Aged garlic extract enhances the production of β‑defensin 4 via activation of the Wnt/β‑catenin pathway in mouse gingiva

Periodontal disease is recognized as a chronic multifactorial inflammatory condition initiated by dysbiosis within subgingival plaque biofilms. Antimicrobial peptides exhibit a wide spectrum of antimicrobial action, and thus, provide one of the first lines of host defense against oral pathogens. Aged garlic extract (AGE) is effective for preventing the progression of periodontal disease. The present study examined whether AGE affects the production of antimicrobial peptides in mouse gingiva. Reverse transcription-quantitative PCR analysis demonstrated that oral administration of AGE in mice increased the mRNA level of <i>Defb4</i> in gingival tissue, while the levels of <i>Defb1</i>, <i>Defb14</i> and <i>Cramp</i> remained unaffected. AGE also upregulated the protein levels of β-defensin 4. To explore the underlying mechanism of the increased β-defensin 4 production induced by AGE, a comprehensive phosphoproteomic analysis in gingival tissues was performed. Proteomic profiling revealed activation of the canonical Wnt/β-catenin pathway in gingiva of mice treated with AGE. Treatment of mouse gingival epithelial GE1 cells with AGE resulted in an increase of β-defensin 4 in the culture medium. In support of proteomics experiments, LF3, a specific inhibitor of Wnt/β-catenin signaling, suppressed the AGE-induced production of β-defensin 4. In addition, β-catenin protein was found to accumulate within the nucleus in cells treated with AGE. In conclusion, the present findings suggested that AGE enhanced the production of β-defensin 4 in mouse gingiva through the canonical Wnt signal transduction pathway.