药学学报

No.	Structure type	Compound	Formula	Source
1	Flavone	Hyperoside	C₂₁H₂₀O₁₂	1, 3
2		Rutin	C₂₇H₃₀O₁₆	1, 4, 5
3		Isoquercitrin	C₂₁H₂₀O₁₂	1, 3, 5
4		Cynaroside	C₂₁H₂₀O₁₁	5
5		Baicalein	C₁₅H₁₀O₅	2
6		Baicalin	C₂₁H₁₈O₁₁	2
7		Wogonin	C₁₆H₁₂O₅	2
8		Wogonoside	C₆H₁₀O₇	2
9		Oroxylin A	C₁₆H₁₂O₅	2
10		Chrysin	C₁₅H₁₀O₄	2
11	Alkaloid	Laurolitsine	C₁₈H₁₉NO₄	1
12		Isaidigodione	C₁₈H₁₈N₂O₄	3
13	Lignans	Lariciresinol	C₂₆H₃₄O₁₁	3
14		Phillyrin	C₂₇H₃₄O₁₁	4
15		Phillygenin	C₂₁H₂₄O₆	4
16	Phenylethanoid glycoside	Forsythoside A	C₂₉H₃₆O₁₅	4
17		Verbascoside	C₂₉H₃₆O₁₅	4
18	Organic acid	Chlorogenic acid	C₁₆H₁₈O₉	1, 5
19		Caffeic acid	C₉H₈O₄	1, 4, 5
20		Quinic acid	C₇H₁₂O₆	1, 4, 5
21		Isochlorogenic acid A	C₂₅H₂₄O₁₂	5
22		Anthranilic acid	C₇H₇NO₂	3
23	Iridoid	Loganic acid	C₁₆H₂₄O₁₀	5
24		Sweroside	C₁₆H₂₂O₉	5
25	Nucleoside	Adenosine	C₁₀H₁₃N₅O₄	1, 2, 3, 4, 5

No.	Structure type	Compound	Formula	Source
1	Flavone	Hyperoside	C₂₁H₂₀O₁₂	1, 3
2		Rutin	C₂₇H₃₀O₁₆	1, 4, 5
3		Isoquercitrin	C₂₁H₂₀O₁₂	1, 3, 5
4		Cynaroside	C₂₁H₂₀O₁₁	5
5		Baicalein	C₁₅H₁₀O₅	2
6		Baicalin	C₂₁H₁₈O₁₁	2
7		Wogonin	C₁₆H₁₂O₅	2
8		Wogonoside	C₆H₁₀O₇	2
9		Oroxylin A	C₁₆H₁₂O₅	2
10		Chrysin	C₁₅H₁₀O₄	2
11	Alkaloid	Laurolitsine	C₁₈H₁₉NO₄	1
12		Isaidigodione	C₁₈H₁₈N₂O₄	3
13	Lignans	Lariciresinol	C₂₆H₃₄O₁₁	3
14		Phillyrin	C₂₇H₃₄O₁₁	4
15		Phillygenin	C₂₁H₂₄O₆	4
16	Phenylethanoid glycoside	Forsythoside A	C₂₉H₃₆O₁₅	4
17		Verbascoside	C₂₉H₃₆O₁₅	4
18	Organic acid	Chlorogenic acid	C₁₆H₁₈O₉	1, 5
19		Caffeic acid	C₉H₈O₄	1, 4, 5
20		Quinic acid	C₇H₁₂O₆	1, 4, 5
21		Isochlorogenic acid A	C₂₅H₂₄O₁₂	5
22		Anthranilic acid	C₇H₇NO₂	3
23	Iridoid	Loganic acid	C₁₆H₂₄O₁₀	5
24		Sweroside	C₁₆H₂₂O₉	5
25	Nucleoside	Adenosine	C₁₀H₁₃N₅O₄	1, 2, 3, 4, 5

Target	Common name	Uniprot ID	Degree
Serine/threonine-protein kinase	AKT1	P31749	99
Insulin	INS	P01308	97
Vascular endothelial growth factor A	VEGFA	P15692	86
Interleukin-6	IL-6	P05231	86
Cellular tumor antigen p53	TP53	P04637	84
Tumor necrosis factor	TNF	P01375	79
Transcription factor AP-1	JUN	P05412	72
Caspase-3	CASP3	P42574	71
Matrix metalloproteinase-9	MMP9	P14780	69
Interleukin-8	IL-8	P10145	68
Prostaglandin G/H synthase 2	PTGS2	P35354	67
Proto-oncogene c-Fos	FOS	P01100	66
Tyrosine-protein kinase SRC	SRC	P12931	66
Mitogen-activated protein kinase 8	MAPK8	P45983	65
Estrogen receptor 1	ESR1	P03372	61

Target	Common name	Uniprot ID	Degree
Serine/threonine-protein kinase	AKT1	P31749	99
Insulin	INS	P01308	97
Vascular endothelial growth factor A	VEGFA	P15692	86
Interleukin-6	IL-6	P05231	86
Cellular tumor antigen p53	TP53	P04637	84
Tumor necrosis factor	TNF	P01375	79
Transcription factor AP-1	JUN	P05412	72
Caspase-3	CASP3	P42574	71
Matrix metalloproteinase-9	MMP9	P14780	69
Interleukin-8	IL-8	P10145	68
Prostaglandin G/H synthase 2	PTGS2	P35354	67
Proto-oncogene c-Fos	FOS	P01100	66
Tyrosine-protein kinase SRC	SRC	P12931	66
Mitogen-activated protein kinase 8	MAPK8	P45983	65
Estrogen receptor 1	ESR1	P03372	61

