鬼箭羽通过调节EGFR酪氨酸激酶抑制剂耐药信号通路延缓糖尿病肾病的进展

doi:10.12122/j.issn.1673-4254.2024.07.04

摘要/Abstract

摘要：

目的结合GEO数据库利用网络药理学、分子对接技术及动物实验探究鬼箭羽治疗糖尿病肾病（DKD）的潜在作用机制。方法通过TCMSP、PubChem及Swiss Target Prediction数据库获取鬼箭羽的活性成分和作用靶点。利用GEO数据库找出与DKD相关基因芯片，使用R语言分析筛选出差异表达基因，结合GeneCards、DisGeNet、OMIM以及TTD疾病数据库整合获取DKD的治疗靶点。利用Venny2.1.0平台将药物与DKD相关的靶基因取交，利用STRING平台构建蛋白-蛋白互作网络图，并利用Cytoscape3.8.2软件进行核心靶点的拓扑属性分析以及“药物-成分-靶点-疾病”网络图构建；利用David平台进行KEGG与GO分析。利用Autodock vina V1.2.0软件对核心靶点及主要药效成分进行分子对接，并通过db小鼠动物实验进一步加以验证。结果符合条件的基因芯片为GSE96804、GSE30528和GSE30529，共包含60例患病和45例正常样本数据，R语言分析得到111个差异基因。网络药理学分析显示鬼箭羽相关靶基因共226种，DKD相关靶基因共4252种，二者取交得到鬼箭羽治疗DKD的潜在靶基因共161种；PPI网络图中筛选出关键核心靶基因为SRC、EGFR、AKT1等，“药物-成分-靶点-疾病“网络图得到鬼箭羽的主要核心活性成分为槲皮素、山奈酚、香叶木素、柚皮素。GO和KEGG富集分析显示，鬼箭羽治疗DKD的生物过程与外源刺激的反应、蛋白质磷酸化等有关，可能主要通过调节EGFR酪氨酸激酶抑制剂耐药通路发挥作用。分子对接结果显示鬼箭羽的主要核心活性成分与关键核心靶点具有良好的结合活性。体内动物实验结果证明鬼箭羽能够改善肾脏组织的病理学变化，且显著抑制了SRC、EGFR、AKT1的表达（P<0.05），延缓DKD的进一步恶化。结论鬼箭羽可能是通过槲皮素、山奈酚、香叶木素、柚皮素等多种活性成分调控SRC、EGFR、AKT1等基因表达，从而影响EGFR酪氨酸激酶抑制剂耐药信号通路治疗DKD。

关键词: 鬼箭羽, 糖尿病肾病, GEO数据库, 网络药理学, 分子对接, 信号通路, 动物实验

Abstract:

Objective To explore the therapeutic mechanism of Euonymus alatus for diabetic kidney disease (DKD). Methods TCMSP, PubChem and Swiss Target Prediction databases were used to obtain the active ingredients in Euonymus alatus and their targets. GEO database and R language were used to analyze the differentially expressed genes in DKD. The therapeutic targets of DKD were obtained using GeneCards, DisGeNet, OMIM and TTD databases. The protein-protein interaction network and the "drug-component-target-disease" network were constructed for analyzing the topological properties of the core targets, which were functionally annotated using GO and KEGG pathway enrichment analyses. Molecular docking was performed for the core targets and the main pharmacologically active components, and the results were verified in db/db mice. Results Analysis of GSE96804, GSE30528 and GSE30529 datasets (including 60 DKD patients and 45 normal samples) identified 111 differentially expressed genes in DKD. Network pharmacology analysis obtained 161 intersecting genes between the target genes of Euonymus alatus and DKD, including the key core target genes SRC, EGFR, and AKT1. The core active ingredients of Euonymus alatus were quercetin, kaempferol, diosmetin, and naringenin, which were associated with responses to xenobiotic stimulionus and protein phosphorylation and regulated EGFR tyrosine kinase inhibitor resistance pathways. Molecular docking suggested good binding activities of the core active components of Euonymus alatus with the core targets. In db/db mouse models of DKD, treatment with Euonymus alatus obviously ameliorated kidney pathologies, significantly inhibited renal expressions of SRC, EGFR and AKT1, and delayed the progression of DKD. Conclusion Euonymus alatus contains multiple active ingredients such as quercetin, kakaferol, diosmetin, naringenin, which regulate the expressions of SRC, EGFR, and AKT1 to affect the EGFR tyrosine kinase inhibitor resistance signaling pathway to delay the progression of DKD.

