research article curcumin protects against 1-methyl-4

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 523484 13 pageshttpdxdoiorg1011552013523484

Research ArticleCurcumin Protects against 1-Methyl-4-phenylpyridiniumIon- and Lipopolysaccharide-Induced Cytotoxicities in theMouse Mesencephalic Astrocyte via Inhibiting theCytochrome P450 2E1

Hai-Yan Gui Rui-Ni Chen Yan Peng Jin-Hua Hu Zhao Mao Rui Ning Wei ShangWei Liu Jing Xiong Gang Hu and Jian Yang

Department of Pharmacology Nanjing Medical University 140 Hanzhong Road Jiangsu Nanjing 210029 China

Correspondence should be addressed to Jian Yang jianyangnjmueducn

Received 11 April 2013 Accepted 22 May 2013

Academic Editor Sedigheh Asgary

Copyright copy 2013 Hai-Yan Gui et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Curcumin is extracted from the rhizomes of the ginger family plantCurcuma longa L which has a good protection for liver kidneyand immune system However there is little information about its contribution in protection of astrocytes recently The presentstudy was undertaken to elucidate the protective effect of curcumin an herbal antioxidant on 1-methyl-4-phenylpyridinium ion-(MPP+-) and lipopolysaccharide- (LPS-) induced cytotoxicities as well as the underlying mechanisms by using primary mousemesencephalic astrocytesThe results showed that curcumin protected themesencephalic astrocytes fromMPP+- and LPS-inducedtoxicities along with reducing reactive oxygen species (119875 lt 005) andmaleic dialdehyde (119875 lt 005) sufficientlyMoreover curcuminsignificantly inhibited the cytochrome P450 2E1 (CYP2E1) expression (119875 lt 001 at mRNA level 119875 lt 005 at protein level) and itsactivity (119875 lt 005) sufficiently induced by MPP+ and LPS in the mouse mesencephalic astrocytes And curcumin as well as diallylsulphide a CYP2E1 positive inhibitor amelioratedMPP+- and LPS-inducedmousemesencephalic astrocytes damage Accordinglycurcumin protects against MPP+- and LPS-induced cytotoxicities in the mouse mesencephalic astrocyte via inhibiting the CYP2E1expression and activity

1 Introduction

Curcumin (17-bis[4-hydroxy 3-methoxy phenyl]-16-hepta-diene-35-dione Cur) is extracted from the rhizomes ofthe ginger family plant Curcuma longa (Curcuma longa L)Studies suggest that Cur has antiinflammatory [1] anti-atherosclerosis [2] antitumor [3] antioxidation [4] scaveng-ing-free radical [5] and other pharmacological effects [6]Animal model data show that dietary Cur is a strong candi-date in the prevention or treatment of major disabling age-related neurodegenerative diseases like Alzheimerrsquos diseaseParkinsonrsquos disease (PD) and stroke [7] Several studiesin different experimental models of PD strongly supportthe clinical application of Cur in PD [8 9] However theunderlying mechanisms of Cur treatment of PD are limit-ed

Astrocytes are the most numerous nonneuronal cell-type in central nervous system and make up 50 of humanbrain volume They produce neurotrophic factor removetoxins and debris from the extracellular space and regulateinnate immunity in the central nervous system [10] Inaddition astrocytes are allowed by some specific enzymesystems to metabolize ammonia glutamate free radicalsxenobiotics and metals Hence they protect the neuronsfrom the endogenous and exogenous toxicities On the otherhand evidence also showed that astrocytes play a vital partin the secondary amplification of cell injury in multipleneurodegenerative disorders such as Parkinsonrsquos disease PDis the second most prevalent neurodegenerative disease Themajor pathological features are the loss of dopaminergicneurons in substantia nigra compact the presence of Lewyrsquosbodies and the significant decrease of dopamine level in

2 Evidence-Based Complementary and Alternative Medicine

the striatum [11 12] Although the exact etiology of thePD is unknown many studies suggest that multiple factorsmay be involved such as heredity environment oxidativestress inflammation and decline in growth factors [13]Oxidative stress which results from the imbalance of reactiveoxygen species (ROS) generation and clearance is one ofthe important pathogenesis of PD [14] ROS generation mayvary considerably depending on cytochrome P450 enzymes(CYPs) Compared with other CYPs cytochrome P450 2E1(CYP2E1) exhibits a higher rate of oxidase activity thatcauses the formation of ROS during the catalytic cycle TheROS generation is likely to initiate lipid peroxidation anddamage cell membranes [15 16] In the brain CYP2E1 isconstitutively expressed in hippocampal pyramid neuronsastrocytes and endothelial cells [17] and CYP2E1 has beenfound to be inducible and catalytically active in the centralnervous system [18ndash20] It has been suggested that 1-methyl-4-phenylpyridinium ion (MPP+ the ion form of 1-methyl-4-phenyl-1236-tetrahydropyridine MPTP) and lipopolysac-charide (LPS) increased the expression of CYP2E1 andinduced oxidative stress in astrocytes [18 19] The increasingevidence shows that CYP2E1 is involved in the MPTP-induced mouse model of PD [21 22]

In this study we demonstrated that Cur had the cyto-protection against MPP+- and LPS-induced toxicities in theprimary mouse mesencephalic astrocytes Meanwhile Curdecreased the increased ROS and maleic dialdehyde (MDA)amount in the primary culture mesencephalic astrocytesexposed to MPP+ and LPS Combination with an increaseof CYP2E1 induced by MPP+ and LPS in astrocytes [1819] we hypothesized that Cur protects against MPP+- andLPS-induced mouse mesencephalic astrocyte damage viainhibiting the CYP2E1 expression and activity

2 Materials and Methods

21 Animal and Materials C57BL6J mice were kept underenvironmentally controlled conditions (ambient tempera-ture 22 plusmn 1∘C humidity 40) on a 12-hour light-dark cyclewith food and water ad libitum All the experiments wereapproved by Institutional Animal Care and Use Committee(IACUC) of Nanjing Medical University And efforts weremade tominimize animal suffering and to reduce the numberof animals used for the experiments

Dulbeccorsquos modified eaglersquos medium (DMEM) was fromGibco (Gibco Grand Island NY USA) MPP+ LPS and cur-cumin (98 purity) were purchased from Sigma-Aldrich (StLouisMOUSA) Ethyl alcohol (EtOH)was from Sinopharm(Shanghai China) Antibody to CYP2E1 was obtained fromAbcam (Cambridge UK) SYBR Premix Ex Taq kit waspurchased from Biotechnology Limited Company (TakaraJapan) Tryptase and MTT were purchased from AMRESCO(Solon OH USA) Fetal bovine serum (FBS) was purchasedfrom Sijiqing (Hangzhou China) Trizol was from Invitro-gen Life (Carlsbad CA USA) MLV and RNase inhibitorwere purchased from Promega (Madison WI USA) 2101584071015840-dichlorofluorescein diacetate (DCFH-DA) was purchasedfrom Biyuntian (Shanghai China) Lactate dehydrogenase

(LDH) MDA H2O2 and glutathione peroxidase (GSH-Px)

diagnostic kits were from Nanjing Jiancheng BioengineeringCompany (Nanjing China) and the other chemicals were allobtained from Sigma unless otherwise stated

22 Primary Mesencephalic Astrocyte Culture Primarymouse mesencephalic astrocyte culture was prepared frommidbrain of newborn of C57BL6J mice as describedpreviously [23] with slight modifications Briefly postnatal(P1-P2) mice were killed by rapid decapitation the meningeswere removed and the midbrain parts were separated Themidbrain parts were dissociated with 025 tryptase at 37∘Cand terminated by DMEM supplemented with 10 FBS andpenicillinstreptomycin After centrifugation at 1500 rpmfor 5min the cell pellets were resuspended and seeded onpolylysine coated flask The cultures were maintained at37∘C in a humidified 5 CO

2-95 air atmosphere [24]

Culture medium was replaced 24 h later and then changedevery 2-3 days After reaching a confluent monolayer of glialcells (10ndash14 days) mesencephalic astrocytes were replatedon other polylysine-coated flasks The tertiary cultures aresim95ndash98 astrocytes with only sim2ndash5 microglial cells asdetermined by immunocytochemical staining with anti-GFAP antibody as well as double immunofluorescence forGFAP and microglial marker Iba-1 [24] The mesencephalicastrocytes were seeded on polylysine coated 6- 12- 24- or96-well plates for corresponding experiments

23 Cell Viability Cell viability was detected by 3-(45-dim-ethylthiazol-2-yl) 25-diphenyltetrazolium bromide (MTT)assay Mesencephalic astrocytes were seeded in polylysinecoated 96-well plates at a density of 1 times 104 cellswell pre-treated with Cur diallyl sulphide (DAS) or vehicle (DMSO)and then incubated with MPP+ (100120583M) and LPS (1 120583gmL)for 48 h Then 20120583L of 5mgmL MTT was added to cellsand cells were incubated at 37∘C for another 4 h The culturemedium was discarded and 100 120583L DMSO was used to dis-solve the precipitateThe absorbancewasmeasured at 570 nmusing an Automated Microplated Reader ELx800 (BioTek)The cytotoxicity assessment was exhibited by cytotoxicityindex which was calculated as previously described [25]Each treatment was in quadruplicate And data came fromthree independent experiments

