Augmenter Of Liver Regeneration Attenuates Ischemia/Reperfusion Injury In Fatty Liver Model And Possible Mechanism

Background & Aims:Liver resections and transplantation has been confined as successful strategy to cure the end-staged liver diseases,including malignant liver disease.In this case,an adequate of liver-organ supply becomes a dominant issue to meet demand of liver surgery.Currently,wide discrepancy between patients needed recipient and graft supply is becoming more serious,which promoted the application of fatty liver as an extended criteria donors.Along with the epidemic of obesity,the patients with non-alcoholic fatty liver disease(NAFLD)are worldwide rapidly increasing,therefore,the increasing proportions of fatty liver as donor organ in liver resection will be a consistent problem to be tackled.Fatty liver or hepatic steatosis enhances the susceptibility of hepatocytes to ischemia/reperfusion injury(IRI),thus increasing the risk of postoperative morbidity and hepatic mortality in patients undergoing liver transplantation or liver resection.Augmenter of liver regeneration(ALR),first identified in weanling and regenerating rat livers,was reported to stimulate the regeneration of the remnant liver after partial hepatectomy.In addition to its profound proliferative effect,ALR also protects the liver from various injuries in the clinical context.We have previously reported that the transfection of the ALR gene protected livers from hepatic IRI in healthy mice,mainly by alleviating mitochondrial swelling,preserving the mitochondrial ultrastructure and inhibiting cytochrome c leakage.However,whether the ALR could protect fatty liver from IRI as well remains somewhat unknown.In this study,the ALR gene was administered to mice with liver steatosis before they were subjected to IRI.The effects of ALR on IRI outcome and the possible mechanisms were explored.This research will not only lead to a new understanding of the mechanism of ALR protection,but also for the development of new potential therapeutic agent combating surgery and transplantation of steatotic liver.Methods:In vivo,liver steatosis in mice was prepared by feeding a methionine-choline deficient(MDC)diet for 2 weeks.The mice fed the MCD diet were randomly divided into four groups.The mice in the sham-operated group underwent laparotomy only,without IRI,whereas the mice in the other three groups underwent either ALR-containing viral transfection(designated Ad ALR),vector only transfection(designated Adnull),or an injection of normal solution(NS;as the control transfection)3 days before IRI.Serum ALT,AST,LDH were analyzed.The structure of liver tissue and cell death were observed by HE staining.Hepatocyte apoptosis were examined by TUNEL stainng,immunohistochemistry of cleaved Caspase-3,4-HNE and PCNA.The protein level of Bcl-2,Bax and cleaved Caspase-3 were tested by Western blot.At the same time,we also detected the activity of SOD,GPx,GSH to observe the antioxidant capacity.In an in vitro study,Hep G2 cells were stable transfected with the ALR-containing vector and empty vector(designated ALR-Tx and Vector-Tx).To induce steatosis,the cells were incubated with 0.3 mM oleic acid/palmitic acid combination(OA/PA,ratio 2:1)for 6h,then underwent hypoxia-reoxygenation(H/R)injury,simulation the ischemia-reperfusion injury model In vivo,to observe the ALR protective effect.Cell survival was measured with the Cell Counting Kit 8.Cell apoptosis was evaluated by measuring Caspase-3 activity,TUNEL staining and by detecting expresions of apoptosis-related proteins such as Bcl-2 and Bax.The mitochondrial membrane potential was detected with JC-1.The level of mitochondrial oxygen consumption rate(OCR)was determined with an XFp Extr acellular Flux Analyzer.Finally,the activities of antioxidant enzymes of SOD and GSH,the mRNA and protein level of MnSOD were also tested.Results:1.The serum level of ALT,AST and LDH were lower in AdALR mice than Adnull mice(ALT: 1452 U/L vs.502 U/L,p < 0.01;AST 1138 U/L vs.558 U/L,p < 0.01;LDH: 3237 U/L vs.1891 U/L,p < 0.05).Liver histology index improved and apoptosis cells were reduced significantly(TUNEL positive cells: 26.68% vs.10.77%;cleaved Caspase-3 positive cells: 3.03% vs.0.98%)in AdALR mice than Adnull mice.These results suggested that ALR could attenuate the IRI and inhibite the cell apoptosis of mice with fatty liver.2.Lipid peroxidation was enhancement after IRI treatment in steatosis liver,but the level of lipid peroxidation products of MDA and 4-HNE(25% vs.5%,p < 0.01)in the AdALR mice were lower than Adnull mice.The results of antioxidant capacity showed that,compaired with Adnull mice,the activity of SOD was increased(80% vs.95%,p < 0.05),the content of GSH(0.63?mol/g tissue vs.1.21?mol/g tissue,p < 0.01)and the ratio of GSH/GSSG(0.78 vs.1.28,p < 0.01)were raised.3.The hepatic mRNA levels of TNF-a,IL-1b,IL-6,and CXCL2 increased significantly in the mice subjected to IRI compared with those in the sham-operated mice,and the expression of these mRNAs was significantly lower in the ALR-transfected mice than in the Adnull mice.The number of F4/80-positive cells was significantly lower in ALR-transfected mice than in the Adnull mice after IRI(1.3% vs.0.6%,p < 0.01).These results suggested that ALR could decrease the release of inflammatory cytokines and inhibite the activation of KCs after IRI in fatty liver.The number of the PCNA positive cells in the ALR transfection mice were increased as compared with the Adnull mice(5.3% vs.28.6%,p < 0.01),suggesting that ALR could enhance the cell growth after IRI in fatty liver.4.In vivo,compared with Vector-Tx cells,the cell vitality rate of ALR-Tx cells was increased(74.9% vs.90.9%,p < 0.01),while the activity of Caspase-3 was reduced(1.85% vs.1.42 %,p < 0.01)and the number of TUNEL positive cells decreased significantly(9.5% vs.4 %,p < 0.01).5.Fatty degeneration cells after H/R injury,ALR-Tx cells decreased the content of total ROS(1.75 vs.0.99,p < 0.01)and the mitochondrial ROS(1.36 vs.1.18,p < 0.01),stabilization of the mitochondrial membrane potential,increased the content of ATP(56.7% vs.78.8%,p < 0.01)and the level of mitochondrial OCR compared with Vector-Tx cells(143.9 pmol/min vs.227.9 pmol/min,p < 0.01).6.The antioxidant capacity of H/R injury in steatosis cells was reduced,but ALR could increase the activity of SOD and GSH,the mRNA(1.85 vs.3.71,p < 0.01)and protein level of MnSOD(MnSOD/actin 0.43,vs.0.82,p < 0.01)were obviously higher than the Vector-Tx cells.Conclusions:ALR protected steatotic hepatocytes from IRI by attenuating oxidative stress,improving antioxidant capacity,increasing the content of ATP and the OCR level of mitochondria,reducing the production of ROS,alleviating of mitochondrial dysfunction,suppressing inflammatory reaction,thereby,inhibiting steatotic cells from apoptosis through IRI.Therefore ALR may be used as a potential therapeutic agent combating surgery of NAFLD for the mitochondrial-preserving and antioxidant effect.