二甲双胍预处理对糖尿病大鼠心肌缺血再灌注损伤时AMPK/PINK1信号通路的影响

Effect of metformin preconditioning on AMPK/PINK1 signaling pathway during myocardial ischemia-reperfusion injury in diabetic rats

目的:评价二甲双胍预处理对糖尿病大鼠心肌缺血再灌注损伤时单磷酸腺苷激活蛋白激酶(AMPK)/PTEN诱导假定激酶1(PINK1)信号通路的影响。方法:清洁级健康雄性SD大鼠36只,6周龄,体质量120~160 g,采用随机数字表法分为3组(n=12):糖尿病假手术组(DS组)、糖尿病心肌缺血再灌注组(DI/R组)和糖尿病心肌缺血再灌注+二甲双胍预处理组(DI/R+Met组)。高脂高糖饲料喂养4周后,通过单次腹腔注射1%链脲佐菌素40 mg/kg制备2型糖尿病模型。采用阻断左冠状动脉前降支30 min,再灌注120 min的方法制备大鼠心肌缺血再灌注损伤模型。DI/R+Met组于心肌缺血前1周给予每天1次二甲双胍200 mg/kg灌胃处理。于再灌注120 min时采集股静脉血样,采用ELISA法检测血清CK-MB和cTnI浓度;随后处死大鼠取心肌组织,采用HE染色法观察病理学结果;采用伊文氏蓝和TTC双重染色法确定心肌梗死体积百分比;采用Western blot法检测心肌自噬相关蛋白Beclin-1、PINK1、磷酸化AMPK(p-AMPK)的表达和微管相关蛋白1轻链3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ)的比值。结果:与DS组比较,DI/R组和DI/R+Met组心肌梗死体积百分比、血清CK-MB和cTnI浓度升高,心肌Beclin-1、p-AMPK和PINK1表达上调,LC3Ⅱ/Ⅰ比值升高(P<0.05),病理学损伤加重;与DI/R组比较,DI/R+Met组心肌梗死体积百分比、血清CK-MB和cTnI浓度降低,心肌Beclin-1、p-AMPK和PINK1表达上调,LC3Ⅱ/Ⅰ比值升高(P<0.05),病理学损伤减轻。结论:二甲双胍预处理减轻糖尿病大鼠心肌缺血再灌注损伤的机制与激活AMPK/PINK1信号通路上调线粒体自噬水平有关。

Objective To evaluate the effect of metformin preconditioning on adenosine monophosphate-activated protein kinase(AMPK)/PTEN-induced putative protein kinase(PINK1)signaling pathway during ischemia-reperfusion(I/R)injury in diabetic rats.MethodsThirty-six clean-grade healthy male Sprague-Dawley rats,aged 6 weeks,weighing 120-160 g,were divided into 3 groups(n=12 each)by the random number table method:diabetic sham operation group(DS group),diabetic myocardial I/R group(DI/R group)and diabetic myocardial I/R+metformin preconditioning group(DI/R+Met group).After 4 weeks of feeding a high-fat and high-glucose diet,the model of type 2 diabetes mellitus was induced by a single intraperitoneal injection of 1%streptozotocin 40 mg/kg.The myocardial I/R injury was induced by blocking the anterior descending branch of the left coronary artery for 30 min followed by 120-min reperfusion in anesthetized animals.In DI/R+Met group,metformin 200 mg/kg was given by intragastric gavage once a day within 1 week before myocardial ischemia.Blood samples from the femoral vein were collected at 120 min of reperfusion for determination of the serum creatine kinase isoenzymes(CK-MB)and cardiac troponin I(cTnI)concentrations by enzyme-linked immunosorbent assay.Then the rats were sacrificed and myocardial tissues were obtained for examination of the pathological changes(by HE staining)and for determination of the percentage of myocardial infarct size(by the double staining of Ewan blue and TTC)and expression of myocardial autophagy-related protein Beclin-1,PTEN-induced putative kinase 1(PINK1),phosphorylated 5′-adenosine monophosphate-activating protein kinase(p-AMPK),and ratio of microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ)(by Western blot).ResultsCompared with DS group,the percentage of myocardial infarct size and serum CK-MB and cTnI concentrations were significantly increased,the expression of Beclin-1,p-AMPK and PINK1 in myocardial tissues was up-regulated,the ratio of LC3II/I was increased(P<0.05),and the pathological changes were aggravated in DI/R group and DI/R+Met group.Compared with DI/R group,the percentage of myocardial infarct size and serum CK-MB and cTnI concentrations were significantly decreased,the expression of Beclin-1,p-AMPK and PINK1 in myocardial tissues was up-regulated,the ratio of LC3Ⅱ/Ⅰwas increased(P<0.05),and the pathological changes were significantly reduced in DI/R+Met group.ConclusionsThe mechanism by which metformin preconditioning reduces myocardial I/R injury is related to activation of AMPK/PINK1 signaling pathway and up-regulation of mitochondrial autophagy in diabetic rats.