Ⅰ型组蛋白去乙酰化酶在糖尿病大鼠心肌缺血再灌注损伤中的作用及其与AMPK∕mTOR信号通路的关系

Role of class Ⅰ histone deacetylase in myocardial ischemia-reperfusion injury in diabetic rats and the relationship with AMPK∕mTOR signaling pathway

目的 评价Ⅰ型组蛋白去乙酰化酶( HDAC)在糖尿病大鼠心肌缺血再灌注损伤中的作用及其与AMP依赖的蛋白激酶∕哺乳动物雷帕霉素靶蛋白(AMPK∕mTOR)信号通路的关系.方法SPF级健康成年雄性SD大鼠,体重210~220 g,腹腔注射柠檬酸盐-链脲佐菌素60 mg∕kg,建立Ⅰ型糖尿病模型,8周后用于实验.取糖尿病大鼠48只,采用随机数字表法分为4组( n=12):假手术组(S组) 、心肌缺血再灌注组(I∕R组)、心肌缺血再灌注+I型HDAC抑制剂MS-275组( I∕R+MS组)和心肌缺血再灌注+MS-275+AMPK抑制剂Compound C组( I∕R+MS+CC组).采用结扎冠状动脉左前降支45 min,再灌注180 min的方法制备心肌缺血再灌注损伤模型. I∕R+MS组腹腔注射MS-275 10 mg∕kg,连续7 d,1次∕d,给药结束后制备心肌缺血再灌注损伤模型;I∕R+MS+CC组于缺血前30 min静脉注射AMPK抑制剂Compound C 0.5 mg∕kg.于再灌注结束时,处死6只大鼠,测定心肌梗死体积;另取6只大鼠,右颈内动脉采血后处死,采用ELISA法检测血清CK-MB和LDH的水平,West-ern blot法测定心肌组织磷酸化AMPK (p-AMPK)、AMPK、磷酸化mTOR(p-mTOR)、mTOR、泛素结合蛋白(P62)和微管相关蛋白轻链3Ⅰ(LC3Ⅰ)、LC3Ⅱ的表达水平,计算 p-AMPK∕AMPK 比值、p-mTOR∕mTOR比值和LC3Ⅱ∕Ⅰ比值.结果 与SH组比较,I∕R组、I∕R+MS组和I∕R+M+CC组心肌梗死体积、血清CK-MB和LDH水平升高,I∕R+MS组心肌组织p-AMPK∕AMPK比值和LC3Ⅱ∕Ⅰ比值升高,p-mTOR∕mTOR比值降低,P62表达下调(P<0. 05),I∕R组和 I∕R+ M+CC组心肌组织 p-AMPK∕AMPK比值、p-mTOR∕mTOR比值、LC3Ⅱ∕Ⅰ比值和P62表达差异无统计学意义( P>0. 05);与I∕R组比较,I∕R+MS组心肌梗死体积、血清CK-MB和LDH水平降低,心肌组织p-AMPK∕AMPK比值和LC3Ⅱ∕Ⅰ比值升高,p-mTOR∕mTOR比值降低,P62表达下调(P<0. 05),I∕R+M+CC组上述指标差异无统计学意义(P>0. 05);与I∕R+MS组比较,I∕R+M+CC组心肌梗死体积、血清CK-MB和LDH水平升高,心肌组织p-AMPK∕AMPK比值和LC3Ⅱ∕Ⅰ比值降低,p-mTOR∕mTOR比值升高,P62表达上调( P<0. 05).结论 Ⅰ型HDAC通过增强AMPK∕mTOR信号通路调控的细胞自噬水平,参与糖尿病大鼠心肌缺血再灌注损伤.

Objective To evaluate the role of classⅠhistone deacetylase (HDAC) in myocardial ischemia-reperfusion (I∕R) injury in diabetic rats and the relationship with adenosine monophosphate-acti- vated protein kinase (AMPK)∕mammalian target of rapamycin (mTOR) signaling pathway. Methods SPF healthy adult male Sprague-Dawley rats, weighing 210-220 g, were used in this study. Type I diabetes mellitus was induced by single intraperitoneal injection of streptozotocin dissolved in citrate buffer 60 mg∕kg, and 8 weeks later the rats with type I diabetes mellitus were used for experiment. Forty-eight diabetic rats were divided into 4 groups (n=12 each) by using a random number table method: sham operation group (group S), myocardial I∕R group (group I∕R), myocardial I∕R plus class I HDAC inhibitor MS-275 group (group I∕R+MS) and myocardial I∕R plus MS-275 plus AMPK inhibitor Compound C group ( group I∕R+MS+CC). Myocardial I∕R was induced by ligation of the left anterior descending branch of the coronary ar-tery for 45 min followed by 180 min of reperfusion in anesthetized rats. In group I∕R+MS, MS-275 10 mg∕kg was intraperitoneally injected once a day for 7 consecutive days, and myocardial I∕R was produced after the end of administration. AMPK inhibitor Compound C 0. 5 mg∕kg was intravenously injected at 30 min before ischemia in group I∕R+MS+CC. Six rats were sacrificed at the end of reperfusion for determina-tion of myocardial infarct size. Another 6 rats were selected at the end of reperfusion and sacrificed for deter-mination of the level of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in serum (by en-zyme-linked immunosorbent assay), expression of AMPK, phosphorylated AMPK ( p-AMPK), mTOR, phosphorylated mTOR (p-mTOR), ubiquitin-binding protein P62 (P62), microtubule-associated protein 1 light chain 3 Ⅰ(LC3 Ⅰ) and LC3Ⅱ in myocardial tissues (by Western blot). The ratios of p-AMPK∕AMPK, p-mTOR∕mTOR and LC3Ⅱ∕Ⅰwere calculated. Results Compared with group S, the myocardial infarct size and levels of serum CK-MB and LDH were significantly increased in I∕R, I∕R+MS and I∕R+M+CC groups, the ratios of p-AMPK∕AMPK and LC3Ⅱ∕Ⅰwere significantly increased, p-mTOR∕mTOR ratio was decreased, and P62 expression was down-regulated in group I∕R+MS (P<0. 05), and no significant change was found in p-AMPK∕AMPK ratio, p-mTOR∕mTOR ratio, LC3Ⅱ∕Ⅰ ratio or P62 expression in I∕R and I∕R+M+CC groups (P>0. 05). Compared with group I∕R, the myocardial infarct size and levels of serum CK-MB and LDH were significantly decreased, the ratios of p-AMPK∕AMPK and LC3Ⅱ∕Ⅰwere in-creased, p-mTOR∕mTOR ratio was decreased, and P62 expression was down-regulated in group I∕R+MS (P<0. 05), and no significant change was found in the parameters mentioned above in group I∕R+M+CC (P>0. 05). Compared with group I∕R+MS, the myocardial infarct size and levels of serum CK-MB and LDH were significantly increased, the ratios of p-AMPK∕AMPK and LC3Ⅱ∕Ⅰ were decreased, p-mTOR∕mTOR ratio was increased, and P62 expression was up-regulated in group I∕R+M+CC ( P<0. 05). Con-clusion Class Ⅰ HDAC is involved in myocardial I∕R injury through enhancing AMPK∕mTOR signaling pathway-regulated level of autophagy in diabetic rats.