二甲双胍促进AMPK、TFEB表达改善自噬减轻大鼠脑缺血再灌注损伤

Metformin promotes the expression of AMPK and TFEB to improve autophagy on cerebral ischemia-reperfusion injury in rats

目的:探究二甲双胍(Met)对大鼠脑缺血再灌注损伤的影响及机制。方法:建立大鼠脑缺血再灌注模型,随机分为假手术组、缺血再灌注组、Met预处理组[造模前3周腹腔注射Met 100 mg/(kg·d)],TTC染色检测各组大鼠脑梗死体积改变,HE染色观察脑组织病理变化;蛋白免疫印迹法检测各组大鼠脑组织中Beclin-1、LC3-Ⅱ/LC3-Ⅰ、p62等自噬相关蛋白以及AMP活化蛋白激酶(AMPK)、p-AMPK(Thr172)、转录因子EB(TFEB)相关通路蛋白含量。结果:与假手术组相比,缺血再灌注组大鼠脑组织病理损伤严重,脑梗死体积增高(P<0.001);与缺血再灌注组相比,Met预处理组大鼠脑梗死体积减少(P<0.01),脑组织病理损伤减轻。与假手术组相比,缺血再灌注组大鼠LC3-Ⅱ/LC3-Ⅰ和Beclin-1蛋白水平表达减少(P<0.05),p62蛋白表达增多(P<0.001);与缺血再灌注组相比,Met预处理组大鼠Beclin-1、LC3-Ⅱ/LC3-Ⅰ蛋白表达均增多(P<0.05),p62蛋白表达减少(P<0.01);与假手术组相比,缺血再灌注组的p-AMPK(Thr172)、TFEB的蛋白表达水平减少(P<0.05);与缺血再灌注相比,Met预处理组的p-AMPK(Thr172)、TFEB的蛋白表达水平均增多(P<0.05);各组AMPK蛋白表达差异无统计学意义(P>0.05)。结论:Met促进AMPK、TFEB表达增强自噬改善脑缺血再灌注损伤。

Objective:To investigate the effect of metformin on and mechanism of metformin in rats with cerebral ischemia-reperfusion injury.Methods:Rat models of cerebral ischemia reperfusion were established,and then randomized to sham operation group,ischemia reperfusion group,and metformin preconditioned group(metformin 100 mg/(kg·d)was intraperitoneally injected 3 weeks before modeling).TTC staining was used to detect the volume change of cerebral infarction in rats in each group,and HE staining was performed to observe the pathological changes of brain tissues.Western blotting was carried out to detect the contents of LC3-Ⅱ/LC3-Ⅰ,Beclin-1,p62 and other proteins as well as AMPK,p-AMPK(Thr172),TFEB proteins in brain tissue of rats in each group.Results:Compared with the sham operation group,pathological damage to the brain tissue was severer,and the volume of cerebral infarction was increased in rats in the ischemia-reperfusion group(P<0.001).Rats in metformin preconditioned group had decreased cerebral infarction volume and minor pathological damage to the brain tissues compared with those in the ischemia reperfusion group(P<0.01).The expression of LC3-Ⅱ/LC3-Ⅰand Beclin-1 protein was decreased in the ischemia-reperfusion group(P<0.05),whereas p62 protein expression was increased compared to the sham operation group(P<0.001).Rats in metformin preconditioned group had upregulated expression of Beclin-1,LC3-Ⅱ/LC3-Ⅰprotein(P<0.05),yet downregulated expression of p62 protein compared with those in the ischemia-reperfusion group(P<0.01).The protein expression of p-AMPK(Thr172)and TFEB was decreased in the ischemia-reperfusion group,whereas protein expression levels of p-AMPK(Thr172)and TFEB were increased in metformin preconditioned group compared to the sham operation group(P<0.05).There was no significant difference in AMPK among groups(P>0.05).Conclusion:Metformin can promote the expression of AMPK and TFEB,enhance autophagy and improve cerebral ischemia-reperfusion injury.