Polyethylene glycols: An effective strategy for limiting liver ischemia reperfusion injury

Liver ischemia-reperfusion injury(IRI) is an inherent feature of liver surgery and liver transplantation in which damage to a hypoxic organ(ischemia) is exacerbated following the return of oxygen delivery(reperfusion). IRI is a major cause of primary nonfunction after transplantation and may lead to graft rejection, regardless of immunological considerations. The immediate response involves the disruption of cellular mitochondrial oxidative phosphorylation and the accumulation of metabolic intermediates during the ischemic period, and oxidative stress during blood flow restoration. Moreover, a complex cascade of inflammatory mediators is generated during reperfusion, contributing to the extension of the damage and finally to organ failure. A variety of pharmacological interventions(antioxidants, anticytokines, etc.) have been proposed to alleviate graft injury but their usefulness is limited by the local and specific action of the drugs and by their potential undesirable toxic effects. Polyethylene glycols(PEGs), which are non-toxic water-soluble compounds approved by the FDA, have been widely used as a vehicle or a base in food, cosmetics and pharmaceuticals, and also as adjuvants for ameliorating drug pharmacokinetics. Some PEGs are also currently used as additives in organ preservation solutions prior to transplantation in order to limit the damage associated with cold ischemia reperfusion. More recently, the administration of PEGs of different molecular weights by intravenous injection has emerged as a new therapeutic tool to protect liver grafts from IRI. In this review, we summarize the current knowledge concerning the use of PEGs as a useful target for limiting liver IRI.

Role of NF-kappaB in Liver Ischemia Reperfusion Injury of Rats

To determine the role of NF-kappaB in ischemia reperfusion (I/R) injury of the rat liver, rats underwent partial hepatic ischemia and reperfusion. The left and median lobes of the liver were subjected to ischemia for 90 min followed by reperfusion for defined times. NF-kappaB activity was analyzed by electrophoretic mobility shift assay (EMSA). Semiquantitative reverse-transcriptase polymerase chain reaction was used to analyze TNF-α and ICAM-1 mRNA levels. Results showed during liver I/R injury, NF-kappaB activation was induced in a time dependent manner. NF-kappaB was activated within 1h and 2h after the initiation of reperfusion and decreased after 4 h. Messenger RNA expression of TNF-α and ICAM-1 were increased after the reperfusion of 2 h. It was concluded that during hepatic I/R injury, NF-kappaB was activated and could bind to special sequence in the promoters of budget genes, which can up-regulate the expression of TNF-α and ICAM-1 mRNA to result in ischemia reperfusion injury of the rat liver.

Heme oxygenase-1 protects donor livers from ischemia/reperfusion injury:The role of Kupffer cells

AIM:To examine whether heme oxygenase (HO)-1 overexpression would exert direct or indirect effects on Kupffer cells activation, which lead to aggravation of reperfusion injury.METHODS: Donors were pretreated with cobalt protoporphyrin (CoPP) or zinc protoporphyrin (ZnPP), HO-1 inducer and antagonist, respectively. Livers were stored at 4℃ for 24 h before transplantation. Kupffer cells were isolated and cultured for 6 h after liver reperfusion.RESULTS: Postoperatively, serum transaminases were significantly lower and associated with less liver injury when donors were pretreated with CoPP, as compared with the ZnPP group. Production of the cytokines tumor necrosis factor-α and interleukin-6 generated by Kupffer cells decreased in the CoPP group. The CD14 expression levels (RT-PCR/Western blots) of Kupffer cells from CoPP-pretreated liver grafts reduced.CONCLUSION: The study suggests that the potential utility of HO-1 overexpression in preventing ischemia/reperfusion injury results from inhibition of Kupffer cells activation.

