四逆汤多组分代谢网络调控效应PK-PD结合模型构建的方法学研究

To establish the integrated “pharmacokinetics (PK) system –pharmacodynamics (PD) system” model of Traditional Chinese Medicine (TCM) is an effective way to profile the effective components of TCM and their bioactivities. So far, it is not clear which components of Sini decoction (SND) play a bioactive role in treating myocardial infarction. In order to clarify the problem, we will establish the integrated PK-PD model of Sini decoction multiple components’ metabolic network regulation. In the integrated PK-PD model, we will select the SND components targeting cardiac muscle cell membrane as PK-markers based on our previous two-dimensional cell membrane chromatography for screening of bioactive compounds of SND, on the other hand, we will select the effect-related biomarkers obtained form the previous metabonomics study of the action mechanism of treating myocardial infarction of SND as PD-markers of metabolic network regulation. Meanwhile, we will apply the combination of microdialysis and liquid chromatography/mass spectrometry for the synchronous collection and determination of PK-markers and PD-markers. By the integrated model, we can find the effective components that can well fit "concentration-effect-time" three-dimensional relationship model and establish the connective network of effective components from SND and its biomarkers to reveal the effective components of SND. The project will lay the foundation for developing SND into a new Chinese medicine compound against myocardial infarction. In this project, it is expected that a novel approach will be developed to study the integrated “PK system-PD system” model of TCM multiple components’ metabolic network regulation based on the combined techniques of cell membrane chromatography and microdialysis as well as metabonomics, in order to provide an effective way for the correlation characterization of TCM components and their activities and a new reference for studying the effective components of TCM.

中药“PK系统-PD系统”结合模型构建是表征中药药效物质基础和生物活性的有效手段。本项目针对四逆汤治疗心肌梗死药效物质基础不清的问题，在四逆汤心肌细胞膜二维活性组分筛选和药效作用机制代谢组学研究的基础上，以四逆汤心肌细胞膜结合成分为药代指标体系，以代谢组学获取的药效相关生物标志物为网络调控效应指标体系，采用微透析-液质联用技术为药动学、药效学指标同步采样和表征手段，建立多组分代谢网络调控效应PK-PD结合模型，探寻良好拟合"浓度-效应-时间"三维关系模型的效应物质，构建四逆汤效应物质和生物标志物的关联网络，揭示四逆汤治疗心肌梗死的药效物质基础，为四逆汤发展成为治疗心肌梗死的新型复方中药奠定基础；以期建立基于细胞膜色谱-微透析-代谢组学组合技术的中药多组分代谢网络调控效应 “PK系统-PD系统” 结合模型研究新方法，为中药成分与活性的关联表征提供有效手段，也为中药药效物质基础研究提供新借鉴。

本项目针对四逆汤治疗心肌梗死药效物质基础不清的问题，采用“系统-系统”的研究策略，开展了基于细胞膜色谱、自由活动大鼠微透析取样、代谢组学等组合技术的中药四逆汤“PK系统-PD系统”的相关性研究。建立了心肌细胞膜色谱柱-整体柱二维液相色谱-飞行时间质谱活性成分筛选方法，鉴定了四逆汤中14种心肌细胞膜结合成分；建立了清醒活动SD大鼠微透析取样－超高效液相色谱－质谱分析系统，对四逆汤中7种主要心肌细胞膜结合成分(包括苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰海帕乌头原碱、异塔拉定、附子灵、尼奥灵、塔拉乌头胺)在健康大鼠和心肌梗死大鼠的体内代谢过程分析，发现在心肌梗死大鼠体内7种生物碱达峰时间较健康对照延迟，7种生物碱的半衰期在心肌梗死大鼠体内明显延长，同时消除减慢，而且健康对照组中它们的曲线下面积AUC接近心肌梗死大鼠体内的4倍，研究结果为四逆汤的临床合理应用提供了科学依据；建立了超高效液相色谱-飞行时间质谱的血清代谢组学方法，以异丙肾上腺素造模后2小时和4小时血清代谢谱相互验证，鉴定22种异丙肾上腺素心肌梗死模型的生物标志物，证实四逆汤主要通过调节支链氨基酸分解代谢、脂肪酸β-氧化、酪氨酸代谢、色氨酸代谢、苯丙氨酸代谢、鞘脂代谢和磷脂代谢等途经，发挥其抗心肌梗死的作用；将四逆汤相关代谢谱和四逆汤诱导代谢谱相关联，建立与四逆汤多组分特点相适应的活性成分群与多靶点药效学的“系统-系统”关联方法，将22种心肌梗死相关生物标志物与22种四逆汤入血相关成分关联，构建了四逆汤药效成分与生物标志物的关联网络，确定了四逆汤中16中抗心肌梗死的药效成分。本项目为四逆汤成分与活性的关联表征提供了有效手段，也为中药药效物质基础研究提供新借鉴。项目研究已经发表论文7 篇，其中SCI 论文6篇，申请专利1项。另外，还有两篇SCI论文待发表。