Term ID	Term description	Observed gene count	False discovery rate
hsa04657	IL-17 signaling pathway	21	1.16E-19
hsa04659	Th17 cell differentiation	16	6.57E-13
hsa04080	Neuroactive ligand-receptor interaction	22	1.89E-12
hsa04010	MAPK signaling pathway	22	5.02E-12
hsa04151	PI3K-AKT signaling pathway	23	1.36E-11
hsa04919	Thyroid hormone signaling pathway	15	2.00E-11
hsa04668	TNF signaling pathway	14	1.03E-10
hsa04115	p53 signaling pathway	12	1.18E-10
hsa04210	Apoptosis	15	1.23E-10
hsa04066	HIF-1 signaling pathway	13	3.83E-10
hsa04915	Estrogen signaling pathway	14	9.94E-10
hsa04024	cAMP signaling pathway	16	1.39E-09
hsa04071	Sphingolipid signaling pathway	13	2.04E-09
hsa04722	Neurotrophin signaling pathway	13	2.04E-09
hsa04660	T cell receptor signaling pathway	12	3.99E-09
hsa04068	FoxO signaling pathway	13	6.38E-09
hsa04510	Focal adhesion	15	9.74E-09
hsa04664	Fc epsilon RI signaling pathway	10	1.45E-08
hsa04750	Inflammatory mediator regulation of TRP channels	11	1.89E-08
hsa04620	Toll-like receptor signaling pathway	11	4.65E-08
hsa04370	VEGF signaling pathway	9	6.56E-08
hsa04621	NOD-like receptor signaling pathway	13	6.99E-08
hsa04658	Th1 and Th2 cell differentiation	10	1.25E-07
hsa04022	cGMP-PKG signaling pathway	12	3.52E-07
hsa04012	ErbB signaling pathway	9	7.94E-07
hsa04020	Calcium signaling pathway	12	1.03E-06
hsa04912	GnRH signaling pathway	9	1.22E-06
hsa04015	Rap1 signaling pathway	12	3.40E-06
hsa04310	Wnt signaling pathway	10	6.52E-06
hsa04924	Renin secretion	7	1.31E-05
hsa04064	NF-kappa B signaling pathway	8	1.52E-05
hsa04672	Intestinal immune network for IgA production	6	2.02E-05
hsa04662	B cell receptor signaling pathway	7	2.43E-05
hsa04060	Cytokine-cytokine receptor interaction	12	3.37E-05
hsa04630	Jak-STAT signaling pathway	9	8.01E-05
hsa00590	Arachidonic acid metabolism	6	9.31E-05
hsa04622	RIG-I-like receptor signaling pathway	6	0.000 18
hsa04014	Ras signaling pathway	10	0.000 2
hsa04670	Leukocyte transendothelial migration	7	0.000 29
hsa04611	Platelet activation	7	0.000 49
hsa04062	Chemokine signaling pathway	8	0.000 86
hsa04650	Natural killer cell mediated cytotoxicity	6	0.002 6
hsa04540	Gap junction	5	0.003 1
hsa04150	mTOR signaling pathway	6	0.005 8
hsa04612	Antigen processing and presentation	4	0.007 1
hsa04530	Tight junction	6	0.009 7

Term ID	Term description	Observed gene count	False discovery rate
hsa04657	IL-17 signaling pathway	21	1.16E-19
hsa04659	Th17 cell differentiation	16	6.57E-13
hsa04080	Neuroactive ligand-receptor interaction	22	1.89E-12
hsa04010	MAPK signaling pathway	22	5.02E-12
hsa04151	PI3K-AKT signaling pathway	23	1.36E-11
hsa04919	Thyroid hormone signaling pathway	15	2.00E-11
hsa04668	TNF signaling pathway	14	1.03E-10
hsa04115	p53 signaling pathway	12	1.18E-10
hsa04210	Apoptosis	15	1.23E-10
hsa04066	HIF-1 signaling pathway	13	3.83E-10
hsa04915	Estrogen signaling pathway	14	9.94E-10
hsa04024	cAMP signaling pathway	16	1.39E-09
hsa04071	Sphingolipid signaling pathway	13	2.04E-09
hsa04722	Neurotrophin signaling pathway	13	2.04E-09
hsa04660	T cell receptor signaling pathway	12	3.99E-09
hsa04068	FoxO signaling pathway	13	6.38E-09
hsa04510	Focal adhesion	15	9.74E-09
hsa04664	Fc epsilon RI signaling pathway	10	1.45E-08
hsa04750	Inflammatory mediator regulation of TRP channels	11	1.89E-08
hsa04620	Toll-like receptor signaling pathway	11	4.65E-08
hsa04370	VEGF signaling pathway	9	6.56E-08
hsa04621	NOD-like receptor signaling pathway	13	6.99E-08
hsa04658	Th1 and Th2 cell differentiation	10	1.25E-07
hsa04022	cGMP-PKG signaling pathway	12	3.52E-07
hsa04012	ErbB signaling pathway	9	7.94E-07
hsa04020	Calcium signaling pathway	12	1.03E-06
hsa04912	GnRH signaling pathway	9	1.22E-06
hsa04015	Rap1 signaling pathway	12	3.40E-06
hsa04310	Wnt signaling pathway	10	6.52E-06
hsa04924	Renin secretion	7	1.31E-05
hsa04064	NF-kappa B signaling pathway	8	1.52E-05
hsa04672	Intestinal immune network for IgA production	6	2.02E-05
hsa04662	B cell receptor signaling pathway	7	2.43E-05
hsa04060	Cytokine-cytokine receptor interaction	12	3.37E-05
hsa04630	Jak-STAT signaling pathway	9	8.01E-05
hsa00590	Arachidonic acid metabolism	6	9.31E-05
hsa04622	RIG-I-like receptor signaling pathway	6	0.000 18
hsa04014	Ras signaling pathway	10	0.000 2
hsa04670	Leukocyte transendothelial migration	7	0.000 29
hsa04611	Platelet activation	7	0.000 49
hsa04062	Chemokine signaling pathway	8	0.000 86
hsa04650	Natural killer cell mediated cytotoxicity	6	0.002 6
hsa04540	Gap junction	5	0.003 1
hsa04150	mTOR signaling pathway	6	0.005 8
hsa04612	Antigen processing and presentation	4	0.007 1
hsa04530	Tight junction	6	0.009 7

基于网络药理学的复方鱼腥草合剂清热解毒药效物质基础及作用机制研究

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韩彦琪 ¹^,²^,^# , 陈志霖 ³^,^# , 刘耀晨 ⁴ , 胡江宁 ⁵ , 许浚 ¹^,² , 张洪兵 ¹^,² , 刘建庭 ¹^,² , 张杨 ¹^,² , 张铁军 ¹^,²^,^* , 刘昌孝 ¹^,²^,^*

药学学报 | 研究论文 2021,56(6): 1653-1662

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药学学报 | 研究论文 2021, 56(6): 1653-1662

基于网络药理学的复方鱼腥草合剂清热解毒药效物质基础及作用机制研究

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韩彦琪¹^,²^,^#, 陈志霖³^,^#, 刘耀晨⁴, 胡江宁⁵, 许浚¹^,², 张洪兵¹^,², 刘建庭¹^,², 张杨¹^,², 张铁军¹^,²^,^*, 刘昌孝¹^,²^,^*

作者信息

1.天津市中药质量标志物重点实验室, 天津 300462

2.释药技术与药物代谢动力学国家重点实验室 (天津药物研究院), 天津 300462

3.浙江康恩贝中药有限公司, 浙江丽水 323400

4.天津医科大学, 天津 300070

5.浙江康恩贝制药股份有限公司, 浙江杭州 310052

通讯作者:

*张铁军, Tel: 86-22-23006295, E-mail: zhangtj@tjipr.com;

刘昌孝，liucx@tjipr.com

Pharmacodynamic material basis and mechanism of Fufang Yuxingcao Mixture for the treatment of heat-clearing and detoxification based on network pharmacology

Yan-qi HAN¹^,², Zhi-lin CHEN³, Yao-chen LIU⁴, Jiang-ning HU⁵, Jun XU¹^,², Hong-bing ZHANG¹^,², Jian-ting LIU¹^,², Yang ZHANG¹^,², Tie-jun ZHANG¹^,²^,^*, Chang-xiao LIU¹^,²^,^*

