Effects of Rhein lysinate on H2O2-induced cellular senescence of human umbilical vascular endothelial cells

Aim: To observe the effect of Rhein lysinate (RHL) on cellular senescence of human umbilical vascular endothelial cells (HUVECs) and elucidate its action mechanism. Methods: Cell viability was determined using MTT assay. The expression levels of Sirt1 mRNA and protein were measured by RT-PCR and Western blot, respectively. Senescence associated (SA)-beta-galactosidase activity was detected to evaluate cell senescence. Apoptosis and cell cycle progression were determined using flow cytometry. Results: Treatment with RHL (10 mu mol/L) for 48 h significantly increased the proliferation of HUVECs. In contrast, treatment with H2O2 (25, 50 and 100 mu mol/L) for 6 d dose-dependently increased beta-galactosidase positive cells. Spontaneous cell senescence appeared as the cell passage increased. Pre-treatment with RHL (10 mu mol/L) reversed H2O2 or increased cell passage-induced cell senescence. H2O2 (100 mu mol/L) significantly arrested HUVECs at G(1) phase (73.8% vs 64.6% in the vehicle group), which was blocked by RHL (10 mu mol/L). RHL (5 and 10 mu mol/L) enhanced both mRNA transcription and protein expression of Sirt1. H2O2 (100 mu mol/L) significantly decreased Sirt1 expression, and induced up-regulation of p53 acetylation and p16(INK4a), which were blocked by pre-treatment with RHL (10 mu mol/L). Interference with siRNA for Sirt1 abolished the effect of RHL. H2O2 (100 mu mol/L) did not induce HUVEC apoptosis. The expression of apoptosis-associated proteins, such as p53, p21, Bcl-2, and Bax, did not significantly change in the presence of H2O2 (100 mu mol/L) or RHL (10 mu mol/L). Conclusion: RHL protected HUVECs against cellular senescence induced by H2O2, via up-regulation of Sirt1 expression and down-regulation of the expression of acetyl-p53 and p16(INK4a).