Oral submucosal fibrosis induced by active components in areca nut: a network pharmacology-based analysis and validation of the mechanism

doi:10.12122/j.issn.1673-4254.2024.05.15

Abstract

Abstract:

Objective To explore the pharmacologically active components in areca nut that induce oral submucosal fibrosis (OSF) and the possible mechanism. Methods The chemical components in areca nut were analyzed using Thermo QE plus liquid chromatography tandem high-resolution mass spectrometer and Compound discover 3.2 data processing software. The chemical activity of the top 20 compounds was analyzed based on Chinese Pharmacopoeia (2015), PubChem, Chemical book, and SciFinder databases. The potential active components, core targets, biological functions and signaling pathways affecting OSF were analyzed by network pharmacology. The targets of OSF were obtained by integrating Genecards and KEGG databases. The compounds acting on the targets were selected from the Systematic Pharmacology Technology Platform of Traditional Chinese Medicine (TCMSP), and the target-compound, compound-TCM, target-compound-TCM network was constructed. Molecular docking was used to analyze the component-target binding. Immunohistochemistry was used to examine the expressions of key proteins in the PI3K-Akt and MAPK pathways in clinical samples of OSF. Results The core intersection target genes between the top 10 active ingredients in areca nut extract and OSF involved mainly the PI3K-Akt and MAPK pathways. In the clinical samples, the expressions of PI3K protein decreased and the expressions p-PI3K, AKT1 and P-Akt all increased significantly in OSF tissue, where increased JNK protein expression and enhanced activity of c-Jun and c-Fos transcriptional factors were also detected. The OSF patients had significantly elevated plasma levels of IL-6 and IL-8 compared with healthy individuals. Conclusion The main active ingredients including arecoline, arecaine, and guvacine are capable of activating the PI3K-Akt and MAPK pathways to promote the expressions of inflammatory mediators IL-6 and IL-8 and induce collagen hyperplasia, thus leading to the occurrence of oral submucosal fibrosis.

Key words: areca nut, oral submucosal fibrosis, network pharmacology, molecular docking

Ruibo LI, Ge GAO, Xi XIE, Haibin LUO. Oral submucosal fibrosis induced by active components in areca nut: a network pharmacology-based analysis and validation of the mechanism[J]. Journal of Southern Medical University, 2024, 44(5): 930-940.

Figures/Tables 8

References 31

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NO.	Molecule	Molecular formula	QD (ppm)	Mw	RT (min)	Matching score	Peak area	Relative content (%)
1	Arecoline	C₈H₁₃NO₂	-5.75	155.09374	3.758	90.9	68518632210	45.473
2	Quinic acid	C₇H₁₂O₆	-2.88	192.06283	1.539	92.6	22692914797	15.060
3	Epicatechin	C₁₅H₁₄O₆	-3.42	290.07805	18.93	94.7	14748028849	9.788
4	Trigonelline HCl	C₇H₇NO₂	-5.85	137.04688	1.547	87.3	12273994597	8.146
5	Citric acid	C₆H₈O₇	-2.05	192.02661	1.672	85.3	11231967076	7.454
6	Procyanidin B1	C₃₀H₂₆O₁₂	-4.2	578.14	18.324	92.4	7264204843	4.821
7	p-Coumaric acid	C₉H8O₃	-5.71	164.04641	4.772	85	3213100960	2.132
8	(+)-Catechin hydrate	C₁₅H₁₄O₆	-3.73	290.07796	19.914	95.2	857006365.1	0.569
9	Uridine	C₉H₁₂N₂O₆	-2.72	244.06887	4.959	93.6	835052793.2	0.566
10	Isorhamnetin-3-O-nehesperidine	C₂₈H₃₂O₁₆	-3.49	624.16686	22.33	92.2	760984003.5	0.505
11	Sucrose	C₁₂H₂₂O₁₁	-3.26	342.1151	1.575	93.4	721956624	0.479
12	4'-O-Glucosylvitexin	C₂₇H₃₀O₁₅	-4.08	594.15604	20.961	87	608955062.8	0.404
13	Procyanidin B2	C₃₀H₂₈O₁₀	-5.78	578.13909	19.473	90.1	563763212.9	0.374
14	Rosarin	C₂₀H₂₈O₁₀	-2.75	428.16707	22.833	79.7	480609598	0319
15	Isoguanosine	C₁₀H₁₃N₅O₅	-2.41	283.09099	10.894	83.8	44491969.7	0.295
16	Nicotinic acid	C₆H₅NO₂	-4.82	123.03143	2.533	78	432658200.9	0.287
17	5-Hydroxymethylfurfural	C₆H₆O₃	-5.63	126.03098	1.912	73.9	431787858.3	0.287
18	2-Pyrrolidinecarboxylic acid	C₅H₉NO₂	-5.99	115.06264	1.547	74.7	397002912.3	0.263
19	L-Tyrosine	C₉H₁₁NO₃	-2.26	181.07348	4.815	88.5	373792777.1	0.248
20	Benzoic acid	C₇H₆O₂	-6.16	122.03603	18.932	88.4	277514777.6	0.184

