从NLRP3炎症复合信号通路探讨“宣肺益肾、化痰解痉”法调控中性粒细胞性哮喘固有免疫通路分子机制研究

Neutrophilic asthma(NA) is the common phenotypic of asthma.The clinical epidemiological investigation and study showed that the course of NA were long and different curring,the syndrome study performance "shenqi kuixu,futan nanhua" as the characteristics. The method of ventilating lung and tonifying kidney and getting spasmolysis and reducing phlegm could control it effectively.Its airway inflammatory response with PMN inflammation and pathological mechanism of NA is different from the eosinophils asthma, hormone therapy can not acquire good effect. The study found that external factors activated the innate immune system, activating the NLRP3 inflammation(NLRP3/ASC/Caspase-1-IL-1β)composite high signaling pathways leading to PMN high expression in theairway may be the key mechanism of NA.We assume that the method of ventilating lung and tonifying kidney and getting spasmolysis and reducing phlegmthe can treat NA,the mechanism may be associated with composite regulating NLRP3 inflammatory signaling pathways ,reducing the high expression of PMN.. To proof the hypothesis,the issue carry out randomized controlled animal experiment , using the TCM of ventilating lung and tonifying kidney and getting spasmolysis and reducing phlegm ,IL-1 receptor antagonist is used as randomized controlled.From the change of lung tissue pathology ,cell counting, cell classification, NLRP3 inflammasome and genes involved and inflammatory factors associated, observating the data in different levels.. NA as the common phenotype of refractory asthma,its treatment is as the breakthrough point in this study. ,we try to combine the phenotype of asthma with TCM syndrome treatment, making standard for Chinese medicine treatment of NA,and provide experimental basis for clinical application.

中性粒细胞性哮喘（NA）是哮喘常见表型，临床流调显示其中医病机关键为“肾气亏虚、伏痰难化”，前期临床研究证实“宣肺益肾、化痰解痉”法能有效控制其病情。NA气道炎症反应以中性粒细胞浸润为主，病理机制不同于嗜酸性粒细胞性哮喘，激素治疗不理想。研究发现病理因素激活NLRP3炎症复合信号通路导致中性粒细胞气道高表达是NA的关键机制。我们提出“宣肺益肾、化痰解痉”法能够调控NLRP3炎症复合信号通路，减少中性粒细胞在气道的高表达起到治疗作用。. 为验证假说拟开展宣肺益肾、化痰解痉中药与IL-1β受体阻断剂随机对照的动物实验，围绕NLRP3炎症复合信号通路，从肺组织病理学改变，细胞计数及分类、相关基因及下游炎性因子等不同层面观察分析研究数据。. 本研究以难治性哮喘的常见表型NA为切入点，将中医治法与哮喘表型相结合，明确其疗效机制，为规范NA的中医治疗，深入临床研究提供实验学依据。

中性粒细胞（NA）性哮喘气道炎症以NA浸润为主，激素治疗不理想。病理因素激活NLRP3炎症复合信号通路导致NA气道高表达是NA的关键机制，其中医病机关键为“肾气亏虚、伏痰难化”，临床研究证实“宣肺益肾、化痰解痉”法能有效控制其病情。我们提出“宣肺益肾、化痰解痉”法能够调控NLRP3炎症复合信号通路，减少NA在气道的高表达起到治疗作用。开展宣肺益肾、化痰解痉中药与IL-1β受体阻断剂随机对照的动物实验：围绕NLRP3炎症复合信号通路，从肺组织病理学改变，细胞计数及分类、相关基因及下游炎性因子开展研究。本研究成功建立NA哮喘模型，研究显示：“宣肺益肾、化痰解痉”法能够有效降低气道细胞总数及NA表达，治疗第14天与治疗1天比较，宣肺组、西药组细胞总数自身比较差异有统计学意义（P＜0.05）；治疗14天较治疗1天，宣肺组、西药组、宣肺补肾组，NA自身比较差异有统计学意义（P＜0.05）；从病理组织观察可见宣肺补肾能够减少组织炎细胞浸润；Q-PCR肺组织检测，宣肺组、西药组、宣肺补肾组，治疗14天较治疗1天，ASC自身比较差异有统计学意义（P＜0.05）；caspase-1、NLRP3、caspase-1治疗后较模型组均有统计学意义（P＜0.05）；宣肺组自身前后差异有统计学意义（P＜0.05）；血清IL-1β、ELISA、IL-8治疗后西药组、宣肺组及宣肺补肾组较模型组差异均有统计学意义（P＜0.05）；宣肺组自身前后差异有统计学意义（P＜0.05）；IL-33治疗后西药组、宣肺组及宣肺补肾组与模型组差异均有统计学意义（P＜0.05）；宣肺组及宣肺补肾组自身比较差异有统计学意义（P＜0.05）。Western blot检测小鼠肺组织中caspase-1，治疗第7、14天西药组、宣肺组及宣肺补肾组较模型组比较差异有统计学意义（P＜0.05）。“宣肺益肾、化痰解痉”法治疗NA哮喘可能通过阻断NLRP3炎症复合通路来有效减少肺组织及血清中IL-1β、IL-33、IL-8 及TNFα等炎症因子的表达，减少肺组织中NLRP3的表达，减轻气道炎症，减少肺组织中NA等炎性细胞的浸润。IL-1受体拮抗剂可有效阻断NLRP3- IL-1炎症通路，可改善理化指标，用于临床治疗NA型哮喘有待于进一步研究。