research article tempol, a membrane-permeable radical ...research article tempol, a...

Research ArticleTempol a Membrane-Permeable Radical ScavengerExhibits Anti-Inflammatory and Cardioprotective Effects inthe Cerulein-Induced Pancreatitis Rat Model

Andrzej Marciniak1 Beata Walczyna2 Grahyna Rajtar3 Sebastian Marciniak3

Andrzej Wojtak4 and Katarzyna Lasiecka5

1Chair and Department of Pathophysiology Medical University of Lublin 20-090 Lublin Poland2Chair and Department of Clinical Pathomorphology Medical University of Lublin 20-090 Lublin Poland3Department of Pharmacology Medical University of Lublin 20-093 Lublin Poland4Chair and Department of Vascular Surgery and Angiology Medical University of Lublin 20-081 Lublin Poland5Department of Pharmacology University of Life Sciences in Lublin 20-033 Lublin Poland

Correspondence should be addressed to Andrzej Marciniak andrzejmarciniakumlubpl

Received 4 June 2015 Revised 30 August 2015 Accepted 31 August 2015

Academic Editor Swaran J S Flora

Copyright copy 2016 Andrzej Marciniak et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

To date it remains unclear whethermild formof acute pancreatitis (AP)may causemyocardial damagewhichmay be asymptomaticfor a long time Pathogenesis of AP-related cardiac injury may be attributed in part to ROSRNS overproduction The aim of thepresent study was to evaluate the oxidative stress changes in both the pancreas and the heart and to estimate the protective effectsof 1-oxyl-2266-tetramethyl-4-hydroxypiperidine (tempol) at the early phase of AP Cerulein-induced AP led to the developmentof acute edematous pancreatitis with a significant decrease in the level of sulfhydryl (ndashSH) groups (oxidation marker) both in heartand in pancreatic tissues as well as a substantial increase in plasma creatine kinase isoenzyme (CK-MB) activity (marker of theheart muscle lesion) which confirmed the role of oxidative stress in the pathogenesis of cardiac damage The tempol treatmentsignificantly reduced the intensity of inflammation and oxidative damage and decreased the morphological evidence of pancreasinjury at early AP stages Moreover it markedly attenuated AP-induced cardiac damage revealed by normalization of the ndashSHgroup levels and CK-MB activity On the basis of these studies it is possible to conclude that tempol has a profound protectiveeffect against cardiac and pancreatic damage induced by AP

1 Introduction

Despite great advancement in therapeutics treatment foracute pancreatitis (AP) has not significantly changed formany years and the illness characterizes itself with a sub-stantial percentage of complications and mortality with itspathogenesis is not fully recognized yet Acute pancreatitis(AP) is an illness which may lead to general inflammatoryreaction which in turn may result in the development ofmultiple organ dysfunction syndrome [1 2]Thepathogenesisof systemic complications of AP is still under intensive inves-tigation The clinical and experimental researches conductedso far have revealed the complex pathogenesis of AP and

a serious role of oxidative and antioxidative processes in thedevelopment of this fatal illnessThe uncontrolled generationof reactive oxygen and nitrogen species (ROSRNS) duringAP may lead to oxidative damage both in the pancreas anddistant organs [3ndash5] ROSRNS are implicated in the patho-physiology of diverse cardiovascular diseases These alsoappear to be common mediators of apoptosis directly or viathe formation of lipid peroxidation and lipid hydroperoxides[6ndash8]

To date it remains unclear whether mild form of APmaycause myocardial damage which may remain asymptomaticfor a long time Hypothetical mechanisms have been pro-posed to explain pathogenesis of AP-related cardiac injury

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 4139851 7 pageshttpdxdoiorg10115520164139851

2 Oxidative Medicine and Cellular Longevity

which may be attributed in part to ROSRNS and inflam-matory cytokines overproduction [9ndash12] Thus cytopro-tectiveantioxidant treatment may prevent development ofcardiac injury at the early phase of AP

4-Hydroxy-2266-tetramethylpiperidine-1-oxyl (tempol)is a membrane-permeable aminoxyl-type free radical scav-enger with unique antioxidant properties This compoundexhibits a superoxide dismutase mimetic action to degradesuperoxide radical thereby preventing the formation ofperoxynitrite The advantageous effect of aminoxyl action isalso caused by inhibiting Fentonrsquos reaction catalyzed by Fe2+ions [13] Preclinical studies using a variety of rat modelshave shown the potential for many clinical applicationsincluding control of hypertension andweight gain protectionagainst ionizing radiation and DNA damage and preventionof doxorubicin treatment-related cardiomyopathy [13ndash16]

Therefore the aim of the present study was to evaluate theoxidative stress in the pancreas and the heart and to estimatethe protective effects of tempol at the early phase of cerulein-induced AP

2 Materials and Methods

21 Reagents Thereagents hydrochloric acid ferric chloridesodium acetate glacial acetic acid and ferrous sulphatewere of analytical or HPLC grade 246-Tris(2-pyridyl)-s-triazine (TPTZ 99) cerulein and 4-hydroxy-TEMPOwere purchased from Sigma-Aldrich Co (St Louis MOUSA) Hydrochloric acid sodium acetate sodium carbonateiron(III) chloride hexahydrate (FeCl

3sdot6H2O) iron(II) sulfate

heptahydrate (FeSO4sdot7H2O) and methanol (998) were

obtained from POCH (Gliwice Poland) Redistilled waterwas used for the preparation of solutions

