基于肠-肺黏膜免疫及其信号转导的葛根芩连汤治疗轮状病毒湿热泻机理研究

Rotavirus diarrheal disease is the second-leading cause of childhood deaths in developing countries. In the treatment of the disease, traditional Chinese medicine has shown its unique advantages. Based on some preliminary research findings, it is found that the Gegen Qinlian decoction can effectively eliminate fever and improve on signs of diarrhea since the functioning of the lung and the large intestine are both interiorly and exteriorly related, which, in turn, means that rotavirus related diarrhea is virtually dependent on the relationship between the two organs. The relationship between the mucous membrane of the lung and the large intestine as part of immune system has also been proved by modern medical studies. The present research project hence suggests that the immune relation between mucous membrane of the lung and the large intestine should be regarded as the major biological reason for "the interior-exterior relationship between the lung and the large intestine"; and to enhance the function of the mucous membranes of the two and their signal transduction becomes accordingly the chief pharmacological hypothesis of the Gegen Qinlian decoction. The project aims to set up and model rotavirus related diarrhea research on small rats to find out the effects of the decoction on the immune system in the treatment of the disease, particularly the effects on the immune relationship between mucous membrane of the lung and the large intestine; and to observe tissue pathological changes to demonstrate the biological significance of "the exterior-interior relationship between the lung and the large intestine", while explicating at the same time the ways that the decoction helps to improve the immunity of the membranes and their signal transduction pathway.

轮状病毒腹泻是发展中国家导致婴幼儿死亡的第二大疾病。中医治疗轮状病毒腹泻具有独特优势。前期研究已发现葛根芩连汤能显著退热、改善腹泻。肺与大肠相表里，轮状病毒腹泻与肠肺的脏腑关系密切相关。而近年来现代医学研究表明肠-肺黏膜存在着免疫相关性。为此，本课题提出了肠-肺黏膜的免疫相关是"肺与大肠相表里"的重要生物学基础，推测调节肠肺黏膜及其信号转导是葛根芩连汤的重要药理环节。拟建立和运用轮状病毒腹泻小鼠模型，研究经方葛根芩连汤治疗轮状病毒腹泻对机体免疫反应，尤其是肠肺黏膜免疫的影响，观察组织病理变化，在论证"肺与大肠相表里"的生物学内涵基础上，阐释葛根芩连汤影响肠-肺黏膜免疫及相关信号转导通路的机制。

目的：分析葛根芩连汤治疗轮状病毒腹泻生物学机制，研究“肺与大肠相表里”与肠-肺黏膜免疫分子机制促进活性物质调控相关性，HVEM、NOD2活化NF-kB及STAT3信号转导通路，寻找调控肠-肺黏膜免疫改变的主要信号通路。.方法：BLAB/C小鼠，SPF级，8周龄，雌雄各半。随机分9组，每组10只。Ⅰ组：大肠湿热证模型组；Ⅱ：大肠湿热证轮状病毒模型组；Ⅲ：正常对照组；ⅠV：模型中药组；V：模型西药组；Ⅵ：模型中西医结合组；VII：轮状病毒模型中药组；VIII：轮状病毒模型思密达西药组；IX：轮状病毒模型葛根芩连汤+思密达中西医结合组。观察症状和体征。收集各组肺泡灌洗液(BALF)和原代分离肠系膜淋巴细胞，流式分选BALF中和肠系膜淋巴细胞中各细胞亚群比例测定。ELISA法测定肺泡灌洗液和肠黏膜黏液中SIgA、IL-1β、IL-2、IL-6、IL-8、TNF-a含量。采用FITC标记葡聚糖实验，检测肠黏膜屏障功能。小鼠大肠、肺福尔马林固定后切片，HE染色，观察病理变化。采用Western Blot和荧光染色方法检测STAT1、pSTAT1、STAT3、pSTAT3、IkBa、pIkBa、pPKC、PKCd和TAK1表达。.结果：随造模时间推移，大部分动物出现发热、便溏、肛周红肿、嗜卧等，体重逐渐减轻。模型组数值较正常对照组升高，组间存在显著性差异（P<0.05）。肺模型组肺泡灌洗液中IL-1β、IL-2、IL-6、IL-8、RVvb、SIgA、TNF-a浓度均高于肠模型组中对应组肠黏膜黏液中各抗体和细胞因子浓度。葛根芩连汤、思密达均可降低湿热证小鼠肠黏膜通透性，葛根芩连汤+思密达共用效果最佳。葛根芩连汤+思密达组结肠、回肠黏膜完整，细胞形态无异常，黏膜下层散在分布少量淋巴细胞。急性炎症反应明显减弱，黏膜下层淋巴细胞浸润数量急剧下降，疗效最佳，西药组疗效好于中药组。葛根芩连汤、思密达均可增加HVEM阳性率。NOD2阳性率与HVEM呈相反趋势。葛根芩连汤、思密达均可增加HVEM、pIkBa蛋白表达量阳性率。其他指标与HVEM、pIkBa呈相反趋势。.结论：肠-肺之间相互联系，主要通过黏膜免疫细胞迁移而使两者黏膜相互联系实现。葛根芩连汤具有治疗湿热证作用，NF-kB及STAT3是调控肠-肺黏膜免疫改变的主要信号通路。