Investigation Of The Mechanism Underlying EGF Promoted Skeletal Muscle Post-injury Regeneration

Skeletal muscle is an essential organ of the human body,a ccounting for about 40% of the body mass.It plays vital roles in various aspects of human movement,breath and metabolism.The development and regeneration of skeletal muscle is a highly complex process influenced by various factors.Among them,epidermal growth factor(EGF)as a multifunctional cytokine that plays important roles in the growth and development of skeletal muscle.However,the molecular mechanism of EGF influenced on skeletal muscle injury and regeneration still remains little known.In this study,a mouse skeletal muscle post-injury regeneration model and C2C12 myoblasts cell line were used to elucidate the molecular mechanism by which EGF promotes myoblasts proliferation and differentiation then improves skeletal muscle post-injury regeneration.This will support the treatment of skeletal muscle injury,which is of great value in resolving muscle health problems such as muscular atrophy and scarcepenia.The contents and results were showed as follows:(1)Bupivacaine hydrochloride was injected into the anterior tibialis muscle of mice to establish an animal model of skeletal muscle injury and regeneration accompanied by EGF protein injection.The morphological of skeletal muscle tissues during the process of post-injury regeneration were detected by HE technique on days 0,1,3,5,7,and 14 after the injury.The results showed that the progress of skeletal muscle was accelerated in EGF injection group than that of control groups.Western blotting results indicated that the expression of Pax7,(marker of myoblast proliferation).Desmin and Myo G,(markers of myoblasts differentiation),were all significantly up-regulated after EGF injected.Immunohistochemical staining was used to detect the localization of EGF and Pax7 expression during the skeletal muscle post-injury regeneration in mice.The results showed that EGF and Pax7 were abundantly expressed at the site of m yofibrillar lysis and around the damaged muscle fibers on the 3rd day of severe injury.In contrast,on day 5after injury,EGF was mainly expressed in newborn muscle fibers,indicating that EGF may regulate the proliferation and differentiation of muscle satellite cells in vivo and promote skeletal muscle post-injury regeneration in mice.(2)Western blotting was used to detect the expression pattern of EGF protein during C2C12 cell proliferation.The results showed that EGF gradually increased during the proliferation,indicating that EGF may be involve in C2C12 cell proliferation.Different concentrations of EGF(20 ng/m L,50 ng/m L,100 ng/m L,150 ng/m L)were added into C2C12 cell culture medium Western blotting results showed that 20 ng/m L of EGF significantly up-regulated the expression of Pax7,PCNA,and CCNB1.The results of Ed U also showed that 20 ng/m L of EGF significantly promoted the proliferation of C2C12 cell.Si RNA was used to interfere the expression of EGF.Western blotting results showed that the expression of Pax7,PCNA,and CCNB1 decreased significantly with EGF interference.The results of Ed U showed that the cell pro liferation rate of C2C12 cell was also decreased significantly with EGF interference.These results indicated that EGF could regulate C2C12 cell proliferation.(3)Therefore,the molecular mechanism of EGF promotes myoblasts proliferation was further investigated in this study.The expression of Pax7,PCNA,CCNB1 and phosphorylated p38 were significantly up-regulated or down-regulated by EGF supplementation or EGF interference.Western blotting results showed that the expression of Pax7,PCNA,and CCNB1 could not be upregulated when the activity of EGFR was inhibited by AG-1478 even though the EGF was supplied.It indicates that EGF affects p38-MAPK signaling pathway activity through EGFR to regulate C2C12 cell proliferation.(4)Western blotting was used to detect the expression pattern of EGF protein during C2C12 cell differentiation.The results showed that EGF gradually increased during the differentiation,indicating that EGF may involve in C2C12 cell differentiation.Different concentrations of EGF(20 ng/m L,50 ng/m L,100 ng/m L,150 ng/m L)were added into C2C12 cell culture medium.Then the cell were induced to differentiation at 72 h.Western blotting results showed that 50 ng/m L of EGF significantly upregulated the expression of Myo G and Desmin,the statistical results showed that 50 ng/m L of EGF significantly could also upregulate the myotube fusion rate of C2C12 cell by Desmin immunofluorescence staining.Si RNA was used to interfere the expression of EGF.Western blotting results showed that the expression of Myo G and Desmin were decreased significantly with EGF interference.Meanwhile,the myotube fusion rate of C2C12 cell was also decreased with the inhibition of EGF expression.These results indicated that EGF could regulate C2C12 cell differentiation.(5)Therefore,the molecular mechanism of EGF promotes myoblasts differentiation was further investigated.The expression of Myo G,Desmin,p-PI3 K,p-AKT,and p-m TOR were all significantly up-regulated or down-regulated by EGF supplementation or EGF interference.Western blotting results showed that the expression of Myo G and Desmin could not be upregulated when the activity of EGFR was inhibited by AG-1478 even though the EGF was supplied.This indicated that EGF affects PI3K/AKT/m TOR signaling pathway activity through EGFR to regulate C2C12 cell differentiation.In summary,this study demonstrates that the injection of EGF c an promote skeletal muscle post-injury regeneration in mice.It also elucidates the molecular mechanism of EGF regulates the activity of p38-MAPK and PI3K/AKT/m TOR signaling pathways through EGFR respectively,and thus promotes myoblasts proliferation and differentiation.This enriches the theoretical system of skeletal muscle injury regeneration and provides new ideas for resolving muscle health problems,such as the therapy of muscle injury and regeneration,which has important application values.