Abstract: Objective: To investigate the role and mechanism of nod-like receptor protein 3 (NLRP3) inflammasome in primary human gingival fibroblasts (hGFs). Methods: NLRP3 silencing RNA (siRNA) transfection was performed to silence NLRP3 in hGFs to induce inflammation by co-culturing with P.gingivalis (Pg) and intervened with 1 μmol/L BMS-986299, an NLRP3 agonist. Flow cytometry was used to detect apoptosis in each group. Enzyme linked immunosorbent assay (ELISA) was used to measure interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Western blot was adopted to evaluate NLRP3, cysteinyl aspartate specific proteinase-1 precursors (pro-Caspase-1), cysteinyl aspartate specific proteinase-1 (Caspase-1), apoptosis associated speck-like protein containing a caspase activating recruitment domain (ASC), Interleukin-1β precursors (pro-IL-1β), nuclear factor kappa-B phosphorylation (p-NF-κB)/nuclear factor kappa-B (NF-κB), and Gasdermins D (GSDMD). And immunofluorescence was performed to detect the NLRP3 localization. Results: Flow cytometry confirmed that NLRP3 silencing suppressed cell apoptosis. ELISA analysis demonstrated that NLRP3 silencing successfully reduced the expression of Pg-induced inflammation factors. Western blot and immunofluorescence revealed that the blocking of NLRP3 significantly reduced NLRP3 inflammatory body activation, inhibited pro-IL-1β expression, NF-κB phosphorylation, and the activity of GSDMD-mediated pyroptosis. However, this beneficial effect was partially restored with the administration of BMS-98629. Conclusion: NLRP3 activates NLRP3 inflammatory bodies and inflammation-related pathways and promotes pyrolysis in hGFs.

Key words: gingival fibroblasts, NLRP3 inflammasome, P.gingivalis, peri-implantitis