Affiliations

1. Tianjin Key Laboratory of Quality Marker of Traditional Chinese Medicine, Tianjin 300462, China

2. State KeyLaboratory of Drug Delivery and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin 300462, China

3. Zhejiang CONBA Chinese Medicine Co., LTD., Lishui 323400, China

4. Tianjin Medical University, Tianjin 300070, China

5. Zhejiang CONBA Pharmaceutical Co., LTD., Hangzhou 310052, China

出版时间: 2021-06-12 doi: 10.16438/j.0513-4870.2021-0085

文章导航

摘要

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探索复方鱼腥草合剂清热解毒功效的药效物质基础及网络调控机制。首先利用网络药理学的方法，选取复方鱼腥草合剂中25个化合物为研究对象，依据反向药效团匹配方法和TCMSP、UniProt等数据库预测化合物作用靶点，借助Omicsbean软件及KEGG数据库对获得靶点进行基因本体（gene ontology，GO）功能富集分析、信号通路分析，利用Cytoscape软件构建“化合物-靶点-通路-药理作用-功效”网络药理图。最后采用脂多糖（lipopolysaccharide，LPS）诱导的RAW264.7细胞炎症模型，验证复方鱼腥草合剂及方中10个重要单体成分的抗炎作用。网络药理预测实验发现，25个化合物可通过211个靶点干预97条信号通路，其中RAC-α丝氨酸/苏氨酸蛋白激酶（RAC-alpha serine/threonine-protein kinase，AKT1）、胰岛素（insulin，INS）、血管内皮生长因子α（vascular endothelial growth factor A，VEGFA）、白介素-6（interleukin-6，IL-6）、细胞肿瘤抗原p53（cellular tumor antigen p53，TP53）、肿瘤坏死因子（tumor necrosis factor，TNF）、转录因子AP-1（transcription factor AP-1，JUN）、半胱氨酸蛋白酶-3（caspase-3，CASP3）、基质金属蛋白酶-9（matrix metalloproteinase-9，MMP9）、白介素-8（interleukin-8，IL-8）、前列腺素G/H合酶2（prostaglandin G/H synthase 2，PTGS2）、原癌基因c-fos（proto-oncogene c-Fos，FOS）、酪氨酸蛋白激酶SRC（tyrosine-protein kinase SRC，SRC）、丝裂原活化蛋白激酶-8（mitogen-activated protein kinase 8，MAPK8）和雌激素受体1（estrogen receptor 1，ESR1）等15个核心靶点和核转录因子κB信号通路（NF-kappa B signaling pathway）、Toll样受体信号通路（Toll-like receptor signaling pathway）、丝裂原活化蛋白激酶信号通路（MAPK signaling pathway）、白介素-17信号通路（IL-17 signaling pathway）、花生四烯酸代谢（arachidonic acid metabolism）、环磷酸腺苷信号通路（cAMP signaling pathway）、T细胞受体信号通路（T cell receptor signaling pathway）、钙离子信号通路（calcium signaling pathway）、TRP通道炎性介质调节（inflammatory mediator regulation of TRP channels）、趋化因子信号通路（chemokine signaling pathway）、Th1和Th2细胞分化（Th1 and Th2 cell differentiation）、自然杀伤细胞介导的细胞毒性（natural killer cell mediated cytotoxicity）等46条信号通路与抗炎、解热、免疫调节、镇痛等过程相关，推测复方鱼腥草合剂通过干预以上过程发挥清热解毒功效，体现了其多成分、多靶点、多途径的治疗作用。体外细胞实验表明，复方鱼腥草合剂及10个单体成分（异槲皮苷、木犀草苷、黄芩素、汉黄芩素、黄芩苷、连翘苷、连翘酯苷A、绿原酸、异绿原酸A、獐牙菜苷）均能显著降低LPS诱导的RAW264.7炎性细胞上清中一氧化氮（nitric oxide，NO）、TNF-α和IL-6的表达（P < 0.05），推测以上10个成分可能为复方鱼腥草合剂的关键药效物质基础。

关键词

复方鱼腥草合剂 / 网络药理学 / 药效物质基础 / 作用机制 / 清热解毒

Abstract

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We explored the pharmacodynamic material basis and network regulatory mechanism of Fufang Yuxingcao Mixture (FYM) for the treatment of fever and inflammation. Targets of the 25 compounds in FYM were predicted according to the reverse pharmacophore method and TCMSP, UniProt database. Gene ontology (GO) function enrichment and pathway analysis of the targets was analyzed by Omicsbean software and the Kyoto Gene and Genome Encyclopedia (KEGG) database. A "compound-target-pathway-pharmacological action-effect" network was established with Cytoscape 3.6.1 software. The lipopolysaccharide (LPS)-induced RAW264.7 cell inflammation model was used to verify the anti-inflammatory effects of FYM and its 10 important components. The network pharmacology experiment showed that 25 compounds affected 97 pathways through 211 targets, of which 15 key targets [including RAC-alpha serine/threonine-protein kinase (AKT1), insulin (INS), vascular endothelial growth factor A (VEGFA), interleukin-6 (IL-6), cellular tumor antigen p53 (TP53), tumor necrosis factor (TNF), transcription factor AP-1 (JUN), caspase-3 (CASP3), matrix metalloproteinase-9 (MMP9), interleukin-8 (IL-8), prostaglandin G/H synthase 2 (PTGS2), proto-oncogene c-Fos (FOS), tyrosine-protein kinase SRC (SRC), c-Jun N-terminal kinase 1 (MAPK8), estrogen receptor 1 (ESR1)] and 46 pathways (including NF-kappa B signaling pathway, Toll-like receptor signaling pathway, MAPK signaling pathway, IL-17 signaling pathway, arachidonic acid metabolism, cAMP signaling pathway, T cell receptor signaling pathway, calcium signaling pathway, inflammatory mediator regulation of TRP channels, chemokine signaling pathway, Th1 and Th2 cell differentiation, natural killer cell mediated cytotoxicity, etc.) were related to anti-inflammatory, antipyretic, immune regulation, and analgesia. In vitro cell experiments showed that FYM and the 10 components (including isoquercitrin, luteoloside, baicalein, wogonin, wogonoside, phillyrin, forsythoside A, chlorogenic acid, isochlorogenic acid A, and sweroside) could significantly reduce the expression of nitric oxide (NO), TNF-α and IL-6 in cell supernatants, indicating that the above 10 components may be the key pharmacodynamic material basis of FYM.

