过氧化氢诱导血管平滑肌细胞内质网应激及其自噬作用

Autophagy and endoplasmic reticulum stress of primary vascular smooth muscle cells triggered by hydrogen peroxide

目的观察过氧化氢(H_2O_2)诱导血管平滑肌细胞(VSMC)内质网应激及其自噬的作用机制。方法体外培养VSMC细胞,加入H_2O_250,100,200,400,600,800,1200和1600μmol·L^(-1)分别作用12和24 h后,MTT法检测H_2O_2对VSMC的抑制率;倒置相差显微镜观察H_2O_2对VSMC细胞形态的影响;采用间接免疫荧光法检测自噬相关蛋白微管相关蛋白轻链3(LC3)和泛素结合蛋白p62的表达,采用Western蛋白印迹法检测自噬相关蛋白人卷曲螺旋肌球蛋白样BCL2相互作用蛋白/自噬基因(Beclin-1)和LC3、内质网应激相关蛋白葡萄糖调节蛋白78(GRP78)和CCAAT-增强子结合蛋白同源蛋白(CHOP)及自噬上游信号通路哺乳动物西罗莫司(雷帕霉素)靶蛋白(m TOR)的表达。结果 MTT结果显示,H_2O_250~1600μmol·L^(-1)作用VSMC细胞12和24 h,VSMC细胞存活率明显降低,12和24 h的IC50分别为(597.2±2.3)和(447.4±1.7)μmol·L^(-1)。倒置相差显微镜观察可见H_2O_2400μmol·L^(-1)组VSMC收缩变圆。H_2O_2400μmol·L^(-1)作用VSMC 12 h时,细胞存活率由(97.5±0.1)%下降至(74.4±1.0)%;作用VSMC 24 h,下降至(56.8±0.9)%(P<0.01)。激光共聚焦显微镜检测发现,H_2O_2400μmol·L^(-1)作用VSMC 8 h,细胞浆中LC3和p62蛋白随H_2O_2作用时间的延长表达增加。LC3与p62的共定位显示,H_2O_2400μmol·L^(-1)作用8 h最明显。Western蛋白印迹结果表明,H_2O_2诱导内质网应激相关蛋白GRP78,CHOP和自噬相关蛋白Beclin-1,LC3Ⅱ/LC3Ⅰ的表达显著增加(P<0.01),m TOR的表达显著降低(P<0.01)。结论 H_2O_2可能通过诱导VSMC内质网应激激活自噬。

OBJECTIVE To observe the effect of the autophagy and endoplasmic reticulum stress （ERS） in vascular smooth muscle cells （VSMCs） with hydrogen peroxide （H202）. METHODS The VSMCs were incubated with different concentrations of H202（50, 100, 200, 400, 600, 800, 1200 and 1600 μmol. L-1） for 12 and 24 h. The cell viability was determined by MTT- assay. The cell morphology was observed under an inverted phase contrast microscope. The expression of autophagy related protein ubiquitin binding protein p62 and microtubule-associated protein light chain 3 （LC3） was detected by indirect immunofluorescence. Western blotting was used to detect autophagy related protein human coiled-coil myosin-like BCL2-interacting protein （Beclin-1 ）, LC3 and mammalian target of sirolimus Rapamycin（mTOR）, as well as the expression of endoplasmic reticulum stress （ERS） related protein glucose regulated proteins 78 ku （GRP78） and C/EBP homologous protein （CHOP）. RESULTS MTT results showed that H2O2 inhibited the growth of VSMCs cells. The half inhibitory concentration （IC50） was 597.2±2.3 and （447.4±1.7）lJmol. L-1 at 12 and 24 h, respectively. The results of the inverted phase contrast microscope showed that VSMCs in H202 group shrinked and turned into smaller round cells. The cell survival rate declined from （97.5±0.1）% in normal control group to （74.4±1.0）% and （56.8±0.9）% in H2O2 400 μmol, L-1 at 12 and 24 h, respectively. The results of the laser scanning confocal micro- scope showed that H202 400 μmol. L-1 increased the expression of LC3 and p62 protein in a cytoplasmic time-dependent manner, and increased the colocalization of LC3 and p62, especially at 8 h. The results of Western blotting demonstrated that H2O2 increased the expression of ERS related protein GRP78 and CHOP, autophagy related protein Beclin-1 and LC3 II/LC3 I （P〈0.01）, and decreased mTOR（P〈0.01 ） in VSMCs. CONCLUSION H2O2 induces autophagy through ERS in VSMCs.