清肺通络敷胸膏干预IV、RSV诱导的肺部炎性损伤及MAPKs信号通路分子靶向研究

Respiratory viral infections due to pneumonia in children are increasing year by year. The pathology characteristics that pulmonary inflammatory injury (PIFI) induced by the most common respiratory viruses IV, RSV are mainly owing to increased permeability of pulmonary microvascular endothelial cells (PMVECs). Early clinical research found that Fuxiong Paste had effect of clearing lung heat and meridians, promoting pneumonia rale absorption, and improving pulmonary microcirculation. Therefore,we proposed hypothesis that there is a certain correlation of traditional Chinese medicine external treatment with skin-meridians pathway and PMVECs permeability regulation. The purpose of this study will apply monoclonal technology in researching effective substance groups of Fuxiong Paste as the breakthrough point,carry the research on pharmacokinetics by UPLC/MS-MS and microdialysis technique to explore the substance basis and targets of the external medicine. Furthermore, Estabilsh mouse model of PIFI, and observate the dynamic changes of transduction pathway (MAPKs) by the technology of Western-blot and so on. Moreover, reveal the specific molecular mechanism of PMVECs permeability increase induced by respiratory viral, and from the disease-network, investigate Fuxiong Paste (main, auxiliary medicine) potency differences, developing the integrated mechanism of "medicine and auxiliary symbiosis" and provide the scientific experimental basis and foundation for the external treatment of Chinese medicine in the clinical application of viral lung inflammatory injury.

呼吸道病毒感染所致小儿肺炎有逐年增高趋势。最常见呼吸道病毒IV、RSV诱导的肺部炎性损伤的病理基础，主要是肺微血管内皮细胞(PMVECs)通透性升高所致肺损伤。前期临床发现清肺通络敷胸膏能促进肺部啰音吸收，实验研究表明改善肺微循环。因而提出假说"基于肺热络瘀理论的清肺通络法干预病毒性肺部炎性损伤与PMVECs通透性调控存在一定相关性"。为此本研究以敷胸膏有效物质群为切入点，采用单克隆抗体技术定量分析。结合UPLC/MS-MS以及微透析技术进行整体和局部药动学研究，探寻物质基础及作用靶器官。通过析因设计，以肺部炎性损伤小鼠模型为对象，采用Western-blot等技术方法，观察MAPKs信号转导通路的动态变化，阐明呼吸道病毒所致PMVECs通透性升高的具体分子机制，探讨敷胸膏（主、辅药）药能效果差异，揭示"药辅共生"整合调节机制，为外治中药在病毒性肺部炎性损伤临床应用提供实验依据与基础。

本研究研究初期首先对清肺通络外治法的基础理论进行了梳理，创新性的提出皮部络脉为清肺通络敷胸膏的生物传输途径；其次采用正交实验设计，确定了6倍量75%乙醇，提取2次，每次3 h为其最佳提取工艺，并应用HPLC法对其进行了定量分析；最后采用HPLC /MS-MS法确定了大黄素、大黄酸、黄芩苷为其的药效物质基础，其体外经皮渗透均符合零级动力学过程，其作用的靶器官为肺。研究中期首先建立IV、RSV诱导的小鼠肺炎模型，并从一般状态、肺组织病理形态及病毒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“MAPKs”亚家族（pERK、pJNK、pP38、MMK4）中信号传导通路关键分子mRNA及蛋白的表达及TNF-a、IL-1、IL-4、IL-6、INF-γ等免疫因子群的动态变化，明确了IV、RSV诱导幼鼠肺部炎性损伤的具体信号分子机制；最后采用2D-DIGE及MALDI-TOF/TOF MS技术对呼吸道合胞病毒诱导幼龄大鼠肺炎肺组织的差异蛋白进行了研究，为进一步研究RSV的致病机制奠定了基础。后期通过观察清肺通络敷胸膏及拆方干预IV、RSV诱导的幼鼠肺部炎性损伤的实验研究，确定了其治疗幼鼠IV、RSV肺炎的时效、量效关系、不同发病时相的最佳组方及药辅共生机制，明确了其通过影响MAPKs信号传导通路和TLR4信号通路关键分子mRNA及蛋白的表达及TNF-a、IL-1、IL-4、IL-6、INF-γ等免疫因子群的动态变化治疗幼鼠肺炎的多靶点药效机制。本研究为探讨中药防治小儿病毒性肺炎、中医外用药作用途径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。