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二甲双胍对H9c2心肌细胞缺氧复氧损伤中FUNDC1介导线粒体自噬的影响及其机制
作者:王鑫1  刘乃丰2 
单位:1. 东南大学 医学院, 江苏 南京 210009;
2. 东南大学附属中大医院 心血管内科, 江苏 南京 210009
关键词:心肌细胞 缺血再灌注 线粒体自噬 线粒体外膜蛋白 氧化应激 二甲双胍 
分类号:R972;R541
出版年·卷·期(页码):2020·39·第二期(156-162)
摘要:

目的:探讨二甲双胍对缺氧复氧条件下心肌细胞内线粒体外膜蛋白介导线粒体自噬的影响,明确二甲双胍对心肌细胞保护的作用机制。方法:对H9c2心肌细胞进行缺氧/复氧(H/R)干预建立心肌缺血再灌注损伤模型,利用二甲双胍预处理H9c2心肌细胞,通过免疫印迹法检测H/R组、二甲双胍组及对照组线粒体自噬受体蛋白FUNDC1、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、半胱天冬氨酸蛋白酶9(caspase9)、线粒体内膜转移酶23(TIM23)的表达,检测细胞线粒体膜电位及活性氧(ROS)水平,利用激光共聚焦检测细胞内线粒体自噬。结果:与对照组比较,H/R组的促凋亡蛋白caspase9表达增加,FUDNC1表达水平下降,细胞内ROS水平显著增加,线粒体膜电位降低;二甲双胍可逆转上述H/R导致的损伤作用,敲减FUNDC1的表达可抑制二甲双胍的保护作用;抑制腺苷酸活化蛋白激酶(AMPK)活性后,可逆转二甲双胍对FUNDC1表达的上调作用。结论:二甲双胍可通过AMPK/FUNDC1途径上调H/R条件下心肌细胞内被抑制的线粒体自噬,维持心肌细胞内线粒体膜电位,减轻细胞内的氧化应激,缓解H/R条件下心肌细胞的凋亡。

Objective: To investigate the effect and mechanism of metformin on FUNDC1 induced mitophagy in H9c2 cardiomyocytes during hypoxia and reoxygenation injury. Methods: The myocardial ischemia reperfusion injury model was established by hypoxia/reoxygenation(H/R) intervention on H9c2 cardiomyocyte. H9c2 cardiomyocytes were pretreated with metformin. The protein expressions of FUNDC1, p-AMPK, caspas9 and TIM23 were detected by Western blot. The mitochondrial membrane potential and reactive oxygen species(ROS) production were detected. Laser confocal was used to detect the number of mitochondrial autophagosomes. Results: The caspase9 and TIM23 protein expressions were increased in H/R group, while the protein expression of FUNDC1 was downregulated compared with the normal group. Meanwhile, the intracellular ROS level was significantly increased, and the mitochondrial membrane potential was lower in the H/R group. Furthermore, the mitophagy was significantly inhibited after H/R injury. Nevertheless, treatment of metformin reversed these effects of H/R injury on H9c2 cardiomyocytes. Knockdown of FUNDC1 expression could inhibit the protective effects of metformin. Inhibition of AMPK activity reversed the metformin induced FUNDC1 protein expression. Conclusion: Metformin can protect H9c2 cardiomyocytes from H/R injury via upregulating the FUNDC1 mediated mitophagy through AMPK/FUNDC1 pathway.

参考文献:

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