药学学报

Protein target	Common name	Uniprot ID	Frequency
Mitogen-activated protein kinase 8	MAPK8	P45983	13
Oxysterols receptor LXR-beta	NR1H2	P55055	7
Proto-oncogene tyrosine-protein kinase Src	SRC	P12931	5
Neutrophil gelatinase-associated lipocalin	LCN2	P80188	4
Phosphatidylinositol 4, 5-bisphosphate 3-kinase catalytic subunit gamma isoform	PIK3CG	P48736	4
Macrophage migration inhibitory factor	MIF	P14174	3
Triggering receptor expressed on myeloid cells 1	TREM1	Q9NP99	3
Hematopoietic prostaglandin D synthase	HPGDS	O60760	3
Plasminogen	PLG	P00747	2
Mitogen-activated protein kinase 14	MAPK14	Q16539	1
Cathepsin K	CTSK	P43235	1
Peroxisome proliferator-activated receptor delta	PPARD	Q03181	1
Peroxisome proliferator-activated receptor gamma	PPARG	P37231	1

Protein target	Common name	Uniprot ID	Frequency
Mitogen-activated protein kinase 8	MAPK8	P45983	13
Oxysterols receptor LXR-beta	NR1H2	P55055	7
Proto-oncogene tyrosine-protein kinase Src	SRC	P12931	5
Neutrophil gelatinase-associated lipocalin	LCN2	P80188	4
Phosphatidylinositol 4, 5-bisphosphate 3-kinase catalytic subunit gamma isoform	PIK3CG	P48736	4
Macrophage migration inhibitory factor	MIF	P14174	3
Triggering receptor expressed on myeloid cells 1	TREM1	Q9NP99	3
Hematopoietic prostaglandin D synthase	HPGDS	O60760	3
Plasminogen	PLG	P00747	2
Mitogen-activated protein kinase 14	MAPK14	Q16539	1
Cathepsin K	CTSK	P43235	1
Peroxisome proliferator-activated receptor delta	PPARD	Q03181	1
Peroxisome proliferator-activated receptor gamma	PPARG	P37231	1

Protein target	Common name	Uniprot ID	Frequency
Aldo-keto reductase family 1 member C2	AKR1C2	P52895	30
Androgen receptor	AR	P10275	24
Tyrosine-protein phosphatase non-receptor type 1	PTPN1	P18031	21
Serum albumin	ALB	P02768	20
Prothrombin	F2	P00734	20
Mitogen-activated protein kinase 1	MAPK1	P28482	19
Glutathione S-transferase P	GSTP1	P09211	19
Aldose reductase	AKR1B1	P15121	17
Aromatase	CYP19A1	P11511	15
cGMP-specific 3', 5'-cyclic phosphodiesterase	PDE5A	O76074	15
Amine oxidase [flavin-containing] B	MAOB	P27338	14
Mitogen-activated protein kinase 8	MAPK8	P45983	13
Thyroid hormone receptor beta	THRB	P10828	13
Liver carboxylesterase 1	CES1	P23141	12
Complement factor B	CFB	P00751	10
Glucosylceramidase	GBA	P04062	10
Estrogen receptor beta	ESR2	Q92731	8
Oxysterols receptor LXR-beta	NR1H2	P55055	6
Glycine amidinotransferase, mitochondrial	GATM	P50440	6
Nuclear receptor subfamily 1 group Ⅰ member 3	NR1I3	Q14994	4
Vitamin D-binding protein	GC	P02774	4
Macrophage migration inhibitory factor	MIF	P14174	3
Phosphoenolpyruvate carboxykinase, cytosolic [GTP]	PCK1	P35558	2
Nuclear receptor ROR-alpha	RORA	P35398	2
Estrogen-related receptor gamma	ESRRG	P62508	2
Heat shock protein HSP 90-alpha	HSP90AA1	P07900	2
Glycogen synthase kinase-3 beta	GSK3B	P49841	2
Retinoic acid receptor RXR-alpha	RXRA	P19793	1
C-1-tetrahydrofolate synthase, cytoplasmic	MTHFD1	P11586	1
cGMP-inhibited 3', 5'-cyclic phosphodiesterase B	PDE3B	Q13370	1
Peroxisome proliferator-activated receptor delta	PPARD	Q03181	1
Peroxisome proliferator-activated receptor gamma	PPARG	P37231	1
Dihydrofolate reductase	DHFR	P00374	1
Arylsulfatase A	ARSA	P15289	1

Protein target	Common name	Uniprot ID	Frequency
Aldo-keto reductase family 1 member C2	AKR1C2	P52895	30
Androgen receptor	AR	P10275	24
Tyrosine-protein phosphatase non-receptor type 1	PTPN1	P18031	21
Serum albumin	ALB	P02768	20
Prothrombin	F2	P00734	20
Mitogen-activated protein kinase 1	MAPK1	P28482	19
Glutathione S-transferase P	GSTP1	P09211	19
Aldose reductase	AKR1B1	P15121	17
Aromatase	CYP19A1	P11511	15
cGMP-specific 3', 5'-cyclic phosphodiesterase	PDE5A	O76074	15
Amine oxidase [flavin-containing] B	MAOB	P27338	14
Mitogen-activated protein kinase 8	MAPK8	P45983	13
Thyroid hormone receptor beta	THRB	P10828	13
Liver carboxylesterase 1	CES1	P23141	12
Complement factor B	CFB	P00751	10
Glucosylceramidase	GBA	P04062	10
Estrogen receptor beta	ESR2	Q92731	8
Oxysterols receptor LXR-beta	NR1H2	P55055	6
Glycine amidinotransferase, mitochondrial	GATM	P50440	6
Nuclear receptor subfamily 1 group Ⅰ member 3	NR1I3	Q14994	4
Vitamin D-binding protein	GC	P02774	4
Macrophage migration inhibitory factor	MIF	P14174	3
Phosphoenolpyruvate carboxykinase, cytosolic [GTP]	PCK1	P35558	2
Nuclear receptor ROR-alpha	RORA	P35398	2
Estrogen-related receptor gamma	ESRRG	P62508	2
Heat shock protein HSP 90-alpha	HSP90AA1	P07900	2
Glycogen synthase kinase-3 beta	GSK3B	P49841	2
Retinoic acid receptor RXR-alpha	RXRA	P19793	1
C-1-tetrahydrofolate synthase, cytoplasmic	MTHFD1	P11586	1
cGMP-inhibited 3', 5'-cyclic phosphodiesterase B	PDE3B	Q13370	1
Peroxisome proliferator-activated receptor delta	PPARD	Q03181	1
Peroxisome proliferator-activated receptor gamma	PPARG	P37231	1
Dihydrofolate reductase	DHFR	P00374	1
Arylsulfatase A	ARSA	P15289	1