24 Determination of Lactate Dehydrogenase Release Mes-encephalic astrocytes injury (cytotoxicity) was quantitativelyassessed by themeasurement of lactate dehydrogenase (LDH)leakage LDH release in culture medium in the presence ofDMSO or Cur for 30min followed by the addition of MPP+and LPS for 48 h was measured using an LDH diagnostickit according to themanufacturerrsquos instruction LDH amountwas calculated by measuring absorbance at 450 nmThe datawere represented as a percentage of LDH release of controlgroup Each treatment was in quadruplicate And data camefrom three independent experiments

25 Hoechst 33342 Staining To quantify apoptotic mesen-cephalic astrocytes mesencephalic astrocytic monolayer was

Evidence-Based Complementary and Alternative Medicine 3

stained with Hoechst 33324 Mesencephalic astrocytes wereseeded in polylysine coated 24-well plates and pretreatedwithCur or vehicle (DMSO) for 30min and then incubated withMPP+ (100 120583M) and LPS (1 120583gmL) for 48 h The cells werefixed with 4 paraformaldehyde for 20min washed withPBS and then incubated with Hoechst33342 (1120583gmL) for10min After washing with PBS the morphological featuresof apoptosis (chromatin condensation) were monitored byfluorescent microscope (Olympus Japan) (Acquisition soft-ware DP2-BSW) Cells with condensed nuclei or nuclearcondensations were scored as apoptotic mesencephalic astro-cytes [26] and each treatment was performed in triplicate

26 Measurement of Reactive Oxygen Species GenerationFormation of ROS was evaluated using 2101584071015840-dichloro-fluorescein diacetate (DCFH-DA) a membrane-permeableprobe After different treatments mesencephalic astrocyteswere loaded with DCFH-DA (50120583M final concentration) inDMEM for 30min in the dark and fixed by 4 formaldehydeAfter rinsing cells with PBS three times cells were observedusing a fluorescentmicroscope At least 400 cells from 12 ran-domly selected fields per dish were counted Then the cellswere collected to measure the fluorescence intensity at theexcitationwavelength of 485 nmand the emissionwavelengthof 530 nm [27] Each treatment was performed in triplicateAnd data came from three independent experiments

27 Determination of Lipid Peroxidation Products Mesen-cephalic astrocytes were seeded in polylysine coated 24-well plates at a density of 2 times 105 cellswell Cells werepretreated with Cur or vehicle (DMSO) for 30min and thenincubated with MPP+ (100 120583M) and LPS (1 120583gmL) 400120583Lfor 48 h Lipid peroxidation products namely the amountof MDA formed by the 2-thiobarbituric acid reaction asthiobarbituric acid reactive substances in the supernatant[28] were measured by using a commercial MDA kit Thespectrophotometric absorbance was assessed at 532 nm inaccordance with the manufacturerrsquos instructions The resultswere expressed as nmolmL in 2 times 105 cells Each treatmentwas in quadruplicate And data came from three independentexperiments

28 The H2O2Production Assay Mesencephalic astrocytes

were seeded in polylysine coated 12-well plates at a densityof 5times106 cellswell Cells were pretreated with Cur or vehicle(DMSO) for 30min and then incubated withMPP+ (100120583M)and LPS (1 120583gmL) 800120583L for 48 h The supernatants wereused to measure H

2O2production using a H

2O2diagnostic

kit essentially according to the manufacturerrsquos manual TheH2O2products were calculated with a calculation formula

provided by the manufacturerrsquos instruction The H2O2prod-

uct was represented as 120583molL in 5times105 cells Each treatmentwas in quadruplicate And data came from three independentexperiments

29 GSH-Px Activity Assay Mesencephalic astrocytes wereseeded in polylysine coated 12-well plates at a density of 5times106cellswell Cells were pretreated with Cur or vehicle (DMSO)

for 30min and then incubated with MPP+ (100 120583M) and LPS(1 120583gmL) for 48 hThemedia were discarded And cells wererinsed with PBS and harvested in 200120583L of PBS The cellsuspension was sonicated by a sonifier and cell debris wasremoved by centrifugation at 12000 g for 15min at 4∘C [29]TheGSH-Px activity of cells wasmeasured by using aGSH-Pxassay kit essentially according to the manufacturerrsquos manualTheGSH-Px activity of cells was calculated with a calculationformula provided by the manufacturerrsquos instruction TheGSH-Px activity was represented as unitmingram proteinEach treatment was in quadruplicate And data came fromthree independent experiments

210 Immunocytochemistry Mesencephalic astrocytes wereseeded in polylysine coated 24-well plates at a density of5 times 10

4 cellswell in growth medium After pretreated withCur (3 120583M) or DMSO for 30min cells were incubated withMPP+ LPS and EtOH for 48 h For immunocytochemistrythe cells were fixed with 4 paraformaldehyde for 30minand then permeabilized with 002 Triton X-100 containing01 BSA for 1 h Immune complexes were formed by incu-bating cells with primary antibodyCYP2E1 (1 400) overnightat 4∘C followed by incubation with anti-rabbit secondaryantibody (1 800) for 1 h at 37∘C and visualized by DABCell morphology was monitored using an inverted lightmicroscope (Olympus Japan Acquisition software DP2-BSW) Negative control staining was performed without theprimary antibodies EtOH treatment was a positive controlof CYP2E1 inducer After immunocytochemical staining theastrocytes were captured at a magnification of 200x Threevisions from each well were randomly selected in order toanalyze immunoreactive productions of CYP2E1 by usingLeica Image Processing and Analysis System Each treatmentwas performed in triplicate And data came from threeindependent experiments

211 Quantitative Real-Time Polymerase Chain ReactionTotal RNA was prepared from mesencephalic astrocytesusing Trizol reagent according to the manufacturerrsquos instruc-tion The first-strand cDNA was synthesized using totalRNA (2 120583g) at 70∘C for 5min 42∘C for 60min and 95∘Cfor 10min by using an oligo (dT) primer MMLV reversetranscription RNase inhibitor and dNTP mix in a totalvolume of 20 120583L The cDNAs were quantitative PCR wasconducted with SYBR Premix Ex Taq kit (Takara Japan)20120583L of PCR mix contained 10 120583L of SYBR Green PCRmaster mix (2times) 1 120583L of each primer 2120583L of cDNA astemplate and 6120583L of water The PCR amplification andquantificationwere done in anAppliedBiosystems 7300Real-Time PCR System with SDS software (Applied BiosystemsWarrington UK) After initial denaturation at 95∘C for 30 samplifications were carried out for 40 cycles at 95∘C for5 s and 60∘C for 31 s The signals from each target genewere normalized based on the signal from the correspondingGAPDH PCR primers used in this study were as follows (1)


GAPDH Forward 51015840-GTATGTCGTGGAGTCTACTGG-TGTC-31015840 Reverse 51015840-GGTGCAGGATGCATTGCTGAC-ATTC-31015840 (2) CYP2E1 Forward 51015840-AAGCGCTTCGGG-CCAG-31015840 Reverse 51015840-TAGCCATGCAGGA CCACGA-31015840Each treatment was performed in triplicate And data camefrom three independent experiments

212 Cytochrome P450 2E1 (CYP2E1) Activity CYP2E1 activ-ity was measured based on the rate of oxidation of p-nitrophenol (PNP) to p-nitrocatechol in the presence ofNADPH by modification of the methods of Reinke andMoyer [30] and Koop [31] Briefly after treatment cells wererinsed with PBS and harvested in 50 120583L of 100mMpotassiumphosphate buffer pH74The cell suspensionwas sonicated bya sonifier (Nanjing China) and the cell debris was removedby centrifugation at 12000 g for 15min at 4∘C The proteinconcentration of the samples was estimated with the BCAkit Standards and blanks were assessed simultaneously Theactivitywas determined in a final volumeof 100120583L containing100 120583g of protein 100mM of potassium phosphate pH 7401mM of PNP 1mM of NADPH and 5mM of MgCl

2

followed by incubation at 37∘C for 10min The reaction wasterminated by the addition of 20120583L of chilled trichloroaceticacid (20 wv) and centrifuged at 10000timesg for 5min Afterbeing centrifuged 100 120583L of the supernatant was added to10 120583L 10 N of sodium hydroxide 4-Nitroatechol formationwas then spectrophotometrically determined at 510 nm Astandard curve with 4-nitrocatehol was used to calculateCYP2E1 activity The results were expressed as nmol perminute per mg of protein Each treatment was in quadrupli-cate Several controls were performed including incubationprotein or regeneration system And data came from threeindependent experiments

213 Statistical Analysis All data were presented as themean plusmn SD (standard deviation) Statistical analysis betweencontrol group and Cur treatment group was performed withtwo-tailed independent Studentrsquos t-tests using SPSS 100 forWindows (SPSS Inc Chicago IL USA) And statisticalanalysis for multiple comparisons was performed by a one-way ANOVA test with Turkeyrsquos corrections Differences wereconsidered statistically significant when the 119875 value was lessthan 005 001 or 0001

3 Results and Discussion

31 Results

311 Cur at Low Concentration Had No Cytotoxicity to theMouse Mesencephalic Astrocytes As shown in Figure 1(a)the viability of the mesencephalic astrocytes was unchangedwhen cells were treated with Cur from 1120583M to 10 120583M com-pared to that of the control However a significant decreaseof the mesencephalic astrocytes viability was observed whencells were treated with 30120583M and 50 120583M of Cur LDHrelease in culture medium was employed to indicate cellmembrane damage Similar to MTT assay (Figure 1(b)) Curat low concentrations (1 120583Mminus10 120583M) did not change the

