【作者】 王旭；

【导师】 王巧；

【作者基本信息】 河北医科大学 ， 中西医结合基础， 2020， 博士

【摘要】 苓桂术甘汤源自张仲景《金匮要略》,是由茯苓、桂枝、白术和甘草组成的经典名方,临床上现常用于心力衰竭(简称心衰)的治疗,疗效显著。但因苓桂术甘汤成分复杂,其治疗心衰的作用机制尚未完全阐明。传统药理学在作用机制研究中,多为“点对点”式,即单一成分针对单一靶点或通路,难以整体上阐述中药多组分针对多靶点的作用机制。代谢组学和网络药理学基于整体观的研究方法与中药研究所提倡的系统观理念相一致,因而特别适合中药复杂体系的作用机制研究。目的:本研究将结合代谢组学和网络药理学方法,从代谢层面和基因层面共同阐述苓桂术甘汤治疗心衰的作用机制。方法:采用超高效液相色谱-四极杆飞行时间串联质谱技术(UHPLC-Q-TOF-MS),使用化学对照品、化合物保留时间、质谱信息及裂解规律等对苓桂术甘汤中的化学成分进行鉴定。利用在线平台Swiss Target Prediction预测各鉴定成分的靶点,与在线数据库Dis Ge NET等获取的心衰相关靶点取交集,得到共有靶点。利用Cytoscape软件构建成分-共有靶点网络及共有靶点蛋白-蛋白互作(PPI)网络,得到核心靶点及相关成分。利用在线平台Metascape对所得靶点进行富集分析及通路分析,预测苓桂术甘汤治疗心衰的作用机制。采用UHPLC-Q-TOF-MS代谢组学方法研究苓桂术甘汤治疗心衰的作用机制。首先,采用非靶向代谢组学方法对正常小鼠灌胃给予苓桂术甘汤后血清代谢轮廓进行表征,筛选差异代谢物并分析代谢通路。其次,利用多柔比星诱导的心衰小鼠模型,结合超声心动检查、组织病理切片染色和生化指标检测评价苓桂术甘汤的药效,同时采用非靶向代谢组学方法对苓桂术甘汤治疗心衰小鼠的血清代谢轮廓进行表征,筛选差异代谢物分析代谢通路,并结合网络分析筛选核心代谢物,预测苓桂术甘汤作用核心靶点。最后,采用脂质组学方法对苓桂术甘汤治疗心衰小鼠的血清中脂质化合物进行表征,筛选差异脂质化合物并分析代谢通路。结果:共鉴定出苓桂术甘汤中78种化学成分,预测得到194个成分靶点,搜索获取814个心衰靶点,取交集后获得46个共有靶点。成分-共有靶点网络及共有靶点PPI网络结果显示,苓桂术甘汤通过多种活性成分,作用于AKT1、TNF、PTGS2(COX-2)、EGFR、PPARG、STAT3、ACE、AGTR1等关键靶点,涉及IL-17、TNF、花生四烯酸多条信号通路。正常小鼠非靶向代谢组学研究共鉴定得到83个差异代谢物,主要涉及α-亚麻酸、鞘脂类及甘油磷脂等代谢通路改变。苓桂术甘汤治疗心衰小鼠非靶向代谢组学研究共鉴定得到54个差异代谢物,甘油磷脂代谢及花生四烯酸代谢发生了显著性变化。通过可视化网络分析,对苓桂术甘汤治疗心衰的潜在靶点进行了预测,包括PLA2、PLD、PLB、ALT、AST、TAT、苯丙氨酸-4-单加氧酶和过氧化物酶。苓桂术甘汤治疗心衰小鼠脂质组学研究共推断性鉴定了90个差异脂质代谢物,涉及甘油磷脂及花生四烯酸代谢,揭示苓桂术甘汤可能抑制LOX途径和COX途径激活,调控脂质代谢。结论:本研究通过整合苓桂术甘汤的代谢组学及网络药理学结果,发现苓桂术甘汤治疗心衰主要涉及花生四烯酸及甘油磷脂代谢,同时综合各部分靶点预测结果,提示苓桂术甘汤可能通过多成分协同作用于PLA2-LOX/COX-2代谢途径中PLA2、COX-2及LOX等靶点,发挥治疗心衰作用。该研究为今后苓桂术甘汤治疗心衰的作用机制提供了理论基础。更多还原

