Astragaloside â…£ Protects H9c2 Cells Against H₂O₂-induced Oxidative Injury Through PI3K/Akt Signaling Pathway

Objective:The purpose of this study was to investigate the cardioprotective effects and mechanism of astragalosideⅣ(AST) on H2O2-induced oxidative injury in H9c2 cells.Methods:1. H2O2 was administered to establish the oxidative injury model of H9c2 cells.H9c2 cells were cultured 24 hours, then replaced serum-free medium with different concentrations of H2O2 for 3,6,12 hours respectively. H9c2 cells were divided randomly into 4 groups randomly:①Control group:high glucose DMEM medium;②H2O2100 group:H2O2100μmol/L;③H2O2200 group:H2O2200μmol/L;④H2O2 400 group:H2O2400μmol/L. The cell viability was measured using MTT method to select the right concentration and time of H2O2 for establishing the oxidative injury model of H9c2 cells.2. Effects of AST on H9c2 cells in H2O2 model and its relationship with PI3K/Akt signaling pathway.H9c2 cells were cultured for 24 hours, then replaced serum-free medium with different reagents for 6 hours. H9c2 cells were divided randomly into 7 groups randomly:①Control group:high glucose DMEM medium;②H2O2 group:H2O2 200μmol/L;③AST10+H2O2 group:AST 10 mg/L and H2O2200μmol/L;④AST20+H2O2 group:AST 20 mg/L and H2O2200μmol/L;⑤LY294002+H2O2 group: LY29400210μmol/L pretreatment for 10min, then add H2O2200μmol/L;⑥LY294002+AST10+H2O2 group:LY29400210μmol/L pretreatment for 10min, then add H2O2200μmol/L and AST 10 mg/L;⑦LY294002+AST20+H2O2 group: LY29400210μmol/L pretreatment for 10 min, then add H2O2200μmol/L and AST 10mg/L. The cell viability was measured using MTT method. Apoptosis of H9c2 cells was assessed using Hoechst staining, agrose electrophoresis of DNA and caspase-3 activity assay. The expression of p-Akt and t-Akt was determined by Western blot analysis.Results:1. H2O2 was administered to establish the oxidative injury model of H9c2 cells.Treatment with H2O2for 3 hours, cell viabilities of 100,200,400μmol/L H2O2 groups cell viability were 94.6%±4.59,84.7%±15.2,66.3%±14.9; Treatment with H2O2 for 6 hours, cell viabilities of 100,200,400μmol/L H2O2 groups cell viability were 92.5±5.33,63.9%±10.1,30.8%±9.83; Treatment with H2O2 for 12 hours, cell viabilities of 100,200,400μmol/L H2O2 groups cell viability were 69.4%±10.9,24.7%±3.06, 24.7%±2.61; compared with control group, the cell viability of each group was decreased significantly; Under 200μmol/L H2O2 treatment for 6 hours, the vaibility of H9c2 cells is suitable for the following study.2. Effects of AST on H9c2 cells in H2O2 model.In H2O2 group, the viability of H9c2 cells was decreased significantly (72.9%±3.09 vs. 100%±0, P<0.01); the activity of caspase-3 was increased significantly (310%±25.3 vs.100%±0, P<0.01); The Hoechst staining positive H9c2 cells and the typical DNA Ladder were increased significantly.Compared with H2O2 group, the vitality of H9c2 cells was increased by 10 mg/L and 20 mg/L of AST (86.2%±6.49,88.2%±4.62 vs.72.9%±3.09, P<0.01); the activity of caspase-3 (198.7%±12.7,178.4%±31.4 vs.310%±25.3, P<0.01) was decreased significantly; The Hoechst staining positive H9c2 cells and the typical DNA Ladder were diminished significantly.3. AST protects H9c2 cells in H2O2 model through PI3K/Akt signaling pathway.Compared with 10 mg/L or 20 mg/L of AST, pretreatment with LY294002, an inhibitor of PI3K/Akt, the viability of H9c2 cells was decreased significantly (73.9%±6.00 vs.86.2%±6.49,71.8%±3.62 vs.88.2%±4.62, P<0.01); the activity of caspase-3 was increased significantly (288.4%±29.9 vs.198.7%±12.7,287.7%±27.6 vs. 178.4%±31.4, P<0.01); The Hoechst staining positive cells and the typical DNA Ladder formation were significantly increased.Western blot showed that both 10 mg/L and 20 mg/L of AST could increase the expression ofρ-Akt (P<0.01), which was markedly abolished by pretreating H9c2 cells with LY294002(P<0.01).Conclusions:1. It is successful that H9c2 cells oxidative injury be induced by H2O2. Under the conditions of 200μmol/L H2O2 treatment for 6 hours, the vaibility of H9c2 cells is suitable for model standard.2. AST protects H9c2 cells against oxidative injury in H2O2 model.3. AST protects H9c2 cells against oxidative injury in H2O2 model through PI3K/Akt signaling pathway.