Sanguiin H-6, a constituent of Rubus parvifolius L, inhibits receptor activator of nuclear factor-[kappa]B ligand-induced osteoclastogenesis and bone resorption in vitro and prevents tumor necrosis factor-[alpha]-induced osteoclast formation in vivo.
Background: Osteoclasts are multinucleated bone-resorbing cells that differentiate in response to receptor activator of nuclear factor-[kappa]-B (NF-[kappa]B) ligand (RANKL). Enhanced osteoclastogenesis contributes to bone diseases, such as osteoporosis and rheumatoid arthritis. Rubus parvifolius L. is traditionally used as an herbal medicine for rheumatism; however, its detailed chemical composition and the molecular mechanisms responsible for its biological action have not been elucidated.
Purpose: To investigate the mechanisms by which R. parvifolius L. extract and its major constituent sanguiin H-6, inhibit osteoclastogenesis and bone resorption.
Methods: Cell proliferation, cell differentiation, and bone resorption were detected in vitro. Inhibition of signaling pathways, marker protein expression, and protein nuclear translocation were evaluated by western blot analysis. Tumor necrosis factor-[alpha] (TNF-[alpha])-mediated osteoclastogenesis was examined in vivo.
Results: R. parvifolius L. extract inhibited the bone-resorption activity of osteoclasts. In addition, sanguiin H-6 markedly inhibited RANKL-induced osteoclast differentiation and bone resorption, reduced reactive oxygen species production, and inhibited the phosphorylation of inhibitor of NF-[kappa]B alpha (I[kappa]B[alpha]) and p38 mitogen-activated protein kinase. Sanguiin H-6 also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), cathepsin K, and c-Src. Moreover, sanguiin H-6 inhibited the nuclear translocation of NFATc1, c-Fos, and NF-[kappa]B in vitro, as well as TNF-[alpha]-mediated osteoclastogenesis in vivo.
Conclusions: Our data revealed that R. parvifolius L. has anti-bone resorption activity and suggest that its constituent, sanguiin H-6, can potentially be used for the prevention and treatment of bone diseases associated with excessive osteoclast formation and subsequent bone destruction.
Receptor activator of nuclear factor-[kappa] b
Nuclear factor of activated T cells
Osteoclasts are multinucleated bone-resorbing cells differentiated from monocyte/macrophage lineage precursors. Many bone diseases such as osteoporosis, Paget's disease, and rheumatoid arthritis, are caused by enhanced osteoclastogenesis. Osteoclastogenesis is controlled by two key cytokines, macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-[kappa]B (NF-[kappa]B) ligand (RANKL). RANKL interacts with its receptor RANK. Subsequently, NF-[kappa]B, activator protein-1, phosphatidylinositol 3-kinase /Akt, and mitogen-activated protein kinase (MAPK) signaling pathways including p38 MAPK, extracellular signal regulated kinase (ERK), and c-jun N-terminal kinase (JNK) are activated (Boyle et al., 2003). Furthermore, RANKL activates nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), a central transcription factor for osteodastogenesis (Takayanagi et al., 2002). NFATc1 translocates to the nucleus and induces the expression of multiple osteoclast markers, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K, and cellular tyrosine protein kinase-Src (c-Src) (Boyle et al., 2003).
Previous studies have shown that reactive oxygen species (ROS) regulate MAPK activation and stimulate osteodastogenesis (Lee et al., 2005). Recently, we reported that fisetin, a strawberry flavonoid, decreases ROS production by upregulation of heme oxygenase-1 (HO-1), a phase II cytoprotective enzyme that prevent oxidative stress, thereby inhibiting osteodastogenesis (Sakai et al., 2013). Furthermore, suppression of HO-1 gene expression by RNA interference enhances osteodastogenesis, while HO-1 overexpression inhibits osteodastogenesis (Sakai et al., 2012). These results suggest that HO-1 inducers could be used as therapeutic agents against bone diseases.
Rubus parvifolius L., widely distributed in East and South Asia, is traditionally used as herbal medicine for fever, angina, enteritis, hepatitis, concretion, eczema, and rheumatism. Although previous studies reported that R. parvifolius L. extract exhibits antioxidant capacity (Gao et al., 2011), the detailed chemical composition and the molecular mechanisms responsible for its biological action remain unclear. Sanguiin H-6 is a hydrolysable tannin isolated from R. parvifolius L, R. fruticosus L., R. idaeus L, and Sanguisorba officinalis L (Tanaka et al., 1985). Sanguiin H-6 has a scavenging effect on lipopolysaccharide-stimulated nitric oxide (NO) production in macrophages (Yokozawa et al., 2002). Furthermore, S. officinalis extracts upregulate HO-1 expression in lung tissue (Lee et al., 2010). However, little is known about the effects of sanguiin H-6 on osteodastogenesis or HO-1 regulation.