Key words

Fufang Yuxingcao Mixture / network pharmacology / pharmacodynamic material basis / mechanism / heat-clearing and detoxification

引用本文

韩彦琪, 陈志霖, 刘耀晨, 胡江宁, 许浚, 张洪兵, 刘建庭, 张杨, 张铁军, 刘昌孝. 基于网络药理学的复方鱼腥草合剂清热解毒药效物质基础及作用机制研究. 药学学报, 2021 , 56 (6) : 1653 -1662 . DOI: 10.16438/j.0513-4870.2021-0085

Yan-qi HAN, Zhi-lin CHEN, Yao-chen LIU, Jiang-ning HU, Jun XU, Hong-bing ZHANG, Jian-ting LIU, Yang ZHANG, Tie-jun ZHANG, Chang-xiao LIU. Pharmacodynamic material basis and mechanism of Fufang Yuxingcao Mixture for the treatment of heat-clearing and detoxification based on network pharmacology[J]. Acta Pharmaceutica Sinica, 2021 , 56 (6) : 1653 -1662 . DOI: 10.16438/j.0513-4870.2021-0085

正文

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复方鱼腥草合剂由鱼腥草、黄芩、板蓝根、连翘和金银花5味药材组成, 具有清热解毒之功效, 用于外感风热引起的咽喉肿痛、急性咽炎、扁桃体炎有风热证候者, 收录于2020版《中国药典》一部。该方以鱼腥草为君药, 具有清热解毒、利尿除湿和清热止痢的功效。黄芩为臣药, 具有清热燥湿、泻火解毒之功。其余为佐药, 共同协助发挥药效, 板蓝根具有清热解毒、凉血利咽功效; 连翘具有清热解毒、消肿散结功效; 金银花具有清热解毒、疏散风热功效。目前, 针对复方鱼腥草合剂的文献报道多为其用于治疗小儿肺炎的临床观察以及主要成分的定量分析研究^[1-5], 而关于其发挥清热解毒功效的药效物质基础及其多成分、多靶点、多途径的网络调节机制尚未见报道。

网络药理学是运用网络可视化等技术揭示药物、基因、疾病、靶点之间复杂的生物网络关系的学科, 能从分子、基因水平上分析药物作用于不同靶点、细胞和器官的行为, 系统地预测和揭示药物的作用及机制^[6]。运用网络药理学, 根据药物之间的结构、功效来构建药物－靶点网络, 可有效地预测中药及其复方制剂的药效成分及作用机制^[7]。因此, 本研究利用网络药理学方法, 对复方鱼腥草合剂中多个化合物进行了作用靶点和通路预测, 通过构建“化合物-靶点-通路-药理作用-功效”网络探讨复方鱼腥草合剂清热解毒作用机制, 同时利用脂多糖(lipopolysaccharide, LPS) 诱导的RAW264.7细胞炎症模型, 验证复方鱼腥草合剂及方中10个重要单体成分的抗炎作用。

材料与方法

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主要材料及仪器 Swiss Target Prediction服务器(http://new.swisstargetprediction.ch/); 中药系统药理学数据库与分析平台(TCMSP, http://lsp.nwu.edu.cn/tcmspsearch.php); UniProt数据库(http://www.uniprot.org/); 京都基因与基因组百科全书(Kyoto Gene and Genome Encyclopedia, KEGG) 通路数据库(http://www.genome.jp/kegg/); STRING 10数据库(http://string-db.org/); Omicsbean在线分析软件(http://www.omicsbean.cn/); Omicshare Tools在线制图软件(https://www.omicshare.com); ChemBioOffice2010; Cytoscape3.6.1软件。

RAW264.7小鼠腹腔巨噬细胞系购于上海生科院。地塞米松、LPS (美国Sigma公司); MTS (美国Promega公司); 白介素-6 (interleukin-6, IL-6)、肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α) 试剂盒(上海西唐生物科技有限公司); 一氧化氮(nitric oxide, NO) 试剂盒(上海碧云天生物技术有限公司); 异槲皮苷标准品(天津士兰科技有限公司); 木犀草苷、连翘酯苷A、异绿原酸A、獐牙菜苷标准品(成都曼斯特生物科技有限公司); 连翘苷标准品(成都普思生物科技股份有限公司); 黄芩素、汉黄芩素、黄芩苷、绿原酸标准品(中国食品药品检定研究院)。酶标仪(德国Berthold公司); 倒置显微镜、细胞培养箱(日本Olympus公司)。

目标化合物的选取 对于本实验目标化合物的选择主要基于以下原则: 首先, 其必须是复方鱼腥草合剂中的主要成分; 其次, 中药虽有众多成分, 但有效成分必须被吸收进入血液才会在体内起作用, 所以还应选择其入血成分进行研究; 再者, 兼顾药材中的主要结构类型, 应选取各类型的代表性化合物。因此, 在本网络药理学实验研究中, 根据以上3个原则并结合本课题组化学物质组及血中移行成分实验结果, 选取了25个关键化合物为研究对象, 详细信息见表 1。

目标化合物靶点的预测分析 登录Swiss Target Prediction服务器, 在线绘制复方鱼腥草合剂25个化合物的化学结构, 采用反向药效团匹配方法得到虚拟匹配筛选结果。由于检索到的药物靶标存在命名不规范等问题, 因此, 本实验使用UniProt数据库中UniProtKB搜索功能, 通过输入蛋白名称并限定物种为“human”, 将检索得到的所有蛋白校正为其官方名称; 另外, 在TCMSP数据库检索, 获取相关靶点信息并整合二者得到的蛋白靶点。

构建蛋白互作网络(protein-protein interaction, PPI) 及筛选关键靶点蛋白 将整合的靶点信息导入STRING10数据库中, 物种限定为“Homo sapiens”, 获得蛋白间相互作用信息, 将结果文件导入Cytoscape 3.6.1软件中绘制PPI网络, 分析其网络拓扑参数, 筛选出关键蛋白靶点。

生物信息学分析 运用Omicsbean软件对靶点蛋白进行生物信息学分析, 探究靶点蛋白在生物过程(biological process)、分子功能(molecular function) 和细胞组分(cellular component) 方面的GO (gene ontology) 功能富集分析。然后, 通过STRING 10数据库得到与靶点相关的通路过程, 利用KEGG数据库以及查阅相关文献, 对得到的通路进行分析。

“化合物-靶点-通路-药理作用-功效”网络构建 根据上述复方鱼腥草合剂25个化学成分的靶点及通路预测结果, 在Excel表格中建立化合物-靶点、靶点-通路、通路-药理作用、药理作用-功效的相互对应关系, 然后导入Cytoscape 3.6.1软件中构建网络, 并运用其插件Network Analyzer计算网络的特征。网络中节点(node) 表示化合物、靶点以及作用通路、药理作用、功效。边(edge) 表示化合物-靶点、靶点-通路以及靶点-药理作用、药理作用-功效相互作用。经处理后, 得到25个成分的相关靶点、通路预测图, 以该图来表示复方鱼腥草合剂“化合物-靶点-作用通路-药理作用-功效”之间的相互关系。

复方鱼腥草合剂及单体化合物对LPS诱导RAW264.7细胞炎症反应的影响 RAW264.7细胞用DMEM高糖完全培养基(含1%双抗和20%胎牛血清) 培养于37 ℃、5% CO₂的细胞培养箱。取生长至80%~90%的RAW264.7, 调整细胞密度为每毫升2×10⁵个均匀接种于96孔板, 于37 ℃、5% CO₂培养箱培养过夜后吸去上清, 按实验分组, 每组设3个复孔, 空白对照组(control) 每孔加入100 μL含2%血清的DMEM, 模型组加入100 μL终浓度为0.1 μg·mL^-1 LPS, 阳性药地塞米松组(Dex) 为加入100 μL终浓度为100 μmol·L^-1地塞米松和0.1 μg·mL^-1 LPS的混合溶液, 复方鱼腥草合剂给药组中每孔加入100 μL终浓度为稀释100、500、2 500倍的复方鱼腥草合剂及0.1 μg·mL^-1 LPS混合溶液, 异槲皮苷、木犀草苷、黄芩素、汉黄芩素、黄芩苷、连翘苷、连翘酯苷A、绿原酸、异绿原酸A和獐牙菜苷溶液给药组每组加入100 μL终浓度为1、10、100 μmol·L^-1样品和0.1 μg·mL^-1 LPS混合溶液。各组细胞处理后, 置于37 ℃、5% CO₂培养箱中培养24 h。收集细胞培养上清液, 用试剂盒检测NO、TNF-α、IL-6的含量变化, 具体实验步骤参照试剂盒说明书。