Protective effect of curcumin against liver warm ischemia/reperfusion injury in rat model is associated with regulation of heat shock protein and antioxidant enzymes

AIM： To investigate the hypothesis that the protective effects of curcumin in hepatic warm ischemia/reperfusion （I/R） injury are associated with increasing heat shock protein 70 （Hsp70） expression and antioxidant enzyme activity. METHODS： Sixty Sprague-Dawley male rats were randomly divided into sham, I/R, C ＋ I/R groups. The model of reduced-size liver warm ischemia and reperfusion was used. Curcumin （50 mg/kg） was administered by injection through a branch of superior mesenteric vein at 30 min before ischemia in C ＋ I/R group. Five rats were used to investigate the survival during 1 wk after operation in each group. Blood samples and liver tissues were obtained in the remaining animals after 3, 12, and 24 h of reperfusion to assess serum alanine aminotransferase （ALT）, aspartate aminotransferase （AST）, liver tissue NO2- ＋ NO3-, malondialdehyde （MDA） content, superoxide dismutase （SOD）, catalase （CAT）, nitricoxide synthase （NOS） and myeloperoxidase （MPO） activity, HspT0 expression and apoptosis ratio. RESULTS： Compared with I/R group, curcumin pretreatment group showed less ischemia/reperfusioninduced injury. CAT and SOD activity and Hsp70 expression increased significantly. A higher rate of apoptosis was observed in I/R group than in C ＋ I/R group, and a significant increase of MDA, NO2^- ＋ NO3^- and MPO level in liver tissues and serum transaminase concentration was also observed in I/R group compared to C ＋ I/R group. Curcumin also decreased the activity of inducible NO synthase （iNOS） in liver after reperfusion,but had no effect on the level of endothelial NO synthase （eNOS） after reperfusion in liver. The 7 d survival rate was significantly higher in C ＋ I/R group than in I/R group. CONCLUSION： Curcumin has protective effects against hepatic I/R injury. Its mechanism might be related to the overexpression of Hsp70 and antioxidant enzymes.

Fish oil alleviates liver injury induced by intestinal ischemia/reperfusion via AMPK/SIRT-1/autophagy pathway

AIM To evaluate whether fish oil(FO) can protect liver injury induced by intestinal ischemia/reperfusion(I/R) via the AMPK/SIRT-1/autophagy pathway.METHODS Ischemia in wistar rats was induced by superior mesenteric artery occlusion for 60 min and reperfusion for 240 min. One milliliter per day of FO emulsion or normal saline was administered by intraperitoneal injection for 5 consecutive days to each animal. Animals were sacrificed at the end of reperfusion. Blood andtissue samples were collected for analyses. AMPK, SIRT-1, and Beclin-1 expression was determined in lipopolysaccharide(LPS)-stimulated HepG2 cells with or without FO emulsion treatment.RESULTS Intestinal I/R induced significant liver morphological changes and increased serum alanine aminotransferase and aspartate aminotransferase levels. Expression of p-AMPK/AMPK, SIRT-1, and autophagy markers was decreased whereas tumor necrosis factor-α(TNF-α) and malonaldehyde(MDA) were increased. FO emulsion blocked the changes of the above indicators effectively. Besides, in LPS-stimulated HepG2 cells, small interfering RNA(siRNA) targeting AMPK impaired the FO induced increase of p-AMPK, SIRT-1, and Beclin-1 and decrease of TNF-α and MDA. SIRT-1 siRNA impaired the increase of SIRT-1 and Beclin-1 and the decrease of TNF-α and MDA.CONCLUSION Our study indicates that FO may protect the liver against intestinal I/R induced injury through the AMPK/SIRT-1/autophagy pathway.

Protective effect of pyrrolidine dithiocarbamate on liver injury induced by intestinal ischemia-reperfusion in rats

BACKGROUND: The nuclear translocation of transcription factors may be a critical factor in the intracellular pathway involved in ischemia/reperfusion(I/R) injury. The aim of the study was to evaluate the role of nuclear factor-kappa B (NF-κB) in the pathogenesis of liver injury induced by intestinal ischemia/reperfusion (IIR) and to investigate the effect of pyrrolidine dithiocarbamate (PDTC) on this liver injury. METHODS: Male Wistar rats were divided randomly into three experimental groups (8 rats in each): sham operation group (control group); intestinal/reperfusion group(I/R group): animals received 1-hour of intestinal ischemia and 2-hour reperfusion; and PDTC treatment group (PDTC group): animals that received I/R subject to PDTC treatment (100 mg/kg). The histological changes in the liver and intestine were observed, and the serum levels of tumor necrosis factor-α (TNF-α), alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver superoxide dismutase (SOD), and nitrite/nitrate (NO) were measured. The immunohistochemical expression and Western blot analysis of liver NF-κB and intercellular adhesion molecule-1(ICAM-1) were observed. RESULTS: IIR induced liver injury characterized by the histological changes of liver edema, hemorrhage, polymorphonuclear neutrophil (PMN) infiltration, and elevated serum levels of AST and ALT. The serum TNF-α level was significantly higher than that of the control group(P<0.01) and a high level of liver oxidant product was observed (P<0.01). These changes were parallel to the positive expression of NF-κB and ICAM-1. After the administration of PDTC, the histological changes after liver injury were improved; the levels of SOD and NO in the liver were elevated and reduced, respectively (P<0.01). The expressions of ICAM-1 and NF-κB in the liver were weakened (P<0.01). CONCLUSION: NF-κB plays an important role in the pathogenesis of liver injury induced by HR. PDTC, an agent known to inhibit the activation of NF-κB, can reduce and prevent this injury.

