基于“肺肾同源”论研究阿那其根醇提取物治疗咳嗽变异性哮喘作用机制

Cough variant asthma(CVA)is a special type of bronchial asthma. The mechanisms of CVA are unclear yet and the conventional therapy is limited. In our previous study Anacyclus Pyrethrum capsule was found to play an important role in the treatment of 100 CVA patients in our hospital. Ethanol extract of Anacyclus Pyrethrum DC root(EEAP) not only can decrease the cough frequency, but also has anti-inflammatory and antioxidant effect onseveral inflammatory models. In this subject, we aim to clarify the mechanism of protective effects of EEAP against CVA via anti-inflammatory, immuneand antioxidant processes. First, we will establish clinical, in vivo, and in vitro models. And then, we will investigate the relationship between oxidative stress and Wnt/β-catenin by immunocytochemistry, Rt-PCR and western blotting. We expect to explain the mechanisms of EEAP via Wnt/β-catenin and TLR4/NF-κB signal pathways. Futher, we will clarify the important interaction of the innate immunity and acquired immune response in CVA inflammation by measuring the expression of inflammatory factors related toT1and T2 lymphocyte. Taken together, based on the research mentioned above , this study can provide some evidence for further clinical application in CVA patients .

咳嗽变异性哮喘（CVA）是支气管哮喘特殊类型。发病机制不清楚，常规治疗效果欠佳。前期研究发现阿那其根胶囊能有效治疗CVA患者。其醇提物(EEAP)可减少CVA大鼠咳嗽次数，对多种炎症模型有抗炎抗氧化作用，其作用机制尚不明确。为明确这一机制，我们认为EEAP可能通过肺肾Wnt/β-catenin和TLR4/NF-κB通路以及免疫调节发挥抗氧化和抗炎作用。为验证该假说，本项目结合临床、动物和细胞实验，采用蛋白免疫印记、免疫荧光、荧光定量PCR等技术，研究CVA模型肺肾组织中氧化应激与Wnt/β-catenin通路的关系，考察Wnt/β-catenin和TLR4/NF-κB通路相关分子含量。采用流式细胞术检测树突细胞表面CD80、CD86共刺激分子的表达，通过ELISA法测定Th1及Th2淋巴细胞相关炎症介质的表达，明确阿那其根免疫调节在哮喘炎症中的重要作用，为其临床应用提供依据。

变异性咳嗽又称咳嗽变异性哮喘（cough variant asthma，CVA），是以慢性咳嗽为主要或唯一临床表现的一种特殊类型支气管哮喘。CVA 病程较长，是哮喘发展的初期阶段。治疗以糖皮质激素为主，治愈难度较大。由于其发病原因目前尚不完全清楚，主要研究认为该病不仅与气道高反应、气道重塑、炎症、免疫学有关，还与遗传学及环境、感染等因素有关。阿那其根为新疆地产民族药，有较好的临床治疗作用。本项目以“肺肾同源”为理论依据，探讨阿那其根醇提取物（EEAP）治疗咳嗽变异性哮喘机制，为临床治疗CVA提供新思路。.试验结果表明，EEAP能促进淋巴细胞(T、B 细胞)体外增殖，对增强机体免疫功能具有重要的意义。临床CVA患者外周血ELISA 结果显示，EEAP能降低 L-4、IL-5、IL-6、IL-13、TNF-α、GM-CSF及 MDA 、4-HNE含量，升高IFN-γ、SOD 含量和Trx 的含量，统计学结果有显著性差异(P<0.05)。动物试验中，采用ELISA法、实时荧光定量PCR法、蛋白免疫印记法和免疫荧光法等技术通过三种途径证实阿那其根醇提取物对CVA的作用。首先通过升高IFN-γ，下调GM-CSF及DC表面的CD80、CD86共刺激分子的表达，初步完成CVA豚鼠体内的免疫调节；再者，通过下调TLR4/NF-κB的信号通路表达，抑制促炎因子IL-4、IL-5、IL-6、IL-13、TNF-α的释放和炎症蛋白TLR4mRNA、NF-κB（p65）mRNA的表达；其次，通过下调ρ-GSK-3β、β-catenin的蛋白表达，降低体内MDA、4-HNE含量，上调GSK-3β、ρ-β-catenin的蛋白表达，升高SOD、Trx的水平，达到抗炎、抗氧化作用。动物试验也再次证实，CVA发生时，肺、肾TLR4、Wnt信号通路相关因子呈现高度相关（p＜0.01）。为“肺肾同源”理论在防治CVA进一步夯实基础。