Integrated Identification of Macrophage NF-κB, p38 MAPK and AKT1 Signalling as Candidate Targets of Zerumbone in Experimental Periodontitis

Characterised by progressive alveolar bone loss, periodontitis arises from immune dysregulation and is classified as a chronic inflammatory condition. Zerumbone (Zer) has anti-inflammatory properties; however, its mechanism within the periodontal context remains unclear. We investigated the therapeutic effects and potential molecular targets of Zer by integrating in vivo efficacy assessment (using male C57BL/6 mice), network pharmacology, molecular docking, and molecular dynamics simulations, together with analysis of public single-cell transcriptomic data, with mechanistic validation using RAW 264.7 macrophages stimulated with LPS. In vivo, Zer attenuated bone resorption, preserved collagen integrity, reduced the number of TRAP-positive osteoclasts, and mitigated inflammation without hepatorenal toxicity. Network pharmacology predicted NFKB1, MAPK14, and AKT1 as potential core targets, while IL-17 pathway enrichment suggested the involvement of IL17A; single-cell data localised NFKB1/MAPK14/AKT1 to macrophages and showed that IL17A is predominantly expressed in T cells. In vitro, Zer dose-dependently suppressed LPS-driven phosphorylation of NF-κB p65, p38 MAPK and AKT1, correlating with reduced pro-inflammatory cytokine release and an M1-to-M2 phenotypic shift. In vivo, Zer also reduced gingival IL-17A expression. However, as IL-17A is primarily produced by T cells and our in vitro experiments were conducted exclusively in macrophages, this observation remains correlative and does not establish a direct T-cell-mediated effect. Collectively, these findings suggest that Zer ameliorates periodontitis partly through suppression of macrophage NF-κB and p38 MAPK signalling, with reduced AKT1 phosphorylation observed alongside these effects; however, whether AKT1 causally contributes to these effects remains to be determined. In addition, the potential impact on T-cell-associated IL-17A responses warrants further investigation.