Remote ischemic perconditioning prevents liver transplantation-induced ischemia/reperfusion injury in rats: Role of ROS/RNS and e NOS

AIM To investigate the underlying mechanisms of the protective role of remote ischemic perconditioning (RIPerC) in rat liver transplantation. METHODS Sprague-Dawley rats were subjected to sham, orthotopic liver transplantation (OLT), ischemic postconditioning (IPostC) or RIPerC. After 3 h reperfusion, blood samples were taken for measurement of alanine aminotransferase, aspartate aminotransferase, creatinine (Cr) and creatinine kinase-myocardial band (CK-MB). The liver lobes were harvested for the following measurements: reactive oxygen species (ROS), H2O2, mitochondrial membrane potential (Delta psi m) and total nitric oxide (NO). These measurements were determined using an ROS/H2O2, JC1 and Total NOx Assay Kit, respectively. Endothelial NO synthase (eNOS) was analyzed by reverse transcription-polymerase chain reaction (RTPCR) and western blotting, and peroxynitrite was semiquantified by western blotting of 3-nitrotyrosine. RESULTS Compared with the OLT group, the grafts subjected to RIPerC showed significantly improved liver and remote organ functions (P < 0.05). ROS (P < 0.001) including H2O2 (P < 0.05) were largely elevated in the OLT group as compared with the sham group, and RIPerC (P < 0.05) reversed this trend. The collapse of Delta psi m induced by OLT ischemia/reperfusion (I/R) injury was significantly attenuated in the RIPerC group (P < 0.001). A marked increase of NO content and phosphoserine eNOS, both in protein and mRNA levels, was observed in liver graft of the RIPerC group as compared with the OLT group (P < 0.05). I/R-induced 3-nitrotyrosine content was significantly reduced in the RIPerC group as compared with the OLT group (P < 0.05). There were no significant differences between the RIPerC and IPostC groups for all the results except Cr. The Cr level was lower in the RIPerC group than in the IPostC group (P < 0.01). CONCLUSION Liver graft protection by RIPerC is similar to or better than that of IPostC, and involves inhibition of oxidative stress and up-regulation of the PI3K/Akt/eNOS/NO pathway.

