基于比较代谢组学研究二妙丸类方抗高尿酸血症整体作用机制与药效物质基础

Ermiao Wan, Sanmiao Wan and Simiao Wan are the commonly used prescriptions in treatment of gout induced by hyperuricemia (HUA) in clinic. The current efficacy evaluation could not comprehensively and objectively reflect their overall mechanism of action and clinical benefit. Our previous research of metabonomics has shown that the metabolic profiling of HUA animal models orally administrated with Simiao Wan headed back to the normal level, exhibiting the different corrective actions of the whole metabonomics from the chemical drug allopurinol. Based on the HUA animal models, the method of metabonomics combined with random forest and other chemometrics methods are applied for multivariate statistical analysis of the metabonomics data of Ermiao Wan and associated prescriptions, screening the biomarkers and metabolic pathways influenced by different prescriptions. Then, a quantitative index for the evaluation of the corrective actions the whole metabonomics can be established, and the overall metabolism mechanism of different prescriptions will be compared. With the method of serum pharmacochemistry, the substances in the serum of HUA animal models orally administrated with different prescriptions are respectively elucidated, and their corresponding prototypes in the prescriptions are regarded as the potentially effective constituents. Then the established technology of chemical constituents fishing and knockout is applied for preparing of any interested peak(s) of potentially effective constituents and the knockout portions. The corrective actions of the whole metabonomics of different samples are evaluated based on the HUA animal models, and the effective constituents are screened out according to the evaluation feedback. Finally, the effective components of Ermiao Wan and associated prescriptions can be elucidated and their prescription composing rules can be illustrated.

二妙丸、三妙丸和四妙丸是临床治疗高尿酸血症所致痛风病的常用良方，但现有药效评价方法不能全面客观反映其整体作用特点与临床优势。前期代谢组学研究结果显示四妙丸能使高尿酸血症大鼠代谢轮廓整体回调，表现出与西药别嘌呤醇不同的代谢纠偏效应。本项目拟基于高尿酸血症大鼠模型，运用代谢组学的方法并结合用随机森林等化学计量学手段对二妙丸类方代谢组学数据进行多元统计分析，筛选不同方剂影响的生物标志物和代谢通路，构建反映整体代谢纠偏作用的代谢组学量化指标，比较二妙丸类方的整体代谢作用机制。进一步采用血清药物化学方法，追溯入血成分群对应的复方原型成分群为候选活性成分群，运用“化学成分缺失/捕获”技术制备候选成分（群）样品及其敲除的提取物样品，基于建立的代谢组学量化指标，评价候选成分（群）在整体代谢纠偏效应中的贡献，反馈筛选调控整体代谢的活性成分群，阐明二妙丸类方抗高尿酸血症的药效物质基础与组方科学内涵。

高尿酸血症（HUA）是由于体内嘌呤代谢紊乱，尿酸生成过多或排泄减少所致血尿酸水平升高的代谢性疾病，是诱发痛风的生化基础和直接病因，已成为危害人类健康的重要代谢性疾病之一。二妙丸类方（主要指二妙丸、三妙丸和四妙丸）是临床治疗高尿酸血症所致痛风病的常用良方。现有基于生化指标的药效评价方法不能全面客观反映其整体作用特点与疗效优势，其降尿酸作用机制还有待系统阐明。本项目首先采用色谱质谱联用技术，分别对二妙丸类方的体内外化学物质基础进行了研究。然后基于高果糖摄入联合氧嗪酸钾腹腔注射构建高尿酸血症大鼠模型，开展二妙丸类方抗高尿酸血症的药效研究，研究结果显示，二妙丸类方能使血浆中尿酸和肌酐水平明显降低，尿液中尿酸排泄量明显增加，并能缓解高尿酸血症引起的肝、肾组织病理性损伤。基于UPLC-Q-TOF/MS技术，开展了二妙丸类方对高尿酸血症大鼠代谢紊乱的整体调控作用研究。运用代谢组学技术并结合随机森林化学计量学手段对二妙丸类方代谢组学数据进行多元统计分析，筛选二妙丸类方影响的潜在生物标志物和代谢通路，确定反映整体代谢纠偏作用的潜在内源性代谢标志物，研究二妙丸类方的整体代谢作用机制。研究共筛选和鉴定出与高尿酸血症相关差异性代谢物35个。二妙丸类方对高尿酸血症相关失衡代谢通路的影响呈现出同中有异的特点，提示由于组方不同，其抗高尿酸血症的整体作用存在明显的差异。二妙丸类方能使高尿酸血症大鼠代谢轮廓整体回调，表现出与西药别嘌呤醇不同的代谢纠偏效应。与模型组相比，随机森林算法筛选出的潜在生物标志物水平在给药二妙丸类方后产生了明显回归空白组水平的趋势，且回归的趋势大小为：四妙丸>三妙丸>二妙丸，该趋势与二妙丸类方降低血尿酸的经典药效指标趋势一致。以上结果将有助于探索二妙丸类方调节整体代谢的变化规律，为阐明二妙丸类方抗高尿酸血症的整体作用机制开辟了新的途径。