周期性机械应力诱导的自噬抑制caspase通路依赖的成肌细胞凋亡

目的:观察大鼠成肌细胞(L6 myoblast,L6)在周期性机械应力作用下细胞凋亡与自噬的水平变化,探讨机械应力诱导的细胞自噬对凋亡的作用.方法:构建大鼠成肌细胞力学刺激模型,加载参数为细胞拉伸形变率15%,加力周期包括3s拉伸及3s舒张,加力时间为6、12、24h,以不加力0h组为对照组.通过Hoechst染色及AnnexinV-F1TC/PI染色观察各组细胞凋亡情况;通过MDC染色及透射电镜(transmission electron microscopy,TEM)评价各组细胞自噬变化;应用Western免疫印迹检测各组凋亡相关蛋白caspase-3、caspase-9、Bcl-2、Bax及自噬相关蛋白LC3Ⅱ、LC3 Ⅰ、P62表达变化.加入自噬抑制剂3-MA后重复实验,检测各组细胞凋亡情况.采用SPSS 17.0软件包对数据进行统计学分析.结果:Hoechst染色及AnnexinV-FITC/PI流式结果表明,大鼠成肌细胞在周期性应力诱导下发生凋亡,24 h时达到凋亡最大值.Western免疫印迹结果显示,凋亡相关蛋白表达水平随加力时间延长而逐渐升高,caspase抑制剂z-VAD-fmk有效抑制应力诱导的细胞凋亡.MDC染色及TEM扫描电镜显示,大鼠成肌细胞在周期性应力诱导下发生自噬,24h细胞自噬达到最大值.Western免疫印迹结果显示,LC3Ⅱ/LC3 Ⅰ随加力时间延长而升高,P62的表达随着加力时间延长而降低.加入自噬抑制剂3-MA后重复试验,发现应力诱导的成肌细胞凋亡被明显抑制.结论:周期性机械应力诱导大鼠成肌细胞发生自噬与caspase通路依赖的细胞凋亡,细胞自噬作为一种代偿机制,保护性地抑制应力诱导的细胞凋亡.

Autophagy induced by cyclic mechanical stretch inhibited caspase-dependent apoptosis of myoblasts

PURPOSE: The aim of this study was to investigate the molecular mechanism of autophagy and apoptosis induced by cyclic mechanical stretch and the potential role of autophagy in stretch-induced apoptosis of myoblasts. METHODS: Loading model of L6 myoblasts was established in vitro. The cells were then subjected to cyclic mechanical stretch involving 3 s of 15% stretch alternating with 3 s of relaxation. The cells were collected after mechanical stretch for 6 h, 12 h and 24 h, respectively. Control cells were cultured on the same plates without mechanical strain. Apoptosis of myoblasts was assessed by Hoechst 33258 staining and Annexin V binding and propidium iodide staining. Autophagy was determined by MDC staining and transmission electron microscopy(TEM). The level of proteins associated with apoptosis and autophagy was detected by Western blot. The data were analyzed with SPSS 17.0 software package. RESULTS: The results of Hoechst 33258 staining and Annexin V binding and propidium iodide staining indicated that mechanical stretch notably induced apoptosis of myoblasts. Caspase inhibitor z-VAD-fmk effectively abrogated apoptosis of myoblasts, indicating mechanical stretch induced caspase-dependent apoptosis. In addition, the results of TEM, MDC staining and Western blot proved that mechanical stretch elicited autophagy of myoblasts. Inhibition of autophagy using 3-MA enhanced caspase-dependent apoptosis induced by mechanical stretch. CONCLUSIONS: Cyclic mechanical stretch induced apoptosis and autophagy of myoblasts time-dependently. Protective autophagy, acting as the compensatory mechanism, inhibited caspase-dependent apoptosis induced by mechanical stretch.