The Effects Of Mechanical Stretch On The Proliferation Of Rat Skeletal Muscle Satellite Cells And Its Possible Pathways

Objective: Skeletal muscle satellite cells are myogenic stem cells.They are generally in a resting state.Movement or damage can activate the resting skeletal muscle satellite cells.Skeletal muscle satellite cells proliferate and differentiate and eventually fuse with damaged muscle fibers to repair the muscle.Mechanical stretch has been studied through simulations.Previous studies have used muscle cell lines more than muscle-derived stem cells to study the effects of muscle injury repair.Studies have shown that mechanical traction has contributed to satellite cells.The role of activation and pro-proliferation,but the specific mechanism of action has not yet been studied clearly,this experiment by exploring the role of different stretch modes on the proliferation of rat skeletal muscle satellite cells and further explore its possible role in pathogenesis for skeletal muscle injury repair and Cellular hypertrophy provides a theoretical basis.Methods: Two-step enzymatic digestion method combine with differential adherent attachment technique were used to separate and purify satellite cells.At the third passage,satellite cells were divided into 5 groups: control group,5% stretch 4h group,5% stretch 6h group,15% stretch 4h group,15% stretch 6h group.Flexcell cell stretch system was performed at a frequency of 1 Hz.The proliferation of satellite cells was detected by CCK-8 after stretch after 24 hours.The activation of Akt,P38,ERK and JNK3 were detected by WB in the immediate stretch and after 24 hours;Myf5,Myo D Protein expression were detected after stretch after 24 hours.Results:1.αstriated muscle actin identification results:α-Striated muscle actin is positively expressed in skeletal muscle,actin is brown,and positive particles are concentrated in the cytoplasm,with different shades of color.In this experiment,a total of 1000 cells were counted,and the number of negative cells was 24.Cell purity(100%)=(1000-24)/1000×100%=97.6%The purity of the cells is ≥ 95%,which meets the requirements for purification.2.CCK8 detection of proliferation results:Compared with the quiet control group,the proliferation of skeletal muscle satellite cells in the 15% stretch group,the 4h stretch group,and the 6h stretch group wassignificantly increased(P<0.01).Among them,the same 15% stretch degree was used to promote the 6h group.The proliferation effect was better than that of the pull 4h group(P<0.05).Compared with the control group,there was no significant difference in the proliferation of skeletal muscle satellite cells in the 5% stretch,stretched 4 h group and 5% stretched 6 h group(P>0.05).3.Immediately after drawing ends:At the end of pulling,the activity of Akt,P38,ERK,and JNK in the 4 h stretch group and the 6 h stretch group was higher than that in the control group(P<0.01)at the15% stretch;with the stretch rate of 15%,the pulling force was 6 h.The activity of Akt,P38 and ERK in the group was higher than that in the 4h group(P<0.01).Compared with the control group,the phosphorylation of Akt,P38,ERK and JNK was not significant in the 5% stretched 4 h group and the 5% stretched 6 h group(P >0.05).4.After pulling finish 24h(1)15% stretch,Myf5 protein expression in the 6h group was higher than that in the control group(P<0.01),and 5% stretched in the 4h group,5% stretch in the 6h group,and 15% in the stretch group.There was no significant change in Myf5 protein expression in Stretched 4h group compared with the control group(P>0.05).At 15% stretch,the expression of Myo D in the 4h and 6h groups was higher than that in the control group(P<0.01),while the Myo D in the 6h group and the 5% stretch in the 5% stretch group.There was no significant change in protein expression compared with the control group(P>0.05).(2)Compared with the control group,the activity of Akt,P38,ERK,and JNK in the4 h stretch group and the 6h pull group increased after 24 h of pull-down(P<0.01);At % stretch,Akt activity was higher in the 6 h group than in the 4 h group(P<0.05).The activities of Akt,P38,ERK,and JNK in the 5% stretching group and the 5%stretching group were not significantly changed(P>0.05).Conclusion(1)Stretching at 15% stretch for 4 hours and pulling for 6 hours both promoted the proliferation of skeletal muscle satellite cells,and the proliferation effect was better at15% stretch for 6 hours.The effect of mechanical traction on the proliferation of skeletal muscle satellite cells is related to the activation of Akt,P38,ERK and JNK pathways.(2)Stretching 4 h at 5% stretch and pulling at 6 h did not promote the proliferation of skeletal muscle satellite cells.