Key words: euonymus alatus, diabetic kidney disease, GEO database, network pharmacology, molecular docking, signaling pathways, animal experiments

王瑾瑾, 崔文飞, 窦雪伟, 尹冰磊, 牛钰琪, 牛羚, 闫国立. 鬼箭羽通过调节EGFR酪氨酸激酶抑制剂耐药信号通路延缓糖尿病肾病的进展[J]. 南方医科大学学报, 2024, 44(7): 1243-1255.

Jinjin WANG, Wenfei CUI, Xuewei DOU, Binglei YIN, Yuqi NIU, Ling NIU, Guoli YAN. Euonymus alatus delays progression of diabetic kidney disease in mice by regulating EGFR tyrosine kinase inhibitor resistance signaling pathway[J]. Journal of Southern Medical University, 2024, 44(7): 1243-1255.

图/表 14

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Mol ID	Molecule Name	Oral bioavailability (%)	Drug-like properties
MOL001040	(2R)-5, 7-dihydroxy-2-(4-hydroxyphenyl) chroman-4-one	42.36	0.21
MOL001755	24-Ethylcholest-4-en-3-one	36.08	0.76
MOL000358	beta-sitosterol	36.91	0.75
MOL000359	sitosterol	36.91	0.75
MOL000422	kaempferol	41.88	0.24
MOL005100	5, 7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl) chroman-4-one	47.74	0.27
MOL000098	quercetin	46.43	0.28
MOL001420	ZINC04073977	38	0.76

Mol ID	Molecule Name	Oral bioavailability (%)	Drug-like properties
MOL001040	(2R)-5, 7-dihydroxy-2-(4-hydroxyphenyl) chroman-4-one	42.36	0.21
MOL001755	24-Ethylcholest-4-en-3-one	36.08	0.76
MOL000358	beta-sitosterol	36.91	0.75
MOL000359	sitosterol	36.91	0.75
MOL000422	kaempferol	41.88	0.24
MOL005100	5, 7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl) chroman-4-one	47.74	0.27
MOL000098	quercetin	46.43	0.28
MOL001420	ZINC04073977	38	0.76

Up-regulated genes	logFC	P	Down-regulated genes	logFC	P
LUM	2.343814499	6.21×10^-12	G6PC	-2.854555923	2×10^-16
MMP7	2.22402567	3.41×10^-9	ALB	-2.203960464	1.2×10^-9
FN1	2.067417931	2.88×10^-10	FOS	-2.069379844	2.37×10^-11
C3	2.028993328	3.53×10^-9	LPL	-1.829387514	1.72×10^-16
IGJ	1.90309721	1.38×10^-7	ZFP36	-1.683736259	1.95×10^-13
VCAN	1.830704374	9.5×10^-8	ALDOB	-1.537200778	4.47×10^-5
MOXD1	1.764184138	8.5×10^-11	FOSB	-1.527474388	2.82×10^-10
COL1A2	1.716058931	5.2×10^-12	EGR1	-1.526816671	1.19×10^-6
C7	1.698285969	1.83×10^-8	CYP27B1	-1.526356948	2.94×10^-11
CCL19	1.538100328	2.34×10^-8	UMOD	-1.494821792	2.77×10^-5
ADH1B	1.527694813	4.53×10^-8	HPGD	-1.491939257	1.49×10^-8
COL6A3	1.45202264	7.49×10^-11	HPD	-1.476264604	7.57×10^-6
MARCKS	1.429551387	1.92×10^-10	EGF	-1.471530504	2.5×10^-11
TNC	1.427538552	1.01×10^-9	KNG1	-1.463898156	3.23×10^-6
MS4A6A	1.397713434	2.25×10^-9	APOH	-1.412843883	6.19×10^-9
LTF	1.389034747	3.58×10^-6	AFM	-1.401540175	1.98×10^-6
IGKC	1.367567925	1.34×10^-6	TPPP3	-1.366144814	8.14×10^-14
THBS2	1.347011952	3.75×10^-10	ESM1	-1.359050399	1.23×10^-9
COL3A1	1.343621855	5.48×10^-8	DUSP1	-1.355440112	4.6×10^-18
CCL21	1.330208775	2.07×10^-6	S100A12	-1.354199988	5.61×10^-8

