Protective Effects Of Dioscin On Cultured Bovine Aortic Endothelial Cells

OBJECTIVE:To investigate the protective effects of dioscin (D) on cultured bovine aortic endothelial cells(BAECs) injured by high glucose and its mechanisms.METHODS:Primary bovine aortic endothelial cells were cultured in vitro by scratching method. The model of oxidative injury was induced by high glucose and dioscin was administered after that. Bovine aortic endothelial cells were randomly divided into five groups:Control group; HG group; dioscin L, M, H group. At the end of experiments, cell viability was determined by MTT assay which shows the changes of cell proliferation. The concentrations of nitric oxide(NO) were determined with nitrate reductase assay. SOD activities were measured by xanthine oxidase assay. MDA production was measured by Thibabituric Acid(TBA) assay. LDH leakage was measured by nitro phenylhydrazine assay.RESULTS:1. endothelial cells cultured in vitro:bovine aortic endothelial cells were cultured in vitro successfully by scratching method.2-4d long fusiform cells, the cells were smaller particles spindle shape; 6-10d adherent cell grouth for the spindle cell latency and increased the number of proliferating; 10-14d colony-like growth into cell fusion, the cells were significantly increased access to growth platform.2. Cell viability:Compared with normal group, cell viability in HG group decreased significantly(69.041±3.5021 vs.92.202±7.1534, P<0.05); compared with HG group, cell viability in dioscin groups(L\M\H) increased significantly(0.684±0.0301vs.0.567±0.0414,0.692±0.0495vs.0.567±0.0414,0.692±0.0495vs.0.567±0.0414, P<0.05). Cell viability in dioscin groups was same as that in C group.3. Concentrations of NO:Compared with normal group, concentration of NO in HG group decreased significantly(69.041±3.5021vs.92.202±7.1534, P<0.05); compared with HG group, concentration of NO in dioscin groups(L\M\H) increased significantly(103.302±8.5809vs.69.041±3.5021, 104.777±9.3255vs.69.041±3.5021,104.342±4.4529vs.69.041±3.5021, P<0.05).4. SOD activities:Compared with normal group, SOD activity in HG group decreased significantly(21.663±1.830vs.26.456±1.446, P<0.05); compared with HG group, SOD activities in dioscin groups(L\M\H) increased significantly(23.853±1.165vs.21.663±1.830,24.011±1.762vs.21.663±1.830,23.577±1.662vs.21.663±1.830, P<0.05).5. MDA production:Compared with normal group, MDA rpoduction in HG group increased significantly(0.5344±0.0661vs.0.3183±0.0418,P<0.05); compared with HG group, MDA rpoduction in dioscin groups(L\M\H) decreased significantly(0.3973±0.0082vs.0.5344±0.0661,0.3811±0.0397vs.0.5344±0.0661, 0.3874±0.0128vs.0.5344±0.0661,P<0.05).6. LDH leakage:Compared with normal group, LDH leakage in HG group increased significantly(1118.702±79.4289vs.760.174±83.2633, P<0.05); compared with HG group, LDH leakage in dioscin groups(L\M\H) decreased significantly(849.322±63.227vs.1118.702±79.4289,907.838±75.662vs.1118 .702±79.4289,801.034±90.8995vs.1118.702±79.4289, P<0.05).7. free calcium concentration(Ca2+i):Compared with normal group, [Ca2+] i in HG group increased significantly (477.16±10.10vs.137.96±9.68); compared with HG group, [Ca2+] i in dioscin groups(L\M\H) decreased significantly (213.68±8.76vs.477.16±10.10,220.38±11.76vs.477.16±10.10, 234.78±8.77vs.477.16±10.10)。8. activity of eNOS:Compared with normal group, the activities of eNOS in HG group decreased significantly; compared with HG group, the activities of eNOS in dioscin groups(L\M\H) increased significantly.CONCLUSIONS:At the cellular level, it is proved that dioscin has important protective effects on endothelial cells induced by oxidative injury. The protective mechanisms may involve:1. SOD activity in damaged endothelial cells was significantly increased with dioscin and the antioxygen function of endothelial cells was improved.2. MDA production in damaged endothelial cells was decreased with dioscin and lipid peroxidation was controlled.3. LDH leakage in damaged endothelial cells was decreased with dioscin and cellular membrane integrity was recovered.4. free calcium concentration ([Ca2+]i) in damaged endothelial cells was significantly increased with dioscin and as a result, the activities of eNOS were increased significantly.