贵州苗族“消疤草”抗肝纤维化的活性成分及作用机制研究

Liver fibrosis is the leading cause of the development of a variety of chronic liver diseases to cirrhosis and the death in patients with liver disease. Prevention or reverse of liver fibrosis is an important means to improve the prognosis of patients with liver diseases, effective prevention and treatment of cirrhosis. The search for safe, effective and inexpensive anti-fibrotic drugs has become a hot topic in the area of liver disease research. Inspired by the "homotherapy for heteropathy" theory of traditional Chinese medicine, we experimentally confirmed, Guizhou Miao's folk medicine "Xiao Bao Cao (herb for scar elimination)" can inhibit the formation of extracellular matrix (ECM) and promote ECM degradation, effectively preventing hepatic fibrosis in rats. This project is intended to determine the active anti-fibrosis ingredient, clarify the material basis of its anti-hepatic fibrosis effect by employing a multi-factor-induced complex hepatic fibrosis pharmacological model in rats and with bioassay-guided fractionation and isolation. In addition, with immunohistochemistry, RT-PCR and Western blot techniques, investigations will be conducted to examine the effect of Xiao Bao Cao's active fractions and chemical entities on serum biochemical indicators, liver fibrosis biomarkers, liver pathology, as well as the activation and apoptosis of hepatic stellate cells and their impact on signal transduction pathways. The outcome will clarify, at the levels of whole animal, cell, and molecule, the mechanism of action of Xiao Bao Cao for its anti-liver fibrosis effect, paving the way for its development and applications.

肝纤维化是各种慢性肝病向肝硬化发展、导致肝病患者死亡的主要原因。阻止或逆转肝纤维化是改善肝病患者预后、有效防治肝硬化的重要手段，寻找安全、有效、价廉的抗肝纤维化药物已成为当前肝病研究的热点。受启于中医"异病同治"理论，我们通过实验证实，贵州民间苗药"消疤草"能较好的抑制细胞外基质（ECM）形成、促进ECM降解，有效预防大鼠肝纤维化。本课题拟以多因素诱导复合肝纤维化大鼠为药理模型，采用活性跟踪导向的分离技术和现代波谱鉴定手段，确定"消疤草"的活性成分，明确其抗肝纤维化的物质基础；运用免疫组织化学、RT-PCR及蛋白免疫印迹等技术，通过考察"消疤草"及其活性成分对大鼠血清生化、肝纤维化标志物、肝组织病理，以及肝星状细胞活化、凋亡及其信号转导通路等的影响，从整体、细胞、分子水平阐明"消疤草"抗肝纤维化作用及其机制，为"消疤草"及其活性化合物的开发利用提供科学依据。

项目背景：肝纤维化是各种慢性肝病向肝硬化发展、导致肝病患者死亡的主要原因。阻止或逆转肝纤维化是改善肝病患者预后、有效防治肝硬化的重要手段，寻找安全、有效、价廉的抗肝纤维化药物已成为当前肝病研究的热点。受启于中医“异病同治”理论，我们通过实验证实，贵州民间苗药“消疤草”能较好的抑制细胞外基质（ECM）形成、促进ECM降解，有效预防大鼠肝纤维化。主要研究内容：本课题以多因素诱导复合肝纤维化大鼠为药理模型，运用现代科学研究方法，从整体、蛋白、细胞、分子水平对“消疤草”抗肝纤维化的药效学及作用机理进行研究。并在此基础上，采用活性跟踪导向的分离技术和现代波谱鉴定手段，明确“消疤草”抗肝纤维化的药效物质基础，从中发现活性单体化合物，初步探讨其作用机制。重要结果与关键数据：1、明确“消疤草”种属鉴定为罂粟科紫堇属植物地锦苗；2、明确“消疤草”对抗肝纤维化有很好的预防及治疗作用，并初步探讨了其作用机制可能为抑制 Smad3活性、上调 Smad7表达来阻断TGF-β1表达信号通路，从而抑制肝星状细胞（HSC）的活化来减少 ECM的形成；3、从“消疤草”中分离得到6个有效化合物，分别鉴定为：①原阿片碱、②β-卡波林、③降氧化北美黄连次碱、④对乙氧基乙酰苯胺、⑤4-甲基-3,6-二氢吡啶-2-酮、⑥去氢催吐萝芙木醇。其中化合物2-6为首次从该植物中分离得到；4、明确了“消疤草”提取物“原阿片碱”对肝星状细胞（LX-2）的抑制作用，可诱导LX-2细胞发生早期及晚期凋亡，可降低Smad3、a-SMA 和Collagen Ⅲ 的蛋白表达量，亦可使MMP-2蛋白的表达量增高。意义：为“消疤草”的开发利用奠定基础，为寻找新型治疗肝纤维化药物的研究提供了方向，对苗族地道药材的开发具有重要意义。