调控自噬对H9c2心肌细胞缺氧／复氧损伤的影响

目的探讨调控自噬水平对H9c2心肌细胞缺氧／复氧（H／R）损伤的影响及意义。方法将H9c2心肌细胞缺氧2h／复氧4h，建立H／R损伤模型。以3-甲基腺嘌呤（3-MA）为自噬特异抑制剂和雷帕霉素为自噬增强剂，试验随机分为四组：正常对照组（C组）、H／R组、H／R＋100mol／L3-MA组（M＋H／R组）、H／R＋100nmol／L雷帕霉素（R＋H／R组），应用MTT法检测细胞活力，透射电镜检测心肌细胞自噬小体，流式细胞技术检测细胞凋亡比例，Westernblot法检测自噬相关蛋白LC3、Beclin1，凋亡相关蛋白Bcl-2、Bax及下游活性片段Caspase-9、Caspase-3蛋白表达。结果H／R组明显诱导H9c2心肌细胞自噬发生、细胞活力下降、凋亡增加（P〈O．01）．Westernblot检测发现，Bax、Caspase-9、Caspase-3活性片段蛋白表达明显增加，Bcl-2表达明显抑制，Bax／Bcl-2比值、活化蛋白Caspase-3、Caspase-9表达增加（P〈O．01）；M＋H／R组H／R损伤作用明显减弱，线粒体凋亡通路及下游蛋白表达抑制（P〈O．01）；而R＋H／R组线粒体凋亡通路进一步激活，促进细胞凋亡发生（P〈O．01）。结论自噬在H9c2心肌细胞H／R损伤中起到致命性作用，抑制自噬可保护心肌细胞H，R氧化应激损伤，其机制与抑制线粒体凋亡通路有关。

The Effects of Regulation of Autophagy on H9c2 Cardiomyocytes Exposed to Hypoxia / Reoxygenation Injury

Objective To investigate the effects of regulation of autophagy on H9c2 cardiomyocytes exposed to hy- poxia/reoxygenation （H / R） injury and its significance. Methods H9c2 cardiomyocyte H / R injury model was estab- lished by hypoxia for 2 hours and reoxygenation for 4 hours. 3-methyladenine （3-MA） was used as autophagy inhibitor, and rapamycin as autophagy inducer. Cultured cardiomyocytes were randomly divided into four groups： sham group, H / R group, H / R＋ 3MA-pre-treated group（100 mol / L 3-MA） and H / R＋ rapamycin-pre-treated group （100 nmol / L ra- pamycin）. Cell viability was measured by MTT, autophagic vacuoles by transmission electron microscopic analysis, apop- tosis rate of cardiomyocyte by flow cytometry analysis, and protein expressions of autophagy-related proteins LC3 and Beclin 1, apoptosis-related proteins Bcl-2, Bax and cleaved Caspase-9, Caspase-3 by Western blot. Results H / R strongly upregulated autophagy, induced myocyte apoptosis, and reduced cell viability （P〈0.01）. Western blot showed that H/R inhibited Bcl-2 expression, promoted Bax, caspase-9 and -3 activation expression （P〈0.01）. Autophagy in- hibitor 3-MA significantly attenuated H / R-induced injury, inhibited mitochondria mediated apoptosis and downstream protein expression （P〈0.01）. Autophagy inducer rapamycin exacerbated cell apoptosis via mitochondrial apoptotic path- way（P〈0.01）. Conclusion Enhanced autophagy plays detrimental role during H9c2 cardiomyocyte H / R injury. Inhibit- ing autophagy with 3-MA may protect against H / R injury through attenuating mitochondria apoptotic pathway.