mTOR信号通路诱导的自噬在心肌细胞缺氧损伤中的作用研究

目的:探讨自噬在心肌细胞缺氧损伤中的作用及分子机制。方法:体外分离培养乳鼠心肌细胞,体外建立缺氧/去血清(H/SD)模型以模拟在体的缺血环境。分别给予自噬抑制剂3-甲基腺嘌呤(3MA,5 mM)和mTOR抑制剂雷帕霉素(1.0μg/L)调节心肌细胞自噬水平。分别采用TUNEL染色检测心肌细胞凋亡,Western blot方法检测心肌细胞蛋白表达水平。结果:H/SD损伤可以显著诱导心肌细胞自噬水平(P0.05),并且细胞自噬水平可以被3-MA及雷帕霉素调节。同时,H/SD可以显著增加心肌细胞凋亡(P0.05),而给予3-MA抑制自噬水平可以减少细胞凋亡(P0.05)。相反,雷帕霉素增加自噬同样可以加重缺氧导致的心肌细胞凋亡(P0.05)。H/SD损伤过程中,心肌细胞mTOR信号通路被激活,而自噬抑制剂3-MA可以显著提高缺氧条件下心肌细胞中p-mTOR(Ser2448)的表达水平(P0.05),并增加mTOR下游分子p-p70S6k(P0.05)和p-S6(P0.05)的表达。结论:mTOR信号通路诱导的细胞自噬可能参与了缺氧损伤诱导的心肌细胞凋亡。

The Effect of mTOR induced Autophagy on the Apoptosis of Cardiomyocytes induced by Hypoxia

ABSTRACT Objective: To investigate the potential role of autophagy on apoptosis in cardiomyocytes(CMs) induced by hypoxic in- jury. Methods: CMs were cultured in normal or hypoxia/serum deprivation (H/SD) medium for 24 h. The autophagy state was regulated by 3-Methyladenine (3MA) and rapamycin administration. Furthermore, TUNEL assay was performed to determine apoptosis in CMs. Moreover, Western blot assay was performed to assess the expressions of signaling proteins. Results: Hypoxic stress increased autophagy and apoptosis in CMs. Meanwhile, hypoxia increased the activation of mTOR signaling. Moreover, the apoptosis in CMs induced by hy- poxia injury was abolished by 3-MA, whereas was aggravated by rapamycin. Conclusion: Our data provide evidence that mTOR signal- ing induced autophagy may play an essential role in the apoptosis of CMs induced by hypoxic injury.