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本文旨在探讨促存活信号通路Raf/Mek/Erk1/2是否参与了葡萄糖调节蛋白75(glucose-regulated protein75,GRP75)对缺糖诱导的细胞凋亡的抑制作用。GRP75过表达的PC12细胞给予Raf/Mek/Erk1/2通路抑制剂U0126预处理之后,无糖培养6、12和24h,同时以DMSO预处理的GRP75过表达PC12细胞组为对照。Western blot检测Erk1/2的磷酸化和表达水平,MTT实验检测细胞存活率,Hoechst 33258染色观察凋亡细胞核的形态学改变,流式细胞仪检测细胞亚二倍体峰,免疫荧光检测细胞色素c(cytochrome c,Cytc)向胞浆的弥散情况。结果显示:U0126在没有影响Erk1/2表达水平的前提下,阻断了GRP75对Erk1/2磷酸化水平的维持;U0126处理组的凋亡率明显高于对照组;U0126处理组Cytc从线粒体向胞浆释放的时间明显早于对照组,同时Cytc向胞浆的弥散程度大于对照组。以上结果提示,U0126通过抑制Erk1/2磷酸化,阻断了缺糖状态下GRP75对Cytc释放和细胞凋亡的抑制作用,这表明GRP75是通过Raf/Mek/Erk1/2级联反应抑制缺糖诱导的线粒体凋亡途径的进程。
This study aimed to investigate whether the pro-survival signaling pathway Raf / Mek / Erk1 / 2 is involved in the inhibitory effect of glucose-regulated protein 75 (GRP75) on glucose-induced apoptosis. After pretreatment with Raf / Mek / Erk1 / 2 pathway inhibitor U0126, PC12 cells overexpressing GRP75 were cultured for 6, 12 and 24 hours in the absence of glucose, respectively. Meanwhile, PC12 cells treated with DMSO pretreated with GRP75 were used as controls. The phosphorylation and expression of Erk1 / 2 were detected by Western blot. The cell viability was detected by MTT assay. The morphological changes of apoptotic nuclei were observed by Hoechst 33258 staining. The sub-diploid peak was detected by flow cytometry. The cytochrome c (cytochrome c, Cytc) to the cytoplasm of the dispersion. The results showed that U0126 blocked the phosphorylation of Erk1 / 2 by GRP75 without affecting the expression of Erk1 / 2. The apoptosis rate of U0126-treated group was significantly higher than that of the control group. Cytoplasmic release time was significantly earlier than the control group, while Cytc diffusion to the cytoplasm was greater than the control group. The above results suggest that U0126 blocks the Erk1 / 2 phosphorylation and blocks the inhibitory effect of GRP75 on Cytc release and apoptosis in the absence of glucose, which indicates that GRP75 inhibits glucose deprivation through the Raf / Mek / Erk1 / 2 cascade Induction of the mitochondrial apoptotic pathway.