Shockwave Mediated Myoblast Differentiation Of Myoblast Cells Was Used To Repair Skeletal Muscle Injury In Rats

ObjectiveTo observe the effect of shock wave on the proliferation and differentiation of myoblasts and repair of injured skeletal muscle in rats,and to lay the foundation for clinical application of shock wave in the treatment of skeletal muscle disease.Methods1、In vitro experiments:The primary isolation and culture of rat skeletal muscle myoblasts was performed by mixed enzyme digestion.Shock waves of different intensity were used to stimulate myoblasts to select the optimal intensity.Based on this,the experiment was divided into a blank group,an experimental group(the optimal intensity of the shock wave),and a positive control group(the myogenic inducer)to verify the effect of the shock wave on the myoblast differentiation effect.Morphological examination of myotube formation;MTT assay of myoblast proliferation.Flow cytometry was used to detect myoblast apoptosis.Western Blot was used to detect the expression of transcription factors Pax7,myoblast differentiation factor(Myo D),myogenic determinant(Myf5),myogenic determinant(Myf6),and myogenin(Myo G).Immunofluorescence was used to detect the expression of desmin,Pax7 and Myo D.2、In vivo experiments:A model of skeletal muscle blunt contusion was established.After successful modeling,the experiment was divided into shock wave treatment group and control group.The treatment group received shock wave treatment at 1 day after injury.The treatment group and the control group were treated with tissue staining at 1d,4d,and 7d for HE staining to observe the morphology of the tissue.Immunohistochemistry was used to detect the expression of myostatin(Mstn)and use Image analysis software was used to analyze average optical density(AOD);Western Blot was used to detect the expression levels of myogenic determinant(Myo D)and Mstn protein.Results1、In vitro experiments:Primary isolation and culture of rat skeletal muscle myoblasts was successfully performed.MTT results showed that the viability of myoblasts was dose-dependent on shock wave stimulation.The higher the energy density,the lower the cell survival rate.At a medium energy density,the cells showed the highest viability at 500 shocks.Flow cytometry results showed that when the shock wave stimulation intensity was too high,the apoptosis rate was significantly higher than that of the blank group(P<0.05).Immunofluorescence showed that shock wave can promote the increase of Myo D protein expression and decrease the expression of Pax7.Western blot results showed that shock waves could significantly increase the expression levels of Myo D,Myf5,Myo G,and Myf6(P<0.05).Pax7 protein expression decreased with time,and the difference was most obvious on the 1d and 3d groups(P<0.05).The expression levels of Myo D protein in the two groups gradually increased with time,and the expression level of the shock wave group was higher than that of the blank control group,and there were differences between the two time periods(P<0.05).The morphological observation of myotube fusion showed that myoblasts showed multiple nuclei under shock wave stimulation and had obvious myotube formation.The myotube fusion index after shock wave stimulation and the control group had statistically significant differences compared to the blank group(P<0.05).2、In vivo experiments:Rat skeletal muscle blunt contusion model was successfully constructed.The HE staining results showed that a large number of inflammatory cells infiltrated and the muscle fibers were disordered in the control group.In the shock wave treatment group,muscle fiber atrophy gradually reduced,inflammatory cell infiltration decreased,and a large number of new muscle cells were seen during the period.The results of Mstn immunohistochemical staining showed that the AOD value,the expression levels of the control group and the treatment group showed a trend of first increase and then decrease at 1 d,4 d,and 7 d.The expression levels of the treatment group at each time point were lower than those of the control group(P<0.05).Western blot results showed Myo D protein expression.The expression of Myo D protein in the shock wave group increased with increasing intensity and then decreased.The intensity of 0.14 m J/mm~2 was the highest and was higher than the control group(P<0.05).The treatment group at 1 d,4 d,and 7 d,the time was higher than the control group(P<0.05).The expression of Mstn protein showed that the protein expression of each group at different times increased to the highest level at 4 d after injury,and then began to decrease.The expression levels of each group showed an upward trend and then a downward trend with time.The expression level was highest at 4 d,but the expression level of each intensity group of the shock wave was lower than that of the control group.As the impact strength increases,the expression level gradually decreases,and the energy flow density of 0.14 m J/mm~2decreases significantly.Conclusions1、ESW has an effect on the viability of myoblasts,and can promote cell fusion under appropriate strength.2、ESW affects the biological characteristics of myoblast proliferation and differentiation by regulating the expression of Pax7,Myo D,Myf5,Myf6,Myo G and other proteins.3、ESW treatment significantly increased the number of newborn cells and reduced the infiltration of inflammatory cells after skeletal muscle injury in rats,which is beneficial for repairing damaged skeletal muscle.4、This topic lays the theoretical foundation for ESW as an emerging treatment method to reduce pain and promote repair of skeletal muscle injury.