Objective  To explore the mechanism of isoliquiritigenin in the treatment of oral submucosal fibrosis by network pharmacology, molecular docking technology and cell experiments.

Methods  The relevant action targets of isoliquiritigenin were searched in the TCMSP, Comparative Toxicogenomics Database and PharmMapper database; the disease targets of oral submucosal fibrosis were found in the GeneCards, DisGeNet and OMIM databases. The common targets of the two were screened by the Venny website and the protein-protein interaction (PPI) network analysis of these targets was carried out using the STRING database. The common targets were input into the DAVID database for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) bioinformatics analysis. Molecular docking verification was performed on isoliquiritigenin and core targets using AutoDock Vina 1.2.x software. After culturing human immortalized keratinocytes (HaCaT cell line) in vitro, the effect of different concentrations of isoliquiritigenin on the survival rate of HaCaT cells was studied by the CCK-8 method. According to the CCK-8 results, 20 μM, 40 μM and 80 μM isoliquiritigenin concentrations were selected for subsequent experiments. The cells were divided into blank group, model group, low, medium and high dose isoliquiritigenin groups. The mRNA expression levels of MMP9, EGFR, TGFB1, TNF and MAPK3 in each group of cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR).

Results  A total of 391 potential action targets of isoliquiritigenin, 969 related targets of oral submucous fibrosis, and 65 intersection targets of the two were screened out. The top-ranked active components of the isoliquiritigenin action targets in terms of degree value included IL6, MMP9, TGFB1, TNF, AKT1, etc. GO analysis indicated that the biological processes (BP) mainly involved the insulin-like growth factor receptor signaling pathway, the cellular response to reactive oxygen species, negative regulation of the apoptotic process, the cellular response to cadmium ions, extracellular matrix degradation, etc. The cellular components (CC) mainly involved the cytoplasm, receptor complex, extracellular matrix, mitochondria, focal adhesion, etc. The molecular functions (MF) mainly involved homotypic protein binding, enzyme binding, endopeptidase activity, protein homodimerization activity, protein kinase activity, etc. The KEGG analysis results demonstrated that the treatment of oral submucous fibrosis by isoliquiritigenin was related to pathways in cancer, the FoxO signaling pathway, the HIF-1 signaling pathway, the MAPK signaling pathway, the AGE-RAGE signaling pathway, etc. The molecular docking results showed that isoliquiritigenin had a strong binding ability to targets such as MMP9, EGFR, TGFB1, TNF and MAPK3. The in vitro cell experimental results showed that, compared with the blank group, the mRNA levels of MMP9, EGFR, TGFB1, TNF, and MAPK3 in the model group were significantly increased (P<0.01). Compared with the model group, the mRNA levels of MMP9, EGFR, TGFB1, TNF, and MAPK3 in the low-dose, medium-dose, and high-dose isoliquiritigenin groups were significantly decreased (P<0.01).

Conclusion  The molecular mechanism of isoliquiritigenin in the treatment of oral submucous fibrosis might be related to the inhibition of the expression of cytokines such as MMP9, EGFR, TGFB1, TNF, and MAPK3, involving processes such as cell proliferation and differentiation, extracellular matrix synthesis and deposition, epithelial-mesenchymal transition, the initiation of the inflammatory response and intracellular signal transduction.

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