Roles And Mechanisms Of Macrophages On Skeletal Muscle Regeneration After Acute Injury

Purpose:Skeletal Muscle injuries are common musculoskeletal problems encountered in Sports medicine,the research on the repair mechanism is no longer focus on the muscle satellite cells.The role of immune cells,especially macrophages are gradually being recognized,However,interaction between macrophages and myogenic cells and the molecular mechanisms remain to be clarified.Therefore,to establish models of acute gastrocnemius muscle contusion and macrophage depletion;to determine the skeletal muscle regeneration and spatiotemporal regulation of related cytokines;to illustrate the possible regulatory mechanisms of macrophages on skeletal muscle regeneration after acute injury.Methods:8-10 week male C57BL6 mice,weight 18.2 to 22.9 g,were randomized to acute-contused group(PBS-liposomes,every eight mice in PBS-liposomes,were respectively sampled 12 hours,1 day,3 days,5 days,7 days and 14 days after the contusion.),macrophage-depleted contusion group(Clodronate-liposomes,every eight mice in Clodronate-liposomes,were respectively sampled 12 hours,1 day,3 days,5 days,7 days and 14 days after the contusion.),the control group for PBS-liposomes(Scond,n=8),and the control group for Clodronate-liposomes(Tcon,n=8).The model of skeletal muscle acute contusion was made by a ball(16.8g,1.59cm in diameter)dropped from 100 cm above the gastrocnemius muscle.The model of macrophage depletion was established through intraperitoneal inject 0.2mL/10g weight clodronate-liposomes 3 days before acute contusion,then 0.1mL on day 0,3,6,9 and 12 after injury.Acute-contused groups were intraperitoneally injected with PBS-liposomes according to the same methods and procedures.H.E.staining was used to evaluate of regeneration process after skeletal muscle contusion;Masson Trichrome Staining was used to identify the fibrosis and scar formation;Verification of the model of macrophage depletion was acquired by flow cytometry and Western Blotting;Immunohistochemistiy was used to analyze the time-course of localization expression of Macrophage-specific marker(F4/80),profibrotic factor(Myostatin),and specific markers for proliferation and differentiation of muscle satellite cells(MyoD and Myogenin)in skeletal muscle after contusion;Time-Course Analysis of the proteins expression of profibrotic factor(Myostatin),extracellular matrix markers(PDGFRa and a-SMA),specific markers for proliferation and differentiation of muscle satellite cells(MyoD and Myogenin),myocyte regeneration factors(IGF-1,uPA,HGF/C-met and COX-2)and Akt/mTOR pathway in skeletal muscle after contusion by Western BlottingResults:In the PBS group,the protein level of F4/80 in skeletal muscle increased instantly,reaching a peak at day 3 after contusion,F4/80 immunoreactivity was transferred from the epimysium to the injured fibers gradually.Less F4/80 immunoreactivity and proteins were found in the Clodronate-liposomes group when compared with that in the PBS groupThe area of collagen fibers and the levels of profibrotic factor(Myostatin)and extracellular matrix markers(PDGFRa and a-SMA)proteins in the Clodronate-liposomes group was significantly higher than that in the PBS group 14 days after contusionIn the PBS group,the protein levels of MyoD and Myogenin expression reached a peak on day 1 and day 3 respectively,MyoD-positive myonuclei expressed along the margin of the normal muscle cells,and gradually migrated into the injured fibers,Myogenin-positive myonuclei expressed on new myoblast nucleus for the first time.The trend for protein levels and localization expression of MyoD and Myogenin in the Clodronate-liposomes group were similar to those of the PBS group,but delayed for two days,the peak protein levels of MyoD and Myogenin were lower in the Clodronate-liposomes group than that in the PBS group.In the PBS group,immunofluorescence localization of F4/80 and myocyte regeneration factors(IGF-1,uPA,HGF/C-met and COX-2)in skeletal muscle were obsersed on day 3 after contusion respectively,and,the protein levels of those cytokines were higher at early stage after contusion.In addition to COX-2,the protein levels of muscle regeneration regulatory factors did not significantly change from the Tcon group.the protein levels of Akt/mTOR pathway were higher at early stage after contusion in the PBS group,but there were no difference between Clodronate-liposomes groups and the Tcon group.Conclusion:Macrophages depletion impaired skeletal muscle regeneration,leading to fibrosis.Macrophages participate in the various phases of skeletal muscle healing by regulating a variety of mechanisms,such as promoting the proliferation and differentiation of muscle satellite cells,suppressing profibrotic factors and inflammatory factors,and activating protein signaling pathway as a potential source of myocyte regeneration factors.