EGCG-exosomes通过miR-30a/Beclin-1轴抑制自噬减轻心肌缺血/再灌注损伤

EGCG-exosomes attenuated myocardial ischemia/reperfusion injury by inhibiting autophagy through miR-30a/Beclin-1 axis

  • 摘要: 目的:探讨表没食子儿茶素没食子酸酯(EGCG)预处理心肌细胞分泌的外泌体(exosomes)对心肌缺血/再灌注(I/R)损伤的作用及分子机制。方法:体外培养H9c2心肌细胞,给予或不给予EGCG预处理,建立缺氧/复氧模型。提取、纯化外泌体,采用在体基因干扰或基因敲除技术,构建antagomiR-30a 和Beclin-1+/-小鼠,建立I/R 模型,缺血30 min 心肌注射外泌体,研究EGCG预处理心肌细胞分泌的exosomes(EGCG-exosomes)对I/R的作用。采用双荧光素酶报告基因实验检测miR-30a与Be-clin-1的相互作用,TTC染色法检测心肌梗死面积,苏木精—伊红(HE)染色观察心肌细胞形态变化;酶联免疫吸附试验(ELI-SA)法检测血清心肌肌钙蛋I(cTnI)含量;透射电子显微镜观察自噬小体数量;实时荧光定量聚合酶链式反应(RT-qPCR)、蛋白印迹法(western blotting法)检测miR-30a与自噬相关基因和蛋白的表达。结果:与I/R组相比,exosomes组自噬体数量减少,心肌损伤减轻。与exosomes组相比,EGCG-exosomes组心肌梗死面积缩小,cTnI含量降低,自噬体数量减少,Beclin-1和LC3-Ⅱ基因及蛋白表达下调,cathepsin D蛋白表达下调,p62基因及蛋白表达上调;沉默miR-30a促进心肌I/R自噬发生,给予EGCG-exosomes则逆转上述作用;此外,敲低Beclin-1与EGCG-exosomes有协同抑制自噬作用(均P< 0.05)。结论:EGCG-exosomes通过miR-30a/Beclin-1轴抑制自噬,减轻心肌I/R损伤。

     

    Abstract: Objective: To explore the effect and molecular mechanism of epigallocatechin gallate (EGCG) pre-treatment of exosomes secreted by cardiomyocytes on myocardial ischemia/reperfusion (I/R) injury by inhibiting autophagy through miR-30a/Beclin-1 axis.Methods: H9c2 cardiomyocytes were cultured in vitro and pretreated with or without EGCG to establish hypoxia/reoxygenation model.Exosomes were extracted and purified, and an-tagomiR-30a and Beclin-1+/-mice were constructed using in vivo gene interference or gene knockdown techniques to establish I/R models.Exosomes were injected into myocardium at 30 min after ischemia and the effect of EGCG pretreatment of cardiomyocyte-secreted exosomes(EGCG-exosomes) on I/R was studied.The interaction between miR-30a and Beclin-1 was detected by double luciferase reporter assay.TTC staining was used to detect the area of myocardial infarction.Hematoxylin-eosin(HE) staining was used to observe the morphological chang-es of myocardial cells.The content of cardiac troponin I(cTnI) in serum was detected by enzyme-linked immuno-sorbent assay(ELISA).The number of autophagosomes was observed by transmission electron microscopy.Real-time fluorescence quantitative polymerase chain reac-tion (RT-qPCR) and western blotting were used to de-tect the expressions of miR-30a and autophagy related genes and proteins.Results: Compared with the I/R group, the number of autophagosomes decreased in the exosomes group, and myocardial injury was attenuated; compared with the exosomes group, myocardial in-farct size was reduced, cTnI content and autophagosomes decreased, the expressions of Beclin-1 and LC3-II gene and protein and the expression of cathepsin D protein were down-regulated, and the expression of p62 gene and protein was up-regulated in the EGCG-exosomes group; silencing miR-30a promoted autophagy of myocardial I/R, while administration of EGCG-exosomes reversed the above effect; in addition, knockdown of Beclin-1 and EGCG-exosomes had a synergistic inhibitory effect on autophagy (all P< 0.05).Conclusion: EGCG-exosomes inhibit autophagy through miR-30a/Beclin-1 axis and attenuated myocardial ischemia/reperfusion injury.

     

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