Abstract: Objective: To investigate the formation and potential regulatory mechanisms of diacylglycerol (DG)-induced neutrophil extracellular traps (NETs), and to assess the effect of NETs on the gingival epithelial keratinization and barrier. Methods: Lipidomics data from periodontitis and healthy subjects were obtained from the MTBLS4684 database, normalized and analyzed for differences of DG content between the two groups. Human peripheral blood-derived polymorphonuclear neutrophils (PMNs) were treated with DG, followed by detection of citrullinated histone H3 (CitH3) expression by Western blot, extracellular nucleic acid changes by Sytox green staining, and changes of CitH3 expression and distribution by immunofluorescence. After pretreatment of PMNs with protein kinase C (PKC) inhibitor and then stimulated with DG, NETs formation was detected by Western blot, Sytox green staining and immunofluorescence. Human gingival keratinocytes (HGKs) were stimulated by extracted NETs, and changes in the expression of loricrin (LOR) and claudin-2 (CLDN2) were examined by qRT-PCR and Western blot. The levels of LOR and CLDN2 expression in the gingival epithelium of the healthy and periodontitis groups were detected by immunohistochemistry, and the markers of NETs, CitH3 and myeloperoxidase (MPO), in epithelium were assayed by Western blot. Results: Multiple DG was raised in the gingival epithelium of patients with periodontitis. After stimulation of PMNs by DG, CitH3 protein expression was enhanced (P<0.01), nucleic acids were released into the extracellular zone, and CitH3 fluorescence expression was elevated with its distribution to the extracellular region; the above processes were effectively blocked by the application of PKC inhibitor. After stimulation of HGKs by NETs, the expressions of keratinization marker LOR and barrier marker CLDN2 were decreased at the transcriptional and protein levels (P<0.05). In the periodontitis group, the levels of LOR and CLDN2 were reduced, and the expressions of CitH3 and MPO were increased in the gingival epithelium (P<0.05). Conclusions: DG promotes the formation and accumulation of NETs by activating PKC in PMNs, which in turn exerts a destructive effect on keratinization and barrier of epithelium.

Key words: periodontitis, neutrophil extracellular traps, diacylglycerol, protein kinase C, keratinization barrier