Abstract

The effect and molecular mechanism of melatonin on the vascular smooth muscle cells proliferation and vascular smooth muscle cells migration induced by oxidized low density lipoprotein was explored in this paper. The first scheme: induce vascular smooth muscle cells with series of oxidized low density lipoprotein concentrations for 48 h (0 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL and 100 μg/mL). The second scheme: 50 μg/mL oxidized low density lipoprotein was used to induce vascular smooth muscle cells s for 0 h, 6 h, 12 h, 24 h and 48 h. The third option: first induce vascular smooth muscle cells with 50 μg/mL oxidized low density lipoprotein for 24 h, then treat vascular smooth muscle cells with series of melatonin concentrations for 48 h (1 nmol/L, 1 μmol/L, 0.1 mmol/L, 1 mmol/L). Cell Counting Kit-8 method was used to detect cell proliferation. Cells were randomly divided into blank control group, melatonin group (0.1 mmol/L melatonin treatment vascular smooth muscle cells 48 h), oxidized low density lipoprotein group (vascular smooth muscle cells were induced by 50 μg/ml oxidized low density lipoprotein for 24 h), oxidized low density lipoprotein +melatonin group (50 μg/ml of oxidized low density lipoprotein induced vascular smooth muscle cells for 24 h and then treated vascular smooth muscle cells s with 0.1 mmol/L of melatonin for 48 h). After relative treatment, cell cycle was detected by flow cytometry and cell migration was detected by scratch test. Proliferating cell nuclear antigen, alpha smooth muscle actin, cyclin A and extracellular regulatory protein kinase were detected by immunofluorescence staining and Western blot 1/2 (extracellular regulatory protein kinase 1/2) signal pathway related protein expression. Compared with the blank control group, vascular smooth muscle cells s treated with 50 μg/ml oxidized low density lipoprotein could significantly induce cell proliferation. After 24 h of induction by oxidized low density lipoprotein (50 μg/ml), the number of cells in S phase and G1 phase was decreased significantly and the number of cells in G2 phase was increased significantly. Cell migration was promoted and proliferating cell nuclear antigen, cyclin A, Cyclina, p-extracellular regulatory protein kinase 1/2 protein expression was up-regulated, down regulating the p-p21 protein level. The difference was statistically significant (p<0.01). The melatonin with series concentrations (1 nmol/L, 1 μmol/L, 0.1 mmol/L and 1 mmol/L) could inhibit the proliferation of vascular smooth muscle cells induced by oxidized low density lipoprotein to varying degrees. After vascular smooth muscle cells treatment with oxidized low density lipoprotein (50 μg/ml) for 24 h and melatonin (0.1 mmol/L) for 48 h, the cells in G1 and S phase increased significantly, the cells in G2 phase decreased significantly, the cell migration was inhibited, which were compared with oxidized low density lipoprotein oxidized low density lipoprotein group. The expression of proliferating cell nuclear antigen, cyclin A, Cyclina, p-extracellular regulatory protein kinase 1/2 protein was decreased and the expression of p-p21 protein was up-regulated, the difference was statistically significant (p<0.01). Melatonin can significantly inhibit the proliferation and migration of vascular smooth muscle cells induced by oxidized low density lipoprotein and its mechanism may be related to activating p21 and blocking extracellular regulatory protein kinase 1/2 signaling pathway.