线粒体自噬在糖尿病因素影响七氟醚后处理心肌保护作用中的机制

Mechanism of Mitophagy in Diabetes Influencing Myocardial Protective Effect of Sevoflurane Postconditioning

目的探讨线粒体自噬在糖尿病因素影响七氟醚后处理心肌保护作用中的机制,为心肌保护提供新的策略.方法清洁级健康雄性SD大鼠32只,7~8周龄,体质量250~300g,采用数字表法随机分为4组(n=8),即非糖尿病缺血再灌注组(NI/R组)、非糖尿病七氟醚后处理组(NSP组)、糖尿病缺血再灌注组(DMI/R组)和糖尿病七氟醚后处理组(DMSP组).以高脂高糖饲养和腹腔注射链脲佐菌素的方法制备糖尿病模型.采用结扎左冠状动脉前降支30min再灌注120min的方法制备大鼠心肌缺血再灌注损伤模型,其中NSP组和DMSP组于再灌注前1min吸入七氟醚,维持呼气末浓度2.5%,持续5min.于再灌注120min后颈动脉采血取样,ELISA法测定血清肌钙蛋白(cTnI)浓度;采用1%2,3,5氯化三苯基四氮唑(TTC)染色测定心肌梗死面积;透射电镜下观察心肌细胞超微结构;运用Western blot法检测心肌组织LC3Ⅱ/LC3Ⅰ、p62和Parkin的表达.比较糖尿病因素与七氟醚后处理两者对心肌缺血再灌注损伤的影响.结果糖尿病大鼠组的心肌缺血再灌注损伤较非糖尿病大鼠组严重,七氟醚后处理组的心肌缺血再灌注损伤较非七氟醚后处理组轻微,糖尿病因素与七氟醚后处理组在心肌缺血再灌注损伤方面存在交互作用(P<0.05).与NI/R组比较,NSP组血清cTnI浓度降低,心肌梗死面积减少,心肌组织LC3Ⅱ/LC3Ⅰ和Parkin的表达下调,p62的表达上调(P<0.05);与NI/R组比较,DMI/R组血清cTnI浓度升高,心肌梗死面积增加,心肌组织LC3Ⅱ/LC3Ⅰ和Parkin的表达下调,p62的表达上调(P<0.05);与NSP组比较,DMSP组血清cTnI浓度升高,心肌梗死面积增加,心肌组织LC3Ⅱ/LC3Ⅰ和Parkin的表达下调,p62的表达上调(P<0.05).结论糖尿病因素可以减弱七氟醚后处理的心肌保护作用,其机制可能与糖尿病大鼠的心肌线粒体自噬水平降低有关.

Objective To explore the mechanism of mitophagy in diabetes influencing myocardial protective effect of sevoflurane postconditioning,and provide a new strategy for myocardial protection.Methods Thirty-two clean healthy male Sprague-Dawley rats,aged 7-8 weeks,weighing 250-300g,were randomly divided into 4 groups(n=8):non-diabetic ischemia-reperfusion group(NI/R group),non-diabetic sevoflurane postconditioning group(NSP group),diabetes mellitus ischemia-reperfusion group(DMI/R group)and diabetes mellitus sevoflurane postconditioning group(DMSP group).Diabetes mellitus models were prepared by feeding high fat and high sugar diet and intraperitoneal injection of streptozotocin.The rat model of myocardial ischemia-reperfusion injury was established by ligation of the left anterior descending coronary artery for 30min and reperfusion for 120min.NSP group and DMSP group were inhaled sevoflurane 1 min before reperfusion to maintain the end-tidal concentration of 2.5% for 5mins.Carotid blood sampling was performed at time of 120 min after reperfusion,serum troponin(cTnl)concentration was determined by ELISA;myocardial infarct size was determined by 1% 2,3,5 triphenyltetrazolium chloride(TTC)staining;The ultrastructure of cardiomyocytes was observed with transmission electron microscopy.The expression of LC3Ⅱ/LC3Ⅰ,p62 and Parkin in myocardial tissue were detected by Western blot.The effects of both diabetes and sevoflurane postconditioning on myocardial ischemia-reperfusion injury were compared by analysis of variance of two-factor factorial design data.Results Myocardial ischemia-reperfusion injury in diabetic rats group was more serious than non-diabetic rats,myocardial ischemia-reperfusion injury in sevoflurane postconditioning group was milder than non-sevoflurane postconditioning group,and there was an interaction between diabetes and the sevoflurane postconditioning in myocardial ischemia-reperfusion injury(P<0.05).Compared with NI/R group,serum cTnl concentration was decreased,myocardial infarct size was decreased,myocardial tissue LC3Ⅱ/LC3Ⅰ and Parkin expression were down-regulated,p62 expression was up-regulated in NSP group(P<0.05).Compared with NI/R group,serum cTnI concentration was increased,myocardial infarct size was increased,myocardial tissue LC3Ⅱ/LC3Ⅰ and Parkin expression were down-regulated,p62 expression was up-regulated in DMI/R group(P<0.05).Compared with NSP group,serum cTnl concentration was increased,myocardial infarct size was increased,myocardial tissue LC3Ⅱ/LC3Ⅰ and Parkin expression were down-regulated,p62 expression was up-regulated in DMSP group(P<0.05).Conclusion The protective effect of sevoflurane postconditioning on diabetic rats was weakened,and the mechanism may be related to the decrease of myocardial mitophagy level in diabetic rats.