| Objective: Sevoflurane,a commonly used inhaled anesthetic,has been shown to exert cardioprotection by inhibiting apoptosis or attenuating autophagic flux injury,while the role of necroptosis in it remains to be further clarified.Diabetes not only aggravates myocardial ischemia-reperfusion injury,but also abolishes the myocardial protective functions of sevoflurane,the mechanisms of which have not been elucidated.In this paper,we compared the effects of sevoflurane on the extent of myocardial injury and the expression of proteins associated with necroptosis in the presence or absence of diabetes to investigate the role of sevoflurane postconditiong on myocardial ischemia-reperfusion injury in diabetic rats and its relationship with RIPK3 mediated necroptosis.Methods:Part Ⅰ:Sixty SD rats of SPF grade weighing 300±20g were randomly divided into three groups: sham operation group(group SHAM),myocardial ischemia-reperfusion group(group I/R),and sevoflurane postconditioning group(group Sevo-Pc).The model of myocardial ischemia-reperfusion injury(MIRI)in rats was established by ligating the anterior descending branch of left coronary artery for 40 min and then reperfusion for 120 min.At the end of surgery,samples were classified and collected according to the experimental requirements.LDH and CK-MB concentrations were measured by centrifugal serum,myocardial infarct size was compared by TTC staining,the expression of RIPK3,p-Ca MKII and p-MLKL in myocardial tissue was determined by western blot,and myocardial ultrastructure was observed by transmission electron microscopy to investigate the role of sevoflurane postconditiong on myocardial ischemia-reperfusion injury in non-diabetic rats and its relationship with RIPK3 mediated necroptosis.Part Ⅱ:Ninety SD rats of SPF grade weighing 90-100 g were fed high-fat diet for 4 weeks,then intraperitoneal injection of 1% streptozotocin 40 mg/kg to construct type 2 diabetic rat model.After excluding the rats with failed modeling,80 rats were finally selected and divided into four groups randomly: sham operation group(group DS),myocardial ischemia-reperfusion group(group DIR),sevoflurane postconditioning group(group DSevo),and sevoflurane postconditiong plus RIPK3 inhibitor GSK-872 group(group GSK).The model of myocardial ischemia-reperfusion injury(MIRI)in diabetic rats was established in a same way as the Part Ⅰ.At the end of surgery,samples were classified and collected according to the experimental requirements.Changes in serum LDH and CK-MB concentrations and myocardial infarct size were compared between non-diabetic and diabetic rats groups(group SHAM,gourp I/R,group DS and group DIR)by biochemical reactions and TTC staining to investigate the effects of diabetes on myocardial susceptibility after myocardial ischemia-reperfusion in rats.Then,the Serum LDH and CK-MB concentrations were measured by biochemical reactions,myocardial infarct size was compared by TTC staining,the protein expression of RIPK3,p-Ca MKII and p-MLKL in myocardial tissue was determined by western blot,myocardial ultrastructure was observed by transmission electron microscopy to investigate the role of sevoflurane postconditioning on myocardial protection abolished by diabetes and its relationship with RIPK3 mediated necroptosis.Results:Part Ⅰ:1.Compared with group SHAM,the serum LDH and CK-MB concentrations were increased in group I/R and group Sevo-Pc(P<0.05).Compared with group I/R,the serum LDH and CK-MB concentrations were decreased in group Sevo-Pc(P<0.05).2.Compared with group SHAM,the percentage of myocardial infarct size increased in group I/R and group Sevo-Pc(P<0.05).Compared with group I/R,the percentage of myocardial infarct size decreased in group Sevo-Pc(P<0.05).3.Compared with group SHAM,the expression of myocardial RIPK3,p-MLKL and p-Ca MKII in group I/R and group Sevo-Pc was increased(P< 0.05).Compared with group I/R,the expression of myocardial RIPK3,p-MLKL and p-Ca MKII in group Sevo-Pc was decreased(P< 0.05).4.Observation of myocardial ultrastructure in nondiabetic rats by transmission electron microscopy: In group I/R,the mitochondria of cardiomyocytes were moderately swollen,the structure of cristae was partially destroyed,a few of them showed mitochondrial membrane rupture,and the myofibrils and sarcomeres were disorganized;In group Sevo-PC,the mitochondria of cardiomyocytes were slightly swollen,the arrangement of myogenic fibers was disorganized,but the distribution of light and dark bands was visible;In group SHAM,the structure of cardiomyocytes and their mitochondria was uniform and intact,showing no significant abnormal changes.Part Ⅱ:1.Compared with the group SHAM,the LDH concentrations were increased in group DS(P<0.05),the CK-MB concentrations were not significantly different in group DS(P>0.05).Compared with the group I/R,the LDH concentrations were not significantly different in group DIR(P>0.05),the CK-MB concentrations were increased in group DIR(P<0.05).2.compared with the group DS,the serum LDH and CK-MB concentrations were increased in the group DIR,group DSevo and group GSK(P<0.05).Compared with group DIR group,the serum LDH and CK-MB concentrations were not significantly different in the group Dsevo(P>0.05),the serum LDH and CK-MB concentrations were markedly decreased in the group GSK(P<0.05).Compared with the DSevo group,the serum LDH and CK-MB concentrations were observably decreased in the group GSK(P<0.05).3.Compared with group SHAM,group DS showed no significant difference in myocardial infarct size(P>0.05).Compared with group I/R,the myocardial infarct proportion was significantly increased in group DIR(P>0.05).4.Compared with group DS,the percentage of myocardial infarct size increased in group DIR,group DSevo,and group GSK(P<0.05).Compared with group DIR,the percentage of myocardial infarct size was not significantly different in group DSevo(P>0.05),the percentage of myocardial infarct size decreased in group GSK(P<0.05).Compared with group DSevo,the myocardial infarct size decreased in group GSK(P<0.05).5.Compared with group DS,the expression of RIPK3,p-MLKL and p-CaMKII in myocardial tissue was up-regulated in group DIR,group DSevo and group GSK(P<0.05).Compared with group DIR,the expression of RIPK3,p-MLKL and p-Ca MKII in myocardial tissue was not significantly different in group Dsevo(P>0.05),the expression of RIPK3,p-MLKL and p-Ca MKII in myocardial tissue was down-regulated in group GSK(P<0.05).Compared with group DSevo,the expression of RIPK3,p-MLKL and p-Ca MKII in myocardial tissue was down-regulated in group GSK(P<0.05).6.Observation of myocardial ultrastructure in diabetic rats by transmission electron microscopy: In group DIR and group DSevo,the cardiomyocytes showed severe degenerative lesions,with severe mitochondrial swelling,loss of mitochondrial cristae and intra-membrane matrix,necroptosis manifestations such as mitochondrial membrane rupture and matrix overflow,massive myofibrils and sarcomeres were disorganized;In group GSK,the mitochondria of cardiomyocytes were moderately swollen,some mitochondrial cristae were broken,individual mitochondrial membrane rupture manifestations were observed,myofibrils were broken,sarcomeres were asymmetrically distributed,and the structure of light and dark bands was unclear.In group DS,the mitochondria of cardiomyocytes were mildly swollen,no mitochondrial membrane rupture manifestations were observed,myofibrils were not significantly broken,but the structure of light and dark bands was unclear.Conclusion:Diabetes not only aggravates myocardial ischemia-reperfusion injury,but also abolishes the cardioprotection of sevoflurane by activating RIPK3 mediated necroptosis,destroying the structural and functional stability of cardiomyocytes and their mitochondria. |
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