Water Extract of Dryopteris crassirhizoma Attenuates Bone Loss by Suppressing Osteoclast Differentiation and Function

The rhizome of Dryopteris crassirhizoma has been used as a traditional herbal medicine for treating various inflammatory and infectious diseases such as tapeworm infestation and mumps. In the present study, we investigated the bone protective effect of water extract of the rhizome of Dryopteris crassirhizoma (WEDC). We found that WEDC inhibits osteoclast differentiation via directly acting on osteoclast precursors. In osteoclast precursors, WEDC inhibited receptor activator of nuclear factor-κB ligand- (RANKL-) induced expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1, a key downstream target of c-Fos during osteoclast differentiation. We found that WEDC inhibits RNAKL-induced activation of extracellular-regulated kinase and NF-κB that mediates c-Fos expression and osteoclast differentiation. In addition to the inhibitory effect of osteoclast differentiation, WEDC markedly suppressed bon-resorbing activity of mature osteoclasts, which was accompanied by disruption of actin ring structure. Furthermore, administration of WEDC suppressed RANKL-induced trabecular bone loss in mice. Collectively, our results demonstrate that WEDC inhibits not only osteoclast differentiation by inhibiting RANK signaling pathways in osteoclast precursors but also bone resorption by disrupting actin ring in mature osteoclasts, thereby contributing to its protective effect on bone loss.


Introduction
Bone homeostasis is maintained by the coordinated actions of osteoclast-mediated bone resorption and osteoblastmediated bone formation. An imbalance in favor of bone resorption, most often due to excess osteoclastic activity, leads to bone loss in pathological conditions such as osteoporosis, lytic bone metastases, and rheumatoid arthritis [1].
Osteoclasts are multinucleated bone-resorbing cells that originate from hematopoietic mononuclear precursors. Receptor activator of nuclear factor-B ligand (RANKL) is an essential cytokine that stimulates entire processes for the development of bone-resorbing osteoclasts [1]. The effects of RANKL are physiologically blocked by osteoprotegerin (OPG) which acts as a decoy receptor for RANKL [2]. Stromal cells/osteoblasts regulate osteoclast differentiation by expressing both RANKL and OPG [1]. Thus, RANKL/OPG ratio in osteoblasts is an important determinant of osteoclast differentiation as well as bone mass. RANKL binding to its receptor RANK on osteoclast precursors induces activation of NF-B and mitogen-activated protein kinases (MAPKs) including extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 through recruitment of the adapter molecules such as TNF receptor-associated factor 6 (TRAF6) [3,4]. These signaling cascades lead to the induction and activation of osteoclastogenic transcription factors such as c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) [4][5][6][7][8]. Genetic studies have shown that deficiency in NF-B p50/p52, c-Fos, or NFATc1 results in blockage of osteoclastogenesis [9][10][11].
In the screening of traditional Korean herbal medicines for antiosteoclastogenic activity, we found that water extract of the rhizome of Dryopteris crassirhizoma (WEDC) efficiently inhibits osteoclast differentiation. D. crassirhizoma is a perennial herbaceous plant widely distributed in Asia, and its rhizome is commonly used as an effective vermicide in traditional Chinese medicine. It has also been prescribed for treating respiratory tract infections, uterine bleeding, mumps, and 2 Evidence-Based Complementary and Alternative Medicine feverish illnesses [12]. Previous studies have shown that the rhizome of D. crassirhizoma has antioxidant, anticancer, antiinflammatory, and antibacterial activities [13][14][15][16]. However, there is little information about the pharmacological effect of the rhizome of D. crassirhizoma on bone metabolism. In the present study, we investigated the inhibitory effect and action mechanism of WEDC on osteoclast differentiation and function. The in vivo efficacy of WEDC was also examined in a murine model of bone loss.  [17]. Antibodies against phospho-ERK1/2 (thr202/Tyr204), ERK, phospho-JNK1/2 (Thr183/Tyr185), JNK, phospho-p38 (Thr180/Tyr182), p38, phospho-p65 (Ser536), p65, phospho-I B (Ser32), and I B were purchased from Cell Singling Technology (Danvers, MA, USA). Antibodies against NFATc1, c-Fos, -actin, and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Preparation of WEDC.
The rhizome of D. crassirhizoma was purchased from Yeongcheon Oriental Herbal Market (Yeongcheon, Republic of Korea). A voucher specimen (no. W197) was deposited in the herbal bank of KM-Based Herbal Drug Research Group, Korea Institute of Oriental Medicine. The rhizome of D. crassirhizoma (50 g) was extracted with 500 mL of boiling water for 3 h. The extract was filtered through a testing sieve (150 m) and lyophilized in a freeze dryer. To prepare WEDC, the lyophilized powder (yield: 12.78%) was resuspended in distilled water, centrifuged at 10,000 ×g for 5 min, and filtered through a 0.2 m sterile filter.

