摘要
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。
The purpose of the present study is to investigate whether glucose-regulated protein 75 (GRP75) overexpression inhibits apoptosis induced by glucose deprivation through Raf/Mek/Erk1/2 signaling pathway. After pretreatment with Mek-specific inhibitor U0126, GRP75 overexpressing PC12 cells were incubated in glucose-free DMEM medium for indicated time (6, 12 and 24 h). And DMSO-treated GRP75 overexpressing PC12 cells were applied as control. Western blot was used to determine the expression and phosphorylation level of Erk1/2. MTT assay was used to measure cell viability. Hoechst 33258 staining and flow cytometry using pro- pidium iodide (PI) staining was used to analysis apoptosis. Immunofluorescence with antibody against cytochrome c (Cyt c) was used to detect Cyt c release from mitochondrion. The results showed U0126 prevented the activation of Erk1/2 maintained by GRP75, but the total Erk1/2 expression was not affected. U0126-treated group showed lower cell viability and higher apoptotic rate compared with control group. Immunofluorescence indicated the delay in release of Cyt c was blocked by U0126. These results suggest U0126 pre- vents protective effect of GRP75 on PC12 cells by inhibiting Erk1/2 phosphorylation, which certifies that GRP75 can inhibit the mito- chondria-dependent apoptotic pathway through Raf/Mek/Erk1/2 signaling cascade.
出处
《生理学报》
CAS
CSCD
北大核心
2011年第1期69-74,共6页
Acta Physiologica Sinica
基金
supported by the National Natural Science Foundation of China (No. 81000978)