Effects Of Ginkgo Biloba Extract On Glutathione Peroxidase And Caspase-3of Endothelial Progenitor Cells In The Peripheral Blood Of Diabetes

Objective:To research the changes of GPX and Caspase-3activity in endothelial progenitor cells derived from peripheral blood of diabetic patients, and protection of Ginkgo biloba extract on EPCs..Methods:We chose20type2diabetes mellitus patients in Nanjing Genetal Hospital as experimental group, and10healthy volunteers as control group. Experimental and control groups were set to five groups. Experimental group:group A1was blank group (without drug intervention), group B1was drug intervention with6.25mg/L GBE, C1was drug intervention with12.5mg/L GBE, D1was drug intervention with25mg/L GBE, El was drug intervention with50mg/L GBE. Control group was divided into A2、B2、C2、D2、E2in the same way.The20ml adult peripheral blood was taken in fasting under sterile conditions, and blood mononuclear cells was isolated by Ficoll density gradient centrifugation. The MNCs were seeded on199Medium(20%fetal calf serum、VEGF10ug/L、IGF-12ug/L、bFGF2ug/L、EGF10ug/L、penicillin-Streptomycin500mg/L). Observe EPCs growth and morphological changes by inverted phase contrast microscope to evaluate the growth process. The cells were stained identification on seventh day. By fluorescence inverted microscope to observe cell color reaction. Cytoplasm uptake Dil-acLDL is red, membrane binding FITC-UEA-I shows green, staining double positive cells is yellow and was identified to be being differentiated EPCs.On the sixth day, attached cells were stimulated with different concentrations (Omg/L、6.25mg/L、12.5mg/L、25mg/L、50mg/L)of Ginkgo biloba extract for24hours, then using UV spectrophotometer to detect GPX activity, microplate reader to detect Caspase-3activity. The experimental data was computed with SPSS18.0software. Measurement data expressed as the form of mean±standard deviation (X±S) the comparison in the group by the single factor analysis of variance, the comparison between groups by t test. P<0.05was considered statistically significant.Results:1. Morphological change and Dyeing identification in EPCs:Freshly isolated MNCs were small round, after2days, cells attached. When the third day, cells tend to form colony from the middle to the peripheral radiation. The sixth day, cells were typical spindle. Some cells end-to-end into a funicular, vascular network structure. When the seventh day, the cells were stained identification. Cytoplasm uptake Dil-acLDL is red, membrane binding FITC-UEA-I shows green, staining double positive cells is yellow and was identified to be being differentiated EPCs.2. Effects of GBE on the GPX activity of EPCs:compared with the control group, GPX activity of experimental group significantly decreased in the same concentration of GBE(P<0.05);12.5mg/LGBE,、25mg/LGBE、50mg/LGBE in control group and25mg/LGBE、50mg/LGBE in experiment group can significantly improve EPCs GPX activity(P<0.011或P<0.05). The higher the concentration, the higher GPX activity.3.Effects of GBE on the Caspase-3of EPCs:compared with the control group, Caspase-3activity of experimental group significantly increased in the same concentration of GBE(P<0.001或P<0.05);25mg/LGBE、50mg/LGBE in control group and12.5mg/L、25mg/LGBE、50mg/LGBE in experiment group can significantly decreased EPCs Caspase-3activity(P<0.01或P<0.05). The higher concentration of GBE, the lower activity of Caspase-3.Conclusions:EPCs grew slowly and its number decreased in peripheral blood of diabetes. Decreased GPX activity and increased Caspase-3activity was found in EPCs of peripheral blood from diabetes. GBE can improve GPX activity, reduce Caspase-3activity and cell toxicity, and inhibit oxidative damage and EPCs apoptosis, which provided an evidence of the prevention and treatment GBE on diabetes, especially on diabetic vascular disease.