LDH release But Cur at high concentrations (30120583M and50120583M) significantly increased the LDH release in themesen-cephalic astrocytes Consistently mesencephalic astrocyteswhich were treated with low concentrations (1120583M 3 120583Mand 10 120583M) of Cur possessed mainly rounded or oval bodyand exhibited extensive processes The body and processes ofmesencephalic astrocytes were near normalcy (control) Incontrast the body of mesencephalic astrocytes which weretreated with high concentrations (30120583M 50120583M) of Curbecame more asperous with shrunken morphologies andprocesses of mesencephalic astrocytes were reduced in sizeand decreased in number (Figure 1(c))

The data suggested that Cur at low concentrations hadno cytotoxicity but at high concentrations had some cytotox-icities in the mouse mesencephalic astrocytes So we chosethe low concentrations of Cur (1120583M 3 120583M and 10 120583M) forsubsequent experiments

312 Cur Ameliorated the Cell Damage Induced by MPP+and LPS in the Mouse Mesencephalic Astrocytes It has beenreported that exposure to MPP+ and LPS potentially resultsin astrocytes damage or death [24] We therefore investigatedwhether Cur protected against MPP+ and LPS in the mesen-cephalic astrocytesThe results showed that Cur (1120583M 3 120583Mand 10 120583M) attenuated the reduction of cell viability and theincrease of LDH release induced by MPP+ and LPS in theprimary cultured mouse mesencephalic astrocytes The mostsignificant protective effect of Cur was at 3 120583M (Figures 2(a)and 2(b)) Therefore 3 120583M of Cur was chosen to explore themechanism in the following experiments

The nuclear staining assay was used to assess themorpho-logical changes of apoptosis in the mesencephalic astrocytesThe normal cells exhibited uniformly dispersed chromatinand intact cell membrane The impaired cells appeared typ-ical characteristics of apoptosis including apoptotic nuclearcondensation Similar to the results of cell viability andLDH release Cur attenuated the mesencephalic astrocytesapoptosis induced by MPP+ and LPS (Figure 2(c))

The data suggested that Cur attenuated the mouse mes-encephalic astrocyte damage induced by MPP+ and LPS(Figures 2(a) 2(b) and 2(c))

313 Cur Decreased the ROS andMDAProduction Induced byMPP+ and LPS in the Mouse Mesencephalic Astrocytes SinceROS played an important role in cell death we next investi-gated the intracellular ROS formation using a fluorescent sen-sitive probe (DCFH-DA) As shown in Figures 3(a) and 3(b)when the mesencephalic astrocytes were treated with MPP+and LPS for 48 h the intracellular ROS level significantlyincreased compared with that of the control respectivelyThe results revealed that MPP+ and LPS enhanced ROSgeneration in the primary mouse mesencephalic astrocytesCotreatment with 3120583M of Cur reduced the intracellularROS production and significantly attenuated an increaseof ROS caused by MPP+ and LPS in the primary mousemesencephalic astrocytes (Figures 3(a) and 3(b))

ROS can initiate lipid peroxidation MDA is one of themost frequently used indicators of lipid peroxidation When


0

20

40

60

80

100

120

0 1 3 10 30 50

Cel

l via

bilit

y (

of c

ontro

l)

lowast

lowastlowast

Curcumin (120583M)

(a)

020406080

100120140160180200

0 1 3 10 30 50

LDH

rele

ase (

o

f con

trol)

lowast

lowastlowast

Curcumin (120583M)

(b)

Control Curcumin (1120583M)

Curcumin (3120583M) Curcumin (10120583M)


(c)

Figure 1 Effects of Cur on the cytotoxicity of the primary cultured mesencephalic astrocytes (a) Cell viability of the primary culturedmesencephalic astrocytes (b) LDH release of the primary cultured mesencephalic astrocytes (c) Morphologies of the primary culturedmesencephalic astrocytes Primary cultured mesencephalic astrocytes were treated with the indicated concentrations of Cur (1 3 10 30 and50) (120583M) for 48 hThe cell viability was determined withMTT LDH release was measured by the LDH diagnostic kit Morphological analysiswas taken under bright field (magnification 40x) And data are presented as mean plusmn SD of three independent experimentslowast119875 lt 005 andlowastlowast

119875 lt 001 compared to the control group


0

20

40

60

80

100

120

0 1 3 10

Cel

l via

bilit

y (

of c

ontro

l)

Curcumin (120583M)

lowastlowast lowast

lowastlowastlowastlowastlowast

Control

LPSMPP+

(a)

020406080

100120140160

LDH

rele

ase (

o

f con

trol)

0 1 3 10Curcumin (120583M)

lowast

lowastlowast lowastlowast

Control

LPSMPP+

(b)

Control LPS

Curcumin M + C L + C

MPP+

(c)

Figure 2 Cur protected the mesencephalic astrocytes from cytotoxicity induced by MPP+ and LPS (a) Cell viability of the mesencephalicastrocytes (b) LDH release of the mesencephalic astrocytes (c) Apoptosis of the mesencephalic astrocytes The primary culturedmesencephalic astrocytes were nonpretreated or pretreated with vehicle (DMSO) or Cur (1 3 and 10) (120583M) for 30min and then incubatedwith PBS MPP+ (100120583M) or LPS (1120583gmL) for 48 h The cell viability was determined with MTT LDH release was measured by the LDHdiagnostic kit Nuclear condensation was assessed by staining with Hoechst33342 (magnification 200x) Data are presented as mean plusmn SD ofthree independent experimentslowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared to the control group119875 lt 005 and

119875 lt 001 comparedto the MPP+ or LPS treatment alone group

themesencephalic astrocyteswere exposed toMPP+ andLPSthe MDA amount in culture medium increased significantlyBeing cotreated with Cur the amount of MDA decreasedmarkedly in the mesencephalic astrocytes (Figure 3(c))These data suggested that Cur decreased the increased ROSandMDA amounts induced byMPP+ and LPS in the primarymouse mesencephalic astrocytes

314 Cur and DAS Ameliorated Cell Viability of the PrimaryMesencephalic Astrocytes Induced by MPP+ and LPS It was

reported that curcumin generated H2O2in astrocytes [32] In

order to explore the role of Cur in protecting the astrocyteswe next examined the H

2O2product and GSH-Px activity

in the mouse mesencephalic astrocytes treated with Curalone or combination with MPP+ and LPS As shown inFigure 4 the H

2O2products did not significantly change in

the mouse mesencephalic astroytes after being exposed toMPP+ and LPS as well as to Cur (Figure 4(a)) The GSH-Pxactivity significantly decreased in the mouse mesencephalicastroytes induced by MPP+ not by LPS Cur attenuated the


Control LPS


MPP+

(a)

002040608

112141618

2

Control LPS

Fluo

resc

ence

inte

nsity

( o

f con

trol)

ControlCurcumin

lowast

lowast

lowastlowast

MPP+

(b)

0010203040506070809

1

MD

A co

nten

t (nm

olm

L)

Control LPS

ControlCurcumin

lowastlowast

MPP+

(c)

Figure 3 Cur decreased ROS and MDA productions in primary mesencephalic astrocytes (a) DCFH-DA fluorescence (green) images ofthe primary cultured mesencephalic astrocytes (b) Quantification of DCFH-DA fluorescence intensity (c) MDA assay in medium of themesencephalic astrocytes The primary cultured mesencephalic astrocytes were nonpretreated or pretreated with vehicle (DMSO) or Cur(3120583M) for 30min and then incubated with PBS MPP+ or LPS for 48 hThe cells were loaded with DCFH-DA (50 120583Mfinal concentration) inDMEM for 30min in the dark and fixed by 4 formaldehyde After rinsing cells with PBS three times cells were observed using a fluorescentmicroscope (magnification 200x) MDA amount was measured by using the MDA diagnostic kit Data are presented as mean plusmn SD of threeindependent experimentslowast119875 lt 005 and lowastlowast119875 lt 001 compared to the control group

119875 lt 005 compared to the MPP+ or LPS treatmentalone group

decrease of GSH-Px activity in the mouse mesencephalicastroytes induced byMPP+ (Figure 4(b)) Both Cur and DAS(a CYP2E1 positive inhibitor) significantly ameliorated cellviability of the mouse mesencephalic astroytes after beingexposed to MPP+ LPS and EthOH (Figure 4(c))

The data implied that the effect of Cur on the decreaseof ROS and MDA in the primary mouse mesencephalicastrocytes induced by MPP+ and LPS was not caused bypromoting H

2O2production Cur as well as DAS protected

the mouse mesencephalic astroytes from MPP+- and LPS-induced cytotoxicities

315 Cur Inhibited Both CYP2E1 Expression and Activityin the Mouse Mesencephalic Astrocytes Induced by MPP+and LPS Our previous work showed that CYP2E1 was animportant responsibility for MPP+ and LPS cytotoxicities[24] A CYP2E1 inhibitor protected the mouse astroytes from


0

1

2

3

4

5

6

7

Control Curcumin

H2O2

prod

uct (120583

M)

Control

LPSMPP+

(a)

0

5

10

15

20

25

30

Control Curcumin

GSH

-Px

activ

ity (u

nitm

ing

pro

tein

)

Control

LPS

lowast

MPP+

(b)

0010203040506070809

1

Control Curcumin DAS

Cel

l via

bilit

y (O

D570

nm) lowastlowast

lowastlowast

lowastlowastlowast

Control LPSEtOHMPP+

(c)

