Protective Effects Of Dioscin On Vascular Endothelial Cells Apoptosis Induced By Oxidized Low-Density Lipoprotein

OBJECTIVE:To investigate the protective effects of dioscin (D) on vascular endothelial cells induced by Oxidized Low-Density Lipoprotein (Ox-LDL) and its mechanisms.METHODS:Primary bovine aortic endothelial cells were cultured in vitro by 0.25% trypsinization and scratching method. The model of injury was induced by Oxidized Low-density Lipoprotein and dioscin was administered before that. Bovine aortic endothelial cells were randomly divided into five groups:Control group, Ox-LDL group, dioscin L group, dioscin M group, dioscin H group. At the end of experiments, cell viability was determined by MTT assay which shows the changes of cell prolifer-ation. Flow cytometry assay was adopted to detect the effect of dioscin on cell apoptosis rate. The concentrations of nitric oxide (NO) were determined with nitrate reductase assay. The concentrations of endothelin (ET) were determined with double antibody sandwich method. Western Blot was adopted to analyze the expression of eNOS.RESULTS:1. Endothelial cells cultured in vitro:Bovine aortic endothelial cells were cultured in vitro successfully by 0.25% trypsinization and scratching method. The culturing cells grew on the wall appeared after 2-4 hours and in round shape. The monolayer cells were formed after 5-7 days and presented as "pavement stone" arrangement. The nucleus in round or ellipse shape and have 1-2 nucleoli.The antigens of VIII factor were positive by immunohistochemisty.2. Cell appearance:Compared with normal group, the cells in Ox-LDL group become round and decreased size, the interspaces widen, and some of them ablate; compared with Ox-LDL group, cells in dioscin groups (L\M\H) become almost normal.3.Cell viability:Compared with normal group, cell viability in Ox-LDL group decreased significantly(64.89±1.02vs100±0,P<0.01);compared with Ox-LDL group, cell viability in dioscin groups(L\M\H) increased significantly(72.92±5.24vs 64.89±1.02,83.89±4.41vs64.89±1.02,92.21±6.78vs64.89±1.02,P<0.01,P<0.05).4. Cell apoptosis:Compared with normal group, cell apoptosis in Ox-LDL group increased significantly(49.6±10.44vs30.0±3.27, P<0.01); compared with Ox-LDL group, cell apoptosis in dioscin groups (L\M\H) decreased significantly(30.4±4.48 vs49.6±10.44,36.5±8.29 vs49.6±10.44,46.7±10.76 vs49.6±10.44, P<0.01, P<0.05).5.Concentrations of NO and ET:Compared with normal group, concentration of NO in Ox-LDL group decreased significantly(27.03±3.12vs59.46±16.19, P<0.01), concentration of ET in Ox-LDL group increased significantly(25.89±2.07vs 16.93±1.64, P<0.05);compared with Ox-LDL group, concentration of NO in dioscin groups(L\M\H) increased significantly(29.05±5.99vs27.03±3.12,38.94±6.34vs 27.03±3.12,49.19±19.24vs27.03±3.12,P<0.01,P<0.05),concentration of ET in dioscin groups(L\M\H) decreased (23.92±1.05vs25.89±2.07,21.30±1.42vs25.89±2.07,17.74±1.58vs25.89±2.07,P<0.05).6. Activity of eNOS:Compared with normal group, the activities of eNOS in Ox-LDL group decreased significantly; compared with Ox-LDL group, the activities of eNOS in dioscin groups (L\M\H) increased significantly.CONCLUSIONS:1. It is successful that BAECs injury induced by Ox-LDL. Under the conditions of 150μg/ml treatment for 6 hours, the viability of BAECs is suitable for model standard.2. Dioscin against BAECs apoptosis in Ox-LDL model, and present concentration dependent.3. Dioscin against BAECs apoptosis induced by Ox-LDL maybe through inhibiting the secretion of ET, raiseing the expression of eNOS, and increaseing the synthesis of NO.