统计学方法 使用GraphPad Prism 5软件进行数据统计, 计量资料用均数±标准差(x±s) 表示, 多组间比较采用单因素方差分析(oneway analysis of variance, one-way ANOVA), P < 0.05认为差异有统计学意义。

结果

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1 化合物潜在靶点分析结果

通过Swiss Target Prediction服务器反向对接实验和TCMSP数据库检索, 得到25个化合物共211个相关靶点。从STRING 10数据库中获得蛋白间相互作用关系(PPI), 然后利用Cytoscape 3.6.1软件构建PPI网络图, 见图 1。对网络进行分析发现, 处于复方鱼腥草合剂PPI网络中心的15个靶点蛋白(表 2) 即丝氨酸/苏氨酸-蛋白激酶(serine/threonine-protein kinase, AKT1)、胰岛素(insulin, INS)、血管内皮生长因子(vascular endothelial growth factor A, VEGFA)、白介素-6 (interleukin-6, IL-6)、细胞肿瘤抗原p53 (cellular tumor antigen p53, TP53)、肿瘤坏死因子(tumor necrosis factor, TNF)、转录因子AP-1 (transcription factor AP-1, JUN)、半胱氨酸天冬氨酸蛋白酶3 (caspase-3, CASP3)、基质金属蛋白酶-9 (matrix metalloproteinase-9, MMP9)、白介素-8 (interleukin-8, IL-8)、前列腺素G/H合酶2 (prostaglandin G/H synthase 2, PTGS2)、原癌基因c-fos (proto-oncogene c-Fos, FOS)、酪氨酸蛋白激酶SRC (tyrosine-protein kinase SRC, SRC)、丝裂原活化蛋白激酶-8 (mitogen-activated protein kinase 8, MAPK8)、雌激素受体1 (estrogen receptor 1, ESR1) 度值较高, 拥有较多相互作用关系, 推测其为蛋白网络的核心靶点, 这些蛋白涉及炎症反应、免疫调节和中枢神经系统等方面, 提示复方鱼腥草合剂发挥清热解毒作用的药效机制与这些蛋白密切相关。

2 GO功能分析

利用OmicsBean分析软件对相关靶点蛋白进行GO功能注释分析, 包含细胞组分、分子功能和生物过程3个方面, 选取P值最小的前10个进行作图呈现, 见图 2。结果发现, 这些蛋白在细胞组分方面主要参与构成细胞质、胞质溶胶、细胞囊泡等过程; 在分子功能方面主要参与酶结合、大分子复合物结合以及蛋白结合绑定等功能; 在生物过程方面主要涉及对细胞过程的正调控、有机物或含氧化合物的应激反应等过程, 推测复方鱼腥草合剂清热解毒功效可能与以上功能过程相关。

3 通路分析

利用STRING 10数据库得到false discovery rate < 0.01的通路共97条, 通过KEGG通路分析及相关文献查阅, 得到46条相关信号通路, 具体信息见表 3。推测复方鱼腥草合剂清热解毒的机制可能与干预这些通路过程有关。

其中与炎症相关的信号通路: 白介素-17信号通路(IL-17 signaling pathway)、丝裂原活化蛋白激酶信号通路(MAPK signaling pathway)、花生四烯酸代谢(arachidonic acid metabolism)、核转录因子κB信号通路(NF-kappa B signaling pathway) 等; 与免疫调节相关的信号通路: T细胞受体信号通路(T cell receptor signaling pathway)、B细胞受体信号通路(B cell receptor signaling pathway)、Th1和Th2细胞分化(Th1 and Th2 cell differentiation)、自然杀伤细胞介导的细胞毒性(natural killer cell mediated cytotoxicity)、抗原处理和呈递(antigen processing and presentation) 等; 与镇痛相关的信号通路: 环磷酸腺苷信号通路(cAMP signaling pathway)、TRP通道炎性介质调节(inflammatory mediator regulation of TRP channels)、钙离子信号通路(calcium signaling pathway)、神经组织的配体-受体相互作用(neuroactive ligand-receptor interaction) 等; 与解热相关的信号通路: 环磷酸腺苷信号通路、花生四烯酸代谢等。

4 “化合物-靶点-通路-药理作用-功效”网络构建

根据对应关系, 在Cytoscape 3.6.1软件中构建“化合物-靶点-通路-药理作用-功效”的网络关系图, 见图 3 (蓝色正方形表示化合物, 绿色圆形表示靶点, 紫色菱形表示通路, 红色三角形表示药理作用, 黄色六边形表示功效)。运用软件的Network Analyzer插件分析发现, 该网络有287个节点(包括25个化合物、211个靶点、46条通路、4个药理作用和1个功效) 和884条边。网络拓扑学结构分析显示, 网络平均度值为6.048, 其中大于度值中位数的化合物有12个, 分别为汉黄芩素、黄芩素、千层纸素A、异绿原酸A、依靛蓝酮、木犀草苷、连翘苷、绿原酸、异槲皮苷、黄芩苷、白杨素、连翘酯苷A; 靶点有9个, 分别为AKT1、丝裂原活化蛋白激酶-9 (mitogen-activated protein kinase 9, MAPK9)、转录因子p65 (transcription factor p65, RELA)、蛋白激酶C α (protein kinase C alpha, PRKCA)、MAPK8、蛋白激酶C β (protein kinase C beta, PRKCB)、丝裂原活化蛋白激酶14 (mitogen-activated protein kinase 14, MAPK14)、JUN、TNF。网络中既存在一个分子与多个靶点蛋白的相互作用, 也存在同一个靶点蛋白与不同分子作用的现象, 显示了复方鱼腥草合剂的多成分与多靶点之间共同作用的机制, 符合中药复方的作用特点。

