GRα Regulates Tβ10-induced Oxidative Stress In Porcine Skeletal Muscle Cells

Stress response is a relatively important factor affecting the quality of meat which lead to the body’s secretion of a large number of stress hormones mainly in cortisol.We have demonstrated that adding cortisol during the feeding of animal diets can cause severe damage to muscle tissue.Compared with the control group,the number of apoptotic cells greatly increased and the muscle fibers increased.However,the current mechanism of how stress hormones affect meat quality and the ways in which meat quality changes is not fully understood.Current research shows that glucocorticoids pass through the cell membrane and bind to glucocorticoid receptor alpha to activate gene expression.Here we study the effect of GRα on skeletal muscle cells growth and the role in the process of producing inferior meat molecular regulatory mechanisms.The results are as followings:1.DEX causes muscle cell oxidative stressDEX has obvious toxicity to muscle cells.Compared with the control group,the number of reactive oxygen free radicals in the cells of the DEX group increased significantly,resulting in cellular oxidative stress.ROS in cells decreased significantly after treated with RU486.MTT results show that: DEX inhibits muscle cell proliferation.The expression of relationship between DEX and oxidative stress was detected using QPCR and Western blot,and the results showed that DEX could suppress the expression of HSP90 both at mRNA and protein level,the expression level of HSP90 in PSC cells can be inhibited after treated with RU486.2.The effects of GRα on the function of PSC cellsThe effect of GRα on cell function to was detected using flow cytometer,and the results showed that the overexpression of GRα could promote cell apoptosis and inhibi cell proliferation.Because of the rise of oxygen free radicals indicates that GRα causes the cells to be in an oxidative stress state.The expression of cell stress and apoptosis factors was detected using QPCR,and the results showed that overexpression of GRα could suppress the expression of BAX,HSP90,CAST and GPX2 at mRNA level.GRα siRNA-transfected had the opposite effect on PSC cells.Glutathione peroxidase activity is detected after overexpression and interference with GRα,the results showed that the overexpression of GRα could promote peroxide glutathionease activity.GRα siRNA-transfected had the opposite effect on PSC cells.3.Screening of GRα target genesGRα siRNA-transfected in PSC cell screening its target genes.The results showed that overexpression of GRα could suppress the expression of Tβ10,HSP90 and HSP27 at mRNA level.GRα siRNA-transfected had the opposite effect on PSC cells.We predicts the binding sites for GRα and Tβ10 on the predicted site.Therefore,we speculate that Tβ10 may be the target gene of GRα.The relationship between GRα and Tβ10 was detected using ChIP and luciferase reporter assays,the results showed that Tβ10 may be a potential target gene for GRα.The effect of Tβ10 on the apoptosis of PSC cells was detected using flow cytometer,showed that the overexpression of Tβ10 could promote cell apoptosis.The experiments found ROS had a significant increase in PSC cell,showed that Tβ10 causes the cells to be in an oxidative stress state.The expression of apoptosis factors was detected using QPCR,and the results showed that overexpression of Tβ10 could suppress the expression of BAX,HSP90,CAST and HSP27 at mRNA level.4.APS alleviates oxidative stress by inhibiting GRα expressionCompared with the overexpression of GRα group,adding different concentrations of(0.1 mg/mL,1 mg/mL,10 mg/mL,100 mg/mL)APS relieved cell stress,and the optimal concentration was 10 mg/mL by detecting the oxygen radical content.After the addition of 10 mg/mL APS,the ROS levels were significantly reduced,and it was consistent with the control group.The effects of APS on PSC cells proliferation was detected using MTT assay,the results showed that APS significantly promote cell proliferation.After adding APS,the activity of peroxide glutathionease was significantly lower than that of GRα group in order to maintain the body’s stability.To determine whether APS relieved cell apoptosis is with associated oxidative stress-related genes and apoptosis-related genes,we measured the expression of these genes using QPCR and Western blot.Compared with the overexpression of GRα group,the APS group had significantly lower expression levels of strss genes and apoptosis-related genes(BAX,HSP90,GRα,CAST and Tβ10).The results showed that APS relieves the body’s stress by inhibiting the expression of GRα.