Experiment On The Mechanism Of Human Umbilical Cord-mesenchymal Stem Cells-derived Exosomes Repairing Muscle Injury In Mice

Background: The occurrence of skeletal muscle injury and degeneration seriously affects the quality and function of muscles,often leading to adverse clinical consequences.Rotator cuff tear is the most common disease of the musculoskeletal system in clinical practice.Degeneration of the rotator cuff muscle often leads to poor postoperative outcomes and the occurrence of retear of the rotator cuff,causing huge mental and economic burden to patients.However,the mechanism of muscle degeneration is not clear,and there is no effective treatment methods in clinical practice besides changing patients’ lifestyle and functional exercise.Human umbilical cord mesenchymal stem cells(hUC MSCs)have the most advantage among all stem cells,and their secreted exosomes have been reported to have biological functions similar to those of maternal stem cells and play a good role in regeneration and repair in various traumatic diseases.However,their therapeutic effects in muscle injury and degeneration are still unclear.Purpose: Explore the regenerative and repair effects of exosomes derived from hUC-MSCs on glycerol induced muscle injury models,their ability to alleviate muscle degeneration,and their mechanisms of action.Methods:(1)Create a glycerol induced muscle injury model,and evaluate the muscle damage,fibrosis,fat infiltration,and atrophy at 0,4,7,and 14 days using HE staining,Masson staining,oil red O staining,and measurement of muscle wet weight;(2)Enzymatic extraction of primary hUC-MSCs,identification of surface markers by flow cytometry,identification of osteogenic and adipogenic differentiation ability by alizarin red staining and oil red O staining;The exosomes were extracted by ultracentrifugation,and their morphology and particle size distribution were identified by transmission electron microscopy and nanoparticle size distribution detection,respectively.The specific markers on the exosomes’ surface were detected by WB;After local transplantation of extracellular vesicles into a glycerol model,HE staining,Masson staining,Immunofluorescence staining(IF),and wet muscle weight measurement were used to evaluate their effects on muscle morphology,regeneration,fibrosis,and atrophy;(3)Western blot(WB)and real-time fluorescence polymerase chain reaction(q PCR)were used to detect the expression of follistatin(FST)in the exosomes of hUC-MSCs;PKH26 staining labeled C2C12 cells,IF detection of C2C12 uptake of extracellular vesicles;The CCK8 experiment detects the optimal incubation concentration and time of exosomes on C2C12 cell lines;In vitro,WB and IF staining were used to verify the effects of exosomes on the regeneration,fibrosis,and atrophy related factors of C2C12 cell lines with myotube atrophy,as well as their effects on FST and MST;Using WB and IF in vitro to verify the effects of FST knockout in exosomes on muscle regeneration,fibrosis,atrophy related factors,as well as the effects on FST and MST;HE staining,Masson staining,WB,IF,and measurement of wet muscle weight were used to verify the effect of FST knockout in exosomes on muscle regeneration,fibrosis,and atrophy in vivo;WB and q PCR were used to verify in vivo whether exosomes exert muscle regeneration,anti fibrosis,and atrophy effects through FST mediated Smad2/AKT signaling pathway.WB and q PCR were used to verify in vitro whether extracellular vesicles exert muscle regeneration,anti fibrosis,and atrophy effects through the Smad2/AKT signaling pathway.(3)Results:(1)Compared with the Sham group,significant damage in muscle morphology were observed on the 4th day after glycerol modeling,accompanied by fibrosis and muscle regeneration.Fat infiltration began on the 7th day,and after 14 days of modeling,significant fibrosis,fat infiltration,and atrophy were observed in the muscles;(2)Flow cytometry showed positive expression of CD29,CD44,CD73,and CD90 in hUC-MSCs,while negative expression of CD34 and CD45.Alizarin red staining and oil red O staining showed that hUC-MSCs had osteogenic and adipogenic differentiation ability;After local transplantation exosomes into the glycerol model,compared to the Sham group,the change rate of wet muscle weight showed that the exosomes group could significantly alleviate muscle atrophy.HE staining showed an improvement in muscle morphology and significant muscle regeneration in the exosomes group.Masson staining showed a significant reduction in muscle fibrosis in the exosomes group,while IF showed an improvement in muscle morphology and significant muscle regeneration in the exosomes group.(3)High expression of FST in exosomes of hUC-MSCs;IF shows that C2C12 cells can uptake exosomes;The CCK8 experiment showed that the optimal concentration and time for incubating C2C12 cells with exosomes was 100ug/ml for 48 hours;In vitro,WB results showed that exosomes can promote the expression of FST and regeneration factors(My HC,Myo D)in C2C12 cell lines,reducing the expression of fibrosis and atrophy related molecules such as α-SMA,Collagen I,Mu RF1,MAFbx,as well as MST,and IF showed an increase in the expression of muscle regeneration factor My HC;In vitro,after knocking down FST in exosomes,WB showed a decrease in the expression of FST and muscle regeneration factors(My HC,Myo D),while the expression of MST as well as fibrosis and atrophy related factors α-SMA,Collagen I,Mu RF1,MAFbx were increased,and IF showed the expression of muscle fibrosis factors α-SMA and Collagen I Increased;In vivo,after knocking down FST in the exosomes,the change rate of wet muscle weight showed no improvement in muscle atrophy,HE staining showed no improvement in muscle morphology,weakened muscle regeneration,Masson staining showed no improvement in fibrosis,WB showed reduced expression of FST and muscle regeneration factors(My HC,Myo D),while the expression of MST as well as fibrosis and atrophy related molecules such as α-SMA,Collagen I,Mu RF1,MAFbx were increased,while IF showed a decrease in the expression of muscle regeneration factors(My HC,Myo D);In vivo,q PCR and WB showed that exosomes can inhibit the activation of Smad2 and promote the expression of AKT and m TOR.After knocking down FST in the exosomes,the inhibitory effect on Smad2 is weakened,while the promoting effect on AKT and m TOR is weakened;In vitro WB and q PCR results showed that exosomes can inhibit the activation of Smad2,promote the expression of AKT and m TOR,thereby promoting the expression of regeneration factors(My HC,Myo D)and reducing the expression of fibrosis and atrophy related factorsα-SMA,Collagen I,Mu RF1,MAFbx.Conclusion: hUC-MSCs-Exo overexpresses FST.After local transplantation of hUC-MSCs-Exo into a glycerol induced mouse muscle injury model,FST can promote the regeneration and repair of damaged muscles in mice by activating the Smad2/AKT signaling pathway,and alleviate fibrosis and atrophy during the injury process.