树豆内酯A调控PXR/NF-κB信号通路抗溃疡性结肠炎的作用机制研究

Ulcerative colitis(UC) is one of the serious gastrointestinal diseases, but its exact mechanism is still not understood. Previous studies have shown that PXR exerts a positive therapeutic role in UC through mechanisms involving feedback with NF-κB signaling pathway. PXR has been a novel effective target for UC therapy. The importance of PXR in UC is becoming increasing clear. Traditional Chinese Medicine (TCM) considered the major pathogensis in ulcerative colitis are blood-stasis and heat-dampness. It has been recognized that many Chinese material medicas for clearing heat-dampness, invigorating blood circulation have the potential therapeutic value in the treatment of UC. In our previous work, a novel stilbene cajanolactone A, isolated from the leaves of Canjanus cajan (L.) Millsp with efficacy in clearing heat-damp as well as activating blood circulation to dissipate blood stasis, has been showed to ameliorate UC effectively in mouse models. The mechanism is probably related to the intestinal PXR activation and NF-κB inhibition by cajanolactone A. Therefore, we have hypothesized that cajanolactone A could attenuate UC through activating the transcriptional activity of intestinal PXR and subsequently inhibiting the NF-κB inflammatory pathway. Firstly, the present study is, using co-transfection assay, transactivation reporter assay, overexpression and gene silencing, mammalian two-hybrid assay, TR-FRET PXR comprtitive binding assay as well as molecular modeling and docking technology, designed to demonstrate that cajanolactone A is the agonist of PXR. And then we investigate the effects and mechanism of cajanolactone A for treatment of UC via the intestinal PXR/NF-κB pathway on cellular and mouse model levels. It is also designed to follows the thought of molecule - cellular - mouse model. The results will make contribution to illuminate the scientific connotation of clearing heat-damp, activating blood circulation to dissipate blood stasis for UC therapy, and will be constructive for the clinical application of TCM.

溃疡性结肠炎（UC）是重大消化道疾病之一，但其发病机制仍不完全明确。近来发现孕烷X受体（PXR）能调控NF-κB信号通路治疗结肠炎，是治疗UC的重要新靶点。而中医认为血瘀和湿热是UC的重要病机，临床上清热利湿、活血化瘀中药治疗UC疗效确切。我们研究也发现，清热利湿、活血化瘀中药树豆叶成分树豆内酯A在动物体内具有显著的抗UC作用，并能激活结肠细胞中PXR靶基因CYP3A4启动子和抑制NF-κB活性。因此我们假设“树豆内酯A可通过激活肠PXR调控NF-κB通路治疗UC”。首先，项目拟通过共转染、PXR转录激活、过表达和基因敲减、双杂交系统、竞争性配体结合、分子建模与对接等实验，在细胞水平验证树豆内酯A是PXR激动剂；接着，从细胞和动物两个层次，探讨树豆内酯A活化肠PXR调控NF-κB通路治疗UC的作用机制，初步阐明树豆叶“清热利湿、活血化瘀”防治UC的科学内涵，并为中医药临床应用提供参考依据。

溃疡性结肠炎（UC）是重大消化道疾病之一，也是诱发结直肠癌的高风险致病因素，其特征是肠道内有非特异性炎症。然而目前为止其发病机制仍不完全明确。近年来发现孕烷X受体（PXR）能调控NF-κB信号通路治疗结肠炎，是治疗UC的重要新靶点。而中医认为血瘀和湿热是UC的重要病机，临床上清热利湿、活血化瘀中药治疗UC疗效确切。我们前期研究发现，清热利湿、活血化瘀中药树豆叶成分树豆内酯A在动物体内具有显著的抗UC作用，并能激活结肠细胞中PXR靶基因和抑制NF-κB活性。因此，本项目旨在研究树豆叶活性成分树豆内酯A调控PXR/NF-κB信号通路在防治UC中的作用机制。首先，通过分子对接和一系列体外实验证明了树豆内酯A一定程度上可激活肠道PXR，是PXR激动剂。接着，我们在细胞水平上发现树豆内酯A可通过激活肠PXR而抑制NF-κB炎症通路。基于此，我们通过动物实验发现树豆内酯A可有效改善DSS和TNBS诱导的两种UC小鼠的结肠炎症状，主要表现为改善结肠缩短现象和降低炎症反应。并且在此两种小鼠模型上，树豆内酯A可通过激活肠PXR及其靶基因Cyp3a11和Mdr1而抑制NF-κB信号通路。研究结果表明树豆内酯A在一定程度上可改善UC症状，其作用机制可能与树豆内酯A激活肠PXR抑制NF-κB信号通路密切相关。研究结果初步阐明了树豆叶“清热利湿、活血化瘀”防治UC的科学内涵，也为中医药临床应用提供科学依据。