基于“数学拆方-液质联用-高内涵筛选”三级体系的芎芍止痛方治疗偏头痛的药效物质基础研究

Migraine, the 7th highest cause of disability worldwide, is a common and neurovascular disorder. Traditional Chinese medicine has been acknowledged by more and more patients with migraine for its advantages of high cure rate, no dependence and few adverse reactions compared with western medicine. Xiongshaozhitong Formula which comes from the experience of the master of traditional Chinese medicine (TCM) has a good clinical effect. Based on the previous research, this project is planned to find its effective constituents by the mathematical method, UPLC-Q-TOF/MS, and high content screening (HCS). The mathematical method which combined with SAS and the TCM theory was used to guide the disassembling prescription and find out the optimal combination for treating the migraine. The constituents was studied by UPLC-Q-TOF/MS and multivariate statistical analysis. The calcitonin gene-related peptide (CGRP), a sign of trigeminal vascular system activation, has played an important role in the development of migraine. Since the role of CGRP is closely related to its receptor CGRP-R, HCS was used for component screening. A three-level system of “mathematical method of disassembling prescription -UPLC-MS- HCS” as a new model on the research of TCM formula was established for find the effective constituents of Xiongshaozhitong Formula on migraine. These new ideas and methods would lead the clinical application and transformation and also promote the modernization of TCM.

偏头痛是一种常见的神经血管性疾病，被称为世界上第7大类致残性疾病，与西药相比，中医药治疗偏头痛具有治愈率高、复发率低、无依赖性的优势，受到各国患者的认可。芎芍止痛方来源于名老中医的经验方，临床疗效确切，本课题以此方为例，采用数学方法指导药效拆方，结合多元回归分析与中医理论，确定治疗偏头痛的最优组合；采用液质联用结合多元统计分析研究化学成分，推断主要有效成分群；降钙素基因相关肽（CGRP）是三叉神经血管系统激活的标志，在偏头痛发生、发展过程中具有极其重要的作用，CGRP的作用与其受体CGRP-R密切相关，因此本课题以CGRP-R为切入点，采用高内涵技术筛选有效成分，从而建立一种“数学方法指导药效拆方-液质联用研究化学成分-高内涵筛选有效成分”的三级筛选体系，明确芎芍止痛方治疗偏头痛的物质基础。通过本课题的研究为中药复方药效物质研究提供一种参考模式，指导中医药临床应用与转化，推进中医药现代化。

芎芍止痛方来源于名老中医经验方，治疗偏头痛临床疗效确切，但是其药效物质基础不明确，制约了其进一步应用。本项目采用数学方法指导其药效拆方，寻找方出中主要药味为丹参、土茯苓、芥子、僵蚕、苍耳子，并对其治疗偏头痛的药效进行了验证。利用液质联用技术对芎芍止痛方主要药味的体外-入血成分进行研究，分析鉴定出其体外成分58种，入血成分18种，推断出其治疗偏头痛的潜在有效成分群。通过网络药理学预测，芎芍止痛方治疗偏头痛的作用机制与炎症、NO、CGGP等信号通路密切相关。采用硝酸甘油型偏头痛大鼠模型，证实芎芍止痛方主要药味可通过调节NOS/NO-NF-κB信号通路治疗偏头痛。同时，基于前期研究，确定nNOS和CGRP为其治疗偏头痛的靶标蛋白。采用表面等离子共振技术，分别以nNOS和CGRP为靶标，筛选出丹参酮ⅡA、阿魏酸、欧前胡素等19个化合物与nNOS蛋白具有较强的亲和力，丹酚酸B、芍药苷、丹参酮ⅡA与CGRP蛋白具有较强的亲和力，进一步筛选出芎芍止痛方治疗偏头痛的有效成分。通过LPS诱导PC12细胞炎症模型，对丹参酮ⅡA、阿魏酸、欧前胡素等化合物的药效及作用机制进行验证，结果表明，隐丹参酮、阿魏酸等6个化合物均可显著降低细胞NO含量，同时存在剂量依赖效应。欧前胡素和花椒毒素可显著抑制细胞nNOS及NF-κB蛋白的表达，对NOS/NO-NF-κB信号通路具有显著的调节作用。.通过上述研究，揭示了芎芍止痛方治疗偏头痛的药效物质基础，为偏头痛新药研发提供了依据，同时建立了一种“数学方法指导药效拆方-液质联用研究化学成分群-表面等离子共振筛选有效成分”的中药复方药效物质筛选体系，为中药复方药效物质基础研究提供了新思路与新方法。此外，已协助将芎芍止痛方研发为芎芍止痛颗粒，完成了成果转化，目前正在按照中药创新药1.1类申请临床试验，将极大的推动芎芍止痛方的临床应用。