药学学报

Rank	Uniprot ID	Protein target	Frequency
1	P56817	β-Secretase 1	18
2	P14061	Estradiol 17-β-dehydrogenase 1	16
3	P01112	GTPase HRas	15
4	Q9BZX2	Uridine-cytidine kinase 2	14
5	P10114	Ras-related protein Rap-2a	14
6	P15121	Aldose reductase	14
7	P23919	Thymidylate kinase	13
8	P11473	Vitamin D3 receptor	13
9	P15121	Aldose reductase	12
10	Q06187	Tyrosine-protein kinase BTK	12
11	P09211	Glutathione S-transferase P	12
12	P50225	Sulfotransferase 1A1	12
13	P49841	Glycogen synthase kinase-3β	12
14	P00492	Hypoxanthine-guanine phosphoribosyltransferase	12
15	P31749	RAC-αserine/threonine-protein kinase	12
16	P06213	Insulin receptor	11
17	Q96C86	Scavenger mRNA-decapping enzyme DcpS	11
18	Q16836	Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial	11
19	Q16222	UDP-N-acetylhexosamine pyrophosphorylase	11
20	Q9BZ11	ADAM 33	10
21	O76054	SEC14-like protein 2	10
22	P29373	Cellular retinoic acid-binding protein 2	10
23	Q16539	Mitogen-activated protein kinase 14	10
24	P08473	Neprilysin	10
25	Q15075	Early endosome antigen 1	10
26	P07900	Heat shock protein HSP 90-α	10
27	P02766	Transthyretin	10
28	Q02127	Dihydroorotate dehydrogenase, mitochondrial	9
29	P04150	Glucocorticoid receptor	9
30	Q16772	Glutathione S-transferase A3	9
31	P27707	Deoxycytidine kinase	9
32	P08246	Leukocyte elastase	9
33	P12268	Inosine-5-monophosphate dehydrogenase 2	9
34	P07858	Cathepsin B	9
35	P35228	Nitric oxide synthase, inducible	9
36	P43235	Cathepsin K	9
37	P24941	Cell division protein kinase 2	8
38	Q13231	Chitotriosidase-1	8
39	P18031	Tyrosine-protein phosphatase non-receptor type 1	8
40	P62942	Peptidyl-prolyl cis-trans isomerase FKBP1A	8
41	P11362	Basic fibroblast growth factor receptor 1	8
42	P07686	β-Hexosaminidase β chain	8
43	Q08499	cAMP-specific 3, 5-cyclic phosphodiesterase 4D	8
44	Q08188	Protein-glutamine γ-glutamyltransferase E	8
45	P19367	Hexokinase-1	8
46	P00918	Carbonic anhydrase 2	8
47	O15530	3-Phosphoinositide-dependent protein kinase 1	7
48	P11766	Alcohol dehydrogenase class-3	7
49	P17931	Galectin-3	7
50	P20248	Cyclin-A2	7
51	P42330	Aldo-keto reductase family 1 member C3	7
52	P08581	Hepatocyte growth factor receptor	6
53	O14757	Serine/threonine-protein kinase Chk1	6
54	P04278	Sex hormone-binding globulin	6
55	Q10588	ADP-ribosyl cyclase 2	6
56	P20248	Cyclin-A2	6
57	Q00534	Cell division protein kinase 6	5
58	P03372	Estrogen receptor	5
59	P29218	Inositol monophosphatase	5

Rank	Uniprot ID	Protein target	Frequency
1	P56817	β-Secretase 1	18
2	P14061	Estradiol 17-β-dehydrogenase 1	16
3	P01112	GTPase HRas	15
4	Q9BZX2	Uridine-cytidine kinase 2	14
5	P10114	Ras-related protein Rap-2a	14
6	P15121	Aldose reductase	14
7	P23919	Thymidylate kinase	13
8	P11473	Vitamin D3 receptor	13
9	P15121	Aldose reductase	12
10	Q06187	Tyrosine-protein kinase BTK	12
11	P09211	Glutathione S-transferase P	12
12	P50225	Sulfotransferase 1A1	12
13	P49841	Glycogen synthase kinase-3β	12
14	P00492	Hypoxanthine-guanine phosphoribosyltransferase	12
15	P31749	RAC-αserine/threonine-protein kinase	12
16	P06213	Insulin receptor	11
17	Q96C86	Scavenger mRNA-decapping enzyme DcpS	11
18	Q16836	Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial	11
19	Q16222	UDP-N-acetylhexosamine pyrophosphorylase	11
20	Q9BZ11	ADAM 33	10
21	O76054	SEC14-like protein 2	10
22	P29373	Cellular retinoic acid-binding protein 2	10
23	Q16539	Mitogen-activated protein kinase 14	10
24	P08473	Neprilysin	10
25	Q15075	Early endosome antigen 1	10
26	P07900	Heat shock protein HSP 90-α	10
27	P02766	Transthyretin	10
28	Q02127	Dihydroorotate dehydrogenase, mitochondrial	9
29	P04150	Glucocorticoid receptor	9
30	Q16772	Glutathione S-transferase A3	9
31	P27707	Deoxycytidine kinase	9
32	P08246	Leukocyte elastase	9
33	P12268	Inosine-5-monophosphate dehydrogenase 2	9
34	P07858	Cathepsin B	9
35	P35228	Nitric oxide synthase, inducible	9
36	P43235	Cathepsin K	9
37	P24941	Cell division protein kinase 2	8
38	Q13231	Chitotriosidase-1	8
39	P18031	Tyrosine-protein phosphatase non-receptor type 1	8
40	P62942	Peptidyl-prolyl cis-trans isomerase FKBP1A	8
41	P11362	Basic fibroblast growth factor receptor 1	8
42	P07686	β-Hexosaminidase β chain	8
43	Q08499	cAMP-specific 3, 5-cyclic phosphodiesterase 4D	8
44	Q08188	Protein-glutamine γ-glutamyltransferase E	8
45	P19367	Hexokinase-1	8
46	P00918	Carbonic anhydrase 2	8
47	O15530	3-Phosphoinositide-dependent protein kinase 1	7
48	P11766	Alcohol dehydrogenase class-3	7
49	P17931	Galectin-3	7
50	P20248	Cyclin-A2	7
51	P42330	Aldo-keto reductase family 1 member C3	7
52	P08581	Hepatocyte growth factor receptor	6
53	O14757	Serine/threonine-protein kinase Chk1	6
54	P04278	Sex hormone-binding globulin	6
55	Q10588	ADP-ribosyl cyclase 2	6
56	P20248	Cyclin-A2	6
57	Q00534	Cell division protein kinase 6	5
58	P03372	Estrogen receptor	5
59	P29218	Inositol monophosphatase	5

