Of Astragalus Polysaccharide Type 2 Diabetes Patients With Peripheral Blood Endothelial Progenitor Cells No Concentration And Pi3k/akt/enos Signaling Pathway

Objective:To observe the effect of Astragalus Polysaccharides on the production of nitric oxide(NO) and the expression of protein kinase B (PKB)/the endothelial nitric oxide synthase (eNOS) of endothelial progenitor cells(EPCs)from peripheral blood in patients with type 2 diabetes(T2DM),and to investigate the mechanism of signal transduction of Astragalus Polysaccharides in promoting the proliferation of EPCs from peripheral blood with T2DM.Methods:Total mononuclear cells were isolated from patients with T2DM (n=20)and age-matched control subjects(n=20)by Ficoll density gradient centrifugation,and then the cells were plated on rat tail collagen coated culture flasks.After 7 days of culture,EPCs were characterized by flow cytometry and direct fluorescent staining under a laser scanning confocal microscope.The effect of Astragalus Polysaccharides on EPCs proliferation was measured by MTT assay.EPCs were treated with various concentrations (0,50,200,800,3200,6400mg/L) and different time(6,12,24,48h)culture of Astragalus Polysaccharides,then treated with LY294002 (a PI3K inhibitor).The NO level was measured using Griess Reaction in cell culture supernatants,the expresstions of phosphorylation of PKB-Ser473 and eNOS-Ser1177 were measured using western blot.Results:The proliferation of EPCs in T2DM is decreased compared with control group(P<0.05). APS increased the number of isolated EPCs in both concentration and time-dependent manner.Apparently increase of EPCs number started at 200mg/l and most evident at 800mg/l(P<0.01).It was also showed that concentration of 800mg/l,APS increase EPCs number after incubation for 6 hours and reached the maximum after incubation for 24 hours and decreased a little at 48 hours,though still significantly higher compared with control group.APS increase the endothelial NO production in a dose and time-dependent manner in cultured EPCs from T2DM,and also increase the expression of the phosphorylation of PKB-Ser473 and eNOS-Ser1177 in a concentration-dependent manner in cultured EPCs(P<0.05).LY294002(a PI3K inhibitor)prevented the NO production and the phosphorylation of eNOS and PKB that induced by APS(P<0.05). Conclusion:The rat tail collagen can be a more cheaper replacement of the fibronectin to culture the EPCs from peripheral blood.Treatment with Astragalus Polysaccharides can increase the NO prouducion and markedly promote the proliferation of EPCs in T2DM through up-regulating the PI3K/PKB/eNOS signal pathways.