Delay Effect Of Daidzein On Senescence Of Rat H9c2 Cells Induced By Hydrogen Peroxide

Objective:To investigate the protective effect of Daidzein（DAI）on rat H9c2 cell senescence induced by hydrogen peroxide（H₂O₂）and its mechanism.Methods:H9c2 cells were treated with different concentrations of H₂O₂ and DAI,and the concentration of H₂O₂ for the construction of aging model in vitro was screened out by MTT method and SA-β-gal staining.The concentrations of DAI for the subsequent experiments were determined by MTT method and SA-β-gal staining.The cells were divided into control group,model group（H₂O₂ 400μmol/L）and DAI low-dose,medium-dose and high-dose groups（20,40,80μmol/L）for subsequent experiments.1.The levels of total antioxidant capacity（T-AOC）,superoxide dismutase（SOD）and malondialdehyde（MDA）were determined by colorimetry.2.ROS changes in H9c2 cells were determined by DCFH-DA fluorescence staining.3.The changes of mitochondrial membrane potential in H9c2 cells were detected by Rh123 fluorescence staining.4.MDC method was used to detect the formation of autophagosomes.5.The protein expressions of p53 and p21,the expression levels of autophagy related proteins p62,Beclin-1,microtubule-associated protein 1 light chain 3（LC3）and the phosphorylation expression levels of phosphatidylinositol 3-kinase（PI3K）,protein kinase B（Akt）and mammalian target of rapamycin（m TOR）in H9c2 cells were determined by Western blot.Results:1.400μmol/L H₂O₂ was used to establish the aging model in vitro.2.DAI20,40 and 80μmol/L were selected as low,medium and high doses.3.DAI decreased the positive rate of SA-β-gal and the protein expression levels of p53 and p21 in aging H9c2 cells.4.DAI increased the level of T-AOC and the activity of SOD in aging H9c2cells,decreased the content of MDA and the level of ROS in cells,and maintained the stability of mitochondrial membrane potential.5.DAI promoted the formation of autophagosomes in aging H9c2 cells,promoted the expression of Beclin-1 and LC3proteins,and the degradation of p62 protein.6.DAI down-regulated the phosphorylation levels of PI3K,Akt and m TOR proteins in H9c2 cells.Conclusion:DAI can inhibit cell senescence by enhancing the antioxidant capacity of H9c2 cells,and activate the autophagy pathway by inhibiting PI3K/Akt/m TOR signaling pathway to play an anti-aging role.