Objective Cellular senescence of endothelial cells has been proposed for its involvement in endothelial dysfunction and atherogenesis. This study investigates the effects of ginsenoside Rb1, a major constituent of ginseng, on H2O2-induced endothelial senescence and molecular changes in primary human umbilical vein endothelial cells.
Methods Prematurely senescent human umbilical vein endothelial cells (HUVECs) were induced by treatment with H2O2 as judged by senescence-associated β-galactosidase assay (SA-β-gal), cell morphological appearance, and plasminogen activator inhibitor-1 (PAI-1. expression. Total nitric oxide (NO) production was measured using Griess reaction. Endothelial NOS (eNOS), PAI RNA expressions were analysed by real time PCR. Total eNOs, pS1177 eNOS and pT495 eNOS protein expressions were analysed by westernblotting.
Results Treatment with 40∼100μM H2O2 caused 26.8∼63.8% of the cells to be SA-β-gal positive. Pretreated with Rb1 markedly inhibited SA-β-gal activity dose-dependently. Also, Rb1 can reduce the expression of PAI which was increased in the H2O2 treated group. In H2O2 treated groups, eNOS mRNA expression decreased, while Rb1 can effectively restore its mRNA expression. eNOS activity of HUVECs was inhibited by decreasing eNOS phosphorylation at Ser-1177 and increasing eNOS phosphorylation at Thr-495 in H2O2 treated groups. While in Rb1 pretreated groups, the both exhibited opposite changes. Consistent with these findings, Rb1 does in fact increase NO levels. All the inhibitory effects of Rb1 on senescence were completely obliterated by L-NAME, the NOS inhibitor.
Conclusion Rb1 can effectively protect HUVEC from senescence through modulating the expression of eNOS.