Classification	Pathway	P-value	Count
Diseases	Influenza A	0.001 7	4
	Shigellosis	0.003 6	3
	Epithelial cell signaling in Helicobacter pylori infection	0.004 0	3
	Chagas disease (American trypanosomiasis)	0.009 3	3
Signal transduction	Prolactin signaling pathway	0.000 1	4
	Toll-like receptor signaling pathway	0.000 3	4
	VEGF signaling pathway	0.003 3	3
	Fc epsilon RI signaling pathway	0.004 1	3
	ErbB signaling pathway	0.006 6	3
	GnRH signaling pathway	0.007 2	3
	TNF signaling pathway	0.009 7	3
Cell processes	Osteoclast differentiation	0.000 1	5
	Progesterone-mediated oocyte maturation	0.006 6	3
Sensory system	Inflammatory mediator regulation of TRP channels	0.000 3	4

Classification	Pathway	P-value	Count
Diseases	Influenza A	0.001 7	4
	Shigellosis	0.003 6	3
	Epithelial cell signaling in Helicobacter pylori infection	0.004 0	3
	Chagas disease (American trypanosomiasis)	0.009 3	3
Signal transduction	Prolactin signaling pathway	0.000 1	4
	Toll-like receptor signaling pathway	0.000 3	4
	VEGF signaling pathway	0.003 3	3
	Fc epsilon RI signaling pathway	0.004 1	3
	ErbB signaling pathway	0.006 6	3
	GnRH signaling pathway	0.007 2	3
	TNF signaling pathway	0.009 7	3
Cell processes	Osteoclast differentiation	0.000 1	5
	Progesterone-mediated oocyte maturation	0.006 6	3
Sensory system	Inflammatory mediator regulation of TRP channels	0.000 3	4

Classification	Pathway	P-value	Count
Diseases	Pathways in cancer	0.001 0	8
	Insulin resistance	0.001 0	5
	Prostate cancer	0.005 7	4
	Thyroid cancer	0.006 4	3
Signal transduction	Insulin signaling pathway	0.000 2	6
	PPAR signaling pathway	0.002 7	4
	Prolactin signaling pathway	0.003 1	4
Cell processes	Progesterone-mediated oocyte maturation	0.005 6	4

Classification	Pathway	P-value	Count
Diseases	Pathways in cancer	0.001 0	8
	Insulin resistance	0.001 0	5
	Prostate cancer	0.005 7	4
	Thyroid cancer	0.006 4	3
Signal transduction	Insulin signaling pathway	0.000 2	6
	PPAR signaling pathway	0.002 7	4
	Prolactin signaling pathway	0.003 1	4
Cell processes	Progesterone-mediated oocyte maturation	0.005 6	4

Target name	PDB ID	Ingredient	Docking score
MAPK8	1UKI	Berberine	6.374
		Geniposide	6.403
		Baicalin	6.207
NR1H2	1PQ9	Berberine	6.397
		Geniposide	6.747
		Baicalin	6.397
MIF	1CA7	Berberine	6.209
		Geniposide	6.395
		Baicalin	6.439
PPARD	3DY6	Berberine	6.527
		Geniposide	7.024
		Baicalin	6.640
PPARG	3B3K	Berberine	6.550
		Geniposide	6.470
		Baicalin	6.641

Target name	PDB ID	Ingredient	Docking score
MAPK8	1UKI	Berberine	6.374
		Geniposide	6.403
		Baicalin	6.207
NR1H2	1PQ9	Berberine	6.397
		Geniposide	6.747
		Baicalin	6.397
MIF	1CA7	Berberine	6.209
		Geniposide	6.395
		Baicalin	6.439
PPARD	3DY6	Berberine	6.527
		Geniposide	7.024
		Baicalin	6.640
PPARG	3B3K	Berberine	6.550
		Geniposide	6.470
		Baicalin	6.641

基于网络药理学的黄连解毒汤调节巨噬细胞炎症反应和代谢的作用机制研究

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衡霞 ¹^,² , 朱宝杰 ¹^,² , 孙莉敏 ¹^,² , 潘林梅 ¹^,² , 段金廒 ¹^,² , 张启春 ¹^,²^,^* , 朱华旭 ¹^,²^,^*

药学学报 | 专题报道 2018,53(9): 1449-1457

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药学学报 | 专题报道 2018, 53(9): 1449-1457

基于网络药理学的黄连解毒汤调节巨噬细胞炎症反应和代谢的作用机制研究

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衡霞¹^,², 朱宝杰¹^,², 孙莉敏¹^,², 潘林梅¹^,², 段金廒¹^,², 张启春¹^,²^,^*, 朱华旭¹^,²^,^*

作者信息

1.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

2.南京中医药大学江苏省方剂高技术研究重点实验室, 江苏南京 210023

通讯作者:

