基于“微透析-多维动态谱效-代谢组学”的气血双补方治疗慢性心力衰竭炮制增效机理研究

Pre-project study found that after some drugs in Shuangbu Prescription(QSP) were processed and compatible, the whole characteristic chromatogram of the prescription changed significantly, and clinical studies have found that the efficacy of processing in treating chronic heart failure has been significantly enhanced, but the mechanism of the synergistic effect of the processed prescription is still unclear. When the chemical substance system of Chinese medicine compound is complex, it is difficult to fully explain its processing mechanism by studying the change rule of one or more chemical components before and after processing. Therefore, this project has changed from the standardization strategy of traditional Chinese medicine（TCM） processing “first chemical composition changes, then pharmacodynamic changes” to reverse research. A new holistic dynamic research method was used to study the synergistic mechanism of QSP in treating chronic heart failure based on the difference of effects caused by changes of chemical composition groups before and after processing, and the technical method of “Microdialysis-Multidimensional Dynamic Spectrum Efficiency-Metabolomics” was used to study the synergistic mechanism of QSP in treating chronic heart failure. Through microdialysis, the “multi-dimensional Spectrum-Effect relationship” and “metabonomics” of “real-time dynamic” were demonstrated. The effective chemical composition groups and multi-component multi-target synergistic mechanism of QSP in treating chronic heart failure before and after processing were explored, and the mechanism of synergistic effect of processing was explained. This project interprets the scientific connotation of synergistic effect of TCM processing from multiple levels and angles, further reveals the influence of TCM processing on TCM compound prescription, and provides scientific basis for safe and rational drug use in TCM clinic.

项目前期研究发现气血双补方中部分药物经炮制配伍后，整个复方特征图谱发生了显著变化，且临床研究发现炮制后治疗慢性心力衰竭功效明显增强，但目前其炮制增效的作用机制尚不清楚。当中药复方化学物质体系较复杂时，对炮制前后一个或多个化学成分变化规律的研究，很难全面阐释其炮制机理。因此，本项目由传统中药炮制“先化学成分变化，后药效变化”的标准化策略转变为逆向研究。采用新的整体动态研究方法，从炮制前后化学成分群改变引起的效应差异入手，基于“微透析-多维动态谱效-代谢组学”的技术方法来研究气血双补方治疗慢性心力衰竭炮制增效机理。通过微透析展现“实时动态”的“多维谱效关系”和“代谢组学”，探索气血双补方炮制前后治疗慢性心力衰竭的有效化学成分群及多成分多靶点协同机制，阐释其炮制增效的作用机理。多层次多角度地诠释中药复方炮制增效的科学内涵，进一步揭示中药炮制对中药复方的影响，为中医临床的安全合理用药提供科学依据。

本项目针对中药复方化学物质体系复杂，仅对其炮制前后一个或多个化学成分变化规律研究难以全面阐释其炮制机理的科学问题。以气血双补方治疗慢性心力衰竭为例，创新性提出“微透析-多维动态谱效-代谢组学”相结合的研究思路阐释其炮制增效作用机制。通过微透析结合质谱联用技术，构建了气血双补方炮制前后体内外动态的UHPLC-Q-TOF-MS/MS及GC-MS/MS指纹图谱。以慢性心衰大鼠模型和心肌损伤细胞模型为多维药效评价体系，建立了炮制前后的“多维动态谱效关系”。进一步筛选出气血双补方炮制前后差异性原型活性成分15个，并对其展开在慢性心衰模型大鼠体内的药代动力学研究，研究结果表明中药炮制影响气血双补方中主要活性成分在体内的过程。将微透析与代谢组学相结合分析气血双补方炮制前后治疗慢性心力衰竭的代谢谱，共筛选得到6个差异性代谢物。将炮制前后差异性成分通过网络药理学进一步对其增效机制展开预测，筛选出与气血双补方治疗慢性心力衰竭作用相关的潜在核心靶点113个。根据靶点PPI网络的度值，选取前6个关键靶点（SRC, NOS2, IL6, STAT3, EGFR and BCL2）与差异性活性成分进行分子对接验证。研究结果表明芒柄花苷与SRC靶点、毛蕊异黄酮葡萄糖苷与NOS2靶点及芍药苷与IL6靶点均有较好的结合能力，其可能成为气血双补方治疗慢性心衰炮制增效潜在的活性成分与靶点。本研究在方法学上具有动态、综合及分析于一体的特点，与中医的整体观和辨证论治不谋而合，更为科学合理地阐释中药复方炮制增效的科学内涵。