars represent mean data of 12 wells for 2 separate experiments. Black bar
represents YOPRO-1 fluorescence in the presence of 0.01 % Triton X-100 and grey bar
represents fluorescence in presence of 0.01 % Triton X-100 + 100 µM MINO. Data was
quantified as area under dye uptake curve (baseline corrected). Error bars represent S.E.M, ns
denotes not significant, P<0.05 calculated by one-way ANOVA (Dunnett’s post-test).
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3.4.6 Investigating the pharmacological properties of MINO towards P2X7 and P2X4.
The pharmacological measurements were further extended to perform concentration-responses
for ATP in the absence or presence of increasing concentrations of MINO (10, 50, 200 µM)
for both human P2X7 (Figure 3.16) and human P2X4 (Figure 3.17). MINO showed a non-
competitive inhibition of ATP responses at both P2X receptors and did not fully inhibit
responses at either channel at concentrations up to 200 µM. The IC50 value for the inhibitory
action of MINO for hP2X7 was found to be 23.2 µM (95 % confidence interval - 14.5 to 36.8).
The IC50 value for MINO in hP2X4 was found to be 53.8 µM (95 % confidence interval - 32.4
to 89.5).
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Figure 3.16 MINO acts as a non-competitive antagonist at human P2X7
A concentration-response curve for ATP-induced YOPRO-1 uptake in a HEK-hP2X7 stable
cell line in the absence and presence of increasing concentration of MINO (10 – 200 µM) was
generated. MINO decreased the amplitude of ATP responses with no obvious competitive
effects. Dose responses were generated as non-linear fit of transformed data using GraphPad
Prism software. EC50 for ATP at hP2X7 was 116.9 µM (95 % confidence interval 164.1 – 533.8
µM). (B) An IC50 plot for MINO acting on human P2X7 using a maximum concentration of
ATP (500 µM). Experiments were performed on 4 separate occasions and data collated. Dose
responses were generated as non-linear fit of transformed data using GraphPad Prism software.
IC50 for MINO was 32.6 µM (95 % confidence interval 7.9 – 135 µM).
76
Figure 3.17 MINO acts as a non-competitive antagonist at human P2X4 receptors
(A) Concentration-response curve for ATP-induced YOPRO-1 uptake in a HEK-hP2X4 stable
cell line in the absence and presence of increasing concentration of MINO (0.01 – 10 µM).
MINO decreases the amplitude of ATP responses with no obvious competitive effects.
The EC50 for ATP at hP2X4 was 0.2 µM (95 % confidence interval 0.2 – 0.3 µM). (B) An IC50
plot for MINO acting on human P2X4 using a maximum concentration of ATP (10 µM).
Experiments were performed on 4 separate occasions and data collated. Dose responses were
generated as non-linear fit of transformed data using GraphPad Prism software. IC50 for MINO
was 53.8 µM (95 % confidence interval 32.4 – 89.4 µM).
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3.7 Investigating the effect of tetracycline and its structural analogues on P2X7
Investigations were made into the action of three other tetracycline antibiotics on P2X
receptors. The parent compound tetracycline (TET) and the clinically relevant antibiotics
doxycycline (DOX) and tigecycline (TIGE) were chosen. DOX is a second-generation
tetracycline class antibiotic also known to have inhibitory effects on microglia and anti-
inflammatory effects in diseases such as rheumatoid arthritis, COPD and Lyme disease (276,
318, 319). DOX displayed a similar inhibitory effect on hP2X7 (Figure 3.18) whereas TET and
TIGE had no inhibitory effect (Figure 3.18). There is evidence that DOX is as effective as
MINO in its anti-inflammatory properties. DOX is said to be clinically more suitable as it does
not exhibit any common side effects associated with MINO usage (320-322). Keeping this in
mind, the pharmacology of DOX was further investigated on P2X7 responses. A concentration-
dependent response of DOX on hP2X7 responses was performed (Figure 3.19) and DOX was
found to non-competitively inhibit hP2X7 responses with an IC50 of 36.9 µM (95 % confidence
interval 10.7 – 127 µM).
78
Figure 3.18 Effect of tetracycline class antibiotics on ATP-induced dye uptake in HEK-
P2X7.
YOPRO-1 uptake was induced in HEK-hP2X7 expressing cells by the addition of 1 mM ATP
in low divalent physiological solution. Drugs (MINO, DOX, TIGE and TET) were prepared in
low divalent buffer containing 2 μM YOPRO-1. Cells were pre-incubated with drugs for 30
minutes at 37 oC before ATP injection on Flexstation 3. DMSO (0.01 %) was added as vehicle
control for TIGE and TET. The bar charts represent mean data from 25-30 wells from 4-5
separate experiments. The effects of MINO on P2X7 responses is shown in panel A, TIGE is
shown in panel B, DOX is shown in panel C and TET responses are shown in D. Data was
calculated as the area under the dye uptake curve at 50-300 second time point (baseline
corrected). Error bars represent S.E.M, ** denotes P<0.05 calculated by one-way ANOVA
(Dunnett’s post-test).
79
Figure 3.19 TET analogue DOX also attenuates P2X7 mediated responses
(A) Concentration-dependent responses were measured for ATP-induced YOPRO-1 uptake in
a HEK-hP2X7 stable cell line ± DOX (10 – 200 µM). DOX reduced the amplitude of ATP
responses with no obvious competitive effects. EC50 for ATP at hP2X7 was 110.8 µM. An IC50
plot for MINO acting on human P2X7 using a maximum concentration of ATP (500 µM). The
dose response curves were generated as non-linear fit of transformed data using GraphPad
prism software. Experiments were performed on 4 separate occasions and data was collated.
The IC50 for MINO was 36.9 µM (95 % confidence interval 10.7 – 127 µM).
80
3.8 Validating the effect of MINO and DOX on P2X7 responses using primary
macrophages
In order to further validate the above results, the effect of MINO and DOX on P2X7 responses
was investigated in primary macrophages isolated from the peritoneum of Wistar rats.
Macrophages, just like microglia, endogenously express the purinergic receptors P2Y1, P2Y2,
P2Y4, P2Y11-14, P2X4, and P2X7 (323). Various concentrations of DOX and MINO were
tested on primary macrophages activated by 500 µM ATP. It was observed that both MINO
and DOX dose dependently inhibited rat P2X7 mediated Ca2+ influx in Fura-2 AM loaded cells.
The IC50 for DOX in rat macrophages is 16.0 µM (95 % confidence interval 1.8- 14 µM). The
IC50 for MINO in rat macrophages is 19.1 µM (95 % confidence interval 4.7 – 77 µM) (Figure
3.20).
81
Figure 3.20 MINO and DOX have an inhibitory effect on P2X7 mediated Ca2+ influx in
primary peritoneal macrophages
Concentration response effects were measured for MINO and DOX acting on human P2X7 in
primary peritoneal macrophages activated by 500 µM ATP. Experiments were performed on 2
separate occasions using macrophages isolated from 3 Wistar rats per experiment and data was
collated. The Ca2+ influx was measured by loading the cells with ratiometric Ca2+ indicator
Fura-2 AM. The fluorescence was measured on a Flexstation 3 immediately following the
injection of ATP in cells pre-incubated with MINO or DOX. (A) The graph represents the non-
linear transform of normalised sustained Ca2+ response (100-300 seconds) in rat macrophages
pre-incubated for 15-30 minutes with varying concentrations of MINO (1µM – 100 µM) . The
IC50 for MINO was 19.09 µM (95 % confidence interval 4.7 – 77 µM). (B)The graph represents
the non-linear transform of normalised sustained Ca2+ response (100-300 seconds) in rat
macrophages pre-incubated for 15-30 minutes with varying concentrations of DOX (1 µM-
100 µM). The IC50 for DOX was found to be 16.04 µM (95 % confidence interval 1.8- 14 µM).
82
3.4.9 Investigating the effect of MINO and DOX on P2Y1 and P2Y2 receptors
To establish the selectivity of MINO towards purinergic receptors expressed in microglia, the
effect of MINO (100 µM) was tested on non-transfected HEK-293 cells endogenously
expressing P2Y1 and P2Y2 receptors. The receptors were activated by the P2Y1 agonist ADP,
and the P2Y2 agonists, ATP and UTP (10 µM for each). The effect of pre-incubated MINO
was measured on P2Y1 or P2Y2-mediated Ca2+ responses in HEK-293 cells using Fura-2 AM.
The peak responses were measured, and it was observed that MINO has a significant inhibitory
effect on both P2Y1 as well P2Y2 mediated Ca2+ release from intracellular stores (Figure 3.21).
A mean difference in Fura-2 fluorescence of 97.2 (95 % confidence interval 86 to 108) was
calculated between ATP and ATP + 100 µM MINO treated cells. The mean difference of Fura-
2 fluorescence for ADP activated HEK-293 cells, was found to be 96.9 (95 % confidence
interval 85.9 to 108.1). Finally, the mean difference between UTP and UTP + MINO Ca2+
response was 61.5 (95 % confidence interval 48.6 to 74.4). These results indicate that MINO
inhibits the P2Y mediated release of Ca2+ from intracellular stores.
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Figure 3.21: MINO inhibits P2Y1 and P2Y2 mediated intracellular Ca2+ influx in HEK-
293 cells
The effect of MINO on P2Y1 and P2Y2 mediated Ca2+ responses in HEK-293 cells
endogenously expressing these receptors were investigated. The HEK-293 cells were loaded
with 2 µM Fura –2 AM Ca2+ indicator and stimulated with either 10 µM ATP (P2Y1, P2Y2
agonist) or 10 µM ADP (P2Y1 agonist) or 10 µM UTP (P2Y2 agonist). Responses were
measured as peak Ca2+ influx from 0-100 sec. Bars represents mean of normalised data from
18-20 wells from 3 separate experiments. Data was calculated as maximum peak value
(baseline corrected). , Error bars represent S.E.M, * P<0.05 calculated by two-way ANOVA
(Sidak’s multiple comparison test).
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3.4.10 Investigating the effect of MINO and DOX on TRPV-1 ion channels
The above results indicate that MINO (as well as DOX) may inhibit P2X mediated membrane
pore formation. Even though membrane pore formation is a characteristic feature of P2X7
receptor activation, there are other membrane receptors, which display similar properties. One
such ion channel is TRPV-1 (324). To evaluate whether MINO affects P2X membrane
specifically, drugs were tested on T-REx- HEK-293 cells stably expressing rat TRPV-1 using
the YOPRO-1 dye uptake assay (Figure 3.22). T-REx- HEK rat TRPV-1 cells were plated
overnight. The cells were stimulated with 1µg/ml tetracycline for 4 hours in CO2 incubator.
YOPRO-1 was induced by the addition of 100 nM capsaicin in low divalent physiological
solution. MINO (100 µM) was prepared in low divalent buffer containing 2 μM YOPRO-1.
Cells were pre-incubated with MINO for 30 minutes at 37 OC before application of agonist
ATP using Flexstation 3. DMSO (0.01 %) was added as vehicle control. Capsaicin activated
TRPV-1 membrane formation (indicated by YOPRO-1 uptake) which was effectively
attenuated by the TRPV-1 antagonist CPZ. MINO (100 µM) also inhibited rat TRPV-1
responses to capsaicin (Figure 3.22). A mean decrease of 51.9 % dye uptake was observed in
rat TRPV-1 pre-incubated with MINO before activation by capsaicin.
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Figure 3.22: MINO inhibits dye uptake in T-Rex-HEK rat TRPV-1 cells
The effect of MINO on capsaicin-induced dye uptake in T-REx-HEK-293 cells stably
expressing rat TRPV-1was investigated. The bar charts represent mean data from 12-15 wells
from 3 separate experiments. Data was calculated as the area under the dye uptake curve from
50-300 second (baseline corrected). The black bar represents the TRPV-1 agonist capsaicin
mediated dye uptake in untreated control cells. This dye uptake is significantly inhibited by
MINO (grey bar) and TRPV-1 antagonist capsazepine (CPZ, grey patterned bar). The error
bars represent S.E.M, * P<0.05 calculated by one-way ANOVA (Dunnett’s post-test).
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3.5 Key Points
➢ MINO inhibits P2X7 and P2X4 mediated dye uptake and Ca2+ influx at higher
concentrations.
➢ MINO has an inhibitory effect on P2X7 mediated IL-1β secretion and cell proliferation.
➢ There is no difference in the inhibitory effect of MINO among the different species (rat and
humans) of P2X7.
➢ The P2X7 and P2X4 mediated responses are reduced but not completely blocked by the
drugs.
➢ Pre-incubation of P2X7 and P2X4 expressing cells with MINO was required for the
inhibition of responses.
➢ Among other tetracycline derivatives tested, DOX has similar efficacy as MINO.
➢ Rat TRPV-1 mediated dye uptake is inhibited by MINO.
3.6 Discussion
This study provides evidence in support of the inhibitory action of MINO on P2X mediated
responses. Membrane pore formation is a characteristic feature of P2X7 receptors, but a similar
increase in membrane permeability is also displayed by the structurally homologous P2X4
receptors. The formation of membrane pores was monitored by stimulating the BV-2 microglial
cell line with ATP and then the dye uptake was measured in these cells in the presence and
absence of MINO. A significant inhibition in the dye uptake was observed in the presence of
MINO (Figure 3.3 and Figure 3.4). These results indicate that MINO may be acting on P2X7
and P2X4 receptors which are expressed endogenously in microglia.
The activation of P2X and P2Y receptors is known to cause the entry of extracellular Ca2+ into
the cells (325). Microglia are electrically non-excitable cells which maintain their Ca2+
homeostasis primarily by Ca2+ release from intracellular stores and with Ca2+ entry through
87
plasma membrane via ligand gated and store operated ion channels (118, 326). Stimulation of
microglial cells with low concentration of (< 100 µM) ATP activates the P2X4-mediated Ca2+
entry while activation by high concentration of ATP (1 mM) activates P2X7-mediated Ca2+
influx as well. The P2X4 receptors have higher affinity and the Ca2+ entry response mediated
by these receptors is quick, large and reversible whereas activation with higher ATP causes an
irreversible sustained Ca2+ response (probably via P2X7 mediated membrane pore formation).
The sustained change in Ca2+ was measured by using the Fura-2 AM dye and it was observed
that MINO blocked the increase in intracellular Ca2+ associated with purinergic receptor
activation (Figure 3.5). This increase in Ca2+ is essential, although not adequate, for activation
of the cytotoxic and inflammatory actions of microglia (327). The P2X7 antagonist
AZ10606120 was able to abolish the dye uptake and Ca2+ influx in these assays, supporting our
hypothesis that MINO is targeting P2X7 to inhibit microglial activation. MINO also inhibited
the Ca2+ influx mediated by different subtypes of metabotropic P2Y receptors expressed
endogenously in HEK-293 cells. Activation of P2Y receptors leads to increase in cytosolic
Ca2+ levels via the phosphoinositol turnover dependent Ca2+ release from intracellular
organelles (328). The inhibitory effect of MINO on P2Y-mediated Ca2+ entry indicates that
there may be a common signalling pathway mediated by purinergic receptor system in
microglia which is being targeted by MINO (Figure 3.20).
The involvement of P2X7 receptors in activation of inflammatory pathways in microglia is
well established (303). It is known that P2X7 activation induces a range of downstream
signalling events including elevation of intracellular Ca2+ to mitochondrial effects, caspase
activation, inflammasome activation, ROS production, PLA2 and PLD activation, MMP9
activation, NF-ĸB activation and microglial proliferation (136, 178). The reduction in IL-1β
release and cell proliferation in the present study provide a reliable evidence for the inhibitory
effect of MINO in P2X associated downstream signalling in microglia. There appears to be
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substantial overlap between signalling pathways activated by P2X7 and the signalling
pathways blocked by MINO. Moreover, there is a high similarity in the diseases where P2X7
is thought to play a role in pathogenesis, for example rheumatoid arthritis, ischaemic stroke,
depression, pain (136, 329, 330) and diseases where MINO has a proven beneficial effect.
After establishing the inhibitory effect of MINO on P2X7 responses in BV-2 microglia, the
direct effect of the drug on P2X7 and P2X4 was established by over-expressing these receptors
in HEK-293 cells. The HEK-293 cells expressing these receptors provide a good model for
testing the effect of drug on each receptor type in isolation. MINO inhibited the dye uptake in
both P2X7 and P2X4 expressing cells in a non-competitive, concentration-dependent manner
(Figures 3.9 to Figure 3.13).
The inhibitory action of MINO requires -incubation with the drugs for 15-30 minutes prior to
application of the agonist. Prolonged exposure of receptor to drugs may sometimes lead to the
internalisation of the receptor, causing downregulation of the associated responses. The data
from immunofluorescence study indicates that there was no difference in the surface expression
of P2X7 in cell with MINO pre-treatment. The possibility of interference of MINO with
fluorescence intensity was ruled out by addition of MINO after YOPRO-1 uptake. No reduction
in fluorescence confirmed that inhibitory effect of the drug is due to its effect on the receptor
and not because it quenches the fluorescence (Figure 3.16 and Figure 3.17). Also, 0.01 % Triton
X-100 was used to lyse the cell membrane in order to obtain maximum dye uptake. HEK-P2X7
cells pre-incubated with MINO did not show inhibition in dye uptake providing evidence that
MINO acts via P2X mediated responses and not on fluorescence of the dye itself (Figure 3.15).
In addition to P2X receptors, other receptors such as transient receptors , TRPV-1 and TRPA-
1 have been reported to show increase in membrane permeability on activation by their ligand
(331-333). MINO has been reported to inhibit the capsaicin-induced membrane pore in TRPV-
1 expressing cells. These results were confirmed in the present study in which MINO reduced
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the rat TRPV-1 mediated YOPRO-1 uptake (Figure 3.21). These results suggest that MINO
might be acting on a common pore dilation mechanism.
To further validate the results obtained with cell lines, MINO and DOX were tested in primary
peritoneal macrophages, which express P2X and P2Y receptor similar to microglia. The data
from these experiments were consistent with our data from previous experiments with BV-2
microglia and the HEK-293 cell system.
Taken together, the results from the above study suggest that MINO may be inhibiting the
formation of macropores by P2X receptors in microglia. The process of macropore formation
has been extensively studied for P2X7 receptors, but also occurs in response to activation of
P2X4 receptors in microglia (315).
An effect of MINO on the long term, pro-inflammatory functions of P2X receptors, rather than
the short-term signalling functions is consistent with the relative lack of side effects of this
drug when given systemically. For instance drugs blocking the P2X4 receptor may have cardiac
side effects due to the signalling role of P2X receptors in the heart (334). Thus, compounds
which block pore formation function of P2X receptors may be useful in therapeutic applications
where MINO has shown promise.
MINO is known to play a role in attenuating the symptoms of a variety of pathological
conditions and a number of mechanisms have been proposed to explain its mode of action.
However, despite the mounting need for development of better drugs with similar efficacy, the
direct molecular targets of MINO that mediate its in vivo actions still remain elusive. In the
present study, we propose a novel target of MINO’s inhibitory action on microglia, the
membrane pore forming purinergic receptors, P2X7 and P2X4.
In the study, we also tested tetracycline (TET) and its analogues doxycycline (DOX) and
tigecycline (TIGE) in order to understand the structure-function relationship of the drug
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towards the receptor (Figure 3.17). While DOX mimicked MINO its efficacy in inhibiting P2X
mediated macropore formation, parent compound TET and the analogue TIGE had no
inhibitory effect at all. MINO and DOX have a polyphenolic vitamin E like structure with a
number of different substituent groups (335). The presence of dimethylamino group on C-7 is
suggested to improve the steric hindrance of these compounds (254). This structural similarity
between MINO and DOX could be a major contributor to their receptor binding and their
superior inhibitory action on P2X7 and P2X4 responses.
The concentrations of both MINO and DOX required for inhibiting P2X responses are in the
high micromolar range. However, many of the in vitro studies using MINO also report the use
of micromolar concentrations (336, 337). Such concentrations are likely achievable in vivo,
with typical serum concentrations in humans reaching > 0.0087 µM (338). In addition, both
MINO and DOX are highly lipophilic and can cross the blood-brain barrier; therefore, CSF
concentrations may be higher than serum concentrations. The higher concentrations of these
drugs in brain can explain their capacity to activate purinergic receptors in microglia. The mean
levels of MINO in the brain have been reported to be three fold more than DOX (339). There
are some common side effects related to long term MINO uptake which include nausea,
dizziness, hyperpigmentation, and symptoms of systemic lupus erythematosus (340). It is
observed that in most cases the symptoms disappear once the drug intake is discontinued. Due
to these reasons, the British National Formulary instructs a follow up after every 3 months in
case long term MINO treatment is administered to a patient. Even though MINO is well
accepted by most patients, current therapy is moving towards using DOX as a substitute for
MINO as it has similar efficacy but reduced incidence of side effects such as nausea, hyper
pigmentations and hypertoxicity often associated with MINO treatment (254). Typically,
MINO and DOX are thought to be safe drugs with limited side effects but one of the goals of
understanding how these second-generation tetracyclines exert their beneficial anti-
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inflammatory and neuroprotective actions is that better, high affinity drugs can be developed.
The results from our study suggest that antagonising P2X receptors may have a similar effect
as MINO and this idea certainly warrants further investigation.
Mechanism of action of MINO via P2X receptors
To witness the inhibitory response of MINO, exposure of cells to the drug prior to receptor
activation is essential. This observation leads us to the question whether MINO acts directly
on the receptor channel protein or not. To understand whether MINO affects the P2X mediated
ion channel activity or the membrane pore formation; we have to first consider the mechanism
underlying the membrane pore formation. P2X receptors are trimeric protein complexes with
intrinsic ion channel activity. Each constituent subunit has two transmembrane domains and
there are three ATP binding pockets on the interface of each of these subunits. On ATP binding
to the binding pockets, the extracellular domain of the receptor undergoes a conformational
change, making way for the small cations to pass through. Ser342 and Leu351 provide the
physical gate of the ion permeation pathway in the crystal structure of rat and human P2X7
(341). Extensive studies have been conducted in order to elucidate the exact mechanism of pore
formation but no unified mechanism has so far been accepted (149, 152, 342-344). However,
two different mechanisms have gained general popularity. According to the first one,
membrane pore formation is an intrinsic property of the ion channel and longer agonist binding
leads to dilation of the small permeation pathway (141). The second hypothesis suggests the
involvement of another class of membrane proteins such as pannexin-1 (343, 344). There are
studies that have provided data for or against each of these hypotheses. The biophysical
approach for studying these mechanisms involves the use of N-methyl D-glucosamine
(NMDG+) for measuring P2X receptor currents using whole cell patching (345). The whole-
cell patch recordings from this method show that there is an increase in NMDG+ permeability
as the time of receptor activation increases (346). In the present study, confirmation was not
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obtained to support the effect of MINO on channel current by measuring patch clamping. In
the FluxIon automated patch clamp experiments attempted, the pre-incubation of cells with
MINO for 15-30 minutes prior to running measuring current, did not result in robust response
(probably due to failure of giga-seal formation). This pre-incubation did not affect any other
assays as the cells in all those cases were sticking to the poly-L lysine coated plate surface.
The second experimental method popularly used for measurement of P2X dependent large pore
formation is the measurement of agonist induced intracellular accumulation of fluorescence
dyes such as ethidium and YOPRO-1. This technique was employed in the present study to
measure P2X7 pore formation and the effect of tetracyclines on that activation. Biophysical
studies conducted on the basis of the crystal structure of P2X4 (elucidated by Hattori and
Gouaux in 2012) have established that the physical gate constituting Cα atoms of three Ser342
residues is 6.4 Å from the central axis (137). YOPRO-1 has a molecular size of 7 Å x 8 Å x 19
Å (347). Thus, either the channel has to dilate as wide as 14 Å or there has to be formation of
pores by P2X7 receptors. Pore formation via P2X4 receptors in microglia also appears to be
independent of other pore forming proteins such as pannexins (315). In the present study, a
linear increase in dye uptake was observed for a period of 5 minutes and this dye uptake was
seen to be reduced in cells pre-exposed to MINO. This observation ascertains that MINO has
an effect on increased membrane permeability associated with prolonged P2X activation. This
could suggest direct action on the channel, since ATP responses in these cells are not pannexin
dependent, even when pannexin proteins are co-expressed (348).