* 张启春, Tel/Fax:86-25-85811509, E-mail:zhangqc@njucm.edu.cn;

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

Network pharmacology-based study on mechanisms of Huanglian Jiedu Decoction impact on macrophage inflammation response

Xia HENG¹^,², Bao-jie ZHU¹^,², Li-min SUN¹^,², Lin-mei PAN¹^,², Jin-ao DUAN¹^,², Qi-chun ZHANG¹^,²^,^*, Hua-xu ZHU¹^,²^,^*

Affiliations

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

2. Jiangsu Province Key Laboratory of High Technology Research, Nanjing University of Chinese Medicine, Nanjing 210023, China

出版时间: 2018-09-15 doi: 10.16438/j.0513-4870.2018-0276

文章导航

摘要

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运用网络药理学方法探究黄连解毒汤调节巨噬细胞炎症反应、糖酵解、鞘脂代谢和谷氨酰胺代谢等方面的多成分、多靶点、多通路的相互作用规律和调控网络，为创新药物研究奠定基础。通过TCMSP数据库筛选黄连解毒汤活性成分，PharmMapper数据库预测靶点蛋白，DAVID数据库进行通路注释和分析，Cytoscape 3.2.1软件构建“活性成分-靶点-通路”网络图，GENEMANIA数据库进行蛋白相互作用分析，Systems Dock Web Site数据库进行分子对接验证。预测结果表明，黄连解毒汤中共筛选出84个活性成分，作用靶点111个，其中与巨噬细胞炎症相关的靶点蛋白13个，涉及代谢通路14条；与糖酵解、鞘脂代谢、谷氨酰胺代谢相关的靶点蛋白34个，涉及代谢通路8条。炎症相关蛋白和代谢相关蛋白通过物理相关性、蛋白共表达等方式互相作用，小檗碱、黄芩苷和栀子苷与5个重要靶点均能较好结合。黄连解毒汤可能通过作用于糖酵解、鞘脂代谢和谷氨酰胺代谢的相关靶点，影响其代谢过程中的代谢产物和酶，从而调节巨噬细胞炎症反应。

关键词

网络药理学 / 黄连解毒汤 / 炎症 / 代谢

Abstract

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This study was designed to explore the impact of Huanglian Jiedu Decoction (HLJDT) on macrophage inflammation reaction using the network pharmacology method. Glycolysis, sphingolipid metabolism and glutamine metabolism were also investigated for "multi-component, multi-target and multi-pathway", which supports a foundation for drug innovative research. The TCMSP database was used to screen the active components of HLJDT, the target protein predicted by PharmMapper database and the DAVID database for pathways annotation and analysis. The Cytoscape 3.2.1 software was used to construct the active componenttarget-pathway network map and GENEMANIA database for protein interaction analysis. System Dock Database Site is used in verification of molecular docking. The results showed that 84 active ingredients were screened in HLJDT with a total of 111 target targets. Fourteen pathways are affected according to 13 macrophage-related inflammatory proteins, and 8 pathways including 34 target proteins from glycolysis, sphingolipid metabolism and glutamine metabolism. Inflammation-related proteins and metabolism-related proteins can interact with each other through physical correlation, protein co-expression, etc. Berberine, baicalin and geniposide combined well with 5 important targets. Huanglian Jiedu Decoction may act on the glycolysis and sphingolipid pathways to regulate macrophage inflammatory responses.

Key words

network pharmacology / Huanglian Jiedu Tang / inflammation / metabolism

引用本文

衡霞, 朱宝杰, 孙莉敏, 潘林梅, 段金廒, 张启春, 朱华旭. 基于网络药理学的黄连解毒汤调节巨噬细胞炎症反应和代谢的作用机制研究. 药学学报, 2018 , 53 (9) : 1449 -1457 . DOI: 10.16438/j.0513-4870.2018-0276

Xia HENG, Bao-jie ZHU, Li-min SUN, Lin-mei PAN, Jin-ao DUAN, Qi-chun ZHANG, Hua-xu ZHU. Network pharmacology-based study on mechanisms of Huanglian Jiedu Decoction impact on macrophage inflammation response[J]. Acta Pharmaceutica Sinica, 2018 , 53 (9) : 1449 -1457 . DOI: 10.16438/j.0513-4870.2018-0276

正文

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中药复方基于“整体观”、“辨证论治”的理论, 具有多成分、多靶点、多途径协同作用的特点, 作用机制复杂, 采用“一靶点、一药物”的模式阐述其作用机制困难较多^[1]。然而, 随着公共生物医学数据的迅速发展, 网络药理学方法应运而生, 为阐明中药复方作用机制提供了一条新途径^[2]。网络药理学是基于系统生物学、多向药理学和分子网络分析的一门新兴学科, 网络药理学技术的运用对于理解中药复方的整体性、互补性和协同性具有重要作用。

黄连解毒汤是清热解毒的传统名方, 该方由黄连、黄芩、黄柏和栀子四味中药组成, 具有清热泻火、解毒的功效。大量研究表明, 黄连解毒汤在肿瘤、糖尿病、关节炎、缺血性脑卒中和肝脏疾病等多种疾病中发挥着重要作用, 而代谢重编程启动的免疫细胞活化和炎症反应则参与了上述疾病的发生发展过程^[3-6]。基于代谢调控网络调节免疫细胞功能有可能是黄连解毒汤抗炎作用的潜在生化机制, 本文将利用网络药理学分析技术, 对黄连解毒汤调节巨噬细胞炎症反应、糖酵解、鞘磷脂代谢和谷氨酰胺代谢等方面的多成分、多靶点、多通路的相互作用规律和调控网络进行了挖掘和分析, 以期为后续深入研究黄连解毒汤调控炎症的作用机制和发展新的有价值的药物奠定基础。

