败酱草总皂苷介导PXR抗炎症性肠病物质基础及作用机制研究

The incidence of inflammatory bowel disease (IBD) in our country increased year by year, to bring great harm to people's health. Prescriptions in which Patrinia acts as the king herb in the clinic have showed ameliorative effect against IBD, but the mechanism underlying is still unknown. Xenobiotics nuclear receptor PXR as IBD therapeutic target has caused widespread concern. In our prevenient studies, we found that the Patrinia saponin can downregulate the expression of inflammatory cytokines including TNF-α and IL-1β, which are regulated by NF-κB, and induce the expression of intestinal drug-metabolizing enzymes, resultingt in maintain cell barrier integrity. Based on this, we proposed " Patrinia saponin can activate PXR receptor, and upregulate intestinal metabolic enzymes and inhibition of NF-κB mediated inflammatory gene expression, promote and restore intestinal mucosal epithelial barrier integrity, to improve the symptoms of IBD.” To further reveal the effect of specific components of Patrinia saponin on PXR and its mechanism, around PXR action on intestinal metabolic enzyme regulation and NF-κB inflammatory pathways, we will adopt the comprehensive state-of-the art techniques, such as metabolomics, plasmid construction , transient transfection , reporter gene, Gene Silencing and so on to observe the PXR introduction effect and acquisision/disfunction PXR, siRNA interference and co-immunoprecipitation, to demonstrate the material basis of Patrinia and anti-IBD mechanism from in vivo, in vitro and molecular levels. This project aims to provide the experimental basis and scientific evidence for IBD treatment and further development of Patrinia.

炎症性肠病（IBD）发病率逐年上升，给人们健康带来巨大危害。以败酱草为主药的组方抗IBD疗效显著，但机理不明，PXR作为IBD治疗靶点引起普遍关注。我们前期发现败酱草总皂苷可抑制NF-κB调控的TNF-α、IL-1β等炎症因子表达，并可诱导肠道药物代谢酶表达而维持细胞屏障完整性。基于此，提出“败酱草总皂苷可特异性活化肠PXR上调肠道代谢酶和抑制NF-κB炎症基因表达，促进并恢复肠道粘膜上皮细胞屏障完整性，改善IBD症状”。为进一步揭示败酱草总皂苷具体成分对PXR的影响及机制，我们围绕PXR对肠道代谢酶调控以及介导NF-κB炎症通路，通过代谢组学、质粒构建、瞬时转染、报告基因分析、基因沉默等技术观察败酱草总皂苷及其主要成分对PXR诱导作用；通过获得/失去PXR功能的研究、siRNA干扰和免疫共沉淀等技术，从细胞-分子-整体动物水平验证上述假说，深入阐明败酱草总皂苷抗IBD物质基础和作用机制。

项目在前期基础上，验证败酱草总皂苷可特异性活化肠PXR上调肠道代谢酶和抑制NF-κB炎症基因表达，促进并恢复肠道粘膜上皮细胞屏障完整性，改善IBD症状。项目执行过程中通过工艺优化，确定了败酱草总皂苷的最佳提取精制工艺参数，申报国家专利1项。项目构建了大鼠溃疡性结肠炎模型并采用代谢组学研究发现，败酱草总皂苷可明显改善大鼠溃疡性结肠炎症状，代谢组学分析各组间区分明显，发表中文核心论文1篇。为了阐明药效物质基础，我们全面分析了败酱草总皂苷部位的化学成分，分离鉴定了31个化学成分，其中12个成分首次发现，主要为皂苷类成分，发表了1篇SCI论文。我们的研究初步阐明了败酱草抗炎症性肠病的物质基础，同时也完善了对败酱草化学成分的认识，为药材及其组方中成药作用机制的阐释、科学质量标准的建立奠定了基础。. 项目分别从体外实验和动物实验上对代表性成分黄花败酱皂苷C的抑炎作用和相关机制进行了研究。体外实验发现黄花败酱苷C对LPS诱导CRL-1790细胞释放IL-8和核内NF-kBp65表达水平均具有显著抑制作用，能显著提高CRL-1790细胞PXR蛋白及其下游相关代谢酶CYP3A4、 CYP1A2表达水平。提示了黄花败酱苷C通过上调PXR蛋白及其下游相关代谢酶CYP3A4、 CYP1A2表达水平而抑制NF-kBp65核转位，最终抑制炎症因子分泌。体内实验以TNBS诱导的结肠炎小鼠为研究对象，发现黄花败酱皂苷C显著抑制结肠炎小鼠血清中TNF-α、IL-6分泌以及肠道组织中mRNA表达水平，且对结肠组织NF-kBp65表达水平也具抑制作用，可促进小鼠肠道粘膜上皮细胞屏障恢复并显著提高小鼠结肠组织PXR、CYP3A的mRNA表达水平。结合现有文献报道及本项目研究结果，我们可以发现黄花败酱皂苷C具有抗炎作用，其作用机制是通过上调炎症细胞或组织中PXR及其下游相关代谢酶表达水平而抑制调控炎症因子释放的关键转录因子NF-κB表达或核转位，进而抑制炎症因子释放，促进组织修复。通过该研究，初步阐明了败酱草皂苷类活性成分抗IBD的作用机制，为科学阐释中药败酱草的作用机理提供了有益的借鉴。