22 Animals and Experimental Model The study was carriedout on 24 male Wistar rats 8ndash10 weeks old weighing 180ndash200 g and obtained from a licensed breeder The study wasapproved by the Ethical Committee for Animal Experimen-tation of the Medical University of Lublin (Licence number136) The rats were kept on standard rat chow and fastedovernight before the experiment with water ad libitum

Acute pancreatitis was induced as previously described[17ndash19] with minor modifications Four subcutaneous injec-tions of cerulein (15 120583g kgminus1 body weight) or vehicles (normalsaline) were given consecutively at hour intervals

Rats were randomly divided into four groups (119899 = 6 pergroup) Group I (control) Group II (tempol) treated withtempol (given in the 1st 2nd and 3rd hours after the firstsaline injection) Group III (cerulein) cerulein-induced pan-creatitis without treatment and Group IV (tempol + ceru-lein) cerulein-induced pancreatitis treated with tempol (3 times100mgkg given in the 1st 2nd and 3rd hours after the firstcerulein injection)

We used three multiple injections of tempol to providea maximal protective effect against cerulein-induced pan-creatitis The dose of tempol was chosen on the basis of aprevious animal studies that showed that similar high doseswere well tolerated [13 20ndash22]

After 6 hours the rats were anaesthetized with thiopentalsodium (50mgkg) the abdominal cavity was opened andblood was collected from the abdominal aorta into tubescontaining EDTA or serum-separating gel The blood wascentrifuged at 3000timesg for 10min Plasma and serum werefrozen and kept at minus80∘C until assayed After blood with-drawal the pancreas and heart were carefully dissected outrinsed with saline blotted on paper weighed and dividedinto samples For biochemical analyses tissue blocks of bothpancreas and heart were snap-frozen in liquid nitrogen andstored at minus80∘C before homogenate preparation

The specific parameters that were evaluated to assess thedegree of pancreatic inflammation included serum amylaselevels pancreatic water content and histological characteris-tics of pancreatic tissue (by fixation and staining with hema-toxylin and eosin (HampE) followed by examination by lightmicroscopy) Plasma creatine kinase isoenzyme (CK-MB)and markers for oxidative damage ferric reducing ability ofplasma (FRAP) andmalondialdehyde and 4-hydroxynonenal(MDA + 4-HNE) and total sulfhydryl (ndashSH) groups in pan-creas and heart were determined

23 Measurement of Parameters for Evaluating AP

231 Determination of Pancreatic Water Content The watercontent of the pancreas was used as an index of pancreaticedema It was quantified by weighing the freshly harvestedtissue (wet weight) and compared with the weight of the samesample after desiccation at 160∘C for 48 h (dry weight) [23]The results were calculated and expressed as a percentage((wet weight minus dry weight) times 100wet weight)

232 SerumAmylase Serum amylase wasmeasured by stan-dard photometric reaction with a commercially available kit(Alpha Diagnostics) using a Shimadzu spectrophotometer(Shimadzu Corporation Japan) according to the manufac-turerrsquos instructions

233 Histology The pancreas was immediately rinsed andfixed in 10 buffered formalin solution Pancreatic scrapeswere embedded in paraffin cut and stained with hematoxylinand eosin (HampE) and graded in a blinded manner Forquantification of edema vacuolization inflammation (neu-trophil infiltration) and necrosis a modification of the scoreoriginally described by Tani et al [24] was used ranging from0 to 3 (edema vacuolization inflammation and necrosis)

24 Indices of Oxidative Stress

241 The Antioxidant Capacity of Plasma The antioxidantcapacity of plasma samples was determined by the ferric-reducing antioxidant power (FRAP) assay of Benzie andStrain [25] This method measures the ability of the antiox-idants contained in a sample to reduce ferric to ferrous ionwhich at low pH forms a coloured ferrous-tripyridyltriazinecomplex that absorbs light at 593 nm

Oxidative Medicine and Cellular Longevity 3

242 Determination of Lipid Peroxidation The levels ofmalondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) inthe tissues of the pancreas and the heart were determined toassess lipid peroxidation In brief samples of tissues weighingabout 300mg were homogenized in the phosphate buffer(20mM pH 74) Prior to homogenization 10 120583L of 05molLbutylated hydroxytoluene in acetonitrile was added to every1mL of tissue homogenate for each sample to prevent tissueoxidation Tissue samples were centrifuged (3000timesg at 4∘Cfor 10min) and the supernatant was frozen at minus80 untilassayMDA+ 4-HNEweremeasured spectrophotometricallyusing a commercial kit Bioxytech LPO-586 (Oxis PortlandUSA) according to the manufacturerrsquos protocol This assay isbased on the reaction of a chromogenic reagent N-methyl-2-phenylindole with MDA and 4-HNE at 45∘C This reactionyields a stable chromophore with maximal absorbance at586 nm [26]

243 Total Sulfhydryl (ndashSH) Groups Assay The total ndashSHgroup content in homogenates wasmeasured usingDTNB (221015840-dinitro-551015840-dithiodibenzoic acid) as described by Ellman[27] DTNB can react with the thiol group to produce acolored compound that absorbs light at 405 nm The rate ofchromophore production is proportional to the concen-tration of total ndashSH within the sample The values wereexpressed as nmolmg protein

25 Creatine Kinase Isoenzyme (CK-MB) Activity PlasmaCK-MB activity was measured by an automatic chemistryanalyser using a Vitros CK slides (Ortho-Clinical Diagnos-tics Inc Rochester NY) to monitor possible cardiotoxicityeffect of AP

