The Role And Mechanism Of ARRB1 In Hepatic Ischemia/Reperfusion(I/R) Injury

Background and Aims:Hepatic ischemia/reperfusion(I/R)injury is a major clinical problem during liver surgical procedures,which usually lead to early transplantation failure and higher organ rejection rate,and current effective therapeutic strategies are still limited.Therefore,in-depth exploring of the molecular mechanism of liver I/R injury is key to the development of new therapeutic methods.ARRBs are multifunctional proteins serving as important signaling scaffolds in numerous physiopathological processes,including liver specific diseases.However,the role and underlying mechanism of ARRBs in hepatic I/R injury remains largely unknow.Methods:We constructed mice with hepatocyte-specific ARRB1 overexpression by injecting C57BL/6 mice intravenously with adeno-associated virus expressing ARRB1(AdARRB1)or the control virus(Ad-GFP).ARRB1 knockout mice were also constructed.Mice partial liver ischemia surgery was conducted to investigate ARRBs function in vivo.Primary hepatocytes isolated from Ad-ARRB1,Ad-GFP,ARRB1 KO and WT mice were cultured and subjected to hypoxia/reoxygenation(H/R)challenge in vitro.The mRNA and protein expression of related genes were detected by qRT-PCR and western blotting.The damage in liver was examined by H&E staining and Suzuki's criteria.Serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured by ELISA kit.Cell apoptosis status of hepatocytes were examined by TUNEL assays.The production of inflammatory cytokines was examined by ELISA assays and qRT-PCR.The infiltration of inflammatory cells was detected by flow cytometry and immunofluorescence assays.Co-immunoprecipitation,pulldown and ubiquitination assays were preformed to examine the interaction between ARRB1,TRAF6 and ASK1.Results:Here,we showed that only ARRB1,but not ARRB2,was downregulated during liver I/R-injured injury.Hepatocyte-specific overexpression of ARRB1 significantly ameliorated liver damage,as demonstrated by decreases in serum aminotransferases,hepatocellular necrosis and apoptosis,infiltrating inflammatory cells and secretion of pro-inflammatory cytokines relative to control mice.Whereas,experiments with ARRB1 knockout mice gotten opposite effects.Mechanistically,ARRB1 directly interacts with ASK1 in hepatocytes,and inhibits its TRAF6-mediated Lysine 6-linked polyubiquitination,which then prevents the activation of ASK1 and its downstream signaling pathway during hepatic I/R injury.In addition,inhibition of ASK1 remarkably abolished the disruptive effect result from ARRB1 deficiency in liver I/R injury in vivo,indicating that ASK1 was required for ARRB1 function in hepatic I/R injury.Conclusion:We proposed that ARRB1 is a novel protective regulator during liver I/R injury,and modulation of the regulatory axis between ARRB1 and ASK1 could be a novel therapeutic strategy to prevent this pathological process.