**3. Results**

#### *3.1. E*ff*ect of FST on Cell Viability in RAW 264.7 Cells*

RAW 264.7 cells were treated with various concentrations of FST for 72 h and then 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. Figure 1A shows that FST had no cytotoxicity on the cells at concentrations up to 800 μg/mL but relatively cytotoxic effect was observed in the 1000 μg/mL treatment group as compared with untreated controls. In the presence of 100 ng/mL RANKL or 100 ng/mL osteoprotegerin (OPG), a decoy receptor for RANKL that inhibits osteoclastogenesis [33,34], cell viability was not significantly reduced by FST at concentrations up to 800 ng/mL compared to that of control groups (Figure 1B). Hence, non-toxic concentrations (<800 μg/mL) were used to investigate the effect of FST on RANKL-induced osteoclast differentiation.

**Figure 1.** Effects of fermented sea tangle extract (FST) and receptor of activator of nuclear factor kappa-B ligand (RANKL) on the viability of RAW 264.7 mouse macrophage-like cells. Cells were treated with desired concentrations of FST in the absence (**A**) or presence (**B**) of 100 ng/mL RANKL and/or 100 ng/mL OPG for 72 h. H2O2 was used as a positive control. Cell viabilities were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Relative cell viability is expressed as percentages compared to treatment of naïve control cells. Results are presented as means ± SD of three independent experiments. \* *p* < 0.05 and \*\*\* *p* < 0.005 indicates significant difference compared to the untreated control cells. OPG: osteoprotegerin; +: cells treated the reagent; -: cells untreated the reagent.

#### *3.2. FST Suppresses RANKL-Induced Osteoclastogenesis in RAW 264.7 Cells*

In order to examine the effect of FST on RANKL-induced osteoclastogenesis, RAW 264.7 cells were treated with FST in the presence of different concentrations of RANKL. As shown in Figure 2A, FST treatment markedly inhibited RANKL-induced osteoclast-like morphological changes. TRAP staining demonstrated that FST suppressed cell fusion and the conversion of RAW 264.7 cells into osteoclasts (Figure 2B FST suppressed numbers of TRAP-positive osteoclasts as compared with RANKL treated cells, dose-dependent manner (Figure 2C). These reductions in TRAP-positive osteoclast number were paralleled by the inhibition of TRAP activity (Figure 2D). As expected, RANKL-induced osteoclast differentiation and TRAP activity were completely suppressed in the presence of OPG.

#### *3.3. FST Disrupts RANKL-Induced Formation of F-Actin Rich Adhesive Structures in RAW 264.7 Mouse Macrophage-Like Cells*

Formation of the F-actin rich adhesive structures by osteoclasts is an essential step in bone resorption [35,36]. Figure 3 indicated that staining with FITC-conjugated phalloidin showed RANKL (100 ng/mL) stimulation increased well-defined F-actin sealing rings with a higher intensity ring height. However, the size of rings formed by RANKL-treated cells was remarkably and concentration-dependently reduced in cells co-treated with FST. Furthermore, OPG treatment complementally inhibited the F-actin sealing ring formation in RANKL-stimulated cells.

**Figure 2.** Inhibition of RANKL-stimulated osteoclast differentiation by FST in RAW 264.7 mouse macrophage-like cells. Cells were stimulated with 100 ng/mL RANKL in the presence or absence of FST or 100 ng/mL OPG for 5 days. (**A**) Representative photographs of the morphological changes are presented. (**B**) Cells were fixed and stained for TRAP and examined under an inverted microscope. (**C**) TRAP-positive multinucleated cells were counted to determine osteoclast numbers. (**D**) Supernatants were collected from cells grown under the same conditions and TRAP activities were measured using an ELISA reader. Results are presented as means ± SD of three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 indicates significant difference compared to RANKL-treated cells. RANKL: receptor of activator of nuclear factor kappa-B ligand; FST: fermented sea tangle extract; OPG: osteoprotegerin; TRAP: tartrate-resistance acid phosphatase; +: cells treated the reagent; -: cells untreated the reagent.

**Figure 3.** Suppression of F-actin ring formation by FST in RANKL-induced RAW 264.7 mouse macrophage-like cells. The cells were co-treated with 100 ng/mL RANKL in the presence or absence of FST or 100 ng/mL OPG for 5 days and stained for F-actin rich adhesive structures with fluorescein isothiocyanate (FITC)-phalloidin and <sup>4</sup>,6-diamidino-2-phenylindole (DAPI). The photographs are representative of the morphological changes observed under a fluorescence microscope. RANKL: receptor of activator of nuclear factor kappa-B ligand; FST: fermented sea tangle extract.

