基于肝脏炎症－肝纤维化－肝癌轴的白首乌生物学行为研究

The human health was severely damaged by hepatic fibrosis induced with chronic inflammation, which would probably develop to hepatic carcinoma. The study on the associativity of hepatic fibrosis and hepatic carcinoma attracted much attention following the Hepatic Inflammation-Fibrosis-Cancer (IFC) Axis theory suggested. TLR4-MyD88-NF-κB signaling pathway was proved correlated with IFC axis closely in recent research. Although Chinese medicine showed significant effect on hepatic inflammation, hepatic fibrosis and hepatic carcinoma in clinical treatment, the regulation of which on IFC axis was still largely unknown. The total glycosides of baishouwu showed evident effect on hepatic inflammation, hepatic fibrosis and hepatic carcinoma in our early studies, the typical active component displayed apparent hepatic target. According to our previous studies, the project would reveal the action character and biological entity of baishouwu on protecting liver, preventing hepatic fibrosis and inhibiting hepatic carcinoma based on IFC axis, and find out the molecule network mechanism of baishouwu targeting on liver by TLR4-MyD88-NF-κB signaling pathway. The studies woud provide scientific evidence for development and clinical application of bashouwu.A new idea and method for studying on treating liver disease with Chinese medicine based on Hepatic Inflammation-Fibrosis-Cancer Axis would be built in the studies.

慢性肝脏损伤后炎症导致的肝纤维化继而发展为肝癌严重危害人类健康。随着肝脏炎症-肝纤维化-肝癌(IFC)轴理论的提出，肝纤维化与肝癌相关性的研究日益受到关注，最新研究证实TLR4-MyD88-NF-κB通路与IFC轴密切相关。中医药在治疗肝脏炎症、肝纤维化及肝癌方面疗效显著，但对于IFC轴的调控作用则未见报道。课题组前期研究证实白首乌总苷对肝脏炎症、肝纤维化及肝癌均有显著防治作用，同时体内过程研究发现白首乌甾苷类代表性活性成分有明显的肝靶向特性。本项目在前期研究基础上，基于IFC轴阐明白首乌总苷保护肝脏、防治肝纤维化及肝癌的功效特点和生物学本质，通过对TLR4-MyD88-NF-κB信号通路的研究深入探明其作用于靶器官肝脏的分子网络机制，从而为白首乌药用开发和临床应用提供科学依据。本项目研究将建立一种基于肝脏炎症-肝纤维化-肝癌轴的中药防治肝病研究新思路和新方法。

本项目基于肝脏炎症-肝纤维化-肝癌轴对白首乌中C21甾苷类组分保护肝脏及防治肝癌的作用功效进行了系统研究，并通过对TLR4-MyD88-NF-κB相关信号通路的研究探明了其作用于靶器官肝脏的作用机理。(1)从药材综合利用的角度，进行了白首乌不同药用部位的不同提取物体外抗肝癌活性比较及其Ｃ21甾苷类重要成分的定量分析研究。结果表明，白首乌的抗肿瘤活性可能与其C21甾苷类成分含量密切相关，白首乌去皮的块根有较强的体外抗肿瘤活性，各提取部位的作用均明显强于根皮相对应提取部位，但作为生产废弃物的根皮中含有一定量的活性成分，因而也有抗肿瘤活性，将其作为废弃物处理会造成资源浪费。(2)通过整体动物实验研究了白首乌C21甾苷类组分对肝脏炎症-肝纤维化-肝癌轴的干预作用及其机理。采用二乙基亚硝胺构建大鼠肝脏炎症-肝纤维化-肝癌动态模型，分时段动态观察白首乌C21甾苷类组分对肝脏炎症、肝纤维化、肝癌的干预作用，评价药物疗效。结果发现，白首乌总苷对二乙基亚硝胺诱导的大鼠肝脏炎症-肝纤维化-肝癌动态模型的干预作用体现在保肝、抗肝纤维化和防治肝癌等方面，其功效作用可能与其下调TLR4、MyD88、TRAF6、TGF-β1及α-SMA的蛋白和基因表达、抑制NF-κB活化及抑制炎症因子TNF-α和IL-6的释放有关。(3)体外实验研究了白首乌C21甾苷类组分干预肝脏炎症-肝纤维化-肝癌轴的分子机制。分别选用人正常肝细胞株L02、人肝星状细胞株HST-LX2以及人肝癌细胞株HepG2作为细胞模型，研究了白首乌C21甾苷类组分对人正常肝细胞损伤、肝星状细胞活化和胶原产生以及肝癌细胞增殖和凋亡的干预作用。结果发现白首乌C21甾苷类组分体外能明显保护肝细胞，减轻细胞损伤，抑制炎症因子IL-6、TNF-α的释放，抑制人肝星状细胞的活化和胶原分泌，下调TLR4、MyD88、TRAF6的基因和蛋白表达，并且能明显抑制人肝癌细胞增殖，诱导其凋亡。体外共培养研究发现，人肝星状细胞能明显促进肝癌细胞的增殖和侵袭，而白首乌C21总甾苷对其有一定的抑制作用，而这与下调TLR4、MyD88、TRAF6的蛋白表达及抑制核因子NF-κB的激活有关。