Calycosin entered HUVECs and ameliorated AGEs-promoted cell apoptosis via the Bcl-2 pathway

Endothelial cell (EC) apoptosis plays a pivotal role in the progression of diabetic complications. Abundant studies have demonstrated the pivotal role of advanced glycation end products (AGEs) on the development of diabetes. The aim of the present study was to investigate the effect of calycosin, a phytoestrogen, on AGEs-induced human umbilical vein endothelial cell (HUVEC) apoptosis. Fluorescence polarization and fluorescence absorption assays indicated that calycosin interacted with AGEs in a time-dependent manner. Further studies found that calycosin entered the cells as detected by HPLC. The MTT method demonstrated that calycosin ameliorated AGEs-induced HUVEC apoptosis in a dose-dependent manner, and statistical significance was observed at 1 × 10(-8) M of calycosin; this behavior was further demonstrated by acridine orange/ethidium bromide staining in that the presence of calycosin dramatically reduced AGEs-induced red staining in HUVECs. Further studies found that pre-incubation with calycosin at 1 × 10(-8) M dramatically increased anti-apoptotic Bcl-2 while decreased pro-apoptotic Bax and Bad expressions as detected by immunocytochemistry, and the effect of calycosin on rebalancing the ratio of Bcl-2/Bax was more significant than that of its glycoside, calycosin-7-O-β-D-glucopyranoside (CG). Furthermore, calycosin slightly reversed AGEs-induced cell oxidative stress at 1 × 10(-8) M, but its antioxidative stress effect was less significant than that of CG. The present study strongly indicates that calycosin can enter the cell and modulate endothelial cell dysfunction by ameliorating AGEs-induced cell apoptosis.