Here, we studied whether R. parvifolius L. extract inhibits the bone-resorbing activity of osteoclasts. Moreover, we investigated the inhibitory mechanisms of sanguiin H-6, a major constituent of R. parvifolius L., on osteodastogenesis in vitro and in vivo.
Materials and methods
Reagents and antibodies
Various reagents for cell culture were prepared as described previously (Sakai et al., 2013). Recombinant TNF-[alpha] was purchased from R8iD systems (Minneapolis, MN, USA). The following antibodies (Abs) were purchased: /J-actin (#A5060, rabbit, SigmaAldrich, St. Louis, MO, USA), Src (#05-184, mouse, Upstate Biotechnology, Lake Placid, NY, USA), HO-1 (#SPA895, rabbit, Stressgen, Ml, USA). Anti-RANK (#sc-9072, rabbit), anti-Lamin A/C (#sc-6215, goat), anti-NF-[kappa]:B p65 (#sc-8008, mouse), and anti-NFATc1 (#sc7294, mouse) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Abs specific for phospho-ERKl/2 (#9101, rabbit), phospho-Akt (#9271, rabbit), phospho-JNK (#9751, rabbit), phospho-p38 MAPK (#9211, rabbit), phospho-inhibitor of NF-[kappa]B alpha (IvBa) (#2859, rabbit), c-Fos (#2250, rabbit), and phosphocFos (#5348, rabbit), were purchased from Cell Signaling Technology (Danvers, MA, USA), Cathepsin K Ab was prepared as described previously (Kamiya et al., 1998). Ellagic acid was purchased from AbcamBiochemicals (Cambridge, United Kingdom). Other reagents, including phenylmethylsulfonyl fluoride and protease inhibitor cocktail, were obtained from Sigma-Aldrich.
Fresh leaves and stem of Rubus parvifolius L. were collected in the Bunkyo campus of Nagasaki University, Nagasaki, Japan, in May 2013, and identified by associate professor Koji Yamada at Division of Laboratory in Medical Plants Garden, Nagasaki University and Professor Takashi Tanaka at Division of Natural Product Chemistry, Nagasaki University Graduate School of Biomedical Sciences. A voucher specimen (NAP0520-13/05) was deposited in Graduate School of Biomedical Sciences, Nagasaki University.
Preparation of plant extract
The fresh leaves and stem of R. parvifolius L. (66 g) were homogenized in ethanol: [H.sub.2]O and acetone: [H.sub.2]O (both 6:4 v/v, 300 ml) using a Waring blender. The homogenate (5.0 g) was filtered and then lyophilized. The plant extract was standardized to the authentic sanguiin H-6 using HPLC fingerprinting (Tanaka et al., 1985). The R. parvifolius L. extract (24 p-g/ml) was equivalent to 1.0 [micro]M of sanguiin H-6.
Sanguiin H-6 isolation
The fresh leaves and stem (360 g) were homogenized with acetone-[H.sub.2]O (7:3, v/v, 2.2 L) using Warning blender. After filtration, the filtrate was concentrated and resulting aqueous solution was subjected to Sephadex LH-20 (5 cm i.d. x 18 cm) column chromatography with [H.sub.2]O containing increasing proportions of methanol (20% stepwise elution, each 200 ml) and then methanol containing increasing proportions of 50% aqueous acetone (stepwise elution of 9:1, 8:2, 7:3, and 6:4, each 200 ml). The fractions were monitored by silica gel TLC (developing solvent toluene-ethyl formate-formic acid, 1:5:2, v/v; the spots were visualized by spraying 2% Fe[Cl.sub.3] in ethanol), and the tannin-containing fractions were combined. The tannin fraction was further separated by Sephadex LH-20 (5 cm i.d. x 18 cm) column chromatography with methanol containing increasing proportions of 50% aqueous acetone (stepwise elution of 9:1, 8:2, 7:3, and 6:4, each 300 ml) to yield sanguiin H-6 (1.38 g). The identity of sanguiin H-6 was confirmed by comparing HPLC retention time (Supplementary content SI) and 'H NMR (400 MHz in acetone-d6) data (Supplementary content S2) with those of the authentic sample (Tanaka et al., 1985). Sanguiin H-6 purity was 78% (Supplementary content Sib). The chemical structure and chemical name of sanguiin H-6 are shown in Fig. 2a and Supplementary content S2, respectively.
