system: [SYSTEM]

user: 3. 结果 3.1. 活性成分的筛选 通过TCMSP数据库，检索得到18个葛根的化学成分，以ADME参数(OB ≥ 30%和DL ≥ 0.15)为标准，共筛选得到4个活性化合物，结果见表1。 Table 1 Kudzu root active compounds and target Mol ID 化学成分 靶标数目 OB (%) DL MOL000392 formononetin 39 69.67 0.21 MOL000358 beta-sitosterol 38 36.91 0.75 MOL002959 3'-Methoxydaidzein 19 48.57 0.24 MOL003629 Daidzein-4，7-diglucoside 1 47.27 0.67 表1. 葛根活性化合物及靶标数 3.2. 药物靶点和疾病靶点数据的筛选 通过TCMSP数据库，共筛选得到92个葛根活性化学成分对应的作用靶点，结果见表1。通过OMIM数据库，查找得到49个解酒相关的靶点。通过网络药理学构建出葛根解酒作用的交互网络，采用不同颜色和形状图形使其可视化后，可直观的看出活性化学成分与疾病靶点之间的网络关系，结果见图1。其中，黄色方形代表药物与疾病的作用靶点，也是葛根解酒最重要的靶点蛋白；黄色圆点代表解酒直接作用靶点；红色三角形代表葛根中被预测出的活性化学成分；蓝色圆点代表葛根活性化学成分的直接作用靶点；紫色圆点代表连接葛根活性化学成分与疾病靶点的交互蛋白。 图1. 葛根解酒的“成分-靶点-疾病”交互网络 3.3. 葛根解酒的直接作用靶点拓扑参数分析 通过Cytoscape 3.6.1软件进行交互网络分析，得到葛根活性成分相关联的蛋白靶点。通过计算得到网络中三个拓扑参数(Degree、Betweenness centrality和Closeness centrality)分别为：1.00、0和0.167。将上述筛选出的靶点作为葛根解酒的潜在靶点蛋白，共得到8个靶点满足以上条件，结果见表2。将潜在靶点蛋白进行蛋白相互关系分析，结果见图2。蛋白相互关系分析说明葛根解酒的潜在靶点蛋白相互关联和彼此调节。 Table 2 Topological parameters related to the direct action target of Pueraria Radi Uniprot ID Protein names Gene names Closeness Centrality Degree Betweeness Centrality P08172 Muscarinic acetylcholine receptor M2 CHRM2 0.28 1 0.00 P13569 Cystic fibrosis transmembrane conductance regulator CFTR 0.22 2 0.01 P14416 D(2) dopamine receptor DRD2 0.22 1 0.00 P21917 D(4) dopamine receptor DRD4 0.22 2 0.01 P47869 Gamma-aminobutyric acid receptor subunit alpha-2 GABRA2 0.28 1 0.00 Q01959 Sodium-dependent dopamine transporter SLC6A3 0.00 0 0.00 P31645 Sodium-dependent serotonin transporter SLC6A4 0.36 2 0.01 V9HWC9 Superoxide dismutase [Cu-Zn] HEL-S-44 0.00 0 0.00 表2. 葛根活性成分解酒的直接作用靶点相关拓扑参数 图2. 葛根解酒作用的潜在靶点蛋白相互关系图 3.4. GO生物学功能分析 将8个潜在靶点映射到DAVID数据库中，进行生物学功能富集。GO富集分析，得到222个生物学进程、39个细胞组成和67个分子功能，其中P值 ≤ 0.001的生物进程共有35条，结果见表3。结果表明，这些靶点与多种生物进程相关，包括对雌二醇反应、突触传递、胆碱能、腺苷酸环化酶抑制G蛋白偶联乙酰胆碱受体信号通路、磷脂酶C激活G蛋白偶联乙酰胆碱受体信号通路、细胞色素c激活参与凋亡过程的半胱氨酸型内肽酶活性等，且这些生物进程可能与解酒的作用密切相关。同时揭示出了中药发挥药效作用涉及体内多个生物进程的调控。 Table 3 Results of GO biological enrichment analysis of Pueraria hangover effec Category Term Count Count% P-Value GOTERM_BP_DIRECT response to drug 12 18.5 1.8E−08 GOTERM_BP_DIRECT response to estradiol 8 12.3 5.2E−08 GOTERM_BP_DIRECT synaptic transmission， cholinergic 6 9.2 0.00000027 GOTERM_BP_DIRECT adenylate cyclase-inhibiting G-protein coupled acetylcholine receptor signaling pathway 4 6.2 0.0000018 GOTERM_BP_DIRECT phospholipase C-activating G-protein coupled acetylcholine receptor signaling pathway 4 6.2 0.0000029 GOTERM_BP_DIRECT extrinsic apoptotic signaling pathway in absence of ligand 5 7.7 0.0000082 GOTERM_BP_DIRECT response to nicotine 5 7.7 0.000012 GOTERM_BP_DIRECT angiogenesis 8 12.3 0.000022 GOTERM_BP_DIRECT G-protein coupled acetylcholine receptor signaling pathway 4 6.2 0.000023 GOTERM_BP_DIRECT response to lipopolysaccharide 7 10.8 0.000037 GOTERM_BP_DIRECT response to hydrogen peroxide 5 7.7 0.000042 GOTERM_BP_DIRECT positive regulation of cell proliferation 10 15.4 0.000064 GOTERM_BP_DIRECT positive regulation of smooth muscle contraction 4 6.2 0.000067 GOTERM_BP_DIRECT positive regulation of transcription from RNA polymerase II promoter 14 21.5 0.000069 GOTERM_BP_DIRECT gamma-aminobutyric acid signaling pathway 4 6.2 0.000077 GOTERM_BP_DIRECT memory 5 7.7 0.000091 GOTERM_BP_DIRECT peptidyl-serine phosphorylation 6 9.2 