Study On The Mechanism Of Bradykinin B2 Receptor Signaling Pathway Inhibiting P53 Expression In Endothelial Progenitor Cells Under High Glucose Environment

Objective: To investigate the expression levels of CD34+ cells,B2 R and P53 in peripheral blood of diabetic patients.And the effect of bradykinin on mitochondrial function of endothelial progenitor cells and the regulation of P53 gene expression in high glucose environment.Methods:1.Among the patients with negative coronary angiography in Department of Cardiology of Yijishan Hospital,20 patients with type 2 diabetes and 20 patients without diabetes were selected.Non-diabetic patients with negative coronary angiography were used as the control group.The patient’s consent was obtained and informed consent was signed.20 ml of arterial blood was extracted through the radial artery.Monocytes were isolated from whole blood samples with lymphocyte isolation solution,CD34+ cells were labeled by FITC,B2 R and P53 were labeled by antibody.The number of CD34+ cells,the expression of B2 R and P53 were detected by flow cytometry.The production of superoxide anion in cells was detected by the superoxide end-point colorimetric quantitative detection kit.2.Cultivation in vitro in C57 BL / 6 j mice bone marrow derived endothelial progenitor cells(epcs),25 mmo/L d-glucose BK environment using different concentration(0.1 n M,1 n M)intervention,testing oxygen free radical generation and mitochondrial function in cells,B2 R,P53 protein expression and molecular level,AKT/P-AKT protein expression,confirmed BK to sugar impact of endothelial progenitor cells(epcs)mitochondrial function,and the expression of P53 gene regulation.3.The B2 R signaling pathway was blocked with the signal blocker HOE-140 and BK was used to intervene at 25 MMO /L D-glucose.Oxygen free radical generation,mitochondrial function,B2 R,P53 protein and molecular level expression,and Akt/p-Akt protein expression in cells were detected.4.PI3 K antagonist LY294002 was used to block the PI3 K signaling pathway in EPC,and BK was used to intervene in 25 MMO /L D-glucose environment.Intracellular oxygen free radical generation,mitochondrial function,protein and molecular expressions of B2 R,P53,and Akt/p-Akt protein expression were detected,indicating that BK reduces oxygen free radical generation,protects mitochondria and down-regulates the expression of P53 gene through B2R/ Akt signaling pathway.Results:1.There was no significant difference in CD34+ expression between diabetic patients and non-diabetic patients(0.2611±0.12879 vs 0.3191±0.11405,P<0.05),and the expression ratio of CD34+ cells in the total population ranged from 0.16% to 0.54%(median:0.26%),the expression of H2 R in circulating CD34+ cells of patients with negative coronary angiography was lower in diabetic patients than in non-diabetic patients(0.2453±0.11177 vs 0.5130±0.12797,P<0.05),and the expression of P53 was higher in non-diabetic patients(0.33474±0.175353 vs 0.10457±0.78290,P<0.05).2.Fluorescence test box and Western Blot showed that hyperoxic environment inhibited the expression of B2 R in EPCs and promoted oxidative stress and P53 expression.BK inhibits oxidative stress and P53 expression in high oxygen environment,and BK inhibits oxidative stress and P53 expression in cells through B2R-dependent PI3K/Akt signaling pathway.Conclusion:1.The expression of B2 R in peripheral blood CD34+ cells of diabetic patients was lower than that of non-diabetic patients,and the expression of P53 was higher than that of non-diabetic patients.2.BK inhibits oxidative stress in endothelial progenitor cells through B2R/PI3K/ Akt signaling pathway,down-regulates P53 expression,and protects mitochondrial function.