Seipin敲低对内质网应激通路诱导细胞凋亡的影响

Effect of Seipin Knockdown on Apoptosis Induced by Endoplasmic Reticulum Stress Pathway

目的:探讨Seipin敲低对肾上腺嗜铬细胞瘤(PC12)细胞凋亡的影响及其相关机制,初步探索Seipin敲低在帕金森病(PD)模型中的可能调控机制。方法:对细胞给予不同浓度的6-羟多巴胺(6-OHDA,0、25、50、75、100及125μmol/L)处理,用CCK8法检测细胞活性筛选6-OHDA最佳造模浓度;选取阴性对照细胞和Seipin敲低细胞分别用6-OHDA诱导体外PD模型作为阴性模型组和Seipin模型组,同时设阴性正常组和Seipin正常组;4组细胞培养18 h时,采用蛋白质免疫印迹法(Western blot)检测内质网应激(ERS)蛋白(GRP78和CHOP)和凋亡相关蛋白Bax水平。结果:CCK8法检测细胞活性,6-OHDA最适作用浓度为75μmol/L;Western blot结果显示,与阴性正常组比较,阴性模型组、Seipin模型组GRP78、CHOP及Bax蛋白水平明显增加,差异有统计学意义(P<0.05);而与阴性模型组相比,Seipin模型组GRP78、CHOP及Bax蛋白水平明显增加,差异有统计学意义(P<0.05)。结论:敲低Seipin诱导细胞凋亡可能涉及ERS蛋白的增加,敲低Seipin后导致6-OHDA诱导的细胞凋亡更加严重。

Objective:To investigate the effect of Seipin knockdown on apoptosis of PC12 cells and its related mechanisms,and to explore the possible regulatory mechanism of seipin knockdown in Parkinson's disease(PD)model.Methods:PC12 cells were treated with different concentrations of 6-hydroxydopamine(6-OHDA,0,25,50,75,100,and 125μmol/L),and the cell viability was measured by CCK8 to select the optimal concentration of 6-OHDA.The control cells and Seipin-knockdown cells were used to induce PD model in vitro with 6-OHDA as the negative model group and seipin model group respectively,while the negative normal group and Seipin normal group also set up.The cells of 4 groups were cultured for 18 h,Western blot were used to detect the levels of endoplasmic reticulum stress proteins(GRP78 and CHOP)and apoptosis-related protein-Bax.Results:The optimal concentration of 6-OHDA was 75μmol/L by CCK8.Western blot results showed that the levels of GRP78,CHOP and Bax proteins in the negative model group and the Seipin model group increased significantly(P<0.05)compared to the negative normal group.The levels of GRP78,CHOP and Bax proteins in the Seipin model group were significantly increased(P<0.05)compared to the negative model group.Conclusion:The apoptosis induced by knockdown of seipin may involve the increase of endoplasmic reticulum stress.Knockdown of Seipin leads to more serious apoptosis induced by 6-OHDA.