二甲双胍通过mTOR信号通路调节自噬减轻心肌梗死小鼠心肌损伤

Metformin alleviates myocardial injury in mice with myocardial infarction by regulating autophagy through mTOR signaling pathway

目的通过在体实验探究二甲双胍(Met)通过mTOR信号通路调节自噬减轻小鼠心肌梗死(MI)损伤中的作用。方法将80只8周龄C57BL/6小鼠随机分为假手术组(Sham组)、单纯二甲双胍[300 mg/(kg·d)]处理组(Met组)、MI组和二甲双胍[300 mg/(kg·d)]干预MI组(MI+Met组)。采用冠状动脉左前降支结扎术制备MI模型,计算模型各组死亡率差别,绘制生存分析曲线,HE染色观察心肌组织形态学变化,试剂盒检测各组血清中肌酸激酶同工酶(CKMB)的含量,TUNEL染色检测各组细胞凋亡情况,蛋白印迹法(Western-blot)检测自噬相关蛋白p62、Beclin1和mTOR磷酸化的表达,免疫荧光染色法检测LC3B的表达。结果与Sham组相比,Met组各项指标均无明显变化;MI组心肌纤维断裂明显、排列紊乱,纤维化程度增加、血清中CKMB含量明显增加(P<0.05)、TUNEL阳性细胞明显增多(P<0.05)、Beclin1表达显著升高(P<0.05)、磷酸化mTOR的表达降低(P<0.05)、p62表达显著降低(P<0.05)、免疫荧光结果显示LC3B表达增加(P<0.05);Met+MI组可明显减轻心肌纤维的断裂及纤维化、降低血清中CKMB含量(P<0.05)、减少TUNEL阳性细胞数(P<0.05)、减少Beclin1的表达(P<0.05)、降低免疫荧光染色中LC3B的表达(P<0.05)。结论二甲双胍通过调节mTOR信号介导的自噬可减轻小鼠心脏MI损伤。

Objective To investigate the role of metformin(Met)in mitigating myocardial infarction injury in mice by regulating autophagy through mTOR signaling pathway in vivo.Methods A total of 80 eight-week-old C57 BL/6 mice were randomly divided into sham group(Sham),metformin alone(300 mg/kg d)treatment group(Met),myocardial infarction group(MI)and metformin(300 mg/kg d)intervention group(MI+Met).Myocardial infarction model was established by ligation of left anterior descending branch of coronary artery.Mortality difference of each group was calculated.Survival analysis curve was drawn.Myocardial histomorphological changes were observed by HE staining.CKMB content in serum of each group was detected by kit.Apoptosis was detected by TUNEL staining and Western-blot.The phosphorylation of autophagy-related proteins p62,Beclin1 and mTOR was detected,and the expression of LC3 B was detected by immunofluorescence staining.Results Compared with Sham group,there was no significant change in all indexes in Met group.Myocardial fibrosis in MI group was disordered with increased fibrosis degree,and serum CKMB content increased significantly(P<0.05),TUNEL positive cells increased significantly(P<0.05)and Beclin1 expression increased significantly(P<0.05),while phosphorylated mTOR expression decreased(P<0.05).The expression of p62 was significantly decreased(P<0.05),and the expression of LC3 B was increased(P<0.05)by immunofluorescence.Met could significantly reduce the breakage and fibrosis of myocardial fibers,decrease the content of CKMB in serum(P<0.05),the number of TUNEL-positive cells(P<0.05),the expression of Beclin1(P<0.05),and the expression of LC3 B in immunofluorescence staining(P<0.05)during myocardial infarction injury.Conclusion Metformin can alleviate myocardial infarction injury in mice by regulating autophagy mediated by mTOR signal.