材料与方法

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软件与数据库 TCMSP数据库(http://lsp.nwsuaf.edu.cn/tcmsp.php)、Pubchem数据库(https://pubchem.ncbi.nlm.nih.gov/)、PharmMapper数据库(http://lilab.ecust.edu.cn/pharmmapper/index.php)、OMIM数据库(http://www.omim.org/)、DAVID数据库(https://david.ncifcrf.gov/)、Kyoto Encyclopediaof Genes and Genomes (KEGG)通路数据库(http://www.Genome.jp/kegg/)、GENEMANIA数据库(http://genemania.org/)、System Dock Database Site数据库(http://systemsdock.unit.oist.jp)、Cytoscape 3.2.1软件。

黄连解毒汤中活性成分的筛选 黄连解毒汤包含黄连、黄芩、黄柏、栀子四味中药, 将这四味中药以中文名称作为关键词分别输入TCMSP数据库进行检索, 所得结果按照口服吸收利用度(OB) > 30%、药物相似性(DL) > 0.18进行筛选, 筛选后所得成分作为黄连解毒汤的活性成分。

黄连解毒汤中活性成分潜在靶点预测 将黄连解毒汤中筛选得到的活性成分以成分名作为关键词输入Pubchem数据库中进行检索, 查找并保存各活性成分的3D结构式为SDF (.sdf)格式, 再将各活性成分的3D结构式提交到PharmMapper数据库中进行靶点预测, 依次选择参数^[7]: 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, 得到与各化合物相关的靶点名称(target name)、基因名称(gene)、Uniprot ID、匹配值(fit score)等结果。根据匹配值筛选前10个靶点作为该化合物的重要靶点蛋白。

炎症巨噬细胞靶点和代谢靶点预测 分别将“inflammation”、“macrophages”作为关键词输入OMIM数据库检索与炎症、巨噬细胞相关的靶点蛋白, 将“glycolysis”、“sphingolipid metabolism”、“glutamine metabolism”作为关键词输入OMIM数据库检索与糖酵解、鞘脂代谢、谷氨酰胺代谢相关的靶点蛋白, 整理这些靶点蛋白并与筛选得到的活性成分的靶点蛋白进行匹配, 得到黄连解毒汤中与炎症巨噬细胞相关靶点和代谢靶点。

相关通路的注释和分析 以标准基因名的形式将活性成分的相关靶点基因列表导入DAVID数据库, 依次勾选OFFICIAL_GENE_SYMBOL (官方基因标志)、Gene list、Submit list, 选择基因功能分类工具, 修改背景为人类, 选择Pathway得到“KEGG- Pathway”相关数据, 保存数据并挑选P < 0.01的通路作为可靠通路, 结合KEGG数据库进行通路注释和分析。

构建黄连解毒汤中抗炎和代谢作用的“药效成分-靶点-通路”网络关系图 将黄连解毒汤中的活性成分、对应预测靶点和通路分析结果, 在Excel表中分别构建“活性成分-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“活性成分-靶点”、“靶点-通路”网络关系图, 利用软件中Merge功能, 将两个网络图合并得到“活性成分-靶点-通路”网络。图中以活性成分、靶点蛋白和通路为3类节点(node), 它们之间的相互关系分别用边(edge)相连表示。利用软件中网络分析功能对所得网络进行分析, 以度(degree)、边介数(edge betweenness)分别反映网络中节点的大小和边的粗细。

蛋白质相互作用关系分析 将筛选得到的炎症巨噬细胞靶点蛋白和代谢靶点蛋白以标准基因名的形式上传到GENEMANIA数据库, 进行蛋白与蛋白之间的相互作用分析, 得到蛋白质相互作用网络图, 整理网络布局并保存图片。

主要活性成分-靶点分子对接 利用Systems Dock Web Site数据库, 将黄连解毒汤中主要药效成分小檗碱、黄芩苷和栀子苷与蛋白相互作用分析得到的5个共有靶点进行分子对接验证。登陆Systems Dock Web Site数据库, 依次上传靶点名称/PDB ID、小檗碱、栀子苷、黄芩苷SDF (.sdf)格式的3D结构式, 成功后开始对接, 整理对接结果中的Docking Score并分析。

结果

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1 黄连解毒汤中有效成分筛选

将黄连、黄芩、黄柏和栀子作为关键词分别输入TCMSP数据库进行检索后, 共得到429个成分, 其中黄连48个、黄芩143个、黄柏140个、栀子98个。根据各个成分的吸收、分布、代谢、排泄(ADME)参数中口服吸收利用度OB > 30%、药物相似性DL > 0.18进行筛选, 共得到84个活性成分。将这84个活性成分进行结构分类, 其中属于生物碱类的成分有17个, 包括小檗碱、表小檗碱、巴马汀等; 属于环烯醚萜类成分有1个, 为京尼平1-β-龙胆二糖苷; 属于黄酮类的成分有33个, 包括汉黄芩苷、去甲基汉黄芩苷、槲皮素等; 其他类成分有33个, 包括豆甾醇、谷甾醇、山奈酚等。

2 黄连解毒汤中活性成分潜在靶点蛋白信息

黄连解毒汤中筛选得到的84个活性成分共涉及111个靶点蛋白, 与炎症相关的靶点蛋白有20个, 其中炎症靶点中与巨噬细胞相关的靶点蛋白有13个, 出现频度最多的靶点蛋白为丝裂原活化蛋白激酶8 (MAPK8)、氧化型胆固醇受体LXRβ (oxysterolsreceptor LXR-beta)等, 详细信息见表 1。将这13个靶点蛋白对应84个活性成分, 其中有38个活性成分可作用于这些靶点。

与糖酵解、鞘脂代谢和谷氨酰胺代谢相关的靶点共有34个, 出现频度最多的靶点蛋白为醛-酮还原酶家族1成员C2 (AKR1C2)、雄激素受体(AR)等, 详细信息见表 2。将这34个靶点对应84个活性成分, 显示84个活性成分都可作用于这些靶点。

