Astragaloside Ⅳ Protects H9c2 Cells Against H₂O₂-induced Oxidative Injury Through ERK1/2 Signaling Pathway

To investigate the protective effects of astragaloside IV on H2O2-induced oxidative injury in H9c2 cells and to explore whether the mechanism of protective effects involve in ERK1/2 signaling pathway.H9c2 cells were cultured for 24 hours with high glucose DMEM medium containing 10% FBS, then serum-free medium was replaced with 100,200,400μmol/L H2O2 for 3,6,12 hours respectively. The viability of H9c2 cells was detected using MTT method. The effective concentration and time was choosen to establish the H2O2-induced injury model of H9c2 cells.H9c2 cells were cultured for 24 hours with high glucose DMEM medium containing 10% FBS, then serum-free medium was replaced with different agents for another 6 hours. H9c2 cells were divided randomly into 4 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. The viability of H9c2 cells was detected using MTT method. Activity of lactate dehydrogenase(LDH), total superoxide dismutase(T-SOD) and manganese superoxide dismutase(Mn-SOD), content of malondialdehyde(MDA) in the culture medium were detected using colorimetric method.H9c2 cells were cultured for 24 hours with high glucose DMEM medium containing 10% FBS, then serum-free medium was replaced with different agents for another 6 hours. H9c2 cells were divided randomly into 7 groups randomly:①-④groups:the same treatments as above.⑤PD+H2O2 group:PD98059 50μmol/L pretreatment for 30 min, then add H2O2200μmol/L;⑥PD+AST10+H2O2 group: PD98059 50μmol/L pretreatment for 30 min, then add H2O2200μmol/L and AST 10 mg/L;⑦PD+AST20+H2O2 group:PD98059 50μmol/L pretreatment for 30 min, then add H2O2 200μmol/L and AST 20 mg/L. The viability of H9c2 cells were detected using MTT method. Activity of LDH, T-SOD and Mn-SOD, content of MDA in the culture medium were detected using colorimetric method. Western blot was performed to exam expression of p-ERKl/2 and ERK1/2 in H9c2 cells respectively.Treatment with H2O2 for 3 hours, compared with control group, cell viablities of 100,200,400μmol/L H2O2 groups(94.6%±4.59%,84.7%±15.2%,66.3%±14.9%) were decreased significantly as concentration increasing(P<0.05); treatment with H2O2 for 6 hours, compared with control group, cell viabilities of 100,200,400μmol/L H2O2 groups(92.5%±7.15%,63.9%±10.1%,30.8%±9.83%) were decreased significantly as concentration increasing(P<0.01); treatment with H2O2 for 12 hours, compared with control group, cell viabilities of 100,200,400μmol/L H2O2 groups(69.4%±10.9%,24.7%±3.06%,24.7%±2.61%) were decreased significantly as concentration increasing(P<0.01). Under 200μmol/L H2O2 treatment for 6 hours, the vaibility of H9c2 cells is suitable for the following study.Compared with control group,200μmol/L H2O2 treatment for 6 hours decreased the cell viability significantly (70.3%±6.52% vs.100%±0, P<0.01), the activity of LDH in the culture medium was increased significantly(289.4±15.8 vs.26.7±6.6 U/L, P<0.01), the activity of T-SOD and Mn-SOD was decreased significantly(8.80±1.42 vs.19.68±1.70,3.21±0.65 vs.6.58±1.45 U/ml, P<0.01), the content of MDA was increased significantly(0.955±0.120 vs.0.307±0.056μmol/L, P<0.01).Compared with H2O2 group, in AST10+H2O2 and AST20+H2O2 groups, the cell viability was increased significantly(89.4%±7.57%,90.1%±4.04% vs. 70.3%±6.52%, P<0.01); the activity of LDH in the culture medium was decreased significantly(147.2±34.3,170.4±14.0 vs.289.4±15.8 U/L, P<0.01), the activity of T-SOD(14.99±0.98,13.76±2.06 vs.8.80±1.42 U/ml, P<0.01) and Mn-SOD (6.43±0.69,6.15±0.97 vs.3.21±0.65 U/ml, P<0.01) was increased significantly, the content of MDA was decreased significantly(0.506±0.051,0.542±0.035 vs. 0.955±0.120 umol/L,P<0.01).Compared with AST10+H2O2 and AST20+H2O2 groups respectively, in groups pretreated with PD98059, inhibitor of ERK1/2, the cell viability was decreased significantly(73.5%±5.96% vs.89.4%±7.57%,74.7%±74.7% vs.90.1%±4.04%, P<0.01), the activity of LDH in the culture medium was increased significantly(277.1±27.3 vs.147.2±34.3,275.7±28.7 vs.170.4±14.0 U/L, P<0.01), the activity of T-SOD(10.52±1.56 vs.14.99±0.98,9.99±1.54 vs.13.76±2.06 U/ml, P<0.01) and Mn-SOD(3.93±0.97 vs.6.43±0.69,3.66±0.81 vs.6.15±0.97 U/ml, P<0.01) was decreased significantly, the content of MDA was increased significantly(0.844±0.054 vs.0.506±0.051,0.855±0.043 vs.0.542±0.035μmol/L, P<0.01).Treated with 10 mg/L or 20 mg/L of AST, expression of p-ERK1/2 in H9c2 cells jnjuried from H2O2 was increased significantly(P<0.01), when PD98059 was added, the expression of p-ERK1/2 was inhibited significantly(P<0.01).1. With 200μmol/L of H2O2 for 6 hours, the oxidative model of H9c2 cells induced by H2O2 was established successfully and with good reproducibility.2.10 mg/L or 20 mg/L AST protects H9c2 cells against H2O2-induced oxidative injury by increasing the viability, decreasing the activity of LDH and the content of MDA, increasing the activity of T-SOD and Mn-SOD in the culture medium.3. AST promotes the expression of p-ERKl/2 in H9c2 cells from injury of H2O2. The protection of AST is inhibited by PD98059, the inhibitor of ERK1/2. AST protects H9c2 cells against H2O2-induced oxidative injury through ERK1/2 signaling pathway.