While it is possible that MINO is acting on some other, yet to be identified protein, such a
protein would need to be expressed in HEK-293 cells and to have a similar role in the function
of both P2X4 receptors and P2X7 receptors. While the sustained ATP-induced Ca2+ increase
is initiated by P2X receptors, other mechanisms may contribute, and these are also potential
targets of MINO. Store operated Ca2+ entry (SOCE) does contribute to the initial microglial
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responses to ATP (349), although its role in the sustained Ca2+ responses to prolonged P2X
activation have not been fully investigated and there is evidence that SOCE in microglia is not
inhibited by MINO (350).
MINO is reported to act directly on several molecular targets implicated in mediating its anti-
inflammatory effects (351) including PARP-1 and MMPs, but definitive links between these
targets and functional inhibition, particularly in vivo, have yet to be established (289, 352). The
anti-apoptotic action of MINO is perhaps the exception, having been linked to its direct
inhibition of the mitochondrial membrane pores that lead to release of cytochrome c (297). It
is tempting to speculate that the inhibition of pore formation by MINO is a mechanism common
to its anti-inflammatory and anti-apoptotic actions. While many selective P2X7 antagonists
have already been developed and tested in pre-clinical studies (136), specific compounds for
P2X4 are limited. However, the use of a non-selective purinergic receptor antagonist, suramin,
has recently been demonstrated to have a significant effect in models of autism and fragile X
syndrome (353, 354) suggesting that compounds blocking multiple purinergic receptors can be
advantageous.
3.7 Conclusion
Taken together, the data from this chapter suggests that MINO and DOX inhibit purinergic
receptor mediated responses in microglia. In line with the studies suggesting that the inhibitory
effect of MINO and related drugs on microglia-mediated neuroinflammation may be due to its
effect on multiple targets; we confirm that the modulation of purinergic responses is one of the
mechanisms involved. This study was designed to investigate the possibility of purinergic
receptors being the direct targets of MINO and our results suggest that there may be an action
of the drug on P2X7 and P2X4 receptors. Whether this is the only mechanism involved in the
inhibitory effect of MINO on microglial proliferation is not conclusive.
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CHAPTER 4
PURINERGIC RECEPTOR MODULATION BY GINSENOSIDES
4.1 Introduction
Panax ginseng vern: ginseng (C.A. Mayers) is a Chinese herb which is widely used in
traditional medicine as an immune tonic believed to enhance physical strength, resistance and
longevity (355). The word Panax is derived from “panacea” which means a solution or remedy
for all difficulties or diseases. This botanical nomenclature is an indicator of the range of
demonstrated pharmacological effects of this plant in alternative medicine. Traditionally,
ginseng is used either in a freshly harvested form referred to as “fresh ginseng” or as long
storage dried forms called “white” and “red” ginseng. Around 200 chemical components from
ginseng have been isolated and identified so far (356). The main bioactive components among
these are ginseng saponins, also known as ginseng glycosides or ginsenosides. These
ginsenosides act as a beneficial defence mechanism for the plants because of their
antimicrobial/antifungal properties and their bitter taste makes the plants non-palatable for
grazing animals (357).
Figure 4.1 Chinese herb Panax ginseng has a number of reported medicinal applications
(A) Fresh P. ginseng plant with roots (B) Dried roots
(http ://www.interpath.com.au/matmed/herbs/Panax~ginseng.htm)
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Ginsenosides are said to be efficacious in attenuating the clinical symptoms of many diseases
such as cancer, inflammation, diabetes, ischemia, stroke, neurodegeneration and cardiovascular
disorders (16, 358, 359). Due to its potential in chemoprotection or adjuvant treatment, ginseng
is one of the most widely purchased herbs in the United States and European countries (360).
Generally, ginsenosides are named as ‘RX’ where R stands for root and x describes the
chromatographic polarity in alphabetic order. Thus, ginsenosides Ra is most polar and Rh has
least polarity among the ginsenosides isolated and characterised (361). Structurally, each
ginsenoside is made up of a four-ring steroid structure, each with distinct carbohydrate moieties
attached to it. Depending on the positions of carbohydrate moieties attached to the C-3 and C-
6 carbon atoms of the steroid rings and their aliphatic side chains, the ginsenosides have been
classified as protopanaxadiols (PPDs) with two positions for attachments or protopanaxatriols
(PPTs) with positions for three attachments. Oleanolic ginsenosides and ginsenosides
metabolites make up a third class of compounds (362). The pseudoginsenoside F11 belongs to
the PPT class even though there is a tetrahydrofuran ring instead of a carbon chain at position
20. Some rare ginsenosides with oleanolic acid type structure and ocotillol type structure are
also been identified (363). Table 4.1 illustrates the classification of various ginsenosides based
upon their backbone structure. The substituent groups of some of the well-known PPD and
PPT based ginsenosides in Table 4.2.
The composition and type of bioactive constituent present in the plant depends on number of
factors including species, age of the plant, the plant part used, cultivation, harvesting and
extraction methods (364). In general, aging increases the saponin content of the plant. Air-
drying the ginseng roots yields white ginseng, which is known to have lower saponin content
than other forms. Red ginseng is processed by boiling at 100 oC before drying (365). The heat
transformation and deglycosylation is thought to be an explanation for the higher saponin
profile. Successful efforts have been made to improve the bioactive component content of
ginseng by steaming the roots at 120 oC. This type of ginseng is referred to as sun ginseng. It
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contains more ginsenosides with reported anti-tumour properties than the other types of
ginseng (366).
To maintain consistency among various pharmaceutical preparations, many standardised
ginseng formulations are commercially available. Two of the more commonly used
formulations are G115 and NAGE (367). G115 is manufactured by the pharmaceutical
company Pharmaton SA (Switzerland) using the root of P. ginseng with the total ginsenoside
content adjusted to 4 % (higher Rg1). NAGE is a root extract of P. quadrifolius with a
ginsenoside content of 10 % (high Rb1, Re content) and is manufactured by Canadian
Phytopharmaceuticals Corporation (Canada) (361, 367). The defined ginsenoside content of
commercially available extracts is important in pre-clinical testing and clinical trials.
Most of the natural herbs are consumed orally and are subjected to degradation in the
gastrointestinal tract. The health benefits attributed to a whole plant extract can be credited
only to a few metabolites that are effectively absorbed through the intestines after
biodegradation in the gut. In order to gain a better understanding of the effects of ginsenosides
in humans, the pharmacological properties of bioactive components and metabolites need to be
studied in detail.
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Table 4.1 Classification of ginsenosides based upon their backbone structures [adapted
from Chen et al. 2009 (368)
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Table 4.2: Substituent R groups of some of the common ginsenosides [adapted from
Sergiy Oliynk and Seikwan oh,2013 (369)
Where Glc=Glucose; Araf= arabinofuranose; Araparabinopyranose; Rha = rhamanopyrinoside
99
4.2 Biodegradation and bioavailability of ginsenosides
Owing to the hydrophilic nature of the bioactive constituents of ginseng extract, not all of these
are easily absorbed from the intestines following oral ingestion. On consumption of ginseng
extract, the large molecular weight ginsenosides are encountered by intestinal flora and are
metabolized into different chemical forms by the removal of carbohydrate groups (370).
Compound K (CK) and PPD are the major products following microbial degradation of the
PPD class of ginsenosides within the gut while PPT gets further degraded to F11 (Table 4.3).
Absorption of the microbiologically transformed ginseng metabolites is relatively easier partly
because of their non-polar nature (361). The absorbed metabolites may be responsible for the
pharmacological properties associated with ginseng extracts.
Figure 4.2 Possible biotransformation of ginsenosides by lactic acid bacteria in human
intestines (371)
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The transport of ginsenosides across the intestinal mucosa is an energy dependent and non-
saturable process. The bioavailability of ginsenosides and their metabolites from intestines is
not too high but efforts are being made to find methods of increasing this. Among the strategies
applied for this purpose is dissolving the ginsenoside complex in medium chain fatty acids to
make a lipid preparation (372), adrenalin-ginsenoside co-administration (373), p-glycoprotein
efflux suppression (374).
Compound K (CK) is the major metabolite present in the bloodstream after oral administration
of ginseng extracts with large molecular weight protopanaxadiol ginsenosides Rb1, Rb2, Rc
and Rd. Plasma concentrations of CK have been found to be around 70 ng/ml in humans (375).
CK is well known for its anti-cancer, anti-inflammatory and anti-oxidant affects (376-378) and
the pharmacokinetics of this active metabolite are well studied. After 36 hours ingestion, the
mean maximum plasma concentration of CK is found to be significantly higher than parent
compound Rb1. It has also been observed that the absorption of CK is greater in females than
males and is also accelerated in individuals taking a high fat diet (379). CK is known to be
more bioavailable than other ginsenosides and is related to a plethora of potential health
benefits (380). A deeper understanding of the mechanism of action of CK would determine the
precise molecular targets of the compound, which would aid in obtaining maximum benefits
of this herb in treatment of various disorders.
4.3 Mechanism of ginsenosides action
Ginsenosides are well known for their anti-oxidant action (381-385), their effect on the eNOS
system (386-390), their ability to interact with ion channels and membrane receptors along
with the associated signalling pathways (391-395). It is believed that molecular targets of
ginsenosides are either on the cell membrane or inside the cells, depending upon the
hydrophobicity of the ginsenosides (361) .
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Even though the pharmacological effect of these bioactive saponins are well described, their
precise molecular targets are not well established. Of foremost interest is the effect of
ginsenosides on voltage gated and ligand gated ion channels. Inhibition of Ca2+ influx is
thought to prevent Ca2+ overload and ROS production (396). This has been proposed as a
potential mechanism of protective effect of ginsenosides towards oxidative damage. For
example, in ventricular myocardiocytes, Rb1, Rb2, Rb3 and Rc were found to decrease the
inhibit Ca2+ influx and lowering ROS production in these cells (397). Similarly, ginsenoside
Rg1 elicited a neuroprotective effect in ischemia-hypoxic conditions by inhibiting Ca2+ influx
through voltage gated Ca2+ channels and NMDA receptors (296). Ginsenosides are known to
be smooth muscle and vascular endothelium relaxants. PPD was observed to cause
vasorelaxation in rat aortic rings through the activation of Ca2+ activated K+ channels ,
inhibition of voltage-gated and receptor associated Ca2+ channels (398). Ca2+ activated K+
channels (KCa) in coronary arteries are associated with production of nitric oxide (NO), a
vasodilator. It is suggested that potentiation of KCa channels may have the potential of
improving endothelial cell function in cardiovascular disease (399). Ginsenoside Re was found
to increase the KCa activity in Human Coronary Artery Endothelial Cells (HCAEC) thereby
providing a plausible explanation to the beneficial effects of ginseng on heart functions (400).
Rg3 was found to inhibit voltage gated Ca2+, K+ and Na+ channels (401). Thus, it appears that
ginsenosides may decrease the influx of positively charges ions and enhance the efflux of
negative ions to reduce the excitability of excitable cells like neurons and muscles.
Additionally, it has been suggested that the regulation of ion channels by ginsenosides may be
through action on the extracellular side (402).
Ginsenosides have a structural similarity with steroids; therefore, it has been suggested that
some of their activities might be through non-genomic pathways from inside the cells (403). It
is further speculated that length of treatment may be a determining factor in the ginsenoside
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action. Long term (more than 10 minutes) treatment with ginsenosides may allow them time to
enter cells across the plasma membrane and act via steroid receptors as well as cause changes
in cell proliferation pathways. Short term treatment of 1-2 minutes may just be enough for these
hydrophilic molecules to interact with plasma membrane ion channels/receptors to cause
changes in membrane polarization and downstream signalling (404).
In 2013, Im and Nah reviewed ginsenoside pharmacology with a specific emphasis on
contrasting the effects of ginsenosides and gintonin on ion channels (391). Initial experiments
performed with ginseng extract found a potentiating effect on Ca2+ signalling. Individual
ginsenosides when tested were unable to mimic this response as these were observed to
stabilize the membrane potential by inhibiting Ca2+ influx. Gintonin, a glycosylated protein
complex containing lysophosphatidic acid, on the other hand was found to have high affinity
for Ca2+ and caused potentiation of ion channels. Im & Nah concluded that ginsenosides and
gintonin may be acting as two complementary and opposite factors of ginseng pharmacology
(391). The authors proposed that the anti-cancer and pro-apoptotic properties of ginseng may
be attributed to its gintonin component. Gintonin, even though it causes an increase in Ca2+
influx, is present in very low levels in ginseng extract (405). These observations highlight the
importance of understanding the actions of ginsenosides and other bioactive compounds found
in ginseng both in isolation and in combination. Only then will we be in a position to account
for the overall health benefits or side effects of this herb.
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Table 4.3 Recent studies correlating the physiological effects of ginsenosides have been
correlated with their capability to modulate various signalling pathways.
Ginsenosides Target signalling pathway Physiological effect Reference
PPD PI3K/Akt/ERK &
Raf/MEK/ERK
Enhanced
angiogenesis
(406)
Re PPAR-ɤ signalling Gap junction
remodelling
(407)
Rb3 Endothelial to mesenchymal
transition (EndoMT)
Anti-fibrosis (408)
Rg3 AMP-activated protein
kinase
Mitochondrial
protecting, autophagy
enhancing role
(409)
Rg5 Inflammatory and apoptotic
pathways
Hepatoprotective
Anti-apoptotic
(410)
Rh2 Orphan Nuclear Receptor
Nur77
Pro-apoptotic(anti-
cancer)
(410)
Re PI 3-K/Akt Signalling Neuroprotective (411)
Rg1 Akt-FoxO1 Interaction Gluconeogenesis-
lowering effect(anti-
type II Diabetes)
(363)
Rg3 derivative
HRG
VEGF, b-FGF, MMPs anti-angiogenic effect (412)
Rg3 Eicosanoids Anti-inflammatory (413)
Rg3 STAT5-PPARgamma Anti-obesity (414)
Rd, Re,
FloralGA
Melanin biosynthesis Melanogenesis
inhibitor
(415)
Rh2 Lytic replication of human
gamma herpes virus
Anti-viral (416)
Rg3 Microglial activation ,NF-κβ
pathway
Anti-depressant
Anti-inflammatory
(417)
Rg1 Vaccine adjuvant Anti-cancer (418)
Rb3,Rd iNOS, STAT3/pSTAT3,
Src/pSrc
Anti-cancer
Pro-biotic effect
(419)
Rg3 NF-κβ/ p38MAPK pathway Anti-oxidant and anti-
senescence in
astrocytes
(420)
Rd, Re co-
treatment
Apoptotic signalling, ROS
generation
Anti-oxidant,
neuroprotective
(421)
Rg3 phosphoinositide 3-
kinase/Akt pathway
Anti-apoptosis (422)
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As discussed in the previous chapters, P2X receptors are trimeric, ATP-gated cation channels
that are permeable to Na+, K+ and Ca2+ ions (423). The seven subtypes of the P2X receptor
family have a widespread distribution and are implicated in a number of physiological
processes such as synaptic transmission, nociception, inflammation and taste sensation (306).
P2X4 and P2X2 are known to interact with extracellular ATP to form a large conductance pore
on the cell membrane, similar to closely related P2X7 (136). Upon receptor activation, the
membrane pore change conformation which causes large cation flux across the cell membrane,
resulting in membrane depolarization and activation of a number of signalling pathways related
to physiological processes such as pain and inflammation (424). Involvement of purinergic
receptors in the initiation and progression of inflammatory processes is well documented with
P2X7 being said to have a more prominent role than structurally homologous P2X4 (185). It
has also been suggested that in activation of the inflammasome, P2X4 may act as an initial
trigger while P2X7 in combination with pannexin-1 may amplify the signal (184). More
recently, studies conducted to understand the role of P2X4 in inflammation suggest that this
ion channel may have a greater role to play in inflammatory processes than previously thought.
Inhibition of P2X4 in a collagen-induced model of arthritis can affect the development of
arthritis and autoimmunity in mice, indicating a direct involvement of P2X4 in synovial
inflammation and joint destruction (425). ATP-P2X4R signalling is implicated in airway
inflammation and airway re-modelling in allergic asthma in mice (426). P2X4 is also involved
in release of an early inflammatory mediator prostaglandin E (PGE2) (427). P2X4 deficiency
in mice leads to impaired inflammasome activation, lack of pain hypersensitivity and complete
absence of PGE2 in tissue exudates of these animals (428). There is also evidence that P2X2
antagonism can cause relief from pain-related responses and urinary inconsistence (429).
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All these and more studies provide evidence in support of the view that P2X receptors are key
modulators in inflammatory pathways and finding more pharmacological tools to target these
receptors in order to manipulate the physiological processes they control remain a priority.
4.4 Research Question
In this study, we investigated the effects of ginseng extract and individual purified chemicals
on ATP-gated P2X7 receptors in our quest to find novel pharmacological modulators for this
receptor. Once the activity was established, the specificity of these compounds on other P2X
and P2Y receptors was then examined.
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4.5 Results
4.4.1 Effect of ginseng extract G115 on human P2X7
To establish any potential effects of ginseng extract on P2X7 receptors, the YOPRO-1 iodide
dye uptake assay was utilised. For initial screening of compounds an approximate EC50
concentration of ATP (200 μM) was used in order to see either potentiation or inhibition of the
response. HEK 293 cells expressing human P2X7 (stable expression) were used. A standard
formulation of ginseng known as G115 was tested as it is widely used as an active ingredient
in modern medicine (430). Pre-treatment of HEK-hP2X7 cells for 10 minutes with 100 µg/ml
G115 dissolved in water increased the rate of ATP-induced dye uptake by around two-fold
(Figure 4.3). G115 without ATP did not induce dye uptake in HEK-hP2X7 cells, suggesting
this was not a non-specific effect on the cells (Figure 4.3).
G115 contains a fixed amount (4 % w/w) of eight ginsenosides from both PPD and PPT
chemical classes, namely Rb1, Rc, Rd, Re (PPD ginsenosides) and Rb2, Rf, Rg1, Rg2 (PPT
ginsenosides) (431). After confirming the effect of G115 on P2X7-mediated dye uptake, it was
important to establish whether the observed effects with G115 resulted from one or more
specific ginsenosides(s) in the formulation. Therefore, 14 purified ginsenosides were tested in
a screening assay encompassing the eight ginsenosides in G115 plus the principal intestinal
metabolites, Rh1, Rh2, Rg3, Compound K (CK), and the two aglycones, PPD and PPT. All
ginsenosides (10 μM) were pre-incubated with the cells before the addition of ATP (200 μM).
None of the ginsenosides stimulated dye uptake on their own, however, four PPD ginsenosides,
Rb1, Rd, Rh2 and CK, significantly increased the rate of dye uptake after stimulation by ATP
(Figure 4.4). In contrast, ginsenosides of the PPT series had no significant effect on the ATP-
induced dye uptake (Figure 4.2).
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4.4.2 Potentiation of human P2X7 mediated dye uptake by the ginseng metabolite CK
As CK was found to be the most effective among the ginsenosides tested in the above assay,
the investigation focused on this ginsenoside to further examine the potentiating action on
P2X7 receptors in more detail. To determine if CK was acting as a positive allosteric modulator
of P2X7 receptors, a full concentration-response curve for ATP in the absence and presence of
10 μM CK was performed (Figure 4.5). Potentiation by CK caused a leftward-shift of the
concentration-response curve reducing the EC50 for ATP from 259.6 μM (95 % confidence
interval – 228.8 to 294.5) to 80.7 μM (95 % confidence interval – 69.5 to 94), increased the
maximum ATP response and required a threshold concentration of ATP (∼ 50 μM) (Figure
4.3). In contrast, when BzATP was used as a full agonist at human P2X7 receptors (234), CK
decreased the EC50 value from 20.3 μM (95 % confidence interval – 16 to 25.7) to 5.9 μM
(95% confidence interval – 4.6 to 7.6) (Figure 4.5). The leftward shifts of the dose response
curves in the presence of both ATP and BzATP were statistically significant (P < 0.01). The
response induced by ATP and CK was solely dependent on P2X7 receptors as it could be
completely abolished by pre-treatment with the selective P2X7 receptor antagonist
AZ10606120 (10 μM) (Figure 4.5). Similar to its metabolite CK, the parent compound Rd was
also observed to increase the YOPRO-1 uptake via ATP-activated P2X7. Various
concentrations of Rd were tested on HEK 293 cells stably expressing hP2X7 and it was found
that Rd increased the dye uptake in these cells in concentration-dependent manner (Figure 4.6).
4.4.3 Potentiation of human P2X7 mediated Ca2+ influx by ginseng metabolite CK
Activation of P2X7 receptors can result in a sustained rise in intracellular Ca2+ leading to either
proliferation/activation or cell death, depending on the magnitude and extent of channel
activation (202). Therefore, it was important to establish whether the ginsenoside potentiation
of P2X7 receptor responses observed in dye uptake experiments led to physiologically relevant
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sustained increases in intracellular Ca2+ concentration. Intracellular Ca2+ responses were
measured in HEK-hP2X7 cells using the ratiometric Ca2+ indicator dye Fura-2 AM. An initial
transient rise in intracellular Ca2+ following ATP addition was observed, which is attributed to
the activation of G-protein coupled P2Y receptors. However, an additional sustained elevation
in intracellular Ca2+ on addition of ATP concentrations > 100 μM was observed following the
transient peak (Figure 4.7). This sustained elevation of intracellular Ca2+ is a characteristic
feature of P2X7 receptor activation (136). In the presence of CK, the sustained Ca2+ response
to 200 μM ATP was further increased (Figure 4.8). Quantification of the changes in Fura-2 AM
ratio during the sustained phase of the response (150-300 seconds) showed that CK
significantly increases the ATP-mediated rise in intracellular Ca2+ (Figure 4.8).
4.4.4 Lack of difference in effect of CK among different species of P2X7
The human, mouse and rat P2X7 cDNA have been cloned and the pharmacological properties
of the corresponding proteins are well characterised (432). It has been observed that there is a
significant difference in pharmacological properties of PX7 depending upon the species of
origin. For example, human and rat P2X7s have higher affinities for agonists in comparison to
mouse P2X7 (432). Also, current growth and channel dilation are more rapid in human and rat
P2X7 in comparison to their mouse counterpart (433). Keeping these species differences in
mind, the effect of selected ginsenosides were tested on human and rat P2X7 responses using
the YOPRO-1 dye uptake assay. HEK-hP2X7 and HEK-rP2X7 cells were exposed to various
concentrations of ginsenosides to obtain concentration response curves. It was found that
ginsenosides were able to potentiate YOPRO-1 dye uptake for both P2X7 orthologues, though
(Figure 4.8). EC50 value for each ginsenosides for both P2X7 orthologues is listed below.
109
Table 4.4 Comparative list of EC50 values of ATP for human and rat P2X7 in the
presence of selected ginsenosides
Ginsenoside EC50 human P2X7 EC50 rat P2X7
CK 1.21 µM 0.667 µM
Rd 0.59 µM 0.646 µM
Rb1 1.36 µM 1.8 µM
Rh2 7.85 µM 3.2 µM
4.4.5 Potentiation of P2X7 mediated cell death by ginseng metabolite CK
We next investigated whether this potentiation of ATP-induced P2X7 receptor responses could
be translated to a significant downstream functional effect, such as the induction of cell death.
HEK-hP2X7 treated with ATP and ginsenosides for 24 hours before MTS was added per well,
4 hours before the stipulated 24 hours’ time point. Neither 10 μM CK, DMSO nor 5 - 500 μM
ATP alone induced any reduction in cell viability (Figure 4.9). In contrast, a high concentration
of ATP (3 mM) for 24 h induced a significant reduction of cell viability (Figure 4.9). Treating
HEK-hP2X7 with a combination of 10 μM CK and non-lethal ATP (500 μM) together, now
significantly reduced cell viability (Figure 4.9). The same effect was not observed with lower
concentrations of ATP. CK, therefore, seems to reduce the threshold required for the cells to
commit to cell death pathways.
