基于非靶向代谢组学探讨免疫介导性肠病湿热证实质及华重楼活性成分作用机制

It was concerned for a long time that Chinese medicine could treat inflammatory bowel disease (IBD) with principle of eliminating heat resolving dampness and toxic. However, its mechanism was explained diversely. The substantial basis for damp-heat syndrome is not firmly established. Our previous work indicated that Chinese medicine from different species, i.e. Baicalin, Abelmoschi corolla extracts and Compound Paris polyhoylla decoction, are effective for TNBS induced IBD rats or mice. Their working route involved PI3K/AKT, TNF-α, IFN-γ and TLR4/CXCR3 respectively. Therefore, the research mentioned above demonstrated the Chinese medicine in a bit monotonous way, may not comprehensively evaluate the effects and even underestimated some key targets. This protocol provided the hypothesis that ‘IBD patients have specific metabolites of damp-heat syndrome which are not existing in gluten sensitive enteropathy (GSE) , even though both IBD and GSE are immune mediated enteropathy. Active components of Paris polyhoylla var．Chinensis (PPC) may significantly influence these metabolites.’ As the first step, IBD patients with damp-heat syndrome will be selected as subjects, GSE patients with spleen deficiency syndrome and health adults as controls, biological samples will be collected and analyzed by Untargeted Metabolomics in order to systematically demonstrate metabolites of damp-heat syndrome and screening for drug targets. The second step is to confirm if active components of PPC could significantly influence these metabolites from TNBS induced IBD rats by using Targeted Metabolomics technique. We expect that substantial basis of damp-heat syndrome will be primarily illustrated and the main working route and drug targets of PPC active components, etc. will be approached.

中医药清热化湿解毒法治疗炎症性肠病（IBD）一直备受关注，但具体机制众说纷纭，湿热证的物质基础尚不清楚。前期研究表明，不同种属中药成分，如黄芩苷、黄蜀葵花提取物及蚤休复方（含华重楼）均对TNBS诱导的IBD模型鼠有治疗作用，作用途径分别涉及PI3K/AKT，TNF-ɑ、IFN-γ以及TLR4/CXCR3。但上述研究只在单一水平反映中药治疗作用，不能全面评价，或可疏漏关键靶点。本课题提出“虽同属免疫介导性肠病，IBD具有麦胶敏感性肠病（GSE）所不具备的湿热证特征性代谢物；华重楼活性成分可影响此类代谢物水平”。拟以IBD湿热证患者为研究对象，以GSE脾虚证患者及健康人为对照，利用非靶向代谢组学分析生物样本，系统反映湿热证代谢组特征，筛选相应靶标。进而采用靶向代谢组学，分析华重楼活性成分等物质对IBD模型大鼠此类代谢物的影响，以期初步阐明湿热证实质，获取华重楼活性成分等大致作用路径和靶标。

为阐明中医药清热化湿解毒法治疗炎症性肠病（IBD）湿热证的机制，明确湿热证物质基础，开展本项研究。研究分为临床及动物实验两个部分。临床以健康对照者（Con）、麦胶敏感性肠病（GSE）、功能性腹泻（FD），以及IBD脾虚证患者为对照，采用UHPLC-HRMS和UHPLC-TQMS拟靶向代谢组学研究方法挖掘IBD湿热证特征性代谢物；动物实验以常温空白组（RNC）、湿热空白组（DNC）以及脾虚证组（RST）为对照，以UHPLC-HRMS非靶向代谢组学阐明黄芩苷（DHG）调节TNBS实验性结肠炎大鼠湿热证的差异代谢物；同时以脂质拟靶向代谢组学以及氨基酸靶向代谢组学，确证重楼总皂苷及华重楼主要活性成分偏诺皂苷类物质（含重楼皂苷VI、重楼皂苷VII），对实验性结肠炎湿热证差异代谢物的调节作用。结果表明：1. 乳酸、L-谷氨酸、ACar 18:1是炎症性肠病潜在诊断标志物。2. 血浆差异代谢物ACar 6:0、5b-胆甾烷-3a,7a,12a,24,25-戊醇为鉴别IBD和FD的潜在诊断标志物。3. 比较IBD 及GSE，血浆中筛选出140种差异代谢物，主要包括糖代谢中间产物、甘油磷脂类物质、芳香族氨基酸代谢产物，5-羟基吲哚-2、PC（16:0_19:1）、PC O-36:2-sn1、F6P 5为主要诊断标志物。4. 与脾虚证比较，炎症性肠病湿热证患者血浆吲哚乳酸、酪胺、L-色氨酸、LPC 16:0-2、PC 38:7-sn1、衣康酸等代谢物丰度显著增加；5. 与脾虚组比较，实验性结肠炎湿热证大鼠血浆L-犬尿氨酸、溶血磷脂酰胆碱LPC 16:1、溶血磷脂酸LPA 18:4、甘氨胆酸的丰度显著增加。6. 黄芩苷干预后，血浆LPC16:1表达下调。7. 重楼总皂苷（DRL）及偏诺皂苷类物质（DPL）均可下调PE 34:0, HexCer 40:7-1, PE 44:1, 和 TG(e)54:2。8. 黄芩苷、重楼总皂苷及偏诺皂苷类等物质对模型鼠以甘油磷脂、鞘脂为主的脂质代谢均有显著调节作用，但对芳香族氨基酸代谢无显著影响。研究显示，IBD病证均展现显著代谢特征。甘油磷脂代谢及芳香族氨基酸代谢紊乱是炎症性肠病湿热证的代谢特点，甘油磷脂代谢是重楼活性成分作用的主要通路及靶标。本研究为客观表征IBD湿热证的物质基础做出有益的尝试。