马齿苋新骨架生物碱抑制脂多糖刺激巨噬细胞NF-κB和MAPK信号通路的抗炎机制及其药动学研究

Portulaca oleracea L. is a commonly used Chinese herb with antiinflammatory effect and has a good name of "natural antibiotics". However, the theraputic materials and its antiinflammatory mechanisms have been unclear untill now, and therefore it is necessary to investigate the reasons in depth. Our research group recently found that Portulaca oleracea L. exists a fraction having many new skeleton alkaloids, in which the two new alkaloids have already been isolated and then identified, and the results indicated that the new skeleton alkaloid, named oleracimine in the P. oleracea L. significantly inhibited proinflammatory mediators, ie, nitric oxide (N0) and prostaglandin E2 (PGE2), and proinflammatory cytocine, ie, tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) releasing, and downregulated the gene and protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), we thus speculate that these new skeleton alkaloids in P. oleracea L. maybe play an important role in the antiinflammatory effect of Portulaca oleracea L. The project will exploit the inflammatory model established by LPS-stimulated RAW264.7 cells to screen the alkaloids with antiinflamatory activities isolated from P. oleracea L., to explore the pharmacokinetic profile of the new skeleton alkaloids in P. oleracea L. with antiinflammatory acitivities by the high sensitivity and accuracy High Performance Liquid Chromatorgaphy mass spectrum (LC/MS/MS) method established，and then from the two main signaling transduct pathways of inflammatory reaction of nuclear factor-kappa B (NF-κB) and mitogen -activated protein kinase (MAPK) mediated to investigate the effects of the new skeleton alkaloids of P. oleracea L. on the important link such as the NF-κB inhibitor (IκBα), p38MAPK and theirs downstream inflammatory cascade enzymes and etc., to ascertain the antiinflammatory targets of the new skeleton alkaloids in P. oleracea L., and thus eluciate the antiinflammatory theraputic materials of Portulaca oleracea L. and its antiinflammatory mechanisms.

马齿苋是常用的抗炎中药，有“天然抗生素”的美誉，然而其抗炎药效物质及其抗炎机制至今尚不清楚。本课题组最近研究发现马齿苋中存在一个含有新骨架生物碱的提取部位，现已分离鉴定了两个新生物碱，其中一个新骨架生物碱Oleracimine经实验证实能够显著抑制炎症介质NO、PGE2及炎症细胞因子TNF-α、IL-6的释放，下调iNOS、COX-2 的基因和蛋白表达，由此推测新骨架生物碱成分在马齿苋抗炎作用中扮演重要角色。本研究采用脂多糖诱导小鼠RAW264.7巨噬细胞建立细胞炎症反应模型，筛选出从马齿苋中分离的具有抗炎活性的新骨架生物碱，应用高灵敏、准确的LC-MS/MS分析技术，探索其在大鼠体内药动学特征，然后研究新骨架生物碱对NF-κB和MAPK介导炎症反应的信号转导通路重要环节如IκBα、p38MAPK及其下游炎症级联酶的影响，确定抗炎靶点，揭示中药马齿苋抗炎药效物质及其抗炎机制。

本课题组历经4年研究时间，经课题组团队的共同合作，按照项目原预定的实验方案，高质量顺利完成了本项目各项指标。本课题组已采用水及乙醇提取，经大孔树脂，硅胶，聚酰胺，ODS，葡聚糖等填料分离，并采用制备HPLC及分析HPLC精制，从马齿苋中分离得到150个化合物，其中49个新化合物，37个已知化合物,64个首分化合物。其中含有一系列不同类型生物碱、黄酮、木脂素、有机酸、呋喃类、内酯，以及酚类、薁类等化合物，并首次在马齿苋中发现多种新碳架生物碱、木脂素等新颖化合物。本课题组建立了LPS诱导RAW264.7巨噬细胞的炎症反应模型，采用Griess、ELISA、qPCR及Western blot方法测定马齿苋中5种新生物碱单体对MAPK及NF-kB信号通路炎症介质如NO、PGE2及炎症细胞因子如TNF-α、IL-6、IL-1β释放量以及COX-2、iNOS、p-ERK、p-JNK蛋白和基因表达的影响。采用DPPH自由基清除的方法，对生物碱、高异黄酮等单体化合物进行抗氧化活性研究。采用UHPLC-ESI-MS/MS测定黄酮类成分在大鼠体内药代动力学行为特征，采用UHPLC-ESI-TOF/MS研究多种生物碱在大鼠体代谢特征。相关内容发表中文及科技核心期刊4篇，SCI收录论文21篇、在线发表SCI收录论文4篇，获得国家发明专利9项。研究内容获辽宁省及沈阳市自然科学学术成果奖3项，会议论文2篇，均获优秀论文奖。. 上述研究对马齿苋单体化合物靶点抗炎新药研制具有重要临床意义，但目前仅完成几个单体化合物的体外活性研究，但不能证明体内有效，另外还有近百个结构新颖、具有新药开发前景的化合物需要深入探索。后续课题组拟建立DSS诱导慢性UC模型小鼠，阐明马齿苋活性部位的药效学及调控TLR4-NLRP3/pro-IL-1β信号通路中多靶点治疗UC的作用靶点；然后从马齿苋中分离鉴定的150个单体化合物中初步筛选出体外具有显著抗炎、抗氧化活性的候选单体化合物，建立LPS诱导的RAW264.7巨噬细胞的炎症模型，以体内实验已阐明的作用靶点为靶标，验证候选单体化合物的活性，通过体内外关联研究，揭示马齿苋治疗UC的药效物质，为马齿苋活性部位、单体靶点新药研制及其临床应用提供理论依据。.