化痰法抗肝纤维化作用及细胞内信号转导通路研究

The intervention of hepatic fibrosis is the focus of prevention and treatment on cirrhosis. According to traditional Chinese medicine, that "retention of phlegm and blood stasis in meridian, deficiency of vital Qi" is the basic pathogenesis of this disease. Under the guidance of this basic pathogenesis theory, it's necessary to start experimental research on the effect and mechanism of "the resolving phlegm" for its anti-hepatic fibrosis. Based on preliminary studies, this paper intended to take the phlegm-based medicine "Semen Sinapis Albae" as the main herb which accredited to " eliminate the phlegm below the costal regions, subcutaneous and in the pleurodiaphragmatic space", through the compatability of DANSHEN, E'ZHU and HUANGQI to make up different kinds of treatments. The experiment was divided into animal tests and cell cultures, to intervene rats model of liver fibrosis and rat hepatic stellate cell lines(HSC-T6), by means of HSC proliferation, detection of Hyaluronic acid (HA)、 Laminin (LN)、 pcⅢ-N and TGF-β1、Smad4、NF-κB、FAK and other cytokines in each group, the expression of TGF-β1mRNA、Smad4 mRNA、NF-κB mRNA、FAK mRNA and the change on ultrastructure of the liver and histopathology, combining with the periodic distribution and state of apoptosis in rat hepatic stellate cells, discussing its major role and its mechanism in liver fibrosis, exploring the cell biology support for its basic pathogenesis,deepening the cognition of " retention of phlegm ", and providing experimental basis for the therapy of anti-hepatic fibrosis on phlegm.

肝纤维化的干预阻断是肝硬化防治工作的重点。我们基于中医有关本病"痰瘀阻络、正气不足"基本病机的认识，从 "化痰"角度入手，在前期研究的基础上，拟以"治胁下及皮里膜外之痰的'白芥子'"为主药，通过其与丹参、莪术、黄芪等配伍组成的"化痰法"，分动物实验和细胞培养两部分，对肝纤维化大鼠模型及大鼠肝星状细胞系HSC-T6分别进行干预。通过检测各组的透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原-N及TGF-β1、Smad4、NF-κB、FAK等细胞因子、TGF-β1mRNA、Smad4mRNA、NF-κB mRNA、FAK mRNA的表达和肝脏超微结构、病理组织学变化，结合鼠肝星状细胞周期分布和凋亡情况,初步探讨"化痰法"抗肝纤维化的作用及其主要机制，寻求肝纤维化基本病机的细胞生物学支持，深化对肝纤维化"痰瘀阻络、正气不足"基本病机中"痰阻"的认识，为肝纤维化从"痰"论治提供实验依据。

本项目在对肝纤维化基本病机全面梳理认识的基础上，认为肝纤维化发生、发展的过程中“痰、瘀、虚”贯穿于疾病的始终。在本病的治疗中“化痰” 与“活血化瘀”、“扶养正气”同等重要，不可偏废。鉴于既往的研究多从“虚、瘀”展开，故本研究侧重从“痰阻”病机角度出发，将“化痰”作为该病的主要治法之一，以化痰中药“白芥子”为载体，通过其与活血化瘀的丹参、补气的黄芪、行气活血的莪术等配伍组成的“化痰法”（白芥子药组），通过动物实验和细胞培养探讨其抗肝纤维化的作用和机制。以稽从不同侧面对“化痰”在治疗肝纤维化中的作用进行探索。在体水平，化痰法可改善大鼠肝功能，明显降低肝纤维化大鼠血清肝纤维化指标PⅢ-NP浓度，同时HE及Masson染色显示化痰法明显改善肝纤维化大鼠形态学改变，减少肝脏细胞的脂肪变性，汇管区炎性细胞浸润，减少肝脏组织中胶原纤维的沉积，除此之外，化痰法不同程度降低显著降低肝纤维化大鼠肝脏组织Collage Ⅰ、Collage Ⅲ蛋白表达。离体水平，化痰法含药血清明显降低肝星状细胞系HSC-T6细胞增殖率，促进其凋亡与坏死，同时化痰法含药血清降低肝星状细胞系HSC-T6细胞Collage Ⅰ、Collage Ⅲ蛋白水平及colla1、colla3基因表达的影响 mRNA水平。说明化痰法在在体与离体水平，均表现出明显的抗肝纤维化作用。化痰法作用于TAA诱导的肝纤维化大鼠，大鼠血清 CK、LDH、iNOS 水平明显低，SOD、GSH-Px、NO、cNOS、TNOS 活性升高，不同程度降低显著降低肝纤维化大鼠肝脏组织α-SMA、p-Smad 2,3 、Smad 2,3 、TGF-β1蛋白表达水平，升高TGF-β1, Smad4, acta2, cdh1, mRNA水平；化痰法含药血清降低肝星状细胞系HSC-T6细胞TGF-β1、p-Smad 2,3 、Smad 2,3 、Smad4 、p-NFκB、NFκB、p-AKT、AKT蛋白水平。从离体和在体水平说明化痰法抗肝纤维化的作用可能与减少氧化应激，调节TGF-β1 /Smad 、NFκB、AKT信号转导通路，抑制HSCs的活化，从而减少细胞外基质过度沉积有关。