Cannabinoid Type 2 Receptor Regulates Repair Of Ischemia/Reperfusion-induced Skeletal Muscles And Its Molecular Mechanisms Through Nrf2 Modulation In Mice

Objective:Skeletal muscle is widely distributed in the human body,most of which is located closely to the body surface.Skeletal muscle has a high incidence of injury and disease in daily life.Skeletal muscle ischemia/reperfusion injury（IRI）leads to high mortality and morbidity and is a common type of clinical muscle injury with currently no effective treatment.Severe IRI causes heavy psychological and economic burden to patients.Skeletal muscle IRI is usually secondary to vascular injury,crush syndrome,compartment syndrome,reconstructive surgery,and the application of tourniquets.Cannabinoid type 2 receptor（CB2R）is an important member of the endocannabinoid system and a seven transmembrane G protein-coupled receptor distributed on the surface of cell membrane.Studies have shown that activation of CB2R could reduce ischemia/reperfusion（IR）-induced brain,heart,kidney and liver injury.Taking into account our previous studies in the rat model of skeletal muscle contusion that the CB2R was time-dependent expression after skeletal muscle injury,and that activation of CB2R could play an anti-inflammatory and anti-fibrotic role,thus improving skeletal muscle repair.Therefore,we speculated that CB2R might also have beneficial effects on skeletal muscle IRI in mice.NF-E2-related factor（Nrf2）is an important antioxidant factor that plays multiple roles in regulating cellular redox homeostasis and modulating cell proliferation and differentiation.Studies also have shown that Nrf2 plays an important role in tissue damage repair.In the present study,we used CB2R selective agonist AM1241 and Nrf2 gene knockout（Nrf2-KO）mice in vivo,combination with in vitro culture of C2C12cells experiment to prove whether activation of CB2R plays beneficial roles by decreasing IR-induced skeletal muscle oxidative injury and promoting skeletal muscle early myogenesis after skeletal muscle IRI and its molecular mechanisms through Nrf2modulation.Methods:Young,8-to 12-week-old C57BL/6 mice（wildtype）and Nrf2 gene knockout（Nrf2-KO）mice on a C57BL/6 background were used.Wildtype mice were randomly divided into 4 groups（Sham group,IR group,vehicle group and AM1241 group）,Nrf2-KO mice were also employed and randomly devided into 4 groups（Sham group,IR group,vehicle group and AM1241 group）.Sterile injury was induced by orthodontic rubber bands（ORBs）according to previous reports by Crawford and Sonmez.Mice in different groups were i.p.injected with vehicle or CB2R agonist AM1241（20 mg/Kg）respectively 0.5 h prior to the surgery and once a day after the surgery.Mice were sacrificed by intraperitoneal injection of 2%sodium pentobarbital（30mg/Kg）at 1 and 4 days post-injury.12 mice were used in each group at each time point and 6 healthy mice were used as the control group（Sham group）.Some of the samples were used for morphological observation by H&E staining,immunofluorescent or immunohistochemical staining of MPO,MyoD,myogenin,CB2R and Nrf2.Some samples were used for biochemical detection,including MDA and SOD,to evaluate the level of oxidative stress status in mice after skeletal muscle IRI.Some samples were used for the measurement of W/D ratio to assess the degree of tissue edema.Some samples were used for Western blotting to determine the protein expression levels of Nrf2,HO-1,MyoD and myogenin.In vitro experiment,C2C12 myoblasts were employed and cultured.Differentiation medium（DM）was used to induce C2C12 cells differentiation and H₂O₂ was used to induce oxidative injury of C2C12 cells.Nrf2 gene knockdown（Nrf2-KD）C2C12 cells were produced by lentiviral-based shRNA transduction of Nrf2.During the culture,C2C12 cells were treated with vehicle or AM1241（1μM-30μM）.Cell viability was measured by CCK-8 assay,and the ROS level and cell apoptosis were detected by flow cytometry.mRNA expressions of CB2R,Nrf2,MHC and myogenin were quantified by qRT-PCR.Protein expressions of CB2R,Nrf2,HO-1,MHC,myogenin,and cleaved caspase 3 were detected and assayed by Western blotting.Immunofluorescent staining of Nrf2 and