Bradykinin B2 Receptor Pathway Inhibits Oxidative Stress Induced Senescence Of Endothelial Progenitor Cells And Cardiac Stem Cells

I. Bradykinin Inhibits Oxidative Stress-Induced Senescence of Endothelial Progenitor CellsObjective:The aim of this study is to determine the mechanism of bradykinin (BK) inhibit oxidative stress induced senescence of human endothelial progenitor cells (hEPCs).Methods:B2 receptor (B2R) expressed on circulating CD34+ hEPCs was analysis in diabetes patients by flow cytometer. hEPCs were exposed to 300 μM hydrogen peroxide (H2O2) with or without treatment with BK. β-galactosidase staining (SA-Gal) and dichlorofluorescein diacetate probe (DCFH-DA) were used to detect senescence and intra-cellular oxidative stress oxygen radicals. Senescence related PCR array and qPCR were performed to detect relative signal molecular. B2R antagonist HOE-140, phosphatidylinositol-3-kinase (PI3K) antagonist LY-294002, epidermal growth factor receptor (EGFR) antagonist AG1478 and B2 receptor siRNA were administered to block the B2R, PI3K and EGFR signal pathway. B2R expression of hEPCs was determined by Flow cytometry and qPCR. Western blot was applied to find senescence associated signal protein.Results:The expression of B2R on circulating CD34+cells was significantly reduced in DM patients as compared to health control. B2 expression on CD34+ cells inversely correlated with MPO in DM patients. BK treatment decreased senescence and intracellular oxygen radicals according SA-Gal staining and DCFH-DA. PCR array and qPCR showed that BK treatment lead to lower expression of retinoblastoma (RB) in H2O2 induced senescence hEPCs. B2R expression was decreased by H2O2 induced senescence while BK administered up-regulated B2R. Western blot showed that BK leads to phosphorylated RB, AKT and cyclin D1 elevated compared with H2O2 treated alone. Antagonists and siRNA blocked the protective effect of BK.Conclusion:BK inhibit oxidative stress induced hEPCs senescence though B2R mediate EGFR and PI3K signal pathway.Ⅱ. Bradykinin Inhibits Oxidative Stress-Induced Senescence ofCardiac Stem CellsObjective:The aim of this study is to determine the mechanism of bradykinin (BK) inhibit oxidative stress induced senescence of cardiac stem cells (CSCs).Methods:C-kit positive CSCs were cultured from C57BL/6J mouses. CSCs were exposed to 25mM D-glucose (D-Glu) with or without treatment with BK. β-galactosidase staining (SA-Gal) and dichlorofluorescein diacetate probe (DCFH-DA) were used to detect senescence and intra-cellular oxidative stress oxygen radicals. B2R antagonist HOE-140, phosphatidylinositol-3-kinase (PI3K) antagonist LY-294002, mTOR antagonist Rapamycin, P53 antagonist PFT-αand B2 receptor siRNA were administered to block the B2R, PI3K, mTOR and P53 signal pathway. Concentration of super oxide and ATP was analyzed. B2R expression of CSCs was determined by Flow cytometry. Western blot was applied to find senescence associated signal protein.Results:BK treatment decreased senescence and intracellular oxygen radicals according SA-Gal staining and DCFH-DA. The concentration of super oxide was low and the concentration of ATP was high while BK treatment. B2R expression was decreased by D-Glu induced senescence. Western blot showed that BK leads to phosphorylated AKT, mTOR elevated and P53, P16 declined compared with D-Glu treated alone. Antagonists of B2R, PI3K, mTOR and siRNA blocked the protective effect of BK. P53 antagonist also inhibits the oxidative stress induced CSCs senescence.Conclusion:Bradykinin inhibits oxidative stress-induced senescence of cardiac stem cells through the B2R/PI3K/AKT/mTOR/P53 signal pathways.