110
Figure 4.3 Standardised ginseng extract G115 increases P2X7 mediated YOPRO-1
uptake.
(A) G115 ginseng formulation and purified ginsenosides potentiate ATP-induced responses at
the human P2X7 receptor. ATP-induced dye uptake was measured at 37 °C using YOPRO-1
(2 μM) as the membrane impermeant dye. Relative fluorescence units (RFU) were measured
following excitation at 490 nm and emission recorded at 520 nm using a fluorescent plate
reader (Flexstation 3). HEK-hP2X7 cells were pre-treated with 100 μg/ml G115 in a low
divalent buffer for 10 minutes at 37 °C. ATP (200 μM) was then added to elicit a P2X7 receptor
response. The mean of five individual wells was plotted. (B) Slope of the dye uptake curve data
(n = 10–20 wells) was plotted for buffer control, ATP or ATP in the presence of 100 μg/ml
G115. Error bars are S.E.M. * denotes P<0.05 using with Dunnett's multiple comparison test.
111
Figure 4.4 Some protopanaxadiol ginseng compounds increase P2X7-mediated YOPRO-
1 uptake
Fourteen purified ginsenoside compounds (protopanaxadiols in purple and protopanaxatriols
in green) were tested for potentiation of P2X7 receptor responses at a concentration of 10 μM.
All compounds were prepared in DMSO and were added to the low divalent assay buffer.
Compounds were pre-incubated for 10 minutes prior to the addition of ATP (200 μM). The data
was quantified as the slope of dye uptake curve. Error bars are S.E.M. * denotes P<0.05
using ANOVA with Dunnett's multiple comparison test.
112
Figure 4.5 CK acts as a positive allosteric modulator of human P2X7 receptors
Dye uptake following P2X7 activation using 200 µM ATP was measured at 37 °C using
YOPRO-1 (2 μM) as the membrane impermeant dye. Relative fluorescence units (RFU) were
measured following excitation at 490 nm and emission recorded at 520 nm using a fluorescent
plate reader (Flexstation 3). Black traces represent responses to agonist (ATP) and red traces
represent responses in presence of CK + ATP (B) A concentration-response curve for the
potentiating effect of CK. Concentration-response curves were generated for ATP (10 μM –
1 mM), with or without 10 μM CK. CK decreased the EC50 of ATP for P2X7 activation from
259.6 μM to 80.78 μM.
113
Figure 4.6 CK acts as a positive allosteric modulator of human P2X7 receptors activated
by BzATP.
(A) Dye uptake induced by P2X7 activation using 30 µM BzATP was measured at 37 °C using
YOPRO-1 (2 μM) as the membrane impermeant dye. Relative fluorescence units (RFU) were
measured following excitation at 490 nm and emission recorded at 520 nm using a fluorescent
plate reader (Flexstation 3). Black open circles represent responses to agonist (BzATP) and
red traces represent responses in presence of CK + BzATP (B) Concentration-response curves
were generated for BzATP (1 μM – 0.3 mM), with or without 10 μM CK. Dose response curve
with varying BzATP concentrations is shown in grey while the dose response curve with ATP
+ CK is shown on red. CK reduces the EC50 of BzATP for P2X7 from 20.39 µM to 5.9 µM.
Non- linear dose response curves of transformed data from 3 independent experiments were
fitted using GraphPad Prism.
114
Figure 4.7 Rd enhances the sustained dye uptake associated with hP2X7 receptor
activation.
Dye uptake induced by P2X7 activation using 200 µM ATP with or without ginsenoside Rd
was measured at 37 °C using YOPRO-1 (2 μM) as the membrane impermeant dye. Relative
fluorescence units (RFU) were measured following excitation at 490 nm and emission recorded
at 520 nm using a fluorescent plate reader (Flexstation 3). Black traces represent responses to
agonist (ATP) and blue traces represent responses in presence of Rd + ATP. (B) Data was
quantified as the slope of dye uptake curve. Error bars are S.E.M. *denotes P<0.05
using ANOVA with Dunnett's multiple comparison test.
115
Figure 4.8 CK enhances the sustained Ca2+ response associated with hP2X7 receptor
activation.
(A) Intracellular Ca2+ responses were measured in Fura-2 AM loaded HEK-hP2X7 cells.
Baseline values were recorded for 15 s and then ATP was applied and Ca2+ measured for 300
seconds. Ca2+ influx in the presence of ATP is shown in black circles and Ca2+ influx in the
presence of CK + ATP is shown in red circles. (B) HEK-hP2X7 were treated with 200 μM
ATP, 200 μM ATP + 0.01 % DMSO or 200 μM ATP + 10 μM CK and Fura-2 fluorescence
measured over time. Quantitative measures sustained Ca2+ response (mean fluorescence
between 100 and 300 seconds (baseline fluorescence between 0 – 15 seconds) in HEK-hP2X7
was plotted as bar graph. * denotes P<0.05, significant effect of CK; one-way ANOVA with
Dunnett's post-test.
116
Figure 4.9 There is no difference in the potentiating effect of selected ginsenosides on
P2X7 mediated YOPRO-1 uptake in humans and rats.
(A) Concentration-response YOPRO-1 uptake curve of CK, Rd , Rb1 and Rh2 in ATP (200
µM) activated HEK-293 cells transfected with human P2X7. PPD (green) is included as a
control. (B) Concentration-response curve of CK, Rd, Rb1, Rh2 in ATP (200 µM) activated
HEK-293 cells transfected with rat P2X7 cells. PPD (green) is included as a control. Non-
linear dose response curves of transformed data from 3 independent experiments were fitted
using GraphPad Prism. Error bars represent S.E.M.
117
Figure 4.10 CK enhanced the ability of ATP to cause cell death in HEK-P2X7 cells.
Cell viability was measured using Cell Titre Aqueous ONE solution (Promega) after incubation
HEK-hP2X7 cells with specific treatments for 24 hours.3 mM ATP reduces cell viability 30%
of the untreated controls. The effect is P2X7 mediated as the antagonist AZ10660120 (10 µM)
reverts the cell viability to 81%. 500 µM ATP is considered as a non-lethal dose of ATP, which
does not affect cell viability after 24 hours incubation period. In the presence of 10 µM CK,
500 µM ATP causes cells death equivalent to 3 mM ATP (app. 32%).The same effect is not
observed at lower concentrations. Error bars represent S.E.M, * P<0.05 by one-way ANOVA
(Tukey’s multiple comparison test).
118
4.4.6 Investigating effect of ginsenosides on P2X4
To gain a better insight into the specificity of ginsenosides towards P2X7, these bioactive
saponins were tested on structurally homologous P2X4 and P2X2 receptors share structural
homology with P2X7 and are known to form secondary permeability pathways similar to P2X7
(even though this property is most consistent and conspicuous for P2X7 (434). Unlike P2X7,
P2X4 require a much lower concentration of ATP to be activated, with an EC50 of 2.5 - 3 µM
(435, 436). To investigate ginsenosides on P2X4, a stable HEK-hP2X4 cell line was generated
and the ginseng extract G115 (100 µg/ml) was tested using YOPRO-1 dye uptake as an
indicator of receptor activity (as detailed in Chapter 3). Cells were either treated with 10 µM
ATP alone or with G115 co-injected with ATP. The P2X4 antagonist 5-BDBD and the
potentiator ivermectin (IVM) were used in order to ensure that P2X4-mediated responses were
being measured. An increase in dye uptake was observed with cells treated with ATP + G115
and ATP + IVM compared to ATP alone (Figure 4.11). The ATP-induced YOPRO-1 uptake
response was inhibited with 5-BDBD (Figure 4.11)
4.4.7 Effect of ginsenosides on P2X4 mediated dye uptake
Following this finding that G115 could enhance hP2X4 responses the 14 purified ginsenosides
were tested. Similar to the observations made with P2X7, out of fourteen ginsenosides tested,
four protopanaxadiol ginsenosides enhanced the dye uptake in the rank order Rd > CK > Rb1
> Rh2 (Figure 4.12).
4.4.8 Ginseng metabolite CK increases Ca2+ influx via P2X4 activation.
CK, the ginsenoside metabolite and its parent compound Rd, both of which produced maximum
potentiation in case of P2X7, were studied in further detail for their effect on P2X4. A
concentration response curve was obtained for dye uptake via P2X4 activated at different ATP
concentrations (0.1-100 µM) in the presence of 10 µM CK or 5 µM IVM. 10 µM CK was seen
119
to cause a slight leftward shift in the ATP concentration-response curve, to increase the
maximal response and to decrease the EC50 value for ATP from 2.06 µM to 0.91 µM. IVM, the
positive control also reduced the EC50 for ATP to 1.6 µM (Figure 4.13).
4.4.9 Effect of selected ginsenosides on ATP concentration response curve in HEK-P2X4
cells
To better understand the potency of selected ginsenosides at P2X4, concentration responses
were generated for the four protopanaxadiol ginsenosides. From the data in Figure 4.14, the
EC50 of the ginsenosides tested were 7.55 µM for Rd, 8.55 µM for CK , 10.10 µM for Rb1 and
5.8 µM for Rh2. Rh2 has the lowest effect on the ATP-induced maximal response but a potency
comparable to other ginsenosides tested (Figure 4.14).
4.4.10 Effect of selected ginsenosides on Ca2+ responses in HEK-P2X4 cells
The Ca2+ permeability of hP2X4 is the highest among the P2X family (79) therefore it was
pertinent to evaluate the effect of ginsenosides on intracellular Ca2+ concentrations. ATP-
induced activation of P2X4 leads to Ca2+ influx which was tested experimentally using the
ratiometric dye Fura 2-AM as previously described. The P2X4 receptor was activated using 1
µM ATP (concentration determined from the ATP dose response curve) and the ginsenosides
were co-injected with ATP at a concentration of 10 µM each. The responses were measured on
Flexstation 3 and the data was quantified as mean of sustained Ca2+ response (100-300 seconds)
observed after the initial transient peak response using baseline corrected data. Consistent with
the dye uptake responses, the intracellular Ca2+ was increased by ginsenosides in HEK-hP2X4
cells in the rank order of Rd > CK > Rb1 > Rh2 (Figure 4.15). Further experiments showed
that 10 µM CK shifted the ATP concentration response curve leftwards and reduced the EC50
value from 1.08 µM (95 % confidence interval 0.6 to 1.8 µM) to 0.3 µM (95 % confidence
interval 0.1 to 0.5 µM) (Figure 4.16).
120
4.4.11 Effect of ginsenosides on P2X4 inward currents using high throughput IonFlux-
16 automated patch clamp system.
To investigate the effect of ginsenosides on P2X4 channel, the automated patch clamp FluxIon
system was used. The FluxIon plates were primed before adding the HEK- P2X4 cells to them.
The cells were prepared at a cell density of 4-10 x 106 cells/2 ml extracellular solution , so that
the final cell density is 2.5 x 106 cells/ ml. Cell suspension (250 µl) was added to each “IN”
well of the Ion-flux 16 plate ( Figure 2.2). The plate was loaded on to the FluxIon automated
patch clamp system. The drug solutions were prepared in 2 x concentrations. ATP was applied
first and the responses (current amplitude in pA) were measured. Then the cells were washed
out with running buffer for 2 minutes. ATP was then reapplied in the presence of various
ginsenosides. Current amplitudes were then measured as an average current of 20 cells for each
condition using FluxIon software. Data was expressed as fold change in current amplitude.
P2X4 antagonist 5-BDBD was able to inhibit the current amplitude by 74.3 %. In the presence
of 10 µM CK and 10 µM Rd, ATP mediated increase current amplitude was increased by ~ 3
folds ( > 50 % increase in comparison to 0.01 % DMSO control) in HEK-P2X4 cells. Rh2
caused a 44.7 % increase in current amplitude but Rb1 was not seen to cause an increase in
channel activity in these settings (Figure 4.17).
4.4.12 Effect of P2X4 and ginsenosides on cell viability
To test whether P2X4 potentiation by ginsenosides leads to initiation of apoptotic cell death, a
cell viability assay similar to the one described above for hP2X7 was performed. In these
experiments cell viability was measured as end-point absorbance of MTS solution (490 nM)
for each treatment and data was quantified as percentage of untreated control (designated as
100 % viability). Unlike the observations made with HEK-hP2X7 cells though, no significant
difference was seen in the viability of HEK-P2X4 cells at any ATP concentration tested (Figure
4.18). A high 3 mM concentration of ATP is considered to be a lethal concentration of ATP
121
for P2X7 containing cells. In HEK-293 cells expressing only hP2X4, 3 mM ATP did not cause
any significant reduction in cell viability (cell death) (Figure 4.18). The presence of 10 µM CK
together with ATP did not reduce cell viability (Figure 4.18).
122
Figure 4.11 Effect of ginseng extract G115 on ATP-induced dye uptake in HEK-293
cells expressing human P2X4
(A) YOPRO-1 uptake was induced in HEK-hP2X4 expressing cells by the addition of 5 µM
ATP (denoted by the arrow) in low divalent physiological solution. Control YOPRO-1 uptake
to ATP is shown in black circles. The response was inhibited by P2X4 antagonist 5-BDBD (20
µM) and potentiated by 5 µM IVM, a positive allosteric modulator of P2X4, shown here in
brown circles and black open circles respectively. Fluorescence was measured for 50-300
seconds on Flexstation 3 (Molecular Devices). (B) Bar charts displaying data from 3 separate
experiments. The data was quantified using area under the dye uptake curve, baseline corrected.
The P2X4 mediated dye uptake was significantly attenuated by 5-BDBD and facilitated by
IVM. 100 µg/ml G115 increased dye uptake via ATP activated P2X4. Error bars represent
S.E.M ** denotes P<0.05 by one-way ANOVA (Dunnett’s post-test).
123
Figure 4.12 Effect of ginsenosides on ATP-induced dye uptake in HEK-293 cells
expressing human P2X4
YOPRO-1 uptake in HEK-hP2X4 cells in presence of 5 µM ATP (denoted by the arrow), ATP
+ PPD1 and ATP + CK in low divalent buffer solution. The ginsenosides were co-injected with
agonist ATP. Fluorescence was measured for 50-180 seconds on Flexstation 3. Ginsenosides
Rd, Rb1, Rh2 and CK (10 µM ATP) were seen to potentiate the P2X4 mediated dye uptake
Data has been quantified as area under dye uptake curve (50-180 sec). Bar graphs show data
from 3-5 independent experiments. Error bars represent S.E.M, * denotes P<0.05 by one- way
ANOVA (Dunnett’s post-test).
124
Figure 4.13 ATP-induced dye uptake in HEK-293 cells expressing human P2X4 in the
presence of varying doses of CK
CK increases the potency and efficacy of ATP binding to P2X4. The EC50 of ATP is lowered
from 1.92 µM to 1.215 µM in the presence of 10 µM CK. Various concentrations of ATP (1
µM - 100 µM) were used to activate the P2X4 receptor and the activation was measured by
YOPRO-1uptake on Flex station 3. Controls are shown here in black dots, the dye uptake in
presence of 10 µM CK is shown in red and in presence of P2X4 potentiator ivermectin (IVM)
is shown in blue. The data is taken from 3 independent experiments and transformed data has
been fitted using non-linear fit of regression. The error bars represent S.E.M.
125
Figure 4.14 Concentration dependent effect of ginsenosides on ATP-induced dye uptake
in HEK- human P2X4 cells
Concentration dependent responses of selected ginsenosides on ATP responses in HEK-P2X4
cells- Different concentrations (0.1-50 µM) of ginsenosides were tested for their effect on
YOPRO-1 uptake in HEK-P2X4 cells stimulated at fixed concentration (5 µM) of ATP. The
EC50 of different ginsenosides was in the rank order Rb1 > Rd > Rh2 > CK with values being
0.11 µM, 7.54 µM, 5.8 µM and 8.5 µM. The data is taken from 3 independent experiments and
transformed data has been fitted using non-linear fit of regression. The error bars represent
S.E.M.
126
Figure 4.15 CK enhances the sustained Ca2+ response associated with hP2X4 receptor
activation.
(A) Intracellular Ca2+ responses were measured in Fura-2AM loaded HEK-hP2X4 cells.
Baseline values were recorded for 15 s and then ATP was applied. Ca2+ influx in presence of
ATP is shown in black and Ca2+ influx in presence of ATP + CK shown in red (B) HEK-hP2X4
were treated with 1 μM ATP or 1 μM ATP + 10 μM ginsenosides and Fura-2AM responses
measured over time. Intracellular Ca2+ in ATP activated HEK-hP2X4 is shown in black bar.
10 µM CK and Rd significantly increase P2X4 mediated the Ca2+ influx. Error bars denote
S.E.M; * denotes P <0.05, significant effect of CK; one-way ANOVA with Dunnett's post-
test.
127
Figure 4.16 CK acts a positive modulator of P2X4 receptor
CK acts a positive modulator of P2X4 receptor by shifting the ATP dose response towards left.
The data is quantified as sustained Ca2+ response (mean fluorescence between 100 and 300 s).
The data was taken from 10-12 wells from 2 independent experiments and transformed data
was fitted using non-linear fit of regression. The EC50 of ATP for P2X4 was reduced from 1.08
µM to 0.27 µM in the presence of 10 µM CK. The error bars represent S.E.M.
128
Figure 4.17 Effect of ginsenosides on current amplitude in HEK-hP2X4 cells
The HEK- P2X4 cells were used at a cell density of 4-10 x 106 cells/2 ml of extracellular
solution and added to the primed IonFLux-16 plates. The final cell density is 2.5 x 106 cells/ml.
Cell suspension (250 µl) was added to each “IN” well of the IonFlux 16 plate. The plate was
loaded on to the IonFlux automated patch clamp system. The drug solutions were prepared in
2 x concentrations. 5-BDBD was prepared in running buffer. ATP was applied first and the
responses (current amplitude) were measured. Then the cells were washed out with running
buffer for 2 minutes. ATP was then reapplied in the presence of various ginsenosides. Current
amplitudes were then measured for each condition using IonFlux software. Data from 3
independent experiments was collated and was expressed as fold change in current amplitude.
Error bars represent S.E.M; *denotes P<0.05, One-way ANOVA, Dunnett’s multiple
comparison test.
129
Figure 4.18 ATP does not induce cell death in HEK-hP2X4 cells
Cell viability was measured using Cell Titre Aqueous ONE solution (Promega) after incubation
HEK-hP2X4 cells with specific treatments for 24 hours. 3 mM ATP had no effect on cell
viability. A range of different concentrations (5µM-3mM) was tested on HEK-P2X4 cells in
the presence of CK and P2X4 antagonist 5-BDBD (20 µM). No changes were observed in cell
viability under any conditions after 24 hours incubation period. Error bars represent S.E.M ns
denotes not significant, one-way ANOVA (Tukey’s multiple comparison test).
130
4.4.13 Role of ginsenosides in modulation of P2X2 receptor function
Pharmacological investigations were carried out on another member of the purinergic P2X
family, the hP2X2 receptor. The full sized P2X2 receptor is called P2X2a to differentiate it
from its multiple splice variants which are found naturally in humans (for instance, the splice
variant P2X2b has missing 69 residues in its C-terminal domain) (437). For measuring
YOPRO-1 uptake, the P2X2a receptor was activated by 5 µM ATP (Figure 4.19 A, B) in a low
divalent buffer. Ginsenosides CK, Rd and Rh1 were seen to increase the dye uptake (Figure
4.15). Rb1 on the other hand did not alter the P2X2a response. For Ca2+ influx measurement,
1µM ATP was used to activate the receptor. Similar to the results obtained from dye uptake
experiments, CK caused a robust increase in Ca2+ influx through activated P2X2a but other
ginsenosides Rd, Rb1 and Rh2 did not mimic this potentiating affect (Figure 4.20 A, B). This
observation was different from expected as dye uptake and intracellular Ca2+ measurements
are generally considered as the indicators of P2X2 receptor activity. Therefore, an increase in
dye uptake with Rd and Rh2 would generally translate into increase in Ca2+ influx as well,
which was not seen in these experiments. Since CK is the drug of interest in this study and had
shown an effect on P2X2 mediated dye uptake as well as Ca2+ responses in these experiments,
10 µM CK was tested on ATP dose response using both YOPRO-1 and Fura- 2 measurements
(Figure 4.15 C, D). CK was seen to reduce the EC50 of ATP in both measures. For YOPRO-1
uptake, EC50 of ATP was changed from 0.6 µM to 0.25 µM by CK, with a definite leftward
shift (Figure.4.16 A). For intracellular Ca2+, the EC50 for ATP alone in P2X2a expressing cells
is lowered from 3.03 µM to 2.67 µM in the presence of CK (Figure 4.16 B).
131
Figure 4.19 Effect of ginsenosides on P2X2a dye uptakes in transiently transfected
HEK-293 cells
(A) YOPRO-1 uptake was measured in HEK- 293 cells transiently transfected with P2X2a
plasmid. Non- transfected cells were used a negative control (mock shown in grey patterned
bar). Since, no specific antagonist of P2X2a is available; P2X7 blocker AZ10660120 (grey bar)
and P2X4 potentiator IVM were used to rule out the possibility of a P2X7 or P2X4 response.
Data was quantified as area under the dye uptake curve, baseline corrected. (B) Dye uptake by
ATP activated HEK-P2X2a cells were tested by co-injecting ginsenosides with ATP. CK and
Rd at 10 µM concentration potentiated the dye uptake significantly. The bar chart display data
from 3 separate experiments. Rb1 and Rh2 did not cause a significant change in dye uptake.
The data was quantified using area under the dye uptake curve, baseline corrected. Error bars
represent S.E.M, * denotes P<0.05 by one-way ANOVA (Dunnett’s post-test).
132
Figure 4.20 Effect of ginsenosides on P2X2a Ca2+ influx in transiently transfected HEK-
293 cells
(A) Ca2+ influx was measured in HEK- 293 cells transiently transfected with P2X2a plasmid
using Ca2+ sensitive ratiometric dye Fura-2AM. Data was quantified as mean of sustained Ca2+
response from 100 – 300 seconds. The mean of minimum response (0-15 seconds) was
deducted from the sustained response. (B) Intracellular Ca2+ concentrations in ATP activated
HEK-P2X2a cells were tested by co-injecting ginsenosides with ATP. 10 µM CK potentiated
the dye uptake significantly. The bar chart displaying data from 3 separate experiments. Rd,
Rb1 and Rh2 did not cause a significant change in intracellular Ca2+ levels. Data was quantified
as mean of sustained Ca2+ response from 100 – 300 seconds. The mean of minimum response
(0-15 seconds) was deducted from the sustained response. Error bars represent S.E.M * denotes
P<0.05 by one-way ANOVA (Dunnett’s post-test)
133
Figure 4.21 Effect of ginsenosides on P2X2a Ca2+ influx in transiently transfected HEK-
293 cells
(A) Concentration dependent response of ginsenosides on YOPRO-1 uptake was measured in
HEK-P2X2a cells. The EC50 of ATP when measured by dye uptake is lowered from in the
presence of 10 µM CK. Various concentrations of ATP (0.01 µM - 50 µM) were used to
activate the P2X2a receptor and the activation was measured by YOPRO-1 uptake on
Flexstation 3. Data was quantified as area under dye uptake (50-300 seconds), baseline
corrected. The data is taken from 3 independent experiments and transformed data has been
fitted using non-linear fit of regression. The error bars represent S.E.M; P<0.05. (B) Various
concentrations of ATP (0.01 µM - 50 µM) were used to activate the P2X2a receptor and the
activation was measured by Ca2+ using ratiometric dye Fura-2AM on Flexstation 3. The EC50
of ATP is lowered in the presence of 10 µM CK. The data is taken from 3 independent
experiments and transformed and fitted using non-linear regression
134
4.4.14 Effect of ginsenosides in modulation of P2Y1 and P2Y2-mediated Ca2+ responses
Finally, the ginsenosides were tested on the metabotropic P2Y receptor which is expressed in
immune cells along with the other P2X receptors tested in this study. P2Y receptors are G
coupled proteins (GPCRs) for extracellular nucleotides. The receptors were activated using
either 10 µM ATP (which is an agonist for both P2Y1 and P2Y2), 10 µM ADP (a P2Y1 agonist)
or 10 µM UTP (a P2Y2 receptor agonist). The peak was measured from 0-100 seconds in the
presence and absence of CK. The ginsenosides were found to have no significant effect on
P2Y1 and P2Y2 mediated Ca2+ release from intracellular stores (Figure 4.21).
