基于亲水作用色谱-质谱联用技术和神经脂质组学的柴胡疏肝散抗脑卒中后抑郁的作用机制研究

Post Stroke Depression (PSD) is clinical frequently occurring disease. Chaihu-Shu-Gan-San is classical prescription on the PSD treatment of liver Qi type stagnation syndrome which the safety and efficiency is widely recognized. This paper intends to combine the animal experiment and clinical research on mining the action mechanism of Chaihu-Shu-Gan-San against PSD.The lyophilized powder of Chaihu-Shu-Gan-San water decoction would been prepared as administration form. The PSD rat model would been established by external carotid artery injection of microspheres and long-term feeding. The neurotransmitters and components coming from Chaihu-Shu-Gan-San in rat hippocampus using cerebral microdialysis sampling technology combined with high-performance liquid phase chromatography electrospray tandem mass spectrometry could been dynamic sampled and accurately determinated to establish the PK-PD correlation model. The neurolipidomics test platform would been established using hydrophilic interaction chromatography - mass spectrometry techniques based on the optimization of chromatographic and mass spectrometric conditions and biological samples pretreatment using ultra performance liquid chromatography flight time mass spectrometry instrument as the analysis instrument. The neurolipidomics research of PSD model rat brain region, cerebrospinal fluid from both PSD rat and patients with liver Qi stagnation syndrome which treated Chaihu-Shu-Gan-San, would been carried out. The role of neurolipidome metabolic network signal molecules in the pathogenesis of PSD could been revealed combining biological information and multivariate statistics. This action mechanism and scientific connotation of Chaihu-Shu-Gan-San could been illuminated based on neurolipidomics against PSD.This study is beneficial to provide scientific basis for further research and development and improvement of clinical curative effect of Chaihu-Shu-Gan-San.

脑卒中后抑郁（PSD）是临床多发病，柴胡疏肝散是治疗肝气郁结型PSD安全、有效的经典名方。本课题拟结合动物实验和临床研究挖掘柴胡疏肝散抗PSD的作用机制。. 以柴胡疏肝散水煎液冻干粉给药，采用“颈外动脉注射微球加中长期饲养法”复制PSD大鼠模型。脑微透析技术结合高效液相色谱电喷雾串联质谱仪实现PSD大鼠海马神经递质和柴胡疏肝散入脑成分动态采样、精确测定，建立PK-PD相关性模型。在优化色谱、质谱条件及神经脂质组前处理基础上，建立基于UPLC-Q-TOF/MS的神经脂质组学亲水作用色谱-质谱联用技术平台，进行柴胡疏肝散对PSD模型大鼠脑区、脑脊液、肝气郁结型PSD患者脑脊液的神经脂质组学研究，结合生物信息学和多元统计揭示神经脂质代谢网络信号分子在PSD发病机制的作用，阐述柴胡疏肝散抗PSD的神经脂质组学作用机制和科学内涵，为进一步开发研究和提高临床疗效提供科学依据。

脑卒中后抑郁（PSD）是临床多发病，柴胡疏肝散是治疗肝气郁结型PSD安全、有效的经典名方，加强对其抗PSD作用机制研究具有积极的现实意义。.在制备柴胡疏肝散水煎液冻干物的基础上，对冻干物中柴胡皂苷c、f、a、b2、d及芍药内酯苷、芍药苷、阿魏酸、柚皮苷、橙皮苷、苯甲酰芍药苷、甘草酸、α-香附酮等其他8个成分，分别建立了UPLC-ELSD、UPLC-UV同步检测方法，并进行含量测定，从源头上确保研究质量。.采用线栓法复制局灶性脑缺血大鼠模型，孤养并结合慢性不可预见性温和刺激以复制PSD大鼠模型。神经行为学评分、脑组织TTC染色、体重、旷场实验行为学分析、糖水偏好度等指标显示，PSD大鼠模型复制成功，抑郁症状稳定，模型成功率高。.进行了柴胡疏肝散抗脑卒中后抑郁的动物实验。实验分为正常组、脑卒中后抑郁模型组、柴胡疏肝散高、中、低剂量组、氟西汀阳性对照组等6组，连续给药32天，每8天为一个周期，每个周期、每个组别共8只大鼠。取海马、前额叶、血浆等生物样本，同时采集了部分时间点的大鼠脑脊液。不同时间点分别测定各组大鼠的旷场实验行为学参数、体重、糖水摄入量等指标，评估柴胡疏肝散改善脑卒中后抑郁的效果。.脑微透析采样技术结合神经递质衍生化方法进行PK-PD模型研究。高效液相色谱-质谱联用仪分别检测柴胡疏肝散抗PSD不同时间点脑微透析样品中活性成分阿魏酸、芍药内酯苷、芍药苷、柚皮苷、苯甲酰芍药苷、橙皮苷、甘草酸和多巴胺、去甲肾上腺素、5-羟色胺、γ-氨基丁酸、甘氨酸、瓜氨酸、谷氨酸等神经递质，建立了多成分、多效应的最佳PK-PD结合模型，获得了PK-PD模型参数。.采用超高效液相色谱-飞行时间质谱联用仪进行柴胡疏肝散抗PSD的脂质组学研究。对比了大鼠脑脊液、血液、前额叶、海马等组织甲基叔丁基醚、二氯甲烷-甲醇、三氯甲烷-甲醇不同萃取体系脂质组检测结果的差异性，以选择最优前处理方法。真空冷冻干燥法获得脑脊液冻干物，UPLC-Q/TOF-MS进行了正、负离子模式下柴胡疏肝散抗PSD192例大鼠血样及部分时间点脑脊液、前额叶脂质组学研究，结合生物信息学建立OPLS-DA脂质组学模型，获取了部分脂质差异代谢物。本研究有助于揭示柴胡疏肝散抗PSD神经脂质组学作用机制和科学内涵，为进一步开发研究和提高临床疗效提供科学依据。