5 复方鱼腥草合剂及单体化合物对LPS诱导RAW264.7细胞炎症反应的影响

通过检测, 得到复方鱼腥草合剂及10个代表性单体对RAW264.7细胞上清液中NO、TNF-α和IL-6释放量的影响(图 4、5)。从结果图可以看出, LPS作用于细胞后, 细胞上清液中的NO、TNF-α和IL-6分泌量明显增加, 与空白对照组比较有显著性差异(P < 0.001), 阳性药地塞米松给药浓度为100 μmol·L^-1时能显著抑制NO、TNF-α和IL-6的释放(P < 0.001)。复方鱼腥草合剂各浓度细胞上清液中的NO、TNF-α和IL-6含量明显低于LPS组(P < 0.05), 并且呈现剂量依赖性。与LPS组比较, 汉黄芩素低浓度给药组对细胞上清中NO的释放无显著抑制作用, 黄芩苷低浓度给药组细胞上清中TNF-α的含量无显著性差异, 连翘酯苷A低浓度给药组对细胞上清中IL-6的含量无显著性差异, 獐牙菜苷在低浓度给药组对NO、TNF-α和IL-6的含量均无显著性差异, 其他单体化合物在高、中、低浓度时对细胞上清NO、TNF-α和IL-6的释放均有显著性抑制作用, 并且呈现剂量依赖性。

讨论

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中医“外感”、“感冒”即为西医中急性上呼吸道感染, 其通常是指由病毒和细菌感染引起的咽喉、扁桃体等部位的呼吸道黏膜急性炎症的总称^{[8, 9]}。当病原体侵袭机体时可与免疫细胞上Toll样受体(TLRs) 结合, 然后通过TLR-NF-κB信号通路产生后续一系列特定基因表达, 最后产生诸如IL-1、IL-6、IL-12和TNF-α等炎症因子, 调节机体的炎症反应^{[10, 11]}。同时, IL-1、IL-6和TNF-α的增加将活化B淋巴细胞受体介导的免疫通路, 产生抗体, 伴随进一步激活MAPK、PI3K-AKT和NF-κB信号途径, 从而引起一系列的炎症反应, 诱发全身性免疫应答。IL-6增高可以刺激T细胞增殖, 增强细胞免疫反应^[12]。这些细胞因子一方面可直接或间接地作用于伤害性感受器, 从而在持续性痛或病理性痛中起到关键性作用^[13]; 另一方面, 其还可直接或间接通过中枢介质作用于体温调节中枢, 体温调定点上移, 引起产热增加, 散热减少, 使体温升高^[14]。另外, 在炎症反应中花生四烯酸的代谢起着关键的作用。花生四烯酸经环氧化酶(COX) 途径和脂氧化酶(LOX) 途径分别代谢产生前列腺素(PGs) 及白三烯类(LTs) 等炎性递质^[15]。PGs是重要的致炎、致痛和致热介质, 前列腺素E₂ (PGE₂) 通过其受体和其他前列腺类物质一起以条件依赖方式参与了炎症不同阶段的调节^{[16, 17]}, 并且PGE₂是中枢性正调节介质之一, 很容易弥散透过血脑屏障, 作用于下丘脑温度敏感神经的PGE₂受体, 通过提高细胞内cAMP使调定点升高^[18]。白三烯作为重要的炎症介质参与了支气管哮喘、慢性阻塞性肺疾病、毛细支气管炎和急性肺损伤等多种肺部疾病的病理生理过程^[19]。白三烯B4是重要的白细胞活化物质, 刺激白细胞的趋化、聚集、释放氧自由基及溶酶体酶, 可增加血管壁通透性, 刺激支气管平滑肌细胞分泌黏液, 促进炎症细胞在气道聚集, 从而加剧炎症反应^[20]。

本研究的靶点预测及作用通路分析结果提示, 复方鱼腥草合剂中的黄酮类成分芦丁、金丝桃苷、木犀草苷、汉黄芩素、黄芩素、黄芩苷、千层纸素A、异槲皮苷, 木脂素类成分连翘脂素、连翘苷, 苯乙醇苷类成分连翘酯苷A、毛蕊花糖苷, 有机酸类成分绿原酸、异绿原酸A、生物碱类成分依靛蓝酮及环烯醚萜类成分獐牙菜苷等化合物可通过作用于IL-1B、IL-8、IL-6、TNF、诱导型一氧化氮合酶2 (NOS2)、转录因子p65 (RELA)、转录因子AP-1 (JUN)、环氧化酶(PTGS1、PTGS2)、蛋白激酶C (PRKCA、PRKCB、PRKCD)、丝氨酸/苏氨酸蛋白激酶(AKT1)、丝裂原活化蛋白激酶(MAPK8、MAPK9、MAPK14) 等靶点, 调节如白介素17、MAPK、PI3K-AKT、TNF、Toll样受体、NF-κB、cAMP、花生四烯酸代谢等信号通路, 发挥抗炎及解热镇痛的作用。

研究显示, 黄芩中黄酮类活性成分黄芩素和黄芩苷可以通过干扰花生四烯酸代谢、NF-κB和MAPK等信号通路, 抑制核因子的活性以及细胞因子的分泌、释放而发挥解热镇痛抗炎的作用^[21]。千层纸素A通过阻断NF-κB的活化, 汉黄芩素通过阻止巨噬细胞中c-Jun氨基末端激酶(JNKs)、原癌基因c-Jun蛋白及AP-1的活化使iNOS和COX-2基因表达下调, 减少NO和PGE₂的产生^[22-24]。金银花中的黄酮类成分木犀草苷可抑制IL-1β诱导的兔软骨细胞MAPKs (ERK1/2、JNK和p38) 的磷酸化和NF-κB p65亚基向细胞核的移位, 进而抑制MMPs、NO、PGE₂和促炎细胞因子的释放, 发挥抗炎作用^[25]。有机酸类成分绿原酸、异绿原酸A通过减弱NF-κB/NLRP3和JNK/AP-1信号通路活化, 降低LPS诱导的COX-2和iNOS的表达, 并逆转IL-6、TNF-α、IL-1β、MCP-1和PGE₂的表达, 减轻LPS所致炎症反应^{[26, 27]}; 环烯醚萜类成分獐牙菜苷通过激活SIRT1调控SIRT1/NF-κB和SIRT1/FOXO1信号通路, 改善LPS诱导的RAW264.7细胞炎症反应^[28]。连翘药材中的连翘苷^[29]及连翘酯苷A^[30]通过抑制MAPK和NF-κB信号通路的激活, 降低促炎细胞因子(TNF-α、IL-1β和IL-6) 的表达, 并且连翘酯苷A亦可通过抑制TLR4/MyD88/NF-κB信号通路, 下调参与调节性T细胞(Tregs) 免疫抑制功能的分子Foxp3、IL-10和TGF-β1, 发挥抗内毒素作用^[31]。鱼腥草及板蓝根中的金丝桃苷、异槲皮苷通过抑制LPS诱导的iNOS表达, 抑制NO的产生, 其机制可能是通过抑制p44/42丝裂原活化蛋白激酶(MAPK)、p38MAPK和JNK的磷酸化, 参与了抗炎作用^[32]。