Figure 4 Cur as well as DAS ameliorated cell viability of the primary mesencephalic astrocytes induced by MPP+ and LPS (a) The H2O2

production of the primary cultured mesencephalic astrocytes (b) The GSH-Px activity of the primary cultured mesencephalic astrocytesThe primary cultured mesencephalic astrocytes were nonpretreated or pretreated with vehicle (DMSO) and Cur (3120583M) for 30min and thenincubated with PBS MPP+ or LPS for 48 h The cultured media were measured for H

2O2production with H

2O2assay kit And the lysates

determined GSH-Px activity using GSH-Px assay kit (c) Effect of Cur and DAS on the cell viability of the primary cultured mesencephalicastrocytes induced byMPP+ and LPSThe primary culturedmesencephalic astrocytes were nonpretreated or pretreatedwith vehicle (DMSO)Cur (3 120583M) and DAS (500 120583M) and then incubated with PBS MPP+ LPS or EtOH for 48 h EtOH was as a positive CYP2E1 inducer Dataare presented as mean plusmn SD of three independent experimentslowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared to the control group119875 lt 005 and

119875 lt 001 compared to the MPP+ or LPS treatment alone group

MPP+- and LPS-induced cytotoxicity [24] Presumably themechanism of Cur neuroprotection was probably throughinhibiting CYP2E1 expression and activity in the mesen-cephalic astrocytes In order to test this probability we furtherinvestigated the effects of Cur on the CYP2E1 expression andactivity in the mesencephalic astrocytes induced by MPP+LPS and ethyl alcohol (EtOH) EtOHwas a positive control ofCYP2E1 inducer in this experiment The results showed thatwhen the mesencephalic astrocytes were exposed to MPP+LPS and EtOH the CYP2E1 mRNA increased by 325222 and 292 respectively Furthermore Cur almost abol-ished the increase of CYP2E1 expression induced by MPP+

LPS and EtOH at mRNA level (Figure 5(a)) Similarly theCYP2E1 immunostaining was much stronger in the mousemesencephalic astrocytes after exposure to MPP+ LPS andEtOH (Figure 5(c)) Semiquantitative analysis showed thatthe mean optical density of CYP2E1 increased to 187 161and 175 respectively and Cur almost abolished the increaseof CYP2E1 expression induced by MPP+ LPS and EtOH atprotein level (Figure 5(b)) Since the expression of CYP2E1was induced by MPP+ LPS and EtOH in the mouse mesen-cephalic astrocytes what happened to the activity of CYP2E1inMPP+ LPS and EtOH-exposedmesencephalic astrocytesWe measured the CYP2E1 activity by the hydroxylation of


005

115

225

335

445

5

Control LPS EtOH

Relat

ive C

YP2E

1 m

RNA

ControlCurcumin

lowastlowast

lowastlowast

lowastlowast

MPP+

(a)

ControlCurcumin

0002004006008

01012014016018

Opt

ical

den

sity

Control LPS EtOH

lowastlowast

lowastlowast

lowastlowast

MPP+

(b)

Control LPS EtOH

Curcumin M + C L + C E + C

MPP+

(c)

0

001

002

003

004

005

006

CYP2

E1 ac

tivity

(nm

olm

gm

in)

ControlCurcumin

Control LPS EtOH

lowastlowast

lowastlowast

MPP+

(d)

Figure 5 Cur inhibited theCYP2E1 expression and activity in the primarymesencephalic astrocytes (a) Effect ofCur onCYP2E1mRNA level(b)-(c) Effect of Cur on CYP2E1 protein level (immunocytochemistry images and the optical density) (d) Effect of Cur on CYP2E1 activityPrimary cultured mesencephalic astrocytes were nonpretreated or pretreated with DMSO or Cur (3120583M) for 30min and then incubated withPBS MPP+ LPS or EtOH for 48 h EtOHwas a positive CYP2E1 inducer CYP2E1 mRNA level was determined with real-time PCR CYP2E1protein level was determined with immunocytochemistry described in Section 2 (magnification 200x) And CYP2E1 activity was measuredby hydroxylation rate of p-nitrophenol Data are presented as mean plusmn SD of three independent experimentslowast119875 lt 005 lowastlowast119875 lt 001 comparedto the control group

119875 lt 005119875 lt 001 compared to the MPP+ LPS or EtOH treatment alone group


p-nitrophenol to 4-nitrocatechol The results showed thatthe CYP2E1 activity of the mouse mesencephalic astrocytesincreased by 169-fold 158-fold and 183-fold after exposingto MPP+ LPS and EtOH respectively (Figure 5(d)) Cursubstantially inhibited both the increase of CYP2E1 expres-sion and activity in the mouse mesencephalic astrocytesinduced by MPP+ LPS and EtOH However Cur treatmentalone seemed to have little effect on the constitutive CYP2E1expression and activity

The data implied that Cur as well as DAS inhibited bothCYP2E1 expression and activity in the mouse mesencephalicastroytes induced by MPP+ LPS and EtOH

32 Discussion At present if not all most of the disablingclinical abnormalities of PD are attributed to a profounddecrease in brain dopamine content in the striatum which isinduced by the remarkable loss of dopaminergic neurons inthe substantia nigra pars compacta (SNpc) [12]Therefore themost potent treatment for PD remains the administration oflevodopa (L-dopa) a precursor of dopamine to alleviate PDsymptoms However the chronic administration of L-dopaoften produces motor and psychiatric side effects which maylimit L-dopa use [31] So it is expected to find the naturalcompounds with the protective effect and no or less sideeffects for treatment of PD

Recent studies have proven that Cur is a strong candidatein the prevention or treatment of major disabling age-relatedneurodegenerative diseases like Alzheimerrsquos disease PD andstroke [7 8 33] Cur can effectively protect against ethanol-induced liver damage [34] and ethanol-induced central ner-vous system neurodegeneration in vivo [35] It can directlyprotect the SHSY-5Y cells from MPP+ toxicity [33] Lowdoses of Cur can decrease the 6-hydroxydopamine- anddopamine-induced neurotoxicities in vivo [36 37] Currentstudies show that Cur protects SH-SY5Y and PC12 cellsfrom 120572-synuclein-induced cytotoxicities through antioxida-tion and scavenging-free radical [38 39] However there islittle information about what are the effects of Cur on themesencephalic astrocytes which maintain homeostasis andprotect against the endogenous and exogenous toxicities inthe central nervous system In the present study we investi-gated the effects of Cur on the primary mouse mesencephalicastrocytes which play a crucial role in PD We demonstratedthat Cur significantly protected against MPP+- and LPS-inducedmousemesencephalic astrocytes damage at low con-centration (1 120583Mminus10 120583M) In contrast high concentrations ofCur (30 120583M 50 120583M) were cytotoxicity to the mesencephalicastrocytes (Figures 1(a) 1(b) and 1(c)) Therefore it maynot be a simple stoichiometric reaction and the function ofCur was biphasic Cur had nontoxicity and protective effecton the mesencephalic astrocytes at low concentrations (nomore than 30 120583M) while at high concentrations (more than30 120583M) Cur promoted themesencephalic astrocytes damageThe reason was probably that Cur had multiple actions suchas antioxidation [4] scavenging-free radical [5] and inducingapoptosis [40]

Oxidative stress plays an important role in PD anddopamine-rich areas of the brain such as mesencephalon

are particularly vulnerable to oxidative stress becausemetabolism of dopamine itself leads to enhancement of ROSgeneration [41] Studies showed that Cur served as a chelatorand directly bound to Fe2+ which catalyzes formation offree radicals via the Fenton reactions [42] Our resultsalso showed that Cur attenuated the three toxins-inducedincreases of ROS and MDA (lipid peroxidation production)It has reported that Cur may terminate lipid peroxidation byinduction of enzymatic and nonenzymatic antioxidants [43]Accordingly the reduction of ROS andMDAafforded byCurin the mouse mesencephalic astrocytes induced by MPP+LPS is likely attributable to its antioxidant effects Recentstudy reported that Cur generated H

2O2in astrocytes [32]

However our study showed that Cur did not significantlypromote the H

2O2generation in the mouse mesencephalic

astrocytes The possible reason was that the concentration ofCur that we used was much lower (3 120583M) than that in report[32] CYP2E1 is likely to be involved in the metabolism ofthe endogenous and exogenous compounds which can causeneurodegenerative diseases such as PD [44] In additionCYP2E1 exhibits a high rate of oxidase activity that causes theformation of ROS during its catalytic cycle [16] The CYP2E1activation increases ROS production in astrocytes [24] Inthis study we found that Cur attenuated the primary culturedmousemesencephalic astrocytes damage and apoptosis alongwith the decrease of ROS and MDA production induced byMPP+ and LPS Moreover Cur as well as DAS a CYP2E1positive inhibitor [45] ameliorated the cell viability in themesencephalic astrocytes induced by MPP+ and LPS Thesedata implied that the inhibition of CYP2E1 was involvedin the protection of Cur in the mesencephalic astrocytesTherefore in the next step we investigate the effects of Cur onCYP2E1 expression and activity in mesencephalic astrocytesafter being exposed to MPP+ and LPS Consistently MPP+LPS andEtOHvirtually triggered theCYP2E1 expression andenzymatic activity in mesencephalic astrocytes Here EtOHwas a positive control of CYP2E1 inducer in this experimentAnd Cur significantly lowered the increase of the CYP2E1expression and activity in mesencephalic astrocytes inducedbyMPP+ LPS andEtOHOur previouswork showed that theinhibition of CYP2E1 byDAS completely protected astrocytesfrom MPP+ LPS and EtOH-induced oxidative stress [24] Itwas also found that Cur as well as DAS ameliorated the cellviability of the mesencephalic astrocytes induced by MPP+LPS and EtOH in this study These data imply that Curprotected the mesencephalic astrocytes fromMPP+ and LPSAnd the potential mechanismwas that Cur inhibited CYP2E1expression and activity and decreased the ROS generationsubsequently in the mesencephalic astrocytes (Figure 6) Itwas reported Cur alleviated ethanol-induced hepatocytesoxidative damage involving heme oxygenase-1 (HO-1) induc-tion [46] Cur also potently induced HO-1 expression andactivity in rat astrocytes [47] HO-1 was the rate-limitingenzyme decomposing heme into biliverdin free iron andcarbon monoxide (CO) by adding an oxygen molecule tothe porphyrin ring of heme CO inhibited ethanol-inducedCYP2E1 activity and hepatotoxicity but had no influence