26 Determination of Protein Concentration Sample proteincontents were estimated by Bradfordrsquos method using bovineserum albumin as a standard [28]

27 Statistical Analysis Results are expressed as means plusmnSEM where appropriate The histopathological data areexpressed as median (with low and high ranges) Statisticalanalysis was performed using analysis of variance (ANOVA)followed by Kruskal-Wallis or Tukeyrsquos multiple range test aspost hoc Probability values less than 005 were consideredsignificant Data handling and statistics were performedusing the statistical software package STATISTICA version10 (StatSoft Inc)

3 Results

31 Effects of Tempol on Serum Amylase Level PancreaticWaterContent andHistopathological Changes in the PancreasTo study the role of tempol in cerulein-induced AP bloodamylase levels and pancreatic water content were initiallydetermined as an indicator of severity of AP Repeatedinjections of cerulein produced mild model of AP with theexpected elevation in serum amylase and pancreatic watercontent 6 hrs later both of which are highly significant when

Table 1 Histological findings in the pancreas observed in rats withor without cerulein and treated with saline or tempol

Edema(0ndash3)

Vacuolisation(0ndash3)

Infiltration(0ndash3)

Necrosis(0ndash3)

Control 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0)Tempol 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0)Cerulein 25 (2-3)lowast 25 (1ndash3)lowast 2 (1ndash3)lowast 1 (0ndash2)lowast

Tempol +cerulein 2 (1-2)lowast 1 (1-2)lowast 1 (0ndash2)lowast 0 (0-1)lowast

Median scores with ranges (minndashmax) of the results on 6 animals in eachgroup are shown lowast119901 lt 005 versus control 119901 lt 005 versus cerulein alone(analysis of variance followed by the Kruskal-Wallis test)

0

20

40

60

80

100

120

140

0

3000

6000

9000

12000

15000

Control Tempol Cerulein

Panc

reat

ic w

ater

cont

ent (

)

Seru

m am

ylas

e (U

L)

AmylaseEdema

Tempol +cerulein

lowastlowast

lowast

lowast

Figure 1 Effect of cerulein (15120583gkgh for 4 h) given alone or incombination with tempol on serum amylase activity and pancreaticwater content Mean plusmn SEM lowast119901 lt 005 compared with control 119901 lt005 compared with cerulein given alone

compared to those obtained in control groups (control tem-pol) Administration of tempol (tempol + cerulein) reducedboth cerulein-provoked blood amylase levels and pancreaticwater content from 13882plusmn801 down to 10533plusmn343UL andfrom 881plusmn13 down to 789plusmn15 respectively (Figure 1)

Morphological signs of AP were associated with bio-chemicalmarkers of the severity of acute pancreatitis Normalpancreatic microarchitecture was found in control groups(control tempol Table 1 Figure 2) whereas cerulein chal-lenge caused destruction of the pancreatic tissue structurecharacterized by vacuolization of acinar cells perivascularinfiltration of neutrophils necrosis and pancreatic edemaformation Treatment with tempol significantly reduced boththe extent and the severity of the histological signs of pancreasinjury (119901 lt 005 Table 1 with exemplifying slides shown inFigure 2)

32 Oxidative Stress Evaluation

321 Oxidative Injury Indices Cerulein challenge resulted ina significant decrease in the ndashSH group content in pancreasand heart in comparison to the control group values (775 plusmn42 nmolmg versus 1247 plusmn 176 nmolmg and 1357 plusmn170 nmolmg versus 2216 plusmn 235 nmolmg resp 119901 lt 005)


(a) (b)

(c) (d)

Figure 2 Histologic section of pancreas (a) control ratsmdashnormal pancreatic structures were observed (b) the rats subjected to cerulein-induced pancreatitis (AP) alonemdashevidences of edema vacuolization inflammatory cell infiltrate and necrosis were noted (c d) the APanimals treated with tempolmdashnote the significant reduction in edema vacuolization and inflammation without any evidence of necrosis ascompared to untreated animals (b) HampE staining original magnification times20

020406080100120140

00

02

04

06

08

Control Tempol Cerulein Tempol +cerulein

-SHMDA+HNE

SH g

roup

s(n

mol

mg

prot

ein)

MD

A+4

-HN

E(n

mol

mg

prot

ein)

lowast

lowast

Figure 3 Effect of administration of tempol on oxidative stressparameters in pancreatic homogenates of rats with cerulein-inducedpancreatitis Mean plusmn SEM lowast119901 lt 005 compared with control

Treatment of cerulein-challenged animals with tempol par-tially restored the ndashSH group content in the pancreas (993 plusmn32 nmolmg versus 775plusmn42 nmolmg) and also significantlyenhanced ndashSH group content in the heart as compared tothe cerulein-challenged rats (1911 plusmn 114 nmolmg versus1357plusmn170 nmolmg119901 lt 005) (Figures 3 and 4)These find-ings suggest that tempol could have contributed to limitingoxidative damage caused by cerulein-induced AP HoweverMDA + 4-HNE in heart and pancreatic tissues showed no

04080120160200240

0005101520


lowast

lowast

Tempol +cerulein

SH g

roup

s(n

mol

mg

prot

ein)

MD

A+4

-HN

E(n

mol

mg

prot

ein)