Bone marrow isolation and osteoclast differentiation
Animal experiments were performed per the guide for the care and use of laboratory animals in our facilities under protocols approved by the Nagasaki University Animal Care Committee. Cell culture was performed as described by Sakai et al. (2013). Briefly, marrow cells from femurs and tibias of mice were cultured overnight in a-minimal essential medium (a-MEM) (Wako Pure Chemicals, Osaka, Japan) containing 10% FBS with 10U/ml penicillin and 100 [micro]g/ ml streptomycin in the presence of M-CSF (50ng/ml) at 37 [degrees]C in 5% C[O.sub.2]. Non-adherent cells were harvested to culture stroma-free bone marrow cells with 50ng/ml M-CSF. After 3 days, the adherent cells were harvested as bone marrow macrophages (BMMs). BMMs were replated and further cultured with sanguiin H-6 in the presence of M-CSF (30 ng/ml) and RANKL (50ng/ml) for 72 h. Osteoclasts were also generated from RAW-D cells, a subclone of RAW264 murine macrophage cell line, having excellent ability to differentiate into multinucleated osteoclasts in the presence of RANKL. RAW-D cells were kindly provided by Prof. Toshio Kukita (Kyushu University, Fukuoka, Japan) (Watanabe et al., 2004).
Tartrate-resistant acid phosphatase (TRAP)--staining
Cells were fixed with 4% paraformaldehyde at 4 [degrees]C for 60 min and then treated with 0.2% Triton X-100 in PBS at room temperature for 5 min. Finally cells were incubated with 0.01% naphthol AS-MX phosphate (Sigma-Aldrich) and 0.05% fast red violet LB salt (Sigma-Aldrich) in the presence of 50 mM sodium tartrate and 90 mM sodium acetate (pH 5.0) for TRAP activity. TRAP-positive red-colored cells with three or more nuclei were considered mature osteoclasts.
Cell viability assay
Cell viability was determined by the Cell Counting Kit-8 (CCK8, Dojindo, Kumamoto, Japan) assay based on water-soluble tetrazolium salt, WST-8. Briefly, BMMs were cultured in 96-well culture plates with sanguiin H-6 in the presence of M-CSF (30ng/ml) and RANKL (50ng/ml). After 72 h, cells were washed twice with PBS, to remove sanguiin H-6 to prevent the reduction of CCK-8 reagent by sanguiin H-6. Then, cells were incubated with CCK-8 for 1 h, and the absorbance at 450 nm was measured with a microplate reader (Bio-Rad iMark, Hercules, CA).
The cell viability was also assessed by trypan blue staining by culturing them in a 35-mm dish with sanguiin H-6 in the presence of RANKL (50ng/ml). After 72 h of culture, cells and culture supernatants were collected and combined, and then mixed with 0.4% trypan blue to identify dead cells. Relative number of remaining live cells was calculated.
Western blot analysis
Cells were rinsed twice with ice-cold PBS, and lysed in a cell lysis buffer (50 mM Tris-HCl [pH 8.0], 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM PMSF, and proteinase inhibitor cocktail). For the phosphorylated protein, phosphatase inhibitors (20 mM sodium fluoride and 2 mM orthovanadate) were added into the cell lysis buffer. The protein concentration was measured with BCA Protein Assay Reagent (Thermo Pierce, Rockford, 1L, USA). An equal amount of protein (5 [micro]g) was applied to each lane. After SDS-PAGE, proteins were electroblotted onto a polyvinylidene difluoride membrane. The blots were blocked with 3% nonfat milk solution containing TBS/0.1% Tween 20 for 1 h at room temperature, probed with various Abs overnight at 4 [degrees]C, washed, incubated with horseradish peroxidase-conjugated secondary Abs (Cell Signaling Technology), and finally detected with ECL-Plus (GE Healthcare Life Sciences, Tokyo, Japan). The immunoreactive bands were analyzed by LAS4000-mini (Fuji Photo Film, Tokyo, Japan).
Bone resorption assay
BMMs were seeded onto Osteo assay plates coated with thin calcium phosphate films (Corning, New York, USA), incubated with M-CSF and RANKL for 3 days until multinudeated osteoclasts were formed, and then they were treated with R. parvifolius extract or sanguiin H-6 at the indicated concentrations. After another 3 days of culture, cells were dissolved in 5% sodium hypochlorite. Images of the resorption pit were taken with a reverse phase microscope (Olympus, Tokyo, Japan). The ratios of the resorbed areas to the total areas were calculated using Image J image-analysis software (http://rsbweb.nih.gov/ij/) as described previously (Sakai et al., 2013).
