鉴定红花逍遥片的化学成分谱，从"病-证-症-方"关联角度，探索该中成药品种治疗经前期综合征(premenstrual syndrome，PMS)肝郁气滞血瘀证的作用特点与生物学内涵。采用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱(UHPLC-Q Exactive Orbitrap HRMS)鉴定红花逍遥片的化学成分谱;依据组方原理将其划分为疏肝解郁组、活血调经组和益气健脾组，通过Pharmmapper数据库与中医药整合药理学研究平台(TCMIPv2。0)收集红花逍遥片各功效组所含中药的候选靶标信息;通过中医证候本体及多维定量关联计算平台(SoFDA)数据库、GeneCards、DisGeNET、MalaCards和己发表文献，收集PMS现代医学的临床症状、中医诊疗标准及其临床症状的相关基因集;依据基因间相互作用信息，建立"红花逍遥片候选靶标-PMS肝郁气滞血瘀证相关基因"互作网络，通过计算网络拓扑特征值，筛选核心网络靶标，并基于Kyoto Encyclopedia of Genes and Genomes(KEGG)数据库开展功能挖掘，进一步探索红花逍遥片组方中各功效组在干预PMS中的优势药效环节，并加以动物实验验证。红花逍遥片中共鉴定获得109个化学成分，其中疏肝解郁组含20个化学成分，主要作用于神经系统、雌激素调节与"免疫-炎症"相关通路而发挥疏肝功效;活血调经组含77个化学成分，主要作用于血液循环系统、"免疫-炎症"与雌激素调节相关通路而发挥补血和气、养血柔肝的功效;益气健脾组含30个化学成分，通过调节"免疫-炎症"、消化系统与激素水平而发挥扶正益气、利水渗湿的功效。疾病靶组织生化指标检测结果表明，与溶剂对照组相比，PMS模型大鼠下丘脑组织中的5-HT与DA水平下降，子宫组织中E2、NO、VEGF、RLN水平下降，OT、PROG、IL-6、IL-1β、TNF-α、ET-1水平上升，红花逍遥片给药后能够缓解或逆转PMS大鼠的上述病理改变，表明红花逍遥片能够通过调节雌激素合成与分泌、干预神经递质合成与信号传导、矫正"免疫-炎症"失衡和调节消化系统功能，发挥其舒肝、理气、活血、健脾的综合功效，有助于改善PMS肝郁气滞血瘀证中机体"神经-内分泌-免疫"系统与血液循环紊乱，为明确红花逍遥片治疗PMS肝郁气滞血瘀证的优势药效环节和探索其作用机制提供参考。本实验获得中国中医科学院中药研究所动物伦理委员会批准(批准号:2023B248)。
Identification of chemical constituents in Honghua Xiaoyao Tablet and the analysis of efficacy connotation against premenstrual syndrome based on the"disease-syndrome-symptom-formula"association network
The present study identified chemical constituents of Honghua Xiaoyao Tablet(HXT)and explored its biological connotation and characteristics on the premenstrual syndrome(PMS)treatment from the"disease-syndrome-symptom"association network.UHPLC-Q Exactive Orbitrap HRMS technology was applied to analyze the chemical constituents in HXT.According to the composition principles,the compatible herbs of HXT were divided into the Shugan Jieyu group,Huoxue Tiaojing group and Yiqi Jianpi group.The candidate targets of the corresponding prescriptions of HXT efficacy groups were collected from the Pharmmapper database and Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine(TCMIP)v2.0.The gene set related to the clinical symptoms included in Traditional Chinese and Western Medicine diagnosis and treatment standards were obtained from SoFDA,GeneCards,DisGeNET,MalaCards and literature published.The"HXT candidate targets-PMS(liver depression,Qi stagnation,and blood stasis syndrome)genes"network was constructed based on the gene interaction information,and further,the core network targets were screened out by topological characteristics of calculating network,and the functional exploration was carried out based on Kyoto Encyclopedia of Genes and Genomes(KEGG)for exploring the therapeutic advantages in PMS treatment of HXT efficacy groups,which were further verified experimentally in vitro.A total of 109 components from HXT were identified,including 20 components from Shugan Jieyu group enriched in the neurological system,estrogen regulation,and"immune-inflammation"related pathways,77 components from Huoxue Tiaojing group enriched in the blood-circulation system,"immune-inflammation"and estrogen regulation related pathways and 30 components from Yiqi Jianpi group regulating immune inflammation,digestive system,and hormone levels.The biochemical indicator detection demonstrated that both the levels of 5-HT and DA in the hypothalamus tissues and the levels of E2,NO,VEGF and RLN in the uterine tissues of PMS model rats were lower than those in controls,which the levels of OT,PROG,IL-6,IL-1β and TNF-α in the uterine tissues were increased in PMS group,which were all reversed by the administration of HXT,indicating that this prescription may regulate the synthesis and secretion of estrogen,intervene in neurotransmitter synthesis and signal transduction,reverse the imbalance of"immune-inflammation",and regulate digestive system function through various biological pathways,exerting the liver-smoothing,Qi-regulating,blood-activating and spleen-tonifying comprehensive effects,leading to alleviating the"neuroendocrine-endocrine-immune"system and blood-circulation disorders.The relevant results may provide a reference for clarifying the advantages and efficacy of HXT in treating PMS with liver stagnation,Qi stagnation,and blood stasis syndrome,and exploring its therapeutic advantages.The animal experiment of this study was approved by the Ethics Committee of the Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences(approval number:2023B248).
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