CB2R通过Nrf2调节小鼠骨骼肌挫伤修复及其分子机制研究

Skeletal muscle contusion is one of the common types of soft tissue injuries in human. Our previous studies indicate that cannabinoid receptor type 2 (CB2R) plays important roles in modulation of inflammatory reaction and fibrogenesis by regulating inflammation- and fibrogenesis-related cytokines during repair of skeletal muscle contusion, and we also found that CB2R agonism reveals beneficial effect on skeletal muscle regeneration after contusion. Much evidence has demonstrated that Nrf2 (nuclear factor-erythroid derived 2-related factor 2) is a pivotal factor in modulation against oxidative stress, which plays important parts in inflammatory reaction, apoptosis, necrosis, repair and cell regeneration in organs through pathways modulating anti-oxidative stress, endoplasmic reticulum stress and autophagy. A recent study shows that CB2R agonism may induce myocardial regeneration by PI3K/Akt/Nrf2 signaling pathway activation. It is suggested that CB2R may regulate inflammatory reaction, fibrogenesis, and muscle regeneration by Nrf2. The present project is attempted to study the roles of Nrf2 in modulating oxidative reaction, fibrogenesis, satellite proliferation and differentiation, and muscle regeneration with CB2R agonist intervention in Nrf2-knockout mice and Nrf2-knockdown cells by molecular biological techniques, and investigate the molecular mechanisms by which CB2R regulates repair of contused skeletal muscles through Nrf2 modulation. The study may also provide fundamental data for clinical treatment of traumatic muscle lesions and muscular diseases, and even for research and development for related drugs.

骨骼肌挫伤是人体常见的软组织损伤类型之一。我们前期研究表明，2型大麻素受体（CB2R）在骨骼肌挫伤修复过程中通过调节炎症及纤维化相关因子对炎症反应及纤维化修复起到重要的作用，激动CB2R有益于骨骼肌再生。Nrf2是抗氧化反应的中枢调节因子，调控氧化应激反应、内质网应激和自噬相关的信号通路，在炎症反应、细胞凋亡、坏死、器官损伤修复与再生中发挥重要的作用。研究表明，CB2R可通过PI3K/Akt/Nrf2信号通路促进心肌细胞再生，提示在骨骼肌挫伤中CB2R可能通过Nrf2调控炎症反应、纤维化修复及骨骼肌再生过程。本项目应用Nrf2基因敲除鼠及基因沉默细胞，通过CB2R激动剂干预,采用分子生物学技术研究Nrf2对骨骼肌挫伤后氧化反应、纤维化修复、卫星细胞增殖、分化与骨骼肌再生的调节作用，探讨CB2R通过Nrf2调控骨骼肌挫伤修复的分子机制，为骨骼肌损伤、疾病的治疗及相关药物研发提供基础依据。

骨骼肌挫伤是人体常见的软组织损伤类型之一。我们前期研究表明，CB2R在骨骼肌挫伤修复过程中发挥重要的调控作用，激动CB2R有益于骨骼肌再生。Nrf2是抗氧化反应的中枢调节因子，在炎症反应、细胞凋亡、器官损伤修复与再生中发挥重要的作用。研究表明，CB2R可通过PI3K/Akt/Nrf2信号通路促进心肌细胞再生，提示CB2R可能通过Nrf2调控骨骼肌修复进程。为验证上述猜想，本研究采用8-10周雄性的C57BL6/J WT鼠、CB2R敲除小鼠以及Nrf2敲除小鼠，参照Crawford使用牙齿矫正橡皮圈(orthodontic rubber bands，ORBs)制作骨骼肌缺血再灌注损伤模型。并进行CB2R激动剂（AM1241）干预，研究骨骼肌损伤修复情况。研究发现，CB2R-KO 小鼠较WT小鼠，损伤区浸润的巨噬细胞总量没有差异，但M1型巨噬细胞数量增多，M2型的数量减少。M1相关的标志物和促炎因子iNOS、TNF-α、IL-1β、IL-6的表达水平增加，M2相关的标志物和细胞因子Arg-1、Clec7a、IL-10、IL-13、TGF-β的表达下降。巨噬细胞原代培养实验显示，CB2R-KO导致巨噬细胞对LPS/IFN-γ刺激的反应性增加，促进自身向M1型极化，而对IL-4/IL-10刺激的敏感性降低，抑制自身向M2型极化。骨骼肌损伤后，AM1241 处理可诱导Nrf2 和HO-1 的表达上调。利用Nrf2-KO鼠研究发现，与WT小鼠相比，在同样接受AM1241 处理的情况下，Nrf2-KO小鼠骨骼肌损伤更为严重,组织内 MDA的水平升高，SOD 的活力降低。4 天时，单位面积内再生的肌管数量减少，MyoD和 myogenin 的表达水平降低，MyoD+和 myogenin+的核比率降低。成肌细胞体外培养实验发现，激活 CB2R 以后，细胞融合指数升高，伴随Nrf2、MHC、myogenin 蛋白水平的升高。而Nrf2-KD细胞接受AM1241处理后，MHC、myogenin 蛋白上调不明显，新生肌管形成相对减少。本研究证实小鼠骨骼肌IRI愈合过程中，CB2可通过抑制巨噬细胞向M1型极化，促进其向M2极化而促进骨骼肌损伤修复。并且，激活CB2R可以通过激活Nrf2通路，减弱氧化应激性损伤，增强新生肌管形成而促进损伤修复。