基于代谢组学技术的香附-川芎药对配伍规律和抗抑郁药效活性成分研究

Chinese medicine pairs (CMP) are specific compatibility with traditional Chinese medicine (TCM) theory as principle，which was formed on basis of effect and treatment theory of TCM. So，study on the compatibility effects of CMP can provide scientific basis for the clinical applications of CMP and creation of TCM new drugs. Xiangfu-Chuanxiong herb pair (XC) is a traditional chinese medicine herb pair with confirmed effect in treatment of qi stagnation caused depression and pain in clinic. Our previously work showed that the antidepressive action of XC was evaluated due to the synergy effects of Xiangfu and Chuanxiong. Therefore, experiments to explore the chemical compositions and compatible regularity of XC herb pair designed to make a thorough research based on metabonomics, combining NMR, UPLC-MS/MS, natural products chemistry and pharmacodynamics, et al. In this study, NMR and UPLC-MS-based metabonomics study will be applied to explore the development of the disease of chronic unpredictable stimulation model (CUMS) and the intervention process of Xiangfu-Chuanxiong herb pair through compared the metabolic profiles of rats’ blood and urine. Combined with the multivariate statistical analysis, we will find out the depression related features of metabolome and potential biomarkers regulated by XC herb pair which would be described by the regulation of functional networks. And the active compounds will find out through the correlation analysis between chemical components, depressive biomarkers and pharmacodynamics statistics by multiple regression analysis. This study will provide the reliable scientific basis for material basis and compatible regularity of CMP s which have exact clinical efficacy. The study can not only provide a scientific basis for prescriptions containing XC herb pair, but also can provide new ideas and methods of the compatibility and pharmacodynamic substance foundation researches on CMP.

药对是中医临床遣药组方常用的配伍形式，组成简单却隐含了方剂的配伍特征，作为单味中药与若干方剂之间的桥梁，以相对简单的药对为切入点，研究其组成规律，是揭示中药方剂配伍规律和物质基础的捷径。香附-川芎药对见于越鞠丸、柴胡疏肝散、小温中丸、四白丹和芎附饮等方剂，具有行气疏肝功效，临床上主治气郁、肝郁所致的胸胁疼痛等症。本课题组前期研究发现香附和川芎均具有抗抑郁作用，且二者配伍后作用显著增强。因此，本课题拟以代谢组学和化学指纹图谱相结合，研究香附-川芎药对配伍前后化学成分的改变，以及对慢性应激导致大鼠机体代谢紊乱调控作用的影响，利用数据挖掘技术，进行化学成分-生物标志物-药效相关性分析，结合细胞水平和分子水平验证，阐明香附-川芎药对配伍特征，揭示其抗抑郁药效成分群及其代谢调控的通路和靶点。该研究不仅可为含香附-川芎药对的方剂研究提供科学依据，而且可为药对配伍和药效物质基础研究提供新的思路及方法。

香附-川芎药对具有行气疏肝功效，临床上主治气郁、肝郁所致的胸胁疼痛等症。本课题组前期研究发现香附和川芎均具有抗抑郁作用，且二者配伍后作用显著增强，但其药效物质基础和作用机制尚不清楚。因此，本项目（1）采用LC-MS技术对药对配伍前后化学成分组成及变化进行了研究，共鉴定了药对中40个成分的结构，发现7个成分的含量（洋川芎内酯A/F、阿魏酸、藁本内酯、α-香附酮、丁基苯酞、圆柚酮）在配伍前后发生了显著改变；（2）应用细胞损伤模型和小鼠急性应激模型获得了药对抗抑郁活性部位（95%乙醇冷浸提后药渣50%回流部位），突破了对香附、川芎传统药效部位的认知；（3）应用大鼠慢性应激模型评价了药对抗抑郁药效，发现其可显著提高模型大鼠自主活动次数及糖水偏好；（4）基于代谢组学方法从机体代谢通路调控的角度初步阐释了药对的抗抑郁起效机制，发现药对参与调控色氨酸代谢、胆汁酸代谢、脂肪酸合成及代谢、花生四烯酸代谢等通路代谢紊乱；(5)通过比较单味药和药对参与调控的通路差异，初步阐释了药对的配伍规律：香附侧重神经递质代谢相关的色氨酸代谢、犬尿酸代谢，以及能量代谢相关的牛磺酸代谢、脂类代谢，而川芎侧重于炎症相关的脂肪酸代谢和花生四烯酸代谢，单味药对药对调控的代谢通路具有协同或互补的作用，亦是其药效优于单味药的原因所在；(6) 应用数理统计相关性分析，推测获得11个成分具有良好的相关性，分别为洋川芎内酯A、洋川芎内酯F、欧当归内酯A、蒿本内酯、槲皮素、正丁基苯酞、阿魏酸、圆柚酮、香附醇、莎草酮 ，α-香附酮；(7)应用蛋白免疫印迹法发现药对可以显著上调神经营养相关的ERK/CREB、BDNF蛋白表达，上调5-HTA受体表达，提示药对可以通过营养神经，促进5-HT分泌起到抗抑郁作用。本项目从细胞水平和分子水平验证，阐明香附-川芎药对配伍特征，揭示其抗抑郁药效成分群及其代谢调控的通路和靶点。该研究不仅可为含香附-川芎药对的方剂研究提供科学依据，而且可为药对配伍和药效物质基础研究提供新的思路及方法。