基于Th1/Th2细胞因子平衡调节及药效差示色谱法联合的百部-紫菀药对药效物质基础及作用机制研究

The effective substances and mechanism of traditional Chinese medicines (TCM) formulas were not clear and definite which became hindrance factors on its way going globewide. Couplet medicines, which play important roles between TCM and TCM formulas, have greatly significance to understand and establish a method to illuminate the therapeutical basis and mechanism of TCM formulas after analyzing their effective substances. In this study, couplet medicine of Baibu-Ziwan with different proportion compatibilities would be particularly selected and the pharmacodynamic action and Th1/Th2 cytokine levels in CVA rats would be researched to determine their effect differences and relationship firstly. Then differential efficacy of chromatography would be employed to determine the fingerprint differences in serum and lung with maximum and minimum effect by HPLC-MS method. The effective substances would be determined by the difference analysis of Th1/Th2 cytokine level changes and fingerprint differences. Finally, cytokine levels of TGF-β,Th1/Th2 and gene expressions of T-bet/GATA-3 on CVA and normal rats would be investigated to explore the pharmacodynamic mechanism of Baibu-Ziwan in signal transduction pathway. The success of this research would provide some scientific evidence for the further study on compatibility and mechanism of Zhi Sou San.

中药复方药效物质及作用机制的不明确成为阻碍中医药发展及走向国际的重要瓶颈。药对反映了复方配伍的特殊规律，对其开展相关研究，有利于为进一步的复方药效物质及作用机制研究提供方法学依据。本课题拟在前期研究的基础上，首先通过比较百部-紫菀药对（不同配伍比例）在咳嗽变异性哮喘（CVA）模型大鼠体内的药效作用及Thl/Th2细胞因子水平，分析两者的相互关系并确定药效差别；采用药效差示色谱法利用液质联用技术测定最大、最小药效的血清及肺组织指纹图谱，联合相关的细胞因子水平变化情况（Th1/Th2）明确药效差示成分，对其结构进行解析从而确定药对的药效物质基础；最后测定百部-紫菀药对在CVA及空白大鼠TGF-β、Th1/Th2等细胞因子水平上的影响并通过进一步测试在T-bet/GATA-3基因表达水平上的影响，从信号转导途径探索药对的药效作用机制。本课题旨为止嗽散复方的配伍及作用机制研究提供一定的科学依据。

中药复方药效物质及作用机制的不明确成为阻碍中医药发展及走向国际的重要瓶颈。药对反映了复方配伍的特殊规律，对其开展相关研究，有利于为进一步的复方药效物质及作用机制研究提供方法学依据。本项目通过对百部、紫菀单味药与配伍后药对的体内药效作用研究表明，百部、紫菀配伍后对药效作用产生协同效果；对相关细胞因子平衡（Th1/Th2、Th17/Treg及MMP-9 /TIMP-1）的测定结果表明，百部-紫菀药对具有纠正上述细胞因子平衡的作用；肺组织病理研究明确了百部-紫菀药对的药效作用，并确定了药效差别；上述实验中对配伍比例的研究表明配伍比例1:1效果最佳。基于血清药理学原理并利用液质联用技术对入血成分进行分析，结合药效差示色谱法测定最大、最小药效的血清及肺组织指纹图谱，联合对应的细胞因子水平变化情况明确药效差示成分为原百部碱、对叶百部碱、槲皮素、咖啡酸、绿原酸、山奈酚与紫菀酮；体外溶出实验表明，药对配伍有助于提高药效成分的体外溶出；百部、紫菀单味药及不同配比组方百部-紫菀药对入血成分在正常及病理模型大鼠体内的药动学实验结果表明药对配伍具有促进药效成分吸收，提高相对生物利用度，提高肺组织分布的作用，确认百部-紫菀药对的的药效物质基础为原百部碱、对叶百部碱、槲皮素、咖啡酸、绿原酸、山奈酚与紫菀酮。通过测定百部-紫菀药对在CVA大鼠肺组织中在T-bet/GATA-3基因表达水平上的影响，确定百部-紫菀药对纠正Th1/Th2 细胞因子平衡是基于上调T-bet蛋白与mRNA表达，下调GATA-3蛋白与mRNA表达含量从而达到药效作用，从信号转导途径明确了药对的药效作用机制。课题的成功实施为中药复方的配伍及机制研究探索了一条成功的研究方法。