3 黄连解毒汤中活性成分潜在靶点蛋白的作用通路注释和分析

将黄连解毒汤中与巨噬细胞炎症相关的13个靶点蛋白以标准基因名的形式输入DAVID数据库中进行KEGG通路注释和分析, 筛选P值小于0.01的通路作为潜在通路, 共得到14条代谢通路, 涉及4种类别, 其中与疾病相关的通路有4条, 与信号转导相关的通路有7条, 与细胞过程相关的通路有2条, 与感官系统相关的通路有1条, 详细信息见表 3。

采用相同的方法对与糖酵解、鞘脂代谢、谷氨酰胺代谢相关的靶点蛋白进行通路注释和分析, 得到8条代谢通路, 涉及3种类别, 其中与疾病相关的通路有4条, 与信号转导相关的通路有3条, 与细胞过程相关的通路有1条, 详细信息见表 4。

4 黄连解毒汤中“活性成分-靶点-通路”网络模型图构建

采用Cytoscape 3.2.1软件分别构建炎症巨噬细胞相关“活性成分-靶点-通路”网络模型图(图 1)、代谢相关“活性成分-靶点-通路”网络模型图(图 2), 以度(degree)的大小反映节点的大小, 以边介数(betweenness)反映边的粗细。

如图 1所示为炎症巨噬细胞相关“活性成分-靶点-通路”网络模型图, 图中共有65个节点, 其中38个为活性成分, 13个为靶点蛋白, 14个为代谢通路, 它们由202条边相连构成一个完整的网络。如图 2所示为代谢相关“活性成分-靶点-通路”网络模型图, 图中共有126个节点, 其中84个为活性成分, 34个为靶点蛋白, 8个为代谢通路, 它们通过354条边相连构成一个完整的网络。

5 蛋白质相互作用关系分析

将13个与巨噬细胞相关的靶点蛋白和34个与糖酵解、鞘脂代谢、谷氨酰胺代谢相关的靶点蛋白以标准基因名的形式输入GENEMANIA数据库, 得到蛋白质相互作用关系网络图, 如图 3所示, 位于图片最内侧的为巨噬细胞炎症相关靶点蛋白, 最外侧为糖酵解、鞘脂代谢、谷氨酰胺代谢相关靶点蛋白, 中间5个靶点蛋白为炎症和代谢共有的靶点蛋白, 分别为丝裂原活化蛋白激酶8 (MAPK8)、氧化型胆固醇受体LXRβ (NR1H2)、巨噬细胞移动抑制因子(MIF)、过氧化物酶体增殖物激活受体δ (PPARD)、过氧化物酶体增殖物激活受体γ (PPARG)。蛋白之间相互作用主要通过物理相关性、蛋白共表达、共享蛋白域、预测、共定位、基因相关性和通路表现出来。其中, 物理相关性占34.47%、预测占28.41%、蛋白共表达占13.62%、共享蛋白域占11.30%、共定位占8.32%、基因相关性占2.61%、通路占1.26%。

6 黄连解毒汤中主要活性成分-靶点分子对接结果分析

选择黄连解毒汤中主要的药效成分小檗碱、栀子苷和黄芩苷, 与结果5中得到的5个靶点MAPK8、NR1H2、MIF、PPARD、PPARG分别进行分子对接, 对接结果如表 5所示。一般认为Docking Score值大于4.25表示对接分子与靶点之间有一定的结合活性, 大于5.0表明对接分子与靶点之间有较好的结合活性, 大于7.0则说明具有强烈的结合活性^[8]。分子对接结果显示小檗碱、黄芩苷、栀子苷与MAPK8、NR1H2、MIF、PPARD、PPARG的Docking Score值均大于5.0, 并且黄芩苷与PPARD的Docking Score值为7.024 > 7.0, 由此表明小檗碱、黄芩苷和栀子苷与这5个靶点均有较好的结合活性, 并且黄芩苷与PPARD有强烈的结合活性。

讨论

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黄连解毒汤是清热解毒的传统名方, 具有保护脑神经元、降血糖、抗炎、抗肿瘤等作用^[9-12], 本文利用网络药理学分析技术, 对黄连解毒汤调节巨噬细胞炎症反应、糖酵解、鞘脂代谢和谷氨酰胺代谢等方面的多成分、多靶点、多通路的相互作用规律和调控网络进行了挖掘和分析。由黄连解毒汤“活性成分-靶点-通路”网络可知:同一个活性成分可以通过同一个靶点与不同的代谢通路相连, 也可以通过不同的靶点与相同的代谢通路相连, 这与黄连解毒汤“多成分、多靶点、多途径”的特点符合。

黄连解毒汤中发挥药效作用的成分主要为生物碱、环烯醚萜和黄酮类成分, 本文筛选得到的84个活性成分中属于生物碱类的成分有17个, 环烯醚萜类成分有1个, 黄酮类的成分有33个, 其他类成分有33个。小檗碱、栀子苷和黄芩苷是黄连解毒汤的主要药效成分, 本课题组^[13]在前期对小檗碱、栀子苷和黄芩苷在脑组织中的分布进行研究, 研究表明黄连解毒汤的主要药效成分可以透过血脑屏障进入脑组织发挥作用。

