祛风宣肺方抑制PAR2下调TRPV1活性治疗感染后咳嗽的新机制

Post-infectious cough (PIC) is one of the most common causes of cough, with high incidence,and is difficult to treatment for western medicine.Qufeng Xuanfei decoction which is based on dispelling wind and diffusing the lung has significant curative effects in the treatment of PIC, but the mechanism is not yet clear. The present study shows that TRPV1 plays an important role in the pathogenesis of PIC. Preliminary experiments show Qufeng Xuanfei decoction can significantly reduce PIC mice cough symptoms and decrease TRPV1mRNA and TRPV1 protein expression in lung tissues of mice. The activity of TRPV1 is regulated by G protein coupled receptors, PAR2 is a member of G protein coupled receptor families, when activated it can upregulate TRPV1 activity by Ca2+ together with DAG viaing PKC . Qufeng Xuanfei decoction contains components which inhibits PAR2 directly or indirectly, we hypothesized that Qufeng Xuanfei decoction plays a role in downregulation of TRPV1 activity through inhibition PAR2 . In this study, we establish a mouse model of PIC and give intervention of Chinese drugs, and then detect the expression of PAR2, TRPV1 protein and PAR2, TRPV1mRNA, besides we also measure these indexes in TRPV1 or PAR2 knocked out mice and in cells with gene's interference of TRPV1 or PAR2 after intervented by Chinese drugs respectively and eventually ensure Qufeng Xuanfei decoction in the treatment of post-infectious cough by downregulation of TRPV1 activity through inhibition of PAR2.

感染后咳嗽（PIC）是咳嗽常见病因，发病率高，西医治疗困难。依祛风宣肺法创立祛风宣肺方在治疗PIC取得显著疗效，但作用机制尚未明晰。目前研究显示TRPV1在PIC发病机制中发挥重要作用。预实验显示祛风宣肺方显著减少细菌PIC小鼠模型咳嗽症状，显著降低小鼠肺组织TRPV1mRNA和TRPV1蛋白表达。TRPV1活性受到G蛋白偶联受体的调节,PAR2是其成员之一，PAR2激活后通过Ca2+与DAG一起经PKC上调TRPV1活性。祛风宣肺方含直、间接抑制PAR2成分，推测祛风宣肺法通过抑制PAR2下调TRPV1活性发挥作用。本研究通过体内外PIC模型并予中药干预，检测PAR2、TRPV1蛋白及mRNA表达，并对PAR2、TRPV1基因干扰细胞与基因敲除小鼠PIC模型予中药干预，测定相应TRPV1、PAR2蛋白及mRNA表达的变化，明确祛风宣肺方通过抑制PAR2下调TRPV1活性治疗PIC的新机制

感染后咳嗽是一种呼吸科较为常见的呼吸道疾病，其临床特征往往表现气道的高反应性和咳嗽敏感性增加。现代研究认为感染后咳嗽的气道高反应性和咳嗽敏感性增加与感染后气道持续的神经源性炎症密切相关。而TRPV1和神经源性炎症在气道高反应性和咳嗽敏感性中起着核心的作用。呼吸道的感染造成上皮细胞坏死，各种炎性因子如PGE2、白三烯等释放，白细胞向炎症部位趋化，造成了局部炎性环境。气道炎症环境使C纤维上TRPV1表达和敏感性增加，这时各种理化刺激可以造成TRPV1通道较正常过激的开放。同时有研究显示NGF/trka可以调控TRPV1的表达。TRPV1的开放一方面引起阳离子内流，神经元去极化传递信息至咳嗽中枢，引起中枢反射，通过胆碱能神经通路形成支气管收缩、黏液的大量分泌，并且伴随有气管异物感和咳嗽；另一方面TRPV1激活引起大量Ca2+内流导致神经肽如SP的释放，神经肽的释放又会加重气道炎症。现代医学尚缺乏治疗该病公认有效的药物，药物仍在动物试验和Ⅰ期临床试验中。根据我们前期研究发现在前期的临床对照试验和动物实验中已经发现对感染后咳嗽具有很好的疗效，但其中的相关机制研究甚少，本研究采用中剂量内毒素诱导模拟革兰阴性菌感染的方法创建动物模型，观察感染后小鼠各项生命指标稳定后，给予中药汤剂灌胃，观察LPS感染后小鼠咳嗽反射敏感性、神经源性炎症以及TRPV1的变化，以及中药的对这些病理指标的作用，结果显示中药可以明显降低气道高反应性，我们观察到了TRPV1的表达和神经源性炎症也明显减低，其中TRPV1活性和表达的调节因子NGF及其受体trka在表达上呈现正相关性，显示了祛风宣肺汤可能是通过减少NGF和trKa的的表达，来降低TRPV1的表达和神经源性炎症，并以此来降低气道高反应性。本研究在动物实验上证实了祛风宣肺汤对感染后咳嗽良好的治疗效果，并初步探讨了相关作用机理，为的临床应用提供科学合理的实验，并提示中药复方具潜在的临床应用。