PD-1 Promotes Contused Skeletal Muscle Regeneration By Regulating Treg Cells And Macrophages

Object:Skeletal muscle contusion is a common form of sports skeletal muscle injury and is accompanied by the infiltration of multiple immune cells.Macrophage M1/M2 polarization plays an important role in contusion skeletal muscle repair,while Treg cells can regulate macrophage M1/M2 polarization and promote damaged skeletal muscle repair.However,these specific mechanisms have not been clarified.Some studies have shown that PD-1 can regulate macrophage M1/M2 polarization;Treg cells can express PD-1.At the same time,PD-1 can promote the production of Treg cells in peripheral tissues and is important for maintaining the normal function of Treg cells effects.Therefore,the purpose of this study is to explore the role of PD-1 in contused skeletal muscle regeneration,and to clarify the regulation of PD-1 on Treg cell generation and macrophage M1/M2 polarization,which will deepen our understanding of the relationship between the immune system and skeletal muscle regeneration.Method:Forty male C57BL/6 mice were randomly divided into 1 day,3 days,7 days,and14 days after injury and the non-injury group(WT1,WT 3,WT 7,WT 14,WT _con,n=8);30 male PD-1 knockout C57BL/6 mice were randomly divided into 1 day,3 days,7 days,and 14 days after injury and the non-injury group(P1,P3,P7,P14,P_con,n=6).One day,three days,seven days,and 14 days after skeletal muscle contusion,bilateral gastrocnemius muscles of the mice were taken.Hematoxylin-eosin staining observed morphological changes of skeletal muscle;Masson staining observed skeletal muscle fibrosis;Immunofluorescence detected total macrophages,M1 macrophages,M2macrophages and Treg cells;The expression changes of fibrosis-related factors,pro-inflammatory factors,anti-inflammatory factors,oxidative stress-related factors,and muscle regeneration-related factors were detected by RT-PCR.Result:1.The damages of skeletal muscle contusion repair after PD-1 knock-outHE staining results showed that on the first day after skeletal muscle injury,a large amount of muscle fiber swelling and necrosis and inflammatory cell infiltration occurred in both the WT group and the PD-1^-/-group.On days 3 and 7 after skeletal muscle injury,some central nuclear muscle fibers?regenerated muscle fibers?appeared in the skeletal muscle of the WT group,but only a small amount of regenerated muscle fibers were seen in the PD-1^-/-group.On the 14th day after skeletal muscle injury,there was only a small amount of regenerated muscle fibers in the WT group,but there were still more regenerated muscle fibers in the PD-1^-/-group,and the diameter of the regenerated muscle fibers in the PD-1^-/-group was significantly lower than that of the WT group?P<0.05?.2.The degree of fibrosis of contused skeletal muscle after PD-1 knockoutMasson staining results showed that skeletal muscle fibrosis increased in both WT and PD-1^-/-groups 14 days after injury,but there were no significant differences in fibrosis in WT14 and P14 groups.In addition,the levels of Col1a1 and Col3a1 mRNA in the WT group and the PD-1^-/-group were significantly higher than those in the uninjured group at 1,3,7,and 14 days after injury?P<0.05?.However,there were still no significant differences between the PD-1^-/-group and the WT group?P>0.05?.3.The effects of PD-1 knockout on Treg cells in contused skeletal muscleImmunofluorescence results showed that one day after skeletal muscle contusion,there were no significant differences in the number of Treg cells between WT group and PD-1^-/-group.At 3 and 7 days after skeletal muscle contusion,the number of Treg cells in WT group was significantly higher than that in PD-1^-/-group?P<0.05?.Fourteen days after skeletal muscle contusion,the number of Treg cells in the WT group was close to normal levels,while the number of Treg cells in the PD-1^-/-group remained little changed and more than that in the WT group.In addition,AREG mRNA levels in PD-1^-/-group were significantly lower than those in WT group at 1,3,and 7 days after injury?P<0.05?.4.The effects of PD-1 knockout on macrophages in contusion skeletal muscleImmunofluorescence results showed that the number of M1 macrophages in the PD-1^-/-group was significantly higher than that in the WT group,while the number of M2macrophages was significantly lower than that in the WT group at 1,3,and 7 days after skeletal muscle contusion?P<0.05?.In addition,Mac-2 mRNA levels of total macrophage markers in the WT group and PD-1^-/-group were not significantly different?P>0.05?;The mRNA levels of iNOS and CD86 markers of M1 macrophages in PD-1^-/-group were significantly higher than those in the WT group at 3 and 7 days after injury?P<0.05?.The mRNA levels of Arg1,CD206,and CD163,markers of M2macrophages,in the PD-1^-/-group were significantly lower than those in the WT group3 days after injury?P<0.05?.5.PD-1 knockout can increase the levels of skeletal muscle inflammationThe levels of pro-inflammatory factors IL-1?,IL-6,and TNF-?in the PD-1^-/-group were significantly higher than those in the WT group at 1,7,and 14 days after injury?P<0.05?.But the levels of anti-inflammatory factors IL-4 and IL-10 mRNA in PD-1^-/-group were significantly lower than those in WT group at 1 and 7 days after injury?P<0.05?,and there was no significant difference in TGF-??P>0.05?.6.PD-1 knockout can increase the levels of oxidative stress in contusion skeletal muscleThe levels of oxidative stress factor Nox2 mRNA in PD-1^-/-group was significantly higher than that in WT group at 1,3,and 7 days after injury?P<0.05?.The mRNA levels of oxidative stress factors Prdx1,Sod1,Gpx4 were significantly lower than those in the WT group?P<0.05?.7.PD-1 knockout can reduce the levels of contusion skeletal muscle regenerationAt 1,3,7,and 14 days after injury,the mRNA levels of muscle regeneration factors Pax7,MGF,and IGF-1 in the PD-1^-/-group were significantly lower than those in the WT group?P<0.05?.Conclusion:PD-1 knockdown reduced contused skeletal muscle Treg cells,reduced the expressions of IL-4 and IL-10,and blocked the conversion of M1 macrophages to M2,manifested by down-regulation of muscle regeneration factors,prolonged inflammatory response period,aggravated the levels of oxidative stress and damaged regeneration of contusion skeletal muscle.These results indicated that PD-1 can affect Treg cell production and macrophage polarization to promote contused skeletal muscle regeneration.