0.00011 GOTERM_BP_DIRECT phospholipase C-activating G-protein coupled receptor signaling pathway 5 7.7 0.00012 GOTERM_BP_DIRECT positive regulation of transcription， DNA-templated 10 15.4 0.00014 GOTERM_BP_DIRECT behavioral fear response 4 6.2 0.00018 GOTERM_BP_DIRECT response to cobalt ion 3 4.6 0.00021 GOTERM_BP_DIRECT positive regulation of vasoconstriction 4 6.2 0.00024 GOTERM_BP_DIRECT transcription initiation from RNA polymerase II promoter 6 9.2 0.00028 GOTERM_BP_DIRECT response to toxic substance 5 7.7 0.00031 GOTERM_BP_DIRECT signal transduction 14 21.5 0.00037 GOTERM_BP_DIRECT peptidyl-threonine phosphorylation 4 6.2 0.0004 GOTERM_BP_DIRECT cell proliferation 8 12.3 0.00047 GOTERM_BP_DIRECT response to hypoxia 6 9.2 0.0005 GOTERM_BP_DIRECT activation of cysteine-type endopeptidase activity involved in apoptotic process by cytochrome c 3 4.6 0.00051 GOTERM_BP_DIRECT positive regulation of ERK1 and ERK2 cascade 6 9.2 0.00054 GOTERM_BP_DIRECT positive regulation of neuron apoptotic process 4 6.2 0.00058 GOTERM_BP_DIRECT positive regulation of nitric oxide biosynthetic process 4 6.2 0.00058 GOTERM_BP_DIRECT regulation of cell proliferation 6 9.2 0.00069 GOTERM_BP_DIRECT T cell activation 4 6.2 0.00076 GOTERM_BP_DIRECT intrinsic apoptotic signaling pathway in response to DNA damage 4 6.2 0.00076 表3. 葛根解酒作用的GO生物学功能富集分析结果 3.5. KEGG通路分析 将8个潜在靶点映射到DAVID数据库中，进行KEGG通路富集分析，共得到76条信号通路，其中P值 ≤ 0.001的信号通路共有28条，结果见表4。其中神经活性配体–受体相互作用、钙信号途径、胆碱能突触、心肌细胞中的肾上腺素信号、PI3K-AKT信号通路等、VEGF信号通路、p53信号通路、NAFLD等信号通路可能直接或间接参与葛根解酒的作用。此结果与解酒机制可能紧密相关。同时利用KEGG信号通路数据库中的KEGG Mapper功能将8个葛根与解酒相关联的靶点蛋白，在与GABA能突触最为密切的信号通路上标注出来，结果显示有4个靶点蛋白参与GABA能突触信号通路的相关调控，结果见图3。 Table 4 KEGG pathway enrichment analysis of Pueraria hangover effec Category Term Count Count % P-Value KEGG_PATHWAY Neuroactive ligand-receptor interaction 18 27.7 7.1E−11 KEGG_PATHWAY Pathways in cancer 18 27.7 1.6E−08 KEGG_PATHWAY Calcium signaling pathway 13 20 2.6E−08 KEGG_PATHWAY Hepatitis B 11 16.9 3.3E−07 KEGG_PATHWAY Cholinergic synapse 10 15.4 3.4E−07 KEGG_PATHWAY Colorectal cancer 8 12.3 8.1E−07 KEGG_PATHWAY Morphine addiction 9 13.8 8.8E−07 KEGG_PATHWAY Thyroid hormone signaling pathway 9 13.8 0.0000052 KEGG_PATHWAY Estrogen signaling pathway 8 12.3 0.000019 KEGG_PATHWAY Retrograde endocannabinoid signaling 8 12.3 0.000022 KEGG_PATHWAY Serotonergic synapse 8 12.3 0.00004 KEGG_PATHWAY Small cell lung cancer 7 10.8 0.000079 KEGG_PATHWAY Progesterone-mediated oocyte maturation 7 10.8 0.000091 KEGG_PATHWAY Prostate cancer 7 10.8 0.000097 KEGG_PATHWAY PI3K-Akt signaling pathway 12 18.5 0.00014 KEGG_PATHWAY Adrenergic signaling in cardiomyocytes 8 12.3 0.00016 KEGG_PATHWAY VEGF signaling pathway 6 9.2 0.00016 KEGG_PATHWAY Apoptosis 6 9.2 0.00017 KEGG_PATHWAY Amphetamine addiction 6 9.2 0.00023 KEGG_PATHWAY p53 signaling pathway 6 9.2 0.00025 KEGG_PATHWAY Non-alcoholic fatty liver disease (NAFLD) 8 12.3 0.00028 KEGG_PATHWAY Viral carcinogenesis 9 13.8 0.00033 KEGG_PATHWAY Toxoplasmosis 7 10.8 0.00033 KEGG_PATHWAY Nicotine addiction 5 7.7 0.00035 KEGG_PATHWAY Tuberculosis 8 12.3 0.00073 KEGG_PATHWAY GABAergic synapse 6 9.2 0.00076 KEGG_PATHWAY Salivary secretion 6 9.2 0.0008 KEGG_PATHWAY Amyotrophic lateral sclerosis (ALS) 5 7.7 0.00084 表4. 葛根解酒作用的KEGG通路富集分析结果 图3. 葛根活性成分潜在靶点在关键信号通路上的标注图