二甲双胍调控PI3K/Akt通路对心肌缺血再灌注模型大鼠心肌细胞自噬的影响研究

Effect of Metformin on the Autophagy of Myocardial Cells in Myocardial Ischemia-reperfusion Model Rats by Regulating PI3K/Akt Pathway

目的:探讨二甲双胍调控磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)通路对心肌缺血再灌注模型大鼠心肌细胞自噬的影响。方法:将60只SD大鼠随机分为假手术组、模型组、二甲双胍低(150 mg·kg^-1)、中(300 mg·kg^-1)、高(500 mg·kg^-1)剂量组、自噬激动剂组(雷帕霉素,5 mg·kg^-1),制备心肌缺血再灌注大鼠模型,药物处理组于建模前3 d以不同剂量二甲双胍及雷帕霉素每天灌胃治疗,模型组及假手术对照组采用等体积生理盐水灌胃,持续3 d。造模成功12 h后,处死大鼠,三苯基氯化四氮唑(TTC)染色观察各组大鼠心肌梗死面积,酶联免疫吸附实验(ELISA)试剂盒检测血清中磷酸肌酸同工酶(CK-MB)及肌钙蛋白I(cTnI)含量,免疫组织化学检测自噬相关蛋白Beclin-1、LC3、P62阳性细胞比例,蛋白免疫印迹(Western blot)检测自噬相关蛋白Beclin-1、LC3(包括LC3-Ⅰ、LC3-Ⅱ)、P62及PI3K/Akt通路蛋白p-PI3K、PI3K、Akt、p-Akt表达情况。结果:与假手术组比较,模型组大鼠心肌梗死面积、血清中CK-MB、cTnI含量、心肌组织中P62阳性细胞比例、P62、p-PI3K、p-Akt蛋白表达明显升高,心肌组织中Beclin-1、LC3阳性细胞比例、Beclin-1、LC3-Ⅱ蛋白表达明显降低(P<0.05);心肌组织中LC3-Ⅰ、PI3K、Akt蛋白表达差异无统计学意义(P>0.05)。与模型组比较,各药物处理组大鼠心肌梗死面积、血清中CK-MB、cTnI含量、心肌组织中P62阳性细胞比例、P62、p-PI3K、p-Akt蛋白表达明显降低,心肌组织中Beclin-1、LC3阳性细胞比例、Beclin-1、LC3-Ⅱ蛋白表达明显升高(P<0.05),且二甲双胍各组呈剂量依赖性。二甲双胍低、中剂量组与自噬激动剂组比较,以上各指标差异有统计学意义(P<0.05)。结论:二甲双胍通过促进心肌缺血再灌注模型大鼠心肌细胞自噬,发挥心肌保护作用,其机制可能与下调PI3K/Akt通路有关。

Objective:To investigate the effect of metformin on the autophagy of myocardial cells in myocardial ischemia-reperfusion model rats by regulating phosphatidylinositol 3-kinase(PI3K)/protein Kinase B(Akt)pathway.Methods:Sixty SD rats were randomly divided into the sham-operated group,the model group,metformin low(150 mg·kg^-1),medium(300 mg·kg^-1)and high(500 mg·kg^-1)dose groups and autophagic agonist group(rapamycin,5 mg·kg^-1).Myocardial ischemia-reperfusion rat model was established.The drug-treated groups were given different doses of metformin or rapamycin daily for 3 days before the model establishment.The model group and the sham-operated control group were intragastrically administered with saline with equal volume for 3 days.After 12 hours of successful modeling,the rats were sacrificed.The myocardial infarction area was observed after triphenyltetrazolium chloride(TTC)staining.Enzyme-linked immunosorbent assay(ELISA)kit was used to detect creatine phosphate isoenzyme(CKMB)and troponin I(cTnI)in serum.The positive cell proportion of autophagy-related proteins Beclin-1,LC3 and P62 were detected by immunohistochemistry.Western blot was used to detect the expressions of autophagy-related proteins(Beclin-1,LC3 and P62)and PI3K/Akt pathway proteins(p-PI3K,PI3K,Akt and p-Akt).Results:Compared with those in the sham-operated group,the infarct size,serum CK-MB,cTnI content,P62 positive cell ratio in myocardium and P62,p-PI3K,p-Akt protein expressions in the model group were significantly increased,Beclin-1,LC3 positive cell ratio and Beclin-1 and LC3-II protein expressions in myocardium were significantly decreased(P<0.05);there were no significant differences in the expressions of LC3-I,PI3K and Akt protein in myocardium(P>0.05).Compared with those in the model group,the myocardial infarction area,serum CK-MB,cTnI content,P62 positive cell proportion in myocardium and P62,p-PI3K and p-Akt protein expressions in the treated groups were significantly decreased,the expression of Beclin-1,LC3 positive cell proportion,Beclin-1 and LC3-II protein in myocardium were significantly increased(P<0.05),and metformin groups were dose dependent.Compared with the autophagy agonist group,there were significant differences in the above indicators between the low and medium dose metformin groups and the autophagy agonist group(P<0.05).Conclusion:Metformin can protect myocardium by promoting the autophagy of myocardial cells in rats with myocardial ischemia-reperfusion injury,and the mechanism may be related to the down-regulation of PI3K/Akt pathway.