Taken together, these results suggest that the selected ginsenosides CK, Rd, Rb1 and Rh2
potentiate P2X7 mediated responses but are not completely specific for this receptor as
potentiating effects of these ginseng saponins are observed in the close relatives P2X4 and
P2X2a. However, there is considerable difference in the magnitude of potentiation of these
responses between the different receptors. In all these experiments, the major in vivo metabolite
CK showed a consistent behaviour of potentiating the P2X receptor responses. To compare the
magnitude of the CK effect, the data from the dye uptake and Ca2+ influx experiments for
P2X7, P2X4 and P2X2a was plotted on to one graph. The response for ATP treatment was
designated as 100% and the ATP + CK response for each of these receptors was compared to
their own control. Fold change was calculated for each of these receptors using the following
formula
Fold change=Area under curve Treated- Area under curve control / Area under curve control
It was observed that the magnitude of increase in receptor potentiation by ginsenosides CK was
in the rank order P2X7 >> P2X2a > P2X4. Thus, these pharmacological studies indicate even
though the ginsenosides are not specific for P2X7 responses alone, they definitely elicit their
largest effect via this receptor (Figure 4.22).
135
Figure 4.21 Selected ginsenosides do not alter P2Y mediated Ca2+ influx
HEK-293 cells express P2Y1 and P2Y2 endogenously. Effect of ginsenosides on P2Y mediated
Ca2+ influx was measured using Fura-2 AM. The receptors were activated using either 10 µM
ATP (agonist for both P2Y1 and P2Y2), 10 µM ADP (P2Y1 agonist) or 10 µM UTP (P2Y2
receptor). Cells were treated with agonist, agonist + 10 µM ginsenosides or agonist + antagonist
(suramin). (A) Raw traces of intracellular Ca2+ measurement following activation by agonist.
Baseline measurements were done for 15 second, after which agonist was injected. Black open
squares are responses in presence of non-specific P2Y inhibitor suramin and green triangles
represent Ca2+ responses in presence of agonist and aglycone PPD. (B) Data was quantified as
mean of peak response (15-200 sec). (B) represents the P2Y mediated Ca2+ influx in the
presence of ATP. (C) represents the P2Y mediated Ca2+ influx in the presence of ADP. (D)
represents the P2Y mediated Ca2+ influx in the presence of UTP. Agonist (ATP, UTP or ADP)
responses are represented as black bar in graphs B,C and D. Ginsenosides CK, Rd, Rb1 and
Rh2 have no significant effect on P2Y mediated intracellular Ca2+ influx. Data analysis done
by one-way ANOVA with Dunnett's post-test.
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Figure 4.22 Comparative analyses of ginsenosides on P2X7, P2X4 and P2X2a mediated
YOPRO-1 uptake and Ca2+ responses
(A) HEK-P2X7 cells activated using 200 µM ATP (EC50) with and without 10 µM CK. The
amount of YOPRO-1 uptake was quantified as area under the dye uptake curve (40-300 sec). The
bar graph represents the fold change between controls and with CK after normalising controls as
100% response. Fold change was calculated using the formula (Area under curve Treated- Area
under curve control/ Area under curve control). 10 µM CK potentiate dye uptake in ATP activated
HEK-P2X7 cells by 4.9 folds. Similarly, the HEK-P2X4 cells were stimulated at 5 µM (EC50) in
the absence of ATP and also with CK + ATP. The responses are increased by 0.5 folds. For HEK-
P2X2a (EC50- 5 µM) this increase was 1.3 folds. Thus, CK potentiates P2X7 mediated dye uptake
most. (B) The HEK-P2X7 cells were activated using 200 µM ATP (EC50) with and without 10
µM CK. The amount of Ca2+ was quantified as Fura 2-AM fluorescence (100-300 sec). The bar
graph represents the fold change between controls (ATP) and with CK after normalising controls
as 100% response. 10 µM CK potentiate dye uptake in ATP activated HEK-P2X7 cells by 4.6
folds. Similarly, the HEK-P2X4 cells were stimulated at 5 µM in the absence of ATP and also
with CK + ATP. The responses are increased by 0.7 folds. For HEK-P2X2a (EC50 -5 µM) this
increase was 1.3 folds. Thus, CK potentiates P2X7 mediated Ca2+ influx maximally in
comparison to other purinergic receptors tested.
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4.6 Key Points
➢ Out of 14 ginsenosides tested, 4 ginsenosides potentiate P2X7 mediated dye uptake and
Ca2+ influx in rank order CK > Rd > Rb1 > Rh2.
➢ Presence of at least one carbohydrate group seems to be essential for potentiation as
aglycone PPD has no effect on P2X7 responses.
➢ CK increase the capability of P2X7 to cause cell death.
➢ Selected ginsenosides tested are not specific for P2X7 as closely related P2X4 and P2X2a
are also potentiated.
➢ In comparison to P2X4 and P2X2, the ginsenosides are more efficacious towards P2X7
responses.
➢ Metabotropic P2Y receptors are not affected by ginsenosides Rd, Rb1, Rh2 and ginseng
metabolite CK.
4.7 Discussion
In this study, a significant increase in P2X7 receptor responses was observed in the presence
of certain ginseng saponins (Rb1, Rh2, Rd and CK). To observe an increase in P2X responses
by these selected ginsenosides activation of P2X7 receptors by orthosteric agonists (ATP,
BzATP) was found to be necessary. These effects were characterized in a HEK-293 cell line
expressing P2X7 receptors. As a result of the P2X7-ginsenoside interaction an increase Ca2+
influx and a subsequent decrease in cell viability to lower concentrations of ATP was observed.
Taking into consideration the widespread distribution of P2X7 channels on immune cells and
the fact that effects could be observed in the submicromolar range with one of the principal
metabolites of ginseng CK (from the electrophysiology data obtained in the lab and published
in 2015) (438), it is possible that this mechanism may account for some of the reported immune
modulatory actions of ginseng in vivo. In case of P2X7 receptor responses it appears that this
potentiation is exclusively exhibited by glycosylated PPD ginsenosides and can be observed in
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the low to sub-micromolar range (EC50 of 0.5–1.1 μM), the probability of this effect being a
result of a non-specific interaction due changes in membrane fluidity (as has been suggested
for certain ginsenoside actions reflecting the amphipathic nature of these steroid-like saponins),
is ruled out.
The increase in P2X7 receptor activity by the PPD class of ginsenosides is reported for the first
time in the present study, however there have been previous reports of some PPD ginsenosides
such as Rc and Rd potentiating GABAA and glycine currents in Xenopus oocytes (439, 440).
In the studies mentioned above, the concentrations of ginsenosides required to elicit measurable
enhancement of receptor activity were much higher (approximately 50 μM), but the results
indicated that the PPD class was more potent that PPT ginsenosides. Ginsenoside Rg3 was also
found to enhance the activity of GABA channels containing the γ2 subunit. Nah (2014) has
extensively reviewed the effect of various ginsenosides on membrane receptors and ion
channels (404). There are a number of studies indicating the inhibitory effect of ginsenosides
on voltage gated Ca2+, K+ , Na+ and hERG K+ and KCNQ K+ channels (441-444). The present
study indicates that the aglycone compounds PPD and PPT were not effective at increasing
P2X7 activity which indicates that to have an effect on the P2X7 receptor function, the presence
of at least one sugar moiety may be essential. Interestingly, both PPD and PPT aglycones have
been shown to inhibit rather than potentiate GABAA currents (445).However, no inhibitory
effects on P2X7 receptor responses with any ginsenoside compound were observed in this
study. Further investigations are required to determine if there are any shared structural features
between the ginsenoside binding sites on P2X7 and GABAA channels. This may also apply to
HERG (Kv11.1) channels, as PPD ginsenosides were typically more effective than PPT
ginsenosides in potentiating tail currents and PPT/PPD aglycones were ineffective (443).
Glycosylated PPD ginsenosides, such as the principal ginseng metabolite CK, are novel potent
positive allosteric modulators of human P2X7 receptors. Several other positive modulators of
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P2X7 receptors have been described including clemastine, tenidap, polymixin B and
Ivermectin (210, 446-449). The mechanism of CK on P2X7 channels shares some similarities
with clemestine, in that the action is rapid, reversible, Ca2+- and voltage-independent and likely
use an extracellular site. Similar to CK, modulators such as clemastine had no direct effects on
P2X7 channels and required the presence of the agonist ATP (447). Although in the above
mentioned study, it was reported an augmented alteration in reversal potential occurring over
tens of seconds, reflecting an increased rate in the permeability to NMDG+ in the presence of
ATP, they recognized that this cannot account for the rapid (<1 s) potentiating effect and
reversibility of clemastine (447). The most plausible explanation, which may also be applicable
to PPD ginsenosides, is that such modulators increase the mean-open time of P2X7 channels.
Furthermore, such a net increase in channel activation is known to accelerate pore dilation.
An important consequence of the PPD ginsenoside action on P2X7 channels is enhanced
sustained Ca2+ influx in HEK-hP2X7 cells. The use of CK as a positive allosteric modulator of
P2X7 receptors reduces the concentration of ATP required to generate a sustained
Ca2+ response. Many downstream consequences of P2X7 receptor activation depend on
sustained Ca2+ signalling (136). With regard to cell viability, brief additions of high
concentrations of ATP (<5 min, < 1 mM) can lead to a transient ‘pseudoapoptosis’ that does
not lead to cell death (450) or to a delayed cell death occurring after a number of hours (107).
Higher concentrations of ATP and/or prolonged applications on the other hand lead to cell
death within minutes because of massive Ca2+influx. In contrast, lower concentrations of ATP
(<1 mM) can have the opposite effect, stimulating proliferation and prolonging cell survival
(202). Consistent with this, the present study has shown that enhancing Ca2+ influx via P2X7
receptors in HEK-hP2X7 cells through the use of CK can effectively convert a sublethal dose
of 500 μM ATP into a lethal concentration as measured by a significant decrease in cell
viability after 24 h. However, given the fact that the timing and extent of Ca2+influx via P2X7
receptors can lead to different functional outcomes, further studies are warranted with different
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ATP/ ginsenoside combinations and examination of other parameters in addition to cell
viability. Keeping this mind, the effect of ginsenosides on cell viability and signalling pathway
involved has been evaluated using macrophages (J774 cell line and rat macrophages). The
results of this study have been discussed in Chapter 5.
After establishing the potentiating effect of ginseng metabolite CK on P2X7, the selectivity of
CK and other selected ginsenosides (Rd, Rb1 and Rh2) was tested on structurally homologous
P2X receptors; P2X4 and P2X2a. P2X4 receptors are expressed in immune cells along with
P2X7. Functional coordination between P2X7 and P2X4 is well characterised (451, 452). Here
it was observed that all selected ginsenosides (more so for CK and Rd that Rb1 and Rh2)
increased responses via P2X4 as well as P2X2 activation, showing that ginsenoside action was
not exclusive for P2X7 but targeted closely related purinergic family members (Figure 4.8-
4.12). However, the increase was approximately 0.5 fold for P2X4 and 1.3 fold for P2X2 in
comparison to P2X7 responses which increase approximately 5 fold in the presence of
ginsenosides.
Since there is evidence that P2X4 can interact with and regulate P2X7 dependent downstream
signals (453), the effect of CK on cell viability in HEK-P2X4 cells was tested in a manner
similar to the one used for HEK-P2X7 cells. No change in viability was observed in the
presence of CK + ATP in HEK-293 cells expressing P2X4 receptor (Figure 4.11), suggesting
the lack of involvement of P2X4 in cell death pathway in these cells.
A number of positive allosteric modulators of P2X4 such as Zn2+, cibacron blue, ivermectin,
doramectin, moxidectin and a few others have been identified and characterised (454). The
data from the present study suggests that the positively modulation of P2X4 responses by
selected ginsenosides may be similar to Ivermectin (IVM), a well characterised positive
allosteric modulator of P2X4. Among the purinergic receptors tested, P2X4 is most sensitive
towards IVM (314), although there is some evidence that IVM also increases human P2X7
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responses to a certain extent. IVM (448), the macrocycline lactone derivative of naturally
occurring avermectin B1, is known to act from the extracellular side in order to potentiate P2X4
currents and delay channel desensitization. It is suggested that IVM binds to a high affinity site
on the transmembrane domains. Ivermectin has been found to modulate the sensitivity of
P2X7/P2X4/Panx-1 pathway towards extracellular ATP leading to induction of autophagic and
inflammatory cell death in breast cancer cells (455). IVM has been used as a positive control
in the experiments conducted in the present study and it has been observed that the magnitude
of P2X4 potentiation caused via G115 is similar to IVM (Figure4.7).
Alcohol acts a negative allosteric modulator of P2X receptors (456) and a decrease in P2X4
gene expression has been observed in brain on alcohol consumption (182, 457). Studies in
rodents and humans have shown that IVM binds to P2X4 on a different site than ethanol and
reduces the alcohol induced debilitating effects by elevating the P2X4 responses to normal
levels (458).Furthermore, there are reports showing that ginseng extract G115 reduces ethanol
induced depression in mice (459). A randomised cross over study has shown that red ginseng
can alleviate alcohol consumption and hangover related symptoms in men (460). There is also
a mounting body of evidence that purinergic receptors contribute to neuronal survival as well
as function (461). It has been found that P2X4 expression decreases in neuronal cells in
Alzheimer’s disease (462). There are a number of studies supporting the protective role of
ginseng and its active constituents in this debilitating neurodegenerative disorder (463-465).
Taken together, these studies suggest that potentiation of P2X4 responses by selected
ginsenosides may be involved in the beneficial effects of ginseng in relieving alcohol related
disorders as well as Alzheimer’s disease.
Among the 7 P2X subtypes, P2X4 is the most highly expressed in vascular endothelium (466,
467). ATP release from vascular endothelial cells results in modulation of P2X4 responses
leading to increased Ca2+ influx (468). A loss-of-function mutation in P2X4 has been
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associated with decrease in P2X4 responses leading to increased pulse pressure, an independent
risk factor of cardiovascular diseases. People inheriting the defective P2X4 gene may be more
vulnerable to cardiovascular disorders (469). Potentiation of P2X4 responses by ginsenosides
in people inheriting mutated or dysfunctional P2X4s may be a useful way of preventing
cardiovascular disease conditions such as atherosclerosis and thrombosis.
The present study provides evidence in support of the positive allosteric effect of
protopanaxadiol ginsenosides on hP2X2 receptor. P2X2a is known to be distributed in central
and peripheral nervous system (470).Neurotransmitters such as nicotine, 5-hydroxytryptamine
(5-HT), noradrenaline, adenosine, bradykinin and histamine are known to increase P2X2a
responses at a high concentration of 100 µM (471). Ginsenosides in the present study potentiate
these responses at a much lower concentration (10 µM being the highest concentration tested).
Increase in P2X2a responses can lead to increase in sensory nerve sensitivity. P2X2a receptors
are also extensively distributed in cochlear cells in the ears and play a role in sound
transduction. Under stress conditions, the amount of ATP increases, causing P2X2a activation.
This ATP -P2X2a-mediated shunt conductance can contribute to change in hearing sensitivity
and temporary hearing loss phenomena and also serve a protective role by decoupling the
cochlear amplifier in response to cochlear stressors (472). The reported protective effect of
ginseng towards hearing loss could be explained by increase in sensitivity of cochlear cells
towards stressors via P2X2a activation leading to decoupling the positive feedback mechanism
within the cochlea that provides acute sensitivity in the mammalian auditory system.
Selected ginsenosides when tested were found to have no effect on the activity of these
receptors. These results indicate that the ginsenosides tested may be targeting only purinergic
ion channels and not their G- protein coupled receptor counterparts.
The results presented in this chapter may also shed some light on a possible structure function
relationship between these ginsenosides and their potency towards the receptors. CK and Rd
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have a glucose molecule attached on their C-20 (R2 position) carbon atom while Rh2 and Rb1
have -H at the C-20 position. So, even though Rh2 and Rb1 potentiate P2X responses
significantly but this increase is not as effective as CK and Rd. Thus, from these results it may
be suggested that the glucose group present on C-20 may facilitate the binding of ginsenosides
on P2X receptors and increasing the potency of agonist ATP for its receptor. Overall, the data
from the present study provides evidence that selected ginsenosides can target purinergic
receptors to positively modulate the receptor responses.
4.8 Conclusions
From the present study, it can be suggested that ginseng metabolite CK is a potent allosteric
modulator of purinergic ion channels expressed in immune cells, being most efficacious
towards P2X7. Presence of glucose in the molecular structure may to be essential for
ginsenosides to elicit an increase in P2X7 responses, as aglycone PPD has no effect on the
magnitude of the responses elicited by this receptor. It could also be suggested that the efficacy
of a particular ginsenoside in potentiating P2X response is due to the presence of a glucose
moiety on C-20 carbon of its steroid ring, as CK and Rd are more effective in enhancing
responses than Rb1 and Rd. The identification and characterisation of novel allosteric
modulators of the purinergic system not only provide a credible explanation to the certain
immunomodulatory properties of ginseng but may also hold a promise for development of new
therapeutic tools in order to target purinergic receptors expressed in immune cells involved in
various pathophysiological conditions.
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CHAPTER 5
SELECTED GINSENOSIDES MODULATE P2X7 RECEPTOR
RESPONSES IN MACROPHAGES
5.1 Introduction
Macrophages are special cells of the innate immune system, which are involved in acute as
well as chronic inflammatory processes. In case of infection, macrophages migrate to the site
of invasion and initiate their antimicrobial activity (473). After neutralizing the infection, the
process of repair and resolution is also taken care of by these cells. Macrophages have the
capability to turn from pro-inflammatory to anti-inflammatory depending upon the cytokine
profile in their microenvironment (474). The inflammatory processes are tightly regulated, and
any imbalance can result in development of chronic inflammatory state. Most current therapies
target the initiation and progression phase of inflammatory process, but it has been strongly
suggested that intervention at repair and resolution phase of inflammation may be a better
approach in the conditions related to chronic inflammation (475).
P2X7 is endogenously expressed in macrophages and is the most extensively studied purinergic
receptor from the immunological perspective (262). High concentrations (in millimolars) of
ATP are released by infected, injured or dying cells in the extracellular milieu where it acts on
the P2X7, thereby triggering an inflammatory response in the cells. The eATP-P2X7
interaction is widely accepted as one of the most important signalling triggers in infectious and
inflammatory diseases (54).
The ATP- P2X7 liaison leads to K+ efflux, NLRP3 inflammasome assembly, caspase-1 and
pro-IL-1 β secretion (476). The increase in Ca2+ influx following P2X7 activation is known to
affect the mitochondrial membrane potential causing an increase in ROS production, which
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may further activate the caspase 3/7 leading to an increase in cell death via apoptosis (477,
478). Modulation of P2X7 responses can have a significant effect on the associated signalling
pathways.
In the previous chapter, the effect of selected ginsenosides on P2X receptors expressed in HEK-
293 cells was investigated. This study provided us with an opportunity to look at P2X receptor
responses to ginsenosides in the absence of any other interacting purinergic receptors. From
the previous study, it was established that ginsenosides can modulate P2X responses. The next
conceptual step was to investigate the effect of these ginsenosides in a system, which was
physiologically more relevant, such as macrophages, where these receptors are co-expressed
endogenously along with the signalling pathways they regulate. Therefore, in the present
chapter the selected ginsenosides were tested for their effect on P2X mediated physiological
responses using a J774 macrophage cell line as well as primary primary peritoneal
macrophages.
Lipopolysaccharide (LPS) is a major component of Gram-negative bacteria (also called
bacterial endotoxin) and it acts as primary inflammatory stimuli after binding to TLR-4
receptors expressed on macrophages (479). Humphrey and Dubyak first reported enhancement
of P2X7 mediated cellular responses by LPS in 1996 (480). It is now well established that LPS-
TLR4 binding leads to activation of the NLRP3 inflammasome cascade, causing maturation
and release of the pro-inflammatory cytokine IL-1β and IL-18 (316). However, this LPS
treatment stimulates only a slow rate of caspase-1 activation. Caspase-1 is known to catalyse
the maturation and release of pro-inflammatory cytokines. A slow rate of caspase-1 activity by
LPS leads to the accumulation of the pro-IL1β in the macrophages. It has been observed that
for an accelerated caspase-1 activity, efflux of K+ ions are required from monocytes and
macrophages. Recent studies have shown that P2X7 involvement is critical for the
inflammasome activation to occur as P2X7 membrane pore leads to K+ ion efflux, which causes
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the inflammasome assembly to occur. Therefore, modulation of P2X7 receptor can have an
effect on LPS mediated pro-inflammatory effects. This aspect has been assessed in this study,
where cells pre-exposed to LPS are then incubated with ginsenosides in order to evaluate
changes in the resulting P2X7 mediated inflammatory pathways.
Ginseng has been used traditionally as an immune-boosting tonic with little understanding of
target receptors. This study provides evidence that ginsenosides target P2X receptors and alter
the signalling pathways mediated by them and thus provides an explanation to some of their
immune-modulatory properties. The results from this study may be helpful in developing these
nutraceuticals as therapeutic tools for targeting P2X mediated pathological conditions such as
rheumatoid arthritis, bronchitis, chronic obstructive pulmonary disorder (COPD) and cancer.
5.2 Research Question
In this study, the effects of selected protopanaxadiol ginsenosides CK, Rd, Rb1 and Rh2 on
responses mediate by ATP-gated P2X7 receptors in macrophages to characterise these newly
identified pharmacological modulators was investigated.
5.3 Results
5.4.1 J774 macrophages express functional P2X7
In this chapter, the modulatory effect of ginsenosides was tested in a physiologically suitable
environment by using a J774 macrophage cell line. J774 macrophage cell line is an immortal
cell line derived from the ascites from peritoneal cavity of a mouse
(https://www.atcc.org/Products/All/TIB-67.aspx). The suitability of this cell line as an ideal
model system for studying macrophage responses has been studied and it has been found that
the J774 macrophages cells respond in a manner comparable to primary macrophages and
therefore are a convenient and a reliable mode for investigating signalling pathways distinctive
to macrophages (481-483). Previous reports have confirmed the endogenous expression of
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P2X7 in the J774 macrophages (212, 484-488). It was pertinent to establish the functionality
of these receptors in J774 cell line used in this study. For this purpose, the ATP- P2X7 mediated
dye uptake and Ca2+ influx was measured using YOPRO-1 and Fura 2-AM respectively.
Following activation by ATP, there was a significant increase in dye uptake indicating the
P2X7 mediated membrane pore formation. Attenuation of dye uptake was observed in the
presence of P2X7 antagonist AZ10606120. No increase in YOPRO-1 fluorescence was seen in
the absence of ATP (Figure 5.1 A and Figure 5.1 B). Furthermore, intracellular Ca2+
concentrations measured using ratiometric Ca2+ indicator Fura 2-AM showed an increased
Fura-2 fluorescence on ATP application and inhibition of this fluorescence in presence of
antagonist (Figure 5.1 C and Figure 5.1 D). These results validated the presence of a functional
P2X7 in the J774 macrophage cell line employed in this study.
