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Effect of Neferine on Liver Ischemia-Reperfusion Injury in Rats

J. Wang, Q. Kan, J. Li, X. Zhang, and Y. Qi

ABSTRACT

Introduction. Liver ischemia/reperfusion leads to the formation of reactive oxygenspecies (ROS) that cause liver injury, a critical clinical problem during liver surgery andtransplantation. The aim of the present study was to investigate the hepatoprotective andantioxidant effects of neferine against liver ischemia/reperfusion injury in rats.Materials and Methods. Wistar rats were randomly divided into 4 groups (n � 8): shamgroup; model group, and neferine high and low groups (50 and 25 mg/kg, respectively).After either saline or neferine was orally administered for 5 days rat livers were subjectedto 30 minutes of ischemia followed by 6 hours of reperfusion. Aspartate aminotransferase(AST), alanine aminotransferase (ALT), and hydroxyl radical levels were measured inserum. The liver was removed to assay malondialdehyde (MDA) and carbonyl contents,superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities, as well as toevaluate histopathologic changes.Results. Neferine significantly prevented AST and ALT elevations, reduced hydroxylradical release, inhibited SOD and GPx activities, and decreased MDA and carbonylcontents. At the same time, neferine attenuated the histopathologic changes.Conclusion. Neferine protected against liver ischemia/reperfusion in rats through anti-oxidant mechanisms. However, further studies are needed to verify whether the hepato-

protection of neferine is correlated with anti-inflammatory and anti-apoptotic effects.

SEVERE liver dysfunction and injury resulting fromischemia/reperfusion is a common clinical situation in

patients with some liver diseases and associated with ther-apies such as liver transplantation, resection, and trauma.1,2

Various biological reactions lead to the production ofreactive oxygen species (ROS) during ischemia/reperfusion.Accumulation of ROS may easily overcome endogenousanti-oxidative systems, such as superoxide dismutase(SOD), catalase (CAT), glutathione peroxidase (GPx), andglutathione (GSH), because they exist in relatively lowconcentrations in the liver. Generated ROS, in turn, pro-voke damage to lipids, proteins eventually leading to liverdamage.3,4 For these reasons, treatment with exogenousantioxidants, particularly in the early stages of reperfusion,markedly reduces liver ischemia/reperfusion injury.

The embryos of Nelumbo nucifera Gaertner seeds are usedommonly in Chinese traditional medicine for nervous disor-ers, insomnia, cough, and cardiovascular diseases.5 Neferine

(Fig 1), a bisbenzylisoquinoline alkaloid, has been isolatedfrom these embryos.6 Neferine has a variety of pharmacolog-ical activities, such as anti-amnesic, anti-depressant, anti-

arrhythmia, anti-hypertensive, and anti-diabetic effects.7–11

0041-1345/11/$–see front matterdoi:10.1016/j.transproceed.2011.04.013

2536

Jung et al pointed out that the anti-amnesic effect ofneferine may be mediated via its antioxidant actions. How-ever, there is no report about the effects of neferine on liverischemia/reperfusion.7

In the present study, we sought to investigate whetherneferine protected the liver against ischemia/reperfusionin rats. Thus, we determined serum aspartate amino-transferase (AST), alanine aminotransferase (ALT) hy-droxyl radical content, SOD GPx activities and livertissue malondialdehyde (MDA) and carbonyl levels aswell as histopathological changes.

From the First Affiliated Hospital of Zhengzhou University,Zhengzhou, Henan Province, People’s Republic of China.

The first author is Juan Wang.Address reprint requests to Prof. Quancheng Kan, the First

Affiliated Hospital of Zhengzhou University, Zhengzhou,450003, Henan Province, People’s Republic of China. E-mail:

hqc2011@sina.com

© 2011 by Elsevier Inc. All rights reserved.360 Park Avenue South, New York, NY 10010-1710

Transplantation Proceedings, 43, 2536–2539 (2011)

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EFFECT OF NEFERINE 2537

MATERIALS AND METHODSAnimals

Male Wistar rats (240 � 20 g) were housed in an air-conditionedoom with 12-hour light and 12-hour dark cycles, with constantemperature (23 � 2°C) and relative humidity (65%–70%). Allxperimental protocols were approved by our Committee fornimal Research. Rats were randomly divided into 4 groups (n �

): sham group; model group; and neferine high and low groups (50ersus 25 mg/kg). Neferine was administered once a day for 5 daysefore the ischemic event.

