基于证候病机的逍遥散-肝郁脾虚证方证相关的生物学基础研究

Correlation between formula Xiaoyaosan and syndrome contains TCM discipline of syndrome differentiation and treatment. In the past, researchers always focused on the biological effects of Chineses formula on the body, but ignored the adaptability of state of body's reaction to the formula itself. The key is to focus on the pathogenesis. Based on the previous works, in this project, selected the correlation between Xiaoyaosan and syndrome of stagnation of liver qi and deficiency in the spleen as example, based on the pathogenesis of syndrome of stagnation of liver qi and syndrome of deficiency in the spleen, we put forward the hypothesis that: biological basis of correlation between xiaoyaoan and syndrome of stagnation of liver qi and deficiency in the spleen might be connected with differential gene of emotions - appetite regulating network and differential gene of DNA methylation. The project contents were as follows: (1) fingerprint of Xiaoyaosan was established with UPLS- MS/MS method; (2) under different state of syndrome pathogenesis of syndrome of stagnation of liver qi and deficiency in the spleen, study the effective component of the correlation between the formula and the syndrome in the body fluid and target tissues; (3) study the differential gene of emotions - appetite regulating network and differential gene in DNA methylation by the methods of transcriptomics, epigenetics, and bioinformatics; (4) at a cellular level, identify the material basis of the correlation between the formula and the syndrome using the TCM small molecule monoclonal antibody technology; (5) take depression patients with the syndrome of stagnation of liver qi and deficiency in the spleen as the objects, further explore the biological foundation of the correlation of Xiaoyaosan and syndrome of stagnation of liver qi and deficiency in the spleen. This research will be of great importance in deepened understanding the theoretical knowledge of correlation of formula and syndrome of classic formulae, and will be important of understanding the individualized diagnosis and treatment ideas based on TCM syndrome differentiation and treatment.

“方证相关”蕴涵着中医辨证论治规律，既往多强调方剂对机体生物效应的作用，忽视证候的机体反应状态对方剂的适应性，其关键在于切中病机。本项目结合前期工作，以逍遥散-肝郁脾虚证方证，针对肝郁与脾虚的证候病机，提出“逍遥散-肝郁脾虚证方证相关的生物学基础可能与‘情绪-食欲调节网络’的差异基因和DNA甲基化有关”的假说，开展（1）UPLS-MS/MS联用方法，建立逍遥散指纹图谱（2）研究肝郁脾虚不同证候病机状态下，体液及靶组织中该方证相关的药效成分并进行网络分析（3）转录组学、表观遗传学方法，研究该方证与肝郁脾虚病机密切相关的“情绪-食欲调节网络”差异基因和DNA甲基化差异基因（4）以中药小分子单克隆抗体技术，在细胞水平上求证该方证相关的物质基础（5）临床以抑郁症肝郁脾虚证患者为对象，进一步探索该方证相关生物学基础。这对于深化经典方剂方证相关的理论认识和辨证论治个体化诊疗思想具有重要意义。

项目背景：“方证相关”蕴涵着中医辨证论治规律，其研究关键在于切中病机。本项目结合前期工作，以逍遥散-肝郁脾虚证方证，针对肝郁与脾虚的证候病机，提出“逍遥散-肝郁脾虚证方证相关的生物学基础可能与‘情绪-食欲调节网络’的差异基因和DNA甲基化有关”的假说，进行了以下研究。.主要研究内容：1.建立逍遥散的体外指纹图谱及成分表征；2.研究不同证候状态下体液中逍遥散-肝郁脾虚证方证相关的药效成分；3.利用转录组学及表观遗传学测序方法，研究逍遥散-肝郁脾虚证方证相关的“情绪-食欲调节网络”差异基因和DNA甲基化差异基因；4.在动物实验的基础上，进行细胞和临床的验证实验：利用中药小分子单克隆抗体技术，在靶组织的细胞上进行验证，临床上以轻、中度抑郁症肝郁脾虚型患者为研究对象进行验证。.重要结果：1.药效物质研究：逍遥散进行了指纹图谱研究及成分的定性、定量表征；体内代谢处置特征与关键有效成分筛选研究发现化合物11具有良好的抗抑郁作用；方证对应网络药理学研究筛选出7种化合物及其抗抑郁作用靶点。2.机制研究：建立了肝郁脾虚证动态模型及其多维度评价方法；发现了前额皮层的抗抑郁的关键非编码RNA miR-200a/200b-3p、circle RNA -VPS13A；下丘脑弓状核的Fam111a为逍遥散调控肝郁脾虚证能量代谢的关键基因等。3.验证研究：验证了逍遥散关键成分芍药苷的抗抑郁作用，完成了临床多中心、随机、双盲、对照试验，以及临床样本甲基化及转录组测序。.科学意义：研究结果对于深化经典方剂方证相关的理论认识和辨证论治个体化诊疗思想具有重要意义，为逍遥散治疗抑郁症的靶点研究提供新的数据支持。另外，研究成果还具有一定的应用价值，扩大经方逍遥散的适应症——防治抑郁症；还可以提高中成药逍遥丸质控标准。