Classification	Pathway	Count	P-value
Human diseases	Prostate cancer	8	3.16E-13
	Melanoma	5	3.96E-08
	Endometrial cancer	4	6.87E-07
	Non-small cell lung cancer	4	8.01E-07
	Small cell lung cancer	4	5.21E-06
	Glioma	3	8.81E-05
	Alzheimer's disease	4	8.87E-05
	Renal cell carcinoma	3	1.15E-04
	Chronic myeloid leukemia	3	1.35E-04
	Colorectal cancer	3	1.89E-04
	Amyotrophic lateral sclerosis (ALS)	2	0.002 482 732
	Acute myeloid leukemia	2	0.002 751 467
	Epithelial cell signaling in Helicobacter pylori infection	2	0.003 847 862
	Long-term depression	2	0.003 956 072
	Pancreatic cancer	2	0.004 176 709
Endocrine system	Insulin signaling pathway	6	2.93E-08
	Melanogenesis	2	0.008 294 07
	GnRH signaling pathway	2	0.008 759 851
Amino acid metabolism	Aminosugars metabolism	4	6.16E-08
	Glutathione metabolism	2	0.001 984 888
Xenobiotics biodegradation and metabolism	Metabolism of xenobiotics by cytochrome P450	4	2.29E-06
	Drug metabolismother enzymes	3	4.25E-05
	Drug metabolismcytochrome P450	3	1.19E-04
	Caprolactam degradation	1	0.009 133 748
Cellular processes	Cell cycle	5	5.30E-07
	Adherens junction	4	3.71E-06
	Focal adhesion	5	7.64E-06
	p53 signaling pathway	3	1.05E-04
Immune system	B cell receptor signaling pathway	4	3.01E-06
	Fc epsilon RI signaling pathway	4	3.71E-06
	T cell receptor signaling pathway	4	1.33E-05
	Antigen processing and presentation	2	0.006 140 796
	Toll-like receptor signaling pathway	2	0.007 989 977
Signal transduction	VEGF signaling pathway	3	1.40E-04
	ErbB signaling pathway	3	2.09E-04
	MAPK signaling pathway	4	4.76E-04
	mTOR signaling pathway	2	0.002 144 93
Nervous system	Axon guidance	3	6.79E-04
Carbohydrate metabolism	Glycolysis/gluconeogenesis	2	8.41E-05
	Galactose metabolism	2	5.37E-04
	Fructose and mannose metabolism	2	0.001 032 896
	Starch and sucrose metabolism	1	0.002 144 93
Nucleotide metabolism	Purine metabolism	4	1.78E-06
	Pyrimidine metabolism	4	7.12E-06
Lipid metabolism	Fatty acid metabolism	2	0.001 610 874
Energy metabolism	Methane metabolism	1	0.009 133 748
Metabolism of terpenoids and polyketides	Geraniol degradation	1	0.005 229 374

