Pretreatment with astragaloside IV protects H9c2 cells against hydrogen peroxide-induced apoptosis by scavenging of reactive oxygen species and regulation of Bcl-2 and Bax expression

Abstract

Astragaloside IV (ASI), a saponin isolated from Radix Astragali, has been found to have potent cardioprotective effects. This study was designed to investigate whether ASI prevents cardiomyocytes from hydrogen peroxide (H2O2)-induced apoptosis. H9c2 cells were pretreated with different concentrations of ASI (5, 10, 20 μg/ml) for 24 h and then exposed to 100 μM H2O2 for 24 h. The cell viability were examined by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay, reactive oxygen species(ROS) generation was quantified by the 2',7'-dichlorofluorescin-diacetate (DCFH-DA) method. The activity of antioxidant enzymes, including catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were determined photometrically. Apoptotic cells were detected by Hoechst 33258 staining, annexin-V binding and by assessment of caspase-3. Protein and mRNA expression of both Bax and Bcl-2 were determined by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The cell apoptosis significantly increased after 24 h of H2O2 exposure. Pretreatment of H9c2 cells with ASI significantly increased the activities of antioxidant enzymes, scavenged ROS and reduced malondialdehyde (MDA) production. ASI increased the expression of Bcl-2, decreased the expression of Bax, and ultimately reduced H2O2-induced H9c2 cells apoptosis. In summary, these results suggest that ASI can block H2O2-induced apoptosis in H9c2 cells, and that the underlying mechanism involves in scavenging of ROS and modulating expression of Bcl-2 and Bax. Key words: Astragaloside IV, H9c2 cells, hydrogen peroxide, oxidative stress, apoptosis.