The Effects Of OPG On High Glucose-induced Apoptosis In Cultured Human Umbilical Vein Endothelial Cells

Objective: To investigate the effects of OPG on high glucose-induced apoptosis in cultured human umbilical vein endothelial cells (HUVECs),and to explore whether the protective effects of OPG were related to TRAIL pathway.So as to provide new experimental evidences for protection of vascllar endothelial cells in the clinical state of high glucose.Methods: Cultured HUVECs (ECV-304) were used for the experiment to investi–gate the effects of OPG on high glucose-induced apoptosis and the mechanism.This study was divided into two parts.The first one was to explore the effects of OPG on serum-free-induced apoptosis in HUVECs.Five groups were divided:the normal control group (containing 10% fetal bovine serum),serum-free treatment group, serum-free+OPG(1.0μg/ml) group,serum-free+OPG (1.0μg/ml)+anti-TRAIL(100 ng/ml) group and serum-free +OPG(1.0μg/ml) +TRAIL(200ng/ml) group;The second part was to explore the effects of OPG on high glucose-induced apoptosis in HUVECs.Six groups were divided:the normal control group (containing 10% fetal bovine serum),serum-free treatment group,serum-free+high glucose(25mM Glucose) group,serum-free +high glucose +OPG(1.0μg/ml) group,serum-free+high glucose+ OPG(1.0μg/ml)+anti-TRAIL(100ng/ml) group and serum-free+high glucose +OPG (1.0μg/ml)+TRAIL(200ng/ml) group.Each group incubated for 24h.The morphology of endothelial cells was observed by acridine orange/ethidium bromide (AO/EB) double staining.The rates of apoptosis were detected by flow cytometer( FCM).Results: 1. The effects of OPG on serum-free-induced apoptosis in HUVECs.1.1 AO/EB double staining:The cell nuclei of the normal control group(containing 10% fetal bovine serum) showed uniform and diffuse green fluorescence;Serum-free treatment group showed apoptotic cells.The cell nuclei of early apoptotic cells showed yellow-green condensation-like or chunky.The cell nuclei of late apoptotic cells showed orange condensation or chunky.Compared with serum-free treatment group, serum-free+OPG (1.0μg/ml) group showed a significant decrease in the ratio of apoptotic endothelial cells.Compared with serum-free+OPG(1.0μg/ml) group, serum- free+OPG(1.0μg/ml) +anti-TRAIL(100ng/ml) group showed a further decrease in the ratio of apoptotic endothelial cells. Compared with serum-free+OPG(1.0μg/ml) group, serum-free+OPG(1.0μg/ml)+TRAIL(200ng/ml) group showed a significant increase in the ratio of apoptotic endothelial cells.1.2 FCM :Incubation of HUVECs with serum-free for 24h,the rate of apoptosis of serum-free treatment group was higher than normal control group(17.19±0.64% VS 7.62±0.65%, p<0.05);Compared with serum-free treatment group,serum-free+ OPG(1.0μg/ml) group showed a significant decrease in the rate of apoptosis(12.89±0.63% VS 17.19±0.64%, p<0.05);Compared with serum-free+OPG(1.0μg/ml) group, serum-free+OPG(1.0μg/ml)+anti-TRAIL(100ng/ml) group showed a further decrease in the rate of apoptosis(9.96±0.80% VS 12.89±0.63%, p<0.05);Compared with serum-free+OPG(1.0μg/ml) group,serum-free+OPG(1.0μg/ml)+TRAIL(200ng/ml) group showed a significant increase in the rate of apoptosis(25.38±2.29% VS 12.89±0.63%, p<0.05).2. The effects of OPG on high glucose–induced apoptosis in HUVECs.2.1 AO/EB double staining:Comparing with serum-free treatment group, serum- free+high glucose group showed a significant increase in the ratio of apoptotic endothelial cells.Compared with serum-free+high glucose group,serum-free+high glucose+OPG(1.0μg/ml) group showed a significant decrease in the ratio of apoptotic endothelial cells.Compared with serum-free+high glucose+OPG(1.0μg/ml) group, serum-free+high glucose +OPG(1.0μg/ml)+anti-TRAIL(100ng/ml) group showed a further decrease in the ratio of apoptotic endothelial cells.Compared with serum-free +high glucose+OPG(1.0μg/ml) group,serum-free+high glucose+OPG(1.0μg/ml)+ TRAIL(200ng/ml) group showed a significant increase in the ratio of apoptotic endothelial cells.2.2 FCM:Compared with serum-free treatment group,serum-free +high glucose group showed a significant increase in the rate of apoptosis(31.30±1.76% VS 16.85 ±1.48%, p<0.05);Compared with serum-free+high glucose group,serum-free+high glucose +OPG(1.0μg/ml) group showed a significant decrease in the rate of apoptosis (22.02±0.41% VS 31.30±1.76%, p<0.05);Compared with serum-free+high glucose+ OPG(1.0μg/ml)group,serum-free+high glucose+OPG(1.0μg/ml) +anti-TRAIL(100 ng/ml) group showed a further decrease in the rate of apoptosis(14.18±1.74% VS 22.02±0.41%, p<0.05);Compared with serum-free+high glucose+OPG(1.0μg/ml) group, serum-free+high glucose+OPG(1.0μg/ml) +TRAIL(200ng/ml) group showed a significant increase in the rate of apoptosis (35.59±3.08% VS 22.02±0.41%,p<0.05).Conclusion: 1.High glucose could induce apoptosis in cultured HUVECs;2. OPG could inhibite high glucose induced-apoptosis in HUVECs,and the protective effects of OPG are related to TRAIL pathway.