基于网络药理学研究杜仲叶提取物改善非酒精性脂肪肝的作用及机制

蔡丹红; 周长威; 张肇锋; 赵烨; 卞瑶瑶; 薛梅; 李玉; 刘笑利; 张良

doi:10.14148/j.issn.1672-0482.2022.0703

Eucommia Folium Extracts Alleviate Nonalcoholic Fatty Liver Disease in vivo and in vitro Based on the Network Pharmacology Study

doi: 10.14148/j.issn.1672-0482.2022.0703

1.
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, China

More Information

Corresponding author: 张良, 男, 教授, 主要从事中药药理的研究, E-mail: zhangl_1999@njucm.edu.cn

摘要

摘要: 目的基于网络药理学探讨杜仲叶提取物(Eucommia ulmoides leaf extracts, ELE)改善非酒精性脂肪肝(Nonalcoholic fatty liver disease, NAFLD)的有效成分、作用靶点及可能的作用机制。方法使用CNKI、万方、TCMSP、Pubmed等数据库、获取并筛选杜仲叶活性成分; 使用TCMSP数据库和SwissTargetPrediction数据库检索并预测ELE活性成分的靶点; 使用Genecard数据库和Drugbank数据库收集NAFLD疾病的靶点; 使用String 11.0构建蛋白互作(Protein-protein interaction)网络图, 分析蛋白靶点之间的关系; 使用Cytoscape 3.7.1软件构建化合物-疾病靶点-通路网络图, 分析三者之间相互作用的关系; 使用DAVID数据库, 对靶点进行GO分析和KEGG通路富集分析, 并对关键靶点进行实验验证。结果 ELE改善NAFLD疾病靶点网络主要包含9个化合物和35个靶点, 与脂质代谢相关的靶点有PPARα和CPT-1A, 与NAFLD相关的通路主要有胰岛素抵抗、AMPK信号通路。体内、体外实验发现ELE能通过激活AMPKα及增强靶点PPARα和CPT-1A的表达, 增强脂质氧化, 改善NAFLD。结论 ELE能增强脂质代谢, 改善NAFLD, 其机制主要与AMPK信号通路相关。
- 网络药理学 /
- 杜仲叶提取物 /
- 非酒精性脂肪肝 /
- AMPK信号通路
Abstract: OBJECTIVE To explore the active ingredients, targets and possible mechanism of ELE alleviating NAFLD based on the method of network pharmacology. METHODS The components in ELE were found in CNKI, Wanfang, TCMSP and Pubmed database. The components targets were found in TCMSP and swisstarget prediction database. NAFLD targets were found in genecard database and drugbank database. PPI network was established to analyze the interaction among targets by String 11.0 database. Compounds-targets-pathways network was established by the software of Cytoscape. Go and KEGG analysis were carried by david database. In addition the key targets were verified by cell and animals experiments. RESULTS The network of ELE against NAFLD contains 9 compounds and 35 targets. The targets related to lipid metabolism are PPARα and CPT-1A, and the signaling pathways mainly related to lipid metabolism are insulin resistance and AMPK signaling pathway. In vivo and in vitro experiments indicated that ELE could enhance lipid oxidation and improve NAFLD by activating AMPKα and enhancing the expression of PPARα and CPT-1A. CONCLUSION ELE can enhance lipid metabolism and improve NAFLD mainly related to AMPK signaling pathway.
- network pharmacology /
- Eucommia ulmoides leaf extract /
- NAFLD /
- AMPK signaling pathway

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Figure 1. Protein-protein interaction (PPI) network

Note: (A) Intersection gene of ELE targets and NAFLD related targets. ELE consists 346 targets generated from 9 active compounds. NAFLD consists 180 targets and there are 35 common targets of ELE and NAFLD. (B) Core network (PPI) diagram of ELE and NAFLD. The nodes get larger with increasing degree. Edges: PPIs between putative targets of ELE and their interactive partners.

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Figure 2. The compound-compound targets-pathway network of ELE

Note: active compounds in blue, target in pink, pathway in red

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Figure 3. Gene ontology and pathway enrichment analysis of identified related targets

Note: (A) biological process. (B) molecular function. (C) cellular component. (D) pathway analysis.

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Figure 4. The effect of palmitic acid on cell viability of L02 and HepG2 cells

Note: L02 and HepG2 cells were treated with palmitic acid (200 μmol·L^-1, 250 μmol·L^-1 and 300 μmol·L^-1, respectively.) for 24 h. (A) L02 cell viability was detected by MTT assay (n=6). (B) HepG2 cell viability was detected by MTT assay (n=6).

