Cytokines Secreted by Arecoline Activate Fibroblasts that Affect the Balance of TH17 and Treg.

Oral submucous fibrosis (OSF) is a chronic progressive oral disease with cancerous tendency. Arecoline plays an important role in the pathogenesis of OSF. Fibroblasts (FBs) are the primary cells involved in the pathogenesis of OSF. There is a change in CD4⁺ IL-17⁺ helper T cells (Th17) and CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (Treg) in OSF patients, but the molecular mechanisms of are not clearly understood. In this work, we studied the molecular mechanisms.

Enzyme digestion was used to extract primary FBs and immunofluorescence was used to identify FBs. Cytotoxic experiment was then performed to determine the effect of arecoline on FBs activity. Enzyme-linked immunosorbent assay (ELISA) was used to detect changes in the amount of cytokines. In addition, we treated human peripheral blood mononuclear cells (PBMC) with the above cytokines and detected their changes. Flow cytometry was used to detect the changes of Th17 and Treg and quantitative- polymerase chain reaction (Q-PCR) was used to detect the changes of RORγt and Foxp3.We have found that the stimulation of arecoline on FBs increased Interleukin-2, Interleukin-6, and Interleukin-21 (IL-2, IL-6, and IL-21) while decreased transforming growth cytokine-β (TGF-β). After the cytokine-containing supernatant was co-cultured with PBMC, the cytometry results showed that Th17 was increased, while Treg was significantly decreased and Q-PCR results showed that RORγt expression was increased and Foxp3 expression was decreased.The arecoline can affect inflammatory cytokines produced by FBs, which then act on immune cells Th17 and Treg, and make them change.