NO.	Molecule	Molecular formula	QD (ppm)	Mw	RT (min)	Matching score	Peak area	Relative content (%)
1	Arecoline	C₈H₁₃NO₂	-5.75	155.09374	3.758	90.9	68518632210	45.473
2	Quinic acid	C₇H₁₂O₆	-2.88	192.06283	1.539	92.6	22692914797	15.060
3	Epicatechin	C₁₅H₁₄O₆	-3.42	290.07805	18.93	94.7	14748028849	9.788
4	Trigonelline HCl	C₇H₇NO₂	-5.85	137.04688	1.547	87.3	12273994597	8.146
5	Citric acid	C₆H₈O₇	-2.05	192.02661	1.672	85.3	11231967076	7.454
6	Procyanidin B1	C₃₀H₂₆O₁₂	-4.2	578.14	18.324	92.4	7264204843	4.821
7	p-Coumaric acid	C₉H8O₃	-5.71	164.04641	4.772	85	3213100960	2.132
8	(+)-Catechin hydrate	C₁₅H₁₄O₆	-3.73	290.07796	19.914	95.2	857006365.1	0.569
9	Uridine	C₉H₁₂N₂O₆	-2.72	244.06887	4.959	93.6	835052793.2	0.566
10	Isorhamnetin-3-O-nehesperidine	C₂₈H₃₂O₁₆	-3.49	624.16686	22.33	92.2	760984003.5	0.505
11	Sucrose	C₁₂H₂₂O₁₁	-3.26	342.1151	1.575	93.4	721956624	0.479
12	4'-O-Glucosylvitexin	C₂₇H₃₀O₁₅	-4.08	594.15604	20.961	87	608955062.8	0.404
13	Procyanidin B2	C₃₀H₂₈O₁₀	-5.78	578.13909	19.473	90.1	563763212.9	0.374
14	Rosarin	C₂₀H₂₈O₁₀	-2.75	428.16707	22.833	79.7	480609598	0319
15	Isoguanosine	C₁₀H₁₃N₅O₅	-2.41	283.09099	10.894	83.8	44491969.7	0.295
16	Nicotinic acid	C₆H₅NO₂	-4.82	123.03143	2.533	78	432658200.9	0.287
17	5-Hydroxymethylfurfural	C₆H₆O₃	-5.63	126.03098	1.912	73.9	431787858.3	0.287
18	2-Pyrrolidinecarboxylic acid	C₅H₉NO₂	-5.99	115.06264	1.547	74.7	397002912.3	0.263
19	L-Tyrosine	C₉H₁₁NO₃	-2.26	181.07348	4.815	88.5	373792777.1	0.248
20	Benzoic acid	C₇H₆O₂	-6.16	122.03603	18.932	88.4	277514777.6	0.184

NO.	Molecule ID	Molecule name	OB/%	PubChem ID
A1	MOL005835	Guvacine	98.35	40468028
A2	MOL005833	Arecaine	84.34	6971195
A3	MOL005838	isoguvacine	72.17	7059534
A4	MOL010481	WLN: GR DSWR DG	70.57	6625
A5	MOL000004	Procyanidin B1	67.87	11250133
A6	MOL000676	DBP	64.54	3026
A7	MOL004544	Quinic acid	63.53	37439
A8	MOL001456	citric acid	56.22	19782904
A9	MOL004365	Isomenthol	55.30	19244
A10	MOL000492	Catechin	54.83	9064
A11	MOL002095	DEP	52.19	6781
A12	MOL000555	Homoarecoline	52.03	34167
A13	MOL010483	dehydeoacetic acid	52.02	1712264
A14	MOL000635	vanilline	52.00	1183
A15	MOL000073	ent-Epicatechin	48.96	1822932
A16	MOL010485	EPA	45.66	446284
A17	MOL010482	WLN: 6OVR BVO6	43.74	6786
A18	MOL001749	ZINC03860434	43.59	7057921
A19	MOL000131	EIC	41.90	5280450
A20	MOL004454	ODD	41.70	5282800
A21	MOL000041	PHA	41.62	6925665
A22	MOL010492	Arecoline	40.70	2230
A23	MOL002032	DNOP	40.59	8346
A24	MOL002850	butylated hydroxytoluene	40.02	31404
A25	MOL000198	(R)-linalool	39.80	443158
A26	MOL000234	L-Limonen	38.09	439250
A27	MOL001739	zoomaric acid	35.78	445638
A28	MOL010488	10Z-heptadecenoic acid	34.42	5312435
A29	MOL002372	(6Z,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene	33.55	11975273
A30	MOL000675	oleic acid	33.13	445639
A31	MOL010487	Guvacoline	32.67	160492
A32	MOL000301	2-lauroleic acid	31.42	5282729
A33	MOL010489	Resivit	30.84	71629
A34	MOL003505	Panosorb	30.82	643460