Cell Culture and Osteoclast Differentiation.
Bonemarrow-derived macrophages (BMMs) were prepared from mouse bone marrow cells as described previously [18] and cultured in -MEM complete medium containing 10% FBS, antibiotics (100 U/mL penicillin and 100 g/mL streptomycin), and M-CSF (60 ng/mL). To generate osteoclasts from BMM cultures, BMMs (1 × 10 4 cells/well) were incubated with M-CSF (60 ng/mL) and RANKL (100 ng/mL) for 4 days in 96-well plates. Cell viability of BMMs was measured with Cell Counting Kit-8 assay (Dojindo Molecular Technologies Inc., Rockville, MD, USA), after culturing BMMs (1 × 10 4 cells/well in a 96-well plate) in the presence of M-CSF (60 ng/mL) for 2 days. Mouse calvarial osteoblasts were obtained from calvariae of newborn mice as described previously [18]. For osteoclast differentiation from the coculture of mouse osteoblasts and bone marrow cells, bone marrow cells (3 × 10 5 cells/well) and osteoblasts (2 × 10 4 cells/well) were cocultured with 10 nM VitD 3 for 6 days in 48-well tissue culture plates. All cultures were replenished with fresh medium on day 3.

Tartrate Resistant Acid Phosphatase (TRAP) Activity and
Staining. Cells were fixed in 10% neutral buffered formalin and permeabilized with 0.1% Triton X-100. Total TRAP activity was measured spectrophotometrically using p-nitrophenyl phosphate as substrate according to the manufacturer's instructions in a TRAP assay buffer (50 mM sodium tartrate and 0.12 M sodium acetate, pH 5.2). TRAP staining was performed according to the protocol described in BD Biosciences Technical Bulletin no. 445. TRAP-positive multinucleated cells (TRAP(+)MNCs) having more than three nuclei were counted as osteoclasts.

Real-Time Quantitative Polymerase Chain Reaction (QPCR).
Osteoblasts (3 × 10 5 cells/well in a 6-well plate) were preincubated with WEDC for 3 h and then stimulated with VitD 3 for 24 h. BMMs (4 × 10 5 cells/well in a 6-well plate) were incubated with WEDC for 3 h and then stimulated with RANKL for the indicated times. Total RNA was isolated using RNA-spin total RNA Extraction Kit (Bioneer, Daejeon, Republic of Korea), and cDNA was synthesized from 1 g of total RNA using AccuPower RT-PreMix (Bioneer). SYBR green-based QPCR amplification was performed using cDNA diluted to 1 : 3, 10 pmol of primers, and AccuPower GreenStar QPCR Master Mix (Bioneer) in the Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primer sequences used were described previously [18]. The relative mRNA levels of target genes were determined according to the 2-ΔΔCT method using Hprt as a reference gene.

Western Blot Analysis.
Cells were washed twice with icecold PBS and lysed in ice-cold lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, and 0.1% SDS) supplemented with protease inhibitor and phosphatase inhibitor cocktail tablets (Roche Applied Science, Indianapolis, IN, USA). Cell lysates were centrifuged at 10,000 ×g for 15 min at 4 ∘ C. Protein concentration of total cell lysates was determined by suing BCA protein assay kit (Thermo Fisher Scientific Inc.). Equal amounts of proteins (30 g) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and then immunoblotted with the indicated antibodies.

Bone Resorption Assay.
Mature osteoclasts were generated by coculturing mouse bone marrow cells and primary osteoblasts with VitD 3 (10 nM) on collagen gels for 6 days. The generated osteoclasts were seeded on an Osteo Assay Surface plate (Corning Inc., Corning, NY, USA), allowed to settle for 2 h, and then incubated with the indicated concentrations of WEDC for 24 h. Cells were stained for TRAP to detect osteoclasts and photographed. Resorbed pits by osteoclasts were photographed and analyzed by using Image J software, after removing cells with sodium hypochlorite bleach.   Student's -tests, while multiple-group comparisons were performed with analysis of variance followed by Dunnett's test. A value <0.05 was considered statistically significant.

