“司外揣内”活性成分谱桥接生物体代谢响应网络探究附子治疗心阳虚型慢性心衰的药效物质基础及作用机理

Clinical and experimental studies have proved that Fuzi is good at treating heart failure. However, the material basis and action mechanism of Fuzi in treatment of heart failure are unclear which lead to that its safety and effectiveness can’t be well controlled. Research is carried out under the guidance of traditional Chinese rule of “Conjecturing the interior by observing the exterior” and “Treatment based on syndrome differentiation". Rat model of Xin Yang Deficiency chronic heart failure is firstly established. Under the guidance of the overall efficacy, the material basis candidates are screened with the comprehensive analysis of in vitro chemical fingerprinting and in vivo active ingredient map. And the pharmacokinetics study of material basis candidates are carried out. Combined with "syndrome" metabolomics studies, the metabolic networks of Fuzi effect on heart are revealed at the micro level. And pharmacodynamics study is carried out using biomarker as the indexes. The “concentration-time-effect/toxicity” network of Fuzi action on heart is constructed by combining pharmacokinetics and pharmacodynamics study results. Research integrates the methods of metabolomics and network pharmacology, and use network effects as cut-in points to study the material basis and action mechanism of Fuzi on Xin Yang Deficiency chronic heart failure. Then computer-aided design, molecular docking techniques are applied to establish "multi-component multi-target effect network" by docking three-dimensional molecular of Fuzi ingredient and target structure of key response points in network. Studies explore the complex material basis and multi-component, multi-target mechanism of Fuzi in treating Xin Yang Deficiency chronic heart failure from different levels and angles. At the same time, the research is expected to be innovative in ideas and methods of traditional Chinese medicine basic research and to explore the new model of traditional Chinese medicine safety and effectiveness evaluation.

附子治疗心衰确有疗效，然而其药效物质基础以及作用机理却不甚明确，导致安全性和有效性未能得到良好控制。项目在中医司外揣内“辨证施治”的思想指导下，建立心阳虚慢性心衰大鼠模型，以宏观整体药效为导向，整合附子体外化学成分谱和体内活性成分谱筛选可能效/毒物质基础并进行药动学研究；结合“证”的代谢组学研究在微观层面上揭示附子心脏作用的生物体代谢响应网络，以生物标记物群替代药效学指标进行药效学研究，建立附子心脏作用的“量-时-效/毒”关系网络；整合代谢组学和网络药理学的研究方法，将药物效应网络作为药效物质基础和作用机理相关性研究的切入点，采用计算机辅助设计、分子对接等技术，对接附子活性三维分子库和网络关键响应点蛋白靶标结构，构建“多组分多靶点作用网络”。研究旨在从不同层次、不同角度深入探索附子治疗阳虚心衰的复杂药效物质基础以及多成分、多靶点的协同作用机制，同时为中药的安全性和有效性评价探索新模式。

研究在“对证治疗”思想的指导下对附子治疗心衰的毒效物质基础进行了研究。在给药剂量范围内，正常大鼠和模型大鼠附子水溶性生物碱的Cmax、AUClast均随着给药剂量的增加而增加，体内的药代动力学过程符合线性动力学过程。附子水溶性生物碱在慢性心衰大鼠体内吸收增加，分布容积降低，消除速率降低，半衰期延长，从而延长附子水溶性生物碱的吸收时间，增加吸收量；且Tmax有缩短趋势，吸收速度有增加趋势。将附子水溶性生物碱体内的药动学进行整合药动学过程。将血管紧张素Ⅱ、醛固酮、心钠肽、内皮素、脑钠肽进行整合药效学研究，采用ADAPT 5.1软件对整合药动学-药效学曲线进行关联拟合分析，采用Sigmod Emax模型与三室模型进行效应室间联结，拟合较优。从代谢组学角度揭示附子脂溶性生物碱的毒性作用和水溶性生物碱对治疗大鼠慢性心衰的作用机制，在一定程度上阐明附子的毒-效物质基础及作用机制。通过代谢组学的研究，最终得到与附子生物碱不同极性部位的毒/效作用机制相关10个差异代谢物。通路分析推测脂溶性生物碱的毒性可能与泛酸和辅酶A生物合成，β-丙氨酸代谢，丙酸代谢，嘧啶代谢，缬氨酸、亮氨酸和异亮氨酸生物合成，缬氨酸、亮氨酸和异亮氨酸降解，氨酰-tRNA合成等代谢通路有密切关系。水溶性生物碱可通过调节心衰大鼠的氨酰-tRNA合成,精氨酸和脯氨酸代谢,色氨酸代谢, 及赖氨酸降解等代谢途径发挥治疗作用。研究进一步采用网络药理学建立附子水溶性生物碱与心力衰竭的成分-关键靶点网络，共得32个附子水溶性生物碱治疗心力衰竭的关键靶蛋白；并通过构建成分-关键靶点网络、GO功能富集分析和KEGG通路分析显示附子水溶性生物碱活性成分可能通过影响肾素-血管紧张素系统、醛固酮调节的钠重吸收、PPAR信号通路等通路发挥治疗心力衰竭的作用。. 除此以外，还扩展研究了附子与人参配伍治疗心衰，附子与白芍配伍治疗类风湿性关节炎的药效物质基础和机理。. 研究建立了中药多成分、多靶点的整合药动学-药效学研究模型，并为中药功效成分组的概念提出提供了新的实验依据。