调控自噬对H9c2心肌细胞缺氧/复氧损伤的影响

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

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+100 mol/L 3-MA组(M+H/R组)、H/R+100 nmol/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,Western blot法检测自噬相关蛋白LC3、Beclin 1,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<0.01),Western blot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<0.01);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<0.01);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<0.01)。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。

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.