“甘草-桔梗”药对治疗气道炎症的协同作用机理研究

The programme aims to explain scientific interpretation of the compatibility of "drug pairs" in traditional Chinese medicine, the classic "drug pairs"-Radix Platycodonis and Radix Glycyrrhizae for the therapy of respiratory system disease were adopted as the object for the various studies: the anti-inflammatory pharmacodynamics substance foundation of the drug pairs, the network regulation mechanism of compatibility and the explanation the molecular mechanism of guiding drug Radix Platycodonis guides the Radix Glycyrrhizae to move upwards. Bioactive based UPLC-Q/TOF system is used for the anti-inflammatory effective components screening and identification; the anti-inflammatory network was constructed by Pharm Mapper and KEGG databases; RT-PCR and western blot are used for the validation of the key targets and the crucial pathway; the results comprehensively explain the synergistic effect mechanism of the combination of Radix Platycodonis and Radix Glycyrrhizae. The PET technology is used for investigation of the distribution of the effective components in vivo; the target proteins are separated by the functionalized alkynyl magnetic microspheres capture means and the proteins are charactered and identified by the molecular biology methods; the studies of lung targeting distribution of Radix Platycodonis and inhibition the P-glycoprotein efflux by Radix Platycodonis interpret the mechanism of guiding drug Radix Platycodonis guides the Radix Glycyrrhizae upwards to lung. The combination of traditional Chinese medicine guide theory and compatibility theory systematically interpret the mechanism of respiratory system disease therapy by the classic "drug pairs"-Radix Platycodonis and Radix Glycyrrhizae from a multidimensional perspective and explore the new thought and method for the study of the compatibility of "drug pairs" in traditional Chinese medicine.

本课题以解读呼吸系统疾病常用药对"甘草-桔梗"的配伍机制为目标，重点围绕抗炎药效物质基础及配伍协同的网络调控机制，桔梗引甘草上行的动态分布过程及分子机制等方面展开研究。运用生物活性指导的UPLC-Q/TOF 分析技术筛选 "药对"中的抗炎成分，通过Pharm Mapper和KEGG构建抗炎网络，并采用分子生物学方法对关键靶点和通路进行验证，从网络药理学的角度阐释甘草与桔梗协同抗炎的作用机制；采用正电子发射断层显像技术研究活性成分体内分布变化情况，利用磁性捕获技术分离桔梗皂苷的作用靶点蛋白，运用分子生物学手段研究靶点蛋白的肺内分布特点和对P-糖蛋白外排功能的影响，阐释桔梗引甘草入肺的引经的分子机制。将中药引经理论与方剂配伍理论有机结合，多层次、多角度阐释"甘草-桔梗"药对治疗肺部炎症的作用机制，为探索"甘草-桔梗"药对配伍的科学内涵提供新的研究思路和方法。

甘草-桔梗药对来源于汉代张仲景的名方桔梗汤，在治疗呼吸系统疾病中应用广泛。本课题以解读甘草-桔梗药对的配伍机制为目标，重点围绕抗炎药效物质基础及配伍协同的网络调控机制，桔梗引甘草上行的动态分布过程及作用机制等方面展开研究。首先利用LPS诱导的小鼠急性肺损伤模型和TNF-α 诱导的细胞炎症模型从整体动物和细胞水平上证明了“甘草-桔梗”具有显著的抗炎效果；结合NF-κB抑制活性相关的双荧光素酶基因报告系统和液质联用技术从“桔梗-甘草”药对中共筛选出11个抗炎活性成分，其中8个来源于甘草，主要为多酚酸类和五环三萜类成分，3个来源于桔梗，主要为萜类化合物；通过同位素标记定量方法（iTRAQ）对药物干预的小鼠急性肺损伤的肺组织进行分析，找出67个差异蛋白，主要与炎症和免疫功能、肌肉收缩功能以及染色体功能相关。利用生物信息学预测了药对中活性成分的主要作用靶点和通路后发现：甘草主要调控MAPKs通路的表达，进而影响NF-κB通路的表达，桔梗主要通过调控PI3K/AKT通路和GSK3β 靶点发挥抗炎作用的，此部分结果初步阐释了“甘草-桔梗”药对治疗肺部炎症的网络调控作用机制；将甘草次酸进行18F标记后静注小鼠，利用PET技术观察口服桔梗总皂苷后甘草次酸的体内分布情况，发现给药后的2小时内肺部甘草次酸的浓度显著升高，而其它器官没有明显的变化，证明了桔梗能够引甘草上行的作用；对桔梗皂苷在体内的代谢过程进行研究，共鉴定出以桔梗次级皂苷3-O-β-D-glucopyranosylplatycodigenin（GP）和3-O-β-D-glucopyranosylplatyconic acid（GPA）为主的7种入血成分，药代动力学研究表明，两种主要入血皂苷成分的达峰时间在1小时左右，与桔梗引药上行的短效性相吻合，同时利用核染料在细胞水平证明了此类皂苷成分能明显增加细胞膜的通透性，导致进入细胞内的化合物量增加，初步阐明了桔梗“引药上行”作用的体内物质基础及作用途径。