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Figure 5. The effect of ELE on cell viability of L02 and HepG2 cells

Note: L02 and HepG2 cells were treated with ELE (0.25 mg·mL^-1, 0.5 mg·mL^-1 and 1 mg·mL^-1, respectively.) for 24 h. (A) L02 cell viability was detected by MTT assay. (B) HepG2 cell viability was detected by MTT assay. x±s, n=6.

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Figure 6. ELE alleviates lipid accumulation in PA-treated L02 and HepG2 cells

Note: L02 and HepG2 cells were pretreated with 200 μmol·L^-1 PA for 24 h and treated with 0.25 mg·mL^-1, 0.5 mg·mL^-1, and 1 mg·mL^-1 ELE for an additional 24 h. (A) Oil red O staining images of L02 cells (magnification 200×). Photographs are representative images of three independent experiments. (B) Quantitative oil red O contents measured at 510 nm. (C~D) Intracellular TG, TC levels measured using commercial kit. (n=6). (E) Oil red O staining images of HepG2 cells (magnification 200×). Photographs are representative images of three independent experiments. (F) Quantitative Oil red O contents measured at 510 nm. G-H. Intracellular TG, TC levels measured using acommercial kit. x±s, n=6.

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Figure 7. ELE alleviates lipid metabolism by activating AMPKα in PA-treated L02 and HepG2 Cells

Note: L02 and HepG2 cells were pretreated with 200 μmol·L^-1 PA for 24 h and treated with 0.25 mg·mL^-1, 0.5 mg·mL^-1, and 1 mg·mL^-1 ELE for an additional 24 h. (A) The protein expression of P-AMPKα, AMPKα, PPARα, CPT-1A in PA-treated L02 cells (n=3). (B) Densitometric analysis of western blot. (C-D) The gene expression of CPT-1A, PPARα in PA-treated L02 cells (n=3). (E) The protein expression of P-AMPKα, AMPKα, PPARα, CPT-1A in PA-treated HepG2 cells (n=3). (F) Densitometric analysis of western blot in (E). (G-H) Gene expression of CPT-1A, PPARα in PA-treated HepG2 cells (n=3).

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Figure 8. ELE attenuates liver obesity and injury in HFD-fed mice

Note: With the HFD, mice were treated daily with ELE (low dosage group: 1 g·kg^-1, high dosage group: 2 g·kg^-1) or PBS (control group, model group). (A) Macroscopic view of mice liver in different groups (n=3). (B) Effect of ELE on mice liver weight (n=7-8). (C) Effect of ELE on the ratios of liver weight to body weight (n=7-8). (D) The changes of mice body weight (n=7-8). (E) Effect of ELE on belly weight (n=7-8). (F) Effect of ELE on serum ALT (n=7-8). (G) Effect of ELE on serum AST (n=7-8).

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Figure 9. ELE ameliorates hepatic steatosis in HFD-fed mice

Note: With the HFD, mice were treated daily with ELE (low dosage group: 1 g·kg^-1, high dosage group: 2 g·kg^-1) or PBS (control group, model group). (A) Oil red O staining of liver tissue (magnification 400×) (n=3). (B-C) Liver TG, TC levels measured using a commercial kit (n=7-8). (D) The level of fast blood glucose (n=7-8). (E) The level of oral glucose tolerance (n=7-8). (F-H) Serum TG, TC, LDL-C levels measured using a commercial kit (n=7-8).

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Figure 10. ELE alleviates liver lipid metabolism by activating AMPKα in HFD-fed mice

Note: With the HFD, mice were treated daily with ELE (low dosage group: 1 g·kg^-1, high dosage group: 2 g·kg^-1) or PBS (control group, model group). (A) Protein expression of P-AMPKα, AMPKα, PPARα, CPT-1A in HFD-fed mice liver tissue. (B) Densitometric analysis of western blot (n=3). (C-D) The gene expression of CPT-1A, PPARα in HFD-fed mice liver tissue (n=3).

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Table 1. Screening of active componets of Eucommia folium

Mol-ID	Ingredient	Chemical structure	Molecular formula	OB(≥30%)	DL(≥0.18)
MOL000422	Kaempferol		C₁₅H₁₀O₆	41.88	0.24
MOL000098	Quercetin		C₁₅H₁₀O₇	46.43	0.28
MOL000006	Luteolin		C₁₅H₁₀O₆	36.16	0.25
MOL002714	Baicalein		C₁₅H₁₀O₅	33.52	0.21
MOL001668	Geniposidic acid		C₁₆H₂₂O₁₀	19.59	0.41
MOL002813	Aucubin		C₁₅H₂₂O₉	35.56	0.33
MOL001955	Chlorogenic acid		C₁₆H₁₈O₉	11.93	0.33
MOL000497	Licochalcone A		C₂₁H₂₂O₄	40.79	0.29
MOL002928	Oroxylin A		C₁₆H₁₂O₅	41.37	0.23