Nuclear factor-KB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

AIM： To evaluate the protective effect of NF-kB decoy oligodeoxynucleotides （ODNs） on ischemia/reperfusion （I/R） injury in rat liver graft. METHODS： Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-kB decoy ODNs or scrambled ODNs. NF-kB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-kB activity in the liver graft was analyzed by electrophoretic mobility shift assay （EMSA）. Hepatic mRNA expression of TNF-α, IFN-γ and intercellular adhesion molecule-1 （ICAM-1） were determined by semiquantitative RT-PCR. Serum levels of TNF-α and IFN-γ were measured by enzyme-linked immunosorbent assays （ELISA）. Serum level of alanine transaminase （ALT） was measured using a diagnostic kit. Liver graft myeloperoxidase （MPO） content was assessed. RESULTS： NF-kB activation in liver graft was induced in a time-dependent manner, and NF-kB remained activated for 16 h after graft reperfusion. NF-kB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kB decoy ODNs significantly suppressed NF-kB activation as well as mRNA expression of TNF-α, IFN-γ, and ICAM-1 in the liver graft. The hepatic NF-kB DNA binding activity [presented as integral optical density （IOD） value] in the NF-kB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat （2.16±0.78 vs 36.78 ±6.35 and 3.06±0.84 vs 47.62± 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P〈0.001）. The hepatic mRNA expression level of TNF-α, IFN-γ and ICAM-1 rpresented as percent of β-actin mRNA （%）] in the NF-kB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat （8.31 ±3.48 vs 46.37±10.65 and 7.46± 3.72 vs 74.82±12.25 for hepatic TNF-α mRNA, 5.58±2.16 vs 50.46±9.35 and 6.47±2.53 vs 69.72±13.41 for hepatic IFN-γ mRNA, 6.79 ±2.83 vs 46.23±8.74 and 5.28±2.46 vs 67.44±10.12 for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P〈0.001）. Administration of NF-kB decoy ODNs almost completely abolished the increase of serum level of TNF-α and IFN-γ induced by hepatic ischemia/reperfusion, the serum level （pg/mL） of TNF-α and in the NF-kB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat （42.7±13.6 vs 176.7±15.8 and 48.4±15.1 vs 216.8±17.6 for TNF-α level, 31.5±12.1 vs 102.1±14.5 and 40.2±13.5 vs 118.6±16.7 for IFN-γ level after 4 and 8 h of reperfusion, respectively, P〈0.001）. Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-kB decoy ODNs, the hepatic MPO content （A655） and serum ALT level （IU/L） in the NF-kB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat （0.17±0.07 vs 1.12±0.25 and 0.46±0.17 vs 1.46±0.32 for hepatic MPO content, 71.7±33.2 vs 286.1±49.6 and 84.3±39.7 vs 467.8±62.3 for ALT level after 4 and 8 h of reperfusion, respectively, P〈0.001）. CONCLUSION： The data suggest that NF-kB decoy ODNs protects against I/R injury in liver graft by suppressing NF-kB activation and subsequent expression of proinflammatory mediators.

Nigella sativa relieves the deleterious effects of ischemia reperfusion injury on liver

AIM: To determine whether Nigella sativa prevents hepatic ischemia-reperfusion injury to the liver. METHODS: Thirty rats were divided into three groups as sham (Group 1), control (Group 2), and Nigella sati-va (NS) treatment group (Group 3). All rats underwent hepatic ischemia for 45 min followed by 60 min period of reperfusion. Rats were intraperitoneally infused with only 0.9% saline solution in group 2. Rats in group 3 received NS (0.2 mL/kg) intraperitoneally, before isch-emia and before reperfusion. Blood samples and liver tissues were harvested from the rats, and then the rats were sacrifi ced. Serum aspartate aminotransfera-se (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels were determined. Total antioxidant 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 light microscopy. RESULTS: The levels of liver enzymes in group 3 weresignifi cantly lower than those in the group 2. TAC in liver tissue was significantly higher in group 3 than in group 2. TOS, OSI and MPO in hepatic tissue were signifi cantly lower in group 3 than the group 2. Histo-logical tissue damage was milder in the NS treatment group than that in the control group. CONCLUSION: Our results suggest that Nigella sa-tiva treatment protects the rat liver against to hepatic ischemia-reperfusion injury.

Diazoxide attenuates ischemia/reperfusion injuryvia upregulation of heme oxygenase-1 after liver transplantation in rats

AIM:To evaluate the effects of diazoxide on ischemia/reperfusion(I/R)-injured hepatocytes and further elucidate its underlying mechanisms.METHODS:Male Sprague-Dawley rats were randomized(8 for donor and recipient per group)into five groups:I/R group(4 h of liver cold ischemia followed by 6 h of reperfusion);heme oxygenase-1(HO-1)small interfering RNA(siRNA)group(injection of siRNA via donor portal vein 48 h prior to harvest);diazoxide(DZ) group(injection of DZ via donor portal vein 10 min prior to harvest);HO-1 siRNA+DZ group;and siRNA control group.Blood and liver samples were collected at 6 h after reperfusion.The mRNA expressions and protein levels of HO-1 were determined by reverse transcription polymerase chain reaction and Western blotting,and tissue morphology was examined by light and transmission electron microscopy.Serum transaminases level and cytokines concentration were also measured.RESULTS:We observed that a significant reduction of HO-1 mRNA and protein levels in HO-1 siRNA and HO-1 siRNA+DZ group when compared with I/R group,while the increases were prominent in the DZ group.Light and transmission electron microscopy indicated severe disruption of tissue with lobular distortion and mitochondrial cristae damage in the HO-1 siRNA and HO-1 siRNA+DZ groups compared with DZ group.Serum alanine aminotransferase,aspartate transaminase,tumor necrosis factor-αand interleukin-6 levels increased in the HO-1 siRNA and HO-1 siRNA+DZ groups,and decreased in the DZ group.CONCLUSION:The protective effect of DZ may be induced by upregulation of HO-1.By inhibiting expression of HO-1,this protection pretreated with DZ was abolished.