Small polar molecules

EthanolDopamineAnilineBenzene

CYP2E1

ROS

Primary mesencephalic astrocytes damage

LPSO2minus

∙OHROO∙

H2O2Cytotoxic agents(isoquinoline derivatives)

Cur (1ndash10120583M) MPP+

Figure 6 The schematic mechanism of Cur in protecting against MPP+- and LPS-induced cytotoxicities in the primary mesencephalicastrocytes CYP2E1 is a main enzyme catalyzing the small polar molecules into oxgen ions peroxides and other cytotoxic agents such asisoquinoline derivatives MPP+ and LPS activated the CYP2E1 and induced mesencephalic astrocyte damage Cur inhibited the CYP2E1expression and activity reduced the ROS production and protected against MPP+- and LPS-induced cytotoxicities in the mesencephalicastrocytes

on CYP2E1 protein expression [48] Together Cur mightprotect against MPP+- and LPS-induced toxicities in themesencephalic astrocytes not only by increasing the HO-1activity but also by inhibitingCYP2E1 expression and activity

4 Conclusion

Our work points to several important conclusions Firstwe report for the first time that Cur can protect againstMPP+- and LPS-induced cytotoxicities in the primarymousemesencephalic astrocytes And inhibition of both CYP2E1expression and activity is suggested as the mechanism ofprotective effects of Cur against MPP+- and LPS-inducedinjuries in the primary mouse mesencephalic astrocytesSecond Cur decreases the CYP2E1 expression and activity inthe primary mouse mesencephalic astrocytes indicating thatCur affects metabolism of the endogenous and exogenouscompounds in the central nervous system Taken togetherthis study provides evidence to explore the possible use ofCur a very low toxic natural compound as a therapeuticapproach in PD at low concentration and provides newinsight into understanding the role of Cur in the drugmetabolism enzymes in the central nervous system

Abbreviations

Cur CurcuminROS Reactive oxygen speciesPD Parkinsonrsquos diseaseCYPs Cytochrome P450 enzymesCYP2E1 Cytochrome P450 2E1MPP+ 1-Methyl-4-phenylpyridinium ion

LPS LipopolysaccharideMDA Maleic dialdehydeH2O2 Hydrogen peroxide

GSH-Px Glutathione peroxidaseDMEM Dulbeccorsquos modified Eaglersquos mediumMTT 3-(45-Dimethylthiazol-2-yl)

25-Diphenyltetrazolium bromideLDH Lactate dehydrogenaseDAB DiaminobenzidineDAS DiallylsulphideEtOH Ethyl alcoholDCFH-DA 2101584071015840-Dichlorofluorescein diacetateFBS Fetal bovine serumDMSO Dimethyl sulfoxideL-dopa Levodopa

Conflict of Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contribution

H-Y Gui and R-N Chen contributed equally to this work

Acknowledgments

This work is supported by the Natural Science Founda-tion of China (nos 30772616 81173128) the National KeyBasic Research Program of China (no 2009CB521906) the


project funded by the Priority Academic Program Devel-opment of Jiangsu Higher Education Institutions Min-istry of Education Science and Technology DevelopmentCenter (2012323411000) Ministry and Education of PRC(20123234110005) and the National Institutes of Health ofUSA (USA F05AT003019)

References

[1] T Santel G Pflug N Y A Hemdan et al ldquoCurcumin inhibitsglyoxalase 1mdasha possible link to its anti-inflammatory and anti-tumor activityrdquo PLoS ONE vol 3 no 10 Article ID e3508 2008

[2] D Lu T Lin XWo PWang Y Zhou and Z Yang ldquoProteomicresearch on anti-atherosclerosis mechanism of curcumin inRAW2647rdquo Zhongguo Zhongyao Zazhi vol 36 no 9 pp 1207ndash1211 2011

[3] B B Aggarwal A Kumar and A C Bharti ldquoAnticancer poten-tial of curcumin preclinical and clinical studiesrdquo AnticancerResearch vol 23 no 1 A pp 363ndash398 2003

[4] S Toda T Miyase and H Arichi ldquoNatural antioxidants IIIAntioxidative components isolated from rhizome of Curcumalonga Lrdquo Chemical and Pharmaceutical Bulletin vol 33 no 4pp 1725ndash1728 1985

[5] A Barzegar and A A Moosavi-Movahedi ldquoIntracellular ROSprotection efficiency and free radical-scavenging activity ofcurcuminrdquo PLoS ONE vol 6 no 10 Article ID e26012 2011

[6] R K Maheshwari A K Singh J Gaddipati and R C SrimalldquoMultiple biological activities of curcumin a short reviewrdquo LifeSciences vol 78 no 18 pp 2081ndash2087 2006

[7] G M Cole B Teter and S A Frautschy ldquoNeuroprotectiveeffects of curcuminrdquo Advances in Experimental Medicine andBiology vol 595 pp 197ndash212 2007

[8] R B Mythri and M M Srinivas Bharath ldquoCurcumin apotential neuroprotective agent in Parkinsonrsquos diseaserdquo CurrentPharmaceutical Design vol 18 no 1 pp 91ndash99 2012

[9] S S Agrawal S Gullaiya V Dubey et al ldquoNeurodegenerativeshielding by curcumin and its derivatives on brain lesionsinduced by 6-OHDA model of Parkinsonrsquos disease in albinowistar ratsrdquo Cardiovascular Psychiatry and Neurology vol 2012Article ID 942981 8 pages 2012

[10] W Shao S Z Zhang M Tang et al ldquoSuppression of neu-roinflammation by astrocytic dopamine D2 receptors via 120572B-crystallinrdquo Nature vol 494 no 7435 pp 90ndash94 2013

[11] A E Lang and A M Lozano ldquoParkinsonrsquos disease First of twopartsrdquoTheNew England Journal of Medicine vol 339 pp 1044ndash1053 1998

[12] A Siderowf and M Stern ldquoUpdate on Parkinson diseaserdquoAnnals of Internal Medicine vol 138 no 8 pp 651ndash658 2003

[13] A H V Schapira ldquoEtiology of Parkinsonrsquos diseaserdquo Neurologyvol 66 no 10 supplement 4 pp S10ndashS23 2006

[14] C Zhou Y Huang and S Przedborski ldquoOxidative stress inParkinsonrsquos disease amechanismof pathogenic and therapeuticsignificancerdquo Annals of the New York Academy of Sciences vol1147 pp 93ndash104 2008

[15] A A Caro and A I Cederbaum ldquoOxidative stress toxicologyand pharmacology of CYP2E1rdquoAnnual Review of Pharmacologyand Toxicology vol 44 pp 27ndash42 2004

[16] G Ekstrom and M Ingelman-Sundberg ldquoRat liver microso-mal NADPH-supported oxidase activity and lipid peroxida-tion dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)rdquo Biochemical Pharmacology vol 38 no 8 pp 1313ndash1319 1989

[17] T Hansson N Tindberg M Ingelman-Sundberg and CKohler ldquoRegional distribution of ethanol-inducible cytochromeP450 IIE1 in the rat central nervous systemrdquo Neuroscience vol34 no 2 pp 451ndash463 1990

[18] C Montoliu M Sancho-Tello I Azorin et al ldquoEthanolincreases cytochrome P4502E1 and induces oxidative stress inastrocytesrdquo Journal of Neurochemistry vol 65 no 6 pp 2561ndash2570 1995

[19] N Tindberg H A Baldwin A J Cross and M Ingelman-Sundberg ldquoInduction of cytochrome P450 2E1 expression inrat and gerbil astrocytes by inflammatory factors and ischemicinjuryrdquo Molecular Pharmacology vol 50 no 5 pp 1065ndash10721996

[20] M Warner and J A Gustafsson ldquoEffect of ethanol oncytochrome P450 in the rat brainrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 91 no3 pp 1019ndash1023 1994

[21] C Pardini F Vaglini C Viaggi A Caramelli andG U CorsinildquoRole of CYP2E1 in the mouse model of MPTP toxicityrdquoParkinsonism and RelatedDisorders vol 14 no 2 pp S119ndashS1232008

[22] F Vaglini C Pardini C Viaggi C Bartoli D Dinucci andGUCorsini ldquoInvolvement of cytochrome P450 2E1 in the 1-methyl-4-phenyl-1236-tetrahydropyridine-induced mouse model ofParkinsonrsquos diseaserdquo Journal of Neurochemistry vol 91 no 2 pp285ndash298 2004