Effect of WEDC on Osteoclast Differentiation in Bone
Marrow Cell-Osteoblast Coculture. We investigated whether WEDC affects osteoclast differentiation in bone marrow cell-osteoblast coculture system. In the coculture system, osteoclastogenic factors such as VitD 3 and IL-1 stimulate osteoclast differentiation by increasing the RANKL/OPG expression ratio in osteoblasts [1]. VitD 3 stimulated osteoclast differentiation in the coculture, which was inhibited by WEDC in a dose-dependent manner (Figures 1(a) and 1(b)). However, WEDC (50 g/mL), a concentration that markedly inhibited osteoclast differentiation in the coculture system, did not affect RANKL and OPG mRNA expression in basal and VitD 3 -stimulated osteoblasts (Figure 1(c)).

Effect of WEDC on RANKL-Induced Osteoclast Differentiation.
We next investigated the possibility that WEDC inhibits osteoclast differentiation by directly acting on osteoclast precursors. In the presence of M-CSF, RANKL stimulated TRAP activity and osteoclast differentiation in its precursor BMM cultures. Consistent with the results of coculture system, WEDC inhibited RANKL-induced osteoclast differentiation and TRAP activity in a dose-dependent manner with an almost complete inhibition of osteoclast formation at 50 g/mL of WEDC (Figures 2(a)-2(c)). Although a slight reduction of cell viability of BMMs was observed at 100 g/mL of WEDC, 5-50 g/mL of WEDC did not affect cell viability of BMMs, indicating that the inhibitory effect of 6 Evidence-Based Complementary and Alternative Medicine WEDC was not due to cellular toxicity or cell proliferation (Figure 2(d)).

Effect of WEDC on RANKL-Induced c-Fos and NFATc1
Expression in Osteoclast Precursors. NFATc1, the master transcription factor for osteoclastogenesis, is induced during osteoclastogenesis at a transcription level, and its induction is dependent on c-Fos [11]. To gain insights into the molecular mechanisms for the antiosteoclastogenic effect of WEDC, we determined whether WEDC affects c-Fos and NFATc1 expression during osteoclast differentiation. Stimulation of BMMs with RANKL increased c-Fos and NFATc1 expression at both mRNA and protein levels, which was dramatically suppressed by WEDC (Figures 3(a) and 3(b)).

Effect of WEDC on RANKL-Induced Early
Signaling Pathways. MAPK and NF-B signaling pathways are implicated in RANKL-induced c-Fos and NFATc1 expression [5][6][7][8]. To elucidate molecular mechanisms underlying the inhibitory effect of WEDC on c-Fos and NFATc1 expression, we explored whether WEDC affects MAPK and NF-B activation in response to RANKL. Stimulation of BMMs with RANKL rapidly activated ERK, JNK, and p38 MAPKs as well as NF-B signaling pathway (p65 phosphorylation and I B phosphorylation and degradation). WEDC slightly inhibited RANKL-induced ERK activation but not JNK or p38 activation. It also attenuated RANKL-induced p65 phosphorylation and I B phosphorylation and degradation (Figure 4).

Effect of WEDC on Bone-Resorbing Activity of Mature
Osteoclasts. We next investigated the effect of WEDC on osteoclastic bone resorption. When mature osteoclasts were cultured for 24 h on a plate coated with an inorganic crystalline calcium phosphate, numerous resorbed pits by osteoclasts were formed in vehicle-treated control cultures. WEDC decreased the total resorbed area in a dose-dependent manner, without affecting the number of osteoclasts (Figures 5(a)-5(c)). Actin ring is a distinctive cytoskeletal structure in mature osteoclasts, and its formation is essential for osteoclastic bone resorption [19]. Osteoclasts formed a ring-like F-actin structure, actin ring, at the cell periphery on tissue culture plates. Treatment of osteoclasts with WEDC for 30 min resulted in disruption of actin ring structure in a dose-dependent manner ( Figure 5(d)).

Effect of WEDC on RANKL-Induced Bone Loss.
Since WEDC exhibited the inhibitory activity on RANKL-induced osteoclast differentiation as well as bone-resorbing activity of mature osteoclasts, we next investigated whether WEDC has a protective effect on bone loss. When mice were intraperitoneally injected with RANKL twice with a 24 h interval, a severe trabecular bone loss was observed at the distal femoral metaphysis (Figure 6(a)). Micro-CT analysis revealed marked reduction in trabecular volume, thickness, and number, with a decreased trabecular separation in the RANKL-injected mice (Figure 6(b)). Oral administration of WEDC significantly attenuated RANKL-induced bone loss