Intestinal microflora in rats with ischemia/reperfusion liver injury

Objectives: To investigate the intestinal microflora status related to ischemia/reperfusion (I/R) liver injury and explore the possible mechanism. Methods: Specific pathogen free grade Sprague-Dawley rats were randomized into three groups: Control group (n=8), sham group (n=6) and I/R group (n=10). Rats in the control group did not receive any treatment, rats in the I/R group were subjected to 20 min of liver ischemia, and rats in the sham group were only subjected to sham operation. Twenty-two hours later, the rats were sacrificed and liver enzymes and malondialdehyde (MDA), superoxide dismutase (SOD), serum endotoxin,intestinal bacterial counts, intestinal mucosal histology, bacterial translocation to mesenteric lymph nodes, liver, spleen, and kidney were studied. Results: Ischemia/reperfusion increased liver enzymes, MDA, decreased SOD, and was associated with plasma endotoxin elevation in the I/R group campared to those in the sham group. Intestinal Bifidobacteria and Lactobacilli decreased and intestinal Enterobacterium and Enterococcus, bacterial translocation to kidney increased in the I/R group compared to the sham group. Intestinal microvilli were lost, disrupted and the interspace between cells became wider in the I/R group.Conclusion: I/R liver injury may lead to disturbance of intestinal microflora and impairment of intestinal mucosal barrier function,which contributes to endotoxemia and bacterial translocation to kidney.

Nitrite,a novel method to decrease ischemia/reperfusion injury in the rat liver

AIM:To investigate whether nitrite administered prior to ischemia/reperfusion(I/R)reduces liver injury.METHODS:Thirty-six male Sprague-Dawley rats were randomized to 3 groups,including sham operated(n=8),45-min segmental ischemia of the left liver lobe(IR,n=14)and ischemia/reperfusion(I/R)preceded by the administration of 480 nmol of nitrite(n=14).Serum transaminases were measured after 4 h of reperfusion.Liver microdialysate(MD)was sampled in 30-min intervals and analyzed for glucose,lactate,pyruvate and glycerol as well as the total nitrite and nitrate(NOx).The NOx was measured in serum.RESULTS:Aspartate aminotransferase(AST)at the end of reperfusion was higher in the IR group than in the nitrite group(40±6.8μkat/L vs 22±2.6μkat/L,P=0.022).Similarly,alanine aminotransferase(ALT)was also higher in the I/R group than in the nitrite group(34±6μkat vs 14±1.5μkat,P=0.0045).The NOx in MD was significantly higher in the nitrite group than in the I/R group(10.1±2.9μmol/L vs 3.2±0.9μmol/L,P=0.031)after the administration of nitrite.During ischemia,the levels decreased in both groups and then increased again during reperfusion.At the end of reperfusion,there was a tendency towards a higher NOx in the I/R group than in the nitrite group(11.6±0.7μmol/L vs 9.2±1.1μmol/L,P=0.067).Lactate in MD was significantly higher in the IR group than in the nitrite group(3.37±0.18 mmol/L vs 2.8±0.12 mmol/L,P=0.01)during ischemia and the first 30 min of reperfusion.During the same period,glycerol was also higher in the IRI group than in the nitrite group(464±38μmol/L vs 367±31μmol/L,P=0.049).With respect to histology,there were more signs of tissue damage in the I/R group than in the nitrite group,and29%of the animals in the I/R group exhibited necrosis compared with none in the nitrite group.Inducible nitric oxide synthase transcription increased between early ischemia(t=15)and the end of reperfusion in both groups.CONCLUSION:Nitrite administered before liver ischemia in the rat liver reduces anaerobic metabolism and cell necrosis,which could be important in the clinical setting.