[23] T Ivanova and C Beyer ldquoEstrogen regulates tyrosine hydrox-ylase expression in the neonate mouse midbrainrdquo Journal ofNeurobiology vol 54 no 4 pp 638ndash647 2003

[24] C HaoW Liu X Luan et al ldquoAquaporin-4 knockout enhancesastrocyte toxicity induced by 1-methyl-4-phenylpyridiniumion and lipopolysaccharide via increasing the expression ofcytochrome P4502E1rdquo Toxicology Letters vol 198 no 2 pp225ndash231 2010

[25] K J Sipe R Dantzer K W Kelley and J A WeyhenmeyerldquoExpression of the 75 kDA TNF receptor and its role in contact-mediated neuronal cell deathrdquoMolecular BrainResearch vol 62no 2 pp 111ndash121 1998

[26] Y Peng W Liu J Xiong et al ldquoDown regulation of differ-entiated embryonic chondrocytes 1 (DEC1) is involved in 8-methoxypsoralen-induced apoptosis in HepG2 cellsrdquo Toxicol-ogy vol 301 no 1ndash3 pp 58ndash65 2012

[27] Y Yuan X Xue R B Guo X L Sun and G Hu ldquoResveratrolenhances the antitumor effects of temozolomide in glioblas-toma via ROS-dependent AMPK-TSC-mTOR signaling path-wayrdquo CNS Neuroscience andTherapeutics vol 18 no 7 pp 536ndash546 2012

[28] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[29] Y Wang and L Fu ldquoForms of selenium affect its transportuptake and glutathione peroxidase activity in the Caco-2 cellmodelrdquoBiological Trace Element Research vol 149 no 1 pp 110ndash116 2012

[30] L A Reinke andM J Moyer ldquoAmicrosomal oxidation which ishighly inducible by ethanolrdquo Drug Metabolism and Dispositionvol 13 no 5 pp 548ndash552 1985


[31] D R Koop ldquoHydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450 isozyme 3ardquoMolecular Pharmacol-ogy vol 29 no 4 pp 399ndash404 1986

[32] H Erlank A Elmann R Kohen and J Kanner ldquoPolyphe-nols activate Nrf2 in astrocytes via H2O2 semiquinones andquinonesrdquo Free Radical Biology and Medicine vol 51 no 12 pp2319ndash2327 2011

[33] S Yu W Zheng N Xin et al ldquoCurcumin prevents dopaminer-gic neuronal death through inhibition of the c-Jun N-terminalkinase pathwayrdquo Rejuvenation Research vol 13 no 1 pp 55ndash642010

[34] A A Nanji K Jokelainen G L Tipoe A Rahemtulla PThomas and A J Dannenberg ldquoCurcumin prevents alcohol-induced liver disease in rats by inhibiting the expression of NF-120581B-dependent genesrdquo American Journal of Physiology vol 284no 2 pp G321ndashG327 2003

[35] V Rajakrishnan P Viswanathan K N Rajasekharan andV P Menon ldquoNeuroprotective role of curcumin from cur-cuma longa on ethanol-induced brain damagerdquo PhytotherapyResearch vol 13 pp 571ndash574 1999

[36] X X Du H M Xu H Jiang N Song J Wang and J XXie ldquoCurcumin protects nigral dopaminergic neurons by iron-chelation in the 6-hydroxydopamine rat model of Parkinsonrsquosdiseaserdquo Neuroscience Bulletin vol 28 pp 253ndash258 2012

[37] Y Luo A Hattori J Munoz Z H Qin and G S RothldquoIntrastriatal dopamine injection induces apoptosis throughoxidation-involved activation of transcription factors AP-1 andNF-120581B in ratsrdquoMolecular Pharmacology vol 56 no 2 pp 254ndash264 1999

[38] M S Wang S Boddapati S Emadi and M R Sierks ldquoCur-cumin reduces 120572-synuclein induced cytotoxicity in Parkinsonrsquosdisease cell modelrdquo BMC Neuroscience vol 11 article 57 2010

[39] Z Liu Y Yu X Li C A Ross and W W Smith ldquoCurcuminprotects against A53T alpha-synuclein-induced toxicity in aPC12 inducible cell model for Parkinsonismrdquo PharmacologicalResearch vol 63 no 5 pp 439ndash444 2011

[40] H P Lee T M Li J Y Tsao Y C Fong and C HTang ldquoCurcumin induces cell apoptosis in human chondrosar-coma through extrinsic death receptor pathwayrdquo InternationalImmunopharmacology vol 13 no 2 pp 163ndash169 2012

[41] J Lotharius and P Brundin ldquoPathogenesis of Parkinsonrsquos dis-ease dopamine vesicles and alpha-synucleinrdquo Nature ReviewsNeuroscience vol 3 no 12 pp 932ndash942 2002

[42] Y Jiao J Wilkinson IV X Di et al ldquoCurcumin a cancerchemopreventive and chemotherapeutic agent is a biologicallyactive iron chelatorrdquo Blood vol 113 no 2 pp 462ndash469 2009

[43] J Miquel A Bernd J M Sempere J Dıaz-Alperi and ARamırez ldquoThe curcuma antioxidants pharmacological effectsand prospects for future clinical use A reviewrdquo Archives ofGerontology and Geriatrics vol 34 no 1 pp 37ndash46 2002

[44] N P Kennedy and K F Tipton ldquoEthanol metabolism andalcoholic liver diseaserdquo Essays in Biochemistry vol 25 pp 137ndash195 1990

[45] C S Lieber ldquoCytochrome P-4502E1 its physiological andpathological rolerdquo Physiological Reviews vol 77 no 2 pp 517ndash544 1997

[46] W Bao K Li S Rong et al ldquoCurcumin alleviates ethanol-induced hepatocytes oxidative damage involving hemeoxygenase-1 inductionrdquo Journal of Ethnopharmacology vol128 no 2 pp 549ndash553 2010

[47] G Scapagnini C Colombrita M Amadio et al ldquoCurcuminactivates defensive genes and protects neurons against oxidativestressrdquo Antioxidants and Redox Signaling vol 8 no 3-4 pp395ndash403 2006

[48] P Yao L Hao N Nussler et al ldquoThe protective role ofHO-1 and its generated products (CO bilirubin and Fe) inethanol-induced human hepatocyte damagerdquoAmerican Journalof Physiology vol 296 no 6 pp G1318ndashG1323 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the striatum [11 12] Although the exact etiology of thePD is unknown many studies suggest that multiple factorsmay be involved such as heredity environment oxidativestress inflammation and decline in growth factors [13]Oxidative stress which results from the imbalance of reactiveoxygen species (ROS) generation and clearance is one ofthe important pathogenesis of PD [14] ROS generation mayvary considerably depending on cytochrome P450 enzymes(CYPs) Compared with other CYPs cytochrome P450 2E1(CYP2E1) exhibits a higher rate of oxidase activity thatcauses the formation of ROS during the catalytic cycle TheROS generation is likely to initiate lipid peroxidation anddamage cell membranes [15 16] In the brain CYP2E1 isconstitutively expressed in hippocampal pyramid neuronsastrocytes and endothelial cells [17] and CYP2E1 has beenfound to be inducible and catalytically active in the centralnervous system [18ndash20] It has been suggested that 1-methyl-4-phenylpyridinium ion (MPP+ the ion form of 1-methyl-4-phenyl-1236-tetrahydropyridine MPTP) and lipopolysac-charide (LPS) increased the expression of CYP2E1 andinduced oxidative stress in astrocytes [18 19] The increasingevidence shows that CYP2E1 is involved in the MPTP-induced mouse model of PD [21 22]

In this study we demonstrated that Cur had the cyto-protection against MPP+- and LPS-induced toxicities in theprimary mouse mesencephalic astrocytes Meanwhile Curdecreased the increased ROS and maleic dialdehyde (MDA)amount in the primary culture mesencephalic astrocytesexposed to MPP+ and LPS Combination with an increaseof CYP2E1 induced by MPP+ and LPS in astrocytes [1819] we hypothesized that Cur protects against MPP+- andLPS-induced mouse mesencephalic astrocyte damage viainhibiting the CYP2E1 expression and activity

2 Materials and Methods

21 Animal and Materials C57BL6J mice were kept underenvironmentally controlled conditions (ambient tempera-ture 22 plusmn 1∘C humidity 40) on a 12-hour light-dark cyclewith food and water ad libitum All the experiments wereapproved by Institutional Animal Care and Use Committee(IACUC) of Nanjing Medical University And efforts weremade tominimize animal suffering and to reduce the numberof animals used for the experiments

Dulbeccorsquos modified eaglersquos medium (DMEM) was fromGibco (Gibco Grand Island NY USA) MPP+ LPS and cur-cumin (98 purity) were purchased from Sigma-Aldrich (StLouisMOUSA) Ethyl alcohol (EtOH)was from Sinopharm(Shanghai China) Antibody to CYP2E1 was obtained fromAbcam (Cambridge UK) SYBR Premix Ex Taq kit waspurchased from Biotechnology Limited Company (TakaraJapan) Tryptase and MTT were purchased from AMRESCO(Solon OH USA) Fetal bovine serum (FBS) was purchasedfrom Sijiqing (Hangzhou China) Trizol was from Invitro-gen Life (Carlsbad CA USA) MLV and RNase inhibitorwere purchased from Promega (Madison WI USA) 2101584071015840-dichlorofluorescein diacetate (DCFH-DA) was purchasedfrom Biyuntian (Shanghai China) Lactate dehydrogenase

