Myogenic Estrogen Induced By Mechanical Stretch Promote The Expression Of Protein Synthesis Signal Protein In Senescent Myoblasts

Research ObjectivesWomen entering menopause are often accompanied by a loss of muscle mass and strength（sarcopenia）as estrogen levels drop.Our previous studies have found that mechanical stretch promotes myogenic estrogen production and improves skeletal muscle mass,but the effect on aging myoblasts has been unclear.Therefore,in this study,we established an aging cell model based on C₂C₁₂muscle cells to detect the effects of mechanical stretch on the expression of aromatase and estrogen production in aging muscle cells,and to explore the expression of signaling proteins related to protein synthesis in muscle cells,so as to further understand the possible molecular mechanism of exercise improving skeletal muscle protein metabolism under low estrogen conditions.Reserach MethodsThe skeletal muscle cells of mice C₂C₁₂were studied in this study.The cells were divided into control group（group C）and D-galactose group（group D）.Group D was treated with 20g/L D-galactose for 48h to establish the aging cell model.On the basis of D-galactose aging cell model,cells were divided into control group（group C）,stretch group（group S）,anastrozole group（group CA）and stretch+anastrozole group（group SA）according to whether anastrozole was added or not,and whether mechanical stretch was performed.In CA and SA groups,200μg/ml anastrozole was added into the medium for 24 h.After that,the S and SA groups were stretched with15%stretch rate and 0.5Hz frequency for 6 hours.The levels of estrogen in medium and the expression of aromatase protein in muscle cells were detected.Protein expression（Akt,m TOR,p70S6K,S6,Fox O）related to protein metabolism in PI3K/Akt pathway was detected.Results1.D-galactose can induce senescence of muscle cells and establish aging cell model.The cells in group D treated with 20g/L D-Gal showed various aging characteristics.Compared with group C,the multiplication capacity of cck8 cells in group D was significantly decreased（P<0.01）,the staining of SA-β-gal positive cells was increased（P<0.05）,and the expression level of aging protein p53 was significantly increased（P<0.01）.2.Aromatase inhibitor anastrozole can significantly reduce the expression of aromatase,while mechanical stretch can stimulate the expression of aromatase.With aromatase inhibition,the expression of aromatase was significantly decreased in group CA treated with anastrozole compared with group C（P<0.05）;Compared with group S,the expression of aromatase in group SA treated with anastrozole was significantly decreased（P<0.01）.With mechanical stretch,aromatase expression in group S was significantly increased compared with group C（P<0.05）;but there was no significant difference between group SA and group CA（P>0.05）.3.The aromatase inhibitor anastrozole significantly decreased myogenic estrogen production in cells,while mechanical stretch elevated myogenic estrogen production.With aromatase inhibition,estrogen concentration in cells of group CA was significantly decreased compared with that of group C（P<0.01）.Compared with group S,estrogen concentration in cells of group SA treated with anastrozole was significantly decreased（P<0.01）.With mechanical stretch,estrogen concentration in cells of group S was significantly increased compared with group C（P<0.01）.Compared with group CA,estrogen concentration in group SA cells was significantly increased（P<0.01）.4.Results of the expression of the proteins related to protein synthesis signal:aromatase inhibitor Anastrozole decreased the phosphorylation of m TOR and S6,and mechanical stretch increased the phosphorylation of Akt.With aromatase inhibition,compared with group C,the phosphorylation of m TOR in group CA was significantly decreased（P<0.01）and phosphorylation of S6 in group CA was decreased（P<0.05）.Compared with group S,m TOR phosphorylation in group SA was significantly decreased（P<0.01）,and S6 phosphorylation was significantly decreased（P<0.05）.With mechanical stretch,phosphorylation of Akt in group S was significantly increased compared with group C（P<0,01）.Compared with group CA,Akt phosphorylation expression in group SA was increased（P<0.05）.ConclusionsBased on the D-galactose aging cell model,mechanical stretch promotes aromatase expression and estrogen production in C₂C₁₂myoblasts,and influences protein synthesis through the PI3K/Akt signaling pathway.