基于炎症反应-神经受损网络研究栝楼桂枝汤治疗缺血性脑卒中的药效物质及作用机制

Inflammation response is an essential step in the progress of cerebral tissue lesions after ischemic stroke. High mobility group protein B1 (HMGB1), peroxiredoxins (Prxs), and specialized pro-resolving mediators (SPMs) are closely related to the different period of the inflammatory response after cerebral infarction. Gua-Lou-Gui-Zhi decoction (GLGZD), which was effective in the treatment of ischemic stroke in clinic, could lower the level of inflammatory cytokines in MCAO rats depended on its multi-constituents character in our previous " constituents -pharmacokinetics-pharmacodynamics" relationship study. Based on our previous research and literature, the present project aims to establish a qualitative and quantitative technique using ultra performance liquid chromatography with time of flight mass spectrometry and triple quadrupole mass spectrometry to quickly monitor the SPMs and metabolites of endogenous amino acid in MCAO rats after different times of HLJDD administration. Specifically, this study aims to interpret the endogenous metabolic fingerprint along with the development of MCAO rats. In addition, the pro-inflammatory and anti-inflammatory cytokines were measured using a liquid chip technology. At the same time, the influences of GLGZD on the key proteins in the different inflammatory pathways including HMGB1 and Prxs were investigated, and the improvement of neural functional recovery together with pathologic change such as infarct volume and encephaledema were determined. According to the cluster network analysis method combining the aforementioned indexes with the serum concentration of the ingredients in GLGZD, a visual characteristics network including the effects and advantages of the above indexes was built to clarify the action mechanism of GLGZD based on its anti-inflammation effect.

炎症反应是脑卒中后脑组织损伤的关键因素。高迁移率蛋白B1（HMGB1）、过氧化物还原蛋白（Prxs）及多种促炎症消退介质与脑卒中后不同时期的炎症反应密切相关。本课题以治疗中风疗效确切的栝楼桂枝汤为研究对象，将在前期“成分-药动-药效”关联研究基础上，基于脑卒中后炎症反应-神经受损网络，以脑内炎症反应的三种主要表征因素（促炎症消退介质、抗炎因子、促炎因子）为切入点，以氨基酸类内源性物质为交互影响因素，从分子水平研究栝楼桂枝汤对HMGB1、Prxs炎症反应途径中关键蛋白的干预作用，以神经功能恢复及脑梗死体积、脑水肿、神经元凋亡等主要病理特征改善为终点检验指标，结合入血成分群的血药浓度指标，并借助集群网络分析方法构建可视化特征网络，综合分析栝楼桂枝汤多成分、多时点、多环节治疗缺血性脑卒中的综合作用及机制，为其临床合理应用提供实验基础和理论依据。

本课题采用HPLC-Q-TOF-MS和UPLC-QqQ-MS法对栝楼桂枝汤入血及入脑成分进行定性定量分析，并结合多因子检测技术对其中含量较高的5个主要入血成分进行PK-PD研究，确定没食子酸、芍药苷、甘草酸可能是栝楼桂枝汤的主要药效成分。建立GC-MS及UPLC-MS/MS代谢组学分析方法，分析栝楼桂枝汤对MCAO大鼠模型干预不同阶段的尿液、血浆内源性代谢组学特征的影响，确定5个氨基酸类及3个炎症消退介质类主要差异生物标记物；在此基础上，进一步结合体内外实验模型，明确栝楼桂枝汤及其有效部位能够改善脑缺血再灌注损伤，减轻脑内炎症水平，抑制神经元凋亡，其作用可能与调控HMGB1/Prxs炎症信号通路有关，包括调节HMGB1、Prxs、TLR4、NF-κB、p38、p-ERK1/2等关键蛋白表达。本课题相关研究有利于诠释栝楼桂枝汤治疗缺血性脑卒中的科学内涵，可为其临床应用和科学准确用药提供实验基础和理论依据。