**1. Introduction**

Bone remodeling is an active physiological process involving bone deposition and bone resorption by osteoblast and osteoclast, respectively. Imbalance of these processes in favor of resorption may lead to the formation of osteolytic lesions and an increase in bone disease-related disorders and morbidity [1–3]. Receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) are cytokines that play important roles in osteoclast differentiation and maturation. RANKL belongs to the tumor necrosis factor (TNF) superfamily and is regarded as the key promoter of osteoclastogenesis. M-CSF by contrast, is involved in the maintenance of

mature osteoclast survival and mobility [4,5]. The binding of RANKL to its receptor RANK results in the activation of various signaling pathways, including the NF-κB pathway [6,7], which then enhances the activation of nuclear factor of activated T cell c1 (NFATc1), which then in turn promotes osteoclast formation by up-regulating the expression of osteoclast-specific genes [8,9]. In addition, a number of previous studies have shown that reactive oxygen species (ROS) are also critical messengers for osteoclast di fferentiation [10,11] and increased activity of the Nrf2 signaling system can block this activation [12–14]. These findings sugges<sup>t</sup> that suppression of ROS production in combination with increasing activity of Nrf2 may provide a means to block osteoclast activity. Although various drugs have been used clinically to inhibit bone resorption, all have severe side e ffects when used long-term [15] and as a result, research into the prevention and treatment of osteolytic diseases using natural products has greatly increased in recent years.

Many marine algae extract or components of these extracts have been shown to exhibit potential for preventing and treating bone resorption related diseases [16,17] and fermented marine algal extracts have attracted the attention of the food and medical care industries [17,18]. The sea tangle, *Laminaria japonica* Aresch, is one of the most well-known edible brown seaweeds and has long been used as an important food supplement in Pacific and Asian countries [19]. This seaweed is rich in polysaccharides, dietary fiber, minerals, carbohydrates, polyphenols and proteins [20,21] and has been reported to protect against obesity, inflammation and cancer [22–25]. Interestingly, Lee et al. [26] developed a fermented form of sea tangle using *Lactobacillus brevis* with high antioxidant activity and showed that a fermented sea tangle extract (FST) protected against liver damage better than a non-fermented sea tangle extract [27,28]. They speculated that glutamate in the sea tangle which converted to gamma-aminobutyric acid through the fermentation process, was the reason behind the increased antioxidant capacity. It has been reported that FST supplementation reduce obesity and improve stress managemen<sup>t</sup> [29]. Furthermore, previous studies have shown that FST can protect against age-associated short-term memory loss and reduced physical functioning [30–32]. However, the e ffect of FST on bone has not previously been investigated and therefore we decided to investigate whether FST had any inhibitory e ffect on RANKL-stimulated osteoclast di fferentiation using RAW 264.7 mouse macrophage cells.

#### **2. Materials and Methods**

#### *2.1. Reagents and Antibodies*

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and other reagents for cell culture were purchased from WelGENE Inc. (Daegu, Republic of Korea). RANKL and osteoprotegerin (OPG) were obtained from Abcam (Cambridge, MA, USA) and Peprotech (Rocky Hill, NJ, USA), respectively. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), tartrate-resistant acid phosphatase (TRAP) assay kit, bovine serum albumin (BSA), <sup>4</sup>,6-diamidino-2-phenylindole (DAPI), 5,6-carboxy-2,7-dichlorofluorescein diacetate (DCF-DA) and N-acetyl cysteine (NAC) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). NE-PERTM nuclear and cytoplasmic extraction reagents kit and polyvinylidene difluoride (PVDF) membranes were obtained from Pierce Biotechnology (Rockford, IL, USA) and Schleicher & Schuell (Keene, NH, USA), respectively. Fluorescein isothiocyanate (FITC)-phalloidin solution was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Primary and secondary antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), Cell Signaling Technology Inc. (Beverly, MA, USA), Abcam, Novus (Novus Biologicals, LLC., Littleton, CO, USA), Thermo Fisher Scientific and R&D system. Appropriate horseradish-peroxidase (HRP)-linked secondary antibodies and enhanced chemiluminescence (ECL) detection solution were purchased from Amersham Corp. (Arlington Heights, IL, USA). All reagents not specifically identified were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

#### *2.2. Preparation of FST*

FST received from Marine Bioprocess Co. Ltd. (Busan, Korea) was extracted as previously described [30]. In brief, yeas<sup>t</sup> extract and glucose were added to water at a ratio of 1:15 (w/v) and sea tangle (*L. japonica* Aresch) was then added and sterilized in an autoclave at 121 ◦C for 30 min. After autoclaving, culture broth of *L. brevis* BJ20 (accession no. KCTC 11377BP) was added to the mix at a concentration of 1.2% (v/v) and the mixture was incubated at 37 ◦C for 2 days. The fermented product was obtained by filtration and lyophilized. The dried extract (FST) so obtained was dissolved in Milli-Q Water to produce a 10 mg/mL stock solution.