Protective effects of L-arginine against ischemia-reperfusion injury in non-heart beating rat liver graft

BACKGROUND: Although the use of non-heart beating donors (NHBDs) could bridge the widening gap between organ demand and supply, its application to liver transplantation is limited due to the high incidence of primary graft loss. Prevention of liver injury in NHBDs will benefit the results of transplantation. This study was conducted to evaluate the protective effects of L-arginine on liver grafts from NHBDs. METHODS: One hundred and four Wistar rats were randomly divided into 7 groups: normal control (n=8) controls 1, 2 and 3 (C-1, C-2, C-3, n=16), and experimental 1, 2 and 3 (E-1, E-2, E-3, n=16). For groups C-1 and E-1, C-2 and E-2, and C-3 and E-3, the warm ischemia time was 0, 30, and 45 minutes, respectively. Liver grafts were flushed with and preserved in 4 degrees C Euro-collins solution containing 1 mmol/L L-arginine for 1 hour in each experimental group. Recipients of each experimental group were injected with L-arginine (10 mg/kg body weight) by tail vein 10 minutes before portal vein reperfusion. Donors and recipients of each experimental control group were treated with normal saline. Then transplantation was performed. At 1, 3, and 24 hours after portal vein reperfusion, blood samples were obtained to determine the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), nitric oxide (NO) and plasma endothelin (ET). At 3 hours after portal vein reperfusion, grafts samples were fixed in 2.5% glutaraldehyde for electron microscopic observation. RESULTS: At I hour after portal vein reperfusion, the levels of NO in groups E-1, E-2, E-3 and C-1, C-2, C-3 were lower, while the levels of plasma ET, serum ALT and AST were higher than those in the normal control group (P<0.05). At 1, 3, and 24 hours, the levels of NO in groups E-1, E-2, E-3 were higher, while the levels of plasma ET, serum ALT and AST were lower than those in the corresponding control groups (C-1, C-2, C-3) (P<0.05). The levels of NO in groups C-2 and C-3 were lower than in group C-1 (P<0.05), and the level of NO in group C-3 was lower than in group C-2 (P<0.05). At 1, 3 and 24 hours, the levels of plasma ET, serum ALT, and AST in groups E-1, E-2, E-3 were lower than those in the corresponding control groups (C-1, C-2, C-3) (P<0.05). The levels of plasma ET, serum ALT, and AST were lower in group C-3 than in groups C-1 and C-2 (P<0.05). Pathological changes in groups E-1, E-2, E-3 were milder than those in the corresponding experimental control groups (C-1, C-2, C-3). CONCLUSIONS: The imbalance between NO and ET plays an important role in the development of ischemia-reperfusion injury of liver grafts from NHBDs. L-arginine can attenuate injury in liver grafts from NHBDs by improving the balance between NO and ET.

Prophylaxis with carnosol attenuates liver injury induced by intestinal ischemia/reperfusion

AIM： To investigate the possible protective effects of carnosol on liver injury induced by intestinal ischemia reperfusion （I/R）. METHODS： Rats were divided randomly into three experimental groups： sham, intestinal I/R and carnosol treatment （n = 18 each）. The intestinal I/R model was established by clamping the superior mesenteric artery for 1 h. In the carnosol treatment group, surgery was performed as in the intestinal I/R group, with intraperitoneal administration of 3 mg/kg carnosol 1 h before the operation. At 2, 4 and 6 h after reperfusion, rats were killed and blood, intestine and liver tissue samples were obtained. Intestine and liver histology was investigated. Serum levels of aspartate aminotransferase （AST）, alanine aminotransferase （ALT） and interleukin （IL）-6 were measured. Liver tissue superoxide dismutase （SOD） and myeloperoxidase （IvIPO） activity were assayed. The liver intercellular adhesion molecule-1 （ICAM-1） and nuclear factor κB （NF-κB） were determined by immunohistochemical analysis and western blot analysis. RESULTS： Intestinal I/R induced intestine and liver injury, characterized by histological changes, as well as a significant increase in serum AST and ALT levels. The activity of SOD in the liver tissue decreased after I/R, which was enhanced by carnosol pretreatment. In addition, compared with the control group, carnosol markedly reduced liver tissue MPO activity and serum IL-6 level, which was in parallel with the decreased level of liver ICAI-1 and NF-κB expression. CONCLUSION： Our results indicate that carnosol pretreatment attenuates liver injury induced by intestinal I/R, attributable to the antioxidant effect and inhibition of the NF-κB pathway.

