Effects Of MGF On MEK-ERK Signal Pathwray In Skeletal Muscle Satellite Cells

OBJECTIVEInjury is a very common phenomenon in the exercise and training of skeletal muscle,seriously can make the body lose exercise ability.Studies have shown that skeletal muscle cells play an important role in skeletal muscle injury repair process.After injury,SC through proliferation,migration,and ultimately into the damaged muscle cells or the formation of new myotubes and muscle cells and mechano growth factor(MGF)is involved in this process.In this study,we investigated the changes of various proteins after blocking MEK-ERK signal pathway by in vitro experiments,and discussed the regulation mechanism of MGF on the signal pathways of skeletal muscle satellite cells,and provided the theoretical basis for skeletal muscle injury repair.METHODSUnder sterile conditions,we extracted bilateral gastrocnemius and soleus muscle of the Sprague-Dawley rats(two weeks of age,male).Two-step enzymatic digestion(type II collagenase and trypsin)was used to digest the finely divided muscle,to release satellite cells;Use twice differential adhesion method to purify satellite cells(the time is 2h).The activities of the satellite cells was identified using trypan blue staining;the purity of the satellite cells was identified by α-sarcometric actin antibody.The third generation of satellite cells was treated for intervention,divided into control group(cell + growth medium),MGF group(cell + MGF + growth medium),MGF+PD group(cell + MGF + PD98059 + growth medium),and intervention one day.The expression of MEK,p-MEK,ERK,p-ERK,MyoD and Myf5 proteins in each group were detected by Western Blot.RESULTS1、Satellite cell activity test results: the total number of cells in the experiment was 614,the number of coloring was 22,satellite cell activity rate was(614-22)÷614×100%=96.4%.2、Satellite cell purity test results: α-sarcometric actin in the cells were brown,that is positive expression,indicating that the cells were the satellite cells.The purity of satellite cells was 97.7%.3、The total protein content of MGF group was significantly higher than that of control group(p <0.01),the total protein content of MGF + PD group was lower than that of MGF group(p <0.05).4、After 24 hours,the expression of MEK protein in MGF + PD group was higher than that in control group(p <0.05)and MGF group(p <0.05).The expression of ERK protein in MGF group and MGF + PD group was higher than that in control group(p <0.01)and that in MGF + PD group was higher than that in MGF group(p <0.01).5 、 After 24 hours,the expression of p-MEK protein in MGF group was significantly higher than that in control group(p <0.01).And the expression of p-ERK protein in MGF group was also higher than that in control group(p <0.05).6、After 24 hours,the expression of myf5 protein in MGF + PD group was higher than that in control group and MGF group(p <0.01).The expression of Myf5 in MGF + PD group was significantly lower than that in MGF group(p <0.01),but no difference with the control group.CONCLUSIONS1.MGF on skeletal muscle satellite cells can activate the MEK-ERK signaling pathway to promote SC total protein synthesis.2.After blocking MEK-ERK pathway,MGF acts on SC,down-regulation of Myf5 protein expression,upregulate MyoD protein expression.