Autophagy and endoplasmic reticulum stress of primary vascular smooth muscle cells triggered by hydrogen peroxide / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To observe the effect of the autophagy and endoplasmic reticulum stress (ERS) in vascular smooth muscle cells (VSMCs) with hydrogen peroxide (H2O2). METHODS The VSMCs were incubated with different concentrations of H2O2(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)μmol·L-1 at 12 and 24 h,respectively. The results of the inverted phase contrast microscope showed that VSMCs in H2O2 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 400μmol·L-1 at 12 and 24 h, respectively. The results of the laser scanning confocal micro?scope showed that H2O2 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 LC3Ⅱ/LC3Ⅰ(P<0.01),and decreased mTOR(P<0.01)in VSMCs. CONCLUSION H2O2 induces autophagy through ERS in VSMCs.