STUDY ON INTERCELLULAR ADHESIONMOLECULE-1 AND P-SELECTIN IN LIVER ISCHEMIA AND REPERFUSION INJURY

Objective To investigate the role of intercellular adhesion molecule-1 (ICAM-1) andP- selectin in hepatic ischemia/reperfusion injury. Methods The relationship between P- selectin as well asICAM- 1 and liver ischemia/reperfusion was studied, using a 60 minutes ischemia - reperfusion rat liver model.The animals were divided into three groups including: the sham group, the ischemic control group receiving onlythe normal saline and the treated group receiving anti-P-selection monoclonal antibody (Mab) at a dose of2mg/kg 15 minutes before reperfusion. The following indexes were analyzed: liver injury tests, liver histotogy,serum enzymes level (AST, ALT), and the levels of ICAM-1 and P-selectin in the blood of rats. ResultsSerum enzymes levels were significantly increased during sixty minutes of left lobar ischemia and reperfusion(saline control), and the increment was significantly inhibited with P- selectin Mab. Liver pathology observed bylight microscopy was greatly ameliorated by Mab. Furthermore, the elevated levels of ICAM- 1 and P- selectinwere found in the blood of ischemia/ reperfusion rats. Concluhon ICAM- 1 and P- selectin contribute tohepatic ischemia / reperfusion injury,and the P - selectin Mab can protect the liver against the injury.

γ-hydroxybutyrate protects the liver from warm ischemia-reperfusion injury in rat

BACKGROUND: Ischemia-reperfusion (I/R) syndrome remains an important clinical consideration in hepatic sur- gery, hemorrhagic shock, and liver transplantation, -y-hy- droxybutyrate (GHB) has been reported to exert protective effects against ischemia-reperfusion injury to various or- gans. To investigate whether GHB protects the liver from warm ischemia-reperfusion injury, we performed this study in rats. METHODS: Thirty male Wistar rats were randomly divided into a sham-operation group, a control group, and three I/R groups pretreated with GHB, GHB plus naloxone or naloxone. After 30 minutes of partial ischemia, followed by 60 minutes of reperfusion in the liver, histomorphological and enzymological changes, lipid peroxidation, apoptosis, and the plasma level of endothelin-1 were observed. RESULTS: I/R increased the serum levels of alanine ami- notransferase, aspartate aminotransferase and lactate dehy- drogenase and the plasma level of endothelin-1 significantly (P<0.01), in addition to increase of apoptotic index (AI) from 0.28%±0.25% to 17.68%±1.91%. The levels of he- patic malondialdehyde were markedly increased, whereas the activities of superoxide dismutase were markedly de- creased. GHB pretreatment prevented the liver from warm ischemia-reperfusion injury significantly, but naloxone par- tially blocked this effect. CONCLUSION: GHB may significantly protect the liver from hepatic warm ischemia-reperfusion injury via several different mechanisms.

Alleviation of ischemia/reperfusion injury in ob/ob mice by inhibiting UCP-2 expression in fatty liver

AIM： To investigate the protective effect of target suppression of uncoupling protein-2 （UCP-2） on ischemia/ reperfusion （I/R） injury in fatty liver in ob/ob mice. METHODS： Plasmids suppressing UCP-2 expression were constructed, and transfected into fatty liver cells cultured in vitro and the ob/ob mouse I/R injury model. Serum tumor necrosis factor （TNF）-α levels, UCP-2 mRNA expression, alanine aminotransferase （ALT） levels in ob/ob mice were tested, and the pathological changes in fatty liver were observed in experimental and control groups. RESULTS： In ob/ob mouse I/R models, serum TNF-α levels were significantly higher than in normal controls. After the plasmids were transfected into the cultured cells and animal models, expression of UCP-2 rnRNA was significantly reduced as compared with that in the control group （2^1.56± 0.15 vs 2^-0.45± 0.15, p 〈 0.05）. In ob/ob mouse models, in which expression of UCP-2 was suppressed, serum ALT levels were significantly lower than those of other groups, and pathological analysis revealed that injury of liver tissues was significantly alleviated. CONCLUSION： The target suppression of UCP-2 expression in fatty liver can alleviate the I/R injury in the ob/ob mice.

