瑶药小钻抗类风湿关节炎活性成分基于NF-κB信号通路的筛选和分子机制研究

The NF-κB-targeted inhibitors have been a new orientation in the study on rheumatoid arthritis treatment.Signal transduction and nuclear transcription factor (NF-κB) controls the activation of the cytokine, which is related to regulating the occurrence and development of rheumatoid arthritis.The EtOAc fraction of Kadsura Longipedunculata, has been previously identified notably anti-inflammatory, analgesic and suppressive for the expression and activity of NF-κB in CIA (Collagen-Induced Arthritis) at rat ankle Synovium.On the basis of such findings, this project, by the integration of traditional Chinese Pharmacology, and its correlated chemistry, as well as Molecular Biology, etc. is aimed at the systematic separation and structural identification, for the composition of active constituents.Meanwhile, targeted on NF-κB, activity evaluation is also conducted on the composition, in virue of gene analysis ,HEK 293/ KB-luc cells and macrophage models.Compounds proved remarkably active are then sufficiently prepared, and the active anti-rheumatoid arthritis ingredients that suppresses NF-κB are further confirmed by the modeling of CIA with mice.This project is aimed to illustrate the physical basis and mechanism of Kadsura Longipedunculata for anti-rheumatoid arthritis ,and to provide experimental and theoretical foundation for the discovery of rheumatoid arthritis micro-molecule for NF-κB-targeted treatment, thus, to lay scientific foundation of developing this Yao national medicines.

以NF-κ B为靶点的抑制剂已成为抗类风湿关节炎的新研究方向。信号转导和核转录因子（NF-κ B）是调控类风湿性关节炎发生、发展中相关细胞因子激活的“总开关”。在课题组前期发现瑶药小钻的乙酸乙酯部位具有显著的抗炎镇痛作用，能下调CIA大鼠踝关节滑膜组织NF-κ B的表达和活性的基础上，本项目拟整合中药化学、中药药理及分子生物学等相关方法，对小钻活性部位的化学成分进行系统的分离和结构鉴定；以NF-κ B为靶点，利用人胚肾成纤维细胞、巨噬细胞模型及基因分析手段对其化学成分进行活性评估；对活性显著的化合物进行足量制备，采用CIA小鼠模型进一步确定具有抑制NF-κ B的抗类风湿关节炎活性成分。本项目旨在阐明小钻抗类风湿关节炎药效物质基础及作用机制，为发现以NF-κ B为靶点治疗类风湿关节炎的小分子化合物提供理论和实验依据，为该民族药物的开发奠定科学基础。

瑶药小钻为五味子科南五味子属植物长梗南五味子的根和茎，是广西瑶族民间治疗风湿性关节炎的习用药材。本项目选择小钻为研究对象，通过硅胶柱层析和葡聚糖凝胶柱层析等现代色谱技术方法，从小钻乙酸乙酯提取物中共分离得到了15个化合物。根据理化性质和现代波谱技术并辅以化学方法进行结构鉴定，发现2个新化合物和5个从小钻中首次分离得到的化合物。体内活性研究发现，小钻乙酸乙酯提取物具有显著的抗类风湿关节炎作用，能下调CIA大鼠踝关节滑膜组织NF-κB的表达。基于NF-κB信号通路进一步对小钻分离得到的化合物进行抗类风湿关节炎研究发现，小钻化合物安五脂素和戈米辛R能抑制LPS诱导RAW264.7细胞TNF-α、IL-1β、IL-6、NO的分泌，抑制TNF-α、IL-6、IL-1β mRNA的表达水平，上调IκB-α蛋白和下调 NF-κBp65 蛋白的表达。本研究建立了小钻药材的质量标准，对小钻性状、显微鉴别、薄层鉴别、水分、灰分、浸出物进行系统研究，并以其主要活性成分之一戈米辛R作为指标成分进行含量测定。本研究还考察了瑶药小钻乙醇提取物对4种肿瘤细胞（人乳腺癌细胞MCF-7、人结直肠癌细胞LOVO、人肝癌细胞HepG2、小鼠黑色素瘤细胞B16）增殖的影响，发现小钻乙醇提取物对4种肿瘤细胞具有一定的增殖抑制作用。本项目阐明了小钻抗类风湿关节炎药效物质基础及作用机制，本项目阐明了小钻抗类风湿关节炎药效物质基础及作用机制，为该民族药物临床治疗类风湿关节炎提供科学依据，为该药物的开发奠定了实验基础。