基于瘀热病机研究紫七软肝方对肝硬化进程中TLR4 /NF-κB通路的调控机制

Based on the clinic experience of professor ZHOU zhong-ying in treating hepatitis and cirrhosis and the proved prescription of Ziqi Ruangan recipe,previous clinical and experimental research demonstrated that stasis-heat is the key pathogenesis and the network of inflammatory and immune response is important in the progression of cirrhosis,and Ziqi Ruangan recipe can regulate tgf-βsmads signaling pathway and effectively inhibit the proliferation and activation of HSC-T6.However, the upstream mechanism underlying such regulation remains unclear. Based on the previous study,cirrhotic rat model made by CCL4 and high-fat diet,fibrosis rat model prepared by knocking out the gene of TLR4,HSC activation model interfered by KC in vitro will become incorporated into this study.The project is designed to probe the biological foundation which is based on TLR4/NF-κB signaling pathway of Ziqi Ruangan recipe and its disassembled prescriptions in preventing and treating hepatic fibrosis in vitro and in vivo. This research may reveal the biological basis of Ziqi Ruangan recipe intervening the progression of cirrhosis from multiple perspectives,the Identity and difference between Fuzheng Huayu and Ziqi Ruangan recipe,finally provide the scientific evidence of stasis-heat pathogenesis and improving the theory and clinical level of TCM in the prevention and treatment of liver cirrhosis.

基于瘀热病机广泛存在于多种外感内伤急难重症过程中，在前期多项研究中揭示了在肝炎肝硬化进程中多以湿热瘀毒互结为基本病机，并以瘀热病机为关键环节，免疫及炎症反应网络机制是其重要发病学基础，周仲瑛教授辨治肝硬化经验方“紫七软肝方”对TGF-β/Smads表达具有调控作用，有效抑制HSC-T6增殖活化，但其上游机制尚未完全阐明。在前期研究基础上，通过体内和体外实验，采用CCL4+高脂饲料复合因素致大鼠肝硬化模型及体外KC干预的HSC活化模型，考察紫七软肝方及拆方及其含药血清防治肝硬化的生物学基础，基于TLR4/NF-κB信号通路深度解析其作用靶点和分子机制。本项目研究将为今后进一步更为深入的研究奠定前期工作基础，进而从多环节、多角度揭示紫七软肝方干预肝硬化形成和加重进程效应机制的现代医学表达，及其与扶正化瘀法的同一性和差异性，诠释肝硬化以瘀热为核心病机的科学内涵，为提高中医药防治肝硬化疗效提供科学依据。.项目任务书依据专家建议,对项目申请书实验方案作出调整，围绕关键科学问题进行针对性设计，为今后继续研究奠定基础。

本项目旨在基于国医大师周仲瑛教授学术经验，围绕“湿热瘀毒复合病机是肝硬化形成和加重进程中的始动因素和关键环节”科学假说，开展中医复合病机新理论科学内涵的创新性研究。在前期已经相继开展了肝炎-肝硬化进程中湿热瘀毒复合病机的理论基础、临床证候演变规律以及清化湿热瘀毒法效应机制的实验研究的基础上，本项目以“肝炎－肝纤维化－肝硬化”进程中免疫炎症网络机制为切入点，进一步研究了以清化湿热瘀毒法为核心的紫七软肝方对TLR4/NF-κB和MAPK信号通路的调控作用。通过体内和体外实验发现：①紫七软肝方能够抑制LX-2细胞活化；②紫七软肝方对LX-2活化过程中TLR4/NF-κB和MAPK信号通路有抑制作用；③通过阻断TLR4信号通路可以切断其下游NF-κB和MAPK双重信号通路，从而抑制LX-2细胞的活化；④紫七软肝方能够减轻“CCl4+高脂饲料”诱导的大鼠肝纤维化程度；⑤紫七软肝方及拆方对“CCl4+高脂饲料”所致大鼠肝纤维化各时相均有不同程度的调控作用，且与扶正化瘀胶囊相比具有同一性和差异性。同期还围绕名医从瘀热辨治肝硬化合并肝癌病案数据分析，发现湿热瘀毒病机同样存在于肝硬化合并肝癌病程中。这些研究揭示了瘀热病机贯穿于肝硬化形成和加重过程始末，紫七软肝方对肝硬化形成和加重过程中的免疫及炎症反应网络具有较强的调控作用，为中医从瘀热复合病机辨治肝硬化提供科学依据，以期将研究成果向临床实用转化。