Effects And Mechanisms Of Bradykinin On Cardiomyocytes Senescence Induced By Oxidative Stress

Research aims:The effects and possible molecular mechanisms of bradykinin against oxidative stress- induced cardiomyocytes senescence were investigated.Research methods:1. H9C2 cells Cultures. H9C2 cells were purchased from ATCC. They were cultured in growth medium DMEM (containing penicillin , Streptomycin sulfate) containing 10% heat-inactivated FBS in an incubator in an atmosphere of 5% CO₂ at 37°C. Cells were passaged at a 1:3ratio when they reached confluence at passage 4 to 12.2. H₂O₂-induced senescence in H9C2 cells. To induce DNA damage and senescence, 24 hours after seeding onto 6-wells plates at a density of 5000, H9C2 cells were exposed to different concentrations of H₂O₂. After 2 hours, the medium was replaced with normal medium and cultered 3 days. Senescence-associatedβ-galactosidase (sa-β-gal) staining was carried out to measure the number of senescence cells. Caspase-3 activity assay were carrided out to determine the apoptosis in ROS-induced H9C2 cells. Then, the proper concentration of H₂O₂ was determined.3. H9C2 cells senescence detection. Sa-β-gal staining was carried out to measure the number of senescence cells. Western blot was carried out to detect P21 in H9C2 cells. Comet assay was carried out to measure DNA damage.4. The possible effect and mechanism of BK against H₂O₂ induced H9C2 cells senescence. All kinds of concentration of BK were given 30 minutes before the induction of senescence. The B2 antagonist HOE-140 and the blocker of NO (L-NAME) were added 5 minutes before BK treatment. Sa-β-gal staining was carried out to measure the number of senescence cells.Research results:1. The numbers of senescence cells increased in H₂O₂ dose-dependently way at concentration of 20 and 100 u mol/L. It led to apoptosis when it exposed the concentrations of H₂O₂ more than 100 u mol/L. However, 30 u mol/L of H₂O₂ was without effect on caspase-3 activity. Therefore,30 u mol/L of H₂O₂ was performed.2. BK caused a dose-dependent decrease in the number of senescent cells. However, there was no effect of BK on the cells that were not treated with H₂O₂.3. Results showed that BK significantly decreased P21 expression (p<0.05), indicating that BK can protected against H9C2 cells senescence.4. Results showed that H₂O₂ significantly increased the olive tail moment (P<0.05), indicating an increased in DNA damage. BK can significantly decreased the olive tail moment (p<0.05), indicating that BK can protected against DNA damage.5. Results showed that BK B2 receptor antagonist HOE-140 and the blockade of NO (L-NAME) significantly increased the number of senescence cells (P<0.05), indicating that HOE-140 and L-NAME antagonized the protective BK effect.Conclusions: Bradykinin prevents cardiomyocytes from superoxide-induced senescence. The possible mechanism is that bradykinin B2 receptor activates eNOS pathway and protects from DNA damage.