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signicantly lower than those in the group 2. TAC inliver tissue was significantly higher in group 3 thanin group 2. TOS, OSI and MPO in hepatic tissue weresignicantly lower in group 3 than the group 2. Histo-

logical tissue damage was milder in the NS treatmentgroup than that in the control group.CONCLUSION:Our results suggest that Nigella sa-tivatreatment protects the rat liver against to hepaticischemia-reperfusion injury.

2008 The WJG Press. All rights reserved.

Key words:Nigella sativa; Ischemia reperfusion injury;Liver

Peer reviewers: Valentin Fuhrmann, MD, Department ofInternal Medicine 4, Intensive Care Unit, Medical University

Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria;Andrej Khandoga, MD, Institute for Surgical Research Ludwig-

Maximilians-University of Munich, Marchioninistr. 27, Munich

81377, Germany

Yildiz F, Coban S, Terzi A, Ates M, Aksoy N, Cakir H, Ocak

AR, Bitiren M. Nigellasativa relieves the deleterious effects

of ischemia reperfusion injury on liver. World J Gastroenterol

2008; 14(33): 5204-5209 Available from: URL: http://www.

wjgnet.com/ 1007-9327/14/ 5204.asp DOI: http:// dx.doi.

org/10.3748/wjg.14.5204

INTRODUCTION

Ischemia followed by reperfusion (I/R) may cause meta-bolic and structural hepatic damage, and may be due totrauma, sepsis, liver transplantation[1]or hepatic pedicleclamping during liver surgery[2]. This remains a signi-cant problem for surgical procedures, and also remainslimitation of liver transplantation[3].

Oxygen free radicals, produced on reperfusion, play acritical role in the injury caused by ischemia-reperfusion[4].Reactive oxygen radicals lead to an inammatory response

and tissue damage by activating some mediators. It can

also directly damage cell components[5]

. Several attemptsto reduce these mechanisms have been reported in theliterature. Protection against reperfusion injury can be in-duced by assorted treatments including administration ofantioxidants and anti-inammatory drugs[4,6-8].

Fahrettin Yildiz, Alpaslan Terzi,Harran University MedicalFaculty, Department of General Surgery, Sanli Urfa 63300,

Turkey

Sacit Coban, Gaziantep University Medical Faculty, Department

of General Surgery, Gaziantep, 27310, TurkeyMustafa Ates, Malatya State Hospital Department of GeneralSurgery, Malatya 44100, Turkey

Nurten Aksoy, Hale Cakir, Ali Riza Ocak, Harran UniversityMedical Faculty, Department of Biochemistry, Sanli Urfa

63300, Turkey

Muharrem Bitiren, Harran University Medical FacultyDepartment of Pathology, Sanli Urfa 63300, Turkey

Author contributions:Yildiz F, Coban S and Terzi A contributedequally to this work; Yildiz F, Coban S, Terzi A and Ates M

designed research; Yildiz F, Coban S and Terzi A performed

research; Aksoy N, Cakir H and Ocak AR analyzed biochemical

data; Bitiren M evaluated the histopathology, and Yildiz F wrote

the paper.

Correspondence to: Dr. Fahrettin Yildiz, Assistant Professor,Harran University Medical Faculty, Department of GeneralSurgery, Sanliurfa 63300, Turkey. fahrettinyildiz@hotmail.com

Telephone:+90-414-3148410 Fax:+90-414-3148410Received:July 14, 2008 Revised:August 18, 2008

Accepted:August 25, 2008

Published online:September 7, 2008

Abstract

AIM: To determine whether Nigella sativa prevents

hepatic ischemia-reperfusion injury to the liver.

METHODS:Thirty rats were divided into three groupsas sham (Group 1), control (Group 2), and Nigella sati-va(NS) treatment group (Group 3). All rats underwenthepatic ischemia for 45 min followed by 60 min periodof reperfusion. Rats were intraperitoneally infused withonly 0.9% saline solution in group 2. Rats in group 3received NS (0.2 mL/kg) intraperitoneally, before isch-emia and before reperfusion. Blood samples and livertissues were harvested from the rats, and then therats were sacriced. Serum aspartate aminotransfera-se (AST), alanine aminotransferase (ALT), and lactatedehydrogenase (LDH) levels were determined. Totalantioxidant capacity (TAC), catalase (CAT), total oxida-

tive status (TOS), oxidative stress index (OSI) and my-

eloperoxidase (MPO) in hepatic tissue were measured.

