基于“风温伏肺”理论探讨清肺透邪方对支原体肺炎CARDS毒素损伤的保护机制研究

The incidence of Mycoplasma pneumoniae pneumonia (MPP) is increasing in the world. Clinical antibiotic-resistant mycoplasma comes out with refractory and severe cases raising. Community-acquired respiratory distress syndrome toxin (CARDS TX) and Th17/Treg immunoregulation are considered as important risk factor for MPP. The epidemiological characteristics of MPP is similar with that of wind-warm syndrome. On the same time, its clinical manifestations as longer and more refractory course,which is alike to the progress caused by pathogen lying latent in lung. Therefore, we put out our opinion that treatment mycoplasma pneumoniae produced CARDS TX damage treated by Qingfei Touxie detoction based on the theory of wind-warm lying latent in lung.The pathogenesis of MPP is that wind-warm lying in lung causes body fluid impairment and collaterals injury. The treatment principle is strengthening the body resistance and eliminating pathogenic factors. We propose hypothesis that dispersing to eliminate pathogenic factors reduce toxin production and improve lung injury,moistening to strengthen the body resistance and optimize immune response. Beginning with protection mechanism of pulmonary injury, proteomics technologyis designed to draw rat MPP different protein network diagram. We centered on MPP toxin and immunity inbalence to clarify the scientific connotation of wind-warm lying latent in lung. According to different response, we attempt to use Label Free technology to screen the effective biomarker in Qingfei Touxie decoction.MPP infected rat model with factorial design is used to reveal its multi-target pharmacological mechanism.

肺炎支原体肺炎（MPP）发病率在全球处于上升趋势，加之临床上MP耐药株的出现，难治性和重症病例增多。CARDS毒素损伤及Th17/Treg细胞免疫失衡在MPP发生发展中被认为是重要危险因素。MPP的流行病学特点与风温发病相似，临床特征表现为病程长而缠绵又与邪伏肺络的致病过程相同，因此提出基于风温伏肺理论辨治MPP的新见解。其病机特点为风温伏肺致津伤络损，治疗原则为扶正祛邪，进而提出“清透”以祛邪减少肺炎支原体毒素产生，改善肺组织损伤；“清润”以扶正调节免疫失衡的假说。本研究以肺组织损伤/保护机制为切入点，采用蛋白组学技术结合创新实验设计绘制鼠MPP差异蛋白网络图，并以肺炎支原体毒素及免疫失衡为中心，阐明“风温伏肺”的科学内涵。针对人群对治疗反应不同，结合现代Label Free技术，筛选清肺透邪方有效生物标记物，继而通过析因设计以MPP鼠模型为研究对象，揭示其多靶点药效机制。

本研究研究初期首先对风温伏肺理论的理论进行了梳理，创新性的提出清肺透邪法辨治儿童肺炎支原体肺炎；其次采用正交实验设计，优选了清肺透邪方的最佳提起工艺。研究中期首先建立MP诱导的小鼠肺炎模型，并从一般状态、肺组织病理形态及肺炎支原体（MP）载量等方面对模型进行了鉴定；其次采用免疫组化法、免疫印记、RT-PCR、流式细胞术等方法，探索不同时相MPP时CARDS毒素、NLRP3/ASC/Caspase-1通路、MyD88/NF-κB通路蛋白和mRNA的表达及IL-1β、IL-8、IL-17等炎症因子的动态变化，明确了MP诱导幼鼠肺部炎性损伤的具体信号分子机制；最后采用同位素相对标记与绝对定量技术（i TRAQ）对肺炎支原体感染及肺炎支原体肺炎肺组织差异蛋白进行分析，绘制蛋白指纹图谱，从差异蛋白角度分析MP肺炎的致病机制为进一步研究MP损伤/保护机制奠定了基础。后期首先通过观察清肺透邪方及拆方干预MP诱导的幼鼠肺部炎性损伤的实验研究，确定了其治疗幼鼠MP肺炎的时效、量效关系、不同发病时相的最佳组方，明确了其通过影响CARDS毒素、调控Th17/Treg平衡、Notch通路、Foxp3/RORγt通路、NLRP3/ASC/Caspase-1通路、MyD88/NF-κB通路关键分子蛋白和mRNA的表达及IL-1β、IL-8、IL-17等免疫因子群的动态变化治疗幼鼠MP肺炎的多靶点药效机制；其次采用蛋白组学分析技术结合创新的实验设计筛选了清肺透邪方治疗MPP血清有效生物标志物。本研究为探讨中药防治儿童肺炎支原体肺炎及中医证候学研究提供了支撑，对中医药的继承、创新和发展具有重要的理论价值。