基于细胞间相互作用解析扶正化瘀方抗肝纤维化的效应基础

Based on the explicit anti-fibrosis curative effect of traditional Chinese herbal formula Fuzheng-huayufang (FZHY) on cirrhosis patients and its in-depth basic researches previously, which has passed phaseⅡ clinical trials in United States, we proposed the hypothesis "focus on the main active components discovery in traditional Chinese medicine formula effectively regulating the complex pathology of liver fibrosis can resolve the main active ingredients compatibility principle and it’s mechanism of action (MOA) of anti-fibrosis". Cconsidering the complex pathological mechanism in liver fibrosis and the key role of bidirectional transmission between liver sinusoidal endothelial cell (LESCs) injury and hepatic stellate cells (HSCs) activation in pathopoiesis and progression of liver fibrosis, we propose a meaningful research project as follows: To analyze the changes of the contents of main components from FZHY in the samples of different tissues after oral administration to analysis the active components; Separating both primary LESCs and HSCs, and co-culturing those cells in vitro to screen the main effective components with High Content Screening; basing on the systematic analysis in filter results to develop the combination among active ingredients, verify the anti-fibrosis efficacy on liver fibrosis model using uniform design to find the effective formula; then adopting and analyzing on CCl4 and bile duct ligation models with specific gene knockout in LESCs which regulates the regeneration and fibrosis in liver; co-culturing the LESCs and HSCs with active components to explore the synergistic effect mechanism of main components; our results will clarify the main active compounds and anti-fibrosis MOA of FZHY in vitro and in vivo based on the influence in pathomechanism between LESCs injury and HSCs activation. Our research will promote FZHY internationalization.

本课题针对肝纤维化研究的新进展与中药复方抗肝纤维化的优势及存在的问题，提出“发现中药复方复杂成分主要效应化合物的组方是研究其有效调节纤维化复杂病理改变的前提，针对其核心病理变化可解析主要有效成分复方配伍原理”的假说。拟以疗效确切并已通过美国Ⅱ期临床试验、前期基础研究较扎实的FZHY方为对象，以其药效物质基础及对肝窦内皮细胞(LESCs))损伤与肝星状细胞(HSCs)活化之间双向信号传递机制影响为切入点，通过①基于体内过程的药效物质研究；②经LSECs去分化、HSCs活化及其相互作用的活性成分高内涵筛选；③均匀设计和验证实验获得最佳有效成分新复方并解析配伍效应；④新成分复方调控LSECs-HSCs相互作用的抗肝纤维化协同配伍效应机制的解析等四步分层次研究，以期明确该方抗肝纤维化对LESCs损伤与HSCs活化之间相互作用病理机制影响的主要物质基础及其效应的关键通路。为推进其国际化提供坚实基础。

中药复方极其复杂多样的化学成分与抗肝纤维化的综合调控作用，既是中医药的特色优势，也是明晰其效应基础的难点，尤其是针对纤维化复杂的病理生物学变化，如何深入解析复杂成分有效调控复杂病理改变的关键效应基础，已成为阐释中医药治疗复杂性疾病科学理论的重要突破口。本项目以长期临床应用证实有效抗肝纤维化的扶正化瘀方（FZHY）为载体，采用四段式(提取物-门静脉血-肝脏-颈静脉血)方法明晰FZHY多成分在体内关键部位的分布，结合药效筛选，确定其在体内起作用的药效成分群；采用均匀设计，进行两种模型的整体试验及再验证，发现有效成分组合JY5（F01、F02和F03三成分组合）与PA（F01和F02两成分组合）；解析了主要成分组合PA影响细胞相互作用的协同配伍作用机制；证实F01主要作用于肝细胞，F02主要作用于肝星状细胞（HSCs），ECM1是F01发挥抗肝纤维化作用的关键靶标之一，CDK9是F02发挥抗肝纤维化作用的关键靶标；发现F-03主要作用于肝窦内皮细胞（LSECs），可通过调控HIF-1α/CXCR4/PDGF/CXCR7通路，干预LSECs去分化、进而抑制HSCs活化。明确本项目的研究策略是“解决中药复方复杂成分治疗肝纤维化复杂病理变化的效应基础”这一重要科学问题的可行路径。已完成预期研究计划目标，为阐释中医药治疗复杂性疾病科学理论提供借鉴，为推动中药复方创新研究提供部分示范与参考。虫草菌丝是FZHY方的主药，其成分最为复杂，既往的复方体内成分分析尚未明晰其主要有效成分，针对这一难点，本项目采用色质联用技术结合活性导向的成分分离方法，成功发现虫草菌丝抗肝纤维化主要活性成分C02。解决FZHY方中虫草菌丝活性物质的技术瓶颈问题。