#### *3.4. FST Inhibits the RANKL-Induced Nuclear Translocation of NF-*κ*B and I*κ*B*α *Degradation in RAW 264.7 Cells*

Activation of NF-κB through nuclear translocation by RANKL is an essential step for initiation of osteoclast differentiation [6,7]. Therefore, we assessed whether FST affected the activation of NF-κB induced by RANKL. As shown in Figure 4A,B, our immunoblotting results reveal that the expression of NF-κB was markedly increased in the nuclei of RANKL treated cells but the expression of IκBα was reduced in the cytoplasm, which suggested that RANKL stimulated activation of NF-κB. However, FST suppressed the RANKL-mediated degradation of IκBα and the subsequent nuclear accumulation of NF-κB. Furthermore, immunofluorescence studies produced similar results. More specifically, phosphorylated NF-κB p65 was predominantly located in nuclei in RANKL-stimulated cells but not in FST and RANKL co-treated cells (Figure 4C).

**Figure 4.** Effects of FST on the RANKL-induced activation of NF-κB in RAW 264.7 mouse macrophage-like cells. (**A**) After co-treating cells with 100 ng/mL RANKL in the presence or absence of FST for 1 h, nuclear and cytosolic proteins were isolated. The expression of NF-κB and IκB-α were determined by Western blotting. Lamin B and β-actin were used as internal controls for the nuclear and cytosolic fractions, respectively. (**B**) Densitometry quantifications of protein expressions were measured by ImageJ. Statistical analyses were conducted using analysis of variances between groups. \*\*\* *p* < 0.0001 when compared to control. (**C**) Cells grown on gelatin-coated glass coverslips were co-treated with 800 μg/mL FST with or without 100 ng/mL RANKL. Localization of phospho-NF-κ<sup>B</sup> p65 was observed under a fluorescence microscope following staining with anti-phospho-NF-κ<sup>B</sup> p65 antibody (red) and DAPI (nuclear stain; blue). Original magnification ×400. RANKL: receptor of activator of nuclear factor kappa-B ligand; FST: fermented sea tangle extract; IκBα: inhibitory proteins of kappa B, alpha; NF-κB: nuclear factor-kappa B; +: cells treated the reagent; -: cells untreated the reagent.

#### *3.5. FST Down-Regulates RANKL-Induced Osteoclast-Associated Gene Expression in RAW 264.7 Cells*

NFATc1 is considered to be the most important regulator of the transcriptional activation of osteoclast differentiation-associated genes by RANKL [8,9]. To examine in more detail the mechanism of FST-mediated inhibition of osteoclastogenesis, we assessed the expression of NFATc1 in RANKL-stimulated RAW 264.7 cells. Consistent with previous studies, the expression of NFATc1 was significantly increased by RANKL but was down-regulated in a concentration-dependent manner by FST (Figure 5). In addition, we investigated the effects of FST on the levels of specific marker for osteoclast such as TRAP, cathepsin (CTSK), matrix metallopeptidase-9 (MMP-9) and osteoclast-associated receptor (OSCAR). Figure 5 showed that RANKL markedly up-regulated levels of these osteoclast-specific markers, which were effectively attenuated by the addition of FST. Co-treatment with OPG also completely prevented increases in these protein markers.

**Figure 5.** Inhibition of the RANKL-induced expressions of osteoclast-regulatory genes by FST in RAW 264.7 mouse macrophage-like cells. Cells were co-treated with various concentrations of FST or 100 ng/mL OPG in the presence or absence of 100 ng/mL RANKL for 5 days. (**A**) The expression levels of osteoclast-regulatory proteins were assessed by Western blot analysis. β-actin was used as the internal control. The results shown are representative of three independent experiments. (**B**) Densitometry quantifications of protein expression were measured by ImageJ. Statistical analyses were conducted using analysis of variances. \* *p* < 0.05 and \*\*\* *p* < 0.0001 when compared to control. ### *p* < 0.0001 when compared to RANKL treatment. RANKL: receptor of activator of nuclear factor kappa-B ligand; FST: fermented sea tangle extract; OPG: osteoprotegerin; CTSK: cathepsin *K*; MMP-9: matrix metalloproteinase-9; NFATc1: nuclear factor of activated T cells c1; OSCAR: osteoclast-associated receptor; +: cells treated the reagent; -: cells untreated the reagent.

