氯喹对过氧化氢诱导平滑肌细胞内质网应激途径凋亡的影响

Effect of chloroquine on hydrogen peroxide-induced apoptosis of smooth muscle cells via endoplasmic reticulum stress pathway

目的探讨氯喹对过氧化氢诱导平滑肌细胞(SMC)内质网应激途径凋亡的影响。方法体外培养主动脉SMC细胞,分别加入100、400和800μmol/L H2O2培养12h后,MTT法检测SMC细胞生存率。将细胞分为对照组、H2O2组、氯喹组、H2O2+氯喹组,培养12h后采用MTT法检测氯喹阻断自噬后SMC细胞的生存率,倒置相差显微镜观察H2O2对SMC细胞形态变化的影响,间接免疫荧光法检测自噬相关蛋白LC3和p62的表达,Western blotting检测Beclin-1、LC3、GRP78、CHOP、Caspase-3及Cleaved Caspase-3蛋白的表达。结果 MTT检测结果显示,100、400、800μmol/L H2O2作用12h后,SMC细胞存活率明显降低,IC50为447.4μmol/L;氯喹阻断自噬后,SMC细胞存活率明显降低(P<0.05)。倒置相差显微镜观察结果显示,H2O2+氯喹组SMC收缩变圆,细胞密度明显下降。激光共聚焦显微镜观察显示,H2O2与氯喹联合作后,SMC细胞质中LC3和p62蛋白表达均明显增加,且有明显共定位现象。Western blotting检测结果表明,H2O2与氯喹联合作用后,SMC中内质网应激相关蛋白GRP78、CHOP和自噬相关蛋白Beclin-1、LC3Ⅱ/LC3Ⅰ的表达明显增加(P<0.05,P<0.01),凋亡执行分子Caspase-3的裂解形式蛋白表达水平明显升高(P<0.01)。结论氯喹对过氧化氢诱导SMC通过内质网应激途径的凋亡具有促进作用。

Objective To investigate the effect of chloroqnine （CO.） on hydrogen peroxide-induced apoptosis of smooth muscle cells （SMCs） via endoplasmic reticulum （ER） stress pathway. Methods The SMCs were cultured in vitro. They were incubated respectively with 100, 400 and 800μmol/L of H202 for 12h. Cell survival rate was determined with MTT assay. To observe the time course of autophagy induced by cisplatin treatment, four experimental groups were set up, namely control group, H202 group, CO group and H202＋CQ.group. Cell survival rate after autophagy interruption was determined by MTT assay. Inverted phase contrast microscopy was used to observe the effects of H202 on morphological changes in SMCs＇. The expressions of autophagy related protein p62 and LC3 were detected by indirect immunofluorescence. The expressions of beclin-1, LC3, GRP78, CHOP, caspase-3 and cleaved caspase-3 were assayed by Western blotting. Results After treatment with 100-800μmol/L of H202 for 12h, the proliferation of SMCs was markedly inhibited, the ICs0 value was 447.4btmol/L. The survival rate of SMCs was lowered obviously after autophagy interruption by CQ.（P〈0.05）. It was found by inverted microscopy that the SMCs in H2Oz＋CQ.group shrank and became rounded, and the cell density decreased significantly. It was found by laser scanning confocal microscopy that the expressions increased obviously of LC3 and p62 proteins in SMCs of HzO2＋CQ, group, and an obvious phenomenon of co-localization was observed. Western blotting demonstrated that the expressions of both ERs related proteins GRP78, CHOP and autophagy related proteins Beclin-1, LC3Ⅱ/LC3 I increased obviously （P〈0.05, P〈0.01） in H202＋CQ.group, and the expression of lytic form apoptotic molecule of caspase-3 protein was significantly increased （P〈0.01）. Conclusion Chloroquine could induce apoptosis of smoothcells via ER stress pathway induced by hydrogen peroxide.