基于血清蛋白质组学的生脉注射液多组分、多靶点效应PK-PD结合研究

It is important and difficult that how to select and confirm effect indices reflecting integral action of traditional Chinese medicines (TCM) and with which to study the integrated pharmacokinetics-pharmacodynamics (PK-PD) with multiple-components and multiple-targets effect of TCM. This project focuses on the main key question for selecting effect indices in the integrated PK-PD study of TCM. Using Shengmai injection (SMI) as research object, the technology of serum proteomics will be first applied to search biomarkers from serum low abundance protein as candidate effect indices, which are related with SMI’s protection on myocardial ischemia and reflect integral action of TCM. Then, candidate effect indices will be further selected through in vivo effect-time and doses-effect relationship investigation for determining the potential effect indices which can reflect sensitively drug concentration change of SMI in vivo. Quantitative analysis method will also be established for the confirmed effect indices and with which the multiple-targets effect pharmacodynamics study of SMI’s protection on myocardial ischemia will be carried out. Meanwhile, multiple-components pharmacokinetics study of SMI’s protection on myocardial ischemia will be performed using multiple effective components as pharmacokinetics markers. Finally, the integrated PK-PD models of SMI with multiple-components and multiple-targets effect will be established combining the pharmacokinetics and pharmacodynamics results. It might help to reveal effect material basis of SMI’s protection on myocardial ischemia through the integrated PK-PD models analysis. This project would attempt to explore a new path for the selection of effect indices and apply the new thread and method reference in the integrated PK-PD study of TCM.

如何选择和确定反映中药整合作用特点的药效指标，并用于开展多组分、多靶点效应的PK-PD结合研究，是中药PK-PD结合研究的重点和难点。本课题针对该研究中的药效指标筛选存在的主要关键问题，以生脉注射液为研究对象，首先采用血清蛋白质组学技术从血清低丰度蛋白中寻找与生脉抗心肌缺血作用相关，体现中药整合作用的血清生物标志物为候选药效指标。然后进一步通过体内药效-时间关系以及量-效关系考察，从候选药效指标中筛选出能够灵敏反映生脉体内浓度变化的药效指标，并建立定量分析方法，据此开展生脉多靶点效应药效动力学研究。同时，以生脉主要药效成分为药代标记物，开展多组分的药代动力学研究。最后，综合药代及药效动力学结果拟合建立生脉多组分、多靶点效应的PK-PD结合模型，揭示生脉抗心肌缺血的药效物质基础。本课题拟为中药PK-PD结合研究中的药效指标筛选探索一条新的途径并为中药PK-PD结合研究提供新的思路和方法参考。

如何选择和确定反映中药整合作用特点的药效指标，是中药PK-PD结合研究领域的重点和难点，需要结合现代先进的科学技术手段，探索建立符合中药作用特点的PK-PD 结合模型。本项目采用串联质谱标签（Tandem mass tag，TMT）的定量蛋白质组学方法开展了生脉注射液抗异丙肾上腺素诱导大鼠心肌缺血的血清蛋白质组学研究，研究鉴定获得了分别与心肌缺血疾病和生脉注射液调控作用相关的血清差异蛋白227个和100个，经生物信息学分析这些差异蛋白涉及多种生物学功能并参与多条重要的代谢或信号转导通路，生脉注射液抗心肌缺血可能主要与其调节心肌能量代谢效率、抑制心肌细胞凋亡等机制有关。血清蛋白质组学研究帮助在分子水平上进一步阐明了生脉注射液抗心肌缺血的作用机制，同时也为生脉注射液PK-PD结合研究筛选血清药效指标提供了参考。基于血清蛋白质组学和前期研究结果，本项目开展了另外一项研究内容即生脉注射液抗大鼠心肌缺血的PK-PD结合研究，本部分研究在生物样本采集技术上进行了创新，具体是建立了一种清醒、自由活动大鼠在体血液微透析采样系统用于采集心肌缺血大鼠给药生脉注射液后的血液透析液，然后以生脉注射液多种主要药效成分人参皂苷为药代标记物，以血清NO为药效替代指标，建立了一套灵敏、可靠的LC-MS/MS分析技术用于同时测定生脉注射液多种主要活性成分人参皂苷Rg1、Rg2、Re、Rf、Rb1、Rd和Rc在大鼠血液透析液中的药物浓度；同时，建立了HPLC-DAN荧光检测技术（HPLC-FLD）用于测定透析液中NO的主要代谢产物NO2− 和 NO3−（NOx−）浓度。所建立的方法成功地运用于心肌缺血大鼠单剂量给药生脉注射液后的药物浓度和NO药效指标测定，据此建立了血药浓度-时间和药效-时间关系，并拟合建立了PK-PD结合模型。该PK-PD结合模型具有良好的预测能力并帮助揭示了生脉注射液抗心肌缺血的体内浓度-效应关系。