The Role Of RIP1 Mediated Necroptosis Insteatotic Liver Ischemia Reperfusion Injury

Objective Liver transplantation is the first choice and the only therapeutic method for all kinds of end-stage liver diseases.However,during the perioperative period of liver transplantation,ischemia reperfusion injury of the transplanted liver will occur,and the steatotic donor liver,as a marginal donor,is especially sensitive,which will further aggravate the liver injury.RIP1 is a key cell signal transduction center in necroptosis involved in ischemia-reperfusion injury to regulate cellular response and determine apoptosis or necrosis.Necrostatin-1(Nec-1)is a competitive allosteric inhibitor of ATP that antagonizes RIP1 activity.The purpose of this study was to investigate the role of RIP1 mediated necroptosis in steatotic liver ischemia reperfusion by means of Nec-1.Methods The rats were randomly divided into 4 groups,8 of which were fed with normal feed for 12 weeks,and the remaining rats were fed with high fat for 12 weeks to form a model of fatty liver.The rats were randomly divided into 3 groups with 8 rats in each group:SD group(fed with normal fat diet for 12 weeks,open and close abdominal surgery only),HFD group(fed with high-fat diet for 12 weeks,open and close abdominal surgery only),HFD+IR group(fed with high-fat diet for 12 weeks,then established cold IR model),HFD+IR+Nec-1 group(On the basis of HFD+IR,Nec-1 1mg/kg was injected via caudal vein 5min before cold ischemia reperfusion).Liver tissue was collected,and pathological changes of liver tissue were observed by HE staining under light microscope.The levels of TNF-α,IL-1β,MDA and SOD in liver were determined by ELISA.Western blot method was used to determine the expression levels of RIP1,RIP3,MLKL.Results Except for the SD group,the serum concentrations of ALT and AST were increased in all the other groups.Pathological examination showed that the liver tissue was seriously damaged,steatosis was obvious,and inflammatory cells were infiltrated in the portal area.The HFD+IR group showed the most obvious liver damage,with a large number of inflammatory cells infiltrating,enhanced oxidative stress response,and significantly increased expression of programmed necrotic related proteins RIP1,RIP3,and MLKL.After treatment with the inhibitor Nec-1,serum concentrations of ALT,AST,and the index level of oxidative stress were decreased,the degree of liver tissue damage was reduced and inflammatory cells were reduced.Western blot test showed that the levels of RIP1,RIP3 and MLKL proteins in liver tissues were significantly down-regulated after nec-1 application,and the expression of MLKL proteins in liver tissues determined by immunohistochemistry was significantly lower than that in the untreated inhibitor model group.Conclusion Necroptosis is involved in the process of ischemia reperfusion injury in rats on steatotic liver,and necrostatin-1 has a protective effect on steatotic liver ischemia reperfusion injury in rats.