靶点预测结果显示, 黄连解毒汤中与巨噬细胞炎症相关的靶点有丝裂原活化蛋白激酶8、氧化型胆固醇受体LXRβ、原癌基因酪氨酸蛋白激酶Src、中性粒细胞明胶酶相关脂质运载蛋白、磷脂酰肌醇4, 5-二磷酸3-激酶催化亚基γ同种型等13个靶点蛋白, 涉及14条代谢通路, 其中与炎症相关的通路有Toll样受体信号通路、FcεRI信号通路、ErbB信号通路、TNF信号通路。与糖酵解、鞘脂代谢、谷氨酰胺代谢相关的靶点有醛-酮还原酶家族1成员C2、雄激素受体、酪氨酸蛋白磷酸酶非受体1型、血清白蛋白、凝血酶原等34个靶点, 涉及8条通路, 其中与三大代谢相关的通路有胰岛素信号通路、PPAR信号通路、催乳素信号通路。

黄连解毒汤巨噬细胞炎症相关靶点和代谢相关靶点的蛋白质相互作用网络结果显示, 巨噬细胞炎症靶点与代谢靶点可以通过多种途径相互作用, 其中MAPK8、NR1H2、MIF、PPARD、PPARG这5个靶点在巨噬细胞炎症靶点和鞘脂代谢、谷氨酰胺代谢靶点中都存在, 利用分子对接技术, 将黄连解毒汤中的主要药效成分小檗碱、栀子苷和黄芩苷与这5个靶点进行分子对接。对接结果表明小檗碱、栀子苷和黄芩苷与这5个靶点都具有较好的结合活性, 并且黄芩苷与PPARD有强烈的结合活性, 提示黄连解毒汤可能通过这5个靶点影响鞘脂代谢、谷氨酰胺代谢过程, 影响代谢调控网络从而对免疫细胞功能进行调节, 发挥抗炎作用。

通路结果也显示, MAPK8参与调控Toll样受体信号通路、FcεRI信号通路、ErbB信号通路、GnRH信号通路、TNF信号通路等炎症通路, 同时, 还参与调控胰岛素信号通路、催乳素信号通路等能量代谢通路; PPARD和PPARG调控PPAR信号通路。MAPK8是c-Jun氨基末端激酶1 (JNK1), 属于丝氨酸/苏氨酸(Ser/Thr)蛋白激酶, 应激反应发生时主要通过对JNK1调节实现对JNK整个信号通路的调控。研究表明, 炎症状态下可以活化巨噬细胞炎性标志物JNK蛋白磷酸化^[14-16], Guo^[17]研究发现小檗碱可以抑制JNK蛋白磷酸化, 并且通过小檗碱干预后的巨噬细胞调节液与脂肪细胞作用等表明小檗碱可能通过抑制巨噬细胞炎症信号通路及炎性基因表达和分泌, 改善胰岛素抵抗。由此推测, 黄连解毒汤可能通过小檗碱与MAPK8蛋白作用抑制其磷酸化, 从而调控JNK信号通路抑制巨噬细胞炎症发生, 进而影响机体的能量代谢过程。PPARD和PPARG是过氧化物酶体增殖物激活受体PPARs的两种亚型, 是参与体内脂质代谢调节的核转录因子^[18], 不仅广泛参与机体的脂质代谢、糖代谢、能量代谢过程, 在炎症过程中也发挥重要作用^[19]。Jin等^[20]研究表明, 黄连解毒汤干预后的高脂血症大鼠肝脏中PPARG的mRNA表达水平明显升高; Liu等^[21]研究也表明小檗碱可以抑制脂肪细胞分化相关基因PPARG的mRNA和蛋白表达, 抑制脂肪细胞活化, 进而减少脂肪的积聚。并且, 脂肪细胞是产生促炎细胞因子的重要场所, 在肥胖状态下, 脂肪细胞会分泌一系列细胞因子从而诱发免疫和炎症过程^[22]。因此, 黄连解毒汤可能通过上调组织中PPARG的mRNA表达从而调节脂质代谢紊乱, 或者通过抑制脂肪组织中PPARG的mRNA表达, 减少脂肪组织的分化, 从而减少促炎细胞因子的分泌进而减少炎症过程的发生。

上述得出的5个重要靶点均为鞘脂代谢、谷氨酰胺代谢和巨噬细胞炎症中共有的靶点, 因此猜测黄连解毒汤可能通过影响鞘脂代谢和谷氨酰胺代谢过程中代谢产物或相关酶从而影响炎症过程。鞘脂是一类以鞘氨醇为骨架的较复杂的化合物, 在维持细胞生长、信号转导、炎症过程中发挥重要作用, 鞘脂代谢异常会产生包括阿尔兹海默病、动脉粥样硬化、糖尿病、心肌病、癌症等多种疾病^[23-25]。神经酰胺(Cer)是细胞内鞘脂代谢的中心^[26], 具有诱导细胞凋亡、调节细胞分化、细胞免疫、炎症反应^[27]等生物学功能。脑组织中Cer水平的升高, 一方面会对神经元产生毒性作用; 另一方面也会促进细胞因子IL-2和IL-6的产生, 引起神经炎症^{[28, 29]}, 而实验室前期研究^[30]结果表明, 黄连解毒汤可以显著下调APP/PS1模型小鼠血浆和脑组织代谢物中Cer的含量。1-磷酸鞘氨醇(S1P)是具有生物活性的鞘脂代谢产物, 研究表明, 1-磷酸鞘氨醇受体激动剂TFY720可能通过与巨噬细胞表面的S1P受体结合, 抑制IL-23产生, 进而减少IL-17、IL-1β、TNF-α和MMP-3表达, 从而调控炎症反应^[31], 实验室前期研究表明, 小檗碱预处理后的神经元中S1P发生显著上调^[32]。谷氨酰胺是谷氨酰胺代谢的重要代谢物质, 为供能和生物合成提供主要的碳源和氮源。研究表明, 经脂多糖(LPS)刺激的巨噬细胞分泌IL-6和TNF-α的多少依赖于细胞外谷氨酰胺的可用量^[33], 并且多项动物研究也表明谷氨酰胺的利用可以提高动物感染后的存活率, 体外研究和临床结果均表明谷氨酰胺对人免疫细胞数量和功能有促进作用^[34]。由此推测, 黄连解毒汤可能通过调节鞘脂代谢和谷氨酰胺代谢过程中代谢产物的含量, 进而调控炎症过程的发生。

