艰难梭菌相关性腹泻(CDAD)是由于广谱抗生素的滥用引起肠道菌群失衡、艰难梭菌过度生长导致的肠道炎症。由于其有较高的病死率和复发率，严重危害儿童的健康。研究发现CDAD进展与复发与免疫屏障破坏和菌群失衡引起的肠道微环境免疫偏差密切相关，而其中NF-κB通路激活的炎症反应具有核心作用。课题组创立启脾益肺法治疗CDAD，临床疗效确切。结合前期工作基础，提出“启脾益肺法调控NF-κB通路，改善肠道微环境免疫偏差的炎症反应，抑制艰难梭菌增殖是其主要机制”的工作假说。本项目拟以CDAD小鼠为对象，分析启脾益肺法改善肠道微环境免疫偏差及调控NF-κB通路介导的炎症，为启脾益肺法防治CDAD提供实验依据和治疗思路。

Clostridium difficile associated diarrhea (CDAD) is the outcome of the abusive application of broad-spectrum antibiotics, which results in flora imbalance and clostridium difficile overgrowth. Due to its high mortality and recurrence, it has become a threat to the children's health. Research has found the progress and recurrence of CDAD are closely related to intestinal mucosa microenvironment immune deviation caused by destruction of the intestinal mucosal immune barrier and flora imbalance. The activation of NF-κB signaling pathways plays the center role in the inflammatory response of CDAD. Our group created the method of arousing spleen and tonifying lung, which is effective in treating CDAD. Combined with previous work, we put forward the hypothesis that “the main mechanism of the method of arousing spleen and tonifying lung is regulating the NF-κB pathway, improving the inflammation caused by intestinal microenvironment immune deviation and inhibiting the proliferation of clostridium difficile”. The study used CDAD mice to analyze the effect of improving intestinal microenvironment immune deviation and the inflammation mediated by the NF-κB pathway. The aim of this study is to reveal the regulatory mechanism and provide experimental foundation and treatment thoughts of CDAD.

目的：观察启脾益肺法对艰难梭菌相关性腹泻（Clostridium difficile associated diarrhea， CDAD）小鼠模型NF-κB信号通路相关蛋白的影响，以及小鼠血清和结肠灌洗液sIgA的浓度。以期探索启脾益肺中药对CDAD的治疗作用机制。材料与方法：1、将20只C57BL/6小鼠，随机分为对照组、CDAD模型组。参照Chen等的造模方法，建立CDAD模型，观察两组小鼠的生存率、结肠炎症活动程度评分、粪便艰难梭菌毒素检测和小鼠结肠组织病理变化。2、将50只C57BL/6小鼠随机分为正常对照组14只、CDAD模型组20只和启脾益肺（QPYF）组16只。预防性启脾益肺中药治疗20天，观察三组小鼠生存率、结肠炎症程度评分、体重减少、结肠病理、粪便艰难梭菌毒素含量和NF-κB通路NF-κB p65、磷酸化NF-κB p65、TNF α和MCP-1的表达水平。3、用ELISA法检测小鼠血清和结肠灌洗液sIgA含量，比较三组小鼠sIgA的含量变化。结果：1、CDAD组小鼠出现体重下降、腹泻等症状，死亡率较高；与正常组相比，CDAD组结肠病理评分具有统计学差异（p＜0.05）。2、与模型组相比，中药组小鼠的生存率较高（Log-rank test p=0.013）；中药组小鼠相对体重的减少明显低于模型组（p＜0.01）；中药组的结肠炎症程度（DAI）评分较低（p＜0.01）；中药组小鼠结肠病理损伤程度低于模型组（p< 0.01）；中药组艰难梭菌毒素相对含量明显低于模型组（p＜0.01）；中药组小鼠TNF α、 MCP-1、NF-κB p65 和磷酸化NF-κB p65的表达量明显低于模型组小鼠（p＜0.01）；3、与模型组相比，中药组小鼠血清sIgA含量较高（p＜0.05）；与模型组相比，中药组小鼠结肠灌洗液sIgA含量较高（p＜0.05）。结论：启脾益肺中药可以减轻CDAD小鼠的腹泻、体重下降等症状；减轻结肠炎症损伤；有效减少小鼠的死亡率。启脾益肺中药可以减少CDAD小鼠肠道毒素分泌和抑制NF-κB通路上NF-κB p65、磷酸化NF-κB p65、TNF-α和MCP-1的表达，同时增加CDAD小鼠sIgA的分泌，可能是其治疗的作用机制。