-SHMDA+HNE

Figure 4 Effect of administration of tempol on oxidative stressparameters in heart homogenates of rats with cerulein-inducedpancreatitis Mean plusmn SEM lowast119901 lt 005 compared with control 119901 lt005 compared with cerulein given alone

difference among all four groups (119901 gt 005 Figures 3 and4)

322 Total Antioxidant Capacity Cerulein challenge alsoresulted in a significant reduction in plasma FRAP value ascompared with control animals (240 plusmn 11 120583M versus 408 plusmn26 120583M 119901 lt 001) on the contrary tempol treatment revertedsignificantly this reduction in FRAP value caused by cerulein-induced AP (337 plusmn 40 119901 lt 005) (Figure 5)


0

100

200

300

400

500


FRA

P (120583

mol

L)

Tempol +cerulein

lowast

lowast

Figure 5 Effect of administration of tempol on plasma totalantioxidant capacities measured by FRAP of rats with cerulein-induced pancreatitis Mean plusmn SEM lowast119901 lt 005 compared with con-trol 119901 lt 005 compared with cerulein given alone

0

300

600

900

1200


CK-M

B le

vels

(UL

)

lowast

lowast

Figure 6 Effect of administration of tempol on activity of serumcreatine kinase MB (CK-MB) of rats with cerulein-induced pancre-atitis Mean plusmn SEM lowast119901 lt 005 compared with control 119901 lt 005compared with cerulein given alone

33 The Serum Levels of CK-MB Tempol treatment alonedid not induce any increase (119901 gt 005) in CK-MB in com-parison to the control group values Cerulein challengeinduced a significant increase in serum levels of CK-MB (ahighly reliable biomarker of cardiac injury) compared withthe control groups (922 plusmn 167UL versus 566 plusmn 45UL 119901 lt001) Treatment of cerulein-challenged animals with tem-pol (tempol + cerulein) markedly reduced CK-MB levelsas compared to cerulein group (609 plusmn 60UL versus 922 plusmn167UL 119901 lt 001) (Figure 6)

4 Discussion

To date it remains unclear whether mild oedematous formof AP may cause myocardial damage which may be asymp-tomatic for a long time It has been also reported that car-diac disturbances such as electrocardiographic abnormali-ties may be associated with AP Pathomechanisms of theseprocesses have not been fully explained yet Probably oneof the major reasons for cardiac injury is the action of freeradicals freed during the inflammatory processes This leads

to direct oxidative lesion of DNA and proteins and increasedlipid peroxidation [29ndash31] The other potential pathogeneticfactors of cardiac disturbances include electrolyte alterationssystemic inflammatory response or circulating proteolyticenzymes [32ndash35]

To study the involvement of oxidative stress in pancreaticand cardiac damage in the early stage of AP we used arat model of cerulein-induced AP Hyperstimulation withthis CCK ortholog cerulein leads to rapid development ofmild edematous form of experimental AP The suggestedmechanism of cerulein action involves production of ROSactivation of oxidant-sensitive transcription nuclear factor-120581B (NF-120581B) and release of proinflammatory cytokines [3637]

In the present study we have observed that pancreaticoverstimulation with cerulein causes a great intensificationof vacuolization leukocyte infiltration and pancreatic edemaevaluated on the basis of microscopic findings The serumamylase and water content were also markedly elevated Theimportant finding of the present study is the observationthat cerulein-induced AP led to a significant decrease inthe level of sulfhydryl (ndashSH) group (representing the majorpool of low molecular weight antioxidants) in both heartand pancreatic tissues as well as a marked increase of thelevel of CK-MB (marker of myocardial injury) We have alsonoted in cerulein group the decrease in FRAP value whichrepresents the combined potential of various antioxidantsin the body On the other hand MDA + 4-HNE levelsin both heart and pancreatic tissues are not significantlyincreased after 6 hours of experiment This effect seems tobe related to early onset of oxidative stress in the course ofAP Previous studies in the cerulein model found increasedoxidative stress to occur very early within the first 1-2 hours[38 39] It is possible that oxidative stress markers (MDA +4-HNE) may be increased earlier and precede glutathioneand low molecular antioxidantsrsquo decreases Our results are inaccordance with Kruse et alrsquos findings [40]They indicated noincrease inmarkers of oxidative damage to DNA or oxidationof lipidsmeasured by 7-hydro-8-oxo-29-deoxyguanosine andMDA + 4-HNE in cerulein model of acute pancreatitisrespectively

Our study also confirms and extends previous reportsshowing that free radical scavenger tempol has an overallprotective effect against pancreatic injury in a ratmodel of AP[41 42]The beneficial effect of administration of tempol wasmanifested by reduction in pancreatic edema histologicalsigns of pancreatic damage and lower serum amylase levelThis result is in accordance with the earlier reports that tem-pol attenuated the severity of APThe experiments conductedby Sledzinski et al [42] pointed to a cytoprotective action ofthe nitrogen dioxide scavenger in the oxidative stress in thepancreas which was induced with butyl hydroxide

