Mechanism Of Muscle Fibrosis After Contusion Injury

Object:Acute skeletal muscle injury is more common in sports injuries.The damage of skeletal muscle has strong self-repair ability,but the speed of fiber deposition is faster than that of muscle regeneration.When the injury is severe,skeletal muscle is accompanied by the occurrence of fibrosis.Our group’s previous study also observed the presence of fibrosis during contusion skeletal muscle repair.,but the mechanism remains unclear.The skeletal muscles with fibrosis have poor biomechanical properties and are prone to re-injury,affecting exercise capacity.Therefore,we established a blunt trauma skeletal muscle fibrosis model.Explore the relationship between various cytokines including: inflammatory factors,chemokines,oxidative stress factors,Notch signaling pathway regulatory factors,and macrophage expression during skeletal muscle repair and skeletal muscle fibrosis.In addition,we also explored the role of macrophages and transforming growth factor(TGF-β)in the formation of damaged skeletal muscle fibrosis and their possible relationship.Methods:Forty male C57BL/6 mice were randomly divided into control groups(C,n=8)skeletal muscle fibrosis model groups(S,n=32).The bilateral gastrocnemius muscles were harvested at the time points of,3,6,12 and 24d post-injury.Masson trichrome staining was used to observe the deposition of collagen fibers in contused skeletal muscle for 12 d and 24 d.oxidative stress factors,chemokines,macrophage markers,Notch signaling pathway regulatory factors,and transforming growth factor expression was analyzed by real-time polymerase chain reaction.The expression and co-localization of transforming growth factor(TGF-β1)and macrophage marker F4/80 were detected by double immunofluorescence.Results:1.Masson staining of collagen fibers and quantitative real-time PCR detection of collagen in contused skeletal musclesCompared with the control group of mouse skeletal muscle tissue at 12 d and 24 d of injury,collagen fibers were significantly deposited,which was accompanied by different degrees of skeletal muscle fibrosis.And at the 24 d,the deposition of collagen fibers at the 12 d day was higher.Quantitative real-time PCR data analysis showed that collagen I COL1A1 and type III collagen COL3A1 m RNA were significantly increased(p<0.01)at the 3d and 6d day of skeletal muscle injury,and then rapidly decreased.2.TGF-β1 and F4/80 immunofluorescence double-labeled and quantitative real-time PCR detection results during contused skeletal muscle repairImmunofluorescence double-labeling experiments showed that the expression of TGF-β1 and F4/80 was highest at the 3d post-injury compared with the control group and then decreased.It was also found that the coincident yellow areas were also significant at the 3d and 6d post-injury,and then decreased.It showed that macrophages expressed TGF-β1 and expressed at high levels at the 3d and 6d post-injury.Data of quantitative real-time PCR analysis showed that TGF-β1 m RNA was also significantly increased at the 3d and 6d post-injury compared with the control group(p<0.01).The macrophage marker F4/80 m RNA also showed a significant increase at the 3d and 6d post-injury(p<0.01),followed by a rapid decrease.3.Quantitative real-time PCR detection results of chemokines during contused skeletal muscle repairThe RT-PCR results showed that CCL2 m RNA increased significantly(p<0.01)at the 3d post-injury and then began to decline,but at the 6d,12 d d post-injury,the control group still had a significant difference(p<0.01).CCL5 m RNA was significantly increased at the 3d post-injury(p<0.01),reached the highest level at the6 d,and subsequently m RNA levels decreased,but still significantly increased at the12 d compared with the control group(p<0.05).CCR2 m RNA was significantly increased at the 3d post-injury(p<0.01),but the m RNA level was subsequently decreased,but still significantly increased on the at the 6d,12 d in the control group(p<0.01,p<0.05).The expression of CXCL12 at the 3d post-injury was significantly increased compared with the control group(p<0.01),and then decreased rapidly.The expression of CXCR4 m RNA at the 3d and 6d post-injury was significantly increased compared with the control group(p<0.01),peaked at 3d and then began to decline.4.Quantitative real-time PCR detection results of inflammatory factors during contused skeletal muscle repairThe RT-PCR results showed that the expression of TWEAK m RNA was significantly increased compared with the control group at the 3d and 6d post-injury(p<0.01).TWEAK receptor Fin14 expression was significantly increased at the 3d post-injury(p<0.01),then gradually decreased.5.Quantitative real-time PCR detection results of oxidative stress factor during contused skeletal muscle repairThe RT-PCR results showed that the expression of oxidative stress factor gp91 phox m RNA(the key subunit of NADPH oxidase)increased significantly at the3 d and 6d post-injury compared with the control group,and peaked at the 3d post-injury(p<0.01),then began to decline.6.Quantitative real-time PCR detection results of Notch signaling pathway regulatory factors during contused skeletal muscle fibrosisThe data showed that Hes1 m RNA was significantly increased compared with the control group at the 3d and 6d post-injury and 6d post-injury.Hey1 m RNA also increased significantly at the 3d and 6d post-injury,and then decreased.Hey2 m RNA was significantly increased on 12 d compared to the control group.Hey L m RNA decreased rapidly only after a significant increase at the 6d post-injury.Conclusion:(1)Overexpression of TGF-β1 and macrophage infiltration during skeletal muscle injury repair may play an important role in the formation of skeletal muscle fibrosis.In addition,TGF-β1 secreted by macrophages may promote the development of damaged skeletal muscle fibrosis during skeletal muscle repair.(2)Notch signaling pathway regulatory factors Hes1,Hey1,Hey2,HeyL,inflammatory cytokines TWEAK/Fn14,oxidative stress factor gp91-phox,chemokine CCL2,CCL5,CCR2,CXCR4,and CXCL12 during contusion of skeletal muscle fibrosis;At high levels,these factors may be involved in the formation of damaged skeletal muscle fibrosis.