基于NF-KB/Nrf2-ARE通路研究彝族药物固公果根对溃疡性结肠炎的抗炎物质基础和作用机制

As the rising incidence of ulcerative colitis (UC), UC is listed as one of the modern cureless diseases by the World Health Organization. Radix Rosae giganteae is the Yi traditional medicine of anti-diarrhea with astringents. The effective part of Radix Rosae giganteae in treatment of diarrhea was firstly determine in our previous research, which showed significantly effect in the treatment of ulcerative colitis in mice induced by DSS. Exploratory study in our group showed that the effective extract of Radix Rosae giganteae can reduce TNF- and IL-6 levels in inflammation part, inhibit NF-kappa-B and active the Nrf2-ARE pathway with anti-inflammatory and treatment of ulcerative colitis. So we will study the anti-inflammatory material basis and mechanism of Radix Rosae giganteae in the treatment of UC based on the NF- kB/Nrf2-ARE pathway. In this project, the functional components to anti-ulcerative colitis were tracked and isolated by a variety of chromatographic separation methods. At the same time, the anti-inflammatory function and mechanism of this extract were evaluated by the Nrf2 (+/+) ICR and Nrf2 (-/-) ICR mice models at the whole animal level of anti-ulcerative colitis. In addition, the expression of inflammatory mediator mRNA and different expressed protein of regulating and controlling factors were determined by using the DNA-ladder and western-blot in the gene and protein level. The results will provide theoretical and experimental basis for this Yi medicine in the treatment of ulcerative colitis.

溃疡性结肠炎发病率日益上升，被世界卫生组织列为现代难治病之一。固公果根是彝族传统药物，具有涩肠止泻之功。前期我们首次确定固公果根治疗腹泻的有效部位，并发现其能有效治疗DSS诱发的小鼠溃疡性结肠炎。探索性研究发现其通过降低炎症部位TNF-a和IL-6含量，抑制NF-B活性，激活Nrf2-ARE通路，达到抗炎、治疗溃疡性结肠炎作用。因此本项目基于NF-B/Nrf2-ARE通路研究固公果根提取物治疗溃疡性结肠炎的抗炎机制和物质基础。运用多种色谱手段追踪分离固公果根提取物治疗溃疡性结肠炎的物质基础；利用Nrf2 (+/+)ICR和Nrf2 (-/-)ICR小鼠在整体动物水平上评价其对溃疡性结肠炎的防治效果；利用RT-PCR和western-blot在基因和蛋白水平上研究其对炎症介质的mRNA表达和相应调控因子的蛋白表达差异，明确作用机制。研究结果将为该彝族药物治疗溃疡性结肠炎提供理论和试验依据

本研究旨在探讨ROE对UC的保护作用及其机制。建立了葡聚糖硫酸钠(DSS)诱导的实验性结肠炎小鼠模型，研究发现ROE降低LPS诱导的炎症细胞因子（TNF-a、IL-6和IL-1b）。ROE抑制p-NF-kB、p-IKKa/b和Keap1蛋白的表达，增加Nrf2和HO-1蛋白的表达。ROE通过Nrf2/NF-kB途径预防DSS诱导的结肠炎。.固公果抗炎有效部位进行化学成分研究，共分离鉴定106个化合物，其中化合物54，90和104为新化合物，化合物21，23，25，39，56，74和76为首次从蔷薇科分离得到；12个化合物为首次从蔷薇属植物中获得；32个化合物为首次从该种植物中分离得到。.针对固公果根提取物，我们利用高内涵筛选和等离子共振的分子互做仪建立高内涵筛选体系，能够基于Nrf2/Nf-kB 进行筛选，通过抑制NO释放和基于NF-kB的分子对接筛选。明确固公果植物中55种化合物都具有一定抑制NO释放的作用，7种化合物能够和NF-kB结合发挥抗炎作用。固公果的三萜类化合物中筛选出了三种种抗炎效果最好的单体化合物MA、SA和冬青苷B，利用蛋白芯片技术和体内外实验深入研究了MA和SA的干预溃疡性结肠炎的药效及作用机制。.蛋白芯片抗炎靶点筛选结合信号通路图分析发现，MA和SA抑制了NF-κB、PI3K/AKT、JAK/STAT、MAPK信号通路中的相关蛋白；Western-Blot结果表明，MA和SA提高了Nrf2和HO-1蛋白的表达，下调了Keap1、p-NF-κB和p-IKKα/β蛋白表达，并且MA和SA增加了细胞核中Nrf2的表达，抑制了NF-κB的入核。体内实验结果表明，MA和SA能够减缓由溃疡性结肠炎引起的小鼠黏性血便、脾脏肿大、结肠缩短以及结肠损伤，对结肠损伤具有保护作用.本课题阐明固公果根提取物能够基于Nrf2/NF-kB发挥抗炎，治疗溃疡性结肠炎的药效和作用机制；明确天然产物Myrianthic acid、Sericic acid和冬青苷B等三萜化合物是固公果根中具有抗炎、抗溃疡性结肠炎的活性单体化合物；首次通过蛋白芯片抗炎靶点筛选实验和体内实验性溃疡性结肠炎实验，确定了Myrianthic acid、Sericic acid和冬青苷B的抗炎作用机制，即通过激活Nrf2信号通路、抑制NF-κB信号通路发挥其抗炎抗氧化作用。