补肺健脾方通过INS/AMPK/FOXO通路调控线粒体自噬改善COPD大鼠骨骼肌功能障碍的机制

Skeletal muscle dysfunction, the most common extrapulmonary complication of chronic obstructive pulmonary disease (COPD), is the key factor leading to limitation of activity, loss of labor force, disability and poor prognosis, which remains under-treated. Improving the function of skeletal muscle is significant to improving the outcomes and reducing the incidence of disability in COPD patients. In the previous study supported by National Natural Science Foundation of China, Bufei Jianpi granules had been proved effective on improving the mitochondria volume density and reducing mitochondrial swelling, which indicated that the regulation of mitochondrial function might be involved in the mechanism of improving peripheral skeletal muscles function in COPD rats, however, the exact mechanism remains unclear. Accordingly, we proposed the hypothesis: Bufei Jianpi granules improve skeletal muscle mitochondrial function and autophagy and improve skeletal muscle dysfunction via regulating insulin (INS)/adenosine monophosphate activated protein kinase (AMPK)/forkhead box O (FOXO) signaling. In this study, we aim to observe the mechanisms of Bufei Jianpi granules on skeletal muscle dysfunction/atrophy via improving mitochondrial morphology, functions and autophagy, and INS/AMPK/FOXO signaling, and the relevant genes and proteins expressions in quadriceps muscles in COPD rats and cigarette smoke extract (CSE) induced L6 cell line, by using the technologies of laser scanning confocal microscope, flow cytometry, immunohistochemistry, quantitative polymerase chain reaction and western blotting, and to explore new therapeutic targets.

骨骼肌功能障碍为COPD主要肺外并发症，是导致患者活动受限、劳动力丧失、致残及预后不良的关键因素。改善骨骼肌功能对改善COPD患者预后和减少致残有重要意义。前期研究表明，补肺健脾方可提高骨骼肌线粒体体密度和减轻肿胀，但机制尚不清楚。围绕线粒体功能及相关信号通路，本研究提出“补肺健脾方可通过调控INS/AMPK/FOXO信号通路改善COPD骨骼肌线粒体功能和自噬，从而改善骨骼肌功能障碍和萎缩”的假说，拟以COPD稳定期大鼠股四头肌和CSE刺激的L6细胞为研究对象，采用激光共聚焦显微镜、流式细胞、免疫组化、qPCR和Western blotting等技术，观察补肺健脾方对线粒体形态和功能、自噬、INS/AMPK/FOXO信号通路及相关基因和蛋白表达的影响，揭示补肺健脾方治疗COPD骨骼肌功能障碍/萎缩的机制，探索新的治疗靶点。

慢性阻塞性肺疾病（COPD）为重大疑难病，骨骼肌功能障碍作为COPD最主要的肺外并发症之一，是患者活动受限的主要原因，也是加快其病情恶化和死亡率的关键因素。本研究提出“补肺健脾方可通过调控INS/AMPK/FOXO信号通路改善COPD骨骼肌线粒体功能和自噬，从而改善骨骼肌功能障碍和萎缩”的假说。从体内、体外实验两部分展开研究，分别制备COPD稳定期大鼠模型和L6细胞COPD骨骼肌炎症模型。采用病理学方法观察线粒体形态和功能以及线粒体自噬；采用ELISA和荧光探针法检测CS、SDH和ROS含量；检测股四头肌组织线粒体功能；采用实时荧光定量PCR（qPCR）和Western blotting（WB）等技术，INS/AMPK/FOXO信号通路相关基因和蛋白表达。. 结果发现：① 补肺健脾方能够显著增加COPD大鼠体重，改善通气受限，减轻肺组织和股四头肌的病理损伤，提高CS、ROS水平和SDH活性，效果优于氨茶碱；② 补肺健脾方可通过上调AMPK-α/PGC-1α/TFAM线粒体合成通路，下调FOXO1/Atrogin1/MuRF1线粒体分解通路和LC3B/ULK1/PINK1/Parkin自噬相关通路，改善线粒体功能和减少线粒体自噬，从而明显改善COPD大鼠骨骼肌功能障碍。③补肺健脾方可以促进10% CSE诱导的L6细胞增殖，提高CS、ROS水平和SDH活性，提高线粒体功能，提高线粒体合成因子的表达，减少线粒体自噬因子的表达，且疗效要优于氨茶碱。④ 补肺健脾方可通过调控ROS/JNK/Bax信号通路，改善CSE诱导的L6细胞线粒体损伤，提高线粒体功能，减轻细胞氧化应激，抑制细胞凋亡，从而改善COPD骨骼肌功能障碍。