Effects And Mechanism Of PRP Combined With Periodic Mechanical Stretching On C2C12 Cell Differentiation And Myotube Atrophy

Objective1.To study the effect of platelet-rich plasma(PRP)combined with periodic mechanical stretch on C2C12 skeletal muscle cell differentiation and on TNFinduced C2C12 skeletal muscle cell atrophy.2.To further study the relevant molecular mechanism of PRP combined with periodic mechanical stretch on the TNF-induced C2C12 skeletal muscle cell muscle atrophy,and provide new ideas for the clinical treatment of muscle atrophy in the future.Methods1.First,in the experiment,they were divided into blank control groups and different concentration PRP intervention groups,that is,1 %PRP,2 %PRP,and 4 %PRP.Protein samples were extracted after the end of the intervention,and the expression levels of differentiation marker proteins Myo D,Myo G and apoptotic autophagy-related proteins Bax,Bcl2,Beclin-1 were determined to define the optimal PRP concentration.2.To further validate its mechanism of action,The experiments were grouped into blank control groups,PRP + Str groups,and PRP + Str + GDC0068 groups,after the end of the intervention,the expression levels of the key signaling pathway proteins,Akt,p-Akt,p70,and p-p70 involved in protein synthesis decomposition were detected by WB assay.3.In the anti-atrophy experiment,the TNF-α induced the C2C12 skeletal muscle cell atrophy model.Cells were divided into blank control,TNF-α group,TNF-α+ 2%PRP group,protein samples were extracted after the intervention,expression changes of dystrophy marker proteins Mu RF-1 and Fbx32,and myotube diameter of C2C12 cells using Jimesa staining.4.The effect of the 2% PRP combined with periodic mechanical stretch improving C2C12 skeletal muscle cell atrophy was also explored.The experiment was divided into blank control group,TNF-α group,TNF-α+2%PRP group,TNF-α+2%PRP+Str group,and the expression changes of atrophy-related proteins Mu RF-1 and Fbx32 were detected by WB assay.5.To further verify its mechanism of action,the experiments were divided into blank control,TNF-α group,TNF-α+ PRP + Str group,TNF-α group + PRP + Str +GDC0068 group,the expression level changes of key signaling pathway proteins,Akt,p-Akt,p70,p-p70,involved in protein synthesis were detected by WB method.Results1.In the PRP differentiation-promoting experiment,compared with the CON group,the expression levels of cell differentiation marker proteins Myo D and Myo G in the 2% PRP intervention group were significantly increased;but high concentration of PRP inhibited cell differentiation.Compared with the CON group,the expression levels of autophagy-related protein Beclin-1 and apoptosisrelated protein Bcl-2 in the 2% PRP group were significantly increased,and the Bax content was significantly decreased.Therefore,2% PRP was selected as the optimal concentration for subsequent experiments.2.In the PRP combined with periodic mechanical stretch experiment,compared with the CON group,the expression levels of the 2% PRP+Str marker proteins Myo D and Myo G increased more significantly;and Akt,p-Akt,p70,p-p70 increased significantly,and the differentiation-promoting effect was inhibited after the use of Akt inhibitor GDC0068.3.In the anti-atrophy experiment,2% PRP was selected for intervention based on the differentiation experiment.The results of cell-modified Giemsa staining showed that compared with the CON group,the myotube in the TNF-α group was significantly atrophy;the TNF-α+2%PRP group could increase the cross-sectional area of the atrophic myotube.4.In the anti-atrophy experiment of PRP combined with periodic mechanical stretch,compared with the TNF-α group,the expression of Mu RF-1 and Fbx32 in the TNF-α+2% PRP group and the TNF-α+2% PRP+Str group were decreased.The TNF-α+2% PRP+Str group decreased more obviously than that of TNF-α+2%PRP group;after intervention with GDC0068,Akt,p-Akt and p-Akt can be downregulated.,p70,p-p70 protein expression levels,the anti-atrophic effect of PRP combined with periodic mechanical stretch was inhibited.ConclusionStudies have shown that 2% PRP combined with periodic mechanical stretch can effectively promote the differentiation of C2C12 myoblasts,improve TNF-α-induced muscle atrophy,and act through the Akt/p70 signaling pathway.