| Objective:Skeletal muscle injury is one of the most common injuries in sports medicine.Our previous studies have found that macrophage depletion led to the down-regulation of insulin-like growth factor 1(IGF-1),mechanical growth factor(MGF)in contused skeletal muscle and impaired muscle regeneration.We hypothesized that IGF-1 or MGF injection may at least partially improve impaired muscle repair induced by macrophage depletion.Therefore,we explored whether injection of IGF-1 or MGF plays an important role in improving the repair of injured skeletal muscles.Methods:We established the animal models of muscle contusion and macrophage depletion combined with IGF-1 or MGF injection in gastrocnemius muscle of C57BL/6 mice.The mice were randomly assigned to 1 of 4 groups according to the treatment:(1)contusion and placebo treatment(C group),(2)contusion and macrophages depletion treatment(CD group),(3)contusion,macrophages depletion and IGF-1 treatment(CDI group),(4)contusion,macrophages depletion and MGF treatment(CDM group).In C,CD,CDI and CDM groups,each mouse suffered bilateral contusions of the gastrocnemius muscles except for the day 0 in each group(non-contusion mice).The mice were sampled before contusion(Con)and at days 1,3,7 and 14 after contusion.Comprehensive histomorphological and genetic analysis of contused muscles was carried out after macrophage depletion and IGF-1 or MGF injection.Skeletal muscle regeneration(Hematoxylin and eosin staining),fibrosis(Masson trichrome stain),surface markers of macrophages and their subtypes,inflammatory cytokines,chemokines,myogenic regulatory factors(MRFs),angiogenic regulatory factors,oxidative stress factor and matrix metalloproteinase(MMP)were detected in contused skeletal muscle(Relative quantitative real-time PCR and Immunofluorescence staining).Results:(1)Effect of clodronate-containing liposomes on macrophage marker in injured skeletal muscle.Compared with simple muscle contusion,the total number of macrophages(F4/80)after macrophage depletion was significantly inhibited(ρ<0.01).These results indicated that macrophages are effectively depleted.(2)IGF-1 injection did not improve the reduced regenerative myofibers and the aggravated muscle fibrosis induced by macrophage depletion in contusedmuscles.The total number,total area and total diameter of regenerative myofibers in contusion muscle 14 days after macrophage depletion were significantly lower than those after simple muscle contusion(ρ<0.05).After IGF-1 injection,the damaged muscle regeneration was not improved(ρ>0.05).Moreover,macrophage depletion induced a significant increase in fibrosis after muscle contusion(ρ<0.05),but the increased fibrosis was not improved by IGF-1 injection(ρ>0.05).(3)IGF-1 injection could not facilitate the coordination among macrophages after macrophage depletion,thereby not improving the regeneration and repair of contused muscles.The cellular immune response was improved after contusion and macrophage depletion by injection of IGF-1,the total number of macrophages(F4/80),pro-inflammatory macrophages(CD68)and anti-inflammatory macrophages(CD163 and CD206)increased significantly to some extent(ρ<0.05).In the late stage of repair(at 14 d postinjury),total macrophages(F4/80)after IGF-1injection were higher than those after both macrophage depletion and simple muscle contusion(ρ<0.05),but the levels of CD68 and CD206 macrophages after IGF-1injection were higher only than those after simple muscle contusion(ρ<0.05),while the levels of CD163 macrophages were lower than that after simple muscle contusion(ρ<0.01).(4)IGF-1 injection did not improve the delayed repair of contused muscles induced by macrophage depletion as determined by MRFs increases,especially in the late stage of repair.Compared with the expression level after simple muscle contusion,the expression levels of MyoD,Myf5,myogenin,and Myf6 increased significantly at different time points after macrophage knockout(ρ<0.05).After injection of IGF-1,the expression of MyoD and myogenin at 14 d postinjury also increased significantly(ρ<0.001).(5)IGF-1 injection partly improved the inhibition of angiogenesis regulatory factors induced by macrophage depletion but could not improve the impaired regeneration and repair of contused muscles.Compared with simple muscle contusion,the expression of HIF-1 α and VEGF increased significantly at the initial stage of repair after macrophage depletion(ρ<0.01),and during all stages of repair after macrophage depletion,the expression of Angpt-1 was lower than that after simple muscle contusion(ρ<0.01).Compared with that after macrophage depletion,the expression of HIF-1α was reduced significantly during all stages of repair after IGF-1 injection(ρ<0.001),while the expression of VEGF in the late stage andAngpt-1 during all stages of repair after IGF-1 injection were increased significantly(ρ<0.05).However,the expression of HIF-1 α,VEGF and Angpt-1 in the late stage of repair after IGF-1 injection was lower than that after simple muscle contusion(ρ<0.001).