Involvement of the MAPK8-Caspase‑3 Apoptotic Pathway in Arecoline‑Associated Periodontitis

Aims Arecoline, a major betel nut-derived alkaloid associated with betel nut chewing, is linked to increased periodontitis severity, but the molecular mechanisms by which it contributes to periodontitis progression remain unclear. Materials and methods Arecoline- and periodontitis-associated targets were pooled within each category, deduplicated and intersected to construct a compound-target-disease network. Candidate targets were prioritised through PPI topology analysis and a multi‑pipeline machine‑learning workflow, followed by exploratory molecular docking and a 100‑ns molecular dynamics simulation to assess structural plausibility. The correlation between prioritised targets and immune‑cell infiltration was further examined in periodontitis datasets. In vitro, human periodontal ligament cells (PDLCs) were treated with arecoline to evaluate oxidative stress, mitochondrial function, and cytotoxicity, while mRNA and protein expression of core targets were measured by qPCR and Western blotting. Results From 4130 periodontitis‑related and 129 arecoline‑associated targets, 71 common genes were identified. PPI network analysis and ensemble machine learning jointly prioritised caspase‑3 and MAPK8 as candidate targets. Molecular docking yielded a score of –4.8 kcal/mol for both complexes, with single‑trajectory MM‑PBSA estimates of –1.78 ± 2.11 kcal/mol (caspase‑3–arecoline) and –15.80 ± 1.87 kcal/mol (MAPK8–arecoline). Immune infiltration analysis of GSE10334 further revealed correlations between caspase‑3/MAPK8 expression and multiple immune‑cell subsets. In cultured PDLCs, arecoline induced oxidative stress, mitochondrial depolarisation, cytotoxicity and apoptosis, accompanied by upregulation of caspase‑3/MAPK8 mRNA and caspase‑3/JNK protein, alongside increased JNK phosphorylation and caspase‑3 cleavage. Functionally, pharmacological inhibition of JNK with SP600125 significantly attenuated arecoline‑induced apoptosis, supporting a functional association between JNK signalling and apoptosis under these in vitro conditions. Conclusions Our findings suggest that the MAPK8-caspase‑3 pathway is associated with arecoline‑induced apoptosis in periodontal ligament cells. Clinical relevance This study identifies the MAPK8-caspase‑3 pathway as a candidate mechanism associated with PDLCs apoptosis after arecoline exposure, warranting further validation in vivo and in clinical settings.