Objective  To investigate the potential mechanism of puerarin in the treatment of oral submucous fibrosis (OSF) using an integrated strategy combining network pharmacology, molecular docking, molecular dynamics simulation and cellular experiments.

Methods  Putative targets of puerarin were retrieved from the TCMSP, Comparative Toxicogenomics Database and PharmMapper databases. OSF-related targets were collected from GeneCards, DisGeNet and OMIM databases. The overlapping targets between puerarin and OSF were identified, and a protein-protein interaction (PPI) network was constructed to screen for core targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on these core targets. Molecular docking was carried out using AutoDock 4.2.6 software, and the top-ranked complexes were subjected to molecular dynamics simulation using GROMACS 2020.6 software to validate binding stability. After assessing the effects of different puerarin concentrations on HaCaT cell viability, the cells were divided into five groups: blank control, model, low-dose puerarin, medium-dose puerarin and high-dose puerarin groups. The mRNA and protein expression levels of TNF, MMP2, EGFR and MMP9 were detected in each group.

Results  A total of 400 puerarin targets and 969 OSF-related targets were screened, yielding 81 overlapping targets. GO and KEGG analyses indicated that the potential mechanisms primarily involve the positive regulation of cell population proliferation, the extracellular space, and the AGE-RAGE signaling pathway. Molecular docking and molecular dynamics simulation demonstrated strong binding affinity of puerarin to core targets like TNF, MMP2, EGFR and MMP9, with stable conformational binding and persistent hydrogen bond interactions. In vitro experiments confirmed that compared to the blank control group, the mRNA and protein expression levels of TNF, MMP2, EGFR and MMP9 were significantly upregulated in the model group (P<0.01). Puerarin treatment significantly reversed these increases in all dose groups compared to the model group (P<0.05).

Conclusion  The molecular mechanism by which puerarin treated OSF was potentially associated with the suppression of key factors such as TNF, MMP2, EGFR and MMP9. This involved the inhibition of inflammatory responses, regulation of extracellular matrix degradation, and modulation of cell proliferation, differentiation, and epithelial-mesenchymal transition.

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