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Table 2. The main targets of Eucommia folium in the treatment of NAFLD

Gene symbol	Gene name	Degree
MPO	Myeloperoxidase	4
MMP2	Matrix metalloproteinase 2	9
MMP9	Matrix metalloproteinase 9	11
MMP1	Matrix metalloproteinase 1	4
IKBKB	Inhibitor of nuclear factor kappa B kinase beta subunit	10
ELANE	Leukocyte elastase	1
ADORA2A	Adenosine A2a receptor	7
NOS2	Nitric oxide synthase, inducible (by homology)	5
ACE	Angiotensin-converting enzyme	3
TTR	Transthyretin	5
INSR	Insulin receptor	10
IGF1R	Insulin-like growth factor I receptor	6
APP	Beta amyloid A4 protein	9
CFTR	Cystic fibrosis transmembrane conductance regulator	5
FASN	Fatty acid synthase	2
CPT-1A	Carnitine Palmitoyltransferase 1A	3
PPARA	Peroxisome proliferator-activated receptor alpha	3
ADORA1	Adenosine A1 receptor (by homology)	7
NR1H4	Bile acid receptor FXR	1
AKT1	Serine/threonine-protein kinase AKT	13
EGFR	Epidermal growth factor receptor erbB1	12
PIK3CA	PI3-kinase p110-alpha subunit	10
PDGFRB	Platelet-derived growth factor receptor beta	3
VCP	Transitional endoplasmic reticulum ATPase	1
PIK3R1	PIK3R1	14
ESR1	Estrogen receptor alpha	5
TERT	Telomerase reverse transcriptase	4
PTPN11	Protein-tyrosine phosphatase 2C	4
STAT3	Signal transducer and activator of transcription 3	8
MAPK8	c-Jun N-terminal kinase 1	8
CASP8	Caspase-8	6
SIRT1	NAD-dependent deacetylase sirtuin 1	3
F2	Thrombin	4

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Table 3. Information of signaling pathways (10 of 77)

ID	Pathway	Degree	Count	P Value
hsa05200	Pathways in cancer	15	16	6.981 06E-11
hsa05205	Proteoglycans in cancer	10	11	1.781 88E-08
hsa04014	Ras signaling pathway	10	11	5.728 88E-08
hsa04931	Insulin resistance	9	10	1.166 26E-09
hsa04068	Foxo signaling pathway	9	10	8.006 91E-09
hsa04152	AMPK signaling pathway	8	9	7.939 08E-08
hsa05161	Hepatitis B	9	9	2.856 03E-07
hsa04932	Non-alcoholic fatty liver disease (NAFLD)	10	9	3.904 25E-07
hsa05212	Pancreatic cancer	8	8	1.701 12E-08
hsa04066	HIF-1 signaling pathway	7	8	2.626 42E-07

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Table 4. Information of biological process (10 of 132)

ID	Term	Count	P Value
GO: 0043066	negative regulation of apoptotic process	15	8.01E-14
GO: 0048015	phosphatidylinositol-mediated signaling	7	6.39E-08
GO: 0014066	regulation of phosphatidylinositol 3-kinase signaling	6	4.76E-07
GO: 0044267	cellular protein metabolic process	6	3.72E-06
GO: 0050900	leukocyte migration	6	4.38E-06
GO: 0001934	positive regulation of protein phosphorylation	6	5.34E-06
GO: 0010629	negative regulation of gene expression	6	7.73E-06
GO: 0040014	regulation of multicellular organism growth	4	1.91E-05
GO: 0046326	positive regulation of glucose import	4	2.97E-05
GO: 0032355	response to estradiol	5	3.30E-05

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Table 5. Information of molecular function (10 of 42)

ID	Term	Count	P Value
GO: 0005515	protein binding	31	8.612 92E-06
GO: 0042802	identical protein binding	14	9.111 58E-10
GO: 0019899	enzyme binding	9	2.447 57E-07
GO: 0043560	insulin receptor substrate binding	5	4.479 75E-09
GO: 0046934	phosphatidylinositol-4, 5-bisphosphate 3-kinase activity	5	7.042 79E-06
GO: 0043548	phosphatidylinositol 3-kinase binding	4	7.075 77E-06
GO: 0004879	RNA polymerase Ⅱ transcription factor activity, ligand-activated sequence-specific DNA binding	4	5.093 10E-05
GO: 0004175	endopeptidase activity	4	0.000 172 604
GO: 0043559	insulin binding	3	3.92261E-05
GO: 0030235	nitric-oxide synthase regulator activity	3	0.000109417

