基于UPLC-Q-TOF-MS技术及网络药理学方法探讨六味地黄丸入脑成分改善D-gal模型大鼠学习记忆能力的作用机制

Mechanism of Brain-Absorption Components of Liuwei Dihuang Pills in Improving Learning and Memory Abilities of D-galactose Model Rats Based on UPLC-Q-TOF-MS Technology and Network Pharmacology

目的:应用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)技术分析六味地黄丸的入脑成分,结合网络药理学及体外验证实验探讨该成分改善D-半乳糖(D-gal)模型大鼠学习记忆能力的核心靶点、关键通路等潜在作用机制。方法:皮下注射D-半乳糖250 mg/(kg·d)构建模型。实验分为空白组、模型组和给药组[六味地黄丸,1.62 g/(kg·d)]。应用新物体识别、Morris水迷宫2种行为学实验评估各组大鼠学习记忆能力;采用苏木精-伊红(HE)染色观察各组大鼠海马神经元形态;通过UPLC-Q-TOF-MS技术对空白大鼠脑脊液样品及六味地黄丸给药后的大鼠脑脊液样品进行化学成分快速识别及鉴定,确定六味地黄丸原型入脑成分;通过中药系统药理学数据库与分析平台、Swiss Target Prediction等数据库预测六味地黄丸入脑成分作用的潜在靶点,通过治疗靶点数据库、GeneCards等数据库筛选阿尔茨海默病(AD)相关疾病靶点,取药物成分靶点与疾病靶点交集绘制韦恩图,通过STRING构建蛋白质-蛋白质相互作用网络模型,使用Cytoscape 3.7.2软件构建六味地黄丸“入脑成分-靶点-AD”网络关系图,通过Metascape数据库对交集靶点进行基因本体(GO)功能富集分析和京都基因与基因组百科全书(KEGG)通路富集分析,分析关键通路。通过体外细胞实验,检测六味地黄丸脑脊液对D-gal损伤PC12细胞增殖率及β淀粉样蛋白(Aβ)、活性氧(ROS)、白细胞介素6(IL-6)、5-羟色胺(5-HT)和多巴胺(DA)水平的影响。结果:六味地黄丸可显著上调模型组大鼠新物体识别指数、穿越平台次数和靶象限停留时间,显著缩短定位潜伏期,差异均有极显著统计学意义(P<0.01),并恢复海马神经元形态。通过数据分析,六味地黄丸给药后在大鼠脑脊液中发现7个原型入脑成分,分别为莫诺苷、(-)-奎宁酸、阿魏酸、芍药苷、泛酸、5-羟甲基糠醛和尿苷。通过网络药理学筛选六味地黄丸入脑成分潜在作用靶点102个,与AD共同靶点47个,获得44条GO信号通路和14条KEGG信号通路,与Aβ沉积、Tau蛋白磷酸化、氧化应激、炎症反应、能量代谢及神经递质水平密切相关。体外细胞实验结果显示,六味地黄丸脑脊液可显著升高D-gal损伤PC12细胞增殖率,5-HT、DA水平,显著降低Aβ、ROS和IL-6水平,差异均有极显著统计学意义(P<0.01)。结论:六味地黄丸对D-gal模型大鼠的学习记忆能力具有改善作用,可能是六味地黄丸入脑成分通过抑制Aβ沉积、Tau蛋白磷酸化和氧化应激,抗炎、改善能量代谢障碍及调节神经递质水平来实现的。

OBJECTIVE:To analyze the brain-absorption components of Liuwei Dihuang pills by applying ultra performance liquid chromatography-quadrupole rod-time of flight-mass spectrometry(UPLC-Q-TOF-MS)technique,and to explore the core targets,key pathways and other potential mechanisms of these components in improving learning and memory abilities of D-galactose(D-gal)model rats based on network pharmacology and extracorporal validation experiments.METHODS:The model was constructed by subcutaneous injection of D-galactose 250 mg/(kg·d).The experiments were divided into the blank group,model group and administration group[Liuwei Dihuang pills,1.62 g/(kg·d)].The learning and memory abilities of rats in each group were evaluated by two behavioral experiments including new object recognition and Morris water maze;the morphology of hippocampal neurons in each group was observed by hematoxylin-eosin(HE)staining;fast recognition and identification of chemical components were conducted on the cerebrospinal fluid samples of blank rats and rats treated with Liuwei Dihuang pills by UPLC-Q-TOF-MS technique,so as to determine the prototype brain-absorption components of Liuwei Dihuang pills.The potential effect targets of the brain-absorption components of Liuwei Dihuang pills were predicted through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and Swiss Target Prediction database,the Alzheimer’s disease(AD)-related disease targets were screened through the therapeutic target database and GeneCards databases,the intersection of drug component targets and disease targets was taken to draw the Venn diagram,the STRING was used to construct the protein-protein interaction network model,and the Cytoscape 3.7.2 software was used to construct the“brain-absorption components-target-AD”network diagram of Liuwei Dihuang pills,the Metascape database was used to perform gene ontology(GO)functional enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis on the intersecting targets,the key pathways were analyzed.Through the extracorporal cell experiment,the effects of cerebrospinal fluid of Liuwei Dihuang pills on the proliferation rate in D-gal damaged PC12 cells and the levels ofβ-amyloid(Aβ),reactive oxygen species(ROS),interleukin 6(IL-6),5-hydroxytryptamine(5-HT)and dopamine(DA)were detected.RESULTS:Liuwei Dihuang pills can significantly up-regulated the new object recognition index,the number of platform crossing and dwell time of target quadrant,and significantly shorten the localization latency in the model group rats,with statistically significant differences(P<0.01),and restored hippocampal neuron morphology.By the data analysis,seven prototype brain-absorption components were found in the cerebrospinal fluid of rats treated with Liuwei Dihuang pills,including monosidine,(-)-quinic acid,ferulic acid,paeoniflorin,pantothenic acid,5-hydroxymethylfurfural and uridine.Totally 102 potential effect targets of the brain-absorption components of Liuwei Dihuang pills were screened out through network pharmacology,which shared 47 common targets with AD;44 GO signaling pathways and 14 KEGG signaling pathways were obtained,which were closely related to Aβdeposition,Tau protein phosphorylation,oxidative stress,inflammatory response,energy metabolism and neurotransmitter levels.The results of extracorporal ell experiments showed that the cerebrospinal fluid of Liuwei Dihuang pills can significantly increased the proliferation rate of D-gal damaged PC12 cells,5-HT and DA levels,and significantly decreased Aβ,ROS and IL-6 levels,with statistically significant differences(P<0.01).CONCLUSIONS:Liuwei Dihuang pills has improved learning and memory abilities of D-gal model rats,and its effects may be achieved by the brain-absorption components of Liuwei Dihuang pills through inhibiting Aβdeposition,Tau protein phosphorylation and oxidative stress,anti-inflammation,improving impaired energy metabolism and regulating neurotransmitter levels.