## 4 Discussion  
Most studies of metabolic bone disease and bone healing investigate either activation of osteoblasts or suppression of osteoclasts [38; 39]. However, enhancing or recovering osteocalastic activity and differentiation can also play an important role in bone remodeling [40]. [PERSON] reported that low-level laser irradiation enhanced tooth movement and the formation of osteoclasts on the pressure side in vivo [41]. Likewise, there have been a number of studies demonstrating the effectiveness of LLLT in bone healing, and there are some studies showing that it inhibits the differentiation of osteoclasts, but this is still controversial [19; 35]. The purpose of this study was to investigate the effect of LLLT irradiation on osteoclast differentiation through a cytological approach to osteoclast differentiation using LLLT, which is widely used in bone healing. The RAW 264.7 murine macrophages used in this study have been widely used in osteoclast-related studies due to various advantages, such as easy experimental approach,  
Figure 5: Effect of LLLT irradiation on the MAPK pathway in RAW 264.7 cells. (**A**) Only LLLT (15 Scheme 264.7 cells, and the change pattern of MAPK was confirmed for 15 to 120 min. (**B**) RAW 264.7 cells were pretreated with RANKL (10 ng/mL) and LLLT for 15 s and incubated for 24 h and the change in MAPK was confirmed. Figure 6: Activation effects of the c-Fos and NFATc1 pathways by LLLT irradiation. (**A**) Only LLLT (15 s) was applied to RAW 264.7 cells, and the change pattern of MAPK was confirmed for 15 to 120 min. (**B**) In the presence of RANKL, LLL was irradiated for 5 to 30 s, and after incubation for 24 h, the expression of c-Fos and NFATc1 was confirmed. rapid RANKL-mediated formation, large number of osteoclasts, and bone resorption (Figure 2) [42]. Mature osteoclasts differ from the progenitors of the hematopoietic/macrophage lineage in their cell-to-cell interactions with osteoblasts. RANKL binds to its receptor RANK on the precursor osteoclast and induces NFATc1 expression, which is a key regulator of osteoclast differentiation through signaling pathways. RANK recruits TRAF6 and activates NF-\(\kappa\)B, while MAPKs and c-Fos result in NFATc1 activation (Figure 3) [43]. The functional importance of the NF-\(\kappa\)B, JNK, and p38 MAPK pathways in osteoclastogenesis has been extensively studied [44; 45]. NF-\(\kappa\)B is a dimeric transcription factor. It is composed of p65 (RelA), c-Rel, RelB, NF-\(\kappa\)B1 (p50), and NF-\(\kappa\)B2 (p52) subunits. In vivo studies using p50- and p52-deficient mice suggest a critical role for NF-\(\kappa\)B in osteoclastogenesis [46]. During osteoclast differentiation, the binding of RANKL and RANK activates inhibitors of the 1 kB kinase (IKK) complex in the NF-\(\kappa\)B pathway, inducing 1 kB ubiquitination and proteolysis [47]. These events result in the translocation of the NF-\(\kappa\)B dimer to the nucleus, enhancing transcription of the target gene. [12]. In this study, it was found that p65 was translocated from the cytoplasm to the nucleus in RANKL only treatment, while in the LLLT irradiation group, the expression of p65 increased and the translocation to the nucleus was more pronounced than in the RANKL only treatment (Figure 4). Therefore, we suggest that LLLT stimulates osteoclast formation by promoting NF-\(\kappa\)B activation. The effect of LLLT on ERK expression in osteoclasts is still unknown; however, [PERSON] et al. found that the MAPK/ERK pathway plays a role in the activation of dental pulp cells following low-level diode laser irradiation [48; 49]. In this study, phosphorylation of ERK was maximal at 30 min after LLLT; maximum phosphorylation was also achieved after 15 s of LLLT exposure in the presence of RANKL as compared to either treatment alone. ERK expression was enhanced in osteoclastogenesis under LLLT. Laser irradiation appears to stimulate the expression of ERK, regardless of cell type.