Surgery

Animals anesthetized with intraperitoneal ketamine (40 mg/kg)and xylazine (10 mg/kg) underwent a midline laparotomy usingminimal dissection. Total hepatic ischemia was induced for 30minutes by clamping the hepatic artery, portal vein, and bile ductusing a vascular clamp. Thereafter the liver was reperfused for 6hours by removing the clamp. Sham group rats only underwent amidline laparotomy.12

Serum Assays

Blood samples were centrifuged at 1000 rpm for 10 minutes; theserum samples were stored at �80°C. The AST and ALT levels asindicators of liver ischemia/reperfusion injury, were measuredusing commercial kits (Jiancheng Institute of Biotechnology, Nan-jing, China). The formation of methylguanidine, an index ofhydroxyl radical production, was determined using a spectrofluori-metric method.

Tissue Assays

Liver tissues homogenized in cold normal saline were centrifugedat 3000 rpm for 10 minutes. The supernates were used to determineSOD and GPx activities, following the commercial kit instructions(Jiancheng Institute of Biotechnology).

Tissue MDA content was measured by the method of Ohkawa etal,13 with small modifications: 2.5 mL trichloroacetic acid (20%)and 1.0 mL thiobarbituric acid (0.6%) were added to 0.5 mL liverhomogenate in a centrifuge tube before the reaction mixture was

Fig 1. The formula of neferine (C38H44N2O6).

heated for 30 minutes in a boiling water bath. After cooling, 4.0 mL m

n-butanol added to the mixture was vortexed for 1 minute beforecentrifugation at 3000 rpm for 10 minutes. The organic layerabsorbance was determined at 535 nm. Results were expressed asnmol/mg protein.

Protein carbonyl content was determined according to themethod described by Levine et al.14 Supernatant aliquots wereincubated with 1% streptomycin sulfate solution before centrifu-gation at 3000 rpm for 10 minutes. The precipitate was incubatedwith 2,4-dinitrophenylhydrazine (DNPH; 10 mmol/L) in HCl (2mol/L) and vortexed every 10 minutes. The protein was precipi-tated by adding an equal volume of 20% trichloroacetic acid. Aftercentrifugation at 10000 rpm for 5 minutes the pellet was washed 3times with ethanol: ethyl acetate (1:1, V/V) to remove excessDNPH. The precipitated protein redissolved in 6 mol/L guanidinesolution was measured for absorbance at 370 nm. The carbonylcontent, calculated as the extinction coefficient of 22,000 mol/Lcm�1/mg protein, was expressed as nmol/mg protein. The proteinontent was determined according to the Bradford method usingovine serum albumin as the standard.15

Histological Studies

Liver tissues fixed in 10% neutral buffered formalin solution wereembedded in paraffin for 5 �m sections that were stained with

ematoxylin and eosin (H&E). The sections were observed by aathologist unaware of the treatment group using light microscopy.

Statistical Analysis

Statistical analysis was performed using one-way analysis of vari-ance (ANOVA) or the two-tailed unpaired Student t test with SPSSfor Windows (SPSS Inc, Chicago, Ill, United States). A differencewas considered significant at P � .05.

RESULTSAST and ALT Levels

Figure 2 shows the serum marker enzyme levels. Ischemia/reperfusion resulted in a significant increase in AST andALT levels compared with the sham group (P � .01). Oraladministration of neferine at both doses significantly reducedAST and ALT levels (P � .01, and P � .05, respectively).

Fig 2. AST and ALT levels determined after 6 hours of reper-fusion (n � 8). Differences were considered significant at P � .05.P � .05; **P � .01 vs model group; ##P � .01 sham group vs

odel group.

.

2538 WANG, KAN, LI ET AL

Hydroxyl Radical Levels

As compared with the sham group, ischemia/reperfusioncaused an obvious increase in the levels of hydroxyl radicalsin serum (Fig 3). Neferine pretreatment at 50 mg/kgsignificantly decreased hydroxyl radical levels (P � .01).