结论

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通过网络药理学技术, 将黄连解毒汤中四味中药进行活性成分筛选, 根据得到的活性成分进行靶点预测、通路分析、蛋白相互作用分析和分子对接分析, 构建“活性成分-靶点-通路网路”和蛋白相互作用网络, 发现黄连解毒汤可能通过作用于MAPK8、NR1H2、MIF、PPARD、PPARG等糖酵解、鞘脂代谢和谷氨酰胺代谢的相关靶点, 影响代谢过程中的有关代谢产物或酶而影响代谢调控网络, 从而对巨噬细胞炎症反应进行调节。

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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2018年第53卷第9期

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doi: 10.16438/j.0513-4870.2018-0276

接收时间：2018-03-29
首发时间：2026-01-15
出版时间：2018-09-15

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收稿日期：2018-03-29
修回日期：2018-04-25

基金

国家自然科学基金资助项目(81573635)

作者信息

1.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

2.南京中医药大学江苏省方剂高技术研究重点实验室, 江苏南京 210023

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* 张启春, Tel/Fax:86-25-85811509, E-mail:zhangqc@njucm.edu.cn;

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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RefWorks
TxT

Protein target	Common name	Uniprot ID	Frequency
Mitogen-activated protein kinase 8	MAPK8	P45983	13
Oxysterols receptor LXR-beta	NR1H2	P55055	7
Proto-oncogene tyrosine-protein kinase Src	SRC	P12931	5
Neutrophil gelatinase-associated lipocalin	LCN2	P80188	4
Phosphatidylinositol 4, 5-bisphosphate 3-kinase catalytic subunit gamma isoform	PIK3CG	P48736	4
Macrophage migration inhibitory factor	MIF	P14174	3
Triggering receptor expressed on myeloid cells 1	TREM1	Q9NP99	3
Hematopoietic prostaglandin D synthase	HPGDS	O60760	3
Plasminogen	PLG	P00747	2
Mitogen-activated protein kinase 14	MAPK14	Q16539	1
Cathepsin K	CTSK	P43235	1
Peroxisome proliferator-activated receptor delta	PPARD	Q03181	1
Peroxisome proliferator-activated receptor gamma	PPARG	P37231	1

Protein target

Common name

Uniprot ID

Frequency

Mitogen-activated protein kinase 8

MAPK8

P45983

Oxysterols receptor LXR-beta

NR1H2

P55055

Proto-oncogene tyrosine-protein kinase Src

SRC

P12931

Neutrophil gelatinase-associated lipocalin

LCN2

P80188

Phosphatidylinositol 4, 5-bisphosphate 3-kinase catalytic subunit gamma isoform

PIK3CG

P48736

Macrophage migration inhibitory factor

MIF

P14174

Triggering receptor expressed on myeloid cells 1

TREM1

Q9NP99

Hematopoietic prostaglandin D synthase

HPGDS

O60760

Plasminogen

PLG

P00747

Mitogen-activated protein kinase 14

MAPK14

Q16539

Cathepsin K

CTSK

P43235

Peroxisome proliferator-activated receptor delta

PPARD

Q03181

Peroxisome proliferator-activated receptor gamma

PPARG

P37231

Protein target	Common name	Uniprot ID	Frequency
Aldo-keto reductase family 1 member C2	AKR1C2	P52895	30
Androgen receptor	AR	P10275	24
Tyrosine-protein phosphatase non-receptor type 1	PTPN1	P18031	21
Serum albumin	ALB	P02768	20
Prothrombin	F2	P00734	20
Mitogen-activated protein kinase 1	MAPK1	P28482	19
Glutathione S-transferase P	GSTP1	P09211	19
Aldose reductase	AKR1B1	P15121	17
Aromatase	CYP19A1	P11511	15
cGMP-specific 3', 5'-cyclic phosphodiesterase	PDE5A	O76074	15
Amine oxidase [flavin-containing] B	MAOB	P27338	14
Mitogen-activated protein kinase 8	MAPK8	P45983	13
Thyroid hormone receptor beta	THRB	P10828	13
Liver carboxylesterase 1	CES1	P23141	12
Complement factor B	CFB	P00751	10
Glucosylceramidase	GBA	P04062	10
Estrogen receptor beta	ESR2	Q92731	8
Oxysterols receptor LXR-beta	NR1H2	P55055	6
Glycine amidinotransferase, mitochondrial	GATM	P50440	6
Nuclear receptor subfamily 1 group Ⅰ member 3	NR1I3	Q14994	4
Vitamin D-binding protein	GC	P02774	4
Macrophage migration inhibitory factor	MIF	P14174	3
Phosphoenolpyruvate carboxykinase, cytosolic [GTP]	PCK1	P35558	2
Nuclear receptor ROR-alpha	RORA	P35398	2
Estrogen-related receptor gamma	ESRRG	P62508	2
Heat shock protein HSP 90-alpha	HSP90AA1	P07900	2
Glycogen synthase kinase-3 beta	GSK3B	P49841	2
Retinoic acid receptor RXR-alpha	RXRA	P19793	1
C-1-tetrahydrofolate synthase, cytoplasmic	MTHFD1	P11586	1
cGMP-inhibited 3', 5'-cyclic phosphodiesterase B	PDE3B	Q13370	1
Peroxisome proliferator-activated receptor delta	PPARD	Q03181	1
Peroxisome proliferator-activated receptor gamma	PPARG	P37231	1
Dihydrofolate reductase	DHFR	P00374	1
Arylsulfatase A	ARSA	P15289	1

