蒙药尖叶假龙胆木脂素类成分抗肝纤维化的药效物质与作用机制研究

Gentianella acuta (Michx.) Hulten, a Mongolian medicine, is used for the treatment of a variety of liver diseases. In the preliminary study on its chemical consistituents, the research group found that lignans from this plant had anti-fibrosis effects.On this basis, this study intends to systematically evaluate the anti-fibrosis effect of lignans in rats with alcoholic liver fibrosis and Find the direct substances in lignans by serum pharmacochemistry;The biomarkers of hepatic fibrosis will be found by metabolomics and the related metabolic pathways will be analyzed. PCMS software will be used to associate the substances directly acting in the body with the biomarkers to evaluate the anti-liver fibrosis activity intensity of lignans and to clarify the struck-activity relationship of lignans with different structures. Then, lignin monomers which were obtained in previous studies were be compared to determine the anti-liver fibrosis efficacy of lignans. HSCs-T6 cellS model induced by acetaldehyde will be used to determine the relevant indicators of TGF- 1/Smads signaling pathway by MTT, PCR and Western blot, and the anti-fibrosis mechanism of LC will be elucidated at the cellular level.The pharmacodynamic substances and mechanism of LC against liver fibrosis will be elucidated from the level of substances in vivo-metabolism-cells, which will provide a scientific basis for the further application of a kind of Mongolian medicine named Gentianella acuta (Michx.) Hulten in the treatment of liver diseases.

蒙药尖叶假龙胆在蒙医中用于多种肝病的治疗，课题组前期对其进行化学成分研究中发现木脂素类成分（LC）具有抗肝纤维化作用。在此基础上，本研究拟以酒精性肝纤维化大鼠模型，系统评价LC抗肝纤维化作用；运用血清药物化学方法确定LC的体内直接作用物质；采用代谢组学方法发现肝纤维化生物标记物并分析相关生物代谢途径；应用PCMS软件将体内直接作用物质与生物标记物相关联，评价LC抗肝纤维化活性强度，并阐明不同结构LC的构效关系，再以前期研究得到的木脂素单体进行比对，确定LC中抗肝纤维化的药效物质；以活性较强的单体成分为代表，采用乙醛诱导的HSCs-T6细胞模型，应用MTT、PCR和Western blot方法测定TGF-β1/Smads信号通路的相关指标，在细胞水平阐明LC抗肝纤维化作用机制。从体内物质-代谢-细胞水平阐明LC抗肝纤维化的药效物质和作用机制，为蒙药尖叶假龙胆治疗肝病的进一步应用提供科学依据。

尖叶假龙胆被现代药理研究证明具有治疗肝纤维化的作用，疗效确切。但其抗肝纤维化的药效物质及作用机制，有待进一步阐明。本项目首先采用大孔树脂柱，聚酰胺柱等分离手段，通过甲醇、乙醇、水等溶剂洗脱分段，得到木脂素有效部位，并确定了得到的木脂素单体的结构。再以优化后的“白酒-玉米油-吡唑”结合高脂饲料法制备肝纤维化模型，对有效部位治疗肝纤维化的治疗作用进行了有效性评价；应用中药血清药物化学方法对有效部位抗肝纤维化有效状态下的血中移行成分进行表征及结构鉴定，结果表征了 65 个体外成分。基于有效部位有效治疗肝纤维化模型大鼠，从大鼠血清中表征了 19 个原型入血成分，以及 466 个代谢产物；采用代谢组学方法，发现并鉴定肝纤维化模型大鼠血液代谢生物标记物 50 个，正离子模式下36个，负离子模式下14个；涉及15条与肝纤维化模型相关的代谢通路。其中有效部位可回调17个，均具有统计学意义，涉及6条代谢通路。采用乙醛诱导的 HSCs-T6 细胞模型，应用 MTT 和 Western blot 方法测定 TGF-β1/Smads 信号通路的相关指标。发现有效部位能降低HSCT6增殖，并在TGF-β1/Smad信号通路中，有效部位能降低受体型Smad3和通用型Smad4的表达，增加抑制型Smad7的表达，从而抑制了TGF-β1/Smads信号通路，进而抑制了乙醛对HSCT6的增殖。将中药血清药物化学、代谢组学研究和细胞实验研究相结合，证明有效部位治疗肝纤维化的药效物质基础及作用机制。为蒙药尖叶假龙胆治疗肝纤维化的进一步应用提供科学依据，可为的开发利用提供数据支撑和科学依据。