固本止咳中药调控γδT+/Th17细胞分泌的IL17A对 PM2.5所致肺损伤的保护机制研究

Respiratory diseases are induced and aggravated by particulate matter 2.5(PM2.5),there is short of effective prevention and treatment at present.Inflammatory is the key link of the lung injury caused by PM2.5,IL17A is the key factor of the inflammatory.Gubenzhike recipe is from national master of chinese medicine Professor ChaoEnXiang’s clinical experience in respiratory diseases,the efficiency is reinforcing deficiency and strengthening body resistance.In the previous study,we found that Gubenzhike recipe might reduce γδT+ cell in lung,and reduce the secretion of IL-17A,reduce inflammatory and fibrosis in lung,improve pulmonary function.So we propose that Gubenzhike recipe can decrease inflammatory cells ,inhibit inflammatory cytokines secretion by regulating IL17A secreted from γδT+/Th17cell,and then produce protective function of lung injury caused by PM2.5.In this research we will establish lung injury model by intratracheal medication,use three kinds of single gene knockout mice,combined with vitro cell experiments,use the methods of pathology、flow cytometry、molecular techniques to definite that in PM2.5 induced lung injury,IL-17A mediated inflammatory is the key factor.Clear the molecular mechanism of protective effect of Gubenzhike recipe,explain the defense mechanism of“vital qi defense-exterior”in PM2.5 induced lung injury, provide the theory basis for “preventive treatment of disease”that in prevention and treatment for PM2.5 related respiratory diseases.

PM2.5诱发和加重多种呼吸系统疾病，目前缺乏有效防治手段。炎症反应是PM2.5致肺损伤的重要环节，IL17A是炎症反应的关键因子。固本止咳中药源于国医大师晁恩祥教授治疗肺系病的经验，具有补虚扶正功效。我们前期研究提示固本止咳中药减少肺组织中γδT+细胞浸润，减少IL-17A分泌，减轻PM2.5诱导的肺部炎症及纤维化，改善肺功能。因此我们提出假设：固本止咳中药通过调控γδT+/Th17细胞分泌IL17A，减少炎症细胞浸润，抑制炎性因子分泌，对PM2.5致肺损伤产生保护作用。本课题拟采用PM2.5气管内给药致肺损伤模型，使用3种单基因敲除小鼠，结合体外细胞实验，运用病理、流式、分子生物学等技术，明确在PM2.5致肺损伤中，IL17A介导的炎症反应所起的关键作用，探讨固本止咳中药的保护性作用的分子机制，为中医药防治PM2.5相关肺系病的“未病先防，既病防变”的“治未病”思想提供新的科学依据。

吸入空气污染物中直径小于2.5μm的颗粒物（PM2.5）会导致肺损伤，固本止咳中药（GBZK）是治疗慢性阻塞性肺疾病（COPD）的有效中药方剂。然而，GBZK对PM2.5诱导的肺损伤的作用机制仍不明确。本研究通过PM2.5刺激建立小鼠肺损伤模型，同时进行GBZK灌胃治疗，4周后收集小鼠的肺组织进行病理染色以分析损伤程度并用商品化试剂盒检测肺组织中IL-17A水平、髓过氧化物酶（MPO）、丙二醛（MDA）和氧化应激相关因子（SOD、GSH-Px）的活性。此外，收集并检测支气管肺泡灌洗液（BALF）和血清中的中性粒细胞和相关炎症因子（IL-1β、TNF-α、IL-6）的数量，以评估GBZK对炎症的影响，并通过Masson染色检测小鼠肺纤维化程度，用免疫组织化学和western blot检测肺组织中HMGB1和NFκBp65的激活情况。研究发现PM2.5可引起小鼠肺损伤，GBZK灌胃给药可减轻肺损伤程度，并呈浓度依赖性。且GBZK治疗后，小鼠肺组织IL-17A水平、MPO活性、MDA含量和氧化应激水平降低，BALF和血液中炎性细胞因子的表达水平下降。GBZK治疗也可改善小鼠的肺纤维化。此外，我们还发现GBZK能阻止小鼠肺HMGB1/NF-κB通路的上调。所以GBZK可能通过抑制HMGB1/NFκB通路，从而抑制炎症和肺纤维化，对PM2.5诱导的小鼠肺损伤具有保护作用。项目执行期间已发表SCI论文1篇，CSCD期刊论文3篇。本项目的研究为肺损伤的预防和治疗提供了新的策略。