调控自噬对H9c2心肌细胞缺氧/复氧损伤的影响

摘要

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). cellviability 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 cellviability (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 cellapoptosis 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.