Effect Of Glucocorticoid Receptor GR? On Protein Breakdown And Synthesis In Porcine Skeletal Muscle Cells

In livestock production,animals are affected by a variety of factors.In order to cope with these changes,the animal body will have a stress response.The HPA axis activity is enhanced when stress occurs,and the adrenal cortex secretes a large amount of glucocorticoid(GC).GC has two receptors,the glucocorticoid receptor alpha(GR?)plays an important role.Current studies have shown that increased levels of glucocorticoids reduce the body's total protein synthesis rate and decrease the average muscle fiber diameter.Previous studies in our research group have also shown that the addition of cortisol to piglet diets leads to a significant decrease in piglet weight gain.On the basis of these,GR? is the main research object to explore the expression changes of genes affecting protein deposition and the regulation of genes affecting the metabolism of porcine skeletal muscle in porcine skeletal muscle cells under stress.The molecular mechanism of corticosteroid-induced decline in pig weight gain provides a theoretical basis.The main findings of the project are as follows: 1.The effect of dexamethasone on protein deposition genesAfter using dexamethasone(DEX)to treat porcine skeletal muscle cells,the expression levels of the protein degradation genes Atrogin-1,MSTN and FOXO1 were detected.The results showed that the expression levels of these genes were significantly increased,and the gene IGF-1 which promotes protein synthesis was decreased.After adding an equal concentration of the glucocorticoid antagonist mifepristone(Mifepristone/RU486),the results showed that the gene expression level that promotes protein degradation decreased,and the gene expression level that inhibited protein decomposition increased.2.Effect of GR? on protein depositionThe GR? overexpression vector was transfected into the cells to promote the expression of GR?,and the interference fragment of GR? was synthesized to inhibit the expression of GR?.The changes in protein concentration after overexpression of GR? and interference with GR? were detected by BCA method.The results showed that overexpression of GR? decreased the protein concentration and increased the protein concentration after interfering with GR?.In addition,RT-PCR and Western Blotting were used to detect the expression of genes involved in protein degradation.It was found that the expression level of genes that promote protein breakdown after super-expression of GR? increased,and the level of gene expression that promoted protein decomposition after interfering with GR? decreased.3.Screening of downstream genes of GR?Using the longissimus dorsi muscle sample preserved by the research group to detect the up-regulated genes in transcriptome sequencing results and the expression changes of these genes after overexpression and interference with GR?,it was found that SLC2A4 was highly expressed in the cortisol group,and SLC2A4 was overexpressed after GR?.The expression increased significantly.The expression of SLC2A4 was significantly decreased after interfering with GR?,and the online transcription site predicted that there may be binding of transcription factors GR? and SLC2A4.It is speculated that SLC2A4 may be a potential target gene of GR?.The luciferase reporter assay by SLC2A4 and the co-transfection assay with the GR? overexpression vector preliminarily showed that SLC2A4 is a target gene of GR?.4.Effect of SLC2A4 on protein depositionThe expression of the protein deposition gene was detected by constructing the SLC2A4 overexpression vector.The results showed that the expression level of the gene Atrogin-1,which promotes protein decomposition,was significantly decreased after overexpression of SLC2A4.The mTOR signaling pathway plays an important role in protein synthesis.RT-PCR detection of genes in the mTOR signaling pathway revealed increased expression of mTOR and its downstream genes S6K1 and 4EBP1 in the mTOR signaling pathway,and mTOR and S6K1 in the mTOR signaling pathway.Increased protein phosphorylation levels suggest that elevated expression levels of the sugar transporter SLC2A4 partially attenuate the decline in protein deposition levels caused by stress.Conclusion: When the stress occurs,the elevated level of glucocorticoids in the body promotes the gene expression level of protein breakdown,which reduces the level of muscle protein deposition.At the same time,the expression level of the sugar transporter gene SLC2A4 also rises,thus relieving stress.The level of protein deposition is reduced.