We have also noted that tempol treatment prevents thereduction of the radical scavenging activity in AP as mea-sured by FRAP and ndashSH group content in both pancreasand heart Moreover treatment with tempol significantlyattenuated increase in CK-MB activity caused by AP How-ever it should be emphasized that tempol has othermolecular


mechanisms that possibly contribute to the protective effectagainst cerulein-induced AP injury In other studies Cuz-zocrea et al [43] showed that tempol inhibits the formation ofperoxynitrous acid anion which in turn prevents activationof poly(ADP ribose) synthetase (PARS) and lesions of thetissues connected with it Additionally by acting protectivelyon vascular endothelium it inhibits the inflow of leukocytesto the inflammatory site [44] We have observed a similareffect comparing the histological changes in pancreas in thecerulein groups of the animals The degree of leukocyteinfiltration of the pancreas in animals withAP that were giventempol was reduced as compared to the group of untreatedanimals with AP In numerous studies using experimen-tal models of AP activated polymorphonuclear leukocytes(PMN) were implicated as the major source of oxidants inpancreatic and lung tissues [45ndash47] Reduced leukocyteinfiltration in the pancreas and limited activation of inflam-matory cells may inhibit inflammation and oxidative stressleading at the same time to the lowering of the degree of tissuedamage [48]

5 Conclusions

Taken together our study shows that tempol amelioratessignificantly cerulein-induced AP which has been confirmedby reduction of pancreatic edema improvement in pancreatichistology and reduction in serum amylase levels Treatmentwith tempol had also a profound protective effect againstcardiac damage induced by AP These beneficial effects ofthis compound may be attributable in part to suppressionof oxidative stress by alleviating protein oxidation throughfree radical scavenging at the early phase of AP Thereforeantioxidant compoundsmay offer therapeutic approaches forAP and prevention of its cardiac complication These datahighlight the need for further research to identify the possiblecomplication of mild AP and the potential benefits of earlycytoprotective-specific therapies in this disease

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] R P Sah R K Dawra and A K Saluja ldquoNew insights into thepathogenesis of pancreatitisrdquo Current Opinion in Gastroenterol-ogy vol 29 no 5 pp 523ndash530 2013

[2] J J De Waele ldquoAcute pancreatitisrdquo Current Opinion in CriticalCare vol 20 no 2 pp 189ndash195 2014

[3] J Escobar J Pereda A Arduini et al ldquoOxidative and nitrosativestress in acute pancreatitis Modulation by pentoxifylline andoxypurinolrdquo Biochemical Pharmacology vol 83 no 1 pp 122ndash130 2012

[4] H Milnerowicz M Jabłonowska and A Bizon ldquoChange ofzinc copper and metallothionein concentrations and the cop-per-zinc superoxide dismutase activity in patients with pancre-atitisrdquo Pancreas vol 38 no 6 pp 681ndash688 2009

[5] HWang SWangA TangHGong PMa andLChen ldquoCom-bined effects of sivelestat and resveratrol on severe acutepancreatitis-associated lung injury in ratsrdquo Experimental LungResearch vol 40 no 6 pp 288ndash297 2014

[6] A L Farre and S Casado ldquoHeart failure redox alterations andendothelial dysfunctionrdquoHypertension vol 38 no 6 pp 1400ndash1405 2001

[7] T Petit ldquoAnthracycline-induced cardiotoxicityrdquo Bulletin duCancer vol 91 supplement 3 pp 159ndash165 2004

[8] A Blum ldquoHeart failuremdashnew insightsrdquo Israel Medical Associa-tion Journal vol 11 no 2 pp 105ndash111 2009

[9] S E Aune S T Yeh P KuppusamyM L KuppusamyM Khanand M G Angelos ldquoSivelestat attenuates myocardial reperfu-sion injury during brief low flow postischemic infusionrdquo Oxi-dative Medicine and Cellular Longevity vol 2013 Article ID279847 9 pages 2013

[10] B-Q Cheng C-T Liu W-J Li et al ldquoEthyl pyruvate improvessurvival and ameliorates distant organ injury in rats with severeacute pancreatitisrdquo Pancreas vol 35 no 3 pp 256ndash261 2007

[11] X Du Y Cao P Xue Z Lin Z Zeng and Q Xia ldquoProtectiveeffect of intermedin on myocardial cell in a rat model of severeacute pancreatitisrdquo Cellular and Molecular Biology Letters vol16 no 3 pp 462ndash476 2011

[12] K K Kao and M P Fink ldquoThe biochemical basis for the anti-inflammatory and cytoprotective actions of ethyl pyruvate andrelated compoundsrdquo Biochemical Pharmacology vol 80 no 2pp 151ndash159 2010

[13] B P Soule F Hyodo K-I Matsumoto et al ldquoThe chemistryand biology of nitroxide compoundsrdquo Free Radical Biology andMedicine vol 42 no 11 pp 1632ndash1650 2007

[14] C S Wilcox and A Pearlman ldquoChemistry and antihyperten-sive effects of tempol and other nitroxidesrdquo PharmacologicalReviews vol 60 no 4 pp 418ndash469 2008

[15] C S Wilcox ldquoEffects of tempol and redox-cycling nitroxides inmodels of oxidative stressrdquo Pharmacology andTherapeutics vol126 no 2 pp 119ndash145 2010

[16] E Bartha I Solti A Szabo et al ldquoRegulation of kinase cascadeactivation and heat shock protein expression by poly(ADP-ribose) polymerase inhibition in doxorubicin-induced heartfailurerdquo Journal of Cardiovascular Pharmacology vol 58 no 4pp 380ndash391 2011

[17] W Zhou B A Levine and M S Olson ldquoLipid mediator pro-duction in acute and chronic pancreatitis in the ratrdquo Journal ofSurgical Research vol 56 no 1 pp 37ndash44 1994

