慢性咳嗽高敏豚鼠模型的建立及其在咳嗽治疗药物药效评价中的应用

The slow progress of new drugs R&D for chronic cough is related closely with the lack of optional animal models for chronic cough. Because of the absence of airway inflammation, cough reflex hypersensitivity and spontaneous cough, the widely used physiologic cough models are unable to reflect the real condition of chronic cough in clinical practice. By motor vehicle exhaust exposure in tunnel, we have established a guinea pig model of chronic cough with cough hypersensitivity. However, it is difficult to standardize the procedure for this animal model in tunnel condition. This project aims to develop a standard procedure for establishing guinea pig model through indoor motor vehicle exhaust exposure for the first time. The optimal conditions for animal model will be determined through adjusting the exposure time, components, and concentration of exhaust to develop the nonspecific chronic airway inflammation, cough reflex hypersensitivity and spontaneous cough, and increased expression and function of neuropeptides, TRPV1, TRPA1. At the same time, we will establish a new method to assess cough sensitivity in guinea pig model by measuring both spontaneous coughs and stimulated coughs, and determine whether the mixture of TRPV1 agonist capsaicin, TRPA1 agonist AITC, and nonspecific stimulator citric acid, is superior to its single component in evoking cough. Subsequently, the effectiveness of this guinea pigs model of chronic cough will be identified via evaluating the efficacy of available anti-tussives and anti-inflammation drugs. In conclusion, this model will strongly promote the research and development of therapeutic drugs for chronic cough.

慢性咳嗽治疗药物的研究进展缓慢，缺乏理想的慢性咳嗽动物模型是重要原因之一。目前国内外通用的生理性咳嗽模型，因缺乏气道炎症、咳嗽高敏感性与自发性咳嗽，与临床慢性咳嗽相差甚远。本课题组已建立了隧道机动车尾气暴露诱发的慢性咳嗽高敏豚鼠模型，但此模型不易标准化。本研究拟采用机动车尾气室内暴露的方法，首次建立标准化的慢性咳嗽高敏豚鼠模型。通过调节尾气成分、浓度及暴露时间等确定最佳造模条件，使此模型具有与慢性咳嗽患者临床特征相一致的咳嗽高敏感性、自发性咳嗽与非特异性慢性气道炎症，并伴有咳嗽相关的神经肽（SP等）、TRPV1、TRPA1表达和功能增强。同时建立一种新的豚鼠自发性咳嗽评价方法，并探讨TRPV1、TRPA1及非特异性激动剂的组合物能否成为更好的应激性咳嗽激发物。继而，采用此模型对单纯镇咳药物及抗炎药物进行药效学再评价，验证此模型的有效性。本动物模型的建立将有力促进慢性咳嗽治疗新药的研发。

慢性咳嗽治疗药物的研究进展缓慢，一个重要原因就是缺乏理想的慢性咳嗽动物模型。目前国内外通用的生理性咳嗽模型，因缺乏气道炎症、咳嗽高敏感性与咳嗽，与临床慢性咳嗽相差甚远。.本项目首先建立了柴油机尾气室内暴露实验室，确定PM2.5暴露浓度为200μg/m3，为合适造模条件，并成功构建具有气道炎症和咳嗽高敏感性的慢性咳嗽高敏豚鼠模型。模型具有以下特征：辣椒素和异硫氰酸烯丙酯咳嗽敏感性显著升高，咳嗽潜伏期显著缩短、咳嗽频次显著增多；具有非特异性的神经源性气道炎症，表现为肺组织的炎症细胞（中性粒细胞、嗜酸性粒细胞、淋巴细胞）水平增高，肺组织的P物质（SP）、瞬时受体电位阳离子通道蛋白（TRP）A1 水平升高以及结状神经节和颈静脉神经节的TRPA1水平升高；脑内存在有害颗粒物的积蓄和炎症损伤，表现为脑内小胶质细胞（标记物Iba-1）和星形胶质细胞（标记物GFAP）激活明显增加，脑内胶质源性神经营养因子（标记物GDNF）和神经生长因子（标记物NGF）释放明显增加且血管细胞的紧密连接（标记物蛋白Occludin、Z0-1）表达明显减少。.研究发现柴油机尾气诱发豚鼠咳嗽敏感性增高的机制为柴油尾气吸入激活迷走神经元/气道的TRPA1，引起SP的合成、释放增多产生气道神经源性炎症，诱发气道非特异性炎症，最终导致咳嗽敏感性增高。神经型一氧化氮合酶(NOS1)对于减低模型在辣椒素激发下的咳嗽次数有较好的效果。柴油机尾气暴露可引起脑内的炎症损伤，该损伤可能是有害颗粒物直接产生，也可能是通过炎症间接产生，而脑内的炎症损伤是否会对外周气道炎症及咳嗽产生进一步的影响，即“肺脑轴”对呼吸系统和中枢神经系统的调控值得进一步的探讨。.项目成功利用动物模型对中药Fructus Mume治疗慢性咳嗽的活性成分进行筛选和验证，并进一步研究主要活性成分的抗炎和降低咳嗽敏感性等作用机制，目前已获得初步结果，验证了模型的有效性。下一步将利用该动物模型对五味子、细辛等止咳中药进行研究。相信本动物模型将更广泛的获得应用，并助力于慢性咳嗽治疗新药的研发。