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Figure 5.1 Expression of functional P2X7 receptor in J774 macrophages
(A) YOPRO-1 uptake was induced in J774 macrophages by the addition of 500 µM ATP at 40
seconds (denoted by the arrow) in low divalent physiological solution. An increase in YOPRO-
1 fluorescence was observed on addition of ATP (black circles). P2X7 antagonist AZ10606120
inhibited the response (grey triangles). Fluorescence was measured for 50-300 seconds on
Flexstation 3 (Molecular Devices). (B) The data was quantified using area under the dye uptake
curve, baseline corrected. Error bars represent S.E.M, n=3; * denotes P<0.05 by one-way
ANOVA (Dunnett’s post-test). Bar charts displaying data from 3 separate experiments. (C)
Intracellular Ca2+ responses were measured in Fura-2 AM loaded J774 cells. Baseline values
were recorded for 15 s and then ATP was applied. Fura -2 fluorescence was measured for 300
seconds. An increase in Fura -2 fluorescence was observed on addition of ATP (black circles)
in comparison to buffer (light grey diamonds) where no change in fluorescence was seen. P2X7
antagonist AZ10606120 inhibited the response (grey triangles). (D) Data was quantified as
mean of sustained Ca2+ response (100-300 seconds), baseline corrected. n=3 *denotes P<0.05,
significant effect of ATP; one-way ANOVA with Dunnett's post-test.
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5.4.2 CK and Rd enhance P2X7 mediated Ca2+ influx in J774 macrophages
Selected ginsenosides were tested for their effect on P2X7 mediated Ca2+ influx with Fura-2
AM loaded cells. It was observed that Ca2+ influx was 186.90 % more in J774 macrophages
treated with ATP + 10 µM CK than with ATP alone. In the presence of Rd, Ca2+ influx was
increased by 63.17 %. Thus, CK and Rd were able to potentiate P2X7 mediated Ca2+ influx in
J774 macrophages (Figure 5.2) similar to HEK-P2X7 cells (Figure 4.6). However, unlike the
HEK-293 system, ginsenosides Rb1 and Rh2 did not allow the Ca2+ influx in J774
macrophages. This could be due to a number of factors including the presence of other
purinergic receptors in macrophages. From Chapter 4, it was established that ginsenosides are
not selective towards P2X7 as they affect P2X4 as well. In addition, the rank order for P2X7
potentiation is HEK-293 cells was CK > Rd > Rb1 > Rh2. Therefore, depending upon the
number of P2X7 receptors expressed in a particular cell line, the P2X4-P2X7 interaction in
these cells, ginsenoside Rb1 and Rh2 may be unable to replicate the responses in J774
macrophages in comparison to the HEK-293 system where these receptors are over-expressed.
5.4.3 Potentiation of P2X7 mediated YOPRO-1 uptake and Ca2+ influx by CK increases
in a concentration dependent manner
The effect of 10 µM CK was tested on YOPRO-1 uptake and Ca2+ influx in J774 macrophages
activated at different concentrations of ATP. For dye uptake, ATP concentrations from 10 µM
to 1 mM were tested and it was found that the EC50 of ATP for P2X7 was 197.50 µM. In the
presence of CK, the EC50 of ATP was reduced to 69.8 µM (95 % confidence interval 47.7 to
102). Therefore, CK lowers the concentration of ATP required to activate P2X7 (Figure 5.3).
Measurements of Ca2+using Fura -2 AM calcium indicator was done in the presence of 10 µM
CK and different concentrations of ATP (5µM to 1.5 mM) (Figure 5.4). EC50 of ATP for P2X7
activation was found to be 163.2 µM (95 % confidence interval 92 to 287). In the presence of
10 µM CK, the EC50 values was 109.2 µM (95 % confidence interval 92 to 131).
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Various concentrations of CK (10 µM to 1 µM) were tested on J774 macrophages activated by
200 µM of ATP (Figure 5.5). An increase in Ca2+ influx was observed with the increasing
concentration of CK. The EC50 of CK for P2X7 activation was found to be 2.2 µM (95 %
confidence interval 1.2 to 4.5). Therefore, the results obtained for J774 macrophages validate
the data from the HEK-293 system (Chapter 4) that CK allosterically modulates P2X7 ion
channels.
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Figure 5.2 Selected ginsenosides enhance the sustained Ca2+ response associated with
P2X7 receptor activation in J774 macrophages
(A) Intracellular Ca2+ responses were measured in Fura-2AM loaded J774 cells. Baseline
values were recorded for 15 s and then ATP was applied. Increase in fluorescence in response
to 200 µM ATP represented here in black dots while the potentiation of ATP responses in
presence of 10 µM CK shown in red. ATP responses were inhibited by P2X7 antagonist
AZ10606120 (shown here in grey). (B) Changes in Fura-2 fluorescence responses were
quantified as mean of Fura-2 fluorescence between 100 and 300 s. Baseline correction was
done by taking away minimum responses measured from 0-15 seconds mean sustained
responses. The Ca2+ influx in the presence of 200 µM ATP is shown in solid black bar. The
Ca2+ influx in ATP activated J77 macrophages is inhibited by 10 µM AZ10606120 (grey open
bar). Ca2+ influx in the presence of ATP + 10 µM CK is shown as red patterned bar, in the
presence of ATP + 10 µM Rd is shown in blue, ATP + 10 µM Rb1 is shown in green and ATP
+ 10 µM Rh2 is shown in green. The aglycone PPD is shown in green open bar. n=3 *P<0.05,
significant effect of ATP; one-way ANOVA with Dunnett's post-test.
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Figure 5.3 CK increases P2X7 mediated dye uptake in J774 macrophages in a
concentration dependent manner
P2X7 mediated dye uptake was measured in J774 macrophages activated at different
concentrations of ATP (10 µM- 1 mM) in the presence and absence of 10 µM CK. YOPRO-1
uptake in presence of ATP alone is shown in black while ATP+ CK responses are shown in
red. EC50 of ATP is decreased from 197.50 µM to 69.83 µM (95 % confidence interval of 47.5
to 102.2) in the presence of CK. The data was taken from 3 independent experiments and
transformed data was been fitted using non-linear fit of regression. The error bars represent
S.E.M.
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Figure 5.4 Effect of CK on Ca2+ influx in ATP activated P2X7 responses in J774
macrophages
Various concentrations of ATP (2 mM- 10 µM) were used to activate the P2X7 receptor and
the activation was measured by sustained Ca2+ response (Fura-2 fluorescence) on Flexstation
3. CK increased the potency and efficacy of ATP binding to P2X7 in macrophages. The EC50
of ATP was lowered from 163.2 µM to 109.2 µM (95 % confidence interval 91.5 to 131.1) in
the presence of 10 µM CK. The data was taken from 3 independent experiments and
transformed data was fitted using non-linear fit of regression. The error bars represent S.E.M.
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Figure 5.5 Effect of CK on ATP-induced Ca2+ influx in P2X7 responses in J774
macrophages
Different concentrations (0.1- 10 µM) of CK were tested for their effect on Ca2+ influx in J774
macrophages stimulated at fixed concentration (200 µM) of ATP. The EC50 of CK was found
to be 2.2 µM (95 % confidence interval 1.2 to 4.5). Data was taken from 2 independent
experiments (10-12 well). Error bars represent S.E.M.
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5.4.4 LPS does not affect P2X7 channel activity in J774 macrophages
During infections, bacterial endotoxin LPS is secreted by bacteria and acts as the greatest
stimulus for macrophage activation (489). LPS induced infection can also increase the amount
of ATP released in the extracellular matrix, causing activation of P2X7.
Macrophages are known to have a number of LPS binding proteins on their surface including
CD14, CD11b, scavenger receptors and toll like receptors (490-493). Due to the absence of a
transmembrane domain, CD14 is thought to be unlikely to initiate an LPS response on its own.
Evidence from various studies suggests that TLR4s are the most important protein involved in
LPS actions. On binding to TLR4, LPS leads to assembly of the NLRP3 inflammasome, which
catalyses the conversion of pro-IL1β to mature IL-1β. LPS is also known to directly interact
with P2X7. P2X7 has a LPS binding site on its cytoplasmic tail, indicating the possibility of a
direct interaction between the two molecules. Extracellular ATP increases during inflammation
and injury, leading to activation of P2X7. It has been observed that Ca2+ influx and K+ efflux
associated with P2X7 activation are both essential for NLRP3 inflammasome assembly and is
also responsible for mitochondrial ROS secretion upstream of NLRP3 activation (494). P2X7
receptor activation is known to amplify LPS-induced signalling via interleukin-1β release (183,
495). Keeping in mind that LPS/ P2X7 interaction can cause an increased pro-inflammatory
response in macrophages, the next step in this study was to find out the effect of ginsenosides,
the positive modulators of P2X7 on LPS primed cells. To determine if LPS altered ATP
induced responses in J774 cells, the difference in Ca2+ influx was measured for LPS primed
and non-primed Fura 2-AM loaded J774 macrophages. Again, it was seen that LPS treatment
did not influence the influx of Ca2+ ion through activated P2X7 channel (Figure 5.6).
Ginsenosides were now tested for the difference in their P2X7 potentiating effect in LPS
primed and un-primed cells. Similar to observations made in Figure 5.7, only CK and Rd were
able to potentiate P2X7 mediated Ca2+ entry but there was no difference in the magnitude of
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this potentiation between LPS primed and non-primed cells. AZ10606120 (10 µM) inhibited
these responses to an equal extent in both the scenarios. From this result, it could be suggested
that LPS did not alter the P2X7 channel activity directly.
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Figure 5.6 LPS priming of J774 macrophages for 4 hours has no effect on P2X7 mediated
Ca2+ responses.
(A) Incubation of J774 macrophages with 100 ng/ml LPS for 4 hours of ATP (200 µM)
activation did not have any effect on magnitude of Ca2+ responses as evident from raw traces
(baseline corrected) of Fura-2 fluorescence. The Ca2+ responses for LPS primed cells are shown
in orange while Ca2+ responses for non-primed macrophages are shown in black. (B) Data was
quantified as mean of sustained response from 100-300 seconds. Baseline correction was done
by taking away minimum responses measured from 0-15 seconds mean sustained responses.
Sustained Ca2+ influx in non-LPS primed macrophages are shown as black bar, Ca2+ influx
after ATP activation in LPS primed cells is shown as orange patterned bars. In both cases,
P2X7 antagonist, AZ10606120, inhibited the Ca2+ responses to an equal extent. n=3, no
significant effect of LPS; one-way ANOVA with Dunnett's post-test.
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Figure 5.7 Effect of selected ginsenosides on Ca2+ influx in LPS primed J774 macrophages
(A) Intracellular Ca2+ concentration after ATP activation of P2X7 receptors was observed to
be potentiated by ginsenosides CK. The magnitude of this potentiation was unaltered by LPS
priming of cells prior to ATP activation. Ca2+ responses of LPS primed cells are shown in
orange and those for non-primed cells are shown in black. The data was measured as change
in Fura-2 fluorescence on Flexstation 3 (Molecular Devices) (B) Data was quantified as mean
of sustained response from 100-300 seconds. Baseline correction was done by taking away
minimum responses measured from 0-15 seconds mean sustained responses. The Ca2+ influx
was significantly potentiated in presence of ginsenoside CK and Rd but there was no difference
in the degree of this potentiation in cells pre-exposed to LPS in comparison to cells.
Ginsenosides Rb2, Rh2 and PPD1 do not exhibit a significant change in Ca2+ influx after ATP
activation. Sustained responses in presence of without LPS priming is shown as black dotted
bar. Ca2+ responses in ATP activated LPS primed cells is shown as orange patterned bar. In
both cases, P2X7 antagonist, AZ10606120, inhibits the Ca2+ responses to an equal extent. n=3,
* denotes P<0.05, significant effect of ATP; one-way ANOVA with Dunnett's post-test.
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5.4.5 CK and Rd increase the ATP induced cell death
P2X7 is referred to as the “death receptor” as its activation via extracellular ATP following
tissue injury or infection leads to switching on of apoptotic cell death pathways (262). There is
evidence in support of the role of P2X7 in cell death (496-498), so ginsenosides were tested on
their effect on P2X7 mediated cell viability using MTS assay. P2X7 receptor activation
requires relatively high concentrations of ATP. By incubating J774 macrophages with various
concentrations (ranging from 10 µM – 3 mM) of ATP for 24 hours and then testing the cell
viability using tetrazolium salt MTS, it was determined that 3mM ATP was a lethal dose for
these cells as it caused more than 70 % reduction in cell viability. ATP (500 µM) was found to
be the highest non-lethal dose of ATP. The effect is P2X7 mediated as the P2X7 antagonist
AZ10660120 (10 µM) reverts the cell viability to 80 %. So, for testing the effect of
ginsenosides on cell viability, the cells were incubated for 24 hours with 10 µM selected
ginsenosides in the presence of 500 µM of ATP. It was observed that 10 µM CK in the presence
of 500 µM ATP decreased the cell viability of J774 macrophages. The percentage of cell death
observed in the presence of 500 µM ATP + 10 µM CK was found to be equivalent to the
percentage of cell death observed in the presence of 3 mM ATP (app. 30 %) (Figure 5.8 A).
The same effect was not observed with any of the other ginsenosides tested. Therefore, it could
be suggested that ginseng metabolite CK was able to enhance the ability of ATP to cause cell
death in J774 mouse macrophages. The ability of ginsenosides to increase cell death was then
tested in LPS primed cells (Figure 5.8 B). J774 macrophage cells were incubated with 100
ng/ml LPS for 24 hours along with all other treatments. It was observed that 3 mM ATP reduced
cell viability to 30% of the untreated controls (Figure 3.8). The effect was P2X7 mediated as
the P2X7 antagonist AZ10660120 (10 µM) reverted the cell viability to 81 %. 500 µM ATP
was used as a non-lethal dose of ATP as it did not affect cell viability. In the presence of 10
µM CK at 500 µM ATP in LPS treated macrophages, the cells death became equivalent to 3
mM ATP (app. 32 %). The same effect is not observed at lower concentrations of CK. Thus,
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in the case of LPS treated cells, CK replicated its ability to enhance cell death in macrophages.
Ginsenoside Rd which did not have any cell death enhancing effect in non LPS primed cells
caused a decrease in cell viability in the presence of ATP. Rb1 and Rh2 did not show any effect
on cell viability in the presence or absence of LPS.
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Figure 5.8 Effect of LPS priming on cell viability in ATP stimulated J774 macrophages in the
presence of ginsenosides
(A) Cell viability was measured using Cell Titre Aqueous ONE solution (Promega) after
incubation J774 macrophage cells with specific treatments for 24 hours (without LPS priming).
Cell viability in the presence of ATP is shown in dark grey bars, ATP + 10 µM ginsenosides
in black patterned bars and ATP + ginsenoside + AZ10660120 shown in grey patterned bars.
Error bars represent SEM, * denotes P<0.05 by one-way ANOVA (Tukey’s multiple
comparison test). (B) Cell viability measured after incubation of J774 macrophage cells in the
presence of 100 ng/ml LPS with all the other treatments specified in panel A for 24 hours. Cell
viability measured in presence of LPS is shown in black patterned bars, LPS + ATP is shown
in orange and LPS + ATP + AZ10660120 shown in grey patterned bars. Error bars represent
SEM, * denotes P<0.05 by two-way ANOVA (Tukey’s multiple comparison test).
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5.4.6 CK and Rd increase P2X7 mediated cellular ROS production
ROS are considered as the key signalling molecules for inflammatory disorders (499). It is
known that P2X7 activation can cause production of ROS upstream of the inflammasome
assembly (500). The effects of ginsenosides on cellular ROS production were tested by using
CM-H2DCFDA dye. It was observed during the optimisation process that if the macrophages
were incubated with the CM-H2DCFDA ROS indicator prior to adding the drugs (as suggested
by the manufacturer’s instructions), the differences in ROS species were difficult to measure
(as ROS are very short lived).
Therefore, for these experiments, the cells were not incubated with CM-H2DCFDA dye before
to adding drugs or LPS. The media was removed from the cells and replaced with ROS
indicator dye ± drugs. The changes in fluorescence were measured every 15 minutes for up to
2 hours on a Clariostar plate reader. The data were then quantified as fold change from initial
to final reading (Figure 5.9). Consistent with the Ca2+ and cell viability data, in ATP activated
non LPS-primed J774 macrophages (CM-H2DCFDA fluorescence fold increase- 1.5),
ginsenosides CK and Rd increased cellular ROS fluorescence by 11.5 and 7.6 fold respectively
(Figure 5.10). In LPS primed cells, it was observed that the ROS production was more in LPS
+ ATP treated cells (the value being 5.2-fold) in comparison to ATP treated cells only.
Ginsenosides CK and Rd again increased these values to 17.8 and 13.5-fold respectively.
Ginsenosides Rb1 and Rh2 do not show any significant difference in ROS production (Figure
5.11). Thus, from these results it can be suggested that the LPS priming of J774 macrophages
could be causing a greater increase in disruption of the mitochondrial membrane potential in
comparison to ATP treated cells alone. This could lead to an increase in production of cellular
reactive species, causing an enhancement in the pro-inflammatory properties of ginsenoside
CK and Rd.
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Figure 5.9 Optimisation of CM-H2DCFDA assay with H2O2 as positive control in the
presence and absence of LPS priming in J774 macrophages for measurement of cellular
ROS
(A) Cellular ROS production was measured in J774 macrophages as change in CM-H2DCFDA
fluorescence for every 15 minutes up to 90 minutes. Amount of cellular ROS produced over
time by J774 macrophages in the presence of H2O2 (positive control) is shown in grey circles,
LPS + H2O2 responses are shown in orange solid circles, basal responses in presence of buffer
are shown in grey triangles and ROS production in presence of LPS (100 mg/ml) are shown as
orange open circles. ROS production in LPS primed cells in presence of H2O2 is shown in
orange. (B) The data was quantified as fold change in CM-H2DCFDA fluorescence from 0 to
90 minutes. Fold change for buffer shown in grey open bar, for 100 ng/ml LPS shown in orange
open bar, 0.01 % H2O2 shown in grey solid bar and H2O2+ LPS shown in orange and grey
patterned bar. Error bars represent S.E.M, *P<0.05 by one-way ANOVA (Dunnett’s multiple
comparison test).
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Figure 5.10 Effect of selected ginsenosides on cellular ROS production in the presence and
absence of LPS priming in J774 macrophages in CM-H2DCFDA assay.
(A) Ginsenosides CK, Rd, Rb1 and Rh2 were tested for their effect on P2X7 mediated cellular
ROS production using CM-H2DCFDA ROS sensitive dye. Increase in CM-H2DCFDA
fluorescence over time in the presence of P2X7 agonist ATP ± ginsenosides is shown here as
fluorescence (random fluorescence units) at different time points. (B) Data was quantified as
fold change in CM-H2DCFDA fluorescence from 0 to 90 minutes. Data from ATP treated
controls are represented as grey bars with black outlines, ATP + ginsenosides treated cells are
represented as black chequered bars and the ATP + ginsenosides+ AZ10606120 is represented
as grey patterned bars. n=3, error bars represent S.E.M, * denotes P<0.05; two-way ANOVA
with Tukey's post-test.
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Figure 5.11 Effect of ginsenosides on cellular ROS production in the presence and absence of
LPS priming in J774 macrophages.
(A) Ginsenosides CK, Rd, Rb1, Rh2 and control a glycone PPD1 were tested for their effect
on P2X7 mediated cellular ROS production in the presence of 100 ng/ml LPS in J774
macrophages. Increase in CM-H2DCFDA fluorescence over time in the presence of LPS + ATP
is shown in orange solid circles, in presence of LPS + ATP + CK is shown in orange open
circles. (B) Data was quantified as fold change in CM-H2DCFDA fluorescence from 0 to 90
minutes. Data from LPS + ATP treated controls are represented as white patterned bars with
black outlines, LPS + ATP + ginsenosides treated cells are represented as orange chequered
bars and the LPS + ATP + ginsenosides + AZ10606120 is represented as grey patterned bars.
n=3, error bars represent S.E.M, * denotes P<0.05; two-way ANOVA with Tukey's post-test.
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5.4.7 CK and Rd increase P2X7 mediated mitochondrial superoxide (mtSOX)
production
In the cellular systems, there are a number of professional producers of ROS such as NADPH
oxidase (NOX), mitochondria, endoplasmic reticulum and peroxisomes. Mitochondria have
been considered as the chief source of endogenous ROS in living organisms. After establishing
the effect of selected ginsenosides on levels of cellular ROS in J774 macrophages, the effect
was reaffirmed using a mitochondria ROS indicator MitoSOX. CM-H2DCFDA used to
measure cellular ROS is a general ROS indicator, which measures all endogenous ROS
(hydroxal, peroxyl and other reactive species) (Figure 5.9 – Figure 5.11). MitoSOX on the
other hand, measures superoxide radicals produce in the mitochondria. For measuring changed
in MitoSOX fluorescence, the cells were incubated with the dye and drugs all at once and the
fluorescence was measured every 15 minutes up to 2 hours (similar to CM-H2DCFDA assay).
Actinomycin A and FCCP (p-trifluoromethoxy carbonyl cyanide phenyl hydrazone) were used
as positive controls. Actinomycin A inhibits the respiratory chain at complex 3 (between
cytochrome b and cytochrome c1), thereby preventing the oxidation of both NADH and
succinate (501). FCCP, on the other hand, is a respiratory uncoupler which increase proton
permeability of the cell and specifically uncouples electron transport chain from ATP synthesis
(502) Both compounds increase the production ROS from mitochondria, so they are suitable
positive controls for the MitoSOX assay. Both antimycin A (10 µM) and FCCP (5 µM) showed
a consistent increase in ROS production over the period of measurements (Figure 5.12). The
results from the mitochondria superoxide measurements confirmed that ginsenoside CK and
Rd increased the superoxide production by acting on ATP activated P2X7 in macrophages. The
effect was observed to be enhanced in presence of LPS. These results confirmed that CK and
Rd but not Rb1 and Rh2 increased the ROS mainly from mitochondrial source. It was further
observed that LPS enhanced the effect of ATP mediated production of mitochondrial
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superoxide (mtSOX) as well as increased the ability of ginsenoside CK and Rd to accentuate
ATP mediated mtSOX production. Macrophages are known to produce ROS by various
mechanisms such a mitochondrial damage, NADPH oxidases (NOXs) and endoplasmic
reticulum stress. NADPH oxidase is a membrane bound complex which is responsible for
production of superoxide and H2O2. Recent studies have indicated the involvement of NOX2
in the production of mitochondrial superoxide. Thus, the increased production of mitochondrial
superoxide on the exposure of macrophages to LPS (Figure 5.14) could be due to the
involvement of NOX2. In presence of LPS, ATP treated macrophages had 5.2-fold change in
MitoSOX fluorescence, LPS + CK + ATP had a 17.83 fold change (6.31 more fold change
that non-LPS treated macrophages); LPS + Rd + ATP increased ROS production by 13.54 fold
(5.91 more fold change than non-LPS treated cells). P2X7 inhibitor AZ10660120 (10 µM) was
able to inhibit this increase in all treatments affirming that these responses are P2X7 mediated.
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Figure 5.12 Effect of ginsenosides on mitochondrial superoxide (mtSOX) productions in
the presence of FCCP and Actinomycin in ATP activated J774 macrophages.
(A) mtSOX production was measured in ATP activated J774 macrophages every 15 minutes
for 90 minutes. FCCP and Actinomycin A were used as positive controls. Changes in MitoSOX
fluorescence in presence of buffer are shown in grey dots. An increase in ROS production was
seen in presence of 10 µM Actinomycin A (blue dots). (B) Data was quantified as fold change
in fluorescence between time points 0 to end point of 90 minutes. Grey bar represents ROS
production by macrophages in buffer, black patterned bar represents data from 500 µM ATP,
positive controls FCCP and Antimycin A are represented by cyan coloured bar and open blue
bar respectively. n=3, error bars represent S.E.M, * P<0.05; one-way ANOVA with Dunnett’s
multiple comparison test.
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Figure 5.13 Effect of ginsenosides on mitochondrial superoxide productions in ATP
activated J774 macrophages.
