通过ROS-ATP/P2X3通路研究乌梅有机酸对咳嗽高敏感的保护作用及机制

Chronic cough is a most common symptom and among them including about 10 % refractory chronic cough (also characterized cough hypersensitivity syndrome, CHS) with increased cough sensitivity as its significant clinical and pathophysiological feature, and current antitussives have fairly poor efficacy and undesirable side-effects for CHS. The pathogenesis of CHS is still unclear and thought to be related to the hypersensitivity of cough innervated vagal nerve caused by airway exposure to noxious stimuli, such as environmental pollutants, airway inflammation and oxidative stress. Recently, our group found the antitussive effect of Fuctus mume (FM) and its formula for refractory chronic cough patients that unresponsive to conventional chemical drugs therapy. In addition, we found that the organic acids from FM significantly decreased the cough hypersensitivity and reduced the oxidative stress injury induced by diesel exhaust particles (DEPs) exposure in chronic cough guinea pig model. In this study, we intend to further separate, purify and identify the active components, to find the main active compounds in Fructus mume acids, and to explain the protect effects and mechanisms of the constituents through ROS-ATP/P2X3 pathway by established different cell models such as the up-regulation by ROS and activation by α,β-methylene ATP of cough hypersensitivity related ion channel receptor P2X3 in PC12 cells, the ROS production, the release of inflammatory cytokines and the changes of ATP content induced by DEPs in human bronchial epithelioid cells and macrophages, etc. This study aims to elucidate the main active constituents and mechanisms, including decrease the expression and activation of P2X3 receptor, regulation of the ATP production, antioxidant and anti-inflammation activities, of Fructus mume acids on cough hypersensitivity, and will also provide references for the development of new drugs for refractory chronic cough.

慢性咳嗽是临床上最常见的病症，难治性慢性咳嗽以咳嗽敏感性增高为显著的临床与病理生理特征，又称咳嗽高敏感综合征，发病与氧化应激等有害刺激导致的咳嗽神经敏感性增高有关，尚缺乏有效的治疗药物。本课题组近年来发现治久咳的乌梅及其复方对一些难治性慢性咳嗽患者有较好疗效，通过慢性咳嗽动物模型筛选出乌梅有机酸具有明显的抗氧化和降低咳嗽敏感性的作用。本项目拟进一步分析乌梅有机酸中主要成分，采用尾气暴露的慢性咳嗽豚鼠模型研究其药效，建立咳嗽受体P2X3高表达及α,β-meATP诱导其活化的PC12细胞模型，尾气颗粒物影响气道上皮及巨噬细胞中ROS、炎症因子、ATP等生成的细胞模型，探讨乌梅有机酸通过ROS-ATP/P2X3通路对咳嗽高敏感的保护作用及机制。本研究旨在阐明乌梅治久咳的活性成分及其降低外周神经P2X3受体表达及活化、调节ATP生成、抗氧化及抗炎等多靶点作用机制，为难治性慢性咳嗽新药研发提供参考。

本课题组临床中发现，乌梅复方及乌梅单味药能很好地缓解部分难治性慢性咳嗽患者的咳嗽，我国药典亦记载乌梅有治久咳之功效，其用于治久咳已得到了较好的临床验证。然而，乌梅治疗难治性慢性咳嗽的药效物质基础尚不明确。基于以上，我们通过香烟烟雾暴露及尾气暴露诱导的咳嗽高敏感动物模型筛选出乌梅主要活性部位为乌梅有机酸（FA），进一步采用硅胶柱层析、ODS中压柱层析对乌梅有机酸的主要成分进行分离、纯化，TLC、HPLC和LC-MS/Q-TOF等方法分析，确定FA中主要有柠檬酸、苹果酸、新绿原酸、绿原酸等。通过改善咳嗽高敏感和气道炎症等指标，筛选出FA中绿原酸和新绿原酸为主要活性成分，柠檬酸、苹果酸等具有协同作用。作用机制研究表明， FA能明显降低机动车尾气暴露导致的咳嗽高敏感，减少BALF中细胞总数及中性粒细胞、巨噬细胞数，减轻肺组织炎症及结构病变，改善氧化应激损伤和黏蛋白的高分泌，下调肺组织及气道P2X3的蛋白水平。在柴油颗粒物（DEPs）刺激的16-HBE细胞模型中，通过流式细胞术筛选出FA和主要成分对DEPs诱导ROS生成的保护作用，表明FA和新绿原酸、绿原酸能显著减少ROS的生成。通过H2O2诱导的PC-12细胞模型，发现FA能通过下调NF-κB (p65)和减少IκBα降解，降低H2O2诱导的P2X3蛋白表达水平，同时可减少H2O2诱导的ATP生成量。在α, β-meATP诱导的PC12细胞模型中，乌梅有机酸FA能抑制钙离子内流。综上，乌梅有机酸主要通过抗氧化、抗炎、减少气道黏蛋白、降低咳嗽相关受体如P2X3的高表达、减少ROS诱导的ATP生成等多靶点作用改善模型动物的咳嗽高敏感性，该作用与ROS-ATP/P2X3通路相关，主要活性成分为绿原酸、新绿原酸，柠檬酸、苹果酸等具有协同保护作用。本研究在阐明乌梅有机酸的活性成分及作用机制的同时，也为难治性慢性咳嗽发病机制研究和新药研发提供新思路。