Quantitative real-time polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using oligo(dT)15 primer (Promega, Madison, WI, USA) and Revertra Ace (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed using a LightCyder 480 (Roche Diagnostics, Mannheim, Germany). cDNA was amplified using Brilliant III Ultra-Fast SYBR QPCR Master Mix (Agilent Technology, La Jolla, CA, USA).
The following primer sets were used:
5'-ACCCAGATCATGTTTGAGAC-3' forward and
5'-GTCAGGATCITCATGAGGTAGT-3' reverse, Hmox-1 (heme oxygenase-1):
5'-CACGCATATACCCGCTACCT-3' forward and
Assessment of intracellular ROS
Intracellular ROS formation was assessed using the cell-permeable fluorescent probe 5,6-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Invitrogen) as described by Sakai et al. (2013). Cells were treated with sanguiin H-6, vehicle (DMSO) as a negative control, or 10 mM of N-acetylcysteine (NAC) ROS scavenger as a positive control for 12 h, and then incubated with 5 [micro]M CM-[H.sub.2]DCFDA at 37[degrees][degrees]C for 30 min. The samples were subsequently analyzed using a FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The data were collected from 10,000 cells and analyzed using the FACSDiva software (Becton Dickinson).
Nuclear extraction preparation
Cells were incubated with sanguiin H-6 or vehicle for the indicated times in the presence and absence of RANKL (50ng/ml). Nuclear fractions were prepared using a nuclear extraction kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's instruction.
TNF-[alpha]-induced osteoclastogenesis in vivo
Eight-week-old male C57BL/6J mice (CLEA Japan, Inc.) were randomized to three groups: 1) mice receiving an intraperitoneal (i.p.) injection of vehicle (DMSO, 2 [micro]l/body weight (g)/day) and a supracalvaria injection of PBS (n = 4); 2) mice receiving an i.p. injection of vehicle (DMSO, 2 [micro]l/body weight (g)/day) and a supracalvaria injection of TNF-[alpha] (1.5 [micro]g in PBS/day) (n = 4); 3) mice receiving an i.p. injection of sanguiin H-6 (10[micro]g/body weight (g) /day) and a supracalvaria injection of TNF-[alpha] (1.5 [micro]g in PBS/day) (n = 5). After 5 days of daily injections, calvarial tissues were removed, fixed with 4% paraformaldehyde, and decalcified with 20% EDTA for 12 days. Paraffin-embedded sections were stained for TRAP activity and counterstained with hematoxylin. The percentage of bone surface covered by eroded surface (ES/BS) was determined by histomorphometry using Image J.
Results are means [+ or -] standard deviation (SD). The significance of the differences between the mean values was determined using an analysis of variance (ANOVA) and the Tukey-Kramer method using the Excel add-in software, Stated. The differences were considered statistically significant when *P < 0.05 and **P < 0.01.
R. parvifolius L extract inhibits bone-resorbing activity of mature osteoclasts
To evaluate the pharmacological activity of R. parvifolius L., we performed a bone resorption assay. BMMs were seeded into Osteo assay plates and cultured with M-CSF and RANKL for 3 days until multinudeated osteoclasts were formed, and then they were treated with the R. parvifolius L. extract. The vehicle-treated controls exhibited numerous resorption pits (Fig. 1a, 0 [micro]M). However, R. parvifolius L. extract significantly and dose-dependently inhibited pit formation (Fig. la). Furthermore, the calculated resorption area of the R. parvifolius L. extract-treated osteoclasts decreased more markedly than that of the vehicle controls did (Fig. lb).
We analyzed the ethanol extract of R. parvifolius L. using HPLC to determine its chemical profile. As we previously reported an abundance of tannins in the R. parvifolius L. (Tanaka et al., 1985), a major component of R. parvifolius L. extract was identified as sanguiin H-6 (Supplementary content SI, peak 1 and S2). To investigate the pharmacological mechanisms of R. parvifolius L. further, we isolated sanguiin H-6 from the R. parvifolius L. extract. The chemical structure and chemical name of sanguiin H-6 is shown in Fig. 2a and Supplementary content S2.
Sanguiin H-6 inhibits osteoclast formation in BMMs and RAW-D cells
To clarify the effects of sanguiin H-6 on osteoclast formation, BMMs were cultured with indicated concentrations of sanguiin H-6 in the presence of M-CSF and RANKL for 72 h. Sanguiin H6 dose-dependently inhibited multinudeated osteoclast formation (Fig. 2b). The number of TRAP-positive multinudeated osteoclasts decreased after sanguiin H-6 treatment even at low doses (0.1 [micro]M, Fig. 2c), with complete inhibition of osteoclast formation occurring at 5 [micro]M sanguiin H-6. Sanguiin H-6 was not cytotoxic at 10 [micro]M; however, cytotoxicity was observed at 25 and 50 [micro]M (Fig. 2d), suggesting that the inhibitory effects of sanguiin H-6 on osteoclast differentiation were not attributable to cytotoxicity.