(A) Ginsenosides CK, Rd, Rb1 and Rh2 were tested for their effect on P2X7 mediated mtSOX
production. Increase in MitoSOX fluorescence from 0 to 105 minutes in the presence of ATP
are shown in black solid circles and MtSOX production in presence of CK+ ATP is shown in
black open circles. (B) Data was quantified as fold change in MitoSOX fluorescence (by
deducting initial value from final value and the dividing by initial fluorescence value). ATP+
ginsenosides treated controls are represented as black patterned bars; ATP+ ginsenosides +
AZ10606120 treated cells are represented as grey pattered bars. n=3, error bars represent
S.E.M, *represents P<0.05, two-way ANOVA with Tukey's post-test
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Figure 5.14 Effect of ginsenosides on mitochondrial superoxide production in presence of
100 ng/ml LPS in ATP activated J774 macrophages.
(A) Ginsenosides CK, Rd, Rb1 and Rh2 were tested for their effect on P2X7 mediated mtSOX
production in the presence of 100 ng/ml LPS. Increase in MitoSOX fluorescence from 0 to 105
minutes in the presence of LPS + ATP is shown in orange solid circles and mtSOX production
in presence of LPS + CK + ATP is shown in black open circles. (B) Data was quantified as
fold change in MitoSOX fluorescence. LPS + ATP treated controls are represented as orange
patterned bars; ATP + ginsenosides treated cells are represented as grey patterned bars and
responses from LPS+ATP+ ginsenosides + AZ10606120 are shown in grey open bars, n=3
*P<0.05, two-way ANOVA with Tukey's post-test.
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5.8 CK increases caspase 3/7 activity in ATP activated J774 macrophages
The measurement of cell death by MTS assay provided an evidence of extent of cell death but
did not establish the mechanism of cell death pathway involved. The progression of cell death
involves activation of a number of processes before the cell reaches a point-of-no-return and
commits itself to death. The cells on a death route would display a few characteristic features
such as large activation of caspases (503), disruption of mitochondrial membrane potential
(504), permeabilisation of outer mitochondrial membrane (505) and exposure of phosphatidyl
serine. All the above act as important biological responses to initiate phagocytosis (506). P2X7
activation is associated with change in cell morphology including membrane blebbing,
phosphatidylserine exposure and activation of classical apoptotic signalling cascade (79, 450,
507). Wen et al. reported the activation of caspase-3 and PARP-1 in ATP activated apoptosis
in HEK-293 cells expressing P2X7 (508).
In an attempt to establish the signalling molecules that potentiated by CK and Rd via ATP ±
LPS activated P2X7 in macrophages, the activity of caspase 3/7 was measured in J774
macrophages. Caspases are the members of the caspase family of proteases that regulate cell
death pathways including apoptosis (509). Various cell death stimuli such as injury, exposure
to inflammatory stimuli (such as LPS) or any other insults can result in activation of caspases
which, in a systematically coordinated manner, work towards proteolysis of numerous cellular
proteins, ultimately leading to cell death. Caspase 3 and 7 along with caspase 9 work
downstream in the cell death pathways are referred as “effector” caspases. They are known to
handle the bulk of proteolysis implicated in cell death (510). P2X7 is known to mediate
apoptosis/cell death via activation of caspase 3/7/9 (511)
The cell viability results from MTS assay clearly indicate that CK increases cell death at
otherwise non-lethal concentrations of ATP (500 µM), but these results are not indicative of
the signalling molecules that are actually switched on during the process. For the purpose of
172
finding whether caspase 3/7 activity is affected by presence of ginsenosides in ATP-P2X7 axis
in macrophages, CellEvent™ Caspase-3/7 Green Detection Reagent was employed along with
NucBlue™ Live ReadyProbes™ Reagent (for cell number). The caspase 3/7 activity was
observed in live cells by capturing images every 30 minutes for a 12 hours period, using Image
Express (Molecular Devices). Caspase activity was recorded as green fluorescence and the
number of cells was counted on the basis of blue fluorescence from NucBlue™ Live
ReadyProbes™ Reagent. Images taken every 30 minutes were later analysed using
MetaExpress software (Figure 5.16 and Figure 5.18). The data was expressed as percentage of
caspase 3/7 positive cells. 0.5 µM staurosporine (STR) was used a positive control as STR is
known to induce apoptosis by activating caspases and is a recommended positive indicator of
apoptotic cell death (512). It was observed that a robust increase in caspase 3/7 activity took
place in presence of STR in comparison to control cells (only buffer) (Figure 5.15). 500 µM
ATP caused a similar increase in caspase 3/7 and most of the response was inhibited by P2X7
antagonist AZ10606120, confirming that it was a P2X7 mediated effect. Various
concentrations of CK (1, 5, 10 and 20 µM) were tested in order to ascertain the most optimum
concentration for measuring this activity. The criterion for optimum concentration was
choosing a concentration, which was non-toxic on its own and produced a robust, measurable
caspase 3/7 response. It was found that at 20 µM concentration of CK, 100 % cells were caspase
3/7 positive within the first 2 hours of the measurement (Figure 5.15). This was a P2X7
independent effect as AZ10606120 was unable to inhibit the caspase 3/7 activity at this
concentration of CK. All concentrations below 10 µM CK was non-toxic on their own.
Therefore, 10 µM was chosen to be the test dose. Since 20 µM CK gave immediate and very
strong caspase activity signals (with about 93 % of the cells showing caspase 3/7 activity at
time point 6), it was decided to include 20 µM CK as a positive control for all these
experiments. Ginsenosides CK, Rd, Rb1 and Rh2 at a concentration of 10 µM were tested for
their effect on caspase 3/7 activity (Figure 5.17). It was seen that 500 µM ATP on its own took
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6 hours to initiate a caspase 3/7 activity and at the end of the 12 hours measurement period,
50% of the macrophages were caspase 3/7 active. Ginsenosides CK, Rd, Rb1 and Rh2 at a
concentration of 10 µM were then tested ad it was found that in the presence of CK + ATP, at
the end of time point 6 (3 hours) 88 % of J774 macrophages were positive. At the end of the
measurements (12 hours) ,caspase 3/7 were active in 100% of the macrophages. P2X7
antagonist AZ10606120 was able to inhibit the caspase 3/7 activity, indicating that P2X7 was
involved in activation of these caspases in the cells. Rd, Rb1 and Rh2 did not show any
potentiation of ATP- P2X7 mediated caspase activity. Even though all these ginsenosides
elicited high caspase 3/7 activity at 20 µM concentration (in the absence of ATP- in a P2X7
independent manner), at 10 µM concentrations only CK was able to elevate the ATP- P2X7
mediated increase in caspase 3/7 activity. These results indicate that CK increase the pro-
inflammatory ability of ATP by increasing Ca2+ / K+ ion fluxes, increasing ROS production
and resulting in a positive effect on caspase 3/7 activity in J774 macrophages. A similar trend
was seen with control 0.01 % DMSO where approximately 50% of the cells were positive for
caspase activity.
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Figure 5.15 Measurement of caspase 3/7 activity in the presence of apoptotic inducer
staurosporine for optimisation of assay
(A) The graph shows the fluorescence of CellEvent™ Caspase-3/7 Green Detection Reagent
for each time point measured (every 30 minutes for 12 hours). The basal caspase 3/7 activity
indicated by increase in green fluorescence over time in J774 macrophages treated with 0.01
% DMSO is shown in grey circles, caspase 3/7 activity in presence of P2X7 agonist ATP is
shown in black. Caspase 3/7 activity in presence of staurosporine (STR, used here as positive
control for apoptosis) is shown in grey circles with black outlines and finally responses in
presence of 20 µM CK is shown in red open circles. (B) The percentage J774 macrophages
positive for Caspase-3/7 activity at time point 6 of the assays (time point 12; 6 hours) were
then plotted as a bar graph. 500 µM ATP is shown as black solid bar, 20µM CK as red open
bar, 0.5µM STR as red patterned bar and 0.01% DMSO as black open bar. The percentage
positive cells in presence STR are significantly higher than those in presence of ATP on its
own. n=3 *P<0.05, significant effect of 0.5 µM STR and 20 µM CK ; one-way ANOVA with
Dunnet’s multiple comparison test.
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Figure 5.16 NucView 488 3/7 caspase activity in presence of 0.01% DMSO, 0.5 µM STR
and 20 µM CK
Representative images of caspase 3/7 activity of J774 macrophages in presence of 0.01 %
DMSO, 0.5 µM STR taken at time point 12 (after 6 hours) are shown here. The images were
taken under three filters (A) Images taken under transmitted light showed the changes in
morphology of the macrophages through time. The cells were adherent to the surface till the
end of the assay. Some cellular debris could be observed, and the cell membranes started to
look shrivelled for cells treated with 20 µM CK. (B) The NucBlue dye stained the nucleus and
was used to assists in keeping track of the cell number. The cell number represented by blue
fluorescence remained constant throughout the assay. The cell membrane showed some
fragmentation, but the nuclei remained stained. (C) Green fluorescence indicated caspase 3/7
activity. It can be seen that in J774 macrophages treated with 0.01 % DMSO, there is negligible
caspase 3/7 activity at the end of 6 h incubation whereas in macrophages treated with 0.5 µM
STR, approximately 70 % cells showed green fluorescence while in presence of 20 µM CK,
around 95 % cells were positive of caspase 3/7 activity.
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Figure 5.17 Measurement of caspase 3/7 activity in the presence of selected ginsenosides
CK, Rd, Rb1 and Rh2
(A) The graph shows the fluorescence of CellEvent™ Caspase-3/7 Green Detection Reagent
for each time point measured (every 30 minutes for 12 hours). Percentage of macrophages
positive for caspase 3/7 activity (0- 12 hours, recorded every 30 minutes) in presence of 500
µM ATP is shown in black circles, caspase activity in presence of ATP + CK is shown in red
circles, ATP + Rd in blue squares, ATP + Rb1 shown in green triangles and ATP + Rh2 is
shown in purple inverted triangles. (B) The percentage J774 macrophages positive for
caspase3/7 activity at time point 12 of the assays (time point 12; 6 hours) were then plotted as
a bar graph. 500 µM ATP is shown as black solid bar, ATP + 10 µM CK as red patterned bar,
ATP + 10 µM Rd as blue patterned bar, ATP + 10 µM Rb1 as green patterned bar and ATP +
10 µM Rh2 as purple patterned bar as black open bar. The percentage positive cells in presence
ATP+CK are significantly higher than those in presence of ATP on its own. n=3, error bars
represent S.E.M, * denotes P<0.05, one-way ANOVA with Dunnet’s multiple comparison test.
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Figure 5.18 NucView 488 3/7 caspase activity in presence of 500 µM ATP, ATP + CK and
ATP + CK + AZ10606120
Representative images of caspase 3/7 activity of J774 macrophages in presence of 500 µM
ATP, 500 µM ATP + CK and 500 µM ATP + CK + AZ10606120 taken at time point 12 (after
6 hours). The images were taken under three filters (A) Images taken under transmitted light
show the changes in morphology of the macrophages through time. The cells are adherent to
the surface till the end of the assay. Some cellular debris could be observed, and the cell
membranes started to look shrivelled for cells treated with ATP + 10 µM CK. (B) The NucBlue
dye stained the nucleus and was used to assists in keeping track of the cell number. The cell
number represented by blue fluorescence remained constant throughout the assay. The cell
membrane showed some breakdown, but the nuclei are stained. (C) Green fluorescence
indicates caspase 3/7activity. It can be seen that in J774 macrophages treated with 500 µM
ATP, there was negligible caspase 3/7 activity at the end of 6 h incubation whereas in
macrophages treated with 500 µM ATP + CK, approximately 92 % cells show green
fluorescence.
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5.4.9 CK and Rd increase caspase 3/7 activity in the presence of LPS in ATP activated
macrophages
LPS primed J774 macrophages were then tested to determine whether caspase 3/7 activation
kinetics were altered. It was found that LPS caused an increase in the baseline caspase 3/7
activity with more LPS treated cells being activated in comparison to controls from the
beginning of the assay (Figure 5.19). There was an increase in overall caspase 3/7 activity in
all the conditions tested. 10 µM CK replicated its behaviour in the presence of 500 µM ATP
and 100 ng/ml LPS achieving the highest caspase activity at about time point 6 (after 3 hours)
with 100 % cells showing positive for caspase 3/7 from this point onward. Rd, Rb1 and Rh2
did not cause a significant increase in caspase activity in presence of LPS + ATP (Figure 5.20).
In comparison to LPS non-treated cells, more caspase 3/7 activity was observed when Rd, Rb1
or Rh2 was added to LPS treated macrophages (Figure 5.19). The data from cell viability and
caspase 3/7 measurement is indicative of the involvement of ginsenosides CK in potentiating
the pro-inflammatory potential of P2X7 in macrophages confronted with inflammatory stimuli,
which may coerce the cells towards apoptotic cell death pathways. The clear evidence for the
cell death pathway followed by the activation is not precisely evident from these results so
further validation using TUNEL or DNA fragmentation could be employed.
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Figure 5.19 Measurement of caspase 3/7 activity in the presence of selected ginsenosides
CK, Rd, Rb1 and Rh2 in LPS primed J774 macrophages.
(A) The graph shows the fluorescence of CellEvent™ Caspase-3/7 Green Detection Reagent
for each time point measured (every 30 minutes for 12 hours). Data is represented as percentage
of macrophages positive for caspase 3/7 activity (0 - 12 hours, recorded every 30 minutes). (B)
The percentage J774 macrophages positive for caspase-3/7 activity at time point 12 of the assay
(6 hours) were then plotted as a bar graph. The percentage positive cells in presence LPS + 0.5
µM STR, LPS + 20 µM CK and LPS + 500 µM ATP are significantly higher than those in
presence of 100 ng/ml LPS on its own. n=3, error bars represent S.E.M, *denotes P<0.05, one-
way ANOVA with Dunnet’s multiple comparison test.
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Figure 5.20 Measurement of caspase 3/7 activity in the presence of selected ginsenosides
CK, Rd, Rb1 and Rh2 in LPS primed J774 macrophages.
(A) The graph shows the fluorescence of CellEvent™ Caspase 3/7 Green Detection Reagent
for each time point measured (every 30 minutes for 12 hours). Data is represented as percentage
of macrophages positive for caspase 3/7 activity (0- 12 hours, recorded every 30 minutes). (B)
The percentage of J774 macrophage cells positive for caspase 3/7 activity at time point 12 of
the assays (6 hours) were then plotted as a bar graph. The percentage positive cells in presence
LPS+ ATP + CK are significantly higher than those in presence of LPS + ATP on its own. n=3
* denotes P<0.05, one-way ANOVA with Dunnet’s multiple comparison test.
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5.4.10 Effect of selected ginsenosides on P2X7 mediated responses in primary peritoneal
macrophages
The J774 macrophage cell line is a well-characterised and reliable model for immunological
studies (481). J774 is also frequently used because it is readily available, and its use reduces
the number of animals involved in research. Conversely, there are some studies indicating that
responses measured using J774 macrophages may be weaker to or different from those of
primary macrophages (513). Methods involving isolation and maintenance of peritoneal
macrophages in cultures have been previously well defined. It is also known that P2X7 receptor
is endogenously expressed in these cells. Therefore, some of the experiments performed using
J774 macrophages in this study were also validated further using primary peritoneal
macrophages (Figures 5.21, 5.22, 5.23, 5.24 and 5.25). The cells were isolated from the
peritoneum of Wistar rats.
5.4.11 CK and Rd increase Ca2+ influx in ATP activated primary peritoneal
macrophages
In consensus with the data obtained using the J774 macrophage cell line, it was observed that
CK and Rd significantly increased the concentration of intracellular Ca2+ following activation
of P2X7 by ATP (Figure 5.21). The responses were inhibited by P2X7 antagonist
AZ10606120. The potentiation by 10 µM CK was greater in primary peritoneal macrophages
than in the J774 cell line. The magnitude of the data was not significantly different in the two
types of cell sources. The results here corroborated our results obtained with the J774
macrophage cell line. The effect of different concentrations (0.1- 50 µM) of CK on Ca2+ influx
in primary macrophage cells was tested by stimulating these cells at fixed concentration (200
µM) of ATP. The EC50 of CK was found to be 0.5 µM (95 % confidence interval – 0.2 to 1.0)
(Figure 5.22).
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5.4.12 Effect of ginsenosides on cell viability in primary peritoneal macrophages
Primary peritoneal macrophages were plated in 96 well plates overnight (± drugs, ± LPS) and
cell viability was measured using MTS. ATP at 500 µM did not cause any cell death after 24
hours treatment. Instead the cell number was higher in ATP treated cells in comparison to
untreated controls. Results from these experiments confirmed that 10 µM CK could accelerate
the death in ATP activated macrophages (CK + ATP cause ~ 45 % cell death) in a P2X7
mediated manner. 10 µM Rd, on the other hand, was able to potentiate this cell death only in
the presence of pro-inflammatory stimulus 100 ng/ml LPS. The increase in cell death was P2X7
mediated as the effect was reversed in the presence of P2X7 antagonist AZ10606120 (Figure
5.23).
5.4.13 Effect of ginsenosides on mitochondrial superoxide production in primary
peritoneal macrophages
The effect of selected ginsenosides was tested on mitochondrial superoxide production.
Ginseng metabolite, CK, increased ROS production (superoxide radicals) ± LPS in primary
peritoneal macrophages (Figure 5.24). FCCP (carbonilcyanide p-
triflouromethoxyphenylhydrazone), the mitochondrial uncoupler, at a concentration of 5 µM,
was used as a positive control for the measurement of mitochondrial superoxides. The other
ginsenosides increase in ROS production (± LPS) significantly in comparison to ATP treated
controls. LPS priming was observed to cause a general increase in ROS production in all
conditions tested.
5.4.14 Effect of CK on caspase 3/7 activity in primary peritoneal macrophages
Primary peritoneal macrophages were also employed for confirming the caspase 3/7 activity
data obtained using J774 macrophages. The numbers of peritoneal macrophages available for
these experiments were limited, so only the ginsenoside CK, which had consistently shown
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potentiation of P2X7 responses, was selected to be tested on primary peritoneal macrophages.
The data from these experiments confirmed the ability of 10 µM CK to increase the caspase
activity 3/7 (Figure 5.25). The responses are similar in that presence and absence of LPS. In
presence of LPS, 4.4 % more peritoneal macrophages were caspase 3/7 positive in comparison
to cells treated with ATP only. In presence of 0.5 µM STR (± LPS), ~ 45 % of the cells were
caspase 3/7 active and LPS treatment did not cause any significant increase in the caspase
activity. Similarly, 10 µM CK treatments resulted in the caspase 3/7 activation in ~ 99 % cells
after 6 hours of exposure and these responses were similar both in presence and absence of
LPS.
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Figure 5.21 Effect of ginsenosides on Ca2+ influx in primary peritoneal macrophages
(A) Intracellular Ca2+ responses were measured in Fura-2AM loaded primary peritoneal
macrophages (isolated from peritoneum of Wistar rats). Baseline values were recorded for
15 seconds and then ATP was applied. Fura-2 responses were measured over time period of
300 seconds on Flex station 3. (B) Primary peritoneal macrophages were treated with 200 μM
ATP, 200 µM ATP + ginsenosides and 200 μM ATP + 10 µM AZ10606120. Quantitative
measures of sustained Ca2+ response (mean fluorescence between 100 to 300 seconds - mean
of baseline fluorescence; from 0-15 seconds) was done. The responses are inhibited by P2X7
antagonist AZ10606120 (shown here as blue open bar). Data was collated from 2 independent
experiments (6 rats), *P <0.05, significant effect of ATP; one-way ANOVA with Dunnett's post-
test.
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Figure 5.22 Effect of CK on ATP-induced Ca2+ influx in P2X7 responses in primary
peritoneal macrophages
Different concentrations (0.1- 50 µM) of CK were tested for their effect on Ca2+ influx in
primary macrophage cells stimulated at fixed concentration (200 µM) of ATP. The EC50 of CK
was found to be 0.5 µM (95 % confidence interval–0.2 to 1.0). Data is taken from 2 independent
experiments (6 rats).
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Figure 5.23 Effect of selected ginsenosides on cell viability in primary peritoneal
macrophages
(A) Cell viability of rat peritoneal macrophage cells measured using Cell Titre Aqueous ONE
solution (Promega) after incubation specific treatments for 24 hours (without LPS). Cell
viability of primary peritoneal macrophages in presence of ATP/ATP + ginsenosides is shown
in grey solid bars and ATP + ginsenoside + AZ10660120 shown in black patterned bars. Error
bars represent S.E.M , * P<0.05 by one-way ANOVA (Tukey’s multiple comparison test). (B)
Cell viability measured in J774 macrophage cells in the presence of 100 ng/ml LPS with all the
treatments (ATP, ATP + ginsenosides, ATP + ginsenosides + AZ10660120) for 24 hours. Cell
viability measured in presence of LPS + ATP / LPS + ATP + ginsenosides is shown in black
patterned bars and LPS + ATP + AZ10660120 shown in orange patterned bars. Error bars
represent S.E.M, * P<0.05 by one-way ANOVA (Tukey’s multiple comparison test).
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Figure 5.24 Effect of ginsenosides on mtSOX production in primary peritoneal
macrophages
Ginsenosides CK, Rd, Rb1 and Rh2 were tested for their effect on P2X7 mediated mtSOX
production in primary peritoneal macrophages. The change in MitoSOX fluorescence was
measured every 15 minutes for 105 minutes. Data was quantified as fold change in MitoSOX
fluorescence. Data from ATP + ginsenosides treated cells are represented as black dotted bars;
ATP + ginsenosides + LPS treated cells are represented as orange chequered bars. Data
collated from macrophages isolated from 6 rats. Error bars represent S.E.M * denotes P<0.05;
two-way ANOVA with Tukey's post-test
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Figure 5.25 Effect of LPS priming on caspase 3/7 activity in ATP stimulated peritoneal
macrophages in the presence of ginsenosides
(A) The graph shows the fluorescence of CellEvent™ Caspase-3/7 Green Detection Reagent
for each time point measured (every 30 minutes for 12 hours). Data is represented as percentage
of macrophages positive for caspase 3/7 activity (0- 12 hours, recorded every 30 minutes). The
peritoneal macrophages from Wistar rats were treated with 500 µM ± LPS, 0.5 µM STR ± LPS,
10 µM CK + ATP ± LPS. (D) The percentage of J774 macrophage cells positive for caspase3/7
activity at time point 12 of the assays (6 hours) were then plotted as a bar graph. The percentage
positive cells in presence ATP + CK ± LPS are significantly higher than those in presence of
LPS + ATP treated cells. The presence of LPS did not alter the caspase 3/7 activity in any of
the treatments. n=3 * Error bars represent S.E.M * denotes P<0.05; two-way ANOVA with
Tukey's post-test
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5.4.15 Effect of ginsenosides on cytokine secretion in J774 macrophages
To measure the effect of selected ginsenosides on secretion of pro-inflammatory cytokines (IL-
1β, TNF-α and IL-6), the J774 macrophages were treated with ginsenoside ± ATP and ± LPS.
ELISA was performed to measure the quantity of pro-inflammatory cytokines (IL-1β, TNF-α
and IL-6) released. In macrophage cells not primed with 100 ng/ml LPS, treatment of cells
with ginsenosides did not result in any significant change in cytokine secretion (Figure 5.24 A,
Figure 5.25 A and Figure 5.56 A).This observation was consistent with the report that
production of proinflammatory cytokines occur in the presence of inflammatory stimuli (such
as LPS) was essential in addition to a K+ releasing stimulus such as ATP (514).
In LPS primed cells, LPS treatment alone in the absence of ATP did not cause an increase in
cytokine production. This observation was consistent with previous reports that induction of
IL-1β maturation and externalisation by TLR agonists (LPS) is very inefficient in the absence
of a secondary signal such as extracellular ATP (515-517). It is known that LPS priming is a
general prerequisite for activation of caspase-1 in ATP stimulated cells (518).
