| ObjectiveTo study of the protective effects and its mechanisms of Astragaloside Ⅳonglomerular mesangial cell oxidative stress injury, and to further explore the molecularmechanism of Astragaloside Ⅳ.Methods1. Establish glomerular mesangial cell oxidative stress damage modelThe cultured human glomerular mesangial cells （HMC） were randomly divided into6groups: normal control group, H2O2(1×105,2×105,3×105,4×105,5×105nmol L-1) groups （stimulating for4h）, MTT method was used to determine the cell vitality,and the lactate dehydrogenase （LDH） activity was detected in cell cultured medium.2. Protective effect of AstragalosideⅣ on glomerular mesangial cell oxidative injuryThe cultured human glomerular mesangial cells were randomly divided into: normalcontrol group; H2O2model group; AstragalosideⅣ (6.25,12.5,25,50,100nmol L-1)intervention group; antioxidants Tempol (1×105nmol L-1) and Rg1(3×104nmol L-1)groups.Cell viability was detected by MTT method, and the lactate dehydrogenase（LDH） activity, superoxide dismutase （SOD） activity and malondialdehyde （MDA）content were detected by biochemical method.3. The mechanisms of AstragalosideⅣon glomerular mesangial cell oxidative injuryThe cultured human glomerular mesangial cells were randomly divided into：Normal control group; H2O2model group; Astragaloside Ⅳ(12.5,100nmol L-1)intervention groups and Tempol (1×105nmol L-1) group. Dealing with each cell group, the cells apoptosis was detected by Hoechst33258staining; DHE staining was used todetect the generation of reactive oxygen species in the cell; Flow Cytometry was used todetect the cell cycle changes; Expression of cell cycle protein CyclinD1, CyclinA andphosphorylated p38, T-p38was detected by Western blot.Results1. Establishment of Glomerular mesangial cell oxidative damage modelH2O2(1×105,2×105,3×105,4×105,5×105nmol L-1) could induce oxidative injury ofglomerular mesangial cells. The results showed that, compared with control group,H2O2treated groups significantly decreased the cell viability and cell survival rate, andincreased the Lactate dehydrogenase （LDH） activity in cell culture supernatant fluid.2. Protective effects of AstragalosideⅣ on glomerular mesangial cell oxidative injuryThe Astragaloside Ⅳ(6.25,12.5,25,50,100nmol L-1) groups could reducemesangial cells oxidative injury induced by H2O2(3×105nmol L-1). The results showedthat, compare with H2O2treated group, Astragaloside Ⅳgroups significantly increasedthe cell survival rate and cell viability, decreased the lactate dehydrogenase （LDH）activity, increased the superoxide disproportionation enzyme （SOD） activity anddecreaed the malondialdehyde （MDA） content in supernatant fluid.3. The protective mechanisms of AstragalosideⅣon glomerular mesangial cell injuryinduced by H2O2oxidative stressThe results showed that Astragaloside Ⅳ (100nmol L-1) could significantly reducethe glomerular mesangial cell oxidative injury induced by H2O2(3×105nmol L-1).Compared with model group, Astragaloside Ⅳ significantly inhibited the glomerularmesangial cell apoptosis, decreased the the ROS production; regulation the expressionof cell cycle protein Cyclin D1, the percentage of each cell cycle and cell growth, anddecreased the expression of phosphorylated P38. Conclusion1. The optimum condition of glomerular mesangial cells oxidative damage model wasinduced by H2O2(3×105nmol L-1) for4h in vitro.2. Astragaloside Ⅳc ouldprotect the cell oxidative damage induced by H2O2, increasecell viability, decreased the LDH activity, increase SOD activity, and decrease theMDA content.3. The protective mechanisms of AstragalosideⅣon H2O2induced glomerularmesangial cell oxidative injury may be associated with decreasing intracellular ROSgeneration, increasing the expression of cell cycle protein Cyclin D1, and inhibiting thep38/MAPK signal pathways and cell apoptosis. |
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