Classification	Pathway	Count	P-value
Human diseases	Prostate cancer	8	3.16E-13
	Melanoma	5	3.96E-08
	Endometrial cancer	4	6.87E-07
	Non-small cell lung cancer	4	8.01E-07
	Small cell lung cancer	4	5.21E-06
	Glioma	3	8.81E-05
	Alzheimer's disease	4	8.87E-05
	Renal cell carcinoma	3	1.15E-04
	Chronic myeloid leukemia	3	1.35E-04
	Colorectal cancer	3	1.89E-04
	Amyotrophic lateral sclerosis (ALS)	2	0.002 482 732
	Acute myeloid leukemia	2	0.002 751 467
	Epithelial cell signaling in Helicobacter pylori infection	2	0.003 847 862
	Long-term depression	2	0.003 956 072
	Pancreatic cancer	2	0.004 176 709
Endocrine system	Insulin signaling pathway	6	2.93E-08
	Melanogenesis	2	0.008 294 07
	GnRH signaling pathway	2	0.008 759 851
Amino acid metabolism	Aminosugars metabolism	4	6.16E-08
	Glutathione metabolism	2	0.001 984 888
Xenobiotics biodegradation and metabolism	Metabolism of xenobiotics by cytochrome P450	4	2.29E-06
	Drug metabolismother enzymes	3	4.25E-05
	Drug metabolismcytochrome P450	3	1.19E-04
	Caprolactam degradation	1	0.009 133 748
Cellular processes	Cell cycle	5	5.30E-07
	Adherens junction	4	3.71E-06
	Focal adhesion	5	7.64E-06
	p53 signaling pathway	3	1.05E-04
Immune system	B cell receptor signaling pathway	4	3.01E-06
	Fc epsilon RI signaling pathway	4	3.71E-06
	T cell receptor signaling pathway	4	1.33E-05
	Antigen processing and presentation	2	0.006 140 796
	Toll-like receptor signaling pathway	2	0.007 989 977
Signal transduction	VEGF signaling pathway	3	1.40E-04
	ErbB signaling pathway	3	2.09E-04
	MAPK signaling pathway	4	4.76E-04
	mTOR signaling pathway	2	0.002 144 93
Nervous system	Axon guidance	3	6.79E-04
Carbohydrate metabolism	Glycolysis/gluconeogenesis	2	8.41E-05
	Galactose metabolism	2	5.37E-04
	Fructose and mannose metabolism	2	0.001 032 896
	Starch and sucrose metabolism	1	0.002 144 93
Nucleotide metabolism	Purine metabolism	4	1.78E-06
	Pyrimidine metabolism	4	7.12E-06
Lipid metabolism	Fatty acid metabolism	2	0.001 610 874
Energy metabolism	Methane metabolism	1	0.009 133 748
Metabolism of terpenoids and polyketides	Geraniol degradation	1	0.005 229 374

基于网络药理学的黄连解毒汤治疗阿尔兹海默症的作用机制研究

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孙莉敏 ¹^,²^,³ , 刘丽芳 ¹^,^* , 朱华旭 ²^,³^,^* , 朱宝杰 ²^,³ , 张启春 ²^,³

药学学报 | 研究论文 2017,52(8): 1268-1275

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药学学报 | 研究论文 2017, 52(8): 1268-1275

基于网络药理学的黄连解毒汤治疗阿尔兹海默症的作用机制研究

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孙莉敏¹^,²^,³, 刘丽芳¹^,^*, 朱华旭²^,³^,^*, 朱宝杰²^,³, 张启春²^,³

作者信息

1.中国药科大学, 天然药物活性组分与药效国家重点实验室, 江苏南京 210009

2.南京中医药大学, 江苏省中药资源产业化过程协同创新中心, 江苏南京 210023

3.南京中医药大学, 江苏省植物药深加工工程研究中心, 江苏南京 210029

通讯作者:

* 刘丽芳, Tel:86-25-86185136, E-mail:liulifang69@126.com;

朱华旭, Tel/Fax:86-25-85811509, E-mail:huaxu72@126.com

Network pharmacology-based study on intervention mechanism of Huanglian Jiedu decoction in the treatment of Alzheimer's disease

Li-min SUN¹^,²^,³, Li-fang LIU¹^,^*, Hua-xu ZHU²^,³^,^*, Bao-jie ZHU²^,³, Qi-chun ZHANG²^,³

Affiliations

1. The State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China

2. Jiangsu Collaboration Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing 210023, China

3. Jiangsu Botanical Medicine Refinement Engineering Research Center, Nanjing University of Chinese Medicine, Nanjing 210029, China

出版时间: 2017-08-12 doi: 10.16438/j.0513-4870.2017-0144

文章导航

摘要

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建立黄连解毒汤"药效成分-靶标-通路"之间的关系，探究该方治疗阿尔兹海默症（Alzheimer'sdisease，AD）的多成分、多靶点和多途径作用机制，为创新药物研究奠定基础。自黄连解毒汤中筛选出已知的17个具有抗AD作用的药效成分，利用PharmMapper进行靶标预测，建立"药效成分-靶标蛋白"对应关系；通过Molecule Annotation System（MAS 3.0）数据库对获得的靶标蛋白进行Kyoto Encyclopedia of Genes and Genomes（KEGG）通路注释；利用Cytoscape 3.4.0软件构建"药效成分-靶点-通路"网络图。预测结果表明，黄连解毒汤中17个药效成分的作用靶点共59个，涉及通路47条，其中与AD相关的靶点蛋白共4个，与神经炎症相关的通路共2条。通过"药效成分-靶标-通路"分析可知，黄连解毒汤可能通过清除/减少β淀粉样蛋白、抑制Tau蛋白过度磷酸化、抗炎和免疫活性等多途径对AD具有治疗作用。

关键词

网络药理学 / 黄连解毒汤 / 阿尔兹海默症 / 作用机制 / 分子对接

Abstract

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This study was designed to explore the "multi-components, multi-targets and multi-pathways" intervention mechanism of Huanglian Jiedu decoction (HLJDD) in the treatment of Alzheimer's disease (AD) by pharmacological network technology, which may establish a foundation for drug development and innovative research. Seventeen active constituents of HLJDD with anti-AD activities were submitted to PharmMapper and Molecule Annotation System (MAS 3.0) bioinformatics softwares to predict the target proteins and carry out related KEGG pathways annotation respectively. The network of "active compound-target-pathway" was constructed and analyzed using the Cytoscape 3.4.0 software. The results suggest that 47 pathways are affected by the 17 active components through 59 target proteins, in which 4 target proteins are related to AD and 2 pathways related to neuroinflammation, respectively. The effect of HLJDD on AD may be dependent on clearing/reducing β-amyloid protein, inhibiting Tau hyperphosphorylation, anti-inflammation and immunoregulation.

