基于谱-酶抑制信息追踪六味地黄丸协同作用于葡萄糖醛酸苷酶有效成分组及其组合给药预防肝癌机理

Elucidating the potentially synergic effects between ingredients in traditional Chinese medicine (TCM) on drug target and therapeutic efficacy in vivo is crucial for its modernization. We previously identified a novel ingredient - D-glucaro-1,4-lactone（1,4-GL） in Liuwei Dihuang Pill (LWPs) based on metabonomic technology, which is a specific inhibitor to β-glucuronidase (β-GD) and exerts preventive properties against hepatocellular carcinoma. Furthermore, we supposed that there exist any other compounds besides 1,4-GL inhibiting β-GD in LWPs from the experimental results in vitro. Therefore, in this project, we intended to identify 2-3 compounds in LWPs which play a synergic role in inhibiting with 1,4-GL on the basis of the information on HPLC profiling of LWPs – enzyme（β-GD）inhibitory effectiveness. Next, various combinatorial drugs composed of these identified compounds and 1,4-GL would be administered orally to rats which would have been treated by diethylnitrosamine (DEN) as a model of hepatocellular carcinoma. Alterations of serum alpha-fetoprotein (AFP) and the metabolic profilings based on 1HNMR would be employed to evaluate the preventive efficacy of each combinatial drug. As a result, the multi-component drug with synergically preventive property against hepatocellular carcinoma in vivo could be confirmed. The key enzyme related to alterations of metabolites after the combinatorial drug administration would be predicted via quantification of the metabolites in biofluid (serum and urine) associated with energy and bile acid metabolic pathway. Then, the mechanism on preventing hepatocellular carcinoma with the combinatorial drug in vivo could be investigated by molecular biology and metabonomics technologies.

阐明中药成分组对靶点及体内药效的协同作用是中药研究的关键科学问题。本课题前期工作通过代谢组学技术鉴定了六味地黄丸（LWPs）中具有预防肝癌作用的新成分葡萄糖二酸1,4-内酯（1,4-GL），一种β-葡萄糖醛酸苷酶（β-GD）抑制剂，而且，据体外实验结果推测LWPs还含有其他抑制β-GD的化合物。因此，本项目拟利用色谱指纹图谱-酶抑制相关性信息追踪LWPs中与1,4-GL协同作用于β-GD的有效成分组。分离鉴定2-3个与1,4-GL协同抑制β-GD的化合物。以二乙基亚硝胺诱导大鼠为肝癌模型，给予不同配伍组合药物，利用血液甲胎蛋白水平变化及代谢组学技术整体评价上述化合物与1,4-GL配伍是否存在药效协同作用，获得协同预防肝癌的组合药物。定量测定给予此组合药物后动物体液能量代谢和胆酸代谢途径代谢物的含量，则可推测影响代谢物变化的关键代谢酶。结合分子生物学和代谢组学技术探讨组合药物预防肝癌机理。

阐明中药复方中的有机小分子体内药效的协同作用，是中药药效物质基础研究的关键和难点。本项目以六味地黄丸中葡萄糖二酸1，4内酯（1，4-GL）为基础，利用谱-效相关性，分离鉴定了丹皮酚、没食子酸和5-羟基糠醛（5-HMF）等水溶性化合物与β-葡萄糖醛酸苷酶（β-GD）活性相关，除1，4-GL外，5-HMF体外具有最高的（15-20%）的β-GD活性抑制作用。建立了体内嘌呤、胆碱和氨基酸代谢途径大约30种代谢物的色谱、质谱检测方法，并应用于肝癌患者或DEN诱导的肝癌大鼠血清和肝脏中代谢物的测定。结果显示，肝癌大鼠血清甲胎蛋白、肝损伤生化指标、脂质过氧化指标等水平显著上调，Fisher比率和抗氧化指标显著下降，嘌呤代谢通路代谢紊乱，血清和肝脏中尿酸含量显著上升，黄嘌呤氧化酶（XOD）mRNA和活性显著上升；1,4-GL干预后，肝癌大鼠肝组织病理切片可见肝脏细胞排列较规整，但大小不均，异型细胞减少，周围纤维结缔组织减少，成癌率由100%下降到20%；药物代谢组学结果显示部分代谢物含量明显回归，上述血清指标均回归正常水平或有回归正常的趋势，逆转了肝癌大鼠血清和肝脏中XOD活性、mRNA表达等；体外细胞实验亦显示1,4-GL作用于肝癌细胞（HCC-LM3）后，XOD mRNA和活性下调，细胞内尿酸等代谢物明显下降等。另外，体内实验证实1,4-GL和5-HMF在预防DEN诱导肝癌方面药效上具有一定的协同作用，其协同作用机理与1,4-GL和5-HMF在下调XOD基因表达方面具有协同作用相关。1,4-GL和5-HMF成为新的2组合预防肝癌药物值得进一步验证。总之，本项目结果证实1,4-GL（或联合5-HMF）可以通过提高机体抗氧化和抗脂质过氧化能力，调节嘌呤化合物代谢及相关酶如XOD活性、mRNA表达等减少尿酸生成和堆积，从而达到预防或延缓肝癌进展的作用。尿酸代谢的紊乱可能促进一些疾病发生发展如痛风和阿尔茨海默症，到目前为止，有关尿酸代谢紊乱与肝癌进展等方面研究非常少，本项目结果提示靶向调控尿酸代谢可能作为肝癌预防和治疗新的药物靶点，具有重要的科学意义，值得进一步研究。