| Objective: Skeletal muscle ischemia reperfusion(IR)injury is commonly seen in both clinical and forensic practice.The regeneration process after skeletal muscle injury mainly refers to the activation,proliferation and differentiation of satellite cells(SCs),the extension and fusion of myoblasts,the formation of new muscle tubes,and finally the formation of complete skeletal muscle fibers.During the formation of myotubes,the expression of some transcription factors such as Myo D and myogenin changes accordingly,regulating the proliferation and differentiation of satellite cells and the formation of skeletal muscle fibers.Cannabinoid type 2 receptor(CB2R)has been found to protect tissue damage and promote tissue regeneration in many experimental mouse models.Our previous study showed that CB2 R is time-dependently expressed in skeletal muscle,myofibroblasts and macrophages.After administration of CB2 R agonist,the regeneration of muscle cells was significantly improved.Thus,CB2 R plays a protective role in skeletal muscle regeneration.Macrophages are immune cells and have two phenotypes,namely,classically activated M1 and selectively activated M2,depending on their high degree of heterogeneity and plasticity.At present,it has been pointed out that the pro-regeneration effect of macrophages on tissue regeneration is mainly by changing their own polarization.Preliminary experimental results indicate that CB2 R can affect the phenotype of macrophages in the skin injury model,therefore this study focuses on whether CB2 R can manipulate the regeneration of skeletal muscle after ischemia-reperfusion injury through regulating the self-polarization of macrophages.Skeletal muscle regeneration is a long and complicated process,which is often accompanied by the formation of fibrosis and scar.Therefore,it is of great practical significance to study the mechanism of muscle cell regeneration and molecular regulation in skeletal muscle repair.In this experiment,we adopted a time-gradient model to analyze the time-expression pattern of macrophages and their molecular markers,which may provide a biological marker for forensic medicine to infer the time of skeletal muscle injury.At the same time,how does CB2 R change the polarization of macrophages and thus affect skeletal muscle regeneration was studied,providing a potential target for clinical development of skeletal muscle regeneration therapy.Methods: A total of 72 male healthy adult C57BL6/J WT and CB2R-KO mice,weighing 24g-26 g,were used at 8-10 weeks.Using the Mc Giveny nevus ligator,the ORBs ligated rubber trap was placed on the great trochanter of the femur of the left hind limb of the mouse to induce ischemia for 2.5 hours.After that,the rubber ring was released to reperfusion the blood flow,that is the skeletal muscle IR injury model.Experiment was divided into control group and injury group.6 WT and CB2R-KO mice were randomly assigned to each group,in the experimental group,the mice were anesthesiated with carbon dioxide,the blood flow was irrigated with 0.9% normal saline,and gastrocnemius muscle of the injured hind limb was taken as the test material at 1,2,4,7 and 10 days after reperfusion.The control group collected samples on the day of modeling(0d).Half of them were embedded in paraffin and then made into paraffin sections for H&E,immunohistochemistry and immunofluorescence staining.The other half were frozen with liquid nitrogen and then lapped to extract histones for molecular morphological detection.H&E is common staining;Immunohistochemical staining is for macrophages marker of F4/80;Immunofluorescence staining is for M1-type marker CD86,M2-type marker CD206.The protein expression levels of Myo D,myogenin,MHC,Arg-1,IL-10,IL-13,TGF-?,Clec7 a,i NOS,IL-6,IL-1?,TNF-? were detected by Western blot.Macrophages isolated from the abdominal cavity were used for primary culture in vitro.The separated macrophages were first cultured by differential adherent method for 6h.The non-adherent cells were removed,and the adherent cells were left for continued culture.The macrophages were stimulated by LPS/IFN-? or IL4/IL-10 to induce macrophages to type M1 or M2.After 24 hours of stimulation,the stimulating factors were removed.Half of the cells were collected for protein extraction.Western blot was used to detect the expression of Arg-1,TGF-?,Clec7 a,i NOS,IL-6,IL-1?,TNF-?.The other half of the cells were further cultured on a serum-free medium for 24 hours for the preparation of CM.Then,the 90% approximately-confluence C2C12 cells were washed with DPBS and added a new conditioned medium composed of 25% macrophage-CM and 75% differential medium with 2% HS and cultured for 3 days.Myo D and myogenin protein expression was detected.The differentiation of myoblast cells was observed by immunofluorescence.Results: After CB2 R knockout,skeletal muscle regeneration was seriously affected compared with WT group.Histological analysis of gastrocnemius(GA)subjected to IR injury showed that no newly formed myofibers were found throughout the experimental period in the sham groups.Although the number of the regenerated myotubes with central nucleis increased sequentially,the numbers in the CB2 R knockout group were less at the same time point.The cross sectional area(CSA)of the regenerated myotubes decreased,and the mean value of CSA decreased.The newly formed myotubes showed a tendency to be dominated by the smaller.Protein expressions of Myo D and myogenin were significantly decreased,too.There was no significant difference in the number of infiltrated MPs between two groups at different times post-injury.We found the infiltration of M1 macrophages increased at 1 day post-injury,peaked at 2 days and decreased over subsequent days.The number of M2 macrophages markedly increased at 2 days,became predominant at 4 days,then gradually decreased over time.Furthermore,the CB2R-KO groups exhibited a higher number of M1 macrophages,but a lower number of M2 macrophages compared to WT controls at 2 days,4 days,7 days,and no difference was found at 1 day and 10 days.CB2 R knockout resulted in increased protein expression of M1-related markers and cytokines,such as i NOS,IL-6,IL-1?,TNF-?,and decreased protein expression of M2-related markers and cytokines,such as Arg-1,Clec7 a,IL-10,IL-13,TGF-?.Primary culture experiments of peritoneal macrophages showed that CB2 R knockout increased the M1 response to LPS/IFN-? stimulation,while decreased the sensitivity of M2 to IL-4/IL-10 stimulation.The co-culture experiment of macrophages conditioned medium(CM)with C2C12 myoblast cells proved that CM from CB2 R deletion-macrophages significantly inhibited the differentiation of C2C12 cells compared with WT group.Conclusion: CB2 R can promote the regeneration of skeletal muscle cells after ischemiareperfusion injury by inducing the polarization of macrophages to M2. |
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