In Vitro Study Of NSC-EXOs Promoting Proliferation And Differentiation Of C2C12 Cells Inhibited By Dexamethasone Through PTEN/Akt/P70 Pathway

Objective:Denervative muscular atrophy(DMA)is a common complication of lumbar diseases,and muscle atrophy has recovered after the release of nerve compression.The exosomes(NSC-EXOs)secreted by neural stem cells(NSCs)may be one of the mechanisms that promote the recovery of muscle atrophy.The purpose of this experiment is to explore whether NSC-EXOs can promote the growth and differentiation of C2C12 cells after being inhibited by dexamethasone.Methods:NSC primary cells were extracted from mouse cerebral cortex and verified by Nestin fluorescence staining.The exosomes secreted by NSCs were extracted by ultracentrifuges and verified by electron microscopy and Western Blot.When C2C12 cells reached 60-80%fusion,the growth medium was replaced by differentiation medium for 4 days,and the C2C12 cells were treated with dexamethasone(DEX)and dexamethasone plus NSC-EXOs(DEX+EXO)for 48 hours,respectively.Western Blot was used to detect myoD and myogenin protein related to muscle differentiation;The mRNA levels of myhl and myh2 were analyzed by qRT-PCR;MYH14 fluorescence staining related to muscle differentiation was observed by immunofluorescence staining.Next,the PTEN/Akt/P70 signal pathway was detected by Western Blot to explore the relationship between the above phenomena and the signal pathway.The C2C12 cells were transfected with PTEN overexpression lentivirus and its control virus,and then treated with NSC-EXOs after induction of differentiation.With the control group,the expression of PTEN protein was verified by Western Blot,and then the effect of NSC-EXOs was determined by detecting myoD and myogenin protein related to muscle differentiation.Western Blot was used to detect the expression of Akt and P70 and their phosphorylated proteins to prove the effect of NSC-EXOs on Akt/P70 signaling pathway.Results:Compared with the control group,NSC-EXOs can effectively improve C2C12 cells after growth and differentiation were inhibited by dexamethasone.NSC-EXOs significantly reduced the expression of PTEN protein,and then increased the expression of Akt and P70 phosphorylated protein.After transfection with lentivirus,PTEN overexpression lentivirus significantly increased the content of PTEN protein in C2C12 cells,and also decreased the protein expression of p-Akt and p-P70.The control virus group still showed Akt and P70 phosphorylation inhibition due to virus infection.Conclusion:Our study found that NSC-EXOs can significantly enhance the growth and differentiation of C2C12 cells after dexamethasone inhibition,and further study of the mechanism found that NSC-EXOs can repair muscle by reducing the content of PTEN in cells to activate the downstream AKT/P70 pathway.Our research is of great significance to explore the functional mechanism of NSC-EXOs and to develop the biotherapy of muscle atrophy based on exocrine body.But our shortcomings are still great.We have not thoroughly revealed the specific molecular mechanism of NSC-EXOs’ action on C2C12,and have not been further verified by in vivo experiments.However,we are still carrying out further research,and we will continue to update our research conclusions after more experimental results are produced.