Oleanolic acid attenuates liver ischemia reper-fusion injury by HO-1/Sesn2 signaling pathway

BACKGROUND: Ischemia reperfusion injury (IRI) is unavoid-able in liver transplantation and hepatectomy. The present study aimed to explore the possible mechanism and the effect of oleanolic acid (OA) in hepatic IRI. METHODS: Mice were randomly divided into 6 groups based on different treatment. IRI model: The hepatic artery, portal vein, and bile duct to the left and median liver lobes (70% of the liver) were occluded with an atraumatic bulldog clamp for 90 minutes and then the clamp was removed for reperfusion. The mice were sacriifced 6 hours after reperfusion, and blood and liver tissues were collected. Liver injury was evaluated by biochemical and histopathologic examinations. The expressions of Sesn2, PI3K, Akt and heme oxygenase-1 (HO-1) were mea-sured with quantitative real-time RT-PCR and Western blotting. RESULTS: The serum aminotransferases level and scores of he-patic histology were increased after reperfusion. The increase was attenuated by pretreatment with OA (P<0.01). Compared with the IR group, OA pretreatment signiifcantly up-regulated the expression of Sesn2, PI3K, Akt and HO-1 in IR livers (P<0.05). Administration of zinc protoporphyrin (ZnPP), an inhibitor of HO-1, diminished the OA effect on HO-1 and Sesn2 expressions (P<0.05) and the protective effect of OA on IRI. CONCLUSIONS: Our results demonstrate that OA can attenu-ate hepatic IRI. The protective mechanism may be related to the OA-induced HO-1/Sesn2 signaling pathway.

Histological and biochemical alterations in early-stage lobar ischemia-reperfusion in rat liver

AIM： To investigate the structural and biochemical changes in the early stage of reperfusion in the rat livers exposed to lobar ischemia-reperfusion （IR）. METHODS： The median and left lobes of the liver were subjected to 60 min ischemia followed by 5, 10, 30, 45, 60 and 120 min reperfusion. Blood samples were taken at different time intervals to test enzyme activities and biochemical alterations induced by reperfusion. At the end of each reperfusion period, the animals were killed by euthanasia and tissue samples were taken for histological examination and immunohistochemistry. RESULTS： Cell vacuolation, bleb formation and focal hepatitis were the most important changes occur during ischemia. While some changes including bleb formation were removed during reperfusion, other alterations including portal hepatitis, inflammation and the induction of apoptosis were seen during this stage. The occurrence of apoptosis, as demonstrated by apoptotic cells and bodies, was the most important histological change during reperfusion. The severity of apoptosis was dependent on the time of reperfusion, and by increasing the time of reperfusion, the numbers of apoptotic bodies was significantly enhanced. The amounts of lactate dehydrogenase, alanine aminotransferase, aspartate aminotransfrase, creatinine and urea were significantly increased in serum obtained from animals exposed to hepatic IR. CONCLUSION： Inflammation and subsequent apoptotic cell death were the most important changes in early-stage hepatic reperfusion injury, and the number of apoptotic bodies increased with time of reperfusion.

Resveratrol attenuates oxidative stress and histological alterations induced by liver ischemia/reperfusion in rats

AIM： To investigate the effects of resveratrol on liver ischemia/reperfusion （I/R） injury in rats. METHODS： A total of 40 male Sprague-Dawley rats weighing 240-290 g were randomized into four groups of ten： （1） controls： data from unmanipulated animals; （2） sham group： rats subjected to the surgical procedure, except for liver I/R, and given saline; （3） I/R group： rats underwent liver ischemia for 45 rain followed by reperfusion for 45 rain; （4） I-R/Resveratrol group： rats pretreated with resveratrol （10 umol/L, iv）. Liver tissues were obtained to determine antioxidant enzyme levels and for biochemical and histological evaluation. RESULTS： Plasma aminotransferase activities were higher in the I/R group than in the I-R/Resveratrol group. Malondialdehyde levels and the hepatic injury score decreased, while superoxide dismutase, catalase, and glutathione peroxidase levels increased in group 4 compared to group 3. In group 4, histopathological changes were significantly attenuated in resveratroltreated livers. CONCLUSION： These results suggest that resveratrol has protective effects against hepatic I/R injury, and is a potential therapeutic drug for ischemia reperfusionrelated liver injury.