Myogenesis Effect Of Leucine On C2C12 Cells And Mechanism Of Action

Skeletal muscle is an important component of the animal body,not only as a power organ for locomotion,but also as the largest metabolic and endocrine organ of the animal body.Mechanical,chemical,biological,heavy metals,trace elements,aging and other factors can cause damage to skeletal muscle,affecting the function of skeletal muscle.In severe cases,this can lead to skeletal muscle atrophy and loss of locomotor capacity.Therefore,it is significant to study the regeneration of injured skeletal muscle,which is accomplished through the proliferation and differentiation of skeletal muscle satellite cells.Leucine(Leu),an essential amino acid,plays a key role in the development of skeletal muscle and can promote the differentiation of skeletal muscle satellite cells,but its mechanism of action is still unclear.Therefore,C2C12 cells and skeletal muscle injury repair model were chosen to study the mechanism of Leucine in the repair of skeletal muscle injury,and hope to provide a theoretical basis for the prevention and treatment of skeletal muscle injury in clinical practice.The role and mechanism of Leucine during the proliferation,differentiation and repair of skeletal muscle injury in mouse C2C12 in vitro were investigated by CCK8 and hemocytometer,immunofluorescence staining,Western blot,CO-IP,ESI mass spectrometry sequencing,overexpression,interference and other methods.The results of the experiment were as follows:(1)CCK8 and hemocytometer studies showed that different concentrations of Leu had no significant effect on the proliferation of C2C12 cells.(2)Immunofluorescence and Western blot results showed that different concentrations of Leucine significantly increased the rate of myotubular fusion,MYOD,MYOG and MYH2 protein levels in C2C12 cells and promoted the differentiation of C2C12 cells,the optimal effect concentration was 2 mmol/L and the period of action was the early stage of differentiation(0～2 d).(3)CO-IP and ESI mass spectrometry sequencing were applied to screen and verifie the RPN2 interacting with Leucine’s sensor SESN2 in the early stage of C2C12 cell differentiation.(4)Western blot results showed that the level of RPN2 increased with the differentiation of C2C12 cells and the highest level appeared on the 5th day of differentiation,which was consistent with the expression pattern of differentiation-related marker proteins MYOD,MYOG and MYH2,demonstrating that RPN2 may be involved in the regulation of differentiation of C2C12 cells.(5)When RPN2 was overexpressed,the protein levels of differentiation-related marker proteins MYOD,MYOG and MYH2 were significantly increased with RPN2 overexpression at different time points;when RPN2 was interfered,the protein levels of MYOD,MYOG and MYH2 were significantly decreased with RPN2 interference at different time points;the addition of Leucine after RPN2 interference increased the protein levels of MYOD,MYOG and MYH2,reversing the results caused by RPN2 interference,demonstrating Leucine can promote the differentiation of C2C12 cells through RPN2.(6)When RPN2 was overexpressed,the protein level of p-GSK3β was evidently decreased and β-catenin in the nucleus was observably increased at different time points;when RPN2 was interfered,the protein level of p-GSK3β was significantly increased and β-catenin in the nucleus was significantly decreased at different time points;when Leucine was added after RPN2 was interfered,p-GSK3β expression was down-regulated and β-catenin expression in the nucleus was up-regulated,indicating that RPN2 could positively regulate the activity of GSK3β/β-catenin pathway to promote differentiation,and the addition of Leucine could partially reverse the decrease in differentiation caused by RPN2 interference.(7)Addition of Leucine to C2C12 cells induced differentiation for 48 h,the protein levels of RPN2 and β-catenin in the nucleus were significantly upregulated,indicating that Leucine activated the GSK3β/β-catenin signaling pathway and promoted the differentiation of C2C12 cells via upregulated RPN2.(8)After mouse tibialis anterior muscle injury was modeled and supplemented with Leucine by intraperitoneal injection,HE staining results showed that a large number of myofibers were lysed and numerous of satellite cells were generated on day 1 of injury;on day 3 of injury,skeletal muscle repair was accelerated in the experimental group,with more obvious newborn myofibers,and some of them had been repaired;on day 7 of injury,a host of muscle bundles were formed at the injury in the experimental group,and repair was basically completed,while a large number of newborn myofibers just appeared in the control group.Western blot results showed that Pax7,MYOD,RPN2 and β-catenin in nucleus protein level were significantly upregulated at 1 d,3 d and 7 d with the addition of Leucine,indicating that the addition of Leucine could accelerate the repair process through the upregulation of β-catenin protein level up-regulation accelerated the process of skeletal muscle injury repair.Leucine had no obvious effect on the proliferation of mouse C2C12 cells.After the Leucine’s sensor SESN2 in the cytoplasm specifically receives the signal of Leucine entering the cell,it can up-regulate the protein level of RPN2 in the early stage of differentiation,reduce the protein level of p-GSK3β,inhibit the degradation of β-catenin,and high concentration of β-catenin enter the nucleus,this process promotes the differentiation of C2C12 cells and accelerates the repair process of skeletal muscle damage,and the optimal concentration of Leucine is 2 mmol/L.