In the presence of LPS, extracellular ATP caused a significant increase in secretion of cytokines
(Figure 5.24 B and Figure 5.25 B). TNF-α is a pleiotropic cytokine which is secreted by many
cells especially the ones from monocytic lineage (macrophages, microglia Kupffer cells,
Langerhans cells, astroglia) (519). TNF-α is a potent pro-inflammatory mediator that controls
several aspects of macrophage function. It is released following injury, infection (exposure to
bacterial-derived LPS) and is said to be one of the most abundant early mediators of
inflammation (520). Measurements of TNF-α secretion (Figure 5.24) showed the secretion of
this cytokine was 51.8 % more in macrophages treated with LPS + ATP + CK in comparison
to cells treated with LPS + ATP alone. An increase of 54.8 % in TNF-α secretion was observed
in presence of 10 µM Rd+ ATP in LPS primed macrophages. 10 µM Rb1 and Rh2 treatment
increased TNF-α by < 40 % (not statistically significant). Figure 5.25 illustrates the secretion
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of IL-1β from LPS-primed ATP activated J774 macrophages ± ginsenosides (quantities
expressed in pg/mL). It was observed that there was an 82.8 % increase in IL-1β in LPS primed
ATP activated macrophages treated with 10 µM CK. An increase of 81.6% was observed in
the presence of 10 µM Rd and 75.5 % in presence of 10 µM Rb1. An increase of 26.6 % was
observed in presence of 10 µM Rh2 but this enhancement was not statistically significant.
As observed in case of the other two cytokines (TNF-α and IL-1β), ATP activation by ATP
alone did not induce IL-6 secretion but LPS priming before ATP activation increased the IL-6
secretion by 92 % (Figure 5.26 B). Ginsenosides treatment, on the other hand, did not increase
the IL-6 release significantly. CK was seen to cause the maximal increase (~ 20 %) among the
ginsenoside tested (Figure 5.26 B).
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Figure 5.24 TNF-α production by J774 macrophages with and without LPS stimulation
(A) J774 macrophages were incubated with ginsenosides ± 500 µM ATP for 30 minutes.
Supernatants were collected and TNF-α was measured by ELISA. The increase in ATP- P2X7
mediated TNF-α secretion by J774 macrophages in presence of ginsenosides was not
significant. Error bars represent S.E.M, no significant effect of treatments observed.
P<0.05Two-way ANOVA, Tukey’s multiple comparison tests. (B) TNF-α production by LPS-
stimulated J774 macrophages was measured by priming the cells with 100 ng/LPS for 4 hours
before stimulating these cells with 500 µM ATP ± ginsenosides for 30 minutes. Supernatants
were collected precisely after 30 minutes incubation period and TNF-α was measured using
ELISA. TNF-α secretion in LPS primed macrophages was significantly increased in presence
of 10 µM CK and 10 µM Rd. Statistical analysis was done using Graph Pad prism; Graphs are
means (± S.E.M) from 3 independent experiments, and each experiment was performed in
duplicates. Error bars represent S.E.M, * denotes P<0.05 Two-way ANOVA, Tukey’s multiple
comparison test.
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Figure 5.25 IL-1β productions by J774 macrophages with and without LPS stimulation
(A) J774 macrophages were incubated with ginsenosides ± 500 µM ATP for 30 minutes.
Supernatants were collected and IL-1β was measured by ELISA. No significant increase in IL-
1β production was observed after ginsenosides treatment in ATP stimulated J774 macrophages.
Error bars represent S.E.M, Statistical analysis was done using Graph Pad prism; Two-way
ANOVA, Tukey’s multiple comparison test. (B) IL-1β production by LPS-stimulated J774
macrophages was measured by priming the cells with 100 ng/LPS for 4 hours before
stimulating with 500 µM ATP ± ginsenosides for 30 minutes. Supernatants were collected
precisely after 30 minutes incubation period and IL-1β was measured using ELISA. IL-1β
secretion in LPS primed J774 macrophages was significantly increased in presence of 10 µM
CK and 10 µM Rd. This increase in inhibited by P2X7 antagonist AZ10606120. Graphs are
means (± S.E.M) from 3 independent experiments, and each experiment was performed in
duplicates; * denotes P<0.05 Two-way ANOVA, Tukey’s multiple comparison test
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Figure 5.26 IL-6 production by J774 macrophages with and without LPS stimulation
(A) J774 macrophages were incubated with ginsenosides ± 500 µM ATP for 30 minutes.
Supernatants were collected and IL-6 was measured by ELISA. No significant increase in IL-
6 production was observed through ginsenosides treatment in ATP stimulated J774
macrophages. Error bars represent S.E.M, Statistical analysis done using Graph Pad prism;
Two-way ANOVA, Tukey’s multiple comparison test. (B) IL-6 production by LPS-stimulated
J774 macrophages were measured by priming the cells with 100 ng / LPS for 4 hours before
stimulating these cells with 500 µM ATP with or without ginsenosides for 30 minutes.
Supernatants were collected precisely after 30 minutes incubation period and IL-6 was
measured using ELISA. An increase in IL-6 secretion in LPS primed macrophages in
comparison to non-primed macrophages was observed. Selected ginsenosides caused an
increase in IL-6 production in LPS primed ATP stimulated J774 macrophages but this increase
was not statistically significant. Graphs are means (± S.E.M) from 3 independent experiments,
and each experiment was performed in duplicates. ns denotes not significant, Two-way
ANOVA, (Tukey’s multiple comparison test).
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5.4.16 Effect of ginsenosides on cytokine secretion in primary peritoneal macrophages
In order to confirm the results obtained using J774 macrophages, TNF-α, IL-1β and IL-6
secretions were measured in ATP activated primary peritoneal macrophages (isolated from
peritoneum of Wistar rats) with and without LPS priming. Measurements of TNF-α secretion
(Figure 5.27) showed that LPS priming before ATP activation increased TNF-α by 60.5 %. An
increase of < 35 % in TNF-α secretion was observed in presence of all ginsenosides in LPS
primed macrophages but none of this enhancement was statistically significant (Figure 5.27).
This is different from the observation made in J774 macrophages (Figure 5.24). Figure 5.28
shows the measurement of IL-1β in primary macrophages. An increase in IL-1β was observed
in LPS primed ATP stimulated rat macrophages in the presence of all ginsenosides.
Measurement of IL-6 in primary macrophages is shown in Figure 5.29. An increase in IL-6 is
observed in LPS primed primary macrophages in the presence of CK. This is different from
J774 macrophage cell line, where none of the ginsenoside tested caused any significant change
in IL-6 secretion (Figure 5.26).
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Figure 5.27 TNF-α production by primary peritoneal macrophages ± LPS stimulation
(A) Primary peritoneal macrophages isolated from Wistar rats were incubated with
ginsenosides ± 500 µM ATP for 30 minutes. Supernatants were collected and TNF-α was
measured by ELISA. The difference in ATP-P2X7 mediated TNF-α secretion by rat
macrophages in presence of ginsenosides was not significant. Statistical analysis done using
Graph Pad prism; Graphs are means (± S.E.M) from 6 different rats. Error bars represent
S.E.M. Two-way ANOVA, Tukey’s multiple comparison test (B) TNF-α production by LPS-
stimulated rat macrophages by measured by priming the cells with 100 ng/LPS for 4 hours
before stimulating these cells with 500 µM ATP with or without ginsenosides for 30 minutes.
TNF-α secretion in LPS primed macrophages is significantly increase in presence of 10 µM
CK and Rd. Statistical analysis done using Graph Pad prism; Graphs are means (± S.E.M) from
6 independent rats. Error bars represent S.E.M * denotes P<0.05 Two-way ANOVA, Tukey’s
multiple comparison test.
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Figure 5.28 IL-1β production by primary peritoneal macrophages ± LPS stimulation
(A) The peritoneal macrophages isolated from Wistar rats were incubated with ginsenosides
and /or 500 µM ATP for 30 minutes. Supernatants were collected and IL-1β was measured by
ELISA. The difference in ATP- P2X7 mediated IL-1β secretion by rat macrophages in presence
of ginsenosides was not significant. (B) IL-1β production by LPS-stimulated human
macrophages by measured by priming the cells with 100 ng/LPS for 4 hours before stimulating
these cells with 500 µM ATP with or without ginsenosides for 30 minutes. Supernatants were
collected after 30 minutes and IL-1β was measured using ELISA. IL-1β secretion in LPS
primed macrophages is significantly increase in presence of 10 µM CK and Rd. Statistical
analysis done using Graph Pad prism; Graphs are means (± S.E.M) from 6 independent rats.
Error bars represent S.E.M, *denotes P<0.05 Two-way ANOVA, Tukey’s multiple
comparison test.
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Figure 5.29 IL-6 production by rat macrophages with and without LPS stimulation
(A) The peritoneal macrophages isolated from Wistar rats were incubated with ginsenosides
and /or 500 µM ATP for 30 minutes. Supernatants were collected and IL-6 was measured by
ELISA. The difference in ATP- P2X7 mediated IL-6 secretion by rat macrophages in presence
of ginsenosides was not significant. (B) IL-6 production by LPS-stimulated human
macrophages by measured by priming the cells with 100 ng/LPS for 4 hours before stimulating
these cells with 500 µM ATP with or without ginsenosides for 30 minutes. Supernatants were
collected after 30 minutes and IL-6 was measured using ELISA. IL-6 secretion in LPS primed
macrophages is significantly increase in presence of 10 µM CK and Rd. Statistical analysis
done using Graph Pad prism; Graphs are means (± S.E.M) from 6 independent rats. Error bars
represent S.E.M * P<0.05 Two-way ANOVA, Tukey’s multiple comparison.
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5.5 Key results from this study are summarised in Figure 5.30
Figure 5.30 Diagrammatic representations of the experimental findings in the present
study.
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The key results from this study can be summarised from this illustration are:
➢ Out of the 4 ginsenosides tested, CK and Rd potentiated P2X7 mediated Ca2+ influx in
macrophages (both J774 and peritoneal macrophages).
➢ LPS priming did not cause any additional increase in P2X7 mediated Ca2+ influx
potentiated by ginsenosides CK and Rd.
➢ Ginseng metabolite CK increased cell death via P2X7 activation. This ability was not
exhibited by Rd, Rb1 and Rh2.
➢ In the presence of LPS, CK and Rd both decreased the viability of J774 macrophages via
potentiation of P2X7.
➢ CK increased caspase 3/7 activity in ATP activated J774 macrophages.
➢ In presence of LPS, there was a general increase in caspase 3/7 activity by all ginsenosides
tested but the increase was not significant statistically. Time required to achieve 100 %
activity by CK + ATP was reduced by LPS priming in J774 macrophages.
➢ In the presence of LPS, CK and Rd increased the cellular ROS and mitochondrial
superoxide production.
➢ CK and Rd increased the production of IL-1β and TNF-α in ATP activated macrophages
only in LPS primed cells (both J774 macrophages and peritoneal macrophages).
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5.6 Discussion
P2X7 is implicated in signalling between macrophages and other cells of the immune system
(521). Upon association with its endogenous ligand, ATP, there is an increase in P2X7 channel
activity, which allows for ion flux through this channel. The Ca2+ influx, which follows P2X7
activation, is always coupled to K+ efflux (522). The rapid K+ release is a critical trigger for
initiation of a number of pro-inflammatory pathways that are activated inside immune cells
following stimulation by the danger signal eATP (170). The mitochondrial membrane potential
is disrupted due to the rapid decrease in K+ and this leads to increases in the production of ROS
(523). Increased ROS production is a well-recognized event following P2X7activation (524).
This is followed by release of cytochrome c from the mitochondrial membrane complex. The
release of cytochrome c activates the caspase cascade (including caspase 9, 3 and 7) for
executing cell death (525). The optimum activation of all these signals is of prime importance
for stimulating and resolving inflammation. An inability to initiate these signals can result in
the incomplete clearance of infection leading to a state of chronic inflammation (526). The
consequences of modulation of P2X7-related ion flux can affect all the associated pathways,
causing an overall change in the ability of to resolve inflammation. In the previous chapter, it
was described that selected ginsenosides interact with P2X7 after it has been activated by ATP,
by binding to a site other than the ATP binding site (allosteric modulation) and can cause an
increase in intracellular Ca2+ levels. These experiments were performing using P2X7 over-
expressed in HEK-293 cells (chapter 4). The recombinant system was ideal for initial screening
purposes as it provided an opportunity for the receptor to be studied in isolation from other
interacting proteins since HEK-293 cells lack the inflammatory machinery mandatory for
eliciting immune responses following the activation of P2X7. For understanding the effect of
P2X7 on downstream signalling, these events had to be studied in macrophages; the cells
furnished with the appropriate machinery for a complete inflammatory response. Therefore, in
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the study described in this chapter, the events following the potentiation of receptor activity
were studied in detail using the J774 macrophage cell line as well as rat primary peritoneal
macrophages.
The selected ginsenosides were tested for their effect on intracellular Ca2+ concentrations
following ATP-induced activation of P2X7. CK and Rd (10 µM each) caused a substantial
increase in Ca2+ influx in macrophages while Rb1 and Rh2 did not show any effect on P2X7
mediated Ca2+ influx (Figure 5.5).
LPS is the paradigmatic stimulator of inflammation (527). The presence of LPS-binding
domains in the C-terminal domain has been suggested to be responsible for the ability of P2X7
to influence secretion of different immunomodulatory molecules (IL-1β, TNF-α, NO) in LPS-
stimulated macrophages (528). Denlinger et al. demonstrated the ability of certain P2X7
derived peptides to bind to LPS in vitro (529). Furthermore, this binding of LPS to P2X7 was
shown to inhibit activation of ERK1, ERK2 (extracellular signal-regulated kinases) by LPS in
RAW 264.7 macrophages (528). On its own, LPS is known to cause a slow activation of
caspase-1, which causes an accumulation of the inactive form of IL-1β. The decrease in
intracellular K+ concentration following activation of P2X7 provides the additional trigger
(signal 2) for the complete activation of caspase-1. Thus, P2X7-LPS axis may be responsible
for the comprehensive inflammatory response in immune cells such as macrophages.
Therefore, it was expected that treatment of J774 macrophages with LPS would cause an
increase in the extent of potentiation provided by CK and Rd. Instead, it was observed that
there was no increase in the Ca2+ influx for macrophages exposed to LPS for 4 hours before
ATP activation in comparison to non-LPS primed cells. This may suggest that the LPS priming
did not affect the P2X7 channel activity directly in J774 macrophages. The channel activity is
increased by ginsenosides but there is no additional influx of ions through the channel for
macrophages stimulated with LPS. Since LPS treatment did not cause any change in expression
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of P2X7 on the surface of cells, the above observations cannot be attributed to the difference
in number of P2X7 receptors on the cell surface following the priming event of macrophages.
Involvement of P2X7 in cell death events has been asserted in many studies (530-534). It has
been suggested that the transient opening of P2X7 channel by short exposure times to ATP
leads to Ca2+ influx (535), Phospholipase D activation (536), ATP-induced ATP release (537),
cell fusion and proliferation whereas a prolonged exposure (30 minutes) causes greater Ca2+
influx, activation of transcription factors, surface molecule shedding, intracellular parasite
killing and cell death (538). In the present study, macrophages were exposed to ATP for 24
hours in the presence and absence of ginsenosides. The cell viability data from the experiments
suggested that CK enhanced the ability of P2X7 to cause cell death at an otherwise non-lethal
dose of ATP-concentration. It was reported by Mackenzie et al. in 2005 that brief activation of
HEK-P2X7 cells (~ 2 minutes) resulted in “pseudoapoptosis” (450). This term was used
because the events following the exposure of these cells to ATP for a short period of time were
similar to apoptotic events (increases in cytosolic and mitochondrial calcium, membrane
blebbing, actin filament disruption, phosphatidylserine exposure, and disruption of ΔΨm).
However, all of these responses were found to be reversible. It was suggested that prolonged
exposure of HEK-P2X7 cells to ATP was required for cell death to occur (the mechanism of
which was proposed to be calcium independent by the authors) (450). The results in the present
study are similar in that J774 macrophages exposed to ATP (500 µM) exhibited Ca2+ influx,
increases in ROS and caspase 3/7 activity but did not result in cell death after 24 hours. In the
presence of CK, the initial trigger was suitably reinforced so that the cells committed to the cell
death pathway.
The ability of Rd to increase P2X7-dependent cell death was evident only in the presence of
LPS. It is known that LPS is a very strong inflammatory stimulus but by itself does not cause
a complete inflammatory response (in terms of cytokine secretion). In the presence of eATP
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though, P2X7 mediated K+ efflux causes the essential trigger to initiate the inflammatory
pathways. It has been shown that LPS stimulated macrophages from P2X7 knockout mice are
resistant to ATP-induced cell death and cytokine release (103, 104). Therefore, it has been
suggested that the P2X7/LPS axis is responsible for initiation and progression of complete
inflammatory/ cell death pathway in macrophages. From the results obtained from the cell
viability studies with the selected ginsenosides, CK was consistent in its behaviour in the
presence as well as absence of LPS. Rd on the other hand did not have any significant effect
on cell viability in non-primed cells. In LPS primed cells Rd was able to cause cell death in 50
% of the J774 macrophages. This may be explained by keeping in mind that P2X7 activation
causes K+ efflux and LPS leads to NLRP3 inflammasome activation. Rd increased Ca2+ influx
via ATP activated P2X7. Therefore, it also enhances the K+ efflux. The increase in K+ efflux
by Rd along with NLRP3 inflammasome activation by LPS together could be a possible tipping
point for Rd to express its cell death increasing potential. Therefore, it could be suggested that
exposure of macrophages to LPS encourages them to commit to cell death and this event is
accentuated by the presence of the ginsenoside Rd. Other ginsenosides tested (Rb1 and Rh2)
did not have any significant effect on cell viability of ATP stimulated J774 macrophages with
or without LPS priming. From the Ca2+ influx data obtained, this was the expected behaviour
of these compounds. It may be suggested that LPS acts downstream of the channel opening
and lowers the concentration of ATP required for P2X7 activation. Once the ATP threshold is
lowered then Rd opens the channels, causes more Ca2+ influx, and affects cell viability in a
manner similar to CK.
In a recent study, LPS treatment of THP-1 macrophages (± ATP) was found to initiate
“pyroptosis” within the first 2 hours of LPS exposure (539). Pyroptosis is a form of cell death
which involves the maturation and release of IL-1β and IL-18 via activation of caspase-1 (540).
Since a number of different events can happen following ATP activation of P2X7 (depending
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on the time of exposure, the concentration of ATP), further investigations with varying ATP
and Rd concentrations for different time spans are required.
The measurement of Ca2+ influx in this study was carried out over a period of 5 minutes, where
cells were exposed to ATP for 240 seconds (Figure 5.1). The MTS assay for measurement of
cell death was carried out for 24 hours (Figure 5.8). The cell death experiments described in
Chapter 5 show that at 500 µM ATP, the cells do show an increase in the initial Ca2+ influx as
well as other apoptotic parameters such as ROS release as well as cytokine production but are
unable to commit to cell death even after a 24 hours exposure. In the presence of CK, the initial
Ca2+ influx is increased many-fold and this increased Ca2+ concentration seems to increase the
magnitude of all the related signals.
An increase in intracellular Ca2+ is known to stimulate opening of the mitochondrial
permeability transition pore complex (PTPC) and the activation of caspases (541). The
activated caspases can degrade the oxidative phosphorylation complexes in the inner
mitochondrial membrane leading to disruption of mitochondrial membrane potential (ΔΨm)
(542) . An increase in ROS generally accompanies the progressive loss of ΔΨm (543). The
increase in cellular ROS and mitochondrial superoxide production by ginsenoside CK in this
study confirmed that the potentiation of P2X7 caused the mitochondrial Ca2+overload (Figure
5.10 and Figure 5.13). This further led to increased caspase 3/7 activity (CK + ATP results in
100 % cells to be caspase 3/7 active after 6 hours) (Figure 5.17). Thus, in the presence of ATP
concentrations where macrophages activate their stress response but the cells fail to die, the
presence of CK would provide the required additional trigger for them to commit to the cell
death pathway.
In the presence of LPS, another pathway is switched on in parallel. The priming of
macrophages with LPS alone cannot initiate caspase-1 activation (544). For caspase-1 to be
active, an increase in K+ efflux and ROS production caused by ATP-induced P2X7 activation
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is known to act as the required second signal or hit (545). An increase in this second signal
would presumably increase caspase-1 activation as well.
In a recent study, it has been suggested that there is cross-talk between the apoptotic pathway
and pyroptotic pathways (546). If a cell is being challenged by both apoptotic (ATP) and
pyroptotic (LPS) stimuli, this could result in activation of caspase 3/7 (via ATP) and caspase-
1 (via LPS). It has been suggested that in this case, the pyroptotic pathway would be inhibited
and caspase-1 would activate caspase 3/7. Therefore, the pro-inflammatory signals would be
switched off and the cells involved would undergo apoptosis. Therefore, the results described
in Chapter 5 may be understood in light of the observations made by Taabazuing et al. (546).
CK and Rd increase K+ efflux (within few seconds) and ROS production (15- 105 minutes) in
ATP-activated macrophages. In the presence of LPS (4 hours priming), these signals activate
caspase-1 (not directly measured) and cause increased production of pro-inflammatory
cytokines. The caspase 3/7 enzymes are maximally active within 6 hours. Thus the 24 hours’
time frame of the experiment is enough for CK to cause macrophage death even in the absence
of LPS. In the LPS primed cells though, the caspase-1 which was active within the 4 hours 30
minutes time frame (when the cytokines were measured) would now switch off the pyrogenic
pathway and instead activate the caspase 3/7 enzymes further. This could explain the behaviour
of ginsenoside Rd which in the absence of LPS priming is unable to help the cells cross the
“point-of-no-return” but in LPS primed cells, seems to mimic the ability of CK to promote cell
death. This could be because the activate caspase-1 from the pyrototic pathway is providing
the additional push over the tipping point required for Rd treated macrophages to die.
5.7 Conclusion
From the present study, it could be concluded that the major ginseng metabolite CK potentiated
P2X7 responses in macrophages. The potentiation of P2X7 by CK increased the Ca2+ influx
and ROS production, thereby activating the caspase 3/7 in these cells. Activation of caspase
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3/7 by CK then led to macrophage death in the presence of an otherwise non-lethal dose of
ATP (500 µM). These results indicated that CK encouraged the cells to commit to cell death
pathway under stress conditions. In the presence of the pro-inflammatory stimulus, LPS, Rd
also enables cell to undergo death, in a manner similar to CK. Presence of pro-pyroptotic signal
(LPS) along with apoptotic stimulus (eATP) was observed to increase the pro-inflammatory
cytokines secretion and this cytokine release was further enhanced by ginsenosides CK as well
as Rd. The coalition of these enhanced apoptotic and pyroptotic signals in the presence of
ginsenosides may provide a strong enough trigger for the macrophages to die. These results
indicate that the bioactive components of Panax ginseng can modulate P2X7 activity, thus
providing a scientific explanation to the anti-viral, anti-cancer and anti-inflammatory
properties of this medicinal herb.
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CHAPTER 6
GENERAL DISCUSSION
The homomeric P2X7 receptor belongs to P2X receptor family of ligand gated ion channels
sensitive to relatively high concentrations of extracellular ATP ( > 100 µM) (547) . P2X7 has
received considerable attention as a potential drug target because of its involvement in the
pathogenesis of inflammatory disorders as a critical mediator of the inflammasome complex
(185). This has led to the identification and characterisation of a number of novel, structurally
diverse modulators (233). Pharmacological profiles of various characterised modulators are
well defined and the IC50/EC50 values for these compounds vary depending upon the chemical
structure and the mammalian species it is tested in. For instance, A740003 has an IC50 of 0.040
µM for hP2X7 while it is 0.020 µM for rat P2X7 (203), Brilliant Blue G non-competitively
inhibits human and rat P2X7 with IC50 values of 0.2 and 0.001 µM respectively (548).
