Methods: We orally infected male C57BL/6 mice with P.g (Porphyromonas gingivalis) at 8 weeks of age to induce periodontitis. MicroCT analysis was performed to confirm alveolar bone resorption. The mice were administered intraperitoneally with pimonidazole which accumulates in hypoxic cells for immunohistochemistry. We also cultured the gingival epithelial cell line (epi 4) in hypoxic condition (5%O2) with or without IL-1β, and measured IL-6 and IL-8 expression by RT-qPCR and ELISA. Then, we investigated hypoxia inducible factor-1α (HIF-1α) activity by Trans AM kit® and examined the possible involvement of HIF-1α in the hypoxia dependent regulation on cytokine production by treating with deferoxamine (HIF-1α activator), chetomine (HIF-1α inhibitor), and HIF-1α siRNA.
Results: MicroCT analysis revealed the alveolar bone resorption in P.g infected mice, compared to control mice. Immunohistochemistry using anti-pimonidazole antibody demonstrated pimonidazole accumulation in the gingival tissue of P.g infected mice, especially, in the gingival epithelium. RT-qPCR and ELISA experiments revealed that IL-1β-induced IL-6 and IL-8 expressions by epi 4 were suppressed in hypoxic condition. Deferoxamine also suppressed the IL-1β-induced IL-6 and IL-8 expressions. On the other hand, the hypoxia dependent suppression was rescued by treating with chetomine or HIF-1α siRNA transfection.
Conclusions: These findings suggest that periodontal diseases induce hypoxia in gingival epithelium, and the hypoxic condition can modulate the local inflammatory responses of gingival epithelial cells in a HIF-1α dependent pathway.