基于靶向调控乳酸杆菌介导胆汁酸代谢的黄芩汤治疗溃疡性结肠炎作用机制研究

Huangqin decoction (HQD) derived from "Treatise on Febrile Diseases", is a traditional Chinese medicine which is widely used to ameliorate gastrointestinal diseases, such as ulcerative colitis (UC). Literature reports and our previous studies showed that HQD could regulate lactobacillus and bile acids which are closely related to the development of UC. But it is not clear whether Huangqin decoction could treat UC by regulating lactobacillus-mediated bile acid metabolism pathway. According to this, we intend to investigate the mechanism of the effect of HQD in the treatment of UC based on the targeted regulation of lactobacillus-mediated bile acid metabolism pathway. The following works will be carried out: (1) By use of the metagenomic sequencing and targeted metabolomics method of bile acid, the special bacteria / plant intervened by HQD and associated with bile acid would be screened; (2) The effects of HQD intervention on lactobacillus strain and bile acid metabolism would be studied, and the effect of FXR-FGF15 pathway would also be investigated; (3) The potential effects on UC by lactobacillus strain regulated by HQD would be evaluated by vivo and vitro experiments. By the point view of multilevel (intestinal flora, signal pathway, small molecule metabolites) and targeted regulation, the mechanism of UC in the treatment of HQD would be revealed. It is expected to provide a scientific basis for the in-depth research of the mechanism of treatment on UC, the rational clinical application and the deep development of HQD.

黄芩汤出自《伤寒论》，为中医治疗胃肠道疾病的经方，其治疗溃疡性结肠炎（UC）疗效确切。文献报道及前期研究显示黄芩汤能够回调与UC密切相关的乳酸杆菌及胆汁酸代谢。但黄芩汤是否是通过调节乳酸杆菌介导胆汁酸代谢进而治疗UC目前还不清楚。据此，本项目提出基于靶向调控乳酸杆菌介导胆汁酸代谢途径，研究黄芩汤治疗UC的作用机制。拟开展以下工作：（1）利用宏基因组测序手段以及胆汁酸靶向代谢组学方法，筛选与黄芩汤干预作用相关的乳酸杆菌功能菌群/株；（2）明确黄芩汤对乳酸杆菌和胆汁酸代谢的影响，及胆汁酸介导的FXR-FGF15信号通路的作用；（3）从体内外验证黄芩汤靶向调控的乳酸杆菌菌株介导胆汁酸代谢治疗UC的潜力。从多层次（肠道菌群、信号通路、小分子代谢物）以及靶向调控角度揭示黄芩汤治疗UC的分子机制。以期为黄芩汤治疗UC作用机制的深入阐释，临床合理应用及深度开发提供科学依据。

黄芩汤出自《伤寒论》，为中医治疗胃肠道疾病的经方，其治疗溃疡性结肠炎（UC）疗效确切。本课题采用3% DSS溶液自由饮用7天建立UC小鼠模型, 同时给予9.1g/kg的黄芩汤干预。通过DAI分数、组织病理学检查、炎症因子和免疫组化分析等验证UC模型与黄芩汤的药效。结果表明黄芩汤能够显著回调UC小鼠的DAI水平升高，结肠损伤，促炎因子IL-4和TNF-α水平上升，抑炎因子IL-10水平下降以及结肠组织ZO-1、Occludin蛋白表达的降低。确证了黄芩汤对UC的治疗作用。.通过16S rRNA扩增子测序分析各组小鼠粪便中肠道菌群结构组成。结果表明黄芩汤部分逆转了UC小鼠的菌群紊乱，显著增加了有益菌如拟杆菌、乳酸杆菌等菌的相对丰度，降低了有害菌如变形菌的相对丰度。再通过UPLC-MS/MS法测定小鼠粪便中胆汁酸水平。结果表明黄芩汤能够部分逆转UC小鼠的胆汁酸代谢紊乱。.通过抗生素造成伪无菌状态再进行粪菌移植，以体质量、DAI、结肠长度和组织病理学检查为指标研究黄芩汤对UC小鼠的治疗作用与肠道菌群的关系。结果表明伪无菌状态下，黄芩汤对UC小鼠的改善作用几乎消失。粪菌移植实验结果发现，经黄芩汤粪菌移植干预后，逆转了UC小鼠DAI指数水平，改善了结肠损伤。而灭菌后的黄芩汤粪菌液并没有如此效果。通过伪无菌和粪菌移植实验，表明黄芩汤依赖肠道菌群对UC小鼠产生治疗作用。.接下来，研究了黄芩汤活性成分丹皮酚对UC小鼠的肠道菌群及代谢产物影响。结果表明丹皮酚部分逆转了肠道菌群如乳酸杆菌的紊乱，调节了胆汁酸和短链脂肪酸等代谢产物水平。其作用可能是通过介导FXR-SHP/LRH-1和 FXR-FGF15通路来实现。.最后探究了黄芩汤、葛根芩连汤对UC小鼠肠道菌群的影响。结果两种复方均能通过调节肠道菌群平衡从而减轻DSS诱导的UC，其中，黄芩汤在调节有益菌的数目上优于葛根芩连汤。.综上，本课题揭示了黄芩汤依赖肠道菌群治疗UC，其能够通过肠道菌群-胆汁酸代谢通路发挥治疗作用。