Role Of Hypoxia Inducible Factor 1α-Adenosine A2B Receptor Pathway In Ischemia Reperfusion Injury Of Donated After Cardiac Death Rat Liver And The Underlying Molecular Mechanisms

Part I Role of HIFla-A2BR pathway in total hepatic ischemia reperfusion injury and the underlying molecular mechanisms Aim The purpose of this study is to investigate the role of hypoxia-inducible factor-1α(HIF1α)-adenosine A2B receptor(A2BR)pathway in total hepatic ischemia-reperfusion injury in rats.Methods A total of 24 male Wistar rats were randomly divided into 4 groups that consisted ofa normal control group(Control group),a TIRI group(TIRI group),a HIF 1α agonist group(FG-4592 group)and a HIF 1α inhibitor group(2ME2 group),6 rats in each group.The serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured by spectrophotometry.The serum levels of tumor necrosis factor-a(TNF-a)and interleukin-6(IL-6)were measured by enzyme-linked immunosorbent assay.The contents of malondialdehyde(MDA).superoxide dismutase(SOD)and myeloperoxidase(MPO)in liver tissues were detected by thiobarbituric acid assay,xanthine oxidase assay and o-dianisidine method,respectively.qRT-PCR was used to detect the mRN A expression of adenosine Al,A2A,A2B and A3 receptors in liver tissues.Western Blot assay was used to detect the levels of HIF la,A2BR,Bcl-2,and Bax protein in liver tissues.The pathological changes in liver tissues were observed and the in situ apoptotic index of liver tissue was detected bt TUNEL assay.Results In contrast to the Control group,the serum levels of ALT,AST,TNF-α,IL-6 and the activities of MDAand MPO,as well as histopathological damage scores and apoptotic indexes in liver tissues significantly increased in the-TIRI group,while the activities of SOD and Bcl-2/Bax ratio significantly decreased in the TIRI group;Moreover,the protein levels of HIFla and A2BR were also significantly increased in TIRI group and the difference was statistically significant(P<0.05).The serum levels of ALT,AST,TNF-a,IL-6 and the liver activities of MDAand MPO,as well as histopathotogical damage scores and apoptotic indexes in FG-4592 group were lower than that in TIRI group,while the activities of SOD and Bcl-2/Bax ratio and the protein levels of HIF1α and A2BR were significantly higher than that in-TIRI group,indicating that the induction of HIFIa promoted the transcription and translation of A2BR,alleviated TIRI-induced inflammatory responses,oxidative stress injury and cell apoptosis;The serum levels of ALT,AST,TNF-α,IL-6 and the liver activities of MDA and MPO,as well as histopathological damage scores and apoptotic indexes in 2ME2 group were significantly higher than that in TIRI group,while the activities of SOD,Bcl-2/Bax ratio and the protein expression of HIF 1α and A2BR were significantly lower than that in TIRI group.The negative effects of FG-4592 is attributed in part to the suppression of HIF1α and ultimately decreased A2BR production.Conclusion HIF1α alleviated TIRI induced excessive inflammation reaction,cell oxidative stress injury arnd apoptosis by promoting the transcription and translation of A2BRPart II Role of HIF1α-A2BR pathway in hepatic cell oxygen-glucose deprivation/reperfusion-induced injury and the underlying molecular mechanisms Aim To investigate the regulation mechanism of HIFla-A2BR pathway in the oxygen-glucose deprivation/reperfusion(OGD/R)-induced injury in BLR-3A cells.Methods The protein expression levels of HIFla,CD73,and A2BRat various time-points after reperfusion in BLR-3Acells were detected by Western blot The intracellular localization of HIF1 a and A2BR was observed by confocal laser scanning microscopy and the interaction of HIF1 a with A2BR was further confirmed by co-immunoprecipitation(co-IP).Transient small interfering RNA(siRNA)technology was used to silence HIF1αorA2BR gene.Flow cytometry analisis was(?)used to determine the cell levels of reactive oxygen species(ROS)and apoptosis rate,and the activities of MDA and SOD contents as well as the protein expression levels of HIF1α,A2BRand CD73 in cells were also detected.Results We found the protein expression levels of HIFla,CD73 and A2BR were increased synchronously and peaked at 6 h after OGD/R.HIF1 a and A2BR were co-expressed and co-immunoprecipitated in BLR-3Acells after OGD/R.Furthermore,HIFla silencing by siRNA significantly aggravated cellular OGD/R induced cell injury by increased the production of ROS and promoted cell apoptosis.The pharmaceutical activation of HIFla with the use of synthetic HIF agonists FG-4592 significantly alleviated cellular OGD/R induced cell injury but this protective effect was abolished by A2BR siRNA treatment,supported the hypothesis that the protective effects of HIF1α aginst OGD/R induced injury was attributed to the transcription and translation of A2BR.Conclusion HIF1α alleviated oxidative stress induced cellular injury and apoptosis during the OGD/R in BLR-3A cells by regulated the transcription and translation of A2BRPart III Donor Treatment with a HIF-Agonist Prevents DCD Liver Graft Injury in a Rat Isolated Perfusion ModelAim The objective of this study was to test the hypothesis that pharmaceutical stabilization of HIF-1 in DCD donors would result in a better graft liver conditionMethods Male SD rats(6 animals per group)were randomly given the synthetic prolyl hydroxylase domain inhibitor FG-4592(Selleck,6 mg/kg of body weight)or its vehicle(dimethylsulfoxide).Six hours later,cardiac arrest was induced by bilateral pneumothorax Rat livers were retrieved 30 min after cardiac arrest,and subsequently cold stored in University of Wisconsin solution for 24 hours.It were reperfused for 60 minutes with Krebs-Henseleit bicarbonate buffer on an isolated perfused liver model,after which the perfusate and liver tissues were investigated.Results Pretreated with FG-4592 in DCD donors significantly improved graft function with increased bile production and synthesis of adenosine triphosphate,decreased perfusate liver enzyme release,histology injury scores and oxidative stress-induced cell injury and apoptosis after reperfusion with the isolated perfused liver model The beneficial effects of FG-4592 is attributed in part to the accumulation of HIF-1 and ultimately increased A2BRproduction.Conclusion Pretreatment with FG-4592 in DCD donors resulted in the activation of HIF-1 pathway and subsequently protecting liver grafts from warm ischemia and cold-stored injury.These data suggest that the pharmacological HIF-1 induction may provide a clinically applicable therapeutic intervention for prevent injury to DCD allografts.