GR? Regulates T?10-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 GR? 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 GR? on the function of PSC cellsThe effect of GR? on cell function to was detected using flow cytometer,and the results showed that the overexpression of GR? could promote cell apoptosis and inhibi cell proliferation.Because of the rise of oxygen free radicals indicates that GR? 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 GR? could suppress the expression of BAX,HSP90,CAST and GPX2 at mRNA level.GR? siRNA-transfected had the opposite effect on PSC cells.Glutathione peroxidase activity is detected after overexpression and interference with GR?,the results showed that the overexpression of GR? could promote peroxide glutathionease activity.GR? siRNA-transfected had the opposite effect on PSC cells.3.Screening of GR? target genesGR? siRNA-transfected in PSC cell screening its target genes.The results showed that overexpression of GR? could suppress the expression of T?10,HSP90 and HSP27 at mRNA level.GR? siRNA-transfected had the opposite effect on PSC cells.We predicts the binding sites for GR? and T?10 on the predicted site.Therefore,we speculate that T?10 may be the target gene of GR?.The relationship between GR? and T?10 was detected using ChIP and luciferase reporter assays,the results showed that T?10 may be a potential target gene for GR?.The effect of T?10 on the apoptosis of PSC cells was detected using flow cytometer,showed that the overexpression of T?10 could promote cell apoptosis.The experiments found ROS had a significant increase in PSC cell,showed that T?10 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 T?10 could suppress the expression of BAX,HSP90,CAST and HSP27 at mRNA level.4.APS alleviates oxidative stress by inhibiting GR? expressionCompared with the overexpression of GR? 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 GR? 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 GR? group,the APS group had significantly lower expression levels of strss genes and apoptosis-related genes(BAX,HSP90,GR?,CAST and T?10).The results showed that APS relieves the body's stress by inhibiting the expression of GR?.