瑶药别旁茶总苷介导PXR治疗炎症性肠病的有效成分及作用机制研究

Inflammatory bowel disease（IBD）is a refractory diseases in the digestive system，and its pathogenesis is not clear. PXR regulates the metabolism-related proteins in the intestinal mucosa and mediates the expression of inflammatory pathways. Now PXR has become a research hotspot as being the therapeutic targets of IBD. Our previous studies have found that elongate jasmine twig and leaf total glycosides could maintain the integrity of cell barrier by inducing the gene expression of metabolism-related proteins in the intestinal mucosa and inhibit the levels of the related flammatory factors regulated by NF-κB. We propose the hypothesis on this basis that elongate jasmine twig and leaf total glycosides improve even cure IBD through promoting and restoring the integrity of the intestinal epithelial cell barrier，activing the expression and function of PXR in the intestine specifically，increasing the gene expression of the metabolic enzymes in intestinal mucosae and inducing the gene expression of NF-κB. To illustrate this hypothesis further , a succession of methods will be used, such as transient transfection , metabolomics, reporter gene assays, plasmid construction, gene silencing and so on, and technologies also will be used , such as the study of obtaining / losing PXR function. co-immunoprecipitation and siRNA interference. Then the regulatory of PXR on intestinal metabolic enzyme and inflammatory pathways mediated by NF-κB will be focused on. From the perspective of cell - molecular - Animal combination , the regulatory of the active ingredient in elongate jasmine twig and leaf total glycosides effect on PXR will be studied so the active ingredient and mechanism of elongate jasmine twig and leaf total glycosides to treat IBD will be revealed.

炎症性肠病（IBD）是消化系统难治疾病之一，其发病机制尚不明确，PXR调控肠道粘膜上的代谢相关蛋白并介导了炎症通路的表达，目前PXR作为IBD治疗靶点已成为研究热点。我们前期研究发现别旁茶总苷通过诱导肠粘膜代谢相关蛋白的基因表达进而维持细胞屏障完整性，同时可抑制NF-κB调控的相关炎症因子水平。在此基础上，我们提出假说“别旁茶总苷通过促进并恢复肠道粘膜上皮细胞屏障完整性，特异性活化肠PXR的表达及功能，上调肠道粘膜代谢酶和抑制NF-κB炎症基因的表达，以最终达到改善甚至治愈IBD目的”。为进一步阐述本假说，我们采用瞬时转染、代谢组学、报告基因分析、质粒构建、基因沉默等方法，通过获得/失去PXR功能的研究、免疫共沉淀和siRNA干扰等技术，围绕PXR对肠道代谢酶调控以及介导NF-κB炎症通路，从细胞-分子-动物三者结合立体的角度出发，揭示别旁茶总苷治疗IBD的有效成分和作用机制。

本研究是在前期研究的基础上，对我们提出的假说“别旁茶总苷通过促进并恢复肠道粘膜上皮细胞屏障完整性，特异性活化肠PXR的表达及功能，上调肠道粘膜代谢酶和抑制NF-κB炎症基因的表达，以最终达到改善甚至治愈IBD目的”进行了验证。课题组首先采用单因素平行实验的方法确定别旁茶总苷最佳提取精制参数，对药物提取的工艺进行了优化，达到了较好的提取效果。其次对提取的总苷成分进行了分析，共检测到29种化学成分，鉴定了其中的26种，包括橄榄苦苷、Jasminoside、Jaspogeranosaide A、Jasmultiside等环烯醚萜苷类成分11种，黄酮及其黄酮苷类成分4种、木脂素类成分3种和内酯类成分3种等，其中包括橄榄苦苷和Jasminoside在内的25种化学成分为首次从别旁茶中发现。后来观察别旁茶总苷对溃疡性结肠炎小鼠模型干预的药效作用，考察不同分组小鼠的尿液代谢表达谱的差异。代谢组学初步研究显示，别旁茶总苷可明显改善溃疡性结肠炎小鼠模型的病变，PLSDA代谢组学数据处理方法可靠。.研究最后运用了体内和体外实验相结合的方法，阐明了别旁茶总苷治疗炎症性肠病的可能发病机制。体内实验结果提示别旁茶总苷是通过改善小鼠体重减轻、结肠缩短、结肠组织炎性浸润等，同时对结肠上皮细胞间的紧密连接具有保护作用。还可以抑制小鼠结肠上皮凋亡及Stat3的磷酸化，并下调肠道组织中糖基化终产物受体（Rage）、诱导型一氧化氮合酶（iNOS）的表达。体外实验结果显示别旁茶总苷单体是通过上调PXR的表达，抑制NF-kB p65的表达从而减少IL-8的分泌，并上调代谢酶CYP3A4、CYP1A2的表达从而起到保护肠道炎症性损伤的效果。以上的实验结果能够解释我们最初的假说，同时还揭示了别旁茶治疗炎症性肠病的可能发病机制及为药物进一步开发研究提供科学依据和奠定部分实验基础。