Effects of osteoprotegerin on apoptosis of human umbilical vein endothelial cells induced by palmitate acids

Abstract

Objective To explore the effects and mechanism of osteoprotegerin (OPG) on apoptosis of human umbilical vein endothelial cells (HUVECs) induced by palmitate acids (PA). Methods HUVECs were divided into three groups: normal control group (treated with 0.4% bovine serum albumin(BSA)) , PA group (treated with 0.4 mmol/L PA), OPG group(pretreated by OPG(50, 100, 200, 400 μg/L) for 24 h, then treated with 0.4 mmol/L PA). Cells in the 3 groups were cultured for 24 hours, then cell apoptosis was assessed by flow cytometry and Hoechst 33258 staining. HUVECs were divided into 5 groups: normal control group (treated with 0.4% BSA), PA group (treated with 0.4 mmol/L PA), OPG group (pretreated by 400 μg/L OPG for 24 h, then treated with 0.4 mmol/L PA), PA+ OPG+ rapamycin group(pretreated with 10 μg/L rapamycin for 24 h, then treated with 400 μg/L OPG for 24 h, and then treated with 0.4 mmol/L PA), PA+ rapamycin group(pretreated with 10 μg/L rapamycin for 24 h, then treated with 0.4 mmol/L PA). The expression levels of tuberin, phosphorylated tuberin(P-tuberin), S6K, phosphorylated S6K(P-S6K), Bcl-2, Bax and caspase 3 protein were measured by western blotting analysis using specific antibodies. The data were compared among groups by using ANOVA followed by SNK test. Results Compared with normal control group, the early apoptosis rate of HUVECs in PA group was significantly increased (18.31%±0.51% vs 6.88%±0.60%, P<0.05). And the early apoptosis of HUVECs decreased dramatically in OPG group compared with that in PA group (12.58%±0.19%, 9.39%±0.42%, 7.55%±0.17%, 5.90%±0.14%, respectively, all P<0.05) and in a OPG dose dependent manner. Compared with normal control group, the late apoptosis rate of HUVECs increased significantly in PA group (9.55%±0.22% vs 5.28%±0.90%, P<0.05). The late apoptosis of HUVECs decreased dramatically in OPG group compared with that in PA group(7.71%±0.17%, 6.42%±0.18%, 5.24%±0.16%, 4.50%±0.16%, respectively, all P<0.05) and in a OPG dose dependent manner. Compared with normal control group, the expression of P-tuberin/tuberin(2.094 2±0.0163 vs 1.1948±0.0541), P-S6K/S6K(2.0942±0.0163 vs 3.2052±0.0051), Bax/β-actin(0.3868±0.0013 vs 1.2991±0.0026), caspase 3/β-actin(0.2346±0.0009 vs 0.5793±0.0103) increased markedly in PA group (all P<0.05), Bcl-2/β-actin(0.2470±0.0038 vs 0.0716±0.0004)decreased significantly in PA group(P<0.05); Compared with PA group, the expression levels of P-tuberin/tuberin(0.8258±0.0074), P-S6K/S6K(2.5073±0.1403), Bax/β-actin(0.7452±0.0045), caspase 3/β-actin(0.4713±0.0066) in OPG group were significantly lower than those in PA group(all P<0.05), Bcl-2/β-actin(0.1909±0.0021)was significantly higher than that in PA group(P<0.05). Conclusions It suggests that protective effect of OPG on HUVECs cultured with PA may be association with tuberin/mTORC1 pathway. Key words: Osteoprotegerin; Palmitate Acid; Human umbilical vein endothelial cells; Mammalian target of rapamycin