毛菊苣活性成分抗肝纤维化分子机制及其构效关系研究

Cichorium glandulosum Boiss.et Hout is one of the most widely used medicinal materials in prescriptions to treat liver disease by Uighur, and the liver protective and anti-hepatic fibrosis activity of four different polar extracts from its roots were studied previously, results showed that they all were active. As different polar extractions and components of 95% ethanol extract with the best activity had the intervention effects on TGF-β/Smad signaling pathway, we mainly isolated the n-butanol extract and found that three sesquiterpene lactones showed significant activity, which were expected to become antfibrotic lead compounds with a new type of structure. Besides, the content of flavonoids in 70% ethanol extract with better anti-hepatic fibrosis activity was the highest, and flavonoids were enriched, but their activities had not been studied. This study intends to perform qualitative and quantitative determination on the active sesquiterpene lactones and flavonoids of Cichorium glandulosum Boiss et Hout. Three active ingredients were taken as the main subjects, by studying the molecular mechanisms of HSC PPP5C phosphatase mediated MAPK, PI3K/AκT signaling pathway, we attempt to investigate the structure-activity relationship of active sesquiterpene lactones and their anti-hepatic fibrosis activity, and to explain the material basis of multi-target effects, to lay the foundation for the discovery of novel antifibrotic drug candidates. Meanwhile, we also investigate the anti-hepatic fibrosis mechanism of total flavonoids in the whole animal and cellular levels, which are the main active extracts and components of Cichorium glandulosum Boiss et Hout.

毛菊苣是维吾尔医治疗肝病处方中应用最多的药材之一，前期研究了毛菊苣根4种不同极性提取物保肝、抗肝纤维化作用，均具有活性。针对活性最好的95%乙醇提取物的不同极性部位及组分对TGF-β/ Smad 信号转导通路的干预作用，主要分离了正丁醇部位，发现3个倍半萜内酯类成分活性显著，有望成为新结构类型的抗肝纤维化先导化合物。此外抗肝纤维化活性较好的70%乙醇提取物中黄酮含量最高，并富集了黄酮，但未对黄酮活性进行研究。本研究拟对毛菊苣中倍半萜内酯类活性成分及黄酮成分进行定性、定量测定；以3个活性成分为主要目标，通过对HSC PPP5C磷酸酶介导的MAPK、PI3K/AKT信号通路的分子机制研究，探明倍半萜内酯活性成分抗肝纤维化的构效关系，阐释其多靶点效应的物质基础，为发现新型抗肝纤维化候选药物奠定基础；并采用整体动物水平及细胞水平探究毛菊苣中主要活性部位及成分——总黄酮的抗肝纤维化作用机理。

毛菊苣是我国维吾尔医治疗肝病最常用的药材之一，多年的临床应用证明毛菊苣具有治疗肝炎、胆囊炎等肝胆疾病的作用。本项目以毛菊苣为研究对象，开展了毛菊苣化学成分研究；考察毛菊苣正丁醇、乙酸乙酯提取物对由肠炎引起肝损伤及肝纤维化的保护作用；从细胞水平对分离得到的25个化合物进行抗炎初筛，并研究其中的莴苣苦素对RAW264.7细胞增殖，MAPK、iNOS、COX-2蛋白表达和基因表达的影响，对大鼠肝星状细胞（HSC-T6）活化、增殖以及Smad家族蛋白表达的影响；采用正交设计对毛菊苣总黄酮提取进行优化，研究总黄酮对HSC-T6活化、增殖、TGF-β/Smad信号通路的基因表达、蛋白表达影响，通过血清指标、病理切片、免疫组化及western-blot 等方法考察总黄酮的体内抗肝纤维化作用；利用GC-MS对毛菊苣挥发油进行成分含量分析，并研究挥发油对大鼠肝纤维化的保护作用；从抑菌圈、最小抑菌浓度、生长曲线、AKP酶、DPPH以及ATBS对毛菊苣体外抗菌抗氧化活性进行研究。对毛菊苣提取分离得到25个化合物，其中新化合物 4 个，新天然产物 1 个，确定了毛菊苣乙酸乙酯提取物、正丁醇提取物、总黄酮、以及莴苣苦素均具有显著的抗肝纤维化作用，毛菊苣挥发油具有显著的抗肝纤维化作用，主要成分为香茅醇，毛菊苣通过破坏细菌细胞壁的生成发挥杀菌作用，其中乙酸乙酯提取物抗菌作用最强，70%乙醇提取物抗氧化活性最好。本研究的意义在于，得到的25个化合物为毛菊苣物质研究提供了基础；莴苣苦素抑制iNOS、COX-2、IL-6、IFN-β基因表达，抑制 iNOS、 COX-2 蛋白表达，下调 P44/42 MAPK 蛋白表达，有效阻止MAPK信号通路，是发挥抗炎作用的重要机制之一；莴苣苦素抑制HSC-T6中α-SMA、Smad3蛋白表达以及上调Smad7、TLR-4蛋白表达，有效阻断Smad 信号转导通路，是发挥抗肝纤维化作用的重要机制之一；毛菊苣总黄酮通过阻断TGF-β/ Smad 信号转导通路发挥抗肝纤维化作用；本研究结果为莴苣苦素成为抗炎保肝先导化合物提供科学依据，毛菊苣的抗菌作用与菊粉的益生菌作用为课题组今后提出从肠道菌群方面抗肝纤维化提供思路与研究基础。