The effects of sanguiin H-6 were confirmed using osteoclast progenitor cell line RAW-D cells. Treatment of RANKL-inducedRAW-D cells with sanguiin H-6 also inhibited osteoclast formation. The number of TRAP-positive RAW-D derived osteoclasts decreased significantly after treatment with > 0.1 [micro]M sanguiin H-6 (Fig. 2e). We also evaluated the viability of RAW-D cells using trypan blue. We did not detect any cell death after culturing RAW-D cells for 72 h with DMSO (vehicle) in the presence of RANKL (50ng/ml). Cytotoxicity was observed at > 10 [micro]M sanguiin H-6 (Fig. 2f).
Therefore, sanguiin H-6 inhibits osteodastogenesis in RANKL-induced in vitro culture system.
Sanguiin H-6 inhibits bone-resorbing activity of mature osteoclasts
To determine whether sanguiin H-6 decreases bone-resorption activity, we performed a bone resorption assay. In vehicle-treated controls, numerous multinudeated osteoclasts (Fig. 3a 0 [micro]M, arrow) and resorption pits (Fig. 3a 0 [micro]M, arrowhead and Fig. 3b 0 [micro]M) were observed. Sanguiin H-6 impaired the spreading of mature osteoclasts (Fig. 3a, arrow) and significantly and dose-dependently inhibited pit formation (Fig. 3a, arrowhead, and Fig. 3b). The calculated resorption area of sanguiin H-6-treated osteoclasts markedly decreased, even at the lowest concentration, compared to that of the vehicle-treated osteoclasts (Fig. 3c), indicating that sanguiin H-6 inhibits the bone-resorption activity of mature osteoclasts.
Sanguiin H-6 upregulates HO-1 and decreases intracellular ROS production
Next, we investigated the effects of sanguiin H-6 on Hmox1 gene expression in osteoclasts. Twenty-four-hour sanguiin H6 treatment upregulated Hmox-1 mRNA expression (Fig. 4a); it also upregulated HO-1 protein expression dose-dependently (Fig. 4b). Moreover, we monitored intracellular ROS levels in RANKL-stimulated BMMs using flow cytometry. As shown in Fig. 4c, sanguiin H-6 dose-dependently inhibited ROS production. Similarly, the ROS scavenger NAC also inhibited ROS production.
Sanguiin H-6 affects RANKL-stimulated intracellular signaling
To elucidate the molecular mechanisms of sanguiin H-6-mediated inhibition of osteodastogenesis, we investigated the effects of sanguiin H-6 on RANKL-stimulated signaling pathways. I[kappa]B[alpha] and p38 MAPK phosphorylation were significantly inhibited by sanguiin H-6 treatment (Fig. 5); however, ERK phosphorylation was not significantly inhibited. Further, the phosphorylation levels of JNK and Akt increased. These results indicate that sanguiin H-6 primarily inhibits I[kappa]B[alpha] and p38 MAPK phosphorylation.
Sanguiin H-6 downregulates NFATc1 and inhibits the nuclear translocation of NFATc1 phosphorylated-c-Fos, and NF-[kappa]B
To investigate whether sanguiin H-6 inhibits osteodastogenesis by downregulating NFATc1, a crucial transcription factor in osteoclasts, and other osteoclast markers, we analyzed the expression of these proteins in mature osteoclasts by western blotting. Sanguiin H-6 did not significantly alter RANK and c-Fos expression. Both proteins remained at sanguiin H-6 concentrations of up to 25 [micro]M; however, sanguiin H-6 significantly downregulated NFATc1 expression at concentrations above 1 [micro]M (Fig. 6a). Moreover, the expression levels of c-Src and cathepsin K, which are transcriptionally regulated by NFATc1, significantly decreased following sanguiin H-6 treatment. These results indicated that sanguiin H-6 downregulated the expression of NFATc1 and its target proteins, including c-Src and cathepsin K. Next, we examined its effects on the nuclear translocation of NFATc1, phosphorylated-c-Fos, and NF-[kappa]B, which coordinately mediate osteodastogenesis (Boyle et al. 2003). Western blot analysis indicated that sanguiin H-6 strongly inhibited the nuclear translocation of NFATc1, phosphorylated-c-Fos, and NF-[kappa]B 6 and 24 h after M-CSF and RANKL treatment, although M-CSF and RANKL markedly activated these transcription factors (Fig. 6b).