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Table 6. Information of cellular component (10 of 23)

ID	Term	Count	P Value
GO: 0005886	plasma membrane	19	0.000 112 612
GO: 0005737	cytoplasm	19	0.002 464 041
GO: 0005634	nucleus	18	0.010 172 407
GO: 0005829	cytosol	15	0.001 507 867
GO: 0016020	membrane	12	0.001 559 922
GO: 0005739	mitochondrion	10	0.000 567 226
GO: 0005615	extracellular space	9	0.002 795 184
GO: 0045121	membrane raft	5	0.000 563 769
GO: 0043235	receptor complex	4	0.001 689 032
GO: 0005943	phosphatidylinositol 3-kinase complex, class IA	2	0.003 727 965

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参考文献(32)

[1]	LONARDO A, TARGHER G. NAFLD in the 20's. from epidemiology to pathogenesis and management of nonalcoholic fatty liver disease[J]. Curr Pharm Des, 2020, 26(10): 991-992. doi: 10.2174/138161282610200424091752
[2]	COLLIER J. Non-alcoholic fatty liver disease[J]. Medicine, 2007, 35(2): 86-88. doi: 10.1016/j.mpmed.2006.11.010
[3]	COBBINA E, AKHLAGHI F. Non-alcoholic fatty liver disease (NAFLD) — pathogenesis, classification, and effect on drug metabolizing enzymes and transporters[J]. Drug Metab Rev, 2017, 49(2): 197-211. doi: 10.1080/03602532.2017.1293683
[4]	PAVLIDES M, COBBOLD JFL. Non-alcoholic fatty liver disease[J]. Medicine, 2015, 43(10): 585-589. doi: 10.1016/j.mpmed.2015.07.004
[5]	KEI N. Multidisciplinary pharmacotherapeutic options for nonalcoholic fatty liver disease[J]. Int J Hepatol, 2012, 2012: 950693.
[6]	CALDWELL S. NASH Therapy: Omega 3 supplementation, vitamin E, insulin sensitizers and statin drugs[J]. Clin Mol Hepatol, 2017, 23(2): 103-108. doi: 10.3350/cmh.2017.0103
[7]	VERGANI L. Fatty acids and effects on in vitro and in vivo models of liver steatosis[J]. Curr Med Chem, 2019, 26(19): 3439-3456. doi: 10.2174/0929867324666170518101334
[8]	STEFANOVIC-RACIC M, PERDOMO G, MANTELL BS, et al. A moderate increase in carnitine palmitoyltransferase 1a activity is sufficient to substantially reduce hepatic triglyceride levels[J]. Am J Physiol Endocrinol Metab, 2008, 294(5): E969-E977. doi: 10.1152/ajpendo.00497.2007
[9]	DUVAL C, MULLER M, KERSTEN S. PPARα and dyslipidemia[J]. Biochim Biophys Acta BBA Mol Cell Biol Lipids, 2007, 1771(8): 961-971.
[10]	MONTAGNER A, POLIZZI A, FOUCHE E, et al. Liver PPARα is crucial for whole-body fatty acid homeostasis and is protective against NAFLD[J]. Gut, 2016, 65(7): 1202-1214. doi: 10.1136/gutjnl-2015-310798
[11]	JUNG YH, AGRAWAL GK, RAKWAL R, et al. Functional characterization of OsRacB GTPase — a potentially negative regulator of basal disease resistance in rice[J]. Plant Physiol Biochem, 2006, 44(1): 68-77. doi: 10.1016/j.plaphy.2005.12.001
[12]	LEE GH, LEE HY, PARK SA, et al. Eucommia ulmoides leaf extract ameliorates steatosis induced by high-fat diet in rats by increasing lysosomal function[J]. Nutrients, 2019, 11(2): 426. doi: 10.3390/nu11020426
[13]	LEE HY, LEE GH, YOON Y, et al. Rhus verniciflua and Eucommia ulmoides protects against high-fat diet-induced hepatic steatosis by enhancing anti-oxidation and AMPK activation[J]. Am J Chin Med, 2019, 47(6): 1253-1270. doi: 10.1142/S0192415X19500642
[14]	HAO DC, XIAO PG. Network pharmacology: A Rosetta Stone for traditional Chinese medicine[J]. Drug Dev Res, 2014, 75(5): 299-312. doi: 10.1002/ddr.21214
[15]	RU JL, LI P, WANG JN, et al. TCMSP: A database of systems pharmacology for drug discovery from herbal medicines[J]. J Cheminform, 2014, 6: 13. doi: 10.1186/1758-2946-6-13
