CDK5介导的含TRPV1神经纤维可塑性对咳嗽高敏感性的作用及机制

Cough reflex hypersensitivity is a common characteristic of chronic cough. Persistent cough, by itself, is associated with cough reflex hypersensitivity since it may injury the airway wall, and cause airway inflammation and remolding through the mechanical stress. Through preliminary work, we found that cough induced by citric acid could cause cough hypersensitivity, which would be dimished by CDK5 inhibitor. So we hypothesize that airway inflammation induced by chronic cough can enhance cough reflex sensitivity through aggravating the expression and activity of CDK5, and the consequent phosphorylation of TRPV1 and the plasticity of C vagal neuronal fiber in airway.To verify the hypothesis, We plan to compare the changes in cough reactivity and level of substance P in bronchoalveolar lavage fluid from guinea pig model with chronic cough after long-term intervention with antagonists of CDK5, TRPV1 and substance P alone or in combination,to detect the expression of mRNA and protein of CDK5 and TRPV1 in the trachea-bronchial tissues with real time PCR and Western bolt, to observe the quantity and distribution of TRPV1 in trachea-bronchial mucosa under laser scanning con-focal microscope combined with fluorescent immuno-staining. The purpose of the study is to clarify the role of CDK5 in cough reflex hypersensitivity and its underlying mechanism, which may lay a theoretical basis for possible CDK5 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。持续咳嗽对气道壁的损伤可能是慢性咳嗽气道炎症和重构的关键因素，并与咳嗽高敏感性相关。我们前期发现：柠檬酸引起的咳嗽可导致咳嗽高敏感性，这种作用可被CDK5抑制剂减弱。故我们推测：慢性咳嗽导致气道炎症可使CDK5表达、活性增高，通过磷酸化TRPV1使气道C迷走神经纤维可塑性发生变化，导致咳嗽高敏感性。拟单独或组合使用CDK5、TRPV1和P物质抑制剂预处理慢性咳嗽模型豚鼠，比较各组咳嗽反应性和支气管肺泡灌洗液中P物质浓度，应用Real time PCR、Western Blot检测上述预处理后模型豚鼠气管、支气管组织CDK5和TRPV1的mRNA和蛋白表达，用荧光免疫染色结合激光共聚焦显微镜观察气管支气管黏膜中表达TRPV1神经纤维数量和分布。从而初步阐明CDK5在慢性咳嗽高敏感性形成中的作用，并探讨其机制，为将来以CDK5为靶点治疗慢性咳嗽奠定理论基础。

咳嗽高敏感性是慢性咳嗽的共同特征。故我们推测：慢性咳嗽导致气道炎症可使CDK5 表达、活性增高，通过磷酸化瞬时受体电位香草酸亚型1（TRPV1）使气道C 迷走神经纤维可塑性发生变化，导致咳嗽高敏感性。拟单独或组合使用CDK5、TRPV1和P物质抑制剂预处理慢性咳嗽模型豚鼠，比较各组咳嗽反应性和支气管肺泡灌洗液中P 物质浓度，检测上述预处理后模型豚鼠气管、支气管组织CDK5和TRPV1的mRNA 和蛋白表达，观察气管支气管黏膜中表达TRPV1 神经纤维数量和分布。.第一部分研究拟探讨吸入柠檬酸建立存在豚鼠慢性咳嗽模型的方法，分析咳嗽高敏感性的产生及适应机制，为将来慢性咳嗽的治疗及药物药理研究提供方法学保障。第二部分研究拟观察不同浓度CDK5抑制剂预处理对慢性咳嗽模型豚鼠咳嗽反应性的影响，证实CDK5在慢性咳嗽高敏感性中的作用。第三部分研究拟阐明CDK5 - TRPV1 – SP（P物质）通路与咳嗽高敏感性的关系，探讨CDK5介导的气道TRPV1神经纤维可塑性是否为咳嗽高敏感性形成的重要机制。.研究结果显示：1）反复吸入TRPV1激动剂柠檬酸可导致豚鼠的咳嗽次数增高，进一步吸入豚鼠咳嗽次数反而下降，咳嗽高反应性存在一定的适应机制。最佳方法为正常清醒豚鼠每天2次，每次3min雾化吸入0.4M的柠檬酸，共15 天。2）CDK5参与慢性咳嗽高敏感性的发生，CDK5 抑制剂的吸入剂量对咳嗽的抑制作用存在阈值效应，机制可能与激活TRPV1有关。3）吸入高浓度CDK5 抑制剂Roscovitine 可降低咳嗽高反应性，机制可能与激活气道C传入迷走神经纤维上受体TRPV1有关。CDK5 抑制剂和TRPV1拮抗剂在气道C传入迷走神经纤维上呈上下游联合定位分布，CDK5 抑制剂与SP拮抗剂无串联或并联关系。.本研究通过实验首次证实CDK5 在慢性咳嗽高敏感性形成中的作用，机制可能与激活气道C传入迷走神经纤维上受体TRPV1有关，为将来以CDK5为靶点治疗慢性咳嗽奠定了理论基础。