SOD and GPx Activities

Antioxidant enzyme activities of liver tissue were markedlydecreased among ischemia/reperfusion rats (group 2) com-pared with sham rats (Group 1; P � .01). Neferine (50mg/kg) significantly increased SOD and GPx activitiescompared with the model group (P � 0.01; Fig 4).

Fig 3. Hydroxyl radical level determined after 6 hours of reperfu-sion (n � 8). Differences were considered significant at P � .05.*P � .05; **P � .01 vs model group; ##P � .01 sham group vs model

Fig 4. SOD and GPx activities determined after 6 hours ofreperfusion (n � 8). Differences were considered significant at P �05. *P � .05; **P � .01 vs model group, ##P � .01 sham group vsmodel group.

group.

MDA and Carbonyl Contents

Contents of MDA and carbonyl, 2 markers of liver oxidativeinjury, were increased significantly in the model comparedwith the sham group. Neferine at high doses prevented theliver oxidative damage as judged by decreased MDA andcarbonyl contents (Fig 5).

Histopathologic Changes

In the sham group the liver tissue was normal withouthemorrhage or leukocyte infiltration. Compared with thesham group, ischemia/reperfusion caused severe edema,vascular congestion, hemorrhage, and leukocyte infiltration.Neferine effectively reduced the histopathologic changes(Fig 6).

Fig 5. MDA and carbonyl contents determined after 6 hours ofreperfusion (n � 8). Differences were considered significant atP � .05. *P � .05; **P � .01 vs model group; ##P � .01 shamgroup vs model group.

Fig 6. Representative photomicrographs (H & E stain) of sec-

tions of liver after ischemia/reperfusion.

EFFECT OF NEFERINE 2539

DISCUSSION

When the liver is transiently deprived of oxygen and thenreoxygenated, antioxidant enzyme activities quickly de-crease and the ROS burst attacks a variety of cellularmolecules. The resulting state of oxidative stress contrib-utes to the pathogenesis of liver injury. Liver ischemia/reperfusion damage remains an unavoidable clinical prob-lem in liver surgery, transplantation, shock, and trauma.16,17

Many studies have shown ROS to play a key role in theliver ischemia/reperfusion injury.18 The activity of hydroxylradicals is the highest among all ROS; it causes liverischemia/reperfusion damage. Our results showing reducedhydroxyl radical levels indicated that neferine has thepotential to scavenge ROS.

SOD and GPx have been widely studied among theendogenous antioxidants. SOD catalyzes the dysmutationof superoxide anion to hydrogen peroxide and oxygen, buthydrogen peroxide still produces liver oxidative injury. GPxfurther catalyzes the transformation of hydrogen peroxideto form water.19 Ischemia/reperfusion reduces liver antiox-idant capacity as evidenced by down-regulated activities ofSOD and GPx, as shown in previous reports. However, ourstudy revealed that neferine treatment reversed the de-crease in the SOD and GPx activities, implying that thedrug enhanced liver antioxidant capacity.

Lipid peroxidation and protein oxidation, which areresponsible for cell membrane damage and irreversibleprotein modification, have been implicated in the patho-genesis of ischemia/reperfusion damage.20 In our study, themodel group displayed significantly increased contents ofMDA and carbonyl, 2 main products of oxidative stress.Pretreatment with neferine reduced the MDA and carbonylcontents, which may be attributed to a scavenging of ROSwith enhanced antioxidant enzyme activities.

AST and ALT concentrations are commonly used asindirect biochemical indices of liver injury.21 In the presentstudy, ischemia/reperfusion injury produced significant in-creases in the levels of AST and ALT, probably resultingfrom cell membrane damage. The histopathologic investi-gation suggested that ischemia/reperfusion produced severepathological alterations in liver: edema, vascular congestion,hemorrhage, and leukocyte infiltration. Neferine treatmentimproved the histological changes induced by ischemia/reper-fusion that were attributed to its antioxidant efficacy.

In conclusion, the findings in our present study suggestedthat treatment with neferine attenuated ischemia/reperfusion-induced liver injury. The protective effects of neferine mayrelate to its antioxidant actions, such as clearing ROS andincreasing antioxidant enzyme activities. However, furtherstudies are needed to verify whether the hepatoprotection ofneferine correlates with anti-inflammatory or anti-apoptotic

activities.

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