Melatonin inhibits ox-LDL-induced proliferation of vascular smooth muscle cells

Abstract

Objective To explore the effect of melatonin (Mel) on the proliferation of vascular smooth muscle cells (VSMCs) induced by oxidized low-density lipoprotein (ox-LDL) and investigate the potential molecular mechanism. Methods ① ox-LDL at different concentrations (0, 10, 20, 50 and 100 μg/mL) was used to treat VSMCs for 48 h, and cell proliferation was measured with CCK-8 assay. ② The effect of different time points (0, 6, 12, 24 and 48 h) on the cell viability was also detect after the VSMCs was treated with the optional concentration of ox-LDL (50 μg/mL). ③ After the VSMCs were induced by 50 μg/mL ox-LDL for 24 h, the cells were treated with Mel at 1, 1×103, 1×105 and 1×106 nmol/L for 48 h to determine the effect of Mel on cell viability. ④ The VSMCs were divided into 4 groups, that is, blank control group, Mel group (1×105 nmol/L Mel for 48 h), ox-LDL group (50 μg/mL ox-LDL for 24 h), and ox-LDL+Mel group (50 μg/mL ox-LDL for 24 h followed by 1×105 nmol/L Mel for 48 h). CCK-8 assay and flow cytometry were employed to detect cell proliferation and cell cycle, respectively. Immunofluorescence staining and Western blotting were adopted to determine the expression of cell proliferation markers, proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA), cell cycle-related protein cyclin A (Cyclin A) and cyclin-dependent kinase inhibitor p21, as well as extracellular regulatory protein kinase 1/2 (ERK1/2). Results Compared with the blank control group, treatment of 50 μg/mL ox-LDL for 24 h in VSMCs significantly promoted cell proliferation, induced cell arrested at S and G1 phase, decreased the proportion of cells at G2 phase, improved cell migration, up-regulated the expression of PCNA, α-SMA, Cyclin A and p-ERK1/2, and down-regulated that of p-p21 protein (all P < 0.01). Mel of 1, 1×103, 1×105 and 1×106 nmol/L inhibited the proliferative ability of VSMCs induced by ox-LDL treatment. Compared with the ox-LDL group, the ox-LDL+Mel group had significantly reduced proportion of G1 and S phase cells, enhanced proportion of G2 phase cells, decreased protein levels of PCNA, α-SMA, Cyclin A and p-ERK1/2, and increased level of p-p21 (P < 0.01). Conclusion Mel significantly inhibits the proliferation in VSMCs induced by ox-LDL, which might be related to activation of P21 and block of ERK1/2 signaling pathway.

Keywords

• atherosclerosis
• vascular smooth muscle cells
• melatonin
• oxidized low-density lipoprotein
• proliferation