整合PK-PD和代谢组学技术解析丹参红花组分抗脑缺血再灌注损伤的配伍机制研究

Both PK-PD and metabonomics are characterized with holism and dynamicness. By judiciously integrating these two techniques to discuss the compatibility principle of Traditional Chinese medicine prescription, we can better reflect the correlation between the in vivo dynamically changing rule of drug components and the whole effect (including drug effect and biological effect), and make up the deficiency of the existing research modes in the idea of holism and perpetual motion. .Based on the previous study, this project proceeds from chemical constituents of Salvia miltiorrhiza and Safflower, and orthogonally combines sixteen prescriptions of different dosage ratio from effective parts of Salvia miltiorrhiza and Safflower. Then the plasmic content fluctuations of the major compounds and metabolic products after intragastric administration in rats are detected mainly by HPLC/MSn; The potency indicators of anti-cerebral ischemia- reperfusion injury are assessed by ELISA, HPLC/ECD or immunohistochemical method simultaneously; The corresponding plasma metabolite fingerprints and the endogenous biomarkers related to cerebral ischemia in rats are also determined through GC/MS and NMR. Finally a variety of mathematical methods such as multi-componential PK-PD model are applied to analyze the dynamic change processes of chemical constituents, efficacy and metabolome, as well as drug-drug-body interactions. With the aim to decipher the compatibility mechanism of chemical constituents of Salvia miltiorrhiza and Safflower to protect from cerebral ischemia-reperfusion injury, and seek the optimal dosage ratio, this research would provide reference for the mechanism study of prescription compatibility based on PK-PD integrated with metabonomics.

PK-PD和代谢组学技术均具有整体、动态的特征，将两者进行有机整合来探讨方剂配伍原理，可更好地反映药物组分在体内的动态变化规律与整体效应（包括药物效应与生物效应）之间的相关性，弥补现有研究模式在整体、恒动理念上的不足。.本项目拟从丹参红花化学组分入手，基于前期研究，L16(45)正交组合丹参红花主要有效部位，制备16个丹红组分配伍方；重点以HPLC/MSn测定大鼠灌胃给药后不同时相的血浆有效成分及代谢组分的含量变化，ELISA、HPLC/ECD、免疫组化等同步测定抗脑缺血再灌注损伤的药效学指标，GC/MS、NMR等同步测定大鼠的血浆代谢物指纹谱和脑缺血损伤相关的内源性生物标志物；引入多组分PK-PD模型等数理方法，解析丹参红花各化学组分、药效、代谢组的动态变化过程和药物-药物-机体的相互作用，以阐述丹红组分的配伍机制，寻求其最佳配比，为基于PK-PD整合代谢组学的方剂配伍机制研究提供借鉴。

代谢组学整体、动态的特征与方剂多组分、多靶点、动态调节的思维理念相吻合，充分体现了整体观、恒动的中医药特色。本课题以经典配伍丹参红花为示范，整合PK-PD与代谢组学技术探讨丹红组分治疗脑缺血再灌注损伤的配伍机制，寻求最佳组方，为中医方剂配伍机制的阐释提供了借鉴。.课题研究首先建立了相应化学组分的含量测定方法，将人工神经网路、遗传算法和熵权法等应用于丹参和红花主要有效部位（丹参酚酸类、丹参酮类、红花红色素类）和有效成分（脱水红花黄色素B）的制备，构建并优化了丹参酚酸类的闪式-微波提取和HPD-300大孔树脂纯化工艺、丹参酮类的闪式-超声提取和HPD-100大孔树脂纯化工艺、红花红色素类的HPD-400大孔树脂纯化工艺，以及脱水红花黄色素B的高速逆流分离工艺。优化后的参数有效提升了丹红组分的得率和纯度，为进一步的药效评价和代谢组学研究奠定了基础。.在药效评价方面，构建了MCAO/R动物模型和OGD/R细胞模型模拟脑缺血再灌注损伤，证实有效部位丹参酚酸类可调节FoxP3+Treg细胞对抗脑缺血损伤引起的神经炎症，丹参酮类可通过抑制parthanatos对抗脑缺血损伤，红花红色素可激活ERK1/2-Nrf2-GSH通路减轻OGD/R诱导的PC12细胞损伤；红花主要水溶性成分能够调控SIRT1信号通路对抗脑缺血损伤引发的氧化应激和细胞凋亡。.在明确丹红有效部位对脑缺血大鼠代谢组调控作用的基础上，正交组合了4个有效部位不同用量配比的9个组方，测定丹红药对12个有效成分的体内变化情况（PK），同步测定抗脑缺血的药效指标（PD）以及内源性代谢物的波动情况，构建PK-PD-代谢物网络，解析相互关系。结果表明，丹红对脑缺血再灌注损伤有良好的保护作用，两药配伍可协同增效；相同剂量下，红花的药效作用更强；9个正交组方中，组方8具有最佳的调控作用；4个有效部位中，丹参酚酸类和红花黄色素对总量零阶矩的影响较为显著；各有效成分峰值药物浓度和最大效应（包括药效和内源性代谢物）之间存在明显滞后现象；药效指标中的HIF、T-AOC、IMA对整体药效的贡献率高于BDNF；丹红组分对检测的内源性代谢物均有显著的调节作用，BJS、TCJ的改变较BY III、GAS略为明显；12个丹红检测成分的矩阵值均在0.9以上，说明了它们与整体药动学的密切相关性，以及对丹红质量评价的重要性。