The Role And Mechanism Of Tectochrysin On Skeletal Muscle Myogenesis

Skeletal muscle is one of the main organs that affect the lean mass,and it is also the main organ that participates in various movements of the body and maintains the homeostasis of the internal environment.Myogenesis is a key step in the formation of skeletal muscle and is involved in the necessary process of skeletal muscle growth and regeneration after injury.Therefore,by studying the mechanism of regulating skeletal muscle myogenesis,it can provide a theoretical basis for raising lean meat percentage in animal husbandry.In recent years,it has become a feasible measure to improve pork quality by extracting natural compounds from fruits,vegetables and plants.Tectochrysin(TEC)is a flavonoid compound derived from propolis,which has anti-oxidation,anti-cancer,antidiarrheal,anti-pathogen and aromatic hydrocarbon receptor ligand activities.So far,there are no research reports on TEC in skeletal muscle.Exploring the potential role and mechanism of TEC in skeletal muscle myogenesis is expected to provide new ideas for the regulation of skeletal muscle growth.In this study,C57BL/6J mice,C2C12 myoblasts and porcine primary muscle satellite cells were used as the research objects,and the role of TEC in skeletal muscle myogenesis was explored through in vivo and in vitro experiments using molecular biology and cell biology techniques.and mechanism.The main results are as follows:1.Effect of TEC on exercise ability in vivo.To investigate the effect of TEC on exercise ability,C57BL/6J mice were treated with TEC by oral gavage at a dose of 20mg/kg per day.The results showed that TEC treatment did not affect body weight and food intake of mice.Compared with the control group,TEC significantly increased the maximum motor speed and motor time(P<0.05)and grip strength(P<0.05)in the TEC-treated group.At the same time,the protein level of My HC was also significantly increased(P<0.05).It indicated that TEC enhanced the exercise ability of mice.2.TEC promotes differentiation and fusion of myogenic cells during myogenesis.Skeletal muscle myogenesis plays an important role in its functional enhancement.To explore whether TEC affects muscle motor function through skeletal muscle myogenesis,this study used cardiotoxin to establish a mouse model of skeletal muscle injury,and administered TEC orally at a dose of 20 mg/kg daily for two weeks.Histomorphological results showed that TEC treatment significantly promoted the recovery of skeletal muscle after injury compared with the control group.At day 5 after skeletal muscle injury,TEC significantly promoted the formation of new myofibers.At the same time,the expression levels of myogenic marker proteins such as e My HC and Myo G were significantly up-regulated in the TEC treatment group(P<0.05).The key steps of skeletal muscle myogenesis include the activation and proliferation of muscle satellite cells and subsequent myoblast differentiation.The results of immunofluorescence staining showed that TEC did not significantly affect the number of PAX7~+cells on the third day of skeletal muscle injury(P>0.05).The results of in vitro experiments showed that TEC had no significant effect on the proliferation of C2C12 cells(P>0.05).Further studies found that TEC significantly promoted the differentiation and fusion of C2C12 myoblasts.Protein results showed that TEC significantly up-regulated the differentiation marker proteins My HC and Myo G at the stage of myoblast differentiation(P<0.05),and reached a peak on the 6th day.The above results indicated that TEC treatment did not affect the proliferation of myocytes,but could significantly promote the differentiation and fusion of myocytes.3.TEC promotes myogenesis mediated by myogenic differentiation through activation of insulin signaling pathway.In order to further explore the mechanism of TEC affecting skeletal muscle myogenesis,this study used molecular docking software analysis,and found that TEC is the component with the highest affinity activity with insulin receptor in propolis.Further results showed that on day 5 after skeletal muscle injury or in C2C12 myoblasts,TEC could significantly promote the phosphorylation of insulin receptor(IR)(P<0.05),thereby activating the insulin signaling pathway and promoting myogenic differentiation.Inhibition of IR activation by pharmacological means(GSK)can significantly inhibit the recovery effect of TEC on skeletal muscle injury.At the same time,GSK significantly inhibited the activation of insulin signaling pathway by TEC,and inhibited the expression levels of myogenic marker proteins such as e My HC and Myo G(P<0.05).The results of cell thermal shift assay showed that TEC can improve the thermal stability of IRβ.Pull down experiment proved that TEC can directly combine with IRβ.According to the molecular docking results,TEC may bind to IR through GLU1077,MET1079 and ASP1160 sites.When GLU1077 and MET1079 were mutated,TEC lost the ability to bind IRβ.4.TEC promotes myogenic differentiation of porcine primary myosatellite cells.In this study,the effect of TEC on myogenic differentiation of porcine primary myosatellite cells was investigated using porcine primary myosatellite cells as a model.The analysis of immunofluorescence staining results revealed that TEC promoted the differentiation and fusion of porcine primary myosatellite cells,and significantly increased the nuclear distribution and fusion index of porcine primary myosatellite cells(P<0.05).At the same time,My HC and Myo G protein levels were significantly increased(P<0.05).In summary,this study showed the role of TEC in myogenic differentiation through in vivo and in vitro experiments,and demonstrated that TEC promotes myogenesis mediated by myogenic differentiation by targeting IR and then activating insulin signaling pathway.Therefore,this study provides a theoretical basis and scientific rationale for the application of TEC to meat quality improvement in pig industrial production and the treatment of human-related diseases.