Effects of low-level laser irradiation on osteoclastogenesis in prostaglandin E2-stimulated macrophages.

Low-level laser accelerates bone healing by regulating inflammation. In periodontal tissues, excessive mechanical stress induces alveolar bone resorption by producing excessive prostaglandin E₂ (PGE₂), which is an inflammatory agent that induces osteoclast differentiation. In this study, we aimed to investigate the effect of a low-energy Ga-Al-As diode laser (LLL) on PGE₂-induced osteoclast differentiation of RAW264.7 (RAW) cells. RAW cells were stimulated with 10^- 6 M PGE₂ and irradiated with 810 nm LLL at 3.0 mW/cm² for 10 min. After LLL stimulation, the cells were cultured for five days and subjected to tartrate-resistant acid phosphatase staining. Expression levels of the osteoclastogenesis-inducing factors, receptor activator of nuclear factor-κB ligand and nuclear factor of activated T cells 1 (NFATc1), were examined 24 and 48 h after PGE₂ stimulation and LLL irradiation. Extracellular ATP concentration was determined 0, 1, 5, 10, and 20 min after PGE₂ stimulation and LLL irradiation. Additionally, intracellular calcium concentration was measured as the fluorescence intensity of the cultured cells over time (20 s/scan) after 10 min of LLL irradiation. To investigate the nuclear translocation of NFATc1, the cells were fixed after 1 h of PGE₂ stimulation and LLL irradiation and subjected to immunofluorescence analysis. The same experiments were performed using the P2 × 4 receptor (ATP-gated channel) antagonist, 5-BDBD. Small osteoclasts were observed in the LLL irradiation group. Receptor activator of nuclear factor-κB ligand and NFATc1 mRNA levels were not significantly different between the LLL-irradiated and non-irradiated groups. Extracellular ATP release and intracellular Ca²⁺ levels were increased by PGE₂ stimulation but decreased by LLL irradiation and 5-BDBD treatment. Nuclear NFATc1 levels were also increased by PGE₂ stimulation, but this effect was reversed by LLL irradiation and 5-BDBD treatment. Overall, our results suggest that LLL irradiation inhibits PGE₂-induced osteoclast differentiation by inhibiting Ca²⁺-NFATc1 signaling via extracellular ATP release.