To Investigate The Mechanism Of Eccentric Skeletal Muscle Overuse Injury Based On Phenotypic Transition And Distribution Of Macrophages

1.Objective Overuse injury is a gradual injury caused by repeated microtraumas,which seriously affects the athlete's competition level and damages the work and life of workers.In the pathological changes of overuse injury,fibrosis is considered to be the key to causing pain and movement disorders.Studies have shown that the phenotypic transition process of macrophages is both a necessary prerequisite for muscle regeneration and a mediator of fibrosis,but its role in the mechanism of overuse injury is still unclear.In this paper,4 weeks of excessive eccentric training to establish an overuse injury model,observe the phenotypic transition and distribution characteristics of macrophages,and explore the causes of skeletal muscle overuse injury.2.Method 72 male SD rats were randomly divided into control group(group C,n=24);continuous eccentric training group(group E,n=24);intermittent eccentric training group(group I,n=24).Each group was randomly divided into 4 groups,corresponding to 1 week,2 weeks,3 weeks,and 4 weeks.Except for group C,all the other groups were given eccentric training for 1-4 weeks,and the exercise load was in the form of speed and time progression(gradient-16 °,time 60-90 min,speed16-20m/min),Group E trains continuously,5 days a week,and Group I trains every other day,3 days a week(Saturday and Sunday rest).After 36 hours of training every weekend,the ipsilateral vastus intermedius was taken.Subsequently,Masson's triple staining technique was performed to observe the formation process of skeletal muscle fibrosis in different phases;immunohistochemistry technique was used to analyze the number and distribution characteristics of M1(CD68)macrophages and M2 type(CD163)macrophages in different phases.36 hours after the fourth week of training,the vastus intermedius was taken and the skeletal muscle ultrastructure changes were observed by transmission electron microscope.3.Results3.1 The process of skeletal muscle fibrosis:(1)Compared with group C,collagen fiber deposition in group E increased significantly at 1,3,and 4 weeks(p<0.01),but significantly decreased in the second week(p<0.001);There was a significant increase in group I.(2)In group E,2E is the lowest value and 4E is the peak value,both of which are extremely significantly different from other phases(p<0.001);group I shows a continuous decreasing trend,and there are extremely significant differences between 1E,2E,and 3E(p<0.001).(3)Compared with 3I and 4I,3E and4 E both increased significantly(p<0.001).3.2 Skeletal muscle M1/M2 macrophage changes:(1)M1 : Compared with group C,group E and group I are higher than group C.Group E showed an increasing trend,and 4E was significantly different from 1E,2E,and 3E(p<0.001);Group I showed a trend of increasing first and then decreasing,with 2I as the peak value and then decreasing.Compared with 4I,4E increased significantly(p<0.001).(2)M2:Compared with group C,group E and group I are higher than group C.Group E showed an increasing trend,4E was significantly different from 1E,2E,and 3E(p<0.001);Group I showed a trend of increasing first and then decreasing,with 2I being the peak value,4I being the lowest value,and being compared with 1I and 2I respectively.Very significant difference(p<0.001).Compared with 3I and 4I,3E(p<0.01)and 4E(p<0.001)increased significantly.3.3 Distribution of skeletal muscle M1/M2 macrophages:(1)M1: There was no statistical difference between the distribution of group E and group I on the perimysium and the distribution of muscle cells(including endomysium)and group C.The percentage of the number of M1 type in group E increased on the perimysium week by week,and 4E was significantly increased from 1E and 2E respectively(p<0.01),but there was no statistical difference between 4E and 3E,and correspondingly on myocytes decrease week by week;group I showed a week-by-week increase in the perimysium,4I was significantly higher than 1I(p<0.001)and 2I(p<0.01),but there was no statistical difference between 4I and 3I.The decrease in muscle cells week by week.(2)M2: Compared with group C,there is no significant difference in group E at any time;in group I,1I is lower than group C and has a significant difference(p<0.05).The perimysium in group E showed a trend of increasing week by week,but there was no significant difference in each phase(p>0.05);in group I,it increased first and then decreased,with 2I as the peak value,respectively compared with 1I(p<0.01),4I(p<0.05)showed a significant difference.4E is higher than 4I,with a very significant difference(p<0.01);the number of M2 macrophages on muscle cells is distributed in a corresponding reverse trend.3.4 Correlation between perimysium thickness and macrophages: The thickness of perimysium in group E is negatively correlated with M1 in muscle cells(p<0.01,r=?0.698),and negatively correlated with M2(p<0.05,r=?0.48);the thickness of the perimysium in group I was positively correlated with the intramuscular M1(p<0.01,r=0.636),and the M2 was wirelessly correlated(p>0.05,r=?0.304).The correlation between the thickness of the perimysium and the percentage of the number of macrophages on the perimysium is opposite to the above.4.Conclusion4.1 Overuse injury of skeletal muscle is related to the excessive deposition of perimysium collagen fibers,that is,fibrosis.4.2 Excessive use of skeletal muscle not only did not inhibit the conversion of M1/M2 macrophages,but over-converted to M2(CD163).4.3 Overuse of skeletal muscle caused changes in the distribution of M1/M2 macrophages,and the migration of M2(CD163)from perimysium to muscle cells decreased,which was related to the thickness of perimysium.