(6)MGF injection did not exert a protective effect on muscle fiber regeneration after macrophage depletion,but decreased the fibrosis of contused skeletal muscle.Macrophage depletion significantly reduced the total diameter,total number and total area of regenerative myofibers at 14 d postinjury(ρ<0.01).However,MGF injection had no effect on the diameter,number,and area of regenerative myofibers(ρ>0.05).Moreover,compared with the fibrosis in the simple muscle contusion group at 14 d postinjury,the skeletal muscle fibrosis increased significantly in the macrophage depletion group(ρ<0.01).However,compared with the macrophage depletion group at 14 d postinjury,the fibrosis area decreased significantly after MGF injection(ρ<0.05).Meanwhile,the levels of collagens I and III decreased significantly in the MGF injection group compared with the levels in the macrophage depletion group at 1 and 3 days postinjury(ρ<0.01).(7)MGF injection did not influence the functional status of satellite cells in contused skeletal muscle after macrophage depletion.Macrophage depletion did not influence the expression of MyoD(ρ>0.05),but did significantly decrease the expression of myogenin at 3 days postinjury(ρ<0.01).However,injection of MGF did not influence the expression of MyoD and myogenin in injured skeletal muscle after macrophage depletion(ρ>0.05).(8)MGF injection did not influence the expression of macrophage markers in contused skeletal muscle after macrophage depletion,but decreased the expression of inflammatory cytokines and chemokines.Compared with macrophage depletion,there was no significant change in the expression of macrophage markers in contusion skeletal muscle after MGF injection(ρ>0.05).Levels of proinflammatory cytokines(TNF-α,IL-1 β and TGF-β)induced by macrophage depletion were significantly higher than those of simple muscle contusion at 3 days after skeletal muscle injury(ρ<0.05).In contrast,compared with macrophage depletion,MGF injection significantly decreased the levels of TNF-α,IFN-γ,IL-1 β,and TGF-β after injury.Moreover,macrophage depletion significantly increased the expression of chemokines(CCL2,CCL5 and CXCR4)(ρ<0.05).However,MGF injection significantly decreased the expression of the threechemokines(CCL2,CCL5,and CXCR4)in injured muscle tissue after macrophage depletion(ρ<0.05).(9)MGF injection decreased the expression of gp91 phox in injured skeletal muscle after macrophage depletion.Compared with macrophage depletion,MGF injection significantly decreased the expression of gp91 phox at 3 days postinjury(ρ<0.01).(10)MGF injection decreased the expression of matrix metalloproteinases in injured skeletal muscle after macrophage depletion.Macrophage depletion caused significant increases in MMP-1,MMP-2,MMP-9,MMP-10,and MMP-14 levels in injured skeletal muscle compared with the MMPs levels after simple muscle contusion(ρ<0.05).Compared with macrophage depletion,MGF injection exhibited significantly increased levels of skeletal muscle MMP-1,MMP-2,MMP-9,MMP-10,and MMP-14 at 3 days after muscle injury(ρ<0.01).Conclusion:Based on the above research results,the following conclusions can be drawn:(1)With the continuous depletion of macrophages,IGF-1 injection could have promoted partial recovery of macrophage subpopulations(CD206)and expression of VEGF and Angpt-1.(2)IGF-1 injection could not completely remedy the important role of macrophages in muscle regeneration-related regulatory factors,myogenic regulatory factors,and angiogenesis regulatory factors.Consequently,exogenic supplementation with IGF-1 did not improve the impaired regeneration and repair of contused muscle induced by macrophage depletion.(3)Injection of MGF could partly ameliorate the impaired skeletal muscle regeneration and fibrosis induced by macrophage depletion.(4)Injection of MGF could reduce the expression of inflammatory cytokines,chemokines,matrix metalloproteinases and gp91 phox in contused skeletal muscle after macrophage depletion.It is beneficial to reduce the fibrosis of injured skeletal muscle. |
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