线粒体动力相关蛋白1表达与糖尿病因素取消大鼠七氟烷后处理心肌保护作用的关系

Relationship between dynamin-related protein 1 expression and diabetic mellitus-casused abolition of cardioprotection induced by sevoflurane postconditioning in rats

目的评价线粒体动力相关蛋白1(Drp1)表达与糖尿病因素取消大鼠七氟烷后处理心肌保护作用的关系。方法清洁级健康雄性Wistar大鼠36只,14~16周龄,体重300~350 g,采用随机数字表法分为3组(n=12):假手术组(S组)、缺血再灌注组(I/R组)和七氟烷后处理组(SPC组)。雄性GK大鼠36只,14~16周龄,体重300~350 g,采用随机数字表法分为3组(n=12):糖尿病假手术组(DM+S组)、糖尿病缺血再灌注组(DM+I/R组)和糖尿病七氟烷后处理组(DM+SPC组)。I/R组、SPC组、DM+I/R组和DM+SPC组采用结扎大鼠左冠状动脉前降支30 min,再灌注120 min的方法制备心肌缺血再灌注损伤模型。SPC组和DM+SPC组于再灌注前1 min时吸入2.4%七氟烷15 min行后处理。再灌注120 min时,随机取6只大鼠,取动脉血标本,采用比色法测定血清LDH活性;然后处死大鼠,取左心室缺血区域组织,采用比色法测定ATP含量,透射电镜下观察心肌线粒体超微结构,根据Flameng线粒体评分判断线粒体受损程度,采用Western blot法检测Drp1表达。再灌注120 min时,随机处死6只大鼠,取心肌组织,采用TTC染色法确定心肌梗死体积,计算心肌梗死体积百分比。结果与S组比较,I/R组及SPC组血清LDH活性、心肌梗死体积百分比升高,心肌组织ATP含量降低,Flameng线粒体评分升高,心肌线粒体Drp1表达上调(P<0.05);与I/R组比较,SPC组血清LDH活性、心肌梗死体积百分比降低,心肌组织ATP含量升高,Flameng线粒体评分降低,心肌线粒体Drp1表达下调(P<0.05),心肌病理学损伤减轻。与DM+S组比较,DM+I/R组及DM+SPC组血清LDH活性、心肌梗死体积百分比升高,心肌组织ATP含量降低,Flameng线粒体评分升高,心肌线粒体Drp1表达上调(P<0.05);与DM+I/R组比较,DM+SPC组血清LDH活性、心肌梗死体积百分比、心肌组织ATP含量、Flameng线粒体评分、心肌线粒体Drp1表达差异无统计学意义(P>0.05),心肌病理学损伤未见明显减轻。结论糖尿病因素取消七氟烷后处理对心肌保护作用的机制可能与抑制Drp1表达下调有关。

Objective To evaluate the relationship between dynamin-related protein 1 expression(Drp1)and diabetic mellitus-casused abolition of cardioprotection induced by sevoflurane postconditioning in rats.Methods Thirty-six clean-grade healthy adult male Wistar rats,aged 14-16 weeks,weighing 300-350 g,were divided into 3 groups(n=12 each)using a random number table method:sham operation group(group S),ischemia-reperfusion(I/R)group,and sevoflurane postconditioning group(group SPC).Thirty-six clean-grade healthy adult male GK diabetic rats,aged 14-16 weeks,weighing 300-350 g,were divided into 3 groups(n=12 each)using a random number table method:diabetes mellitus sham operation group(group DM+S),diabetes mellitus plus I/R group(group DM+I/R),and diabetes mellitus plus sevoflurane postconditioning group(group DM+SPC).The model of myocardial I/R injury was established by ligating the left anterior descending branch of the coronary artery for 30 min followed by 120-min reperfusion in anesthetized animals.In SPC and DM+SPC groups,2.4%sevoflurane was inhaled for 15 min starting from 1 min before reperfusion.Six rats were randomly selected at 120 min of reperfusion,and arterial blood samples were taken for determination of serum LDH activity by colorimetric method.The rats were then sacrificed,the ischemic regions of the left ventricle were taken,the ATP content was determined by colorimetry,the ultrastructure of myocardial mitochondria was observed using transmission electron microscopy,the degree of damage to mitochondria was determined by Flameng mitochondrial score,and Drp1 expression was detected by Western blot.Another 6 rats were randomly selected at 120 min of reperfusion and sacrificed,and myocardial tissues were taken for determination of myocardial infarct size by TTC staining,and the percentage of myocardial infarct size was calculated.Results Compared with group S,the serum LDH activity and percentage of myocardial infarct size were significantly increased,the ATP content in myocardial tissues was decreased,Flameng mitochondrial score was increased,and myocardial mitochondrial Drp1 expression was up-regulated in I/R and SPC groups(P<0.05).Compared with group I/R,the serum LDH activity and percentage of myocardial infarct size were significantly decreased,the ATP content in myocardial tissues was increased,Flameng mitochondrial score was decreased,and myocardial mitochondrial Drp1 expression was down-regulated(P<0.05),and myocardial pathological damage was attenuated in group SPC.Compared with group DM+S,the serum LDH activity and percentage of myocardial infarct size were significantly increased,Flameng mitochondrial score was increased,the ATP content in myocardial tissues was decreased,and myocardial mitochondrial Drp1 expression was up-regulated in group DM+I/R(P<0.05).Compared with group DM+I/R,no significant change was found in serum LDH activity,percentage of myocardial infarct size,Flameng mitochondrial score,the ATP content in myocardial tissues,and myocardial mitochondrial Drp1 expression(P>0.05),and no significantly attenuated myocardial pathological damage was found in group DM+SPC.Conclusion The mechanism by which diabetes mellitus abolishes cardioprotection induced by sevoflurane post-conditioning may be related to inhibiting down-regulation of Drp1 expression in rats.