Also liver tissue histopathology was evaluated by lightmicroscopy.RESULTS:The levels of liver enzymes in group 3 were

Nigella sativa relieves the deleterious effects of ischemia

reperfusion injury on liver

Fahrettin Yildiz, Sacit Coban, Alpaslan Terzi, Mustafa Ates, Nurten Aksoy, Hale Cakir, Ali Riza Ocak,

Muharrem Bitiren

www.wjgnet.com

RAPID COMMUNICATION

Online Submissions: wjg.wjgnet.com World J Gastroenterol 2008 September 7; 14(33): 5204-5209

wjg@wjgnet.com World Journal of GastroenterologyISSN 1007-9327

doi:10.3748/wjg.14.5204 2008 The WJG Press. All rights reserved.

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Yildiz Fet al. Nigella sativafor liver ischemia reperfusion 5205

Various therapeutic effects, such as antioxidant, anti-inammatory, anticancer[9], antihistaminic[10], antibacterialeffects[11]have been described forNigellasativa. Addition-ally, it has been shown that Nigella sativahas protectiveeffect against ischemia reperfusion injury to various

organs

[12-14]

. Thymoquinone, the active constituent ofNigellasativaseeds, is a pharmacologically active quinone,which possesses several properties including analgesicand anti-inflammatory actions[15]. It has been reportedthat thymoquinone prevents oxidative injury in various invitroand in vivostudies in rats[16,17]. It has been suggestedthat thymoquinone may act as an antioxidant agent andprevents membrane lipid peroxidation in tissues[18]. Themechanism of action is still largely unknown. But, itseems these effects may be related to inhibition of eico-sanoid generation, namely thromboxane B2 and leucotri-enes B4 (by inhibiting cyclooxygenase and 5-lipooxygen-ase, respectively), and membrane lipid peroxidation[13].

Moreover, it has been demonstrated that Nigella sa-tivacan signicantly prevent hepatotoxicity[19]and mighthave protective effects against nephrotoxicity inducedby either disease or chemicals[13]. But, the exact mecha-nism is not clear. There are also several clinical studies.In one study, the prophylactic effect of boiled extract of

N. sativaon asthmatic disease was examined[20]. Similarly,black seed oil was shown to be an effective adjuvant forthe treatment of patients with allergic diseases[21]. In an-other clinical study, signicant benets of Nigella sativaextract in the treatment of acute tonsillopharyngitis wasshown[22]. Also, it was shown that Nigellasativahas anti-

epileptic effects in children with refractory seizures[23]

.Therefore, it seems possib le that the administra-

tion of Nigella sativamight protect the liver against theischemia reperfusion injury; therefore, our aim was toconfirm this hypothesis. We investigated alterations inthe oxidant- antioxidant balance by measuring oxidantparameters such as total oxidative status (TOS), oxida-tive stress index (OSI) and myeloperoxidase (MPO), andantioxidant parameters, such as total antioxidant capac-ity (TAC) and catalase (CAT) in the liver tissue. Also weexamined histopathological changes in the liver paren-chyma.

MATERIALS AND METHODS

Thirty male Wistar-albino rats weighting 190 g to 250 gwere used in this experimental study. All animals weremaintained under standard conditions, and were fed wa-ter and rodent chow adlibitumand treated in compliancewith the National Institutes of Health guidelines. Ratswere deprived of food, but not water, for 24 h beforesurgery.

Animals were divided into three groups, sham group(Group 1), control group (Group 2), and Nigella sativatreatment group (Group 3). All rats were anesthetized

with 0.2 mL/100 g of ketamine hydrochloride intraperi-toneally. After the abdomen was shaved and disinfected,a midline incision was made and rats underwent eithersham surgery or ischemia-reperfusion. Ischemia was car-ried out by exposing the afferent and efferent blood ves-

sels and then clamping for 45 min with a microvascularbulldog clamp. Forty ve minutes later, the ischemicliver was reperfused by opening the clamp, and reperfu-sion was achieved for 60 min. Nigellasativawas given tothe rats in treatment group, before ischemia and before

reperfusion at a dose of 0.2 mL/kg by intraperitonealroute. We chose the dose of this agent according to re-ported studies about I/R and Nigella sativa, as this dosehas been shown to be effective in previous studies[24,25].Rats in the control group were infused only with saline.At the end of the procedures, the rats were killed andblood and liver tissue samples were obtained. A portionof liver was stored at -80for future analyses. Plasmaalanine aminotransferase (ALT), aspartate aminotrans-ferase (AST), and lactate dehydrogenase (LDH) activi-ties were measured for evaluating the liver functions. Toassess oxidative injury, TAC, TOS and OSI levels weredetermined.

