Effect Of Curcumin On Wnt5a Signaling Pathway And Myogenic Regulatory Factors During Myogenic Differentiation And Muscle Regeneration

Background:Skeletal muscle injuries are one of the most common sports injuries in sport and if not treated with effective rehabilitation,they may delay skeletal muscle repair or even lead to cumulative damage.On the one hand,curcumin is a natural polyphenol compound that has been found to promote the repair and regeneration of damaged skeletal muscle.On the other hand,studies have shown that the Wnt5a pathway is closely linked to cell proliferation and development.Objective:This study aims to explore the molecular mechanisms involved in the myogenic differentiation of C2C12 cells and regeneration of skeletal muscle in mice after injury,and to reveal the role of curcumin in myogenic differentiation and muscle regeneration and the effect of curcumin on the molecular mechanisms of myogenic differentiation and muscle regeneration.Methods:Cell experiments:First,horse serum was added to the conventional medium to induce myogenic differentiation of C2C12 myogenic cells for 6 days,and changes in the expression of Wnt5a,Myo D,Myogenin,Myf5 and MHC during differentiation were observed using Western blot,RT-q PCR and immunofluorescence techniques.Second,Wnt5a overexpression and low expression C2C12 cell models were constructed to observe the role of Wnt5a in myogenic differentiation.Third,Ca²⁺inhibitors or agonists were added to Wnt5a overexpression or low expression model to explore the effects of Ca²⁺channel opening or closing on Wnt5a,Ca N,NFAT2,Myo D,Myf5,Myogenin and MHC.Fourth,curcumin was used to intervene on C2C12 myoblasts for 24 hours to observe the effect of curcumin on the expression of Wnt5a,Ca N,NFAT2,Myo D,Myogenin,Myf5 and MHC,while the effect of curcumin intervention on Ca²⁺concentration during differentiation of C2C12 myoblasts was examined using fluorescence microscopy.Fifth,Wnt5a was knocked down and overexpressed on the basis of curcumin intervention to observe the effect of Wnt5a on the effect of curcumin intervention.Animal experiments:First,cardiotoxin was injected at the tibialis anterior muscle of C57BL/6 mice to construct a skeletal muscle injury repair model to observe the expression changes of Wnt5a,Myo D,Myogenin,Myf5 and MHC in muscle regeneration from day 1 to day 21 after modelling;meanwhile,the histomorphological changes during skeletal muscle injury repair were observed by HE staining.Second,a Wnt5a mutant mouse model was constructed by knocking out the Wnt5a gene,and changes in the expression of Wnt5a,Ca N,NFAT2,Myo D,Myogenin,Myf5 and MHC were observed in the mutant mice.Third,the effect of curcumin on the expression of Wnt5a,Ca N,NFAT2,Myo D,Myogenin,Myf5 and MHC was observed after feeding the mice with curcumin for 14 days.Fourth,a Wnt5a mutant mouse model of skeletal muscle injury was constructed and fed with curcumin to observe the changes in the expression of Wnt5a,Ca N,NFAT2,Myo D,Myogenin,Myf5 and MHC in skeletal muscle compared towild-type mice.Results:Cell experiments:First,the expression of Wnt5a,Myo D,Myogenin,Myf5 and MHC all showed a trend of increasing and then decreasing during days 0-6 of myogenic differentiation of C2C12 cells.Second,the expression of Wnt5a,Myo D,Myogenin,Myf5 and MHC was higher in C2C12 myogenic cells after Wnt5a overexpression compared to the horse serum group;in contrast,the expression of Wnt5a,Myo D,Myogenin,Myf5 and MHC was significantly decreased after Wnt5a low expression.Third,Wnt5a overexpression was followed by a significant increase in intracellular Ca²⁺concentrations,on the basis of which the addition of Ca²⁺inhibitors significantly reduced Ca²⁺levels and decreased the expression of Wnt5a,Ca N,NFAT2,Myo D,Myf5,Myogenin and MHC;in contrast,intracellular Ca²⁺concentrations were significantly reduced following low expression of Wnt5a,on the basis of which the addition of Ca²⁺agonists significantly increased Ca²⁺levels and increased the expression of Wnt5a,Ca N,NFAT2,Myo D,Myf5,Myogenin and MHC.Fourth,the expression of Wnt5a,Ca N,NFAT2,Myo D,Myogenin,Myf5 and MHC was significantly increased in C2C12myogenic cells after curcumin intervention compared to the horse serum group.Fifth,knockdown of Wnt5a on the basis of curcumin intervention resulted in a significant decrease in the expression of Wnt5a,Ca N,NFAT2,Myo D,Myogenin,Myf5 and MHC;in contrast,overexpression of Wnt5a on the basis of curcumin intervention resulted in a further increase in theirexpression.Animal experiments:First,the expression of Wnt5a,Myo D,Myogenin,Myf5 and MHC also showed a trend of increasing and then decreasing during days 1 to 21 of skeletal muscle injury repair in mice.HE staining results showed that inflammatory cell infiltration in muscle tissue gradually increased from 1 to 3 days after CTX injection,and cell morphology in the injured area changed and cells lost regular arrangement;on days 7 to 21 after CTX injection,the inflammatory cell infiltrate gradually decreased,the nucleus moved to the center of the cell,and the muscle fibres regained their regular arrangement.Second,compared to wild-type mice,in Wnt5a knockout mice,Wnt5a was not expressed,Myo D,Myogenin and Myf5 expression was significantly decreased,and Ca N and NFAT2 showed a decreasing trend but the difference was not significant.Third,the expression of Wnt5a,Ca N,NFAT2,Myo D,Myogenin,Myf5and MHC in skeletal muscle of mice was significantly higher after 1-14 days of curcumin feeding compared to the model group.Fourth,for the mutant mice,the expression of Wnt5a,Ca N,NFAT2,Myo D,Myogenin,Myf5 and MHC was much lower in both the model and curcumin groups than in thewild-type mice.Conclusion:The results of this study indicate that the Wnt5a/Ca²⁺/Ca N/NFAT2 signalling pathway plays an important role in myogenic differentiation of C2C12 cells and in the repair regeneration of injured skeletal muscle in mice.By promoting the expression of Wnt5a,curcumin activates the Wnt5a/Ca²⁺/Ca N/NFAT2 signalling pathway and upregulates the expression levels of myogenic regulatory factors,thereby promoting myogenic differentiation andskeletal muscleregeneration.