肌纤维转化生长因子-β信号激活与急性肌损伤炎症反应的相关性研究

目的探讨肢体创伤后急性肌损伤炎症反应过程中肌纤维自身转化生长因子-β(TGF-β)信号对肌内炎症的影响。方法取野生C57BL/6小鼠(野生小鼠组)、肌纤维条件性TGF-β受体Ⅱ敲除小鼠(敲除小鼠组)各16只。肌毒素腓肠肌内注射诱导小鼠急性肌损伤。比较两组小鼠在注射后0、4、7、10 d肌内单核-巨噬细胞、巨噬细胞、M1型巨噬细胞、CD4+T细胞、辅助性T细胞(Th)1、2、17等的渗出差异。取新生野生小鼠和敲除小鼠各2只,体外培养原代成肌细胞,分别分为两组:干扰素组(γ-干扰素处理)和对照组(仅加入等量溶剂)。比较两组肌细胞的白细胞介素(IL)-6、IL-10、单核细胞趋化蛋白1(MCP-1)、巨噬细胞炎症蛋白-1α(MIP-1α)、H-2Kb蛋白、H2-Ea蛋白、Toll样受体(TLR)3蛋白、TLR7蛋白表达差异,以及野生小鼠和敲除小鼠干扰素组之间的差异。结果肌毒素注射后4、7 d,敲除小鼠组肌组织内单核-巨噬细胞比例(75.73%±3.62%、45.27%±2.32%)、巨噬细胞比例(38.67%±2.76%、24.87%±2.19%)、M1型巨噬细胞比例(43.21%±0.11%、30.43%±2.19%)、CD4+T细胞比例(20.13%±1.62%、5.67%±0.32%)显著高于野生小鼠组(58.52%±2.43%、29.21%±2.45%,20.63%±2.32%、16.23%±1.25%,24.98%±0.35%、14.23%±1.69%,10.70%±0.43%、2.50%±0.45%),且以Th1和Th17为主,差异均有统计学意义(P<0.05)。体外实验结果显示:与对照组比较,干扰素组IL-6、MCP-1、MIP-1α、H-2Kb蛋白、H2-Ea蛋白、TLR3蛋白显著上调,且敲除小鼠干扰素组上调较野生小鼠干扰素组更显著,差异均有统计学意义(P<0.05)。结论内源TGF-β信号缺失影响肌纤维免疫行为的调节,使肌内炎症加剧,肌细胞修复延迟。

Correlation between activation of transforming growth factor signal in muscle fibers and inflammatory response to acute muscle injury

Objective To investigate the effect of transforming growth factor(TGF-β)signal in muscle fiber itself during inflammation/immunity response on intramuscular inflammation.Methods Sixteen wild C57BL/6 mice(wild group)and sixteen mice with skeletal muscle-specific deficiency of TβRⅡ(knock-out group)between 4-8 weeks of age were selected for this study.Acute muscle injury in mice was induced by injection of myotoxin cardiotoxin(CTX)into gastrocnemius.The differences in intramuscular inflammation were compared between the wild and knock-out groups on 0,4,7 and 10 d after CTX injection by observing exudation of mononuclear phagocytes,macrophages,M1 type macrophages,CD4+T cells and helpers T cells(Th1,2&17).Two newborn C57BL/6 wild mice and 2 SM TGF-βr2-/-knock-out mice were selected to culture primary myoblasts in vitro which were divided into 2 groups:an interferon group subjected to interferon simulation and a control group subjected to addition of an equal amount of solvent.The differences in expression of IL-6,IL-10,MCP-1,MIP-1α,H-2Kb,H2-Ea,Toll-like receptor(TLR)3 and TLR7 were compared between the interferon and control groups,as well as between the wild and knock-out groups.Results On 4&7 d after CTX injection,the ratios of mononuclear/macrophage(75.73%±3.62%,45.27%±2.32%),macrophages(38.67%±2.76%,24.87%±2.19%),M1 macrophages(43.21%±0.11%,30.43%±2.19%),CD4+T cells(20.13%±1.62%,5.67%±0.32%)in the muscle tissue from the knock-out mice were significantly higher than those from the wild mice(58.52%±2.43%,29.21%±2.45%;20.63%±2.32%,16.23%±1.25%;24.98%±0.35%,14.23%±1.69%;10.70%±0.43%,2.50%±0.45%),with a majority of Th1&Th17(P<0.05).In vitro results showed that the levels of IL-6,MCP-1,MIP-1α,H-2Kb,H2-Ea and TLR3 were significantly upregulated in the interferon group compared with the control group and that such upregulation in the nock-out mice was more significant than in the wild mice(P<0.05).Conclusions Endogenous TGF-βsignal activation plays a role in the functional recovery after muscle trauma,because it is involved in the regulation of immune behavior of muscle fibers,thus affecting intramuscular inflammation and muscle regeneration.