LPS是革兰阴性菌致病的主要因素, 能通过与细胞膜受体相互作用, 作用于宿主细胞, 并通过细胞内信号传递级联基因表达发生变化^[33]。当RAW264.7被LPS刺激时, 通过作用于细胞膜上的膜受体, 激活诱导细胞内各种炎症信号通路, 进而调节相关炎症因子基因的表达, 最终合成和释放众多内源性生物活性因子, 如NO、PGE₂、TNF-α、IL-6和IL-1β等^[34] (机制图见图 6)。本研究以LPS刺激RAW264.7细胞作为炎症实验模型, 对复方鱼腥草合剂及异槲皮苷、木犀草苷、黄芩素、汉黄芩素、黄芩苷、连翘苷、连翘酯苷A、绿原酸、异绿原酸A、獐牙菜苷10个关键化学成分的抗炎活性进行验证, 结果表明复方鱼腥草合剂及10个成分均表现出显著的抗炎活性。因此推测, 该10个成分可能为复方鱼腥草合剂发挥抗炎作用的关键药效物质基础。

综上, 本研究通过网络药理学手段及体外细胞实验分析了复方鱼腥草合剂可能的作用靶点及途径, 初步阐释了其清热解毒功效的作用机制及药效物质基础, 为后续进行深入作用机制及质量标志物的研究奠定了基础。

作者贡献: 韩彦琪、陈志霖、许浚共同设计了文章的研究方法与思路; 张洪兵、刘建庭、张杨完成了文献搜索与整理; 韩彦琪、刘耀晨、陈志霖完成了实验工作及初稿与终稿的撰写; 韩彦琪、刘耀晨、胡江宁分析并整理了实验数据; 刘昌孝、张铁军提供了文章主题中心思想并完成了论文的修改。

利益冲突: 所有作者均声明没有利益冲突。

基金

收起

国家自然科学基金资助项目(81830111)
中医药现代化研究重点专项(2019YFC1711200)
天津市自然科学基金资助项目(19JCQNJC12700)
广西中药药效研究重点实验室开放课题(20-065-38)

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2021年第56卷第6期

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文章信息

doi: 10.16438/j.0513-4870.2021-0085

接收时间：2021-01-16
首发时间：2025-12-18
出版时间：2021-06-12

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文章信息

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出版历史

收稿日期：2021-01-16
修回日期：2021-02-26

基金

国家自然科学基金资助项目(81830111)

中医药现代化研究重点专项(2019YFC1711200)

天津市自然科学基金资助项目(19JCQNJC12700)

广西中药药效研究重点实验室开放课题(20-065-38)

作者信息

1.天津市中药质量标志物重点实验室, 天津 300462

2.释药技术与药物代谢动力学国家重点实验室 (天津药物研究院), 天津 300462

3.浙江康恩贝中药有限公司, 浙江丽水 323400

4.天津医科大学, 天津 300070

5.浙江康恩贝制药股份有限公司, 浙江杭州 310052

通讯作者:

*张铁军, Tel: 86-22-23006295, E-mail: zhangtj@tjipr.com;

刘昌孝，liucx@tjipr.com

参考文献

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https://castjournals.cast.org.cn/joweb/yxxb/CN/10.16438/j.0513-4870.2021-0085

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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

No.	Structure type	Compound	Formula	Source
1	Flavone	Hyperoside	C₂₁H₂₀O₁₂	1, 3
2		Rutin	C₂₇H₃₀O₁₆	1, 4, 5
3		Isoquercitrin	C₂₁H₂₀O₁₂	1, 3, 5
4		Cynaroside	C₂₁H₂₀O₁₁	5
5		Baicalein	C₁₅H₁₀O₅	2
6		Baicalin	C₂₁H₁₈O₁₁	2
7		Wogonin	C₁₆H₁₂O₅	2
8		Wogonoside	C₆H₁₀O₇	2
9		Oroxylin A	C₁₆H₁₂O₅	2
10		Chrysin	C₁₅H₁₀O₄	2
11	Alkaloid	Laurolitsine	C₁₈H₁₉NO₄	1
12		Isaidigodione	C₁₈H₁₈N₂O₄	3
13	Lignans	Lariciresinol	C₂₆H₃₄O₁₁	3
14		Phillyrin	C₂₇H₃₄O₁₁	4
15		Phillygenin	C₂₁H₂₄O₆	4
16	Phenylethanoid glycoside	Forsythoside A	C₂₉H₃₆O₁₅	4
17		Verbascoside	C₂₉H₃₆O₁₅	4
18	Organic acid	Chlorogenic acid	C₁₆H₁₈O₉	1, 5
19		Caffeic acid	C₉H₈O₄	1, 4, 5
20		Quinic acid	C₇H₁₂O₆	1, 4, 5
21		Isochlorogenic acid A	C₂₅H₂₄O₁₂	5
22		Anthranilic acid	C₇H₇NO₂	3
23	Iridoid	Loganic acid	C₁₆H₂₄O₁₀	5
24		Sweroside	C₁₆H₂₂O₉	5
25	Nucleoside	Adenosine	C₁₀H₁₃N₅O₄	1, 2, 3, 4, 5

No.

Structure type

Compound

Formula

Source

Flavone

Hyperoside

C₂₁H₂₀O₁₂

1, 3

Rutin

C₂₇H₃₀O₁₆

1, 4, 5

Isoquercitrin

C₂₁H₂₀O₁₂

1, 3, 5

Cynaroside

C₂₁H₂₀O₁₁

Baicalein

C₁₅H₁₀O₅

Baicalin

C₂₁H₁₈O₁₁

Wogonin

C₁₆H₁₂O₅

Wogonoside

C₆H₁₀O₇

Oroxylin A

C₁₆H₁₂O₅

Chrysin

C₁₅H₁₀O₄

Alkaloid

Laurolitsine

C₁₈H₁₉NO₄

Isaidigodione

C₁₈H₁₈N₂O₄

Lignans

Lariciresinol

C₂₆H₃₄O₁₁

Phillyrin

C₂₇H₃₄O₁₁

Phillygenin

C₂₁H₂₄O₆

Phenylethanoid glycoside

Forsythoside A

C₂₉H₃₆O₁₅

Verbascoside

C₂₉H₃₆O₁₅

Organic acid

Chlorogenic acid

C₁₆H₁₈O₉

1, 5

Caffeic acid

C₉H₈O₄

1, 4, 5

Quinic acid

C₇H₁₂O₆

1, 4, 5

Isochlorogenic acid A

C₂₅H₂₄O₁₂

Anthranilic acid

C₇H₇NO₂

Iridoid

Loganic acid

C₁₆H₂₄O₁₀

Sweroside

C₁₆H₂₂O₉

Nucleoside

Adenosine

C₁₀H₁₃N₅O₄

1, 2, 3, 4, 5

Target	Common name	Uniprot ID	Degree
Serine/threonine-protein kinase	AKT1	P31749	99
Insulin	INS	P01308	97
Vascular endothelial growth factor A	VEGFA	P15692	86
Interleukin-6	IL-6	P05231	86
Cellular tumor antigen p53	TP53	P04637	84
Tumor necrosis factor	TNF	P01375	79
Transcription factor AP-1	JUN	P05412	72
Caspase-3	CASP3	P42574	71
Matrix metalloproteinase-9	MMP9	P14780	69
Interleukin-8	IL-8	P10145	68
Prostaglandin G/H synthase 2	PTGS2	P35354	67
Proto-oncogene c-Fos	FOS	P01100	66
Tyrosine-protein kinase SRC	SRC	P12931	66
Mitogen-activated protein kinase 8	MAPK8	P45983	65
Estrogen receptor 1	ESR1	P03372	61

