关联药效的多组分药代动力学（Poly-PK）策略探讨滋膵饮治疗糖尿病肾病药效物质基础

Zicuiyin (ZCY) is a classical prescription to treat diabetic nephropathy (DN). In the previous metabonomics study, we found ZCY was effective for patients who were in the 3rd stage of DN by influencing the metabolism of phospholipids, fatty acids and amino acids. Correlation analysis between the components of ZCY and these metabolic pathways is of great significance for revealing the therapeutic material basis of ZCY. Poly-PK strategy has the characteristics of entirety and efficiency based on the changes of drug multicomponents in vivo and endogenous components, and avoids the fragmentation research of therapeutic material basis. In this study, Poly-PK strategy is used to explore therapeutic material basis of ZCY. First, QTOF/MS will be used to determine ZCY chemical spectrum in vitro. Then, the endogenous components related to DN will be found through metabonomics approach and associated with pharmacodynamics (PD) indicators. Finally, the kinetics of endogenous components associated with PD, the absorbed components and secondary metabolites of ZCY will be determined. Overall, the relationship between the exposure and metabolism of the drug-derived components in the body and the endogenous components related to the PD was integrated. In this study, the endogenous components related to PD will be used as the link to integrate the more comprehensive PK and PD relationships, and the therapeutic material basis of ZCY on DN will be clearly defined.

滋膵饮（ZCY）是治疗糖尿病肾病（DN）经方，前期代谢组学研究发现，ZCY通过影响磷脂、脂肪酸、氨基酸代谢对DN Ⅲ期患者发挥疗效。发现和评价其所含的皂苷、黄酮、多糖等诸多成分与上述机体代谢的相关性，对揭示ZCY药效物质基础具有重要意义。多组分药代动力学（Poly-PK）方法整合中药入血成分和内源性成分信息，避免药效物质基础研究易碎片化的问题，具有整体高效的特点。本课题采用Poly-PK思路，以DN大鼠为模型，首先采用高分辨质谱确定ZCY体外化学成分谱；其次通过代谢组学方法找出与DN相关的内源性成分，并与药效指标相关联；然后确定机体用药后ZCY体内化学成分及次级代谢物及与药效相关内源性成分的经时过程。最终整合体内药源性成分的暴露与代谢及与药效相关内源性成分动态变化的相关性。本课题以与药效相关内源性成分为纽带，整合更为全面的药代和药效关系，为系统的阐明ZCY药效物质基础提供新思路。

滋膵饮（ZCY）是治疗糖尿病肾病（DKD）的经方。本课题采用多组分药代动力学（Poly-PK）结合药效学与代谢组学的研究思路，以DKD大鼠为模型，对ZCY进行药效物质基础与作用机制的研究。采用UPLC-QTRAP MS/MS方法，鉴定出ZCY44个成分，初步建立了ZCY的化学成分谱。采用指纹图谱分析方法确定33个共有峰，10批样品相似度均大于0.979。采用UPLC-Triple Quad MS/MS方法，建立了19种化合物含量测定方法，用以进行ZCY质量控制。采用UPLC-QTRAP MS/MS方法，对灌胃给药后的大鼠血浆样品进行分析，共确认13个入血成分，采用UPLC-Triple Quad MS/MS方法，对DKD大鼠和正常大鼠单次给药后各成分药代动力学进行分析，并比较其差异。网络药理学确定ZCY治疗DKD的关键靶点26个，核心靶点3个。KEGG表明ZCY通过PI3K/Akt、MAPK、Ras、VEGF信号通路与DKD治疗相关。分子对接表明关键入血活性成分与核心靶蛋白均有较好的结合力。采用高脂高糖饮食结合链脲佐菌素尾静脉注射方法建立DKD大鼠模型，随机分为模型组、二甲双胍组、氯沙坦组、ZCY低剂量组、ZCY中剂量组、ZCY高剂量组和正常组，连续观察8周。对各组大鼠的随机血糖、24小时尿蛋白、24小时尿微量白蛋白、血清肌酐、血清尿素氮、肾脏病理染色、相关通路蛋白表达进行测定统计，比较不同剂量ZCY对模型大鼠疾病状态的改善效果，并从PI3K/Akt信号通路角度探讨ZCY可能的作用机制。对正常组、模型组、ZCY低剂量组和高剂量组大鼠粪便进行肠道菌群检测，分析DKD疾病状态与药物干预作用。采用GC-TOF/MS方法，对模型组和正常组大鼠血清样本进行代谢组学分析，确定包括麦芽糖、半乳糖酸、葡萄糖-1-磷酸、葡萄糖-6-磷酸等16种碳水化合物，4种有机酸，胆酸，3-苯基乳酸等化合物是区分疾病与正常大鼠的潜在生物标志物，DKD涉及氨酰tRNA生物合成、甘氨酸、丝氨酸和苏氨酸代谢、乙醛酸和二羧酸代谢、氮代谢、三羧酸循环等代谢通路。对潜在生物标志物与ZCY疗效进行相关性分析，确定与疗效指标相关的内源性成分30种，包括阿糖醇、蛋氨酸亚砜、肌醇、天冬酰胺、N-甲基丙氨酸、果糖-1-磷酸等。本课题整合ZCY体内暴露与药效及内源性代谢物相关性，为系统阐明ZCY药效物质基础提供理论依据。