## 4 Discussion  
Previous studies have shown that _D. fortunei_ is effective in promoting calcium absorption during fracture and preventing pain and hyperlipidemia, as well as being useful for sedation [32; 33]. The mechanism underlying the effect of DFPs on osteoclast differentiation of RANKL-induced BMDMs appears to be the same as that reported by [PERSON] et al. (2012). However, in this study, we found that DFPs significantly inhibited osteoclast differentiation. BMDMs treated for 3 days with DFPs from RANKL-induced cells displayed increased TRAP activity, which is important for osteoclast differentiation. Osteoclast formation was suppressed in a dose-dependent manner by 1-200 \(\upmu\)g/mL DFPs, and cytotoxicity was not observed. These findings were also obtained for polysaccharides of different molecular weights (10, 30, 100, 300, and 1000 kDa). These results imply that DFPs suppress osteoclast differentiation, regardless of their size or molecular weight. Furthermore, our findings are similar to those of [PERSON] et al., in which DFPs significantly increased bone mineral density and bone mineral content index in a rat model of postmenopausal osteoporosis, compared with those of control rats, thereby exhibiting anti-osteoporotic effects [26]. But there were no significant differences in most assays between animals treated with different doses of it. Mechanistic studies, including quantifications of bone calcium, magnesium, and phosphate, are needed to confirm and elucidate these differences. RANKL-activated NF-kB is a key transcription factor in osteoclast differentiation [34]. IxkB kinases phosphorylate IxkB, which initially inhibits NF-kB expression, and IxkB is subsequently degraded by proteolysis through the ubiquitin-proteosome pathway, allowing the translocation of NF-kB to the nucleus [35]. Therefore, the reduced IxkB expression is an important factor in the RANKL-induced differentiation of osteoclasts. We also analyzed the DFP-induced downregulation of the levels of main transcription factors, as well as osteoclast-specific factors (at the mRNA and protein levels). NFATc1 and c-Fos mRNA and protein levels were significantly reduced in BMDMs treated with DFPs (100 \(\upmu\)g/mL) to induce differentiation into osteoclasts. Additionally, DFP-induced suppression of c-Fos and NFATc1 expression downregulated the transcription and translation of osteoclast-specific genes, including those encoding Atp6v0d2, DC-STAMP, and cathepsin K. IxkB degradation was promoted in RANKL-induced BMDMs (vehicle group) but was suppressed in BMDMs treated with DFPs. These results demonstrated that the mechanisms through which DFPs suppress osteoclast differentiation rely on the NF-kB signaling pathway and involve RANKL. Moreover, activating MAPKs is important in RANKL-induced osteoclast differentiation. IxkB reduces NF-kB activity, affecting osteoclast differentiation. DFP treatment suppresses the activation of NFATc1, an important transcription factor in osteoclast differentiation, by suppressing JNK activation [36]. These results support the hypothesis that DFP-induced suppression of NF-kB activation reduces the expression of c-Fos and NFATc1 during RANKL-induced osteoclast differentiation, preventing the differentiation of TRAP-positive osteoclasts. Overall, this study confirms that DFPs exert anti-osteoporotic effects; nevertheless, further studies are required to elucidate whether DFPs bind to cognate receptors on the cell surfaces of progenitor cells and inhibit cell signaling pathways that activate NF-kB and MAPK, or whether DFPs freely enter the cell and directly inhibit NF-kB and MAPK activation. Bisphosphonates and parathyroid hormone differentiate osteoclast precursors into osteoclasts by inducing RANKL expression in osteoblasts co-cultured with osteoclast precursors. Among the most common therapeutics for osteoporosis in clinical settings are bisphosphonates, and more recently, medications containing parathyroid hormone, teripartide, and aloloparatide have also been used.