MHC were also performed.Data were expressed as means±standard deviation and analyzed using GraphPad Prism 6.0 software.One-way analysis of variance followed by Tukey’s Honestly Significant Difference post hoc test or Student’s two-tailed unpaired t-test was used to measure differences between mean values of the different treated groups.Values of p<0.05 were considered statistically significant.Results:In animal experiments,compared with the vehicle group,treatment of IR mice with AM1241 significantly reduced muscle injury,reflected by less sarcoplasm dissolution,a decrease in the loss of nuclei,and a decrease in neutrophil infiltration 1 day after skeletal muscle IRI.The W/D ratio in the AM1241 group significantly decreased compared with the vehicle group.Treatment with AM1241 significantly reduced tissue MDA level and increased tissue SOD activity compared with the vehicle group.Compared with the vehicle group,AM1241-induced CB2R activation significantly increased the number of regenerating myotubes 4 days post-injury.MyoD and myogenin protein expression significantly increased in the AM1241 group 4 days after reperfusion.Immunofluorescent staining showed that the AM1241 group exhibited significant increases in MyoD-and myogenin-positive nuclei compared with the vehicle group.IRI increased the protein expression of Nrf2 and HO-1 in skeletal muscles compared with the sham group.AM1241treatment significantly increased Nrf2 and HO-1 protein expression compared with the vehicle group.Nrf2 knockout（Nrf2-KO）mice were treated with AM1241 1 day after IRI,which resulted in a significant increase in muscle fiber injury,with more sarcoplasm dissolution and neutrophil infiltration and a significant increase in histological damage scores compared with wildtype mice activated by AM1241.After activation of CB2R by AM1241,the skeletal muscle W/D ratio in the Nrf2-KO mice significantly increased compared with wildtype mice.MDA level significantly increased and SOD activity significantly decreased in the Nrf2-KO mice compared with wildtype mice.AM1241treatment 4 days post-injury significantly decreased the number of regenerating myotubes in the Nrf2-KO mice compared with wildtype mice.MyoD and myogenin protein expression significantly decreased in the Nrf2-KO mice compared with wildtype mice after treatment with AM1241.Immunofluorescent staining showed that AM1241-induced CB2R activation significantly decreased MyoD-and myogenin-positive nuclei in the Nrf2-KO mice compared with wildtype mice.The in vitro experiment demonstrated that 1mM H₂O₂ reduced cell viability by approximately 50%.Compared with the H₂O₂ group,pretreatment with AM1241significantly and dose-dependently prevented the H₂O₂-induced reduction of cell viability,decreased ROS generation,decreased protein expression level of cleaved caspase 3 and protected cells against apoptosis.Pretreatment with AM1241 promoted nuclear translocation of Nrf2 and dose-dependently increased protein expression level of Nrf2 and HO-1.AM1241-treated Nrf2-KD cells after H₂O₂ treatment exhibited a decrease in cell viability and an increase in the protein level of cleaved caspase 3 compared with Scr cells subjected to the same treatment.Replacement of proliferation medium（PM）with differentiation medium（DM）resulted in a time-dependent formation of multinucleated myotubes and increase of fusion index.The mRNA expressions and protein expression levels of CB2R,Nrf2,myosin heavy chain（MHC）and myogenin significantly increased during cell differentiation in a time-dependent manner compared with cells cultured in PM.Treatment with AM1241 enhanced multinucleated myotube formation and increased the fusion index,with increases in the protein expression levels of MHC,myogenin,and Nrf2compared with the vehicle group.Knockdown of Nrf2 suppressed the myotube formation and decreased the fusion index,with decreases in the protein expression levels of Nrf2,myogenin,and MHC.Conclusion:Activation of CB2R plays beneficial roles by ameliorating IR-induced skeletal muscle oxidative injury and enhancing skeletal muscle early myogenesis in mice subjected to skeletal muscle IRI,which is partially via Nrf2 signaling.