Inhibitory effect of paeoniflorin on vascular endothelial cell migration and angiogenesis and its related mechanisms

Abstract

Objective To investigate the effects of paeoniflorin on the migration and angiogenesis of human umbilical vein endothelial cell (HUVEC), and the relationship with c-Met receptor and downstream molecules. Methods HUVEC were cultured in vitro with extracellular matrix (ECM) containing 10% fetal bovine serum and 20 ng/ml hepatocyte growth factor (HGF). The toxic effects of paeoniflorin on cells were detected by cell counting kit-8 (CCK-8) method after treatment with 0, 5, 10, 15 and 20 mmol/L paeoniflorin for 24 h. The c-Met plasmid was transfected into HUVEC by Lipofectamine® 3000. Transwell assay was used to detect the migration and invasion of HUVEC. Angiogenesis in HUVEC was measured by angiogenic assay. Western blotting was used to detect the expression of related proteins. Results The proliferation of HUVEC cells was inhibited when the concentration of paeoniflorin was 15 mmol/L (76.667±7.506, P=0.000). Paeoniflorin in concentration of 5, 10 mmol/L inhibited cell angiogenesis (12.667±1.528, P5=0.001; 6.333±1.528, P10=0.000), cell migration and invasin (249.333±42.360, P5=0.001; 97.667±21.032, P10=0.000). Furthermore paeoniflorin inhibited the phosphorylation of c-Met (0.833±0.702, P=0.000) and its downstream molecules phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) (0.420±0.030, P=0.000; 0.370±0.050, P=0.000; 0.730±0.121, P=0.001) and P70S6K (0.420±0.061, P=0.000), and decreased the expression of VEGF (0.317±0.031, P=0.000). After transfection of c-Met plasmid, the expression of c-Met in HUVEC cells was significantly increased (0.793±0.045, P=0.001). Also paeoniflorin inhibited the angiogenesis (20.667±1.528, P=0.000), invasion and migration (269.000±18.520, P=0.000) of HUVEC cells, suppressed c-Met (0.387±0.067, P=0.003) and its downstream pathway were reversed (PI3K: 0.677±0.046, P=0.000; Akt: 0.577±0.055, P=0.000; mTOR: 0.543±0.078, P=0.001; P70S6K: 0.433±0.090, P=0.000), blocked the expression of VEGF (0.763±0.042, P=0.001). Conclusion Paeoniflorin inhibits the migration and invasion of HUVEC cells through c-Met/ PI3K/Akt/ mTOR pathway and regulates the expression of VEGF to reduce the angiogenesis of HUVEC cells. Key words: Paeoniflorin; Human umbilical vein endothelial cells; Angiogenesis; Migration; Invasion