[18] M Buyukberber M C Savas C Bagci et al ldquoTherapeutic effectof caffeic acid phenethyl ester on cerulein-induced acute pan-creatitisrdquo World Journal of Gastroenterology vol 15 no 41 pp5181ndash5185 2009

[19] M Lampel and H F Kern ldquoAcute interstitial pancreatitis in therat induced by excessive doses of a pancreatic secretagoguerdquoVirchows Archiv A Pathological Anatomy and Histology vol373 no 2 pp 97ndash117 1977

[20] A Marciniak M Klonowska M Chomicki E Szpringer KLutnicki and A Wojtak ldquoEffects of a low molecular weightsuperoxide dismutase mimic 4-hydroxy-tempo on the lipidperoxidation in burned ratsrdquo Bulletin of the Veterinary Institutein Pulawy vol 49 no 1 pp 101ndash106 2005

[21] S M Hahn A M Deluca D Coffin C M Krishna and J BMitchell ldquoIn vivo radioprotection and effects on blood pressureof the stable free radical nitroxidesrdquo International Journal ofRadiation Oncology Biology Physics vol 42 no 4 pp 839ndash8421998


[22] M Leach S Frank A Olbrich J Pfeilschifter and C Thiemer-mann ldquoDecline in the expression of copperzinc superoxide dis-mutase in the kidney of rats with endotoxic shock effects of thesuperoxide anion radical scavenger tempol on organ injuryrdquoBritish Journal of Pharmacology vol 125 no 4 pp 817ndash8251998

[23] MM Lerch PHoppe-Seyler andWGerok ldquoOrigin and devel-opment of exocrine pancreatic insufficiency in experimentalrenal failurerdquo Gut vol 35 no 3 pp 401ndash407 1994

[24] S Tani M Otsuki H Itoh et al ldquoHistologic and biochemicalalterations in experimental acute pancreatitis induced by supra-maximal caerulein stimulationrdquo International Journal of Pancre-atology vol 2 no 5-6 pp 337ndash348 1987

[25] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996

[26] I Erdelmeier D Gerard-Monnier K Regnard NMoze-HenryJ-C Yadan and J Chaudiere ldquoReactions of 1-methyl-2-phenyl-indole with malondialdehyde and 4-hydroxyalkenals Analyti-cal applications to a colorimetric assay of lipid peroxidationrdquoChemical Research in Toxicology vol 11 no 10 pp 1176ndash11831998

[27] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[28] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[29] J Y Song J W Lim H Kim T Morio and K H Kim ldquoOxi-dative stress induces nuclear loss of DNA repair proteins Ku70and Ku80 and apoptosis in pancreatic acinar AR42J cellsrdquoJournal of Biological Chemistry vol 278 no 38 pp 36676ndash36687 2003

[30] H Kasai and K Kawai ldquoOxidative DNA damage mechanismsand significance in health and diseaserdquo Antioxidants amp RedoxSignaling vol 8 no 5-6 pp 981ndash983 2006

[31] F M Yilmaz G Yilmaz M F Erol S Koklu and D YucelldquoNitric oxide lipid peroxidation and total thiol levels in acuteappendicitisrdquo Journal of Clinical Laboratory Analysis vol 24 no2 pp 63ndash66 2010

[32] B Yegneswaran J B Kostis and C S Pitchumoni ldquoCardiovas-cular manifestations of acute pancreatitisrdquo European Journal ofEmergency Medicine vol 6 no 1 pp 27ndash29 1999

[33] R Pezzilli B Barakat P Billi and B Bertaccini ldquoElectrocardio-graphic abnormalities in acute pancreatitisrdquo European Journalof Emergency Medicine vol 6 no 1 pp 27ndash29 1999

[34] A Rubio-Tapia J Garcıa-Leiva E Asensio-Lafuente G Robles-Dıaz and F Vargas-Vorackova ldquoElectrocardiographic abnor-malities in patients with acute pancreatitisrdquo Journal of ClinicalGastroenterology vol 39 no 9 pp 815ndash818 2005

[35] C A Albrecht and F A Laws ldquoST segment elevation pattern ofacutemyocardial infarction induced by acute pancreatitisrdquoCar-diology in Review vol 11 no 3 pp 147ndash151 2003

[36] J H Yu J W Lim H Kim and K H Kim ldquoNADPH oxidasemediates interleukin-6 expression in cerulein-stimulated pan-creatic acinar cellsrdquoThe International Journal of Biochemistry ampCell Biology vol 37 no 7 pp 1458ndash1469 2005

[37] J H Yu andH Kim ldquoOxidative stress and inflammatory signal-ing in cerulein pancreatitisrdquoWorld Journal of Gastroenterologyvol 20 no 46 pp 17324ndash17329 2014

[38] D B Gough B Boyle W P Joyce et al ldquoFree radical inhibitionand serial chemiluminescence in evolving experimental pancre-atitisrdquo British Journal of Surgery vol 77 no 11 pp 1256ndash12591990

[39] T Reinheckel J Prause B Nedelev et al ldquoOxidative stressaffects pancreatic proteins during the early pathogenesis of ratcaerulein pancreatitisrdquo Digestion vol 60 no 1 pp 56ndash62 1999

[40] P Kruse M E Anderson and S Loft ldquoMinor role of oxidativestress during intermediate phase of acute pancreatitis in ratsrdquoFree Radical Biology and Medicine vol 30 no 3 pp 309ndash3172001