NO.	Molecule ID	Molecule name	OB/%	PubChem ID
A1	MOL005835	Guvacine	98.35	40468028
A2	MOL005833	Arecaine	84.34	6971195
A3	MOL005838	isoguvacine	72.17	7059534
A4	MOL010481	WLN: GR DSWR DG	70.57	6625
A5	MOL000004	Procyanidin B1	67.87	11250133
A6	MOL000676	DBP	64.54	3026
A7	MOL004544	Quinic acid	63.53	37439
A8	MOL001456	citric acid	56.22	19782904
A9	MOL004365	Isomenthol	55.30	19244
A10	MOL000492	Catechin	54.83	9064
A11	MOL002095	DEP	52.19	6781
A12	MOL000555	Homoarecoline	52.03	34167
A13	MOL010483	dehydeoacetic acid	52.02	1712264
A14	MOL000635	vanilline	52.00	1183
A15	MOL000073	ent-Epicatechin	48.96	1822932
A16	MOL010485	EPA	45.66	446284
A17	MOL010482	WLN: 6OVR BVO6	43.74	6786
A18	MOL001749	ZINC03860434	43.59	7057921
A19	MOL000131	EIC	41.90	5280450
A20	MOL004454	ODD	41.70	5282800
A21	MOL000041	PHA	41.62	6925665
A22	MOL010492	Arecoline	40.70	2230
A23	MOL002032	DNOP	40.59	8346
A24	MOL002850	butylated hydroxytoluene	40.02	31404
A25	MOL000198	(R)-linalool	39.80	443158
A26	MOL000234	L-Limonen	38.09	439250
A27	MOL001739	zoomaric acid	35.78	445638
A28	MOL010488	10Z-heptadecenoic acid	34.42	5312435
A29	MOL002372	(6Z,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene	33.55	11975273
A30	MOL000675	oleic acid	33.13	445639
A31	MOL010487	Guvacoline	32.67	160492
A32	MOL000301	2-lauroleic acid	31.42	5282729
A33	MOL010489	Resivit	30.84	71629
A34	MOL003505	Panosorb	30.82	643460

Main active ingredients		Binding energy with target (kcal/mol)
Molecule name	PubChem ID	AKT1 (4GV1)	INS (1TYL)	EGF (1NQL)	EGFR (3W2S)	VEGFA (4KZN)	TP53 (3Q01)	MAPK3 (4QTB)	MYC (5I4Z)
Guvacine	40468028	-4.35	-4.16	0.00	-4.41	-3.82	0.00	0.00	-4.17
Arecaine	6971195	-4.25	-4.43	0.00	-4.27	-3.74	0.00	0.00	-4.46
isoguvacine	7059534	-4.38	-4.06	0.00	-4.01	-3.78	0.00	0.00	-3.85
WLN: GR DSWR DG	6625	-5.66	-4.89	-5.32	-4.73	-4.38	-4.76	-5.71	-4.54
Procyanidin B1	11250133	-6.86	-6.42	-6.70	-6.68	-5.45	-6.02	-6.60	-5.94
Quinic acid	37439	-4.78	-4.45	0.00	-5.28	-4.11	0.00	0.00	-4.14
ent-Epicatechin	1822932	-6.54	-5.61	-5.72	-6.15	-4.91	-5.51	-6.54	-5.16
Arecoline	2230	-5.28	-4.69	0.00	0.00	-4.47	-4.03	0.00	-4.48