Lactate Regulating Myoblast Differentiation Through ROS Mediated Mechanism

Skeletal muscle has a strong regenerative capacity,it can be in the exercise injury caused by the destruction of muscle structure,rapid regeneration into mature muscle fibers,in a few weeks to complete the restoration of skeletal muscle structure and function.The ability of skeletal muscle to regenerate depends on satellite cells(SCS),which activate cells called Myoblasts,which fuse into myotubes and develop into muscle fibers.C2C12 cells are isolated from mouse skeletal muscle and belong to the substrain of myoblast cell line.Differentiation of skeletal myoblast is a multi-step process associated with cell cycle exit,muscle-specific gene expression and the formation of multinucleated myotubes through myogenic cell fusion.Lactate has long been recognized as a“Metabolic waste product” of exercise,and is responsible for the decline in muscle p H and muscle fatigue.But now it is found that it not only can be used as energy substrate and gluconeogenesis precursor,but also has the characteristic of signal transmission,which plays a role in a variety of physiological and pathological processes.Studies have shown that lactate regulates myoblast differentiation,which is accompanied by changes in Reactive oxygen species(ROS),but whether ROS are involved in the regulation of C2C12 cell differentiation by lactate remains unclear,it remains to be further studied.In this study,C2C12 myoblasts were used as the research object,and the specific mechanism of ROS-mediated lactate regulation of C2C12 cell differentiation was studied by MTT method,fluorescent probe method,cellular immunofluorescence method,real-time quantitative PCR(RT-q PCR)and Western Blot(WB).The main findings are as follows:(1)2% horse serum induced the differentiation of C2C12 cells,established a myoblast differentiation model,and different concentrations of sodium lactate(5 m M,10 m M,20 m M,50 m M)worked for 1,3,5 and 7 days to explore the effect of lactate on myoblast differentiation and ROS production.The results showed that lactate concentrations of 5 m M,10 m M and 20 m M could significantly increase the diameter of the myotube during C2C12 cell differentiation and increase the myotube fusion rate.At the same time,the concentrations of lactate at 5 m M,10 m M and 20 m M increased the content of ROS during the differentiation of C2C12 cells.It was suggested that C2C12 cell differentiation increased with increasing lactate concentration within the physiological range of 0-20 m M,and the increase in ROS level was consistent with the increase in C2C12 myotube.(2)Using the p38MAPK-specific inhibitor SB203580,four groups of control,lactate(10 m M),SB203580(5 μM),lactate(10 m M)+ SB203580(5 μM)were set up,and cells were cultured for 3,5,and 7 days.To investigate the correlation between lactate regulation of C2C12 cell differentiation and p38 MAPK signaling pathway.The results showed that the use of SB203580 significantly inhibited the myotube diameter of C2C12 cells during differentiation and reduced the myotube fusion rate,suggesting that the promotion of C2C12 cell differentiation by lactate was inhibited by SB203580.WB found that phosphorylation of p38 MAPK protein increased in the lactate group,while phosphorylation levels of p38 MAPK protein were significantly inhibited in both the SB203580 group and the lactate + SB203580 group.Studies have also shown that lactate acting myoblasts have a low phosphorylation level on day 3 of early differentiation and a significantly upregulated phosphorylation level by day 7 of differentiation.These results suggest that the stimulating effect of lactate on C2C12 cell differentiation is achieved by upregulating p38 MAPK phosphorylation.(3)The ROS scavenger N-acetylcysteine(n-acetylcysteine,NAC)was used,and four groups of control,lactate(10 m M),NAC(5 m M),lactate(10 m M),lactate(10m M)+ NAC(5 m M)were set up,and cells were cultured for 3,5,and 7 days for experiments.To explore the effect of ROS on the differentiation of C2C12 cells and its possible mechanism.The results showed that compared with the control group,NAC reduced the myotube fusion rate of C2C12 cells on the 5th and 7th days of differentiation.In addition,WB and RT-q PCR were used to detect the expression of myogenic regulators(Myf5,Myo D and Myo G)in regulating C2C12 cell differentiation,and the results showed that Myf5,the early regulator of myoblast differentiation after NAC,was still expressed on the 5th and 7th days of differentiation.Myo D and Myo G,which regulate mid-to-late differentiation factors in cells,were significantly inhibited after NAC,suggesting that C2C12 cell differentiation was significantly delayed after NAC cleared ROS.At the same time,the results showed that the phosphorylation level of p38 MAPK was also downregulated after the use of NAC.The results showed that ROS-mediated lactate promoted the differentiation of C2C12 cells and was closely related to phosphorylation of p38 MAPK.The results showed that lactate promoted the differentiation of C2C12 cells through ROS-mediated regulation of phosphorylation of p38 MAPK,which provided a theoretical basis for the injury repair of skeletal muscle by regulating the differentiation of myoblasts with lactate,and further provided a reference for the physiological role of ROS.