Up-regulated genes	logFC	P	Down-regulated genes	logFC	P
LUM	2.343814499	6.21×10^-12	G6PC	-2.854555923	2×10^-16
MMP7	2.22402567	3.41×10^-9	ALB	-2.203960464	1.2×10^-9
FN1	2.067417931	2.88×10^-10	FOS	-2.069379844	2.37×10^-11
C3	2.028993328	3.53×10^-9	LPL	-1.829387514	1.72×10^-16
IGJ	1.90309721	1.38×10^-7	ZFP36	-1.683736259	1.95×10^-13
VCAN	1.830704374	9.5×10^-8	ALDOB	-1.537200778	4.47×10^-5
MOXD1	1.764184138	8.5×10^-11	FOSB	-1.527474388	2.82×10^-10
COL1A2	1.716058931	5.2×10^-12	EGR1	-1.526816671	1.19×10^-6
C7	1.698285969	1.83×10^-8	CYP27B1	-1.526356948	2.94×10^-11
CCL19	1.538100328	2.34×10^-8	UMOD	-1.494821792	2.77×10^-5
ADH1B	1.527694813	4.53×10^-8	HPGD	-1.491939257	1.49×10^-8
COL6A3	1.45202264	7.49×10^-11	HPD	-1.476264604	7.57×10^-6
MARCKS	1.429551387	1.92×10^-10	EGF	-1.471530504	2.5×10^-11
TNC	1.427538552	1.01×10^-9	KNG1	-1.463898156	3.23×10^-6
MS4A6A	1.397713434	2.25×10^-9	APOH	-1.412843883	6.19×10^-9
LTF	1.389034747	3.58×10^-6	AFM	-1.401540175	1.98×10^-6
IGKC	1.367567925	1.34×10^-6	TPPP3	-1.366144814	8.14×10^-14
THBS2	1.347011952	3.75×10^-10	ESM1	-1.359050399	1.23×10^-9
COL3A1	1.343621855	5.48×10^-8	DUSP1	-1.355440112	4.6×10^-18
CCL21	1.330208775	2.07×10^-6	S100A12	-1.354199988	5.61×10^-8

Core targets	Target protein	TCMSP serial number	Active ingredients	Number of hydrogen bonds	Combination way	Affinity(kJ/mol)
SRC	5MTJ	MOL000098	Quercetin	9	Hydorgen interaction	-6.92
		MOL000422	Kaempferol	4	Hydrophobic interaction, Hydorgen interaction	-5.84
		MOL005100	5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one	4	Hydrophobic interaction, Hydorgen interaction	-5.71
		MOL001040	(2R)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one	6	Hydrophobic interaction, Hydorgen interaction	-6.67
EGFR	6Z4D	MOL000098	quercetin	6	Hydrophobic interaction, Hydorgen interaction	-4.74
		MOL000422	kaempferol	1	Hydrophobic interaction, Hydorgen interaction	-5.65
		MOL005100	5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one	5	Hydrophobic interaction, Hydorgen interaction	-4.80
		MOL001040	(2R)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one	4	Hydrophobic interaction, Hydorgen interaction	-4.87
AKT1	7NH4	MOL000098	Quercetin	7	Hydrophobic interaction, Hydorgen interaction	-5.43
		MOL000422	Kaempferol	6	Hydrophobic interaction, Hydorgen interaction	-4.97
		MOL005100	5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one	3	Hydrophobic interaction, Hydorgen interaction, Π-Cation interaction	-5.59
		MOL001040	(2R)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one	3	Hydrophobic interaction, Hydorgen interaction	-6.52