当归补血汤有效组分抑制肝星状细胞活化的分子机制研究

Activation of hepatic stellate cells (HSC) is the key effectors in hepatic fibrogenesis. Oxidative stress and transforming growth factor (TGF)-β1 are important factor for regulating HSC activation. Danggui Buxue Decoction is effective for patients with hepatic fibrosis, and its active ingredients are almost conclusive. However, little is known about the interaction between effective components of Danggui Buxue Decoction and target molecules. In view of previous our findings, we favor the hypothesis that Astragaloside IV, the active component of Astragali radix, suppresses oxidative stress of activated HSC via GSK-3β/Nrf2 signal pathway, and Ferulaic acid, the active component of Angelica sinensis, down-regulate extracellular matrix-related gene expression by TGF-β1/Smad signal pathway. Astragaloside IV and Ferulaic acid have synergistic effects on inhibition of HSC activation. To test this hypothesis, this project use chronic carbon tetrachloride- and bile duct ligation-induced hepatic fibrosis rats, and culture-activated HSC as the study object, and the quantitative pharmacology method for evaluating synergistic effects of Astragaloside IV and Ferulaic acid on inhibition of HSC activation. The underlying the mechanisms of its action will be explored in the animal, cell and molecular levels. Thus, this project will expand the scientific connotation of potentiation and assistanceof Traditional Chinese medicine, and offer the scientific basis for development of Traditional Chinese medicine components.

肝星状细胞（HSC）活化是肝纤维化发生的病理关键，氧化应激和转化生长因子β1（TGF-β1）是调节HSC活化的重要因素。当归补血汤抗肝纤维化的临床疗效确切，药效物质基本清楚，但当归补血汤有效组分与其作用靶标分子间的相互作用规律尚不清楚。据此，我们基于前期研究，提出黄芪有效组分黄芪甲苷通过GSK-3β/Nrf2信号通路抑制HSC氧化应激，而当归有效组分阿魏酸通过TGF-β1/Smad信号通路下调胶原合成酶表达，黄芪甲苷联合阿魏酸具有协同抑制HSC活化作用的科学假说。为了验证假说，本项目拟采用CCl4和胆管结扎诱导的肝纤维化动物模型和体外培养活化的HSC细胞模型，应用定量药理学方法评价黄芪甲苷和阿魏酸配伍抗肝纤维化和抑制HSC活化的协同作用。从动物、细胞、分子水平研究当归补血汤有效组分抑制HSC活化的分子机制。从而为揭示黄芪-当归相须相使配伍理论的科学内涵和研制组分配伍创新药物提供科学依据。

肝星状细胞活化是肝纤维化发生的病理关键，氧化应激和转化生长因子β1是调节肝星状细胞活化的两个重要因素。当归补血汤抗肝纤维化临床疗效确切，但其抗肝纤维化作用的有效组分和配伍机制尚不清楚。本资助项目按计划在体内和体外探讨黄芪甲苷（黄芪的有效成分）和阿魏酸（当归的有效成分）联合抗肝纤维化、抑制肝星状细胞活化和抗氧化的作用。探讨黄芪甲苷和阿魏酸联合对糖原合成酶激酶3β/ Nrf2信号传导通路和转化生长因子β/Smad信号传导通路的调控作用。我们研究发现：黄芪甲苷联合阿魏酸显著改善大鼠胆汁淤积性肝纤维化，用Compusyn计算软件分析显示体外黄芪甲苷和阿魏酸联合具有协同效应。黄芪甲苷与阿魏酸协同抑制HSC-T6细胞和胆汁淤积性肝纤维化大鼠肝脏组织肝星状细胞活化。黄芪甲苷显著抑制HSC-T6细胞和胆汁淤积性肝纤维化大鼠肝脏组织氧化应激，其机制与诱导Nrf2核转位、上调II相抗氧化酶基因表达和抑制糖原合成酶激酶3β活性有关。阿魏酸对HSC-T6细胞和胆汁淤积性肝纤维化大鼠肝脏组织氧化应激、Nrf2核转位、II相抗氧化酶基因表达和糖原合成酶激酶3β磷酸化无显著性影响。阿魏酸显著抑制HSC-T6细胞和胆汁淤积性肝纤维化大鼠肝脏组织胶原、转化生长因子β1及其受体、Smad4和Smad3表达。黄芪甲苷对HSC-T6细胞和胆汁淤积性肝纤维化大鼠肝脏组织胶原、转化生长因子β1及其受体、Smad4和Smad3表达无显著性影响。黄芪甲苷和阿魏酸联合在体外通过非依赖的方式活化Nrf2和抑制转化生长因子β1表达，p38MAPK在黄芪甲苷与阿魏酸联合应用体外抑制肝星状细胞活化中具有重要作用。本资助项目的研究结果阐明黄芪甲苷和阿魏酸联合抑制肝星状细胞活化的分子机制，揭示黄芪当归相须相使配伍理论的科学内涵，为研制组分配比创新药物提供科学依据。本资助项目研究结果发表论文6篇，其中SCI收录论文3篇。