(LDH) MDA H2O2 and glutathione peroxidase (GSH-Px)

diagnostic kits were from Nanjing Jiancheng BioengineeringCompany (Nanjing China) and the other chemicals were allobtained from Sigma unless otherwise stated

22 Primary Mesencephalic Astrocyte Culture Primarymouse mesencephalic astrocyte culture was prepared frommidbrain of newborn of C57BL6J mice as describedpreviously [23] with slight modifications Briefly postnatal(P1-P2) mice were killed by rapid decapitation the meningeswere removed and the midbrain parts were separated Themidbrain parts were dissociated with 025 tryptase at 37∘Cand terminated by DMEM supplemented with 10 FBS andpenicillinstreptomycin After centrifugation at 1500 rpmfor 5min the cell pellets were resuspended and seeded onpolylysine coated flask The cultures were maintained at37∘C in a humidified 5 CO

2-95 air atmosphere [24]

Culture medium was replaced 24 h later and then changedevery 2-3 days After reaching a confluent monolayer of glialcells (10ndash14 days) mesencephalic astrocytes were replatedon other polylysine-coated flasks The tertiary cultures aresim95ndash98 astrocytes with only sim2ndash5 microglial cells asdetermined by immunocytochemical staining with anti-GFAP antibody as well as double immunofluorescence forGFAP and microglial marker Iba-1 [24] The mesencephalicastrocytes were seeded on polylysine coated 6- 12- 24- or96-well plates for corresponding experiments

23 Cell Viability Cell viability was detected by 3-(45-dim-ethylthiazol-2-yl) 25-diphenyltetrazolium bromide (MTT)assay Mesencephalic astrocytes were seeded in polylysinecoated 96-well plates at a density of 1 times 104 cellswell pre-treated with Cur diallyl sulphide (DAS) or vehicle (DMSO)and then incubated with MPP+ (100120583M) and LPS (1 120583gmL)for 48 h Then 20120583L of 5mgmL MTT was added to cellsand cells were incubated at 37∘C for another 4 h The culturemedium was discarded and 100 120583L DMSO was used to dis-solve the precipitateThe absorbancewasmeasured at 570 nmusing an Automated Microplated Reader ELx800 (BioTek)The cytotoxicity assessment was exhibited by cytotoxicityindex which was calculated as previously described [25]Each treatment was in quadruplicate And data came fromthree independent experiments

24 Determination of Lactate Dehydrogenase Release Mes-encephalic astrocytes injury (cytotoxicity) was quantitativelyassessed by themeasurement of lactate dehydrogenase (LDH)leakage LDH release in culture medium in the presence ofDMSO or Cur for 30min followed by the addition of MPP+and LPS for 48 h was measured using an LDH diagnostickit according to themanufacturerrsquos instruction LDH amountwas calculated by measuring absorbance at 450 nmThe datawere represented as a percentage of LDH release of controlgroup Each treatment was in quadruplicate And data camefrom three independent experiments

25 Hoechst 33342 Staining To quantify apoptotic mesen-cephalic astrocytes mesencephalic astrocytic monolayer was








2O2diagnostic












2




31 Results










0

20

40

60

80

100

120

0 1 3 10 30 50

Cel

l via

bilit

y (

of c

ontro

l)

lowast

lowastlowast

Curcumin (120583M)

(a)

020406080

100120140160180200

0 1 3 10 30 50

LDH

rele

ase (

o

f con

trol)

lowast

lowastlowast

Curcumin (120583M)

(b)




(c)




0

20

40

60

80

100

120

0 1 3 10

Cel

l via

bilit

y (

of c

ontro

l)

Curcumin (120583M)

lowastlowast lowast


Control

LPSMPP+

(a)

020406080

100120140160

LDH

rele

ase (

o

f con

trol)

0 1 3 10Curcumin (120583M)

lowast


Control

LPSMPP+

(b)

Control LPS


MPP+

(c)












Control LPS


MPP+

(a)

002040608

112141618

2

Control LPS

Fluo

resc

ence

inte

nsity

( o

f con

trol)

ControlCurcumin

lowast

lowast

lowastlowast

MPP+

(b)

0010203040506070809

1

MD

A co

nten

t (nm

olm

L)

Control LPS

ControlCurcumin

lowastlowast

MPP+

(c)









0

1

2

3

4

5

6

7

Control Curcumin

H2O2

prod

uct (120583

M)

Control

LPSMPP+

(a)

0

5

10

15

20

25

30

Control Curcumin

GSH

-Px

activ

ity (u

nitm

ing

pro

tein

)

Control

LPS

lowast

MPP+

(b)

0010203040506070809

1


Cel

l via

bilit

y (O

D570

nm) lowastlowast

lowastlowast

lowastlowastlowast

Control LPSEtOHMPP+

(c)










005

115

225

335

445

5

Control LPS EtOH

Relat

ive C

YP2E

1 m

RNA

ControlCurcumin

lowastlowast

lowastlowast

lowastlowast

MPP+

(a)

ControlCurcumin

0002004006008

01012014016018

Opt

ical

den

sity

Control LPS EtOH

lowastlowast

lowastlowast

lowastlowast

MPP+

(b)

Control LPS EtOH


MPP+

(c)

0

001

002

003

004

005

006

CYP2

E1 ac

tivity

(nm

olm

gm

in)

ControlCurcumin

Control LPS EtOH

lowastlowast

lowastlowast

MPP+

(d)

















CYP2E1

ROS


LPSO2minus

∙OHROO∙





4 Conclusion


Abbreviations









Acknowledgments




References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























2O2diagnostic












2




31 Results










0

20

40

60

80

100

120

0 1 3 10 30 50

Cel

l via

bilit

y (

of c

ontro

l)

lowast

lowastlowast

Curcumin (120583M)

(a)

020406080

100120140160180200

0 1 3 10 30 50

LDH

rele

ase (

o

f con

trol)

lowast

lowastlowast

Curcumin (120583M)

(b)




(c)




0

20

40

60

80

100

120

0 1 3 10

Cel

l via

bilit

y (

of c

ontro

l)

Curcumin (120583M)

lowastlowast lowast


Control

LPSMPP+

(a)

020406080

100120140160

LDH

rele

ase (

o

f con

trol)

0 1 3 10Curcumin (120583M)

lowast


Control

LPSMPP+

(b)

Control LPS


MPP+

(c)












Control LPS


MPP+

(a)

002040608

112141618

2

Control LPS

Fluo

resc

ence

inte

nsity

( o

f con

trol)

ControlCurcumin

lowast

lowast

lowastlowast

MPP+

(b)

0010203040506070809

1

MD

A co

nten

t (nm

olm

L)

Control LPS

ControlCurcumin

lowastlowast

MPP+

(c)









0

1

2

3

4

5

6

7

Control Curcumin

H2O2

prod

uct (120583

M)

Control

LPSMPP+

(a)

0

5

10

15

20

25

30

Control Curcumin

GSH

-Px

activ

ity (u

nitm

ing

pro

tein

)

Control

LPS

lowast

MPP+

(b)

0010203040506070809

1


Cel

l via

bilit

y (O

D570

nm) lowastlowast

lowastlowast

lowastlowastlowast

Control LPSEtOHMPP+

(c)










005

115

225

335

445

5

Control LPS EtOH

Relat

ive C

YP2E

1 m

RNA

ControlCurcumin

lowastlowast

lowastlowast

lowastlowast

MPP+

(a)

ControlCurcumin

0002004006008

01012014016018

Opt

ical

den

sity

Control LPS EtOH

lowastlowast

lowastlowast

lowastlowast

MPP+

(b)

Control LPS EtOH


MPP+

(c)

0

001

002

003

004

005

006

CYP2

E1 ac

tivity

(nm

olm

gm

in)

ControlCurcumin

Control LPS EtOH

lowastlowast

lowastlowast

MPP+

(d)

















CYP2E1

ROS


LPSO2minus

∙OHROO∙





4 Conclusion


Abbreviations









Acknowledgments




References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2




31 Results










0

20

40

60

80

100

120

0 1 3 10 30 50

Cel

l via

bilit

y (

of c

ontro

l)

lowast

lowastlowast

Curcumin (120583M)

(a)

020406080

100120140160180200

0 1 3 10 30 50

LDH

rele

ase (

o

f con

trol)

lowast

lowastlowast

Curcumin (120583M)

(b)




(c)




0

20

40

60

80

100

120

0 1 3 10

Cel

l via

bilit

y (

of c

ontro

l)

Curcumin (120583M)

lowastlowast lowast


Control

LPSMPP+

(a)

020406080

100120140160

LDH

rele

ase (

o

f con

trol)

0 1 3 10Curcumin (120583M)

lowast


Control

LPSMPP+

(b)

Control LPS


MPP+

(c)












Control LPS


MPP+

(a)

002040608

112141618

2

Control LPS

Fluo

resc

ence

inte

nsity

( o

f con

trol)

ControlCurcumin

lowast

lowast

lowastlowast

MPP+

(b)

0010203040506070809

1

MD

A co

nten

t (nm

olm

L)

Control LPS

ControlCurcumin

lowastlowast

MPP+

(c)









0

1

2

3

4

5

6

7

Control Curcumin

H2O2

prod

uct (120583

M)

