Effects Of Macrophage Depletion On Skeletal Muscle Regeneration And The Mechanism Involved

Object:Skeletal muscle injury is a common musculoskeletal problem associated with sports activities,high speed vehicle accidents,etc.Though macrophages are essential for skeletal muscle regeneration,which is a complex process,the roles and mechanisms of the macrophages in the process of muscle regeneration are still not fully understood.The objective of this study is to explore the roles of macrophages and the mechanisms involved in the regeneration of injured skeletal muscle.Methods:Eighty C57BL/6 mice were randomly divided into muscle contusion(S,n=32),muscle contusion control(SCon,n=8),macrophages depleted(T,n=32)and macrophages depleted control groups(TCon,n=8)).Their gastrocnemius muscles were harvested at the time points of 1,3,7 and 14d post-injury.The percent of macrophages in injured gastrocnemius muscle was tested by Flow cytometry.The changes of skeletal muscle morphology were assessed by hematoxylin and eosin(HE)stain.The gene and protein expression was analyzed by real-time polymerase chain reaction and western blotting.Results:1.Macrophage depletion impairs skeletal muscle regenerationThe HE results showed that skeletal muscle fibers were significantly impaired and the injured fibers had almost degenerated at 1d and 3d post-injury in both groups.At 7d after injury,the damaged muscle area in the group S had been replaced mostly by newly formed muscle fibers,whereas numerous necrotic myofibers and inflammatory cells dominated the injured muscle regions of group T.The size of regenerating myofibers in macrophage depletion mice was significantly smaller than those observed in the contusion group at 14d post-injury.2.The effects of macrophage depletion on proliferation and differentiation of muscle satellite cellsThe results showed that MyoD and myogenin mRNA increased significantly at the time points of 1d and 3d post-injury(p<0.01).As compared to the contusion group,macrophage depletion significantly inhibited MyoD mRNA level at 7d and significantly increased myogenin mRNA level at 14d post-injury(p<0.05).3.The effects of macrophage depletion on the expression of regulatory factors for muscle regenerationThe data showed that HGF mRNA increased significantly at 1d,3d and 7d post-injury(p<0.01).However,macrophage depletion significantly inhibited HGF mRNA levels at the time points of 1d and 3d post-injury,as compared to the contusion group(p<0.05).The expression of uPA and IGF-1 like HGF,they increased significantly after muscle injury and significantly inhibited in the macrophage depletion group.There was no significant change in the expression of MGF post-injury.However,macrophage depletion significantly decreased MGF mRNA levels at Id and 3d post-injury.Just like others,GDF11 and CB2R mRNA increased significantly in the contusion group,and decreased significantly in the macrophage depletion group.4.The effects of macrophage depletion on the expression of inflammatory cytokinesThe data showed that TNF-? mRNA increased dramatically at 1d,3d,7d and 14d post-injury(p<0.01).However,as compared to the contusion group,macrophage depletion markedly increased the expression of TNF-? at 7d post-injury(p<0.01).IFN-? and IL-6 mRNA increased dramatically after injury,and increased significantly at 14d post injury in the macrophage depletion group,as compared to the contusion group(p<0.05).IL-12 mRNA increased dramatically at Id and 3d post-injury(p<0.01).As compared to the contusion group,there was no significantly change in the macrophage depletion group.TWEAK mRNA increased significantly at the time points of 3d and 7d after injury.However,macrophages depletion significantly reduced the expression of TWEAK mRNA at the time points of 1d and 7d post-injury.In addition,as compared to the contusion group,macrophages depletion significantly increased the expression of TWEAK mRNA at control and 14d post-injury.On the other hand,the mRNA of IL-10(anti-inflammatory cytokine)increased at 1d,3d,and 7d post-injury(p<0.01).Macrophages depletion significantly reduced the expression of IL-10 mRNA at 1d post-injury(p<0.05).5.The effects of macrophage depletion on the expression of chemokinesThe RT-PCR results showed that CCL2,CCL3,CCL8 and CXCR4 mRNA increased significantly at 1d,3d and 7d,and CCL4 reduced significantly at 14d post-injury.CXCL12 increased significantly at 7d after injury(p<0.01).However,as compared to the contusion group,macrophage depletion increased CCL2 mRNA significantly at 14d(p<0.05),increased CCL3 mRNA significantly at 3d and 14d,decreased CCL4 mRNA significantly at Id and increased CCL4 mRNA significantly at 14d,decreased CCL8 mRNA significantly at 1d and 3d and increased significantly at 14d,increased CXCL12 and CXCR4 mRNA significantly at 14d post-injury.6.The effects of macrophage depletion on the expression of oxidative stress factorsThe data showed that gp91phox mRNA increased significantly at 1d,3d and 7d post-injury.However,as compared to the contusion group,macrophage depletion significantly reduced the expression of gp91phox mRNA at 1d,3d and 7d(p<0.01),and significantly increased at 14d post-injury(p<0.05).In addition,gp91phox protein increased significantly at 3d post-injury(p<0.01).Macrophages depletion significantly increased significantly at 14d post-injury.7.The effects of macrophage depletion on the expression of angiogenesis factorsHIF-1? mRNA increased significantly at 1d,3d and 7d post-injury.Macrophage depletion significantly increased the expression of HIF-1? mRNA at 14d post-injury.Angptl mRNA increased significantly at 1d,3d,7d and 14d post-injury(p<0.01).However,as compared to the contusion group,macrophage depletion increased Angptl mRNA before injury and at 14d post-injury(p<0.05).VEGF mRNA decreased significantly at 7d post-injury(p<0.05).However,macrophage depletion significantly increased VEGF mRNA before injury as compared to the contusion group(p<0.05).8.The effects of macrophage depletion on the pathway of protein synthesisWestern blotting analysis showed that p-Akt/Akt and p-mTOR/mTOR increased significantly at 1d,p-p70s6k/p70s6k increased significantly at Id and 3d,p-4EBP1/4EBP1 increased significantly at 1d,3d,7d and 14d post-injury.However,there were no significantly change in the expression of p-Akt/Akt,p-mTOR/mTOR,p-p70s6k/p70s6k and p-4EBP1/4EBP1 in the macrophage depletion group after injury.Conclusion:1.The change of macrophages,inflammatory cytokines,muscle regeneration regulatory factors,oxidative stress factors,chemokines,angiogenesis factors and Akt/mTOR pathway were activated in skeletal muscle after injury,and they may play important roles in the regeneration of muscle contusion.2.Macrophages depletion impaired muscle regeneration,the mechanism may be involved of inhibiting the expression of muscle regeneration regulatory factors,angiogenesis factors and Akt/mTOR pathway activated,increased the expression of inflammatory cytokines,chemokines and oxidative stress factors in the late stage of regeneration.In addition,macrophage play an important role in muscle regeneration after injury.