[41] A Dąbrowska D Jacewicz A Łapinska et al ldquoPivotal par-ticipation of nitrogen dioxide in L-arginine induced acutenecrotizing pancreatitis protective role of superoxide scavenger4-OH-TEMPOrdquo Biochemical and Biophysical Research Commu-nications vol 326 no 2 pp 313ndash320 2005

[42] Z Sledzinski M Wozniak J Antosiewicz et al ldquoProtectiveeffect of 4-hydroxy-TEMPO a lowmolecularweight superoxidedismutase mimic on free radical toxicity in experimentalpancreatitisrdquo International Journal of Pancreatology vol 18 no2 pp 153ndash160 1995

[43] S Cuzzocrea M C McDonald E Mazzon et al ldquoTempol amembrane-permeable radical scavenger reduces dinitroben-zene sulfonic acid-induced colitisrdquo European Journal of Phar-macology vol 406 no 1 pp 127ndash137 2000

[44] S Cuzzocrea M C McDonald H M Filipe et al ldquoEffects oftempol a membrane-permeable radical scavenger in a rodentmodel of carrageenan-induced pleurisyrdquo European Journal ofPharmacology vol 390 no 1-2 pp 209ndash222 2000

[45] HMurakami ANakaoWKishimotoMNakano andH Tak-agi ldquoDetection of O

2

minus generation and neutrophil accumulationin rat lungs after acute necrotizing pancreatitisrdquo Surgery vol118 no 3 pp 547ndash554 1995

[46] A Szuster-Ciesielska J Daniluk and M Kandefer-SzerszeldquoOxidative stress in blood of patients with alcohol-relatedpancreatitisrdquo Pancreas vol 22 no 3 pp 261ndash266 2001

[47] N Tsuji N Watanabe T Okamoto and Y Niitsu ldquoSpecificinteraction of pancreatic elastase and leucocytes to produceoxygen radicals and its implication in pancreatitisrdquoGut vol 35no 11 pp 1659ndash1664 1994

[48] G Chen F Xu J Li and S Lu ldquoDepletion of neutrophils pro-tects against l-arginine-induced acute pancreatitis inmicerdquoCel-lular Physiology and Biochemistry vol 35 no 6 pp 2111ndash21202015

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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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

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


which may be attributed in part to ROSRNS and inflam-matory cytokines overproduction [9ndash12] Thus cytopro-tectiveantioxidant treatment may prevent development ofcardiac injury at the early phase of AP

4-Hydroxy-2266-tetramethylpiperidine-1-oxyl (tempol)is a membrane-permeable aminoxyl-type free radical scav-enger with unique antioxidant properties This compoundexhibits a superoxide dismutase mimetic action to degradesuperoxide radical thereby preventing the formation ofperoxynitrite The advantageous effect of aminoxyl action isalso caused by inhibiting Fentonrsquos reaction catalyzed by Fe2+ions [13] Preclinical studies using a variety of rat modelshave shown the potential for many clinical applicationsincluding control of hypertension andweight gain protectionagainst ionizing radiation and DNA damage and preventionof doxorubicin treatment-related cardiomyopathy [13ndash16]

Therefore the aim of the present study was to evaluate theoxidative stress in the pancreas and the heart and to estimatethe protective effects of tempol at the early phase of cerulein-induced AP

2 Materials and Methods

21 Reagents Thereagents hydrochloric acid ferric chloridesodium acetate glacial acetic acid and ferrous sulphatewere of analytical or HPLC grade 246-Tris(2-pyridyl)-s-triazine (TPTZ 99) cerulein and 4-hydroxy-TEMPOwere purchased from Sigma-Aldrich Co (St Louis MOUSA) Hydrochloric acid sodium acetate sodium carbonateiron(III) chloride hexahydrate (FeCl

3sdot6H2O) iron(II) sulfate

heptahydrate (FeSO4sdot7H2O) and methanol (998) were

obtained from POCH (Gliwice Poland) Redistilled waterwas used for the preparation of solutions

22 Animals and Experimental Model The study was carriedout on 24 male Wistar rats 8ndash10 weeks old weighing 180ndash200 g and obtained from a licensed breeder The study wasapproved by the Ethical Committee for Animal Experimen-tation of the Medical University of Lublin (Licence number136) The rats were kept on standard rat chow and fastedovernight before the experiment with water ad libitum

Acute pancreatitis was induced as previously described[17ndash19] with minor modifications Four subcutaneous injec-tions of cerulein (15 120583g kgminus1 body weight) or vehicles (normalsaline) were given consecutively at hour intervals

Rats were randomly divided into four groups (119899 = 6 pergroup) Group I (control) Group II (tempol) treated withtempol (given in the 1st 2nd and 3rd hours after the firstsaline injection) Group III (cerulein) cerulein-induced pan-creatitis without treatment and Group IV (tempol + ceru-lein) cerulein-induced pancreatitis treated with tempol (3 times100mgkg given in the 1st 2nd and 3rd hours after the firstcerulein injection)

We used three multiple injections of tempol to providea maximal protective effect against cerulein-induced pan-creatitis The dose of tempol was chosen on the basis of aprevious animal studies that showed that similar high doseswere well tolerated [13 20ndash22]

After 6 hours the rats were anaesthetized with thiopentalsodium (50mgkg) the abdominal cavity was opened andblood was collected from the abdominal aorta into tubescontaining EDTA or serum-separating gel The blood wascentrifuged at 3000timesg for 10min Plasma and serum werefrozen and kept at minus80∘C until assayed After blood with-drawal the pancreas and heart were carefully dissected outrinsed with saline blotted on paper weighed and dividedinto samples For biochemical analyses tissue blocks of bothpancreas and heart were snap-frozen in liquid nitrogen andstored at minus80∘C before homogenate preparation

