长期递增式香烟烟雾暴露制备慢性阻塞性肺疾病小鼠骨骼肌功能障碍模型的研究

关键词：慢性阻塞性肺疾病  骨骼肌功能障碍  烟雾暴露  造模

Keywords:chronic obstructive pulmonary disease  skeletal muscle dysfunction  smoke exposure  modeling

摘要：

      摘要 目的：探讨25周递增式烟雾暴露方法能否成功制备慢性阻塞性肺疾病（chronic obstructive pulmonary disease，COPD）小鼠骨骼肌功能障碍模型。 方法：将健康雄性C57BL/6小鼠随机分为模型组（MG，n=6）和对照组（CG，n=6）。MG小鼠全身暴露于香烟烟雾25周，6天/周，每次间隔4h，点燃香烟数量和每日烟熏频次随实验进程逐渐增加，CG小鼠常规饲养。在造模期间，每月测量小鼠体重，每2个月测量小鼠抓力。25周烟雾暴露结束后进行肺功能测试和腓肠肌离体肌力测试。通过HE染色观察肺组织和腓肠肌病理变化并计算肺泡横截面积（cross-sectional area，CSA）、肺泡平均线性截距（mean linear intercept，MLI）和肌纤维CSA。Western Blot法检测腓肠肌MyHC（slow）、MyHC（IID）、Atrogin-1和MuRF-1的表达。 结果：与CG小鼠相比：①MG小鼠一般情况变化明显，体重显著下降（P＜0.05）；②MG小鼠肺组织表现出慢性支气管炎和肺气肿的典型改变，肺泡CSA和MLI显著增加（P＜0.05），肺功能指标FVC、FEV20、MV、PEF和Cydn均显著下降（P＜0.05）；③MG小鼠肌纤维间隙增加，CSA显著降低（P＜0.05），且MyHC（slow）的表达显著下降（P＜0.01），抓力下降（P＜0.05），腓肠肌等长收缩力和35Hz电刺激时肌肉收缩力同样降低（P＜0.05）；④MG小鼠腓肠肌Atrogin-1和MuRF-1的表达水平升高（P＜0.05）。 结论：长期递增式烟雾暴露导致小鼠COPD疾病特征明显，同时降低骨骼肌收缩功能、促进肌肉纤维类型转变和肌肉萎缩，并升高骨骼肌萎缩相关蛋白表达水平，表明25周递增式烟雾暴露可成功制备COPD小鼠骨骼肌功能障碍模型。

Abstract:

      Abstract Objective： To investigate whether the 25-week incremental smoke exposure can successfully establish a mouse model of skeletal muscle dysfunction in chronic obstructive pulmonary disease （COPD）. Method： Healthy male C57BL/6 mice were randomly divided into the model group （MG， n=6） and the control group （CG， n=6）. The MG mice were whole-body exposed to cigarette smoke 6 days/week for 25 weeks， with 4h intervals between sessions. The number of lighted cigarettes and the daily frequency of cigarette smoking were gradually increased throughout the experiment. The CG mice were routinely housed. During the modeling， the body weight of the mice was measured monthly， and the grip strength was measured bimonthly. Lung function tests and isolated gastrocnemius strength were performed at the end of the 25-week smoke exposure. Pathological changes in lung and gastrocnemius were observed by HE staining. Alveolar cross-sectional area （CSA）， alveolar mean linear intercept （MLI）， and myofiber CSA were calculated. Western Blot was performed to detect the expression of MyHC （slow）， MyHC （IID）， Atrogin-1， and MuRF-1 in the gastrocnemius. Result： Compared with CG mice：①MG mice showed significant general condition change and significant reduction in body weight（P<0.05）； ②MG mice showed typical changes of chronic bronchitis and emphysema in lung tissue， with significant increase in alveolar CSA and MLI （P<0.05）, and significant reduction in lung function including FVC， FEV20， MV， PEF and Cydn（P<0.05）； ③MG mice showed increased spacing between muscle fibers, significantly reduced CSA（P<0.05）, decreased expression of MyHC （slow）（P<0.01）, and reduced grip strength（P<0.05）, as were isometric contraction strength and force during 35 Hz electrical stimulation of the gastrocnemius muscle （P<0.05）； ④The expression levels of Atrogin-1 and MuRF-1 in the gastrocnemius were elevated in MG mice （P<0.05）. Conclusion： Long-term incremental smoke exposure led to obvious characteristics of COPD in mice， with decreased skeletal muscle contractile function， promoted muscle fiber type transformation and muscle atrophy， and elevated the expression level of muscle atrophy-related proteins. This suggests that 25 weeks of incremental smoke exposure could successfully establish a mouse model of COPD skeletal muscle dysfunction.