The Mechanism Of Creatine Regulating The Differentiation Of C2C12 Cells

Skeletal muscle or iginates from the paraxial mesoderm and is composed of striated muscle fiber bundles.Its growth and development ar e achieved by gr adually increas ing the expans ion of the nuc leus and the sarcoplasmic domain around the nuc leus in each f iber.In r ecent years,studies have found that creatine plays a very important r ole in skeletal musc le cell differ entiation,prolif eration,improvement of musc le mass,and treatment of musc le atr ophy.Ther efor e,this study thoroughly explor ed the effect of creatine on the diff erentiation of mous e myoblasts from both in vivo and in vitro,aiming to c lar ify the mechanis m of creatine promoting the diff erentiation of skeletal muscle cells.The main contents of the study:(1)Wester n blotting and immunof luor escence results showed that when the concentration of creatine tr eatment was 2 m M,the pr otein expr ess ion levels of myocyte diff erentiation mar kers Myo D and Myo G were s ignif icantly increased,and the myotube fus ion rate was signif icantly increased compared w ith other concentr ation treatment groups.By detecting the cha nges in Myo D,Myo G and My HC gene express ion,the optimal concentr ation to promote the diff erentiation of mouse C2C12 cells was screened.In or der to further c lar ify the promoting effect of creatine on the differentiation of C2C12 cells,we tr eated C2C12 myoblasts by adding cr eatine at diff erent time per iods(0,1,3,5 D),detecting musc le differ entiation mar ker genes and calculating the myotube fusion rate.(2)The mechanis m by which creatine promotes the differ entiation of mouse C2C12 cells :According to the class ical diff erentiation-r elated pathways known in the laboratory,we add creatine to tr eat C2C12 cells in vitro,Western blotting was used to detect the expr ession changes of key molecules in the pathway dur ing the diff erentiation of musc le satellite cells to determine the molecular mechanism of creatine r egulating the diff erentiation of C2C12 cells.(3)The effect of creatine on the Wnt/?-catenin s ignaling pathway: In order to detect the relationship between creatine and the Wnt/?-catenin signaling pathway,we used si RNA interf erence fragments to interfer e with SLC6A8(creatine transport r eceptor)to make creatine Can not enter the cell,detect the changes in the expr ess ion of the diff erentiation mar kers Myo D,Myo G,My HC and the inf luence of the interfer ence of the S LC6A8 gene on the intr acellular localization of ?-catenin.First determine where creatine activates the Wnt/?-catenin signaling pathway.Then,the Wnt/?-catenin signaling pathway was inhibited by adding the Wnt pathway inhibitor XAV939 to verify the differentiation of C2C12 cells and the effect of creatine on ?-catenin protein into the nucleus.(4)The role of creatine in the pr ocess of muscle injur y and repair in mice: According to the data phenomenon obtained from in vitr o exper iments,in order to fur ther study the r ole of creatine in the diff erentiation process of myoblasts,we built a mous e musc le injury r epair model,damaged the tibialis anter ior musc le of the mouse and f ed creatine,us ing HE staining Methods : Obser ve the morphologic al changes dur ing the damage repair proc ess to ver if y the effect of creatine on the damage r epair.(5)The expr ession law of Wnt/?-c atenin s ignal pathway in the process of muscle damage repair : F irst,constr uct a damage r epair model,and us e creatine feeding method to treat the injur ed experimental objects,and detect the effect of creatine on Wnt/?-catenin signaling pathway during the pr ocess of muscle repair in vivo.Obtained exper imental results show that:(1)Wester n blotting and immunof luorescence r esults c lar if ied that the 2m M creatine treatment group signif icantly increased the protein express ion levels of the musc le cel l differentiation mar kers Myo D and Myo G.and the myotube fus ion rate was signif icantly higher than that of other concentration tr eatment groups.C2C12 cells 0 D,1 D,3 D and 5 D were treated by adding 2 m M creatine,Western blotting r esults c lar if ied that after creatine treatment,the pr otein expr ession of myos in heavy chain My HC and myogenic factor Myo G increas ed signif icantly with differentiation,and was The diff erence in the control gr oup was signif icant,The results of immunof luor escence detection were cons istent w ith those of Wester n blotting,and the myotube fusion rate was signif ic antly impr oved.(2)The results of Western blotting clarified that ?-catenin protein enters the nucleus in large quantities af ter adding 2 m M creatine in vitro.The expr ess ion of Notch1 protein was not signif icantly diff erent,and the differ ence of Hes-1 pr otein expr ession was not s ignif icant;Compar ed with the control gr oup,there was no signif icant difference between the phosphorylated PI3 K activity and the phosphorylated AKT activity after creatine treatment.The above results indicate that the r egulation of C2C12 diff erentiation by creatine is c los ely r elated to the Wnt/?-catenin pathway.(3)The results of Western blotting showed that ?-catenin entered the nuc leus in large quantities after creatine treatment,and reached a peak at 48 hours.The express ion of Myo D was consistent w ith it.The expr ession of differ entiation markers Myo G and My HC increased,and the myotube f us ion r ate was signif icantly up-r egulated;After the S LC6A8 gene is dis turbed,the expr ession of Myo G and My HC proteins decreases,the myotube fus ion rate decreas es,and the localization of ?-catenin in the nucleus decreases.This phenomenon has not been r eversed af ter the addition of creatine.T he r esults indicate that creatine enters the cell through S LC6A8 Stimulate ?-catenin into the nuc leus;add inhibitor XAV-939 to tr eat C2C12 cells in vitro.According to the data analys is results,it is found that the amount of ?-catenin pr otein and the muscle-der ived regulatory factors My HC and Myo G proteins in the nuc leus of the exper imental group(XAV-939-,CR+)T he express ion level was s ignif icantly higher than that of the control group(XAV-939-,CR-),and the myotube fus ion r ate was s ignif icantly increased;the amount of?-catenin protein in the nucleus of the experimental group(XAV-939+,CR-)and the protein expr ession of myogenic r egulatory factors My HC and Myo G T he level of myotube f us ion was reduced,and the myotube fus ion rate was signif icantly r educed;there was no s ignif icant differ ence between the XAV-939 and creatine combined treatment gr oup(XAV-939+,CR+)and the XAV-939 single tr eatment group(XAV-939+,CR-).In summary,creatine enters the cell thr ough SLC6A8 to stimulate ?-catenin to enter the nuc leus and promote the differ entiation of mouse C2C12 cells.(4)The results of HE staining and Western blotting showed that musc le f ibers were necros is and myolys is occurred on the f irst day of injury,and satellite cells began to prolif er ate in lar ge numbers on the third day;on the seventh day of injur y,compared with the control group,the number of musc le nuclei in the creatine treatment group was obvious Increased,differ entiation and fusion into new musc le f ibers;on the fourteenth day of injury,the muscle f ibers in the r egener ated tissue of the control group wer e smaller,and the r egenerated tissue s lic es of the creatine treatment group contained more muscle f ibers and the new musc le f ibers were arranged tightly in the f ield of view.Western blotting results clar if ied that the amount of PAX 7 protein increased s ignif icantly in the ear ly stage of injur y,and r eached a peak on the third day.The express ion of Myo D protein was consistent with PAX 7.The expr ess ion of myogenic fac tor Myo G and myos in heavy chain My HC was signif ic antly up-r egulated dur ing the r epair process.(5)Wester n blotting results showed that after creatine treatment,a large amount of ?-catenin gene in the cytoplasm enters the nuc leus to promote the completion of damage repair.