The specific parameters that were evaluated to assess thedegree of pancreatic inflammation included serum amylaselevels pancreatic water content and histological characteris-tics of pancreatic tissue (by fixation and staining with hema-toxylin and eosin (HampE) followed by examination by lightmicroscopy) Plasma creatine kinase isoenzyme (CK-MB)and markers for oxidative damage ferric reducing ability ofplasma (FRAP) andmalondialdehyde and 4-hydroxynonenal(MDA + 4-HNE) and total sulfhydryl (ndashSH) groups in pan-creas and heart were determined

23 Measurement of Parameters for Evaluating AP

231 Determination of Pancreatic Water Content The watercontent of the pancreas was used as an index of pancreaticedema It was quantified by weighing the freshly harvestedtissue (wet weight) and compared with the weight of the samesample after desiccation at 160∘C for 48 h (dry weight) [23]The results were calculated and expressed as a percentage((wet weight minus dry weight) times 100wet weight)

232 SerumAmylase Serum amylase wasmeasured by stan-dard photometric reaction with a commercially available kit(Alpha Diagnostics) using a Shimadzu spectrophotometer(Shimadzu Corporation Japan) according to the manufac-turerrsquos instructions

233 Histology The pancreas was immediately rinsed andfixed in 10 buffered formalin solution Pancreatic scrapeswere embedded in paraffin cut and stained with hematoxylinand eosin (HampE) and graded in a blinded manner Forquantification of edema vacuolization inflammation (neu-trophil infiltration) and necrosis a modification of the scoreoriginally described by Tani et al [24] was used ranging from0 to 3 (edema vacuolization inflammation and necrosis)

24 Indices of Oxidative Stress

241 The Antioxidant Capacity of Plasma The antioxidantcapacity of plasma samples was determined by the ferric-reducing antioxidant power (FRAP) assay of Benzie andStrain [25] This method measures the ability of the antiox-idants contained in a sample to reduce ferric to ferrous ionwhich at low pH forms a coloured ferrous-tripyridyltriazinecomplex that absorbs light at 593 nm







3 Results



Edema(0ndash3)



Necrosis(0ndash3)




0

20

40

60

80

100

120

140

0

3000

6000

9000

12000

15000


Panc

reat

ic w

ater

cont

ent (

)

Seru

m am

ylas

e (U

L)

AmylaseEdema

Tempol +cerulein

lowastlowast

lowast

lowast







(a) (b)

(c) (d)


020406080100120140

00

02

04

06

08


-SHMDA+HNE

SH g

roup

s(n

mol

mg

prot

ein)

MD

A+4

-HN

E(n

mol

mg

prot

ein)

lowast

lowast



04080120160200240

0005101520


lowast

lowast

Tempol +cerulein

SH g

roup

s(n

mol

mg

prot

ein)

MD

A+4

-HN

E(n

mol

mg

prot

ein)

-SHMDA+HNE





0

100

200

300

400

500


FRA

P (120583

mol

L)

Tempol +cerulein

lowast

lowast


0

300

600

900

1200


CK-M

B le

vels

(UL

)

lowast

lowast



4 Discussion









5 Conclusions




References















































2










of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Results



Edema(0ndash3)



Necrosis(0ndash3)




0

20

40

60

80

100

120

140

0

3000

6000

9000

12000

15000


Panc

reat

ic w

ater

cont

ent (

)

Seru

m am

ylas

e (U

L)

AmylaseEdema

Tempol +cerulein

lowastlowast

lowast

lowast







(a) (b)

(c) (d)


020406080100120140

00

02

04

06

08


-SHMDA+HNE

SH g

roup

s(n

mol

mg

prot

ein)

MD

A+4

-HN

E(n

mol

mg

prot

ein)

lowast

lowast



04080120160200240

0005101520


lowast

lowast

Tempol +cerulein

SH g

roup

s(n

mol

mg

prot

ein)

MD

A+4

-HN

E(n

mol

mg

prot

ein)

-SHMDA+HNE





0

100

200

300

400

500


FRA

P (120583

mol

L)

Tempol +cerulein

lowast

lowast


0

300

600

900

1200


CK-M

B le

vels

(UL

)

lowast

lowast



4 Discussion









5 Conclusions




References















































2










of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)


020406080100120140

00

02

04

06

08


-SHMDA+HNE

SH g

roup

s(n

mol

mg

prot

ein)

MD

A+4

-HN

E(n

mol

mg

prot

ein)

lowast

lowast



04080120160200240

0005101520


lowast

lowast

Tempol +cerulein

SH g

roup

s(n

mol

mg

prot

ein)

MD

A+4

-HN

E(n

mol

mg

prot

ein)

-SHMDA+HNE





0

100

200

300

400

500


FRA

P (120583

mol

L)

Tempol +cerulein

lowast

lowast


0

300

600

900

1200


CK-M

B le

vels

(UL

)

lowast

lowast



4 Discussion









5 Conclusions




References















































2










of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

300

400

500


FRA

P (120583

mol

L)

Tempol +cerulein

lowast

lowast


0

300

600

900

1200


CK-M

B le

vels

(UL

)

lowast

lowast



4 Discussion









5 Conclusions




References















































2










of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusions




References















































2










of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































2










of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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