Protective Effect Of Estrogen On Oxidative Damage Of Mouse C₂C₁₂ Myoblasts

Objective:Studies have shown that at each stage of the muscle injury model,there are some potential sites for the generation of free radicals,the oxidative stress caused by the generation of oxygen free radicals is an important factor in the entire process of skeletal muscle injury and repair.Due to its antioxidant structural properties,can estrogen play its antioxidant role in the oxidative stress of cells that occurs during skeletal muscle injury and repair,thereby protecting and preventing muscles from injury and inflammation.Therefore,based on the establishment of hydrogen peroxide-induced oxidative damage to mouse C₂C₁₂ myoblasts,this study explores the protective effect of estrogen supplementation on skeletal muscle cell oxidative damage,and explores related mechanisms for estrogen treatment and prevention of skeletal muscle damage provides experimental evidence at the cellular level.Methods:In this experiment,mouse C₂C₁₂ myoblasts were used as the research object.A model of oxidative stress induced by hydrogen peroxide was established.After the cells were subcultured to 80%confluency,the serum-free medium was replaced for grouping and rewells were set.The experiment was divided into controls Group（C,blank control group）,estrogen group（E,using 1umol/L of 17β-estradiol pretreatment for 1 hour）,hydrogen peroxide group（H,adding 1mmol/L hydrogen peroxide）,estrogen hydrogen peroxide group(EH,pretreatment with 1umol/L 17β-estradiol After 1 hour（after adding 1 mmol/L hydrogen peroxide）.After the intervention,the lactate dehydrogenase（LDH）activity in the culture medium was measured;intracellular reactive oxygen species（ROS）production,superoxide dismutase（SOD）and hydrogen peroxide（CAT）activities;And related protein expression.Results:1.Cytotoxicity（1）Content of lactate dehydrogenase（LDH）in cell culture mediumCompared with the control group（C）,there is no significant difference in the estrogen group（E）（297.33±18.88U/L,304.00±8.72U/L,P=0.352）,the hydrogen peroxide group（H）increased significantly（297.33±18.88U/L,502.67±7.01U/L,P<0.001）,compared with the hydrogen peroxide group（H）,estrogen Hydrogen peroxide group（EH）was significantly reduced（502.67±7.01U/L,403.33±10.25U/L,P<0.001）（2）Cytotoxicity resultsCompared with the control group（C）,Estrogen group（E）showed no significant difference（31.25±1.98%,31.95±0.92%,P>0.05）;the hydrogen peroxide group（H）Significantly increased（31.25±1.98%,52.84±0.74%,P<0.001）;compared with the hydrogen peroxide group（H）,the estrogen hydrogen peroxide group（group EH）was significantly reduced（52.84±0.74%,40.30±5.21%,P<0.001）.2.Detection of relative fluorescence levels of DCF products in each group of cellsCompared with the control group（C）,Estrogen group（E）showed no significant difference（100%,116.67±17.51%,P=0.206）.The hydrogen oxide group（H）increased significantly（100%,606.67±21.60%,P<0.001）;compared with the hydrogen peroxide group,the estrogen hydrogen peroxide group（EH）decreased significantly（606.67±21.60%,251.67±34.30%,P<0.001）.3.Cell viability（1）MTT assay of cell absorbance of each groupcompared with the control group（C）,Estrogen group（E）showed no significant difference（0.36±0.04,0.37±0.03,P=0.504）,and the hydrogen peroxide group（H）decreased significantly（0.36±0.04,0.34±0.04,P<0.01）.Compared with the hydrogen peroxide group（H）,the estrogen hydrogen peroxide group（EH）increased significantly（0.34±0.04,0.352±0.04,P<0.01）.（2）Relative cell viability results:Compared with the control group（C）,the estrogen group（E）has no significant difference（100%,100.38±1.55%,P>0.05）,the hydrogen peroxide group（H）was significantly reduced（100%,94.05±1.30%,P<0.001）;compared with the hydrogen peroxide group,the estrogen hydrogen peroxide group（EH）increased significantly（94.05±1.30%,96.76±1.68%,P<0.01）.4.Intracellular SOD and CAT activity test results（1）Test results of intracellular SOD activityCompared with the control group（C）,there was no significant difference in the estrogen group（E）（38.31±1.00U/mg,37.54±0.56U/mg,P=0.079）.The hydrogen peroxide group（H）was significantly reduced（38.31±1.00U/mg,31.94±0.76U/mg,P<0.001）;compared with the hydrogen peroxide group,the estrogen hydrogen peroxide group（group EH）was significantly increased（31.96±0.77U/mg,34.36±0.44U/mg,P<0.01）.（2）Intracellular CAT activity test resultsCompared with the control group（C）,the estrogen group（E）has no significant difference（25.92±0.67U/mg,25.95±0.52U/mg,P=0.917）,The hydrogen peroxide group（H）was significantly reduced（25.92±0.69U/mg,22.31±0.64U/mg,P<0.001）;compared with the hydrogen peroxide group,the estrogen hydrogen peroxide group（EH）was significantly increased（22.31±0.64U/mg,25.29±0.70U/mg,P<0.001）.5.Intracellular Nrf2,p-Nr2,and HO-1 protein expression test results（1）Nrf2 western blot resultshydrogen peroxide group（H）expression level was reduced compared to the control group（C）（P<0.01）,the estrogen group（E）was significantly enhanced（P<0.01）;compared with the hydrogen peroxide group（E）,the expression of Nrf2 in the estrogen hydrogen peroxide group（EH）was also increased（P<0.01）.（2）Phosphorylated western blot results of Nrf2It showed that the estrogen group（E）and hydrogen peroxide group（H）were increased compared with the control group（C）（P<0.01）;compared with the hydrogen peroxide group（H）,the expression of phosphorylation of Nrf2 in the estrogen hydrogen peroxide group（EH）was also increased（P<0.01）.（3）HO-1 Western Blotting ResultsThe expression level of the hydrogen peroxide group（H）was reduced compared to the normal control group（C）（P<0.01）,and the estrogen group（E）was significantly enhanced（P<0.01）;Compared with the hydrogen peroxide group（E）,HO-1 expression in the estrogen hydrogen peroxide group（EH）was also increased（P<0.01）.Conclusions:1.Estrogen can improve the oxidative damage of mouse C₂C₁₂ myoblasts induced by H2O2.The mechanism is to reduce the generation of intracellular reactive oxygen species and increase the activity of superoxide dismutase（SOD）and catalase（CAT）in cells.Increased expression and phosphorylation of Nrf2 protein2.In the absence of oxidative damage to mouse C₂C₁₂ myoblasts,estrogen can increase the expression and phosphorylation of Nrf2 protein,but cannot reduce the generation of intracellular reactive oxygen species and increase intracellular superoxide dismutase（SOD）,catalase（CAT）activity.