OSI and MPO levelsThe enzyme analyses of liver tissue were performed onthe supernatant fractions of the tissue homogenates.The tissues were homogenized in 150 mmol/L ice-coldKCl to make a 10% homogenate, using a glass Teon

homogenizer. Then, the samples were sonicated on iceten times for 5 s. The homogenates were centrifuged at12 500gfor 30 min at 3, and the supernatant fractionswere obtained.

Tissue samp les for histological staining we reobtained and xed in 10% formalin-phosphate-buffered

saline at 4overnight. The samples were dehydratedand embedded in paraffin. Sections (7 m) were cutand stained with hematoxylin and eosin. A pathologistevaluated the slides in a blinded manner.

Biochemical analysesPlasma was used to measure AST, ALT and LDH as indi-cative parameters of hepatic function. The plasma activi-ties of AST, ALT and LDH were estimated by commer-cially available kits using an autoanalyser (aerosetAbbottLaboratories, Chicago, IL ).

Measurement of the total antioxidant capacityTAC of supernatant fractions was determined using anovel automated measurement method developed byErel[26]. In this method, hydroxyl radical, which is themost potent biological radical, is produced. In the assay,ferrous ion solution, which is present in Reagent 1, ismixed with hydrogen peroxide, which is present in Rea-gent 2. The sequential produced radicals such as browncolored dianisidinyl radical cation, produced by the hyd-roxyl radical, are also potent radicals. Using this method,antioxidative effect of the sample against the potent-freeradical reactions, which is initiated by the produced hyd-roxyl radical, is measured. The assay has excellent preci-

sion values, lower than 3%. The results are expressed asnmol Trolox Equiv./mg protein.

Measurement of total oxidant statusTOS of supernatant fractions was determined using a

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novel automated measurement method, developed byErel[27]. Oxidants present in the sample oxidize the fer-rous ion-o-dianisidine complex to ferric ion. The oxidati-on reaction is enhanced by glycerol molecules, which areabundantly present in the reaction medium. The ferric

ion makes a colored complex with xylenol orange in anacidic medium. The color intensity, which can be measu-red spectrophotometrically, is related to the total amountof oxidant molecules present in the sample. The assayis calibrated with hydrogen peroxide and the results areexpressed in terms of nmol H2O2Equiv./mg protein.

Oxidative stress indexPercent ratio of TOS level to TAC level was acceptedas OSI. OSI value was calculated according to the fol-lowing formula[28]: OSI (Arbitrary Unit) = TOS (nmolH2O2Equiv./mg protein)/TAC (nmol Trolox Equiv./mgprotein).

Determination of myeloperoxidase activityThe MPO (EC 1.11.1.7) activity was determined, usinga 4-aminoantipyrine/phenol solution as the substratefor MPO-mediated oxidation by H2O2 and changes inabsorbance at 510 nm were recorded [29]. One unit ofMPO activity was dened as that which degraded 1 mol

H2O2/min at 25. The results were expressed as mU/gprotein.

Determination of catalase activityLiver catalase activity was determined by Goths colori-

metric method, in which supernatant was incubated inH2O2 substrate, and the enzymatic reaction stopped bythe addition of ammonium molybdate. The intensity ofthe yellow complex formed by molybdate and H2O2wasmeasured at 405 nm[30].

Histopathologic evaluationLiver tissues were embedded in paraffin, cut into 3 to5-m sections, and mounted. After deparaffinization,the tissues were stained with hematoxylin and eosin (HE)for histological examination. Histological examinationwas performed by a pathologist.

Statistical analysisFor statistical analyses, nonparametric independentgroup comparisons were made. For multiple compari-sons, the Kruskal-Wallis was used for comparisons be-tween groups and the Mann-Whitney test used if anystatistical significance was found. A level of 5% (P