The Impact Of Polyol Pathway On Steatotic Liver Subjected To Ischemia-reperfusion

Part one:ARI markedly alleviated the I/R-induced impairments in fatty miceObjective As the first key enzyme of polyol pathway,aldose reductase has been increasingly revealed a deteriorative effect on ischemic heart,brain and kidney.Till now,the underlying mechanism has not been fully elucidated;meanwhile,the impact of aldose reductase on ischemic fatty liver remain unclear.Herein,we conducted this study to investigate how and whereby aldose reductase might impact on ischemic steatotic liver.Methods After intraperitoneally administrating with zopol(ARI group)or saline(Ctrl group),the high-fat-diet-induced steatosis liver mice were subjected to 6h of ischemia and subsequent 6/24h of reperfusion,the mice received intraperitoneal zopol and sham operation were deployed as Sham group.The hepatic TG content and AR protein level were measured to assess the steatosis homogeneity and inhibiting efficiency.liver function was evaluated by serum alanine aminotransferase and aspartate aminotransferase.To assess the histological variations,HE section and scanning electron microscopy were utilized.We next prepared single-hepatocytes suspension to dectect hepatic necrosis and apoptosis by flow cytometry.Meanwhile,the flux of Caspase-dependant apoptosis pathway was evaluated by western blot.Subsequently,cytocolic contents of NAD(P)(H)、GSH/GSSG、MDA and ROS were detected by microplate reader or flow cytometry.Results TG contents presented with no apparent difference among three group while ARI group demonstrated significantly decreased AR protein level as compared with Ctrl and Sham group with a inhibiting effeicency of 58.9%and 45.0%,respectively(P<0.05).In contrast with Ctrl group,ARI markedly mitigated the I/R-induced hepatic function decreasement,histological disintegration and hepatocytes’ necrosis and apoptosis.In protein level,ARI also notably elevated Bcl 2 while inhibited BAX and the activation of Caspase-dependant apoptosis pathway.Furthermore,pretreating ischemic fatty liver with ARI evidently increased intracellular NADPH/NADP,NAD/NADH and GSH/GSSG ratios whereas lowered MDA and ROS contents.Conclusion ARI could protect fatty liver from severe I/R impairments and this effect might be further associated to improved energy and redox status as well as suppressed Caspase-dependant apoptosis pathway.Part two:AR-overexpression worsened H/R impairments in steatosis human hepatocyte L02Objective To investigate the impact of AR-overexpression on steatosis human hepatocyte L02 subjected to hypoxia/reoxygenation.Methods After transfecting with pAR(pAR group)or empty plasmid(GFP)or without transfection,human hepatocyte L02 were exposed to high culture medium to induce steatosis and then subjected to 4h of hypoxia and 6h of reoxygenation.TG content and AR protein level were firstly investigated to evaluate the steatosis homogeneity and transfecting efficiency.We subsequently detected the variation in mitochondrial membrane potential JC-1 staining.In addition,the flux of Caspase-dependant apoptosis pathway was measured by western blot.Results There was no significant variance in the TG contents among three group while pAR revealed significantly elevated AR protein content in contrast with Ctrl and GFP group with a transfecting effeicency of 41.3%and 33.5%,respectively(P<0.05).Compared with Ctrl and GFP group,AR-overexpression in vitro remarkably disrupted the mitochondrial membrane potential in steatosis L02 after hypoxia/reoxygenation.Meanwhile,AR-overexpression markedly elevated Bcl 2 and suppressed BAX and the activation Caspase-dependant apoptosis pathway in protein level.Conclusion AR-expression could render fatty hepatocyte L02 more vulnerability to I/R stress and this trend was further correlated to increased mitochondrial depolarization and flux of Caspase-dependant apoptosis pathway.Part three SDI remarkedly alleviated the ischemia/reperfusion impairments in mice liverObjective To explore the independant role of sorbitol dehydrogenase,the second key enzymes in polyol pathway,on mice liver following ischemia/reperfusion.Methods Wild-type C57BL/6 mice administrated with intraperitoneal SDI(SDI group)or saline(Ctrl group)were subjected to 1h of 70%hepatic blood occulation and 24h reperfusion.The SDH protein level was firstly measured to assess the inhibiting efficiency.Subsequently,the changes in liver function,histology and hepatocytes’ necrostic and apoptotic ratios were investigated via biochemistry measurement,HE section and flow cytometry,respectively.Meanwhile,protein SIRT1 and cleaved-Caspase 3 were quantified by western blot.Furthermore,intrahepatic ATP and NAD(H)contents were also detected to revealed the anaerobic glycolysis during hypoxia phase.Results As compared with Ctrl group and Sham group,SDI significantly decreased liver SDH protein level with inhibiting a efficiency of 46.1%and 41.6%,respectively(P<0.05);meanwhile,alleviated the I/R-induced liver function decreasement,inflammatory factors release,histological disintegration and increasements of hepatic apoptosis and necrosis.In protein level,SDI enhanced and inhibited SIRT1 and cleaved-Caspase 3 respectively.Additionally,SDI group presented with remarkedly higher NAD(H)and ATP level in contrast to Ctrl group.Conclusion SDI-administration could evidently palliated the ischemia/reperfusion impairments in mice liver,and this protective role might be associated to improved energy status and SIRT1 protein leve as well as decreased activation in protein Caspase 3 in ischemic liver.