Discussion
Abnormal activation of osteoclasts attributes to bone loss in many bone destructive diseases including postmenopausal osteoporosis, lytic bone metastases, and rheumatoid arthritis [1]. Accordingly, modulation of osteoclast differentiation and function can be a potent therapeutic target for various bone diseases characterized by excessive bone resorption.
Here we have demonstrated for the first time that WEDC attenuates bone loss by suppressing osteoclast differentiation and function.
To explore the molecular mechanism by which WEDC inhibits osteoclast differentiation, we examined the RANKL/ RANK/OPG system. WEDC did not affect RANKL and OPG Previous studies have shown that activation of MAPKs (ERK, JNK, and p38) and NF-B mediates RANKL-induced c-Fos expression and osteoclast differentiation [5][6][7][8]. It was reported that RANKL activates JNK, p38, and the classical NF-B signaling pathway through the RANK-TRAF6 pathway in a manner dependent on E3 ubiquitin ligase activity of TRAF6 [3,4]. However, it is still unclear how RANKL activates ERK. In the present study, WEDC inhibited RANKLinduced ERK activation without affecting JNK and p38 activation. The classical NF-B signaling pathway involves activation of the I B kinase (IKK) complex that stimulates I B phosphorylation and degradation, allowing nuclear translocation of NF-B heterodimer containing the p50 and p65 subunits [20]. In addition to I B , posttranslational p65 modifications such as phosphorylation, acetylation, and ubiquitination represent another mechanism regulating NF-B transcriptional activity [20]. It has been suggested that p65 phosphorylation at Ser536 in the transactivation domain promotes its transcriptional activity [21,22]. In our study, WEDC suppressed RANKL-induced p65 phosphorylation at Ser536 and I B phosphorylation and degradation. Therefore, our findings suggest that WEDC inhibits osteoclast differentiation, at least in part, by suppressing RANKLinduced activation of ERK and the classical NF-B signaling pathway, without interfering with the RANK-TRAF6 pathway. In addition to the inhibitory effect on osteoclast differentiation, WEDC also decreased resorbed pit area by mature osteoclasts without affecting the number of osteoclasts, indicating that WEDC inhibits bone-resorbing activity of osteoclasts. Given the crucial role of actin ring structure in boneresorbing activity of osteoclasts [19], our findings of WEDCinduced disruption of actin ring structure in osteoclasts suggest that the antiresorptive property of WEDC is due to disruption of actin ring structure. The classical NF-B signalling pathway is involved in bone-resorbing activity of osteoclasts as well as osteoclast differentiation. It has been shown that blocking NF-B pathway by overexpressing a dominant-negative form of IKK beta suppresses boneresorbing activity of osteoclasts, whereas NF-B activation by expression of a constitutively active form of IKK beta upregulates it [23]. Neither inhibition nor activation of NF-B pathway by the IKK beta mutants affected osteoclast survival [23]. In addition, NBD peptide, a selective peptide inhibitor of the classical NF-B pathway, was shown to inhibit bone resorption by disrupting actin ring [24]. Therefore, these observations suggest that the inhibitory action of WEDC on the classical NF-B pathway may contribute to its antiresorptive function.
Consistent with the in vitro results, administration of WEDC protected bone loss induced by RANKL. Since injection of exogenous RANKL rapidly induces trabecular bone loss through stimulating osteoclast differentiation and function [17], the protective effect of WEDC on bone loss is mainly due to the suppression of osteoclast differentiation and function. However, we cannot exclude the possibility that WEDC might affect osteoblastic bone formation. Indeed, WEDC could stimulate matrix mineralization in calvarial osteoblast cultures (data not shown), although its effect on bone formation in vivo remains to be elucidated. Given the crucial role of excessive RANKL activity in pathological bone destruction, our findings strongly suggest that WEDC may be useful in preventing or treating various bone destructive diseases.
Phloroglucinols, triterpenes, flavonoid glycosides, and phenolic compounds have been isolated from the rhizome of D. crassirhizoma [25][26][27][28][29]. Of them, phloroglucinol derivatives have been linked to several biological activities of the rhizome of D. crassirhizoma, including antioxidant, antibacterial, and antitumor activities [13,25,30]. We found the presence of a tetrameric phloroglucinol, dryocrassin ABBA, in WEDC with HPLC analysis (data not shown). Dryocrassin Evidence-Based Complementary and Alternative Medicine 9 ABBA exhibited a marginal inhibitory effect on RANKLinduced osteoclast differentiation at concentrations without affecting cell viability (data not shown). However, the constituents mediating the protective effect of WEDC on bone loss still remain to be elucidated.

Conclusion
We have demonstrated that WEDC inhibits osteoclast differentiation and function by inhibiting RANK signaling pathways in osteoclast precursors and by disrupting actin ring in mature osteoclasts, respectively. Furthermore, WEDC suppressed RANKL-induced bone loss in vivo. These findings suggest that WEDC may be useful in preventing or treating bone diseases associated with excessive bone loss.