The Effect Of OGT Mediated O-GlcN Acylated RIPK3 In Sevoflurane Postconditioning Reducing Necroptosis Induced By Myocardial Ischemia Reperfusion Injury

Background and Purpose:Inhalation anesthetics could exert a clear myocardial protective effect against myocardial ischemia reperfusion injury(MIRI).Previous research have demonstrated that inhalation anesthetic mediated cardioprotective effect against MIRI via?-N-acetylglucosamine(O-Glc NAc)modification of mitochondrial voltagedependent anion channel.O-Glc NAc modification protects against cardiomyocytes necroptosis.Although there are many clinical applications of volatile anesthetics,sevoflurane is still the most widely used volatile anesthetic in cardiothoracic surgery.Cardiothoracic surgery emphasizes the hemodynamically stable during the periope-rative period,especially the stages of anesthesia induction and anesthesia recovery,and the pharmacodynamic characteristics of sevoflurane can be consistent with this.Previous to this,there were many clinical and basic studies on myocardial protection effect of sevoflurane postconditioning(SPC),but the mechanism of its protective effect has not been fully elucidated.The aim of this study was to explore whether the OGT mediated O-Glc NAcylated RIPK3 level up-regulation and necroptosis signaling down-regulation were the important mechanisms of SPC induced cardioprotective effect.Methods:This study established in vivo and in vitro MIRI models.In the in vivo model,sixty rats were randomly divided into four groups: SHAM group,SEVO group,IR group and SPC group.In the Langendorff isolated model,forty-five rats were randomly divided into five groups: SHAM group,IR group,SPC group,SPC +solvent(DMSO)group and SPC + inhibitor(OSMI-1)group.Except for the SHAM group and SEVO group,each rat underwent 30 min ischemia followed by 2 h reperfusion in the other groups.Hemodynamic changes were continuously monitored during the experiment,and recorded at the end of the equilibrium period(T0),30 min(T1),60 min(T2),90 min(T3),and 2 h(T4)after reperfusion.The cardiac function indexes of rats in each group were monitored by small animal echocardiography.At the end of reperfusion,myocardial infarction size was measured by 1% TTC,the concentration of LDH in serum or coronary effusion was detected according to the ELISA method,the myocardial pathology slices of rats in each group were observed under light microscope after HE staining,necrotic myocardium were observed by fluorescence microscopy,the expression levels of O-Glc NAcylation and necroptosis biomarkers proteins were measured by Western Blot.In addition,for the further mechanism research,the combination of O-Glc NAcylation proteins and proteins associated with the necroptosis pathway were measured by Co-immunoprecipitation.Results:1.In vivo MIRI model,rats HR,MAP and RPP in four groups were continuous monitored and recorded at different time points of T0,T1,T2,T3 and T4,the results showed that SPC improved hemodynamics change after MIRI;2.In vivo MIRI model,rats echocardiogram were captured by small animal echocardiography and cardiac function parameters were measured,it showed that SPC could improve cardiac systolic dysfunction,alleviate change of cardiac structure and reverse ventricular hypertrophy after IR injury;3.In vivo MIRI model,the detection of myocardial infarction size and HE staining results in each group demonstrated that SPC could reduce rats' myocardial infarction size after IR injury and improve cardiomyocytes disorder arrangement after IR injury;4.In vivo MIRI model,the serum LDH concentration was monitored,and it was found that SPC could reduce the concentration of serum myocardial necroptosis marker enzyme LDH after IR injury;5.In vivo MIRI model,necroptotic cardiomyocytes were observed by fluorescence microscopy,the results indicated that SPC could reduce the necroptosis cardiomyocytes after IR injury;6.In vivo MIRI model,the results of detection necroptosis related proteins indicated that SPC could down-regulate the expression levels of p-RIPK3,RIPK3,p-MLKL and MLKL protein;7.In vivo MIRI model,the results of detecting glycosylation related proteins demonstrated that SPC could up-regulate the expression level of total protein O-Glc NAc glycosylation and OGT protein after IR injury;8.In vivo MIRI model,the interaction between O-Glc NAc,RIPK3,and MLKL in cardiomyocytes was detected by protein co-immunoprecipitation experiments,and the results showed that SPC could up-regulate the Glc NAcylated RIPK3 level and inhibit the combination of RIPK3 and MLKL after IR injury;9.In vitro MIRI model,the rats HR,LVSP,LVEDP and ±dp/dtmax in five groups at different time points T0,T1,T2,T3 and T4 were continuously monitored and recorded by the monitor.The results showed that SPC improved the hemodynamics after MIRI,but the OGT inhibitor OSMI-1 canceled this protective effect of SPC improving the hemodynamics;10.In vitro MIRI model,myocardial infarction size was determined by 1% TTC staining.The results showed that SPC could reduce myocardial infarction size after IR injury,but the myocardial protection of SPC reducing myocardial infarction size was cancelled after OSMI-1 administration;11.In vitro MIRI model,the LDH concentration of coronary effusion was monitored,it found that SPC could reduce the LDH concentration of coronary effusion after IR injury,but after giving OSMI-1,the LDH concentration of coronary effusion increased significantly;12.In vitro MIRI model,the results of detecting glycosylation related proteins indicated that SPC could up-regulate the total protein O-Glc NAc glycosylation and OGT protein expression levels after IR injury,but the total protein O-Glc NAc glycosylation and OGT protein expression levels are significantly down-regulated after OSMI-1 administration;13.In vitro MIRI model,the results of detecting necroptosis related proteins showed that SPC could down-regulate the expression levels of p-RIPK3 and p-MLKL protein,but the expression levels of p-RIPK3 and p-MLKL protein were significantly up-regulated after OSMI-1 administration.Conclusions:1.SPC played myocardial protection effect via inhibiting RIPK3/MLKL mediated necroptosis when MIRI;2.SPC further up-regulated O-Glc NAcylated RIPK3 level by promoting OGT mediated O-Glycosylation modification when MIRI;3.SPC could up-regulate O-Glc NAcylated RIPK3 level when MIRI,further inhibited the formation of RIPK3/MLKL complex,and ultimately reduced necroptosis caused by MIRI.