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

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 PAkt 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.

Keywords: areca nut; molecular docking; network pharmacology; oral submucosal fibrosis.

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槟榔成分提取流程图 Fig.1 Flow chart of preparing areca nut extract.

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槟榔成分网络药理学分析 Fig.2 Network pharmacological analysis of the components of areca nut. A: Network pharmacology technology flow chart. B: Areca nut active ingredient-disease target for oral submucous fibrosis (OSF). C: Protein-protein interaction. D: Core target acquisition map. E: Intersection of the core target genes of the active ingredients of areca nut and OSF. F: Areca nut-component-target-OSF target interaction map. G: Effect of areca nut active ingredients on GO enrichment of OSF targets. H: KEGG pathway enrichment analysis of the effects of areca nut active ingredients on OSF targets. I: KEGG pathway secondary classification analysis. J: Molecular docking binding energy heat map.

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槟榔成分分子对接 Fig.3 Molecular docking of areca nut components. A: Chemical structures of 10 areca nut active ingredients. B: Visualization of molecular docking of some active ingredients of areca nut with the core targets.

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临床验证 Fig.4 Clinical validation. A: HE staining, Masson, SR staining (Original magnification: ×100 or ×200). B: Fibronectin immunohistochemistry and H-score semi-quantitative analysis (×100 or ×200; n≥5). C: SR staining and differentiation of COL I and COL III under polarized light (×100). Orange and red fluorescence indicate Col I. Green fluorescence indicates COL III. D: Immunohistochemical staining of PI3K-Akt pathway proteins (×100 or ×200; n≥5). E: Immunohistochemistry and H-score semi-quantitative analysis of PI3K-Akt pathway proteins (n≥5). F: Immunohistochemical staining of MAPK pathway protein (×100, ×200 or ×400; n≥5). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

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临床样本血浆IL-6与IL-8分析 Fig.5 Plasma IL-6 and IL-8 levels in patients with OSF (Normal group: n=6; OSF group: n=9). *P<0.05, ****P<0.0001.