Development of arecoline-induced mouse model for oral submucous fibrosis: An in vitro and in vivo study

Objective: To establish an arecoline-induced preclinical model of oral submucous fibrosis (OSMF) and to investigate associated fibrotic and inflammatory mechanisms using in vitro and in vivo approaches. Design: In vitro, hTERT and HaCaT cells were exposed to varying concentrations of arecoline to assess fibrotic markers, inflammatory cytokines, and reactive oxygen species (ROS), quantified by ELISA and qRT-PCR analysis. In vivo, Swiss albino mice were administered with arecoline (1 mg/kg for 25 days or 2 mg/kg for 15 days) via the sub-buccal route. OSMF induction was evaluated by body weight, mouth opening diameter estimation, cytokine expression, histopathological and immunohistochemical analysis, confirming fibrotic and inflammatory activation, characteristic of OSMF. Results: In vitro, arecoline exposure induced dose-dependent increase in intracellular ROS generation, with elevated cytokines (IL-6, IL-13, TNF-α) and fibrosis-related genes (TGF-β1, Col1α2, Col3α1), and downregulated anti-fibrotic (IFN-γ) marker, indicating role of inflammatory activation and oxidative stress in inducing fibrosis. In vivo, arecoline-induced OSMF-like condition in mice showed reduction in body weight and mouth opening diameter. Histopathological findings also revealed epithelial atrophy, subepithelial hyalinization, and collagen accumulation in buccal and tongue tissues in arecoline-treated mice. Immunohistochemical analysis further confirmed overexpression of TGF-β1 and NF-κB in arecoline-treated mice, along with upregulation of TGF-β1, inflammatory cytokines (IL-6, IL-1β, TNF-α), and decline in IFN-γ, as determined by ELISA. Conclusion: The study demonstrates that arecoline induces OSMF-like condition in both cultured cells and mice, providing practical model for preliminary evaluation of potential anti-OSMF therapeutic agents by assessing fibrotic, inflammatory, oxidative stress markers, key molecular regulators of OSMF.