Protein target

Common name

Uniprot ID

Frequency

Aldo-keto reductase family 1 member C2

AKR1C2

P52895

Androgen receptor

P10275

Tyrosine-protein phosphatase non-receptor type 1

PTPN1

P18031

Serum albumin

ALB

P02768

Prothrombin

P00734

Mitogen-activated protein kinase 1

MAPK1

P28482

Glutathione S-transferase P

GSTP1

P09211

Aldose reductase

AKR1B1

P15121

Aromatase

CYP19A1

P11511

cGMP-specific 3', 5'-cyclic phosphodiesterase

PDE5A

O76074

Amine oxidase [flavin-containing] B

MAOB

P27338

Mitogen-activated protein kinase 8

MAPK8

P45983

Thyroid hormone receptor beta

THRB

P10828

Liver carboxylesterase 1

CES1

P23141

Complement factor B

CFB

P00751

Glucosylceramidase

GBA

P04062

Estrogen receptor beta

ESR2

Q92731

Oxysterols receptor LXR-beta

NR1H2

P55055

Glycine amidinotransferase, mitochondrial

GATM

P50440

Nuclear receptor subfamily 1 group Ⅰ member 3

NR1I3

Q14994

Vitamin D-binding protein

P02774

Macrophage migration inhibitory factor

MIF

P14174

Phosphoenolpyruvate carboxykinase, cytosolic [GTP]

PCK1

P35558

Nuclear receptor ROR-alpha

RORA

P35398

Estrogen-related receptor gamma

ESRRG

P62508

Heat shock protein HSP 90-alpha

HSP90AA1

P07900

Glycogen synthase kinase-3 beta

GSK3B

P49841

Retinoic acid receptor RXR-alpha

RXRA

P19793

C-1-tetrahydrofolate synthase, cytoplasmic

MTHFD1

P11586

cGMP-inhibited 3', 5'-cyclic phosphodiesterase B

PDE3B

Q13370

Peroxisome proliferator-activated receptor delta

PPARD

Q03181

Peroxisome proliferator-activated receptor gamma

PPARG

P37231

Dihydrofolate reductase

DHFR

P00374

Arylsulfatase A

ARSA

P15289

Classification	Pathway	P-value	Count
Diseases	Influenza A	0.001 7	4
	Shigellosis	0.003 6	3
	Epithelial cell signaling in Helicobacter pylori infection	0.004 0	3
	Chagas disease (American trypanosomiasis)	0.009 3	3
Signal transduction	Prolactin signaling pathway	0.000 1	4
	Toll-like receptor signaling pathway	0.000 3	4
	VEGF signaling pathway	0.003 3	3
	Fc epsilon RI signaling pathway	0.004 1	3
	ErbB signaling pathway	0.006 6	3
	GnRH signaling pathway	0.007 2	3
	TNF signaling pathway	0.009 7	3
Cell processes	Osteoclast differentiation	0.000 1	5
	Progesterone-mediated oocyte maturation	0.006 6	3
Sensory system	Inflammatory mediator regulation of TRP channels	0.000 3	4

Classification

Pathway

P-value

Count

Diseases

Influenza A

0.001 7

Shigellosis

0.003 6

Epithelial cell signaling in Helicobacter pylori infection

0.004 0

Chagas disease (American trypanosomiasis)

0.009 3

Signal transduction

Prolactin signaling pathway

0.000 1

Toll-like receptor signaling pathway

0.000 3

VEGF signaling pathway

0.003 3

Fc epsilon RI signaling pathway

0.004 1

ErbB signaling pathway

0.006 6

GnRH signaling pathway

0.007 2

TNF signaling pathway

0.009 7

Cell processes

Osteoclast differentiation

0.000 1

Progesterone-mediated oocyte maturation

0.006 6

Sensory system

Inflammatory mediator regulation of TRP channels

0.000 3

Classification	Pathway	P-value	Count
Diseases	Pathways in cancer	0.001 0	8
	Insulin resistance	0.001 0	5
	Prostate cancer	0.005 7	4
	Thyroid cancer	0.006 4	3
Signal transduction	Insulin signaling pathway	0.000 2	6
	PPAR signaling pathway	0.002 7	4
	Prolactin signaling pathway	0.003 1	4
Cell processes	Progesterone-mediated oocyte maturation	0.005 6	4

Classification

Pathway

P-value

Count

Diseases

Pathways in cancer

0.001 0

Insulin resistance

0.001 0

Prostate cancer

0.005 7

Thyroid cancer

0.006 4

Signal transduction

Insulin signaling pathway

0.000 2

PPAR signaling pathway

0.002 7

Prolactin signaling pathway

0.003 1

Cell processes

Progesterone-mediated oocyte maturation

0.005 6

Target name	PDB ID	Ingredient	Docking score
MAPK8	1UKI	Berberine	6.374
		Geniposide	6.403
		Baicalin	6.207
NR1H2	1PQ9	Berberine	6.397
		Geniposide	6.747
		Baicalin	6.397
MIF	1CA7	Berberine	6.209
		Geniposide	6.395
		Baicalin	6.439
PPARD	3DY6	Berberine	6.527
		Geniposide	7.024
		Baicalin	6.640
PPARG	3B3K	Berberine	6.550
		Geniposide	6.470
		Baicalin	6.641

Target name

PDB ID

Ingredient

Docking score

MAPK8

1UKI

Berberine

6.374

Geniposide

6.403

Baicalin

6.207

NR1H2

1PQ9

Berberine

6.397

Geniposide

6.747

Baicalin

6.397

MIF

1CA7

Berberine

6.209

Geniposide

6.395

Baicalin

6.439

PPARD

3DY6

Berberine

6.527

Geniposide

7.024

Baicalin

6.640

PPARG

3B3K

Berberine

6.550

Geniposide

6.470

Baicalin

6.641