Salvia Miltiorrhiza Inhibits The H₂O₂-induced Oxidative Damage On Myocardial Cells Via Activating The Expression Of Nrf2

Objective To observe the protective effect of Salvia miltiorrhiza on oxidative damage induced by H₂O₂ in rat myocardial cells,and explore the molecular mechanism of Salvia miltiorrhiza on inhibiting the oxidative damage.Methods1.Establish oxidative damage model by H₂O₂ :1)Different concentrations of H₂O₂ were added to rat myocardial H9C2 cells which were incubated of same time for SRB detection.The cell growth inhibition rate was calculated to determine the optimal concentration of H₂O₂.2)The same concentrations of H₂O₂ were added to rat myocardial H9C2 cells which were incubated of different time for SRB detection.The cell growth inhibition rate was calculated,and the optimal incubation time of H₂O₂ was determined.2.Establish anti oxidative damage model by Salvia miltiorrhiza:1)Different concentrations of Salvia miltiorrhiza were added to rat myocardial H9C2 cells for same time before the addition of H₂O₂ treatment.Calculated the cell survival rates by SRB detection,and determined the optimal concentration of the Salvia miltiorrhiza.2)The same concentrations of Salvia miltiorrhiza were added to rat myocardial H9C2 cells for different time before the treatment of H₂O₂.Calculated the cell viability by SRB detection,and determined the optimal incubation time of Salvia miltiorrhiza.3.DAPI staining was performed in rat myocardial H9C2 cells.Cell number and cell nucleus morphology were observed under microscope.4.Expression of Myocardin mRNA and Nrf2 mRNA were detected by Real-time PCR in order to observe the effects of H₂O₂ and Salvia miltiorrhizahis on Myocardin mRNA and Nrf2 mRNA in rat myocardial H9C2 cells.5.Expression of Myocardin protein and Nrf2 protein were detected by Western Blotting in order to observe the effects of H₂O₂ and Salvia miltiorrhizahis on Myocardin protein and Nrf2 protein in rat myocardial H9C2 cells.6.Observe the effects of Myocardin gene and Nrf2 gene on cell proliferation: detect cell proliferation when over expressed or down regulated of Myocardin gene IV and Nrf2 gene.7.Observe the effects of changing Myocardin gene on the expression of Nrf2 gene : detected the expression of mRNA and protein of Nrf2 gene by overexpressing Myocardin gene and down regulating Myocardin gene.Results1.Oxidative damage model by H₂O₂ can be determined when cells were incubated for 24 hours by 200 μM H₂O₂ because the cell number was close to LD50.The number of cells observed by microscope was significantly reduced in comparison with the control group,and the nuclear swelling,shrinkage and fragmentation were obvious.2.The oxidative damage of myocardial cells was reduced when cells were incubated by 0.15mg/ml salvia miltiorrhiza for one hour.The cell number of Salvia miltiorrhiza+H₂O₂ group observed by microscope was more than that in the H₂O₂ group,and the status of nuclear uniformity and swelling of the nuclei,pyknosis,fragmentation compared with H₂O₂ group were improved.3.H₂O₂ could inhibit the expression of myocardin mRNA and protein and increase the expression of Nrf2 mRNA and protein,while Salviae miltiorrhiza could further reduced the expression of myocardin mRNA and protein and obviously increase the expression of Nrf2 mRNA and protein.4.Compared with control group,the relative number of living cells which were over-expressed Myocardin gene or down-regulated Nrf2 gene was decreased,while it was increased by down-regulation Myocardin gene or over-expressed Nrf2 gene.5.Nrf2 mRNA and protein expression were significantly down regulated when Myocardin gene was over-expressed,while they were obviously up regulated when Myocardin gene was down-regulated.Conclusion:1.Oxidative damage of myocardial cells caused by H₂O₂ can activate antioxidant damage pathway led by Nrf2.2.Salvia miltiorrhiza can inhibit H₂O₂-induced myocardial oxidative damage.and its mechanism may be achieved by down-regulating the expression of Myocardin and further promoting the expression of Nrf2.3.Nrf2 promoted cell proliferation and Myocardin inhibited cell proliferation.The effects of the two on cell proliferation were opposite.4.Myocardin gene could involved in the regulation of Nrf2 expression.