Rhein inhibits hydrogen peroxide-induced apoptosis of human umbilical vein endothelial cells and its mechanism

Abstract

This study investigated the effect of rhein(RH) on the apoptosis and autophagy of human umbilical vein endothelial cells(HUVECs) induced by hydrogen peroxide(H_2O_2) and its underlying mechanism. The oxidative damage model in HUVECs was established and the cells were divided into different treatment groups. Cell survival rate was detected by MTT assay, apoptosis by Annexin V-FITC/PI double staining and Hoechst 33258 fluorescence staining, autophagy by Ad-mCherry-GFP-LC3 B adenovirus transfection, and protein expression by Western blot. The results showed that RH could protect cells by increasing the cell survival rate in a dose-dependent manner, decreasing the expression of apoptosis-related proteins(Bax and cleaved caspase-3) and the ratio of Bax/Bcl-2, elevating the expression of Bcl-2, up-regulating the expression of microtubule-associated protein 1 light chain 3(LC3)-Ⅱ, and down-regulating the expression of p62. Adenovirus transfection results showed that RH could increase the green and red spots, as well as the yellow spots. However, after the addition of autophagy inhibitor 3-MA, autophagy was reduced and apoptosis was increased. RH could enhance the expression of silent information regulator 2 related enzyme 1(SIRT1). The addition of SIRT1 inhibitor EX-527 reduced the protective effect of RH and cell viability. The addition of 3-MA had no effect on the expression of SIRT1 protein, but the expression of SIRT1 and LC3-Ⅱ proteins decreased and the expression of p62 increased after the addition of EX-527. After RH treatment, the phosphorylation of adenosine monophosphate-activated protein kinase(AMPK) increased, while that of the mechanistic target of rapamycin(mTOR) decreased in a dose-dependent manner. Moreover, this effect could be weakened by the AMPK inhibitor compound C. RH may enhance autophagy through SIRT1/AMPK/mTOR pathway to reduce H_2O_2-induced apoptosis of HUVECs.