Ozone ultrafine bubble water induces the cellular signaling involved in oxidative stress responses in human periodontal ligament fibroblasts

ABSTRACT Periodontitis is a chronic inflammatory disease caused by oral microorganisms in the subgingival biofilm. Stable aqueous ozone ultrafine bubble water (OUFBW) has recently begun to be used as an antiseptic in the treatment of periodontitis. The effectiveness of OUFBW is thought to depend on the bactericidal actions of dissolved ozone exerted via its oxidizing effect. On the other hand, the effects of ozone on the periodontal tissues are largely unknown. In this paper we examined the cellular responses after OUFBW treatment. Human primary periodontal ligament fibroblasts (hPDLFs) or Ca9-22 human gingival epithelial cells were treated with OUFBW or UV-inactivated OUFBW. The production of reactive oxygen species (ROS), the activation of mitogen-activated protein kinase (MAPK) and the nuclear factor-kappa B (NF-κB) activation were analyzed. The transcript profiles of hPDLFs after OUFBW treatment were also analyzed by RNA sequencing (RNA-seq). Our results showed that OUFBW induces oxidative stress by generating ROS, which, in turn, activated the MAPK pathway. OUFBW triggered activation of c-Fos, a major component of the transcription factor activator protein 1 (AP-1), and also nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), which possessed a high sensitivity to oxidative stress. The results of RNA-seq analysis revealed that the numerous genes involved in oxidative stress responses or MAPK signaling pathway were up-regulated after OUFBW treatment. Investigation of the signaling pathways activated by OUFBW highlights another aspect of the biological roles of OUFBW, in addition to its bactericidal activity, in the treatment of periodontitis. GRAPHICAL ABSTRAC