The Roles Of Macrophages In Repairing Skeletal Muscle Contusion

Object:Macrophages play complex roles in the regeneration of skeletal muscle.However,the roles of macrophages,especially the mechanisms involved,in the regeneration of muscle contusion are still not fully understood.therefore,we constructed a muscle contusion injury and followed it up with morphological and gene expression analyses.To observe the expression patterns of pro-fibrogenic factors,muscle regenerative factors,inflammatory factors,chemokines,and matrix metalloproteinases and the morphology of muscle fibers,regenerated muscle fiber size,and collagen deposition during the skeletal muscle contusion repair process.Clarifying the roles of macrophages in repairing skeletal muscle contusion.Methods:Forty male C57BL/6 mice(average body weight 21.3g,purchased Shanghai Jiesijie.Animal Research Center,Shanghai,China).The mice were randomly divided into two groups : a quiet control group(C,n=8)and a skeletal muscle contusion group(S,n=8).According to the time of drawing,the contusion group was divided into 3,6,12 and 24 days after injury(8 at each time point).Their gastrocnemius were harvested at the time point of 3,6,12 and 24 d post-injury.The change of the size of skeletal muscle regenerated muscle fibers were assessed by hematoxylin and eosin(HE)stain.Collagen deposition in contused skeletal muscles were assessed by Masson trichrome staining.The gene expression was analyzed by real-time polymerase chain reaction Results:1.Changes of regenerated myofibers size and expression of cell markers during skeletal muscle injury repair.The HE results showed that skeletal muscle fibers were significantly impaired and the injured fibers had almost degenerated at 1d and 3d post-injury.A large number of regenerated myofibers appeared on the 6th and 12 th day after injury,and the number of central nucleus muscle fibers increased.At the 24 days after injury,the number of central nucleus muscle fibers decreased,and the nucleus began to move from the center to the edge.The results showed that MyoD and myogenin mRNA increased significantly at the time point of 3d and 6d post-injury(p<0.01).The peak value was reached at the 3days after injury,and its expression decreased from the 3rd day to 24 th day after injury,and returned to normal at the 24 days post-injury.2.Fibrosis in repair of skeletal muscle contusionMasson's trichrome staining results showed that on the 12 th day after injury,a large amount of blue collagen fibers surround the newborn muscle fibers.There were less blue collagens on the 24 th day after the injury than 12 th day.At the same time,the gene expression levels of pro-fibrogenic factors in the injured skeletal muscles were assessed.The expression of TGF-?1 mRNA in the injured group was significantly increased at 3,6 and 12 days after injury(p<0.01).There was no significant difference in the expression level of myostatin during skeletal muscle repair.3.The expression of macrophage markers during skeletal muscle regenerationThe fluorescence quantitative PCR results showed that the mRNA expressionlevels of M1 macrophage markers CD68,M2 macrophage markers CD163 and CD206 were significantly higher than those in the control group at the 3 and 6 days after injury(p<0.01).The expression of CD68 mRNA peaked at the 3d after injury.The expression of CD68 mRNA decreased from 3rd day to 24 th day after injury.On the other hand,The expression of CD163 and CD206 mRNA peaked on the 6days after injury.The expression of CD163 and CD206 mRNA showed a decreasing trend from the 6 to 24 daya after injury,and returned to normal at the time of 24 days after injury.4.The expression of muscle regeneration factors in the recovery of skeletal muscle contusionThe PCR results showed that IGF-1 and MGF mRNA peaked at the 6 days after injury(p<0.01),followed by a decreasing trend,but the expression level was still significantly higher on 12 d after injury than that of the normal control group(p<0.05).HGF,uPA,COX-2 are also important muscle regeneration factors.Compared with the control group,their expression increased significantly at the 3 and 6 days after injury,and reached the peak at the time point of 3 days post-injury.Their expression decreased from the 3rd day to the 24 th day,and returned to normal at the time of24 days.5.The expression of inflammatory factors in skeletal muscle repairFluorescent quantitative PCR results showed that the inflammatory factors(IL-6,IL-1?,TNF-?,INF-?,TWEAK,fn14,IL-10)in the skeletal muscle contusion group were significantly increased in the early stage of regeneration(3d and 6d after injury)compared with the control group(p<0.01).The expression levels of IL-6,IL-1?,TNF-?,INF-?,and fn14 mRNA peaked at the 3days after injury,and showed a decreasing trend from the 3days to the 24 days after injury.TWEAK,IL-10 gene expression reached its peak at the 6days after injury,then decreased,and returned to normal level at the 24 days after injury.6.The expression of chemokines in skeletal muscle repairReal-time polymerase chain reaction showed that,compared with the control group,the chemokines(CCL2,CCR2,CXCR4)in the skeletal muscle of the contusion were significantly up-regulated at the 3 days after injury(p<0.01),and reached the peak,the expression level of CCL5 mRNA reached the peak at the time of6 days after contusion,decreased on the 6 days and the 12 days after injury,and returned to the control group on 24 days.7.Expression of matrix metalloproteinases during regeneration of skeletal muscle contusionThe results of fluorescence quantitative PCR showed that the mRNA expression of MMP-2,MMP-9,MMP-10 increased rapidly after injury,reached the peak at the 6days after injury,and the expression of MMP-2,MMP-9,MMP-10 showed a downward trend from 6d to 12 days after injury.The expression level of MMP-2 and MMP-10 at 3d and 12 d after injury was still higher than that of the control group.8.Association of macrophage-specific markers with pro-fibrogenic factors,myogenic regenerative factors,inflammatory factors,chemokines,and matrix metalloproteinasesPearson correlation analysis showed that there were significant correlations between the M1 macrophages(CD68)and muscle regeneration factors(HGF,uPA,COX-2),inflammatory factors(IL-1?,IL-6,TNF-?,INF-?,IL-10,TWEAK),profibrosis Factors(TGF-?1),chemokines(CCL2,CCR2).M2 macrophages(CD163)are associated with inflammatory factors(IL-10,fn14),muscle regeneration factors(COX-2),matrix metalloproteinase(MMP-10).M2 macrophages(CD206)were significantly associated with muscle regeneration factors(IGF-1,MGF,COX-2),chemokines(CCL5),and matrix metalloproteinases(MMP-9,MMP-2).Conclusion:1.the expressions of pro-fibrotic factors,muscle regeneration factors,inflammatory factors,chemokines,and matrix metalloproteinases all changed during the repair of skeletal muscle contusion,suggesting that they may play important roles in skeletal muscle regeneration.2.Macrophages play key roles in skeletal muscle regeneration,and they may be involved in the skeletal muscle repair by regulating or secreting pro-fibrogenic factors,muscle regeneration factors,inflammatory factors,chemokines,and matrix metalloproteinases.