Sanguiin H-6 inhibits TNF-[alpha]-induced osteodastogenesis in vivo
Because sanguiin H-6 effectively inhibited NF-[kappa]B activation, we investigated its effects on in vivo osteodastogenesis induced by TNF-[alpha], a potent osteoclastogenic cytokine that activates the NF-[kappa]B pathway in osteoclasts (Abu-Amer et al. 1998). In agreement with a previous study (Kitaura et al. 2004), we found that the number of TRAP-positive osteoclasts significantly increased in mice treated with TNF-[alpha] alone. In contrast, mice treated with both TNF-[alpha] and sanguiin H-6, the number of TRAP-positive osteoclasts significantly reduced (Fig. 7a and b). TNF-[alpha] injection increased the percentage of the eroded surface (ES) per bone surface (BS); hence, the addition of sanguiin H-6 reduced the percentage of ES/BS (Fig. 7c). These results suggest that sanguiin H-6 potently inhibits TNF-[alpha]-mediated inflammatory osteodastogenesis in vivo.
Ellagic acid inhibits osteoclast formation
Sanguiin H-6 is a hydrolysable, high-molecular weight tannin that does not diffuse through the cell membrane easily. Sanguiin H-6 most likely hydrolyzes into small molecule such as ellagic acid in the medium. We evaluated the inhibitory effects of sanguiin H-6 and ellagic acid on osteoclast formation. BMMs were cultured with indicated concentrations of sanguiin H-6 or ellagic acid in the presence of M-CSF and RANKL for 72 h. Both compounds dose-dependently inhibited multinucleated osteoclast formation (Fig. 8). The concentration of these compounds required for 50% of inhibition of osteoclast formation ([IC.sub.50]) was calculated using concentration-activity curves. The [IC.sub.50] values of sanguiin H-6 and ellagic acid were approximately 0.42 [micro]M and 1.43 [micro]M, respectively.
Here, for the first time, we report the anti-bone resorbing activity of R. parvifolius L. extract. Furthermore, sanguiin H-6 inhibited the osteoclastogenesis in vitro. It also markedly inhibited the bone-resorption activity, upregulated HO-1 expression, decreased intracellular ROS levels, and reduced I[kappa]B[alpha] and p38 MAPK phosphorylation in osteoclasts. Moreover, sanguiin H-6 downregulated osteoclast marker proteins, including NFATc1, c-Src, and cathepsin K, inhibited RANKL-induced nuclear translocation of NFATc1, phosphorylated-cFos, and NF-[kappa]B, as well as TNF-[alpha]-induced osteoclastogenesis in vivo. Taken together, these results demonstrate that sanguiin H-6 has potent inhibitory effects on bone resorption via suppression of osteoclastogenesis.
Osteoclastogenesis is activated by MAPKs, including p38 MAPK, ERK, and JNK. Among these MAPKs, sanguiin H-6 strongly inhibited p38 MAPK activation. Previous studies have reported the importance of the p38 MAPK in RANKL-induced osteoclastogenesis (Matsumoto et al. 2000; Lee et al. 2002). Sharma et al. (2007) reported that the p38 MAPK regulates NFATc1 induction during RANKL-induced osteoclastogenesis. In addition to inhibition of NFATc1 expression, our study showed that sanguiin H-6 inhibits cathepsin K and c-Src expression, which are regulated by NFATc1 (Song et al. 2009). Thus, the finding indicates that sanguiin H-6 inhibits the p38 MAPK-NFATc1-dependent pathway.
Previous reports have shown that genetic suppression of p38 MAPK in embryonic fibroblasts and pharmacological inhibition of p38 MAPK in RAW264.7 macrophage-like cells upregulate HO-1 (Naidu et al. 2009). Consistent with these data, we found that p38 MAPK inhibition promotes HO-1 upregulation in osteoclasts (Sakai et al. 2012). HO-1 contributes to the synthesis of biliverdin and bilirubin, which are endogenous ROS scavengers, and HO-1 silencing induces ROS production (Ryter et al. 2006). In our study, sanguiin H-6 enhanced HO-1 expression and reduced intracellular ROS production, consistent with previous results demonstrating ROS accumulation in HO-1-deficient cells (Poss and Tonegawa, 1997). Therefore, our results suggest that p38 MAPK inhibition by sanguiin H-6 effectively promotes HO-1 upregulation and reduces ROS production.