#### *2.3. Cell Culture and Viability Analysis*

RAW 264.7 cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM containing 10% heat inactivated FBS, penicillin (100 units/mL) and streptomycin (100 g/mL) at 37 ◦C in a humidified 5% CO2 atmosphere and subcultured every 3 days. The viability of the cells was assessed by MTT assay as previously described [14]. Briefly, the cells were treated with the desired concentrations of FST with or without 100 ng/mL RANKL for 72 h and then incubated with 50 μg/mL MTT solution for 3 h. Formazan crystals were dissolved in DMSO and the absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) microplate reader (Dynatech Laboratories, Chantilly, VA, USA) at 540 nm.

#### *2.4. Osteoclast Di*ff*erentiation and TRAP Assay*

Osteoclast formation was measured by quantifying cells positively stained by TRAP. Briefly, the cells were fixed in 4% paraformaldehyde (pH 7.4) at room temperature for 10 min and then stained with commercial TRAP staining kit according to the manufacturer's instructions. Osteoclasts were defined as TRAP-positive multinuclear cells containing 3 or more nuclei, under a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany). TRAP activity was determined in culture media using a TRAP assay kit, in accordance with the manufacturer's instructions. TRAP activities were expressed as percentages of control activities.

#### *2.5. F-Actin Ring Staining*

As described previously, evaluation of actin ring formation was performed [14]. Briefly, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS for 5 min and then stained with an anti-actin antibody at 4 ◦C overnight. After washing with PBS, the cells were incubated with FITC-conjugated phalloidin for 30 min at 37 ◦C and then counterstained with 2.5 μg/mL DAPI for 20 min. F-actin rings were analyzed by fluorescence microscopy (Carl Zeiss, Oberkochen, Germany).

#### *2.6. Western Blot Analysis*

As described previously, total protein was extracted from the cells using the Bradford Protein assay kit [14]. Nuclear and cytosolic proteins were prepared using a NE-PER nuclear and cytoplasmic extraction reagents kit according to the manufacturer's instructions. Equal amounts of protein from samples were loaded and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat skim milk in trisbu ffered saline containing 0.1% Triton X-100 (TBST) for 1 h and probed with specific primary antibodies at 4 ◦C overnight (Table 1). After washing three times with TBST, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies for 2 h. Protein expression was detected by an ECL kit and visualized by Fusion FX Image system (Vilber Lourmat, Torcy, France).


**Table 1.** Information of primary and secondary antibodies.

CTSK: cathepsin K; HO-1: heme oxygenase-1; IκBα: inhibitory proteins of kappa B, alpha; MMP-9: matrix metalloproteinase-9; NFATc1: nuclear factor of activated T cells c1; NF-κB: nuclear factor-kappa B; Nrf2: nuclear factor-erythroid 2-related factor 2; NQO-1: NAD(P)H quinone oxidoreductase 1; OSCAR: osteoclast-associated receptor; TRAP: tartrate-resistance acid phosphatase; HRP: horseradish-peroxidase.

#### *2.7. Immunofluorescence Staining for NF-*κ*B*

RAW 264.7 cells were seeded on gelatin-coated glass coverslips. After it was cultured for 24 h, cells were treated with RANKL in the presence or absence of various concentrations of FST for 24 h, fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 15 min and blocked with PBS containing 5% BSA. Cells were stained with primary antibody against phosphoNF-κ B p65 at 4 ◦C overnight and incubated with a fluorescein-conjugated anti-rat IgG in the dark at 37 ◦C for 1 h. Cells were mounted on slides and then analyzed by fluorescence microscope.

#### *2.8. Measurement of Intracellular ROS Levels*

The production of intracellular ROS was measured by a flow cytometer with DCF-DA as described previously [14]. Briefly, the cells were treated with FST in the presence or absence of 100 ng/mL RANKL. In the last 20 min of treatment, 10 μM DCF-DA was added to the incubated cells in the dark. Following incubation, the cells were washed twice with PBS and 10,000 cells were analyzed for intracellular ROS content by BD Accuri C6 software in a flow cytometer (BD Biosciences) at 480/520 nm. To observe ROS generation by fluorescence microscopy, cells were stimulated with RANKL in the presence or absence of FST for 1 h. Cells were then stained with DCF-DA and then fixed with 4% paraformaldehyde for 2.

## *2.9. Statistical Analysis*

All experiments were performed at least three times. Data were analyzed using GraphPad Prism software (version 5.03; GraphPad Software, Inc., La Jolla, CA, USA) and expressed as the mean ± standard deviation (SD). Di fferences between groups were assessed using analysis of variance followed by ANOVA-Tukey's post hoc test and *p* < 0.05 was considered to indicate a statistically significant di fference.