[16]	KIM S. Getting the most out of PubChem for virtual screening[J]. Expert Opin Drug Discov, 2016, 11(9): 843-855. doi: 10.1080/17460441.2016.1216967
[17]	SAFRAN M, CHALIFA-CASPI V, SHMUELI O, et al. Human gene-centric databases at the Weizmann institute of science: GeneCards, UDB, CroW 21 and HORDE[J]. Nucleic Acids Res, 2003, 31(1): 142-146. doi: 10.1093/nar/gkg050
[18]	SZKLARCZYK D, FRANCESCHINI A, WYDER S, et al. STRING v10: Protein-protein interaction networks, integrated over the tree of life[J]. Nucleic Acids Res, 2014, 43(D1): D447-D452.
[19]	HUANG DW, SHERMAN BT, TAN QN, et al. DAVID Bioinformatics Resources: Expanded annotation database and novel algorithms to better extract biology from large gene lists[J]. Nucleic Acids Res, 2007, 35(suppl_2): W169-W175. doi: 10.1093/nar/gkm415
[20]	LIU C, GUO FF, XIAO JP, et al. Research progress on chemical constituents and pharmacological effects of different parts of Eucommia ulmoides[J]. China J Chin Mater Med, 2020, 45(3): 497-512.
[21]	HAO S, XIAO Y, LIN Y, et al. Chlorogenic acid-enriched extract from Eucommia ulmoides leaves inhibits hepatic lipid accumulation through regulation of cholesterol metabolism in HepG2 cells[J]. Pharm Biol, 2016, 54(2): 251-259. doi: 10.3109/13880209.2015.1029054
[22]	ABDELMALEK MF, DIEHL AM. Nonalcoholic fatty liver disease as a complication of insulin resistance[J]. Med Clin N Am, 2007, 91(6): 1125-1149. doi: 10.1016/j.mcna.2007.06.001
[23]	HERNANDEZ-RODAS MC, VALENZUELA R, VIDELA LA. Relevant aspects of nutritional and dietary interventions in non-alcoholic fatty liver disease[J]. Int J Mol Sci, 2015, 16(10): 25168-25198. doi: 10.3390/ijms161025168
[24]	YU JH, XIAO ZC, ZHAO RZ, et al. Paeoniflorin suppressed IL-22 via p38 MAPK pathway and exerts anti-psoriatic effect[J]. Life Sci, 2017, 180: 17-22. doi: 10.1016/j.lfs.2017.04.019
[25]	HOPKINS AL. Network pharmacology: The next paradigm in drug discovery[J]. Nat Chem Biol, 2008, 4(11): 682-690. doi: 10.1038/nchembio.118
[26]	LAU JKC, ZHANG X, YU J. Animal models of non-alcoholic fatty liver disease: Current perspectives and recent advances[J]. J Pathol, 2017, 241(1): 36-44. doi: 10.1002/path.4829
[27]	WANG CH, TAO QM, WANG XH, et al. Impact of high-fat diet on liver genes expression profiles in mice model of nonalcoholic fatty liver disease[J]. Environ Toxicol Pharmacol, 2016, 45: 52-62. doi: 10.1016/j.etap.2016.05.014
[28]	SUN X, DUAN XP, WANG CY, et al. Protective effects of glycyrrhizic acid against non-alcoholic fatty liver disease in mice[J]. Eur J Pharmacol, 2017, 806: 75-82.
[29]	MI Y, TAN DH, HE Y, et al. Melatonin modulates lipid metabolism in HepG2 cells cultured in high concentrations of oleic acid: AMPK pathway activation may play an important role[J]. Cell Biochem Biophys, 2018, 76(4): 463-470.
[30]	FANG K, WU F, CHEN G, et al. Diosgenin ameliorates palmitic acid-induced lipid accumulation via AMPK/ACC/CPT-1A and SREBP-1c/FAS signaling pathways in LO2 cells[J]. BMC Complement Altern Med, 2019, 19(1): 255.
[31]	MOODY L, XU GB, CHEN H, et al. Epigenetic regulation of carnitine palmitoyltransferase 1 (Cpt1a) by high fat diet[J]. Biochim Biophys Acta BBA Gene Regul Mech, 2019, 1862(2): 141-152.
[32]	PAWLAK M, LEFEBVRE P, STAELS B. Molecular mechanism of PPARα action and its impact on lipid metabolism, inflammation and fibrosis in non-alcoholic fatty liver disease[J]. J Hepatol, 2015, 62(3): 720-733.