西罗莫司对鱼藤酮诱导PC12细胞凋亡的抑制作用

Inhibitory effect of sirolimus on rotenone-induced PC12 cell apoptosis

目的观察西罗莫司干预鱼藤酮诱导PC12细胞凋亡的作用,以探讨西罗莫司用于治疗帕金森病的可能性及机制。方法 PC12细胞处理分为正常对照组、鱼藤酮50nmol.L-1组和西罗莫司25,50,100和200μg.L-1组。PC12细胞加入西罗莫司25,50,100和200μg.L-1预处理12h后,再加入鱼藤酮50nmol.L-1作用24h。采用4′,6-二脒-2-苯基吲哚(DAPI)免疫荧光实验检测细胞凋亡形态变化;AnnexinⅤ-FITC/PI染色法检测细胞凋亡率;罗丹明123检测线粒体膜电位;吖啶橙荧光染色法检测自吞噬体形成;Western印迹法检测胱天蛋白酶3和微管相关蛋白轻链3(LC3)表达。结果与鱼藤酮模型组比较,西罗莫司50,100和200μg.L-1组细胞凋亡率分别降低了20.6%,35.4%和40.2%(P<0.05,P<0.01);西罗莫司25,50,100和200μg.L-1组可以升高鱼藤酮导致的线粒体膜电位降低,分别为1.8,2.2,2.4和3.2倍。西罗莫司100μg.L-1预处理促使LC3由Ⅰ型向Ⅱ型转变,胱天蛋白酶3的20ku活性片段降低16.2%。结论西罗莫司通过增强失调线粒体的自吞噬降解作用,降低线粒体相关的促凋亡因子的释放和激活,进而抑制依赖胱天蛋白酶凋亡途径的激活而对鱼藤酮诱导的细胞凋亡具有保护作用,提示西罗莫司有治疗帕金森病的可能。

OBJECTIVE To investigate the intervention of sirolimus in rotenone-induced apoptosis and its mechanisms.METHODS PC12 cells were divided into normal control,rotenone 50 nmol·L-1 model,and sirolimus 25,50,100 and 200 μg·L-1 groups.Cells were exposed to sirolimus 25,50,100 and 200 μg·L-1 for 12 h,and again to rotenone 50 nmol·L-1 for 24 h,respectively.Morphologic changes in PC12 cells were tested by immunofluorescence method with 4′,6-diamidino-2-phenylindole fluorescein staining.The apoptotic rates and the mi...