UPLC/LTQ-Orbitrap-MS Combined with Network Pharmacology to Explore the Mechanism of Platycodon Grandiflorum in the Treatment of Nonalcoholic Fatty Liver Disease
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摘要:
目的 结合UPLC/LTQ-Orbitrap-MS技术和网络药理学方法预测桔梗治疗非酒精性脂肪肝病(NAFLD)的作用靶点及潜在的作用机制，并进行相关实验验证，为深入揭示桔梗治疗NAFLD的药效物质及作用机制奠定基础。 方法 通过UPLC/LTQ-Orbitrap-MS技术及中药系统药理学数据库与分析平台(TCMSP)数据库筛选桔梗的活性成分；使用Swiss Target Prediction数据库对桔梗的活性成分进行靶点预测；同时通过OMIM、Disgenet、TTD等数据库获取NAFLD靶点；将疾病相关靶点映射到化合物潜在靶点中，获取公共靶点，并将信息导入Cytoscape软件和String在线分析平台分别制作网络图和PPI图，同时进行拓扑学分析；基于R软件使用Bioconductor生物信息软件包进行关键靶基因GO与KEGG功能富集分析。构建NAFLD小鼠模型，通过病理染色切片，qPCR实验验证网络药理学富集分析结果。 结果 结合质谱分析与数据库筛选结果，共获得桔梗活性成分13个，药物靶点278个，疾病靶点1 536个，共同靶点83个，涉及PI3K-AKT、胰岛素抵抗、TNF-α、IL-17、JAK-STAT、T细胞受体等信号通路。实验验证显示关键靶基因存在差异性表达。 结论 该研究初步揭示了桔梗治疗NAFLD的活性成分及其作用机制，为后续的深入研究提供了参考价值。 Abstract: OBJECTIVE Combining UPLC/LTQ-Orbitrap-MS technology with network pharmacology method to predict the target of Platycodon grandiflorum in the treatment of non-alcoholic fatty liver disease (NAFLD) and the potential mechanism, and carrying out relevant experimental verification, in order to reveal the effective substances and mechanism of Platycodon grandiflorum in the treatment of NAFLD.METHODS The active components of Platycodon grandiflorum were screened by UPLC/LTQ-Orbitrap-MS and TCMSP database. Swiss Target Prediction Database was used to predict the active components of Platycodon grandiflorum. Targets of NAFLD were obtained through OMIM, Disgenet, TTD and other databases. The disease-related targets were mapped to the potential targets of compounds to obtain the common targets, and the information was imported into Cytoscape software and String online analysis platform to make network diagram and PPI diagram, respectively, and topological analysis was carried out at the same time. Functional enrichment analysis of key target genes GO and KEGG was carried out by using Bioconductor bioinformatics software package based on R software. NAFLD mouse model was established, the results of network pharmacology enrichment analysis were verified by pathological staining and qPCR.RESULTS Combined with the results of mass spectrometry and database screening, a total of 13 active components, 278 drug targets, 1 536 disease targets and 83 common targets were screened in Platycodon, involving PI3K-AKT, insulin resistance, TNF-α, IL-17, JAK-STAT, T cell receptors and other signaling pathways. Experimental verification showed that the key target genes were differentially expressed.CONCLUSION This study reveals the active components of Platycodon grandiflorum in the treatment of NAFLD and its mechanism of action, it provides reference value for further research. -
表 1 引物序列
基因名 上游引物(5'→3') 下游引物(5'→3') MAPK3 CAGCTCAACCACATTCTAGGTA TCAAGAGCTTTGGAGTCAGATT AKT1 TGCACAAACGAGGGGAATATAT CGTTCCTTGTAGCCAATAAAGG TNF ATGTCTCAGCCTCTTCTCATTC CGATCACCCCGAAGTTCAGTAG GAPDH AGGTCGGTGTGAACGGATTTG GGGGTCGTTGATGGCAACA 
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表 2 负离子模式下部分化合物的鉴定
No. tR/min 加和离子 m/z实测值 m/z理论值 分子式 化合物名称 δ 碎片离子 1 3.04 [M-H]- 1 547.677 98 1 547.674 78 C69H112O38 桔梗皂苷E(Platycoside E) 1.356 1 457.657 48，1 005.555 79，987.701 05 2 11.73 [M-H]- 1091.528 32 1091.526 88 C52H84O24 去芹糖桔梗皂苷D(Deapio-platycodin D) 0.315 681.558 23，723.400 57，663.493 41 3 13.13 [M-H]- 1 223.569 7 1 223.569 14 C57H92O28 桔梗皂苷D(Platycodin D) 0.459 681.647 10，469.152 44，1 133.567 88 4 14.18 [M-H]- 1 265.580 04 1 265.579 7 C59H94O29 桔梗皂苷C(Platycodin C) -0.284 1 223.612 31，1 205.595 95，723.381 04 5 16.51 [M-H]- 1 237.548 58 1 237.548 4 C57H90O29 桔梗二酸A(Platycinic acid A) 0.143 1 207.483 64，1 027.451 42，485.282 23 6 19.74 [M-H]- 1 265.580 08 1 265.579 7 C59H94O29 桔梗皂苷A(Platycodin A) -0.569 1 223.518 80，1 205.570 07，723.426 82
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表 3 核心靶点信息
基因名称 蛋白名称 基因ID AKT1 RAC-alpha serine/Threonine-protein kinase P31749 MAPK3 Mitogen-activated protein kinase 3 P27361 EGFR Epidermal growth factor receptor P00533 TNF Tumor necrosis factor Tumor necrosis factor P01375 MAPK1 Mitogen-activated protein kinase 1 P20482 CASP3 Caspase-3 P42574 STAT3 Signal transducer and activator of transcription 3 P40763 HSP90AA1 Heat shock protein HSP 90-alpha P07900 ESR1 Estrogen receptor P03372 MMP9 Matrix metalloproteinase-9 P14780 MTOR Serine/threonine-protein kinase mTOR P42345 IL-2 Interleukin-2 P60568 PPARG Peroxisome proliferator-activated receptor gamma P37231 MAPK14 Mitogen-activated protein kinase 14 Q16539 AR Androgen receptor P10275 PIK3CA Phosphatidylinositol 4, 5-bisphosphate 3-kinasecatalytic subunit alpha isoform P42336 MMP2 72 kDa type Ⅳ collagenase P08253 JAK2 Tyrosine-protein kinase JAK2 O60674 CYP19A1 Aromatase P11511 APP Amyloid-beta A4 protein N-APP P12023 MPO Myeloperoxidase P05164 GSK3B Glycogen synthase kinase-3 beta P49841 MET Hepatocyte growth factor receptor P08581 MMP1 Interstitial collagenase P03956 CDK4 Cyclin-dependent kinase 4 P11802 F2 Prothrombin P00734 PLG Plasminogen P00747 AHR Aryl hydrocarbon receptor P35869 PARP1 Poly polymerase 1 P09874 PPARA Peroxisome proliferator-activated receptor alpha Q07869
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