靶标代谢组经时谱效关系探究芪玉三龙汤抑制非小细胞肺癌药效物质基础

Qi-Yu-San-Long decoction (QYSLD) has been used to treat non-small cell lung cancer (NSCLC) for over twenty years in clinical practices, and its efficacy is considered credible. Both clinical study and modern pharmacological experiments have confirmed that QYSLD can inhibit NSCLC and improve survival status of the study subjects, but the research on the pharmacodynamic substances of this prescription has not been carried out. Based on the preliminary study on the chemical components of QYSLD, a high resolution mass spectrometry (HRMS)-data independent acquisition (DIA)-post targeted screening strategy will be established to systematic identification of prototype components and metabolites of QYSLD in blood. Previous study on untargeted metabolomics found that QYSLD had a corrective effect on endogenous metabolic disorders induced by NSCLC. Then, Lewis tumor-bearing mice model will be established. A multi-reaction ion monitoring method based on UPLC-QqQ/MS will be explored to achieve the targeted dynamic monitoring of exogenous metabolites ("spectrum") of QYSLD in blood and the metabolic response index ("efficacy") linked to QYSLD inhibiting NSCLC at different time points after administration. A time-dependent spectrum-efficacy relationship analysis will be used to characterize the substances highly associated with therapeutic effects in the exogenous metabolites of QYSLD. The substances highly associated with pharmacodynamics will be validated by the pharmacodynamics experiments and a classical signal transduction pathway. Then, the pharmacodynamic substances of QYSLD will be elucidated. This study will be conducive to the quality control of QYSLD, and reveal its mechanism of inhibiting NSCLC comprehensively and thoroughly.

芪玉三龙汤（QYSLD）是治疗非小细胞肺癌（NSCLC）的名老中医验方。临床和现代药理学研究均证实QYSLD对NSCLC具有抑制作用并能改善研究对象生存质量，但该方药效物质尚未明确。在QYSLD化学成分定性研究基础上，拟建立高分辨质谱-非信息依赖扫描-后靶向筛查策略快速辨识QYSLD血中移行成分；前期非靶标代谢组学研究发现QYSLD对NSCLC诱导的内源性代谢紊乱具有纠偏作用，以Lewis肺癌荷瘤小鼠为模型，拟建立UPLC-QqQ/MS多反应监测方法，从整体代谢调控的角度靶标性监测给药后不同时间点QYSLD血中移行成分（“谱”）与其抑制NSCLC代谢应答指标（“效”）的动态变化；构建经时谱效关系表征QYSLD血中移行成分与药效高度相关的成分，且以药效学和经典信号转导通路验证，阐明QYSLD抑制NSCLC的药效物质。该研究有利于QYSLD的质量控制及全面深入揭示其抑制NSCLC的作用机理。

本课题首先利用UPLC-QTOF/MSE快速分析了芪玉三龙汤（Qi-Yu-San-Long decoction, QYSLD）中166种化学成分，其中皂苷类22个，生物碱类13个，黄酮类27个，萜类32个，氨基酸类20个，苯丙素类16个，有机酸类9个，甾醇类6个，蒽醌类6个，其它类15个。其中16个成分经过对照品验证，明确了QYSLD的化学物质基础。然后成功建立高分辨质谱-非信息依赖扫描-后靶向筛查策略，并将其用于快速分析大鼠血浆、尿液、粪便中QYSLD的原型成分及代谢产物。在大鼠血浆、尿液和粪便中分别鉴定出101种（41种原型成分、60种代谢产物）、85种（20种原型成分、65种代谢产物）和93种（21种原型成分、72种代谢产物）QYSLD外源性代谢物。主要代谢类型有去糖基化、去甲基化、去羟基化、脱水、脱氢、脱羧、甲基化、硫酸化、葡萄糖醛酸化、乙酰化反应及它们相应的组合反应。.通过细胞实验建立“谱-效”关联性分析探究QYSLD中发挥抗氧化作用及抑制A549细胞作用的活性成分群。综合运用GRA、PLSR和BP-ANN模型结合MIV算法评价QYSLD活性成分，结果显示：去乙酰车叶草苷酸、泽漆新苷等8种成分与DPPH自由基清除能力密切相关；去乙酰车叶草苷酸、水晶兰苷等9种成分与FRAP实验结果密切相关；去乙酰车叶草苷酸、毛蕊异黄酮-7-O-β-D-葡萄糖苷等6种成分与抑制A549细胞增殖能力有关；对香豆酸、毛蕊异黄酮-7-O-β-D-葡萄糖苷等22种成分与抑制A549细胞横向迁移能力有关；泽漆新苷、澳洲茄边碱等5种成分与抑制A549细胞纵向迁移能力有关；车叶草苷酸、对香豆酸等12种成分与抑制A549细胞侵袭能力有关。验证实验结果表明：与未能入选活性成分的化学成分比较，谱效关系筛选出的活性成分表现出更好的抗氧化及抑制A549细胞增殖的作用。.最后，构建肺癌大鼠模型，建立靶标代谢组经时谱效关系探究QYSLD抑制NSCLC药效物质。结果显示去乙酰车叶草苷酸、毛蕊异黄酮-7-O-β-D-葡萄糖苷、对香豆酸、泽漆新苷、澳洲茄边碱、车叶草苷酸等QYSLD血中移行成分与其抑制NSCLC的作用密切相关。本研究基于UPLC-Q/TOF-MS成功构建靶标代谢组经时谱效关系探究QYSLD抑制NSCLC药效物质，为QYSLD的整体质量控制奠定了良好的物质基础。