The bone-resorption activity of mature osteoclasts is primarily regulated by the cytoskeletal organization. Upon contact with a mineralized surface, the osteoclast cytoskeleton creates a resorptive microenvironment and secretes bone-degrading molecules, c-Src and cathepsin K are central molecules involved in osteoclastic bone resorption. c-Src is a non-receptor-type tyrosine kinase that regulates the attachment of osteoclasts to the bone surface. Previous studies have shown that Src-deficient mice exhibit an osteopetrotic phenotype (Soriano et al. 1991) and that Src-deficient osteoclasts fail to form ruffled borders, resorb lacunae (Boyce et al. 1992), or have impaired spreading (Lakkakorpi et al., 2000). Herein, sanguiin H-6 significantly suppressed c-Src protein expression in osteoclasts. This may have caused the observed impairment in the spreading of mature osteoclasts and the lack of pit formation following sanguiin H-6 treatment in assayed osteoclasts. Furthermore, cathepsin K is an osteoclast-specific lysosomal cysteine proteinase that dissolves bone matrix proteins, such as type 1 collagen. Cathepsin K gene mutations have been linked to pycnodysostosis, a hereditary bone disorder characterized by defective bone resorption (Gelb et al. 1996), and cathepsin K-deficient mice exhibit osteopetrosis (Saftig et al. 1998). Herein, sanguiin H-6 strongly suppressed cathepsin K and c-Src expression, suggesting that it could be a potent bone resorption inhibitor.
Osteoclast differentiation is regulated by various transcription factors, including NFATc1, c-Fos, and NF-[kappa]B (Boyle et al. 2003). NFATc1 is activated by binding to its promoter region. Both c-Fos and NF-[kappa]B can bind to the promoter region of NFATc1 and synergistically regulate NFATc1 expression (Asagiri et al. 2005; Chuvpilo et al. 2002). Interestingly, c-Fos-deficient mice showed defects in NFATc1 induction (Matsuo et al. 2004). Furthermore, previous studies have demonstrated that ROS activates NF-[kappa]B (Schreck et al. 1992). Our results revealed that sanguiin H-6 decreased ROS production and inhibited I[kappa]B phosphorylation and nuclear translocation of NF-[kappa]B, suggesting that NF-[kappa]B activation was prevented. Taken together, sanguiin H-6 inhibits osteoclastogenesis by suppressing NFATc1, c-Fos, and NF-[kappa]B pathways.
Chronic inflammation progresses bone loss. TNF-[alpha] is a potent osteoclastogenic cytokine involved in the pathogenesis of chronic inflammatory osteolysis, seen in diseases such as rheumatoid arthritis (Cope and Maini, 1995). TNF-[alpha] stimulates bone resorption by increasing osteoclast differentiation and activity through the NF-[kappa]B signaling pathway (Abu-Amer et al. 1998). Herein, sanguiin H-6 suppressed TNF-[alpha]-induced osteoclast formation in vivo. This effect may be due to the suppression of TNF-[alpha]-induced activation of the NF-[kappa]B signaling pathway.
The bioactive components of natural products have been recognized as good sources of novel therapeutic drugs. Although Rubus parvifolius L. has been traditionally used as herbal medicine, there is no published data documenting its use in bone diseases. As seen in the Supplementary content Sib, R. parvifolius L. extract is rich in sanguiin H-6 and lambertianin A. Similar to sanguiin H-6, lambertianin A has anti-osteodastogenic activity (data not shown). These results suggest that R. parvifolius L. could be a good source of therapeutic drugs for bone diseases.
Numerous studies have demonstrated that plant components have inhibitory effects on osteoclastogenesis. Indeed, several phenolic compounds, such as luteolin, quercetin, apigenin, EGCG, and resveratrol, have strong anti-osteoclastogenic potential. A recent study determined the concentrations of luteolin, quercetin, apigenin, acteoside, and EGCG required for 50% inhibition of osteoclast formation ([IC.sub.50]), and found that relatively low concentrations were necessary (2.6, 2.3, 4.8, 5.1, and 6.6 [micro]M, respectively) (Lee et al. 2013). Although luteolin and quercetin strongly inhibited osteoclastogenesis, they were highly toxic at 10 [micro]M. Resveratrol is a polyphenolic phytoestrogen with potent anti-osteoclastogenic activities. Notably, at 10[micro]M, resveratrol completely inhibits osteoclastogenesis; however, it also has cytotoxic effects (He et al. 2010). Since the experimental conditions of each study differed, it is difficult to directly compare our results with those of previous studies. Nonetheless, we did observe strong anti-osteoclastogenic activity and cytotoxicity of resveratrol at 10[micro]M (data not shown). Sanguiin H-6 exhibited the strongest inhibition effect. Additionally, sanguiin H-6 was not cytotoxic even at 5 [micro]M. The [IC.sub.50] value of sanguiin H-6 and ellagic acid on osteoclast formation was approximately 0.42 [micro]M and 1.43 [micro]M, respectively. The data suggested that sanguiin H-6 had anti-osteoclastogenic activity around threefold higher than that of ellagic acid. Sanguiin H-6 most likely hydrolyzes to ellagic acid in medium (1 mol of sanguiin H-6 hydrolyzes to 3 moles of ellagic acid). These results suggest that ellagic acid might act as an anti-osteoclastogenic compound.