Control

LPSMPP+

(a)

0

5

10

15

20

25

30

Control Curcumin

GSH

-Px

activ

ity (u

nitm

ing

pro

tein

)

Control

LPS

lowast

MPP+

(b)

0010203040506070809

1


Cel

l via

bilit

y (O

D570

nm) lowastlowast

lowastlowast

lowastlowastlowast

Control LPSEtOHMPP+

(c)










005

115

225

335

445

5

Control LPS EtOH

Relat

ive C

YP2E

1 m

RNA

ControlCurcumin

lowastlowast

lowastlowast

lowastlowast

MPP+

(a)

ControlCurcumin

0002004006008

01012014016018

Opt

ical

den

sity

Control LPS EtOH

lowastlowast

lowastlowast

lowastlowast

MPP+

(b)

Control LPS EtOH


MPP+

(c)

0

001

002

003

004

005

006

CYP2

E1 ac

tivity

(nm

olm

gm

in)

ControlCurcumin

Control LPS EtOH

lowastlowast

lowastlowast

MPP+

(d)

















CYP2E1

ROS


LPSO2minus

∙OHROO∙





4 Conclusion


Abbreviations









Acknowledgments




References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

0 1 3 10 30 50

Cel

l via

bilit

y (

of c

ontro

l)

lowast

lowastlowast

Curcumin (120583M)

(a)

020406080

100120140160180200

0 1 3 10 30 50

LDH

rele

ase (

o

f con

trol)

lowast

lowastlowast

Curcumin (120583M)

(b)




(c)




0

20

40

60

80

100

120

0 1 3 10

Cel

l via

bilit

y (

of c

ontro

l)

Curcumin (120583M)

lowastlowast lowast


Control

LPSMPP+

(a)

020406080

100120140160

LDH

rele

ase (

o

f con

trol)

0 1 3 10Curcumin (120583M)

lowast


Control

LPSMPP+

(b)

Control LPS


MPP+

(c)












Control LPS


MPP+

(a)

002040608

112141618

2

Control LPS

Fluo

resc

ence

inte

nsity

( o

f con

trol)

ControlCurcumin

lowast

lowast

lowastlowast

MPP+

(b)

0010203040506070809

1

MD

A co

nten

t (nm

olm

L)

Control LPS

ControlCurcumin

lowastlowast

MPP+

(c)









0

1

2

3

4

5

6

7

Control Curcumin

H2O2

prod

uct (120583

M)

Control

LPSMPP+

(a)

0

5

10

15

20

25

30

Control Curcumin

GSH

-Px

activ

ity (u

nitm

ing

pro

tein

)

Control

LPS

lowast

MPP+

(b)

0010203040506070809

1


Cel

l via

bilit

y (O

D570

nm) lowastlowast

lowastlowast

lowastlowastlowast

Control LPSEtOHMPP+

(c)










005

115

225

335

445

5

Control LPS EtOH

Relat

ive C

YP2E

1 m

RNA

ControlCurcumin

lowastlowast

lowastlowast

lowastlowast

MPP+

(a)

ControlCurcumin

0002004006008

01012014016018

Opt

ical

den

sity

Control LPS EtOH

lowastlowast

lowastlowast

lowastlowast

MPP+

(b)

Control LPS EtOH


MPP+

(c)

0

001

002

003

004

005

006

CYP2

E1 ac

tivity

(nm

olm

gm

in)

ControlCurcumin

Control LPS EtOH

lowastlowast

lowastlowast

MPP+

(d)

















CYP2E1

ROS


LPSO2minus

∙OHROO∙





4 Conclusion


Abbreviations









Acknowledgments




References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

0 1 3 10

Cel

l via

bilit

y (

of c

ontro

l)

Curcumin (120583M)

lowastlowast lowast


Control

LPSMPP+

(a)

020406080

100120140160

LDH

rele

ase (

o

f con

trol)

0 1 3 10Curcumin (120583M)

lowast


Control

LPSMPP+

(b)

Control LPS


MPP+

(c)












Control LPS


MPP+

(a)

002040608

112141618

2

Control LPS

Fluo

resc

ence

inte

nsity

( o

f con

trol)

ControlCurcumin

lowast

lowast

lowastlowast

MPP+

(b)

0010203040506070809

1

MD

A co

nten

t (nm

olm

L)

Control LPS

ControlCurcumin

lowastlowast

MPP+

(c)









0

1

2

3

4

5

6

7

Control Curcumin

H2O2

prod

uct (120583

M)

Control

LPSMPP+

(a)

0

5

10

15

20

25

30

Control Curcumin

GSH

-Px

activ

ity (u

nitm

ing

pro

tein

)

Control

LPS

lowast

MPP+

(b)

0010203040506070809

1


Cel

l via

bilit

y (O

D570

nm) lowastlowast

lowastlowast

lowastlowastlowast

Control LPSEtOHMPP+

(c)










005

115

225

335

445

5

Control LPS EtOH

Relat

ive C

YP2E

1 m

RNA

ControlCurcumin

lowastlowast

lowastlowast

lowastlowast

MPP+

(a)

ControlCurcumin

0002004006008

01012014016018

Opt

ical

den

sity

Control LPS EtOH

lowastlowast

lowastlowast

lowastlowast

MPP+

(b)

Control LPS EtOH


MPP+

(c)

0

001

002

003

004

005

006

CYP2

E1 ac

tivity

(nm

olm

gm

in)

ControlCurcumin

Control LPS EtOH

lowastlowast

lowastlowast

MPP+

(d)

















CYP2E1

ROS


LPSO2minus

∙OHROO∙





4 Conclusion


Abbreviations









Acknowledgments




References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control LPS


MPP+

(a)

002040608

112141618

2

Control LPS

Fluo

resc

ence

inte

nsity

( o

f con

trol)

ControlCurcumin

lowast

lowast

lowastlowast

MPP+

(b)

0010203040506070809

1

MD

A co

nten

t (nm

olm

L)

Control LPS

ControlCurcumin

lowastlowast

MPP+

(c)









0

1

2

3

4

5

6

7

Control Curcumin

H2O2

prod

uct (120583

M)

Control

LPSMPP+

(a)

0

5

10

15

20

25

30

Control Curcumin

GSH

-Px

activ

ity (u

nitm

ing

pro

tein

)

Control

LPS

lowast

MPP+

(b)

0010203040506070809

1


Cel

l via

bilit

y (O

D570

nm) lowastlowast

lowastlowast

lowastlowastlowast

Control LPSEtOHMPP+

(c)










005

115

225

335

445

5

Control LPS EtOH

Relat

ive C

YP2E

1 m

RNA

ControlCurcumin

lowastlowast

lowastlowast

lowastlowast

MPP+

(a)

ControlCurcumin

0002004006008

01012014016018

Opt

ical

den

sity

Control LPS EtOH

lowastlowast

lowastlowast

lowastlowast

MPP+

(b)

Control LPS EtOH


MPP+

(c)

0

001

002

003

004

005

006

CYP2

E1 ac

tivity

(nm

olm

gm

in)

ControlCurcumin

Control LPS EtOH

lowastlowast

lowastlowast

MPP+

(d)

















CYP2E1

ROS


LPSO2minus

∙OHROO∙





4 Conclusion


Abbreviations









Acknowledgments




References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

5

6

7

Control Curcumin

H2O2

prod

uct (120583

M)

Control

LPSMPP+

(a)

0

5

10

15

20

25

30

Control Curcumin

GSH

-Px

activ

ity (u

nitm

ing

pro

tein

)

Control

LPS

lowast

MPP+

(b)

0010203040506070809

1


Cel

l via

bilit

y (O

D570

nm) lowastlowast

lowastlowast

lowastlowastlowast

Control LPSEtOHMPP+

(c)










005

115

225

335

445

5

Control LPS EtOH

Relat

ive C

YP2E

1 m

RNA

ControlCurcumin

lowastlowast

lowastlowast

lowastlowast

MPP+

(a)

ControlCurcumin

0002004006008

01012014016018

Opt

ical

den

sity

Control LPS EtOH

lowastlowast

lowastlowast

lowastlowast

MPP+

(b)

Control LPS EtOH


MPP+

(c)

0

001

002

003

004

005

006

CYP2

E1 ac

tivity

(nm

olm

gm

in)

ControlCurcumin

Control LPS EtOH

lowastlowast

lowastlowast

MPP+

(d)

















CYP2E1

ROS


LPSO2minus

∙OHROO∙





4 Conclusion


Abbreviations









Acknowledgments




References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















005

115

225

335

445

5

Control LPS EtOH

Relat

ive C

YP2E

1 m

RNA

ControlCurcumin

lowastlowast

lowastlowast

lowastlowast

MPP+

(a)

ControlCurcumin

0002004006008

01012014016018

Opt

ical

den

sity

Control LPS EtOH

lowastlowast

lowastlowast

lowastlowast

MPP+

(b)

Control LPS EtOH


MPP+

(c)

0

001

002

003

004

005

006

CYP2

E1 ac

tivity

(nm

olm

gm

in)

ControlCurcumin

Control LPS EtOH

lowastlowast

lowastlowast

MPP+

(d)

















CYP2E1

ROS


LPSO2minus

∙OHROO∙





4 Conclusion


Abbreviations









Acknowledgments




References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























CYP2E1

ROS


LPSO2minus

∙OHROO∙





4 Conclusion


Abbreviations









Acknowledgments




References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article curcumin protects against 1-methyl-4

Documents