Attenuation Of Cardiocyte Oxidative Injury Due To Astragalus Membranaceus By Up-regulating Endogenous Cytoprotective Mechanism

Objectives:To investigate and compare the protective effects of two Astragalus membranaceus components Astragalosideâ…£and Quercetin on oxidative injury of hypoxic rat myocardial cells, and determine their dose-effect relationships, then choose a better component to clarify its endogenous mechanisms on attenuation of cardiocyte oxidative injury.Methods:1. SD neonate rat myocardial cells were cultured in vitro and reared in an oxygen deficient milieu (mixture of 94%N₂,5%CO₂,1% O₂) as hypoxia model.2. Cells were divided into the following groups: A, control group; B, hypoxia group; C, hypoxia+QUE (C₁ 100mg/L, C₂ 50 mg/L, C₃ 25mg/L); D, hypoxia+AST (D₁ 50mg/L, D₂ 25mg/L, D₃ 12.5mg/L), and E, hypoxia+VitE (10mg/L). The concentration of AST and QUE used in the article was determined according to previous reports and our preliminary experiments.3.The viability of cells (CCK-8), superoxide dismutase (SOD) activity and levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), active oxygen species (ROS) were detected after hypoxia for 12hr pretreated with different doses of Astragalosideâ…£, Quercetin or VitE.4. According to the results of the experiments, a dose of 25mg/L Astragalosideâ…£was chosen for the later research.5. Label the Astragalosideâ…£molecule with FITC or HRP, then observe whether they can enter the cells and their distributions in the cells under fluorescent microscope, confocal microscope and electron microscope.6. Detect the ability of Astragalosideâ…£to scavenge the hydroxy radical (OH^-)and superoxide anion(O₂^-)in vitro. 7. Block the activation of SOD and detect the scavenge ability of Astragalosideâ…£on intracellular ROS of hypoxic myocardial cells.8. The SOD-1 mRNA expressions of hypoxic and normal myocardial cells treated with Astragalosideâ…£were detected by RT-PCR.9. The SOD-1 protein expressions of hypoxic and normal myocardial cells treated with Astragalosideâ…£were detected by Western blotting.Results:1. After 12h hypoxia, levels of LDH, MDA, ROS in group B increased significantly, while CCK-8,SOD decreased significantly as compared with those in group A. After treated with Astragalosideâ…£, Quercetin or VitE, levels of LDH, MDA, ROS in group C, D and E all decreased, and CCK-8, SOD increased markedly as compared with those in group B. At the same doses, levels of LDH and CCK-8 in Astragalosideâ…£-treated groups were significantly improved than those in Quercetin-treated groups. While there were no significant differences in MDA, SOD and ROS levels between these groups. The protective effects of groups C₂, C₃, D₂ and D₃ are better than that of the group E.2. It was found that the Astragalosideâ…£molecules labeled with FITC entered into the cell and located in the endochylema under the laser confocal microscopy. Astragalosideâ…£molecules labeled with HRP located on the mitochondrial membrane under electronic microscopy.3. The in vitro study showed that the amounts of OH^- decreased significantly, but the quantity of O₂^- remained unchanged after treatment with Astragalosideâ…£.4. After blockage of SOD activity with DDC, the levels of ROS in myocardial cells remained unchanged after Astragalosideâ…£treatment.5. The expression of SOD-1 mRNA increased after Astragalosideâ…£treatment in hypoxic and normoxic myocardial cells.6. The expression of SOD-1 protein increased after Astragalosideâ…£treatment in hypoxic and normoxic myocardial cells.Discussion and Conclusions1. Both Astragalosideâ…£and Quercetin were beneficial in protecting myocardial cells against hypoxia because of attenuating oxidative damage. The protective effects of large and middle dosages are better than that of VitE. 2. The effect of Astragalosideâ…£is better than Quercetin in decreasing the amounts of LDH and increasing the number of viable cells at the same dosage, but the effect on attenuating oxidative damage remains the same.3. Astragalosideâ…£labeled with HRP could enter into the cells and locate on the mitochondrial membrane specifically.4. Astragalosideâ…£could attenuate oxidative stress by increasing the SOD-1 mRNA and SOD-1 protein expressions of normal and hypoxic cells and removing intracellular OH^- directly through reacting with it.5. Astragalosideâ…£could not scavenge the intracellular O₂^- directly, so it could not scavenge ROS in the hypoxic myocardial cells after blocking the activation of SOD.