Inflammation Regulates Delayed Onset Muscle Damage And Repair In Mice Through GCN2

ObjectiveDelayed onset muscle damage can usually cause delayed onset of muscle soreness,such as stiffness,tenderness and pain during exercise,which affects the process of training and competition.Studies have shown that delayed onset muscle damage and repair are related to inflammation,and GCN2 plays an important role in regulating inflammation,which is of great significance for finding effective treatment of muscle damage.This time,we established the model of delayed onset muscle damage through a single bout of downhill running.And we explored the role and mechanism of GCN2 in damage and repair by the GCN2 knockout.In order to provides a theoretical basis for further prevention of injury and promotion of repair.MethodsThe 8-week-old male wild(WT)mice the GCN2 gene knockout(KO)mice performed a single bout of downhill running to establish a model of exercise-induced muscle damage.WT mice were randomly divided into control group(C0 group),tissue collection at 48 hours after downhill running(C48 group),and tissue collection at 120 hours after downhill running(C 120 group),and the GCN2 KO mice were randomly divided into G0 group,G48 group,and G120 group as the standard of WT mice.Complete blood count(CBC)was used to test the changes in blood,and the hematoxylin-eosin staining was used to analysis the histomorphology of the quadriceps muscle.The degree of muscle damage was assessed by modified Armstrong muscle injury method.CBC mainly includs:modulus and percentage of neutrophils,modulus and percentage of lymphocytes,modulus and percentage of monocytes,modulus and percentage of eosinophils,modulus and percentage of basophils,white blood cell count.Western blot was mainly used to detect the changes in GCN2 signaling pathways and inflammatory factors caused by one-bout of downhill running and GCN2 KO.Results(1)Evaluation of the degree of muscle injury in mice of each group1)There was no significant difference between C0 group and G0 group.2)48 hours after exercise,the degree of injury(%injured)in C48 group and G48 group were significantly higher thanC0 group and G0 group(P<0.05),and the G48 group was significantly lower than C48 group(P<0.05).3)After 120 hours,the degree of injury in C120 group and G120 group were significantly higher than C0 group and G0 group(P<0.05),and G120 group was significantly lower than C120 group(P<0.05).(2)CBC of mice in each groupAllogeneic mice:1)WT mice:Compared with C0 group,NEUT#?NEUT%?BASO#and BASO%in C48 group were significantly increased(P<0.05),BASO#and BASO%in C120 group were significantly increased(P<0.05).2)GCN2 KO mice:Compared with G0 group,NEUT#and NEUT%of G48 group were significantly increased(P<0.05),NEUT#,BASO%of G120 group were increased significantly(P<0.05);And NEUT%in G120 group decreased significantly(P<0.05)compared with G48 group.Different genotypes mice:1)Compared with CO group,NEUT#was decreased significantly in GO group at rest(P<0.05).2)48h after exercise,compared with C48 group,NEUT#?LYMPH#?BASO#and WBC were significantly reduced in G48 group(P<0.05).3)120h after exercise,BASO#was significantly reduced in G120 group compared with C120 group(P<0.05).(3)Protein expression levels of pro-inflammatory and anti-inflammatory factors in each groupThe wild type mice:compared with CO group mice,C48 group had significantly lower in expression levels of NF-?B(-62%)?IL-10(-22%)?IL-12(-22%)in mice(P<0.05).Compared with C48 group,the expression levels of IL-6(-22%)in C120 group was significantly decreased(P<0.05),but the expression level of NF-?B(+104%)?IL-10(+104%)?IL-12(+14%)were significantly increased(P<0.05).GCN2 KO mice:compared with G0 group,the expression level of IL-6(-18%)and NF-?B(-63%)in G48 group were significantly decreased(P<0.05),the expression level of IL-10(+462%)and IL-12(+202%)protein in G120 group were significantly increased(P<0.05).Compared with G48 group,the expression level of IL-6(+32%)?NF-?B(+32%)?IL-10(+650%)?IL-12(+650%)in G120 group mice were significantly increased(P<0.05).(4)Protein expression levels of GCN2 signaling pathway in each groupThe wild type mice:Compared with C0 group,the expression level of GCN2(-18%),eIF2? phosphorylation(-22%)and ATF4(-10%)had significantly decrased in C120 group(P<0.05).GCN2 KO mice:The expression level of P-eIF2a(-39%)and ATF4(-40%)in G120 group mice was significantly lower than G0 group(P<0.05).And the level of eIF2?phosphorylation(-45%)and expression level of ATF4(-45%)protein in the G120 group was significantly down regulated than G48 group(P<0.05).ConclusionsA single bout of downhill running downhill running could induce delayed muscle injury in mice and the injury could be repaired 120 hours after exercise,it is related to inflammation.Deficiency of GCN2 gene could inhibit the expression of blood inflammatory cells and pro-inflammatory cytokines and play a protective role in delayed onset muscle damage induced by downhill running.Deficiency of GCN2 gene could promote the multiplication of the expression of anti-inflammatory factors during the recovery.It is indicating that GCN2 plays an important role in the process of repair of delayed onset muscle damage.Maybe it is related to accelerated repairing.