ATP-P2X/P2Y受体-组胺对咳嗽高敏感性的作用及机制

Cough reflex hypersensitivity is a common characteristic of chronic cough, airway inflammation and remodeling are its common pathological features. Adenosine triphosphate (ATP) may be associated with airway inflammation of chronic cough, which could enhance cough reflex sensitivity through the consequent activation of TRPV1 which is on terminal of C fiber. Through preliminary work, we found that cough induced by citric acid could cause cough hypersensitivity, and doses of ATP in bronchoalveolar lavage (BAL) fluid from guinea pig models with chronic cough were significantly higher than that in BAL fluid of control group. So we hypothesize that persistent cough can induce airway epithelial damage， which may cause the release of intracellular ATP combining with the corresponding receptors. Then the subsequent airway inflammation enhance cough reflex sensitivity through the following activation of TRPV1. We plan to verify the hypothesis by comparing the changes in cough reactivity and level of ATP in BAL fluid from guinea pig model with chronic cough after long-term intervention with ATP nucleotidase inhibitor, antagonists of TRPV1 and antagonists of P2X/P2Y receptors alone or in combination; by detecting the expression of mRNA and protein of P2X/P2Y and TRPV1 in the trachea-bronchial tissues, and by observing the quantity and distribution of TRPV1 in trachea-bronchial mucosa under laser scanning con-focal microscope combined with the fluorescent immuno-staining. The purpose of the study is to clarify the role of ATP in cough reflex hypersensitivity, to explore its underlying mechanism, and finally to lay a theoretical basis for a possible ATP intervention as a target in the treatment of chronic cough.

咳嗽高敏感性是慢性咳嗽的共同特征，气道炎症和重构是其普遍病理改变。ATP可能与慢性咳嗽的气道炎症相关，而气道炎症又可能通过活化迷走神经末梢的TRPV1导致咳嗽高敏感性。预试验表明：反复吸入柠檬酸可导致豚鼠咳嗽敏感性增高，此时支气管肺泡灌洗液的ATP浓度高于正常对照组。故我们推测，持续性咳嗽产生周期性作用力造成气道上皮损伤，释放大量ATP与相应受体结合，释放的炎症介质活化TRPV1，增强咳嗽敏感性。本研究拟通过柠檬酸建立慢性咳嗽豚鼠模型，比较对照组、慢性咳嗽组和ATP胞外核苷酸酶抑制剂、P2受体拮抗剂及TRPV1拮抗剂预处理组的气道内ATP水平、咳嗽反应性变化，检测气管和支气管组织中P2X、P2Y及TRPV1的mRNA和蛋白表达，观察黏膜中表达TRPV1神经纤维的数量和分布，并分析与咳嗽反应性的关系。证实ATP参与慢性咳嗽高敏感性的形成，并探讨其机制，为将来以ATP为靶点治疗咳嗽奠定基础。

咳嗽高敏感性是慢性咳嗽的共同特征，气道炎症和重构是其普遍病理改变。ATP可能与慢性咳嗽的气道炎症相关，而气道炎症又可能通过活化迷走神经末梢的TRPV1导致咳嗽高敏感性。故我们推测，持续性咳嗽产生周期性作用力造成气道上皮损伤，释放大量ATP与相应受体结合，释放的炎症介质活化TRPV1，增强咳嗽敏感性。本研究拟通过柠檬酸建立慢性咳嗽豚鼠模型，比较对照组、慢性咳嗽组和ATP胞外核苷酸酶抑制剂、P2受体拮抗剂及TRPV1拮抗剂预处理组的气道内ATP水平、咳嗽反应性变化，检测气管和支气管组织中P2X、P2Y及TRPV1的mRNA和蛋白表达，观察黏膜中表达TRPV1神经纤维的数量和分布，并分析与咳嗽反应性的关系。证实ATP参与慢性咳嗽高敏感性的形成，并探讨其机制，为将来以ATP为靶点治疗咳嗽奠定基础。通过本研究发现长期反复吸入柠檬酸可建立稳定的慢性咳嗽豚鼠模型，为慢性咳嗽的发生机制、药效评价和药理学基础研究提供合适的方法和手段，模型豚鼠的咳嗽高反应性可能与气道内TRPV1激活和神经源性炎症有关，长期致咳刺激诱导豚鼠出现咳嗽适应现象，可能与豚鼠气道神经肽受体敏感性下降有关。P2X3 受体拮抗剂 AF-35333吸入能有效降低柠檬酸吸入慢性咳嗽豚鼠模型的咳嗽高反应性，且呈阈值效应，因此以P2X3 受体拮抗剂为靶点抑制慢性咳嗽高反应性是可行的。P2X3 受体参与慢性咳嗽高反应性的具体机制可能与激活共表达在气道感觉神经纤维上的TRPV1无关，可能通过其他TRP家族如TRPV4的介导而参与慢性咳嗽高敏感性的形成。我们已成功申请国家自然科学基金面上项目，进一步采用在体和离体肺组织记录迷走神经单纤维放电活动的方法，观察豚鼠和TRPV4基因敲除小鼠对上述试剂的放电反应变化，证实TRPV4-ATP-P2X受体信号通路参与咳嗽高敏感性形成，并探讨其机制，为以此治疗咳嗽奠定基础。