This is the first report to show that R. parvifolius L. extract had anti-bone resorption activity. It is also the first to show that sanguiin H-6, a major constituent of R. parvifolius L., inhibited RANKL-induced osteoclast differentiation and function via inhibition of c-Fos and NFATc1 activation in vitro. The inhibitory action of sanguiin H-6 on RANKL-induced activation of the NF-[kappa]B and p38 MAPK pathways was most likely to be involved in its antiosteoclastogenic activity. Further, sanguiin H-6 prevented TNF-[alpha]-induced osteoclast formation in vivo. Thus, our findings strongly suggest that sanguiin H-6 should be further evaluated as a potential therapeutic candidate for various bone diseases associated with excessive osteoclast formation and bone loss.
Received 10 April 2015
Revised 11 March 2016
Accepted 6 April 2016
Conflict of interest
The authors declare that there is no conflict of interest.
We thank Mr. Shun Narahara, Mrs. Haruna Matsushima-Narahara, and Dr. Jin-Ping Hu from the Division of Dental Pharmacology, Nagasaki University, and Mr. Tomomichi Kobayashi from the Biopathology Institute for their help with the in vivo study. This study was partly supported by Grants-in-Aid for Scientific Research from Ministry of Education, Science, and Culture of Japan to E. S. (grant number: 25462892)
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.04.002.
Abu-Amer, Y., Ross, F.P., McHugh, K.P., Livolsi, A., Peyron, J.F., Teitelbaum, S.L., 1998. Tumor necrosis factor-alpha activation of nuclear transcription factor-kappaB in marrow macrophages is mediated by c-Src tyrosine phosphrylation of Ikappa B alpha. J. Biol. Chem. 273 (45), 29417-29423.
Asagiri, M., Sato, K., Usami, T., Ochi, S., Nishina, H., Yoshida. H., Morita, I., Wagner, E.P, Mak, T.W., Serfling, E., Takayanagi, H., 2005. Autoamplification of NFATc1 expression determines its essential role in bone homeostasis. J. Exp. Med. 202 (9), 1261-1269.
Boyce, B.F., Yoneda, T., Lowe, C., Soriano, P, Mundy, G.R., 1992. Requirement of pp60c-src expression for osteoclasts to form ruffled borders and resorb bone in mice. J. Clin. Invest. 90 (4), 1622-1627.
Boyle, W.J., Simonet, W.S., Lacey, D.L., 2003. Osteoclast differentiation and activation. Nature 423 (6937), 337-342.
Cope, A.P, Maini, R.N., 1995. Soluble tumor necrosis factor receptors in arthritis. J. Rheumatol. 22 (3), 382-384.
Chuvpilo, S., Jankevics, E., Tyrsin, D., Akimzhanov, A., Moroz, D., Jha, M.K., Schulze-Luehrmann, J., Santner-Nanan, B., Feoktistova, E., Konig, T., Avots, A., Schmitt, E., Berberich-Siebelt, F., Shimpl, A., Serfling, E., 2002. Autoregulation of NFATd/A expression facilitates effector T cells to escape from rapid apoptosis. Immunity 16 (6), 881-895.
Gao, J., Sun, C., Yang, J., Shi, J., Du. Y, Zhang, Y., Li, J., Wan, H., 2011. Evaluation of the hepatoprotective and antioxidant activities of Rubus parvifolius L. J Zhejiang Univ-Sci B. 12 (2), 135-142.
Gelb, B.D., Shi, G.P., Chapman, H.A., Desnick, R.J., 1996. Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science 273 (5279), 1236-1238.
He, X., Andersson, G., Lindgren, U., Li, Y., 2010. Resveratrol prevents RANKL-induced osteoclast differentiation of murine osteoclast progenitor RAW 264.7 cells through inhibition of ROS production. Biochem. Biophys. Res. Commun. 401 (3), 356-362.