Target

Common name

Uniprot ID

Degree

Serine/threonine-protein kinase

AKT1

P31749

Insulin

INS

P01308

Vascular endothelial growth factor A

VEGFA

P15692

Interleukin-6

IL-6

P05231

Cellular tumor antigen p53

TP53

P04637

Tumor necrosis factor

TNF

P01375

Transcription factor AP-1

JUN

P05412

Caspase-3

CASP3

P42574

Matrix metalloproteinase-9

MMP9

P14780

Interleukin-8

IL-8

P10145

Prostaglandin G/H synthase 2

PTGS2

P35354

Proto-oncogene c-Fos

FOS

P01100

Tyrosine-protein kinase SRC

SRC

P12931

Mitogen-activated protein kinase 8

MAPK8

P45983

Estrogen receptor 1

ESR1

P03372

Term ID	Term description	Observed gene count	False discovery rate
hsa04657	IL-17 signaling pathway	21	1.16E-19
hsa04659	Th17 cell differentiation	16	6.57E-13
hsa04080	Neuroactive ligand-receptor interaction	22	1.89E-12
hsa04010	MAPK signaling pathway	22	5.02E-12
hsa04151	PI3K-AKT signaling pathway	23	1.36E-11
hsa04919	Thyroid hormone signaling pathway	15	2.00E-11
hsa04668	TNF signaling pathway	14	1.03E-10
hsa04115	p53 signaling pathway	12	1.18E-10
hsa04210	Apoptosis	15	1.23E-10
hsa04066	HIF-1 signaling pathway	13	3.83E-10
hsa04915	Estrogen signaling pathway	14	9.94E-10
hsa04024	cAMP signaling pathway	16	1.39E-09
hsa04071	Sphingolipid signaling pathway	13	2.04E-09
hsa04722	Neurotrophin signaling pathway	13	2.04E-09
hsa04660	T cell receptor signaling pathway	12	3.99E-09
hsa04068	FoxO signaling pathway	13	6.38E-09
hsa04510	Focal adhesion	15	9.74E-09
hsa04664	Fc epsilon RI signaling pathway	10	1.45E-08
hsa04750	Inflammatory mediator regulation of TRP channels	11	1.89E-08
hsa04620	Toll-like receptor signaling pathway	11	4.65E-08
hsa04370	VEGF signaling pathway	9	6.56E-08
hsa04621	NOD-like receptor signaling pathway	13	6.99E-08
hsa04658	Th1 and Th2 cell differentiation	10	1.25E-07
hsa04022	cGMP-PKG signaling pathway	12	3.52E-07
hsa04012	ErbB signaling pathway	9	7.94E-07
hsa04020	Calcium signaling pathway	12	1.03E-06
hsa04912	GnRH signaling pathway	9	1.22E-06
hsa04015	Rap1 signaling pathway	12	3.40E-06
hsa04310	Wnt signaling pathway	10	6.52E-06
hsa04924	Renin secretion	7	1.31E-05
hsa04064	NF-kappa B signaling pathway	8	1.52E-05
hsa04672	Intestinal immune network for IgA production	6	2.02E-05
hsa04662	B cell receptor signaling pathway	7	2.43E-05
hsa04060	Cytokine-cytokine receptor interaction	12	3.37E-05
hsa04630	Jak-STAT signaling pathway	9	8.01E-05
hsa00590	Arachidonic acid metabolism	6	9.31E-05
hsa04622	RIG-I-like receptor signaling pathway	6	0.000 18
hsa04014	Ras signaling pathway	10	0.000 2
hsa04670	Leukocyte transendothelial migration	7	0.000 29
hsa04611	Platelet activation	7	0.000 49
hsa04062	Chemokine signaling pathway	8	0.000 86
hsa04650	Natural killer cell mediated cytotoxicity	6	0.002 6
hsa04540	Gap junction	5	0.003 1
hsa04150	mTOR signaling pathway	6	0.005 8
hsa04612	Antigen processing and presentation	4	0.007 1
hsa04530	Tight junction	6	0.009 7

Term ID

Term description

Observed gene count

False discovery rate

hsa04657

IL-17 signaling pathway

1.16E-19

hsa04659

Th17 cell differentiation

6.57E-13

hsa04080

Neuroactive ligand-receptor interaction

1.89E-12

hsa04010

MAPK signaling pathway

5.02E-12

hsa04151

PI3K-AKT signaling pathway

1.36E-11

hsa04919

Thyroid hormone signaling pathway

2.00E-11

hsa04668

TNF signaling pathway

1.03E-10

hsa04115

p53 signaling pathway

1.18E-10

hsa04210

Apoptosis

1.23E-10

hsa04066

HIF-1 signaling pathway

3.83E-10

hsa04915

Estrogen signaling pathway

9.94E-10

hsa04024

cAMP signaling pathway

1.39E-09

hsa04071

Sphingolipid signaling pathway

2.04E-09

hsa04722

Neurotrophin signaling pathway

2.04E-09

hsa04660

T cell receptor signaling pathway

3.99E-09

hsa04068

FoxO signaling pathway

6.38E-09

hsa04510

Focal adhesion

9.74E-09

hsa04664

Fc epsilon RI signaling pathway

1.45E-08

hsa04750

Inflammatory mediator regulation of TRP channels

1.89E-08

hsa04620

Toll-like receptor signaling pathway

4.65E-08

hsa04370

VEGF signaling pathway

6.56E-08

hsa04621

NOD-like receptor signaling pathway

6.99E-08

hsa04658

Th1 and Th2 cell differentiation

1.25E-07

hsa04022

cGMP-PKG signaling pathway

3.52E-07

hsa04012

ErbB signaling pathway

7.94E-07

hsa04020

Calcium signaling pathway

1.03E-06

hsa04912

GnRH signaling pathway

1.22E-06

hsa04015

Rap1 signaling pathway

3.40E-06

hsa04310

Wnt signaling pathway

6.52E-06

hsa04924

Renin secretion

1.31E-05

hsa04064

NF-kappa B signaling pathway

1.52E-05

hsa04672

Intestinal immune network for IgA production

2.02E-05

hsa04662

B cell receptor signaling pathway

2.43E-05

hsa04060

Cytokine-cytokine receptor interaction

3.37E-05

hsa04630

Jak-STAT signaling pathway

8.01E-05

hsa00590

Arachidonic acid metabolism

9.31E-05

hsa04622

RIG-I-like receptor signaling pathway

0.000 18

hsa04014

Ras signaling pathway

0.000 2

hsa04670

Leukocyte transendothelial migration

0.000 29

hsa04611

Platelet activation

0.000 49

hsa04062

Chemokine signaling pathway

0.000 86

hsa04650

Natural killer cell mediated cytotoxicity

0.002 6

hsa04540

Gap junction

0.003 1

hsa04150

mTOR signaling pathway

0.005 8

hsa04612

Antigen processing and presentation

0.007 1

hsa04530

Tight junction

0.009 7