【Abstract】 Lingguizhugan Decoction(LGZGD),documented in Jinguiyaolue written by Zhongjing Zhang,is composed of Fulin,Guizhi,Baizhu and Gancao.It is clinically used in the treatment of heart failure(HF)with remarkable curative effect.Due to the complex constituents of LGZGD,the mechanism of LGZGD in the treatment of HF has not been fully elucidated.The mechanism researches of traditional pharmacology were mostly point-to-point type,which was single component aimed at single target or pathway.It is difficult to expound thse mechanism of traditional Chinese medicine,which were multicomponent aimed at multiple targets.Metabolomics and network pharmacology are approaches of holistic perspective and are in line with the systematic view of traditional Chinese medicine.Therefore,they are particularly suitable for studying the mechanism of traditional Chinese medicine:Objective: To investigate the therapeutic mechanism of LGZGD in HF treatment based on an integration of the metabolomics and network pharmacy at the metabolic level and gene level.Methods: The constituents of LGZGD were recognized and identified by application of ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometer(UHPLC-Q-TOF-MS)technology.The chemical standard,retention time,and cracking rules of MS information were used to identify the constituents.Using online platform Swiss Target Prediction,the targets of constituents of LGZGD were predicted.The targets of HF were obtained from online database Dis Ge NET.The shared targets were intersection of targets of LGZGD and targets of HF.The network of constituents-shared targets and protein-protein interactions(PPI)of shared targets were constructed in Cytoscape.The core targets and their related costituents were obtained then.Enrichment analysis and pathway analysis were applied on Metascape,and the mechanism of LGZGD in treatment of HF were predicted.Metabolomics method based on UHPLC-Q-TOF-MS was used to study the mechanism of LGZGD in treatment of HF.First,non-target metabolomics method was used to analyze serum metabolic profile of normal mice after administration of LGZGD.The differential metabolies were screened out and pathways of them were analysis.Second,non-target metabolomics method was used to analyze serum metabolic profile of doxorubicin-induced HF mice after administration of LGZGD.The efficacy of LGZGD was evaluated by integration of ultrasonic echocardiography,histopathological staining and biochemical assays.The differential metabolies were screened out and pathways of the them were analyzed.In addition,core metabolites were screened out by network analysis to predict the core target of LGZGD.Finally,lipidomics method was used to analyze serum lipid metabolic profile of doxorubicin-induced HF mice after administration of LGZGD.The differential lipid metabolies were screened out and pathways of the them were analysis.Results: Altogether 78 constituents were identified accurately or putatively in LGZGD,194 targets of constituents were predicted and 814 targets of HF were obtained.46 shared targets of LGZGD and HF were gained after intersection.In the network of constituents-shared targets and PPI,it was showed that the key targets,such as AKT1、TNF、PTGS2(COX-2)、EGFR、PPARG、STAT3、ACE、AGTR1 and et al,were possibly regulated by variety of constituents of LGZGD,involving regulation of IL-17,TNF,arachidonic acid signaling pathway and lipolysis regulation of lipids and lipoproteins process and et al.83 differential metabolies were identified in normal mice non-targets metabolomics study.It showed that alpha-linolenic acid,sphingolipid and glycerophospholipid metabolic pathways were involved in LGZGD effect.54 differential metabolies were identified in non-targets metabolomics of LGZGD in treatment of HF study.It showed that arachidonic and glycerophospholipid metabolic pathways were involved in LGZGD effect.After visual network analysis,the potential targets of LGZGD in the treatment of HF were predicted,including PLA2,PLD,PLB,ALT,AST,TAT,phenylalanine-4-mono-oxygenase and peroxidase.90 differential lipid metabolies were identified in lipidomics of LGZGD in treatment of HF study,in which arachidonic and glycerophospholipid metabolic pathways were involved.It is suggested that LGZGD might inhibit the activation of LOX and COX pathways and regulate lipid metabolism.Conclusion: By integration of metabolomics and network pharmacology in this study,it is showed that glycerophospholipid and arachidonic acid metabolism were significant involved as the mechanism of LGZGD in the treatment of HF.By combination all parts of results,it revealed that PLA2-LOX/COX-2 pathway were regulated by varieties of constituents of LGZGD,targeting PLA2,COX-2 and LOX.This study provided a theoretical basis for the mechanism of LGZGD in the treatment of heart failure.更多还原