Similarly, P2X7 agonists ATP and BzATP are 10 times and 100 times more potent in rats than
in mice (549). In spite of a nanomolar/micromolar potency of some of the P2X7 antagonists
tested, there is a lack of specificity for many as these can still affect other purinergic receptors
(550). There is also a great variation in the agonist potency among various species which further
impedes studies relating this receptor (551).
Modulating P2X7 using pharmacological and genetic approaches alters inflammatory cytokine
secretion and cell death in immune cells (552-556). For instance, inhibition of P2X7 by
A438079 reduced the pro-inflammatory effects of heat shock protein (HSP 90) in subarachnoid
haemorrhage (557). Inhibition of P2X7 by LL-37 (an anti-microbial peptide) decreased the
production of pro-inflammatory cytokines (558). P2X selective antagonists NF449 and
A438079 inhibited HIV-1 mediated infection and production of inflammatory cytokines IL-10
and IL-1β in a human tonsil explant model (559). The P2X7 antagonist A8044598 reduced
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inflammation in brain and liver of C57BL/6J mice exposed to chronic ethanol and high fat diet
(560). Blockade of P2X7 by A438079 was also found to inhibit cytokine induced colitis of
human colonic mucosa (553). P2X7 receptors have been reported to trigger phagocytosis and
regulate cell death (561). These studies indicate that modulation of P2X7 related signalling
pathways can have important therapeutic implications in pathological conditions leading to
inflammation and cell death. Therefore, identification and characterisation of novel modulators
that target P2X7 specifically would be beneficial.
In this thesis, an attempt was made to describe the ability of two completely distinct sets of
compounds, tetracycline derivatives (MINO and DOX) and ginseng derived compounds (CK
and Rd) to modulate P2X7 receptor activity. Both these sets of compounds were previously
reported to have a role in the regulation of inflammatory processes, but this study provided
evidence of a possible common molecular target; the P2X7 receptor.
The lipophilic microglial inhibitor minocycline (MINO) is known to inhibit the activation and
proliferation of microglia, thereby down regulating neuroinflammation and neuropathic pain
(562-565). Microglia are believed to exist in two polar states: the immunomodulatory (M1)
state and pro-inflammatory state (M2). It is suggested that MINO selectively inhibits the
microglia from converting to the M2 state (566). Several attempts have been made to identify
and characterise molecular targets of MINO but no protein has so far been established as an
exclusive target for MINO action (301, 337, 352, 567). It has been suggested that microglia
derived Fas-ligand mediates immunoreactive responses and recruits’ microglia to the sites of
lesions via P2X7 (568). MINO was found to downregulate the Fas/P2X7 mediated
microembolic injury during ischaemia (569). It was observed that P2X7 activation led to the
accumulation and subsequent release of Fas ligand in conditions of cerebral ischemia (570). In
this study, pharmacological evaluation of P2X7 as a possible direct target for MINO was not
considered (571). In another recent study, the MINO-mediated reduction in microglia
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activation was reported to be accompanied by reduction in levels of TLR-2, its adaptor protein
MyD88, as well as the NLRP3 components of the inflammasome (572). The P2X7/NLRP3
axis is central to inflammatory signalling. The downregulation of NLRP3 protein levels by
MINO may suggest an action of MINO upstream of the inflammasome assembly.
Chapter 3 described the pharmacological characterisation of MINO in order to evaluate the
possibility of P2X7 being directly affected by this drug. The results of this study indicated that
MINO downregulated P2X7 channel activity (Ca2+ influx, Figure 3.2) and membrane pore
formation (YOPRO-1 uptake, Figure 3.6). The P2X7 associated physiological responses,
including cytokine secretion and cell death, were inhibited by MINO. A detailed
pharmacological evaluation of MINO indicated that this tetracycline derivative inhibited the
efficacy of the agonist ATP towards P2X7 receptor at relatively high concentrations with an
IC50 ~ 32.6 µM. It inhibited P2X7 and P2X4 in non-competitive allosteric manner. It was not
exclusively selective for P2X7 as closely related P2X4 and P2Y responses were also blocked
by the drug. It was suggested inhibition of P2X7 may prevent loss of astroglia by enhancing
PARP1 activity in the molecular layer of the dentate gyrus following status epilepticus.
Another study conducted by Alano et al. in 2006 found that MINO inhibits poly (ADP-ribose)
polymerase-1 (PARP-1) at nanomolar concentrations. This study reported that micromolar or
sub-micromolar concentrations did not affect PARP-1 activity (300). The results in this thesis
(Chapter 3) describe that MINO downregulates P2X7 function at high micromolar
concentrations. At concentrations below 25µM (results not shown), MINO loses its inhibitory
properties towards P2X receptors. Considering all these studies, it could be reasoned that
MINO at higher concentrations targets purinergic receptors, which can then inhibit anti-
apoptotic PARP-1 indirectly and down regulate pro-apoptotic TLR-1 and NLRP-3 proteins,
preserving the M1 phase of microglia. On the other hand, at low concentration, MINO inhibits
PARP-1 and increase pro-inflammatory state (M2). So, even though MINO may not be
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recognized as a selective, specific antagonist of P2X7, it can be inferred that P2X7 is one of
the targets of this multi-target drug.
Conventionally, drugs were intended to target a single cellular component/protein (“on-target”
approach), with the purpose of achieving high selectivity for that distinct protein and reducing
the chances of any side effects (off-target effects) (573). Due to this consideration, compounds
interacting with multiple targets were often discouraged and labelled as “dirty drugs’ (574).
However, the intricacies of the existing incurable diseases such as neurodegenerative disorders,
cancers and mental health problems have clearly proved that such single-target drugs may not
be sufficient for therapeutic purposes (575). On the other hand, studies have shown that multi-
targets drugs may in fact have a safer profile in these complex pathologies (576, 577). Between
the years 2015-2017, 21 % of the total new molecular entities approved by the US Food and
Drug Administration (FDA) were multi-target drugs (574). Therefore, microglial inhibitor
MINO could be targeting an array of yet undiscovered proteins, which are acting at undefined
positions within the P2X7 inflammatory cascade.
An insight into the structure-function relationship was also provided by the interaction of
structural analogues of tetracycline; closely related MINO and DOX as well as the
glycylcycline TIGE with P2X7. Tetracycline and its analogues share a basic chemical structure
made up of tetracyclic naphthacene carboxamide ring system (578). The presence of
dimethylamine group at C4 in the ring A is said to be essential for the antimicrobial activity of
tetracycline (579). The ROS-scavenging abilities of these compounds are generally attributed
to the phenol rings which react with free radicals to generate a resonance stabilised, unreactive
phenolic radical (580). No definite structural attribute has been assigned to the anti-
inflammatory properties of these compounds at this time. In this thesis, the tetracycline
derivatives showing P2X7 inhibitory responses were MINO and DOX. The structural
difference between these two compounds and tetracycline is the modification of C-7 of ring D
211
in MINO and C-6 in ring C in DOX. The methyl group on the upper peripheral zone of these
compounds could be responsible for binding to P2X7 receptor. Addition of the t-
butylglycylamido group on position C-9 in TIGE is assumed to contribute to its better antibiotic
activity, but in this study, TIGE did not inhibit P2X7 activity, indicating that this structural
variation may not contribute to anti-inflammatory properties of tetracyclines. Docking studies
are essential for providing further insight into the structure-activity relationship of these
compounds so that structural analogues with better specificity, strength and selectivity could
be generated to target P2X7 mediated inflammatory action.
Inflammation is a protective response elicited by the body in order to defend the living system
against any incoming disturbance or potential danger. The cells of the immune system
collaborate in a precisely coordinated manner to fight infection as well as resolve and repair
any damage caused by the inflammatory response. Ideally, inflammatory responses are
perfectly balanced, which means that the inflammatory cells and other components involved in
immune system-pathogen combat are cleared immediately after the infection is resolved. In
case of sub-optimal inflammatory responses, resolution of the inflammatory cells is not very
efficient, and the neutrophils and macrophages recruited for eliminating the invading pathogen
persist long after the actual infecting agent has been removed. This imperfect resolution of
inflammation leads to chronic inflammation. Inhibiting P2X7 responses may be beneficial in
suppressing the initial acute inflammatory responses but in case of chronic inflammation, an
up regulation of P2X7 could help in resolving inflammation, thereby helping to ameliorate or
abolish the symptoms related to chronic inflammation. The second part of this thesis (Chapters
4 and 5) describes novel positive modulators of P2X receptors.
Over activation of P2X7 has been reported to have positive effects under certain conditions.
For instance, the ATP-P2X7 pathway activates the p38/JNK/ATF-2 signalling pathway;
thereby protecting bone marrow derived macrophages (BMDMs) and the macrophage cell line
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RAW 264.7 from vascular stomatitis virus (VSV) mediated cell death via decreasing viral
replication in vitro. Similar results were obtained in vivo where a decrease in viral replication
was observed on activation of P2X7 in wild type mice (581). That viral suppression is P2X7
mediated is confirmed by the absence of any anti-viral activity in P2X7 knockout mice (581).
Thus, potentiation of P2X7 could have anti-viral effects. P2X7 activation has been shown to
have anti-bacterial effects as well. A decrease in bacterial load in epithelial cells and
macrophages is observed on activation of P2X7 by extracellular ATP following infection by
various Chlamydieae strains (233). P2X7 knockout mice have been found to be more
susceptible to Chlamydiae infection in comparison to their wild type counterparts (582).
Similarly, P2X7 has been linked with Mycobacterial infections. Loss-of-function mutation of
P2X7 is associated with increased susceptibility to tuberculosis infection (583). It is suggested
that the loss of functional P2X7 receptors impairs the capacity of macrophages to eliminate
intracellular bacteria (584). It has been shown that ATP activation of P2X7 causes bacterial
clearance in infected macrophages by increasing activation of phospholipase D and apoptosis
of infected cells. Genetic deletion of P2X7 was observed to decrease the susceptibility of
infection by H37Rv strain of Mycobacterium tuberculi (585).
P2X7 receptor is also known to play both anti-tumour and pro-tumour roles depending upon
the concentration of ATP in the interstitium of tumours, amount of ectonucleotidases in the
immune cells, tumours and stromal cells as well as the type of purinergic receptors expressed
by the tumour cells (424). Therefore, the role of P2X7 in cancer progression is often debated.
Overstimulation of P2X7 resulting in tumour cell death has been demonstrated in a number of
preclinical studies. (202, 586). In clinical trials though, increasing ATP in the extracellular
matrix has not been shown to have many beneficial effects. It is suggested that tumour cells
may be resistant to extracellular ATP because of disengagement of P2X7 from intracellular
cell death pathways in some cancers. Therefore, potentiation of P2X7 could be a useful
213
approach in combating cancer in scenarios where the receptors are less responsive to
extracellular ATP.
Mello et al. in 2017 demonstrated that potentiation of P2X7 responses by hyperthermia changes
plasma membrane fluidity resulting in an increased membrane pore and dilation following
ATP-P2X7 activation. In combination with chemotherapeutic agents such as cisplatin and
mitomycin C, potentiation of P2X7 via hyperthermia was seen to enhance the efficacy of these
antitumor drugs (587). Potentiation of P2X7 responses by selected ginsenosides characterised
as pharmacological modulators in this thesis (Chapter 4 and Chapter 5) may also function in a
manner similar to hyperthermia and could be used in combination with other anti-tumour
agents.
The results from chapter 4 indicated that selected ginsenosides increased the P2X7 channel
activity and related signalling in HEK-293 (transfected) and macrophages. Not only do these
results provide an explanation for some of the immunomodulatory properties of Panax ginseng,
they also identified two naturally occurring chemical compounds that can decrease the EC50 of
ATP for P2X7 activation, thereby enhancing all downstream events controlled by this receptor.
Of the 4 ginsenosides tested, CK and Rd consistently caused pronounced enhancement of PX7
related effects. The similar efficacy of these two ginsenosides might suggest a common binding
site for both ginsenosides on P2X7. A comparison of the structure of the four ginsenosides
tested and the varying magnitude of their effects on P2X7 can shed some light on the structure-
function relationship of these drugs with the receptor. For instance, positional isomers CK and
Rh2 have similar structures except for the position of one glucose molecule. This subtle change
in structure probably affects the orientation in which the two compounds lodge themselves in
the binding site on P2X7 and this differential binding defines the levels of P2X7 enhancement
elicited by the two compounds. Thus, the in-depth investigation of selected ginsenosides in
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chapter 4 brought forward two new P2X7 modulators and their pharmacological properties
which could be beneficial in future drug development.
In chapter 5, the physiological relevance of the potentiation of P2X7 by ginsenosides CK and
Rd was validated. In the presence of sub-lethal doses of ATP, the apoptotic machinery was
switched on, but the trigger was not large enough for the cell to die. In the presence of
pyroptotic stimuli (LPS), the pro-inflammatory pathway was activated but cell death did not
occur. In the presence of CK (ATP ± LPS), the pro-apoptotic signals were high enough to help
the cells to reach a point-of-no-return and thus die. In light of the recent study suggesting a
cross-talk between pyroptosis and apoptosis (where it has been suggested that caspase-1
stimulates caspase 3/7) (546), it could be suggested that large activation of caspase 3/7 at an
early time point in the presence of CK might help in preventing the pro-inflammatory pathway
and re-routing the cells towards apoptosis. Taken together, the results from Chapter 4 and
Chapter 5 have added to our knowledge of the pharmacological properties and physiological
relevance of bioactive components of ginseng, thereby providing a scientific validation to some
of the reported immunomodulatory properties of ginseng. P2X7 activation is known to have
beneficial anti-viral, anti-fungal, anti-bacterial as well as anti-cancer effects. Generation of
structural analogues of CK and Rd with greater specificity for P2X7 can be considered in order
to generate better therapeutic tools to target P2X7.
Pacheco et al. have shown that the rate of entry of the photosensitizer methylene blue (MB) in
macrophages increases from 4.2 % to 90.2 % following ATP activation of P2X7 membrane
pores (588). The authors of this study have suggested that the pore associated with P2X7R
could as a drug delivery system to increase the passage of hydrophilic drugs into cells
expressing P2X7. Thus, hydrophilic drugs (with molecular mass less than 900 Da) can be
delivered into specific cells by opening up the P2X7 associated membrane pore. Increasing the
215
dilation of this pore further by positive modulators such as CK and Rd may additionally
enhance the efficacy of this drug delivery system.
General Conclusions
In conclusion, the findings from the present study have provided evidence in support of P2X7
as a molecular target for MINO as well as ginseng compounds CK and Rd, even though these
compounds have contrasting modulatory effects on P2X7 activity. The results from these
studies have added to the pharmacological knowledge related to P2X7 and P2X4 receptors.
This knowledge can prove beneficial in drug development. In the physiological context, a
healthy state can be maintained if inflammatory responses are optimum. The inflammatory
responses should be strong enough to deal with the external challenge but should be controlled
so as self-cells are not harmed. Therefore, inhibition of overactive P2X7 by the negative
modulator MINO may have anti-inflammatory effects. On the other hand, potentiation of P2X7
by CK and Rd may be beneficial in boosting immunity in the case of viral, microbial, parasitic,
fungal infections as well in some forms of cancer. This thesis has provided an insight into the
pharmacological properties of novel P2X7 modulators and the information from the studies
described herein may prove useful in generating more specific therapeutic tools to target the
multifaceted inflammatory P2X7 receptor.
Future Directions
The direct effect of MINO on P2X channels has not been established. It is proposed that MINO
might affect the common pore formation pathway mediated by P2X receptors but the
confirmation of the effect of MINO on P2X7 using electrophysiology would be useful in order
to exactly describe the mode of action of this drug. In Chapter 4, the direct effect of
ginsenosides on the P2X4 channel has been established using an automated patch clamp system
(IonFlux). The selectivity of selected ginsenosides for P2X7 was evaluated by testing them
216
P2X4, P2X2 and P2Y. It would be useful to test these drugs on other P2X receptors as well
(P2X1, P2X3, P2X5 and P2X6). This could provide a better understanding of the structure-
function relationship and binding properties of ginsenosides for purinergic receptors.
Computational docking studies would be useful for predicting the energetically and
geometrically feasible binding sites for ginsenosides on these receptors. In Chapter 5, CK and
Rd have been shown to help the macrophages challenged by stress to commit to a cell death
response. The entire cell death pathway has not been completely characterised in this work. A
direct measure of apoptosis using assays such as TUNEL to confirm nicking of DNA would
provide confirmation about the type of cell death induced by these receptors. Also, in the pro-
inflammatory pathway, further testing for any direct effects of ginsenosides on caspase-1
activity would be useful. The data in the present study was not validated on human cells
(macrophages or monocytes). The initial screening was done using HEK-293 cells over-
expressing the receptors of interest. The studies were further validated using immortalised cell
line (BV2 microglial and J774 macrophage cell lines). Apoptotic pathways are often altered in
immortalised cell lines, therefore validating the results in primary cells or in vivo would be a
good approach. The primary macrophages from rat were employed to confirm some of the
results obtained with cell lines (Chapter 3 and 5). Macrophages and microglia express both
P2X7 and P2X4. In order to determine the effect of drugs on one receptor type independently
of the other, CRISPR/Cas9 gene editing system can be used. The CRISPR/Cas9 can silence
one purinergic receptor type (example P2X7) so that the selectivity of the receptor responses
(example P2X4) can be studied independently of the other. In Chapter 4, CRISPR/cas9 can be
used to silence the P2X7 receptors in J774 macrophages or BV2 microglia to measure the effect
of ginsenosides on P2X4 receptors in these cells. Similarly, in Chapter 3 and 5, the P2X4
receptors can be silenced in J774 macrophage cells to test the ginsenosides on P2X7 mediated
signalling pathways. It is also known that drug interactions can be different depending on cells
217
studied and species studied. Therefore, the best possible approach would be to test these drugs
in immune cells of human origin (human macrophages or monocytes).
218
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267
APPENDICES
Cell Line Information
A. HEK-293 cells
Cell line name: HEK-293
Description:
• Species: Human, Homo sapiens
• Tissue: Embryonic kidney
• Age: foetus
Depositor: ATCC (procured by Dr Leanne Stokes)
Biosafety level: 2
Growth mode: Adherent
Morphology: Epithelial-like
Propagation: Complete DMEM: F12 medium: DMEM:F12 + 10 % FBS + 1 % Pen Strep
(+5,000 Units/ml Pen; +5,000 μg/ml Strep)
Sub culturing:
• Ratio: Split 1:8 to 1:10
• Seeding: at 2−4⇥10, 000 cells/cm2. Remove spent medium, rinse with PBS (w/o Ca
and Mg), add fresh 0.05 % trypsin/EDTA, incubate at 37°C until the cells detach. Add
fresh medium, aspirate, and transfer into new flask. Incubate in incubator at 37°C (and
5 % (v/v) CO2).
268
Preservation: FBS 90 %; DMSO 10 % (v/v)
B. J774A.1 macrophage cell line
Cell line name: J774
Description:
• Species: Mus musculus, mouse
• Tissue: ascites
• Disease: reticulum cell sarcoma
Depositor: ATCC (kindly provided by Dr. Ray Helliwell at RMIT)
Biosafety level: 1
Growth mode: Adherent
Morphology: Epithelial-like
Propagation: Complete RPMI medium: RPMI + 10 % FBS + 1 % Pen Strep (+5,000 Units/ml
Pen; +5,000 μg/ml Strep)
Subculturing:
• Ratio: Split 1:10
• Seeding: at 2−4⇥10, 000 cells/cm2. Remove spent medium, rinse with PBS (w/o Ca
and Mg), add fresh 0.05 % trypsin/EDTA, incubate at 37°C until the cells detach. Add
fresh medium, aspirate, and transfer into new flask. Incubate in incubator at 37°C (and
5 % (v/v) CO2).
• Preservation: FBS 90 %; DMSO 10 % (v/v)
269
C. BV-2 microglia cell line
Cell line name: BV-2
Description:
• Species: Mus musculus, mouse
• Cell line status: Transformed ( recombinant reterovirus J2)
Biosafety level: 2
GROWTH MODE: ADHERENT
Morphology:
Propagation: Complete DMEM: F12 medium: DMEM: F12 + 10 % FBS + 1% Pen Strep (+
5,000 units/ml Pen; +5,000 μg/ml Strep)
Subculturing:
• Ratio: Split 1:3 to 1:8
• Seeding: at 2−4⇥10, 000 cells/cm2. Remove spentmedium, rinsewithPBS (w/o Ca and
Mg), add fresh 0.05% trypsin/EDTA, incubate at 37°C until the cells detach. Add fresh
medium, aspirate, and transfer into new flask. Incubate in incubator at 37°C (and 5%
(v/v) CO2).
• Preservation: FBS 90%; DMSO 10% (v/v)
270
D. Buffers
Phosphate Buffer Saline (PBS, pH 7.3)
S.No Reagents Concentration(1X) Amount to be added
1. NaCl 137 mM 8 g
2. KCl 2.7 mM 0.2 g
3. Na2HPO
4 10 mM 1.44 g
4. KH2PO
4 1.8 mM 0.24 g
5. CaCl2
.2H
2O 1 mM 0.133 g
6. MgCl2.6H
2O 0.5 mM 1.0 g
The above salts were weighed and dissolved in 800 ml distilled water. The pH was adjusted to
7.3 with conc. HCl and then the volume was adjusted to 1 L using distilled water. The buffer
was then sterilized by autoclaving at 15 psi for 20 minutes.
Electrolyte Low Divalent Buffer (ELDV, pH 7.3)
S.No Reagents Concentration(1X) Amount to be added
1. NaCl 145 mM 8.47 g
2. HEPES 10 mM 2.38 g
3. Glucose 13 mM 2.34 g
4. KCl 5 mM 5 ml of 1M stock
5. CaCl2
.2H
2O 0.2 mM 200 µL of 1 M stock
The above salts were weighed and dissolved in 800 ml distilled water. The pH was adjusted to
7.3 with 4 M NaOH and then the volume was adjusted to 1 L using distilled water.
271
Physiological buffer (Etotal, pH 7.3)
S.No Reagents Concentration(1X) Amount to be added
1. NaCl 145 mM 8.47 g
2. HEPES 10 mM 2.38 g
3. Glucose 13 mM 2.34 g
4. KCl 5 mM 5 ml of 1M stock
5. CaCl2
.2H
2O 2 mM 2 ml of 1 M stock
6. MgCl2
.6H
2O 1 mM 1ml of 1M stock
The above salts were weighed and dissolved in 800 ml distilled water. The pH was adjusted to
7.3 with 4 M NaOH and then the volume was adjusted to 1 L using distilled water.
Krebs-HEPES buffer (pH 7.3)
S.No Reagents Concentration(1X) Amount to be added
1. NaCl 140 mM 8.18 g
2. KCl 2.8 mM 0.208 g
3. HEPES 10 mM 2.38 g
4. Glucose 10 mM 1.8 g
5. CaCl2
.2H
2O 2 mM 0.294 g
6. MgCl2
.6H
2O 2 mM 0.406 g
The above salts were weighed and dissolved in 800 ml distilled water. The pH was adjusted to
7.3 with 4 M NaOH and then the volume was adjusted to 1 L using distilled water.
272
HBSS-HEPES Buffer (pH 7.3)
S.No Reagents Concentration(1X) Amount to be added
1. HEPES 20 mM 1 ml of 1M solution
2. CaCl2
.2H
2O 2 mM 100 µL of 1M solution
3. MgCl2
.6H
2O 1.2 mM 60 µL of 1M solution
This buffer was prepared fresh (50 ml) for every experiment. The above solutions were added
to 45 ml 1 X HBSS (Ca2+, Mg2+, phenol red free) buffer and the pH was adjusted to 7.3 with
10 M NaOH. The volume was made to 50 ml using HBSS. The buffer was syringe filtered
(0.22 µM Millipore filters) before use.
Ammonium chloride lyse (10X concentration)
S.No Reagents Amount to be added
1. NH4Cl 8.02g
2. NaHCO3 0.84g
3. EDTA (disodium) 0.37g
Volume was made up to 100ml with Millipore water. Working solution was prepared by
diluting 10 X concentrate with 90 ml Millipore water. Solution was refrigerated until use.