清金化痰汤通过免疫应答调节慢性阻塞性肺疾病炎症反应干预气道黏液高分泌的研究

Mucus hypersecretion in airways is relevant to the development and prognosis of chronic obstructive pulmonary disease (COPD). Obstruction of phlegm-heat and qi movement in lung is the syndrome feature of COPD. Our team has demonstrated that Qingjinhuatan Decoction reduced the symptoms of patients in acute exacerbations with COPD by clearing heat, resolving phlegm and regulating qi, reducing inflammation and mucus production. We have demonstrated that Qingjinhuatan Decoction reduced goblet cells metaplasia and mucus secretion through neutrophil elastase in animal models. The unbalance between the recognition and activation of immune response and inflammation by Toll-like receptors (TLRs) and negative regulation by regulatory T cell (Treg cells) is critical in the development of chronic inflammation and mucus hypersecretion in COPD. TLR4- MyD88/ TRIF - NF-κB transduction pathway actives the inflammation in COPD by recognition of innate immunity, while CD4+CD25+Foxp3+ Treg cells, the native regulatory T cells, dampen the intensity of inflammatory cascades. The present study will be conducted to clarify the influence and mechanism of Qingjinhuatan Decoction in reducing mucus production through immune response and inflammation in vivo and in vitro.

气道黏液高分泌是影响慢性阻塞性肺疾病（慢阻肺）病情和预后的独立危险因素，痰热壅肺，气机不畅是其证候特征，前期工作表明，依清热化痰，宣降肺气组方的清金化痰汤具有抗炎作用，可减轻慢阻肺急性加重期患者的症状，改善黏液高分泌；动物实验证明该方可通过干预中性粒细胞弹性蛋白酶，减少杯状细胞化生，减轻黏液高分泌。 Toll样受体识别和激活的固有免疫应答和炎症反应与调节T淋巴细胞（Treg细胞）的负性调控之间的失衡在慢阻肺慢性炎症及黏液高分泌的形成中具有重要作用。TLR4- MyD88/ TRIF - NF-κB是通过固有免疫识别，激活慢阻肺炎症反应的信号传导通路；CD4+CD25+Foxp3+ Treg细胞是天然产生的调节T细胞，可阻断慢阻肺炎症的瀑布效应。本研究通过体内、外实验，从调节免疫应答及炎症反应深入研究清金化痰汤干预慢阻肺气道黏液高分泌的作用机理。

目的：探讨中药复方干预慢阻肺急性加重期大鼠气道粘液高分泌的作用机制。本项目分体内实验与体外实验两部分。体内实验：方法：60只Wistar大鼠随机均分为正常组、模型组、克拉霉素组、清金化痰组、解毒清肺组。采用气道内滴注LPS联合烟熏的方法建立慢阻肺急性加重期大鼠模型，连续30天分别给予蒸馏水、克拉霉素、清金化痰汤、解毒清肺合剂灌胃。实验第31天测肺功能并取材。进行HE染色、ABPAS染色、ELISA检测BALF和血清炎症因子含量，免疫组化、Western Blot、RT-PCR检测肺组织相关蛋白质的表达，流式细胞术检测外周血中Th17、Treg细胞的数量及百分比。结果：与正常组相比，模型组肺功能 FEV0.3/FVC、MVV显著降低，杯状细胞数、MUC5AC、NE、ROR-γt表达显著增加，NF-κB、EGFR-PI3K-AKT磷酸化增加，Foxp3表达显著抑制，细胞因子IL-1β，TNF-α，IL-6，IL-17A表达增加。清金化痰组与模型组相比，肺功能FEV0.3/FVC、MVV明显上升，杯状细胞数量减少，MUC5AC、NE、ROR-γt表达降低，NF-κB与EGFR-PI3K-AKT磷酸化抑制，Foxp3表达上调，IL-1β，TNF-α，IL-6，IL-17A表达降低，具有统计学差异。体外实验：方法：用LPS刺激支气管上皮细胞(NHBE)，同时给清金化痰汤、解毒清肺合剂、克拉霉素。检测细胞上清液中相关指标。用Transwell小室检测中性粒细胞（PMN）趋化功能。 结果：清金化痰汤、解毒清肺合剂、克拉霉素组IL-8、TNFα减少，趋化PMN能力降低；清金化痰汤、解毒清肺合剂TLR4、NF-κB基因、蛋白表达下调，克拉霉素并不显著。结论：清金化痰汤、解毒清肺合剂可能通过抑制TLR4-MyD88/TRIF-NF-κB信号通路的激活，抑制细胞因子分泌缓解炎症反应。克拉霉素可通过抑制NF-κB基因和蛋白的表达抑制炎症细胞因子的合成释放，但可能并非由TLR4-MyD88/TRIF-NF-κB信号通路引起。