## 3 Results
### DFPs Suppress RANKL-Induced Key Osteoclastic Marker Expression  
Based on the potent inhibitory effect of DFPs on RANKL-induced osteoclast precursors, we next elucidated their effects on the mRNA and protein levels of key transcription factors and osteoclast-specific genes. DFP treatment (100 \(\upmu\)g/mL) reduced the NFATc1 and c-Fos mRNA and protein levels during osteoclast differentiation (Figure 4A). Furthermore, downregulation of these key transcription factors by DFPs inhibited the expression of DC-STAMP, Atp6v0d2, and cathepsin K genes (Figure 4A). These results suggest that DFPs suppress early-stage osteoclastingenesis by downregulating osteoclast-specific transcription factors. \begin{table}
\begin{tabular}{c c} \hline \hline
**Parameters** & **Content**1 ** \\ \hline Chemical composition (\%) & \\ Neutral sugar & 65.88 \(\pm\) 2.04 \\ Uronic acid & 23.16 \(\pm\) 0.63 \\
2-Keto-3-deoxy-mannooctanoic acid & 0.35 \(\pm\) 0.01 \\ Protein & 4.47 \(\pm\) 0.34 \\ \hline Component sugar (Mol\(\%\)2) & \\ Arabinose & 15.56 \(\pm\) 0.78 \\ Fucose & 3.73 \(\pm\) 0.11 \\ Galactose & 23.75 \(\pm\) 0.51 \\ Glucose & 14.97 \(\pm\) 0.25 \\ Mannose & 12.93 \(\pm\) 2.02 \\ Rhamnose & 5.18 \(\pm\) 0.13 \\ Ribose & 1.83 \(\pm\) 0.13 \\ Xylose & 3.76 \(\pm\) 0.12 \\ Galacturonic acid & 3.45 \(\pm\) 0.47 \\ Glucuronic acid & 14.84 \(\pm\) 0.25 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Chemical composition of polysaccharides derived from _Drynaria fortunei_. Figure 3: Inhibitory effects of DFPs subjected to ultrafiltration (10–1000 kDa) on osteoclast differentiation of receptor activator of nuclear factor-\(\kappa\)B ligand (RANKL)-stimulated BMDMs and cell viability. The effects of 0–200 ug/mL DFPs were analyzed by measuring the TRAP activity in RANKL-induced BMDMs. Data are presented as mean \(\pm\) SEM. **_p_ < 0.01 compared to the control (one-way ANOVA). Figure 2: Inhibitory effects of DFPs on osteoclast differentiation of receptor activator of nuclear factor-\(\kappa\)B ligand (RANKL)-stimulated bone-marrow-derived macrophages (BMDMs). **(A)** Inhibitory effects of DFPs (0–200 ug/mL) on osteoclast differentiation, determined using tartrate-resistant acid phosphatase (TRAP) staining (100\(\times\) magnification). **(B)** Quantification of TRAP activity and cell viability. Data are presented as mean \(\pm\) SEM. *_p_ < 0.05, **_p_ < 0.01 compared to the control (one-way analysis of variance [ANOVA] with [PERSON]’s post hoc test). To further examine the effect of DFPs on the differentiation mechanism of osteoclasts, BMDMs were pretreated with DFPs and then treated with RANKL each day (0, 1, 2, and 3 days) to determine the levels of specific proteins. Protein levels of c-Fos and NFATc1 were significantly reduced during osteoclast differentiation in the DFP-treated group (100 ug/mL) (Figure 4B), which was similar to the mRNA level results. Subsequently, BMDMs pretreated with DFPs were treated with RANKL at different time points within 1 h (0, 5, 15, and 30 min) to examine the levels and phosphorylation of mitogen-activated protein kinases (MAPKs) (Figure 4B). Increases in the levels of p38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) were observed in BMDMs of the vehicle group stimulated by RANKL, but no significant differences were observed in the DFP-treated group, except for JNK. In the vehicle group, the degradation of nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor (IxB)-\(\alpha\) was increased, whereas in the DFP-treated group, IxB-\(\alpha\) was inhibited (Figure 4B). Figure 4: Molecular mechanism underlying DFP modulation of osteoclast differentiation of bone-marrow-derived macrophages. (**A**) Inhibitory effects of DFPs on the mRNA levels of key transcription factors and osteoclast-specific genes involved in cell fusion and maturation. (**B**) Inhibitory effects of DFPs on c-Fos and nuclear factor of activated T cells 1 (NFATc1) protein levels. \(\beta\)-Actin was used as the control. Data are presented as mean \(\pm\) SEM.