从气道炎症反应和TLR4/MyD88/NF-κB通路探讨血府逐瘀汤干预COPD的效应机制

Chronic obstructive pulmonary disease (COPD), associated with increased mortality and morbidity, has been a serious threat to health worldwide. Airway inflammation is recognized as the central mechanism in the pathogenesis of COPD. Thus inhibiting airway inflammation has been proposed as a key strategy for COPD treatment. The recent studies have shown that TLR4/MyD88/NF-κB signaling pathway is involved in airway inflammation and contributes to the development of COPD. Traditional Chinese formula Xuefu Zhuyu Decoction (XZD), is proved to be effective in improving pulmonary infection in COPD patients, and could significantly inhibit inflammatory reaction. However, it is not clear that whether XZD exhibit protective effect in COPD through inhibiting airway inflammation by regulating TLR4/MyD88/NF-κB pathway. Aiming to study the mechanism of XZD, COPD animal model will be firstly used to investigate the effect of XZD on the secretion of inflammatory mediators and mucus secreted proteins; Secondly, ELISA, RT-PCR, Western blot, are used to study the effect of XZD on expression of TLR4 pathway-related key proteins and genes; Lastly, the relationship between the protection effect of XZD and regulation of TLR4/MyD88/NF-κB pathway is investigated using pathway inhibitor, RNA interference technology and knockout mice. The project would propose a novel therapeutic strategy for COPD, and provide important scientific basis for application and drug development of XZD.

慢性阻塞性肺病（COPD）是一种高发病率及致死率的疾病。气道炎症及其引起的粘液高分泌是COPD发病的重要机制。因此抑制气道炎症是改善COPD的关键环节。研究表明TLR4/MyD88/NF-κB通路与气道炎症密切相关。血府逐瘀汤能改善COPD患者肺部感染，抑制炎症反应发生。但其是否通过调控TLR4/MyD88/NF-κB通路，抑制气道炎症反应而改善COPD尚不明确。本研究采用COPD动物模型，通过ELISA、免疫印迹、PCR、免疫组化等技术，观察血府逐淤汤对气道炎症及气道黏液高分泌的影响，以及对TLR4/MyD88/NF-κB通路相关蛋白表达的调控；并采用通路抑制剂、siRNA干扰等技术，从细胞和整体动物水平研究TLR4信号通路在血府逐瘀汤抑制COPD气道炎症效应中的作用，深入阐释血府逐瘀汤的分子作用机理，为临床COPD治疗提供一个新的思路，亦为后续血府逐瘀汤进一步研究和开发提供科学依据。

本研究通过鼻腔滴入脂多糖（LPS）加熏香烟的方法建立 COPD 小鼠动物模型，应用药理学和细胞分子生物学技术，分析不同浓度血府逐瘀汤对细胞因子和粘液蛋白表达的影响，结果显示血府逐瘀汤对 COPD 小鼠气道炎症及气道黏液高分泌的具有抑制作用并存在剂量效应关系。通过研究高剂量血府逐瘀汤对 COPD 小鼠改善炎症反应和 TLR4/ MyD88/NF-κB 信号通路调节之间的关系以及血府逐瘀汤对 COPD 患者外周血单核细胞 TLR4/ MyD88/ NF-κB 信号通路的调控实验，结果发现血府逐瘀汤对 COPD 小鼠及患者的炎症及气道黏液分泌抑制效应与 TLR4/MyD88/NF-κB 信号转导通路的调控作用相关。通过对体外培养人体气道上皮细胞 NCI-H292 进行香烟提取物(CSE)刺激并 给予 NF-κB 抑制剂、激动剂与高剂量血府逐瘀汤水煎液进行处理，结果显示 NCI-H292 给予高剂量血府逐瘀汤处理后炎症因子和粘液分泌蛋白的表达明显下降；细胞免疫荧光检测显示 NCI-H292 细胞表面 TLR4 表达下降以及 Western blot 检测显示 TLR4、MyD88、IκB 的磷酸化水平及蛋白表达下降与高剂量血府逐瘀汤相关。进而从细胞水平说明血府逐瘀汤对 COPD 保护效应与 NF-κB 间存在相关性。 通过对 TLR4 敲除（TLR4-/-）小鼠和野生型小鼠进行造模，采用 ELISA、 RT-PCR、Western blot 法检测 BALF 、血清及肺、气道组织中炎症细胞因子和MUC5AC、 AQP5 、MyD88、NF-κB 表达。结果显示野生型小鼠给予中高剂量血府逐瘀汤具有降低炎症因子水平、抑制粘液分泌相关蛋白 MUC5AC、 AQP5 表达；在 TLR4 敲除（TLR4-/-）小鼠 COPD 模型中，与野生型小鼠模型相比， BALF 和血清中炎症细胞因子表达下降；TLR4敲除鼠COPD模型中血府逐瘀汤对粘液分泌蛋白、TLR4、MyD88、NF-κB 表达的抑制作用明显减弱。HE 染色显示 TLR4-/- COPD 小鼠相较于野生型小鼠气道塌陷更少.