#### *3.6. FST Attenuates RANKL-Induced Intracellular ROS Accumulation Associated with Activation of Nrf2 in RAW 264.7 Mouse Macrophage-Like Cells*

Overproduction of intracellular ROS plays a critical step in RANKL-mediated osteoclastogenesis [12–14], thereby we examined whether FST inhibits the generation of ROS during RANKL-mediated osteoclastogenesis using DCF-DA, a cell permeant redox-sensitive dye. We demonstrated by flow cytometry that ROS levels were significantly increased by RANKL and that these up-regulation were abolished by FST (Figure 6A,D). Moreover, this effect of FST was supported by our fluorescence microscopic examination (Figure 6B) and further, co-treatment with N-acetyl cysteine (NAC), an intensive ROS scavenger, completely alleviated RANKL-induced ROS generation and F-actin ring formation (Figure 6C). In addition, Figure 6E,F shows that FST has the efficacy of equivalence and/or superiority compared with NAC and it was suggested that FST is a powerful anti-oxidant, thereby it has a suppressed RANKL-mediated ROS generation.

**Figure 6.** Effect of FST on RANKL-induced reactive oxygen species (ROS) generation in RAW 264.7 mouse macrophage-like cells. Cells were co-treated with 100 ng/mL RANKL for 1 h in the presence or absence of 800 μg/mL FST or 10 mM NAC. (**A**,**D**) Cells were stained with 5,6-carboxy-2, 7-dichlorofluorescein diacetate (DCF-DA) and DCF fluorescence was measured by flow cytometry. Results are means of two independent experiments. (**B**) After staining with DCF-DA, images were obtained using a fluorescence microscope. Images are representative of at least three independent experiments. (**C**) Cells cultured under the conditions used to induce osteoclast differentiation were fixed and stained for F-actin ring with FITC-phalloidin solution and imaged under a fluorescence microscope. Representative photographs of the morphological changes observed are presented. (**E**) Cellular proteins were isolated from cells and the expression of NFATc1, phospho-NF-κ<sup>B</sup> and phosphor-Nrf2 by Western blot analysis. β-actin was used as the internal control. The results shown are representative of three independent experiments. (**F**) Statistical analyses were conducted using analysis of variances. \* *p* < 0.05 and \*\*\* *p* < 0.001 when compared to control. ### *p* < 0.0001 when compared to RANKL treatment. RANKL: receptor of activator of nuclear factor kappa-B ligand; FST: fermented sea tangle extract; NAC: N-acetyl cysteine osteoprotegerin; NFATc1: nuclear factor of activated T cells c1; p- NF-κB p65: phosphorylated nuclear factor-kappa B p65; p-Nrf2: phosphorylated nuclear factor-erythroid 2-related factor 2; +: cells treated the reagent; -: cells untreated the reagent.

In addition, we show that FST increased the expression and phosphorylation of Nrf2 in RANKL-stimulated cells, which was associated with an increase in typical Nrf2-dependent cytoprotective enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H: Quinone oxidoreductase 1 (NQO-1) (Figure 7A,B). Furthermore, we observed that Nrf2 translocation to the nucleus was promoted by FST treatment (Figure 7C,D).

**Figure 7.** Activation of Nrf2 signaling pathway by FST in RAW 264.7 mouse macrophage-like cells. Cells were treated with FST with or without 100 ng/mL RANKL for 5 days. (**A**) Total cellular proteins were isolated from cells and the expression levels of Nrf2 and its regulatory proteins were assessed by Western blot analysis. β-actin was used as the internal control. (**C**) The expression of nuclear and cytosol Nrf2 were determined by Western blotting. Lamin B and β-actin were used as internal controls for the nuclear and cytosolic fractions, respectively. The results shown are representative of three independent experiments. (**B**,**D**) Statistical analyses were conducted using analysis of variances between groups. \* *p* < 0.05 and \*\*\* *p* < 0.0001 when compared to control. # *p* < 0.05 and ### *p* < 0.0001 when compared to RANKL treatment. RANKL: receptor of activator of nuclear factor kappa-B ligand; FST: fermented sea tangle extract; Nrf2: nuclear factor-erythroid 2-related factor 2; p-Nrf2: phosphorylated nuclear factor-erythroid 2-related factor 2; HO-1: heme oxygenase-1; NQO-1: NAD(P)H quinone oxidoreductase 1; +: cells treated the reagent; -: cells untreated the reagent.