Key words

network pharmacology / Huanglian Jiedu decoction / Alzheimer's disease / mechanism / molecular docking

引用本文

孙莉敏, 刘丽芳, 朱华旭, 朱宝杰, 张启春. 基于网络药理学的黄连解毒汤治疗阿尔兹海默症的作用机制研究. 药学学报, 2017 , 52 (8) : 1268 -1275 . DOI: 10.16438/j.0513-4870.2017-0144

Li-min SUN, Li-fang LIU, Hua-xu ZHU, Bao-jie ZHU, Qi-chun ZHANG. Network pharmacology-based study on intervention mechanism of Huanglian Jiedu decoction in the treatment of Alzheimer's disease[J]. Acta Pharmaceutica Sinica, 2017 , 52 (8) : 1268 -1275 . DOI: 10.16438/j.0513-4870.2017-0144

正文

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“整体观”和“辨证论治”是中医药两大特色理论体系, 在治疗一些复杂及慢性疾病方面显示出独特的优势^[1]。随着系统生物学、多向药理学和生物信息学等学科的快速发展, 网络药理学作为一种药物研究的新模式应运而生, 其整体性和系统性的特点与中医药“整体观”、“辨证论治”的理论体系相一致^[2], 可在一定程度上诠释中药复方的多成分、多靶点和多途径的作用特点, 为中药复方的作用机制揭示和创新药物研究提供了有力的研究手段。

阿尔兹海默症(Alzheimer’s disease, AD)是一种严重威胁老年人生命健康的中枢神经系统退行性疾病^[3]。现代药理研究表明, AD的常见病理特征有β-淀粉样蛋白斑块(β-amyloid plaques, Aβ斑块)、过度磷酸化Tau蛋白形成神经纤维缠结、乙酰胆碱递质减少、炎症反应、神经元丢失及淀粉样血管病等^[4]。中医理论认为, “毒损脑络”在该病的发生过程中起着重要作用^[5]; 清热解毒可作为老年痴呆新的治疗原则^[6]。而黄连解毒汤作为清热解毒方中的经典方剂, 近年来在AD治疗方面展现出良好的应用前景^[7]。本文以黄连解毒汤中具有抗AD作用的药效成分为研究对象, 应用网络药理学的研究方法, 建立“药效成分-靶标-通路”之间的关系, 探究该方治疗AD的多成分、多靶点和多途径作用机制, 为创新药物研究奠定基础。

材料与方法

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实验材料

ChemDraw Ultra 7.0, ChemBio3D Ultra 12.0, PharmMapper数据库(http://59.78.95.61/pharmmapper/); UniProt数据库(http://www.Uniprot.org/); 生物分子功能注释系统(Molecule Annotation System, MAS 3.0) 数据库(http://bioinfo.capitalbio.com/mas3/); Kyoto Encyclopedia of Genes and Genomes (KEGG)通路数据库(http://www.Genome.jp/kegg/); Cytoscape 3.4.0软件。

黄连解毒汤中抗AD作用的药效成分筛选

黄连解毒汤由黄连、黄芩、黄柏和栀子(3：2：2：3) 组成。参照乙酰胆碱酯酶(acetylcholinesterase, AChE)抑制剂筛选模型, 应用Ellman比色法^[8]从黄连解毒汤中筛选具有抑制AChE的活性成分, 同时查阅文献, 统计黄连解毒汤中具有抗AD的药效成分。

黄连解毒汤中抗AD作用的药效成分潜在靶点预测

首先, 利用ChemDraw Ultra 7.0软件绘制黄连解毒汤中具有抗AD作用的药效成分的分子结构式(图 1), 保存为MOL2 (.mol2) 文件。再将文件导入ChemBio3D中转换为SDF (.sdf)格式保存, 然后将^*.sdf格式的各个活性成分文件分别导入PharmMapper数据库中的Submit Job进行靶点预测, 依次选择参数: Generate Conformers-Yes; Maximum Generated Conformations-100; Select Targets Set-Human Protein Targets Only (2 241); Number of Reserved Matched Targets (Max 1 000)-100。得到与该化合物相关的蛋白质数据库编码(PDB ID)、靶点名称(target name)、频数(number of feature)和匹配值(fit score)等结果。根据匹配值, 筛选前10个靶点作为与该化合物相关的重要靶点蛋白。由于得到的靶点蛋白存在命名不规范的情况。因此, 运用UniProt数据库中的UniProtKB统一靶点编码为“P08473、Q08499和O15530”格式, 方便统计和分析。

相关通路的注释和分析

将获得的活性成分相关靶点导入MAS 3.0数据库中进行通路分析, 得到“pathwayIndexByPathway_kegg”结果, 挑选P < 0.01的通路作为可靠通路, 并进一步采用KEGG数据库对这些通路进行注释。

构建黄连解毒汤中抗AD作用的“药效成分-靶标-通路”网络关系图

将17个药效成分的预测靶点结果和通路分析, 在Excel表中分别构建“药效成分-靶点”、“靶点-通路”之间的相互关系, 然后将其导入Cytoscape软件中建立“药效成分-靶标-通路”网络关系图^[9]。图中以药效成分、靶点蛋白和通路为3类节点(node), 它们之间有相互关系的分别用边(edge)相连, 通过构建药效成分-蛋白-通路、药效成分-蛋白-药效成分、蛋白-药效成分-蛋白、通路-蛋白-通路和蛋白-通路-蛋白等5种连接, 建立完整的网络图。

结果

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1 黄连解毒汤中抗AD作用的药效成分筛选

黄连解毒汤中小檗碱、药根碱、巴马汀、表小檗碱、非洲防己碱和黄连碱具有明显抑制AChE作用(图 2), 半数抑制率分别是11.88、30.34、9.168、30.25、4.784和21.03 μmol·L^-1, 这些结果与文献报道相一致^{[10, 11]}。查阅文献发现, 格兰地新具有抑制β-分泌酶(BACE)的作用^[11]; 黄芩素、黄芩苷、去甲基汉黄芩素和汉黄芩素具有较强的抗氧化活性和清除自由基的活性^{[12, 13]}; 京尼平苷、10-O-乙酰基京尼平苷、红景天苷、(1R, 7R, 10S)-7-羟基-11-O-β-D-吡喃葡萄糖基愈创木烷-4-烯-3-酮、10-(6-O-反-芥子酰基吡喃葡萄糖基)栀子二醇和11-(6-O-反-芥子酰基吡喃葡萄糖基)栀子二醇表现出不同程度地提高老年痴呆转基因果蝇短期学习记忆能力^[14]。

2 黄连解毒汤中抗AD作用的药效成分潜在靶点蛋白信息

黄连解毒汤中筛选出的具有抗AD作用的17个活性成分共涉及59个靶点蛋白, 详细结果见表 1。从得到的靶点信息可知, BACE1 (P56817) 出现的频数最高(18次), 其次是雌二醇17-β-脱氢酶1 (P14061) 和GTP酶HRas (P01112) 等。

3 黄连解毒汤中抗AD作用药效成分潜在靶点蛋白的作用通路注释和分析

将黄连解毒汤中抗AD作用成分预测出的59个靶点蛋白信息导入MAS 3.0中进行KEGG通路注释和分析, 共得到47条代谢通路, 涉及13种类别, 其中与疾病(human diseases)相关的通路共15条; 与免疫系统(immune system)相关的通路共5条; 与信号传导(signal transduction)相关的通路共4条; 与神经系统(nervous system)相关的通路仅1条; 与碳水化合物代谢(carbohydrate metabolism)相关通路共4条; 与脂质代谢(lipid metabolism)相关通路仅1条等(表 2)。

4 黄连解毒汤中抗AD作用“药效成分-靶标-通路”网络构建

采用Cytoscape 3.4.0软件建立黄连解毒汤中抗AD活性成分-靶点蛋白-通路网络模型图, 17个活性成分、59个靶点和47条通路之间的相互关系见图 3。

讨论

收起

通过“药效成分-靶标-通路”网路分析发现: ① 黄连解毒汤中生物碱、黄酮和环烯醚萜3大类成分既可通过不同的靶点与同一作用通路相连接, 也可以通过同一靶点与不同的作用通路相连接, 说明不同成分的作用靶点具有协同作用; ② 黄连解毒汤具有多成分、多靶点和多途径的整体调节特点, 其作用靶点包括BACE1、雌二醇17-β-脱氢酶1和GTP蛋白酶等, 作用通路涉及疾病-内分泌-细胞-神经-免疫-信号传导等多条代谢通路。

反向药效团匹配结果表明, 药效成分作用靶点中与AD相关的靶点蛋白共4个, 分别是BACE1、糖原合成酶激酶-3β (glycogen synthase kinase-3β, GSK-3β)、脑啡肽酶(neprilysin, NEP)和诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS), 其中BACE1为AD的一个病理特征, 在生成Aβ的过程中发挥了关键性作用。因此, BACE1抑制剂是治疗AD的一个靶点^[15]; GSK-3β可促进Tau蛋白过度磷酸化、Aβ异常聚集和神经细胞凋亡, 在AD病变过程中发挥关键作用^[16]; NEP是脑内最主要的Aβ降解酶^[17], 在AD的病理和治疗过程中起到关键作用^[18]; iNOS参与了AD的发病进程^[19]。反向对接实验结果表明: ① 黄芩苷和11-(6-O-反-芥子酰基吡喃葡萄糖基)栀子二醇与BACE1相接, 预测该2种成分通过抑制BACE1活性, 减少Aβ产生, 发挥对抗AD的作用^[20]; ② 11-(6-O-反-芥子酰基吡喃葡萄糖基)栀子二醇与GSK-3β相接, 预测其可通过抑制Tau蛋白过度磷酸化和Aβ异常聚集、减弱神经细胞凋亡发挥抗AD作用; ③ 巴马汀、表小檗碱、非洲防己碱、格兰地新、黄连碱、小檗碱、药根碱和10-O-乙酰基京尼平苷均与NEP相接, 预测这些成分可通过增强NEP活性, 清除脑内Aβ, 发挥抗AD作用; ④ 11-(6-O-反-芥子酰基吡喃葡萄糖基)栀子二醇与iNOS相接, 预测其通过抗炎作用发挥抗AD作用。

通过分子对接发现, 与AD相关的靶点蛋白中GSK-3β与胰岛素信号通路(insulin signaling pathway)相接。研究发现^{[21, 22]}, 胰岛素可以透过血脑屏障进入中枢神经系统, 发挥调节作用。当胰岛素信号通路受损时, 导致脑中葡萄糖代谢衰退, 与AD疾病发展密切相关, 因此AD的另一种假说“AD可能是3型糖尿病”^[23]。研究^{[24, 25]}表明, 胰岛素信号传导通过作用于生长因子受体结合蛋白2 (growth factor receptor bound protein-2, Grb2)、丝裂原活化蛋白激酶(mitogen activated protein kinase, MAPK)信号通路和GSK-3β等, 影响细胞生长、调控β淀粉样前体蛋白(β-amyloid precursor protein, βAPP)的表达、Aβ斑块清除和Tau磷酸化水平等。Li等^[26]研究表明, 黄连解毒汤可有效改善2型糖尿病(T2DM)大鼠学习记忆能力, 对T2DM大鼠海马Tau蛋白Alzheimer病样过度磷酸化修饰具有较好的抑制作用。因此, 黄连解毒汤可能通过胰岛素信号通路发挥清除/减少脑内Aβ、抑制Tau蛋白过度磷酸化而发挥抗AD作用。

据文献^[27]报道, AD主要的病理产物—Aβ斑块和神经元纤维缠结(NFTs)可以引起神经炎症, 对脑组织产生毒性, 而这些神经炎症细胞因子的产生受活化的小胶质细胞和星形胶质细胞调控, 其中小胶质细胞内与神经炎症相关的信号通路包括MAPK信号通路、NF-κB信号通路、Toll样受体信号通路、过氧化物酶体增殖物激活受体-γ (PPAR-γ)、Notch信号通路及JAK-STAT信号通路^[28]。从网络分析结果中发现, 黄连解毒汤中抗AD作用的药效成分与神经炎症相关的信号通路分别是MAPK信号通路和Toll样受体信号通路; 而MAPK信号通路中的ERK信号通路、JNK/SAPK9通路和p38MAPK通路均在AD所造成神经细胞损伤机制中发挥重要作用^[29]; Toll样受体-4 (TLR-4) 信号通路在AD的发病过程中发挥重要作用^[30]。

与MAPK信号通路具有相关性的化合物:黄连碱、药根碱、黄芩苷、京尼平苷、去甲基汉黄芩素、黄芩素、汉黄芩素、11-(6-O-反-芥子酰基吡喃葡萄糖基)栀子二醇、10-O-乙酰基京尼平苷、10-(6-O-反-芥子酰基吡喃葡萄糖基)栀子二醇、(1R, 7R, 10S)-7-羟基-11-O-β-D-吡喃葡萄糖基愈创木烷-4-烯-3-酮和红景天苷。Toll样受体(Toll-like receptors, TLRs)是一类重要的模式识别受体(pattern recognition receptors, PRR), 可特异性识别病原相关分子模式(PAMPs), 参与激活先天性免疫和获得性免疫应答的启动。与Toll样受体信号通路具有相关性的化合物为药根碱。以上预测结果表明, 黄连解毒汤中生物碱、黄酮和环烯醚萜3大类化合物均具有不同程度的抗炎作用, 与本实验室前期研究结果相一致^[31], 该方可能通过抗炎作用而发挥其AD治疗作用。

结论

收起

本文应用网络药理学的研究方法对该方中17个已知的的药效成分进行靶标预测和通路分析, 通过建立“药效成分-靶点-通路”网络图, 发现黄连解毒汤中药效成分可能通过抑制Aβ产生、抑制Tau蛋白过度磷酸化和Aβ异常聚集、减弱神经细胞凋亡、清除脑内Aβ、抗炎和免疫活性发挥抗AD作用, 提示这些可能是该方治疗AD的潜在机制。

基金

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国家自然科学基金资助项目(81573635)

参考文献

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文献

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2017年第52卷第8期

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doi: 10.16438/j.0513-4870.2017-0144

接收时间：2017-02-17
首发时间：2026-01-14
出版时间：2017-08-12

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收稿日期：2017-02-17
修回日期：2017-03-21

基金

国家自然科学基金资助项目(81573635)

作者信息

1.中国药科大学, 天然药物活性组分与药效国家重点实验室, 江苏南京 210009

2.南京中医药大学, 江苏省中药资源产业化过程协同创新中心, 江苏南京 210023

3.南京中医药大学, 江苏省植物药深加工工程研究中心, 江苏南京 210029

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* 刘丽芳, Tel:86-25-86185136, E-mail:liulifang69@126.com;

朱华旭, Tel/Fax:86-25-85811509, E-mail:huaxu72@126.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Rank	Uniprot ID	Protein target	Frequency
1	P56817	β-Secretase 1	18
2	P14061	Estradiol 17-β-dehydrogenase 1	16
3	P01112	GTPase HRas	15
4	Q9BZX2	Uridine-cytidine kinase 2	14
5	P10114	Ras-related protein Rap-2a	14
6	P15121	Aldose reductase	14
7	P23919	Thymidylate kinase	13
8	P11473	Vitamin D3 receptor	13
9	P15121	Aldose reductase	12
10	Q06187	Tyrosine-protein kinase BTK	12
11	P09211	Glutathione S-transferase P	12
12	P50225	Sulfotransferase 1A1	12
13	P49841	Glycogen synthase kinase-3β	12
14	P00492	Hypoxanthine-guanine phosphoribosyltransferase	12
15	P31749	RAC-αserine/threonine-protein kinase	12
16	P06213	Insulin receptor	11
17	Q96C86	Scavenger mRNA-decapping enzyme DcpS	11
18	Q16836	Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial	11
19	Q16222	UDP-N-acetylhexosamine pyrophosphorylase	11
20	Q9BZ11	ADAM 33	10
21	O76054	SEC14-like protein 2	10
22	P29373	Cellular retinoic acid-binding protein 2	10
23	Q16539	Mitogen-activated protein kinase 14	10
24	P08473	Neprilysin	10
25	Q15075	Early endosome antigen 1	10
26	P07900	Heat shock protein HSP 90-α	10
27	P02766	Transthyretin	10
28	Q02127	Dihydroorotate dehydrogenase, mitochondrial	9
29	P04150	Glucocorticoid receptor	9
30	Q16772	Glutathione S-transferase A3	9
31	P27707	Deoxycytidine kinase	9
32	P08246	Leukocyte elastase	9
33	P12268	Inosine-5-monophosphate dehydrogenase 2	9
34	P07858	Cathepsin B	9
35	P35228	Nitric oxide synthase, inducible	9
36	P43235	Cathepsin K	9
37	P24941	Cell division protein kinase 2	8
38	Q13231	Chitotriosidase-1	8
39	P18031	Tyrosine-protein phosphatase non-receptor type 1	8
40	P62942	Peptidyl-prolyl cis-trans isomerase FKBP1A	8
41	P11362	Basic fibroblast growth factor receptor 1	8
42	P07686	β-Hexosaminidase β chain	8
43	Q08499	cAMP-specific 3, 5-cyclic phosphodiesterase 4D	8
44	Q08188	Protein-glutamine γ-glutamyltransferase E	8
45	P19367	Hexokinase-1	8
46	P00918	Carbonic anhydrase 2	8
47	O15530	3-Phosphoinositide-dependent protein kinase 1	7
48	P11766	Alcohol dehydrogenase class-3	7
49	P17931	Galectin-3	7
50	P20248	Cyclin-A2	7
51	P42330	Aldo-keto reductase family 1 member C3	7
52	P08581	Hepatocyte growth factor receptor	6
53	O14757	Serine/threonine-protein kinase Chk1	6
54	P04278	Sex hormone-binding globulin	6
55	Q10588	ADP-ribosyl cyclase 2	6
56	P20248	Cyclin-A2	6
57	Q00534	Cell division protein kinase 6	5
58	P03372	Estrogen receptor	5
59	P29218	Inositol monophosphatase	5

Rank

Uniprot ID

Protein target

Frequency

P56817

β-Secretase 1

P14061

Estradiol 17-β-dehydrogenase 1

P01112

GTPase HRas

Q9BZX2

Uridine-cytidine kinase 2

P10114

Ras-related protein Rap-2a

P15121

Aldose reductase

P23919

Thymidylate kinase

P11473

Vitamin D3 receptor

P15121

Aldose reductase

Q06187

Tyrosine-protein kinase BTK

P09211

Glutathione S-transferase P

P50225

Sulfotransferase 1A1

P49841

Glycogen synthase kinase-3β

P00492

Hypoxanthine-guanine phosphoribosyltransferase

P31749

RAC-αserine/threonine-protein kinase

P06213

Insulin receptor

Q96C86

Scavenger mRNA-decapping enzyme DcpS

Q16836

Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial

Q16222

UDP-N-acetylhexosamine pyrophosphorylase

Q9BZ11

ADAM 33

O76054

SEC14-like protein 2

P29373

Cellular retinoic acid-binding protein 2

Q16539

Mitogen-activated protein kinase 14

P08473

Neprilysin

Q15075

Early endosome antigen 1

P07900

Heat shock protein HSP 90-α

P02766

Transthyretin

Q02127

Dihydroorotate dehydrogenase, mitochondrial

P04150

Glucocorticoid receptor

Q16772

Glutathione S-transferase A3

P27707

Deoxycytidine kinase

P08246

Leukocyte elastase

P12268

Inosine-5-monophosphate dehydrogenase 2

P07858

Cathepsin B

P35228

Nitric oxide synthase, inducible

P43235

Cathepsin K

P24941

Cell division protein kinase 2

Q13231

Chitotriosidase-1

P18031

Tyrosine-protein phosphatase non-receptor type 1

P62942

Peptidyl-prolyl cis-trans isomerase FKBP1A

P11362

Basic fibroblast growth factor receptor 1

P07686

β-Hexosaminidase β chain

Q08499

cAMP-specific 3, 5-cyclic phosphodiesterase 4D

Q08188

Protein-glutamine γ-glutamyltransferase E

P19367

Hexokinase-1

P00918

Carbonic anhydrase 2

O15530

3-Phosphoinositide-dependent protein kinase 1

P11766

Alcohol dehydrogenase class-3

P17931

Galectin-3

P20248

Cyclin-A2

P42330

Aldo-keto reductase family 1 member C3

P08581

Hepatocyte growth factor receptor

O14757

Serine/threonine-protein kinase Chk1

P04278

Sex hormone-binding globulin

Q10588

ADP-ribosyl cyclase 2

P20248

Cyclin-A2

Q00534

Cell division protein kinase 6

P03372

Estrogen receptor

P29218

Inositol monophosphatase

Classification	Pathway	Count	P-value
Human diseases	Prostate cancer	8	3.16E-13
	Melanoma	5	3.96E-08
	Endometrial cancer	4	6.87E-07
	Non-small cell lung cancer	4	8.01E-07
	Small cell lung cancer	4	5.21E-06
	Glioma	3	8.81E-05
	Alzheimer's disease	4	8.87E-05
	Renal cell carcinoma	3	1.15E-04
	Chronic myeloid leukemia	3	1.35E-04
	Colorectal cancer	3	1.89E-04
	Amyotrophic lateral sclerosis (ALS)	2	0.002 482 732
	Acute myeloid leukemia	2	0.002 751 467
	Epithelial cell signaling in Helicobacter pylori infection	2	0.003 847 862
	Long-term depression	2	0.003 956 072
	Pancreatic cancer	2	0.004 176 709
Endocrine system	Insulin signaling pathway	6	2.93E-08
	Melanogenesis	2	0.008 294 07
	GnRH signaling pathway	2	0.008 759 851
Amino acid metabolism	Aminosugars metabolism	4	6.16E-08
	Glutathione metabolism	2	0.001 984 888
Xenobiotics biodegradation and metabolism	Metabolism of xenobiotics by cytochrome P450	4	2.29E-06
	Drug metabolismother enzymes	3	4.25E-05
	Drug metabolismcytochrome P450	3	1.19E-04
	Caprolactam degradation	1	0.009 133 748
Cellular processes	Cell cycle	5	5.30E-07
	Adherens junction	4	3.71E-06
	Focal adhesion	5	7.64E-06
	p53 signaling pathway	3	1.05E-04
Immune system	B cell receptor signaling pathway	4	3.01E-06
	Fc epsilon RI signaling pathway	4	3.71E-06
	T cell receptor signaling pathway	4	1.33E-05
	Antigen processing and presentation	2	0.006 140 796
	Toll-like receptor signaling pathway	2	0.007 989 977
Signal transduction	VEGF signaling pathway	3	1.40E-04
	ErbB signaling pathway	3	2.09E-04
	MAPK signaling pathway	4	4.76E-04
	mTOR signaling pathway	2	0.002 144 93
Nervous system	Axon guidance	3	6.79E-04
Carbohydrate metabolism	Glycolysis/gluconeogenesis	2	8.41E-05
	Galactose metabolism	2	5.37E-04
	Fructose and mannose metabolism	2	0.001 032 896
	Starch and sucrose metabolism	1	0.002 144 93
Nucleotide metabolism	Purine metabolism	4	1.78E-06
	Pyrimidine metabolism	4	7.12E-06
Lipid metabolism	Fatty acid metabolism	2	0.001 610 874
Energy metabolism	Methane metabolism	1	0.009 133 748
Metabolism of terpenoids and polyketides	Geraniol degradation	1	0.005 229 374

Classification

Pathway

Count

P-value

Human diseases

Prostate cancer

3.16E-13

Melanoma

3.96E-08

Endometrial cancer

6.87E-07

Non-small cell lung cancer

8.01E-07

Small cell lung cancer

5.21E-06

Glioma

8.81E-05

Alzheimer's disease

8.87E-05

Renal cell carcinoma

1.15E-04

Chronic myeloid leukemia

1.35E-04

Colorectal cancer

1.89E-04

Amyotrophic lateral sclerosis (ALS)

0.002 482 732

Acute myeloid leukemia

0.002 751 467

Epithelial cell signaling in Helicobacter pylori infection

0.003 847 862

Long-term depression

0.003 956 072

Pancreatic cancer

0.004 176 709

Endocrine system

Insulin signaling pathway

2.93E-08

Melanogenesis

0.008 294 07

GnRH signaling pathway

0.008 759 851

Amino acid metabolism

Aminosugars metabolism

6.16E-08

Glutathione metabolism

0.001 984 888

Xenobiotics biodegradation and metabolism

Metabolism of xenobiotics by cytochrome P450

2.29E-06

Drug metabolismother enzymes

4.25E-05

Drug metabolismcytochrome P450

1.19E-04

Caprolactam degradation

0.009 133 748

Cellular processes

Cell cycle

5.30E-07

Adherens junction

3.71E-06

Focal adhesion

7.64E-06

p53 signaling pathway

1.05E-04

Immune system

B cell receptor signaling pathway

3.01E-06

Fc epsilon RI signaling pathway

3.71E-06

T cell receptor signaling pathway

1.33E-05

Antigen processing and presentation

0.006 140 796

Toll-like receptor signaling pathway

0.007 989 977

Signal transduction

VEGF signaling pathway

1.40E-04

ErbB signaling pathway

2.09E-04

MAPK signaling pathway

4.76E-04

mTOR signaling pathway

0.002 144 93

Nervous system

Axon guidance

6.79E-04

Carbohydrate metabolism

Glycolysis/gluconeogenesis

8.41E-05

Galactose metabolism

5.37E-04

Fructose and mannose metabolism

0.001 032 896

Starch and sucrose metabolism

0.002 144 93

Nucleotide metabolism

Purine metabolism

1.78E-06

Pyrimidine metabolism

7.12E-06

Lipid metabolism

Fatty acid metabolism

0.001 610 874

Energy metabolism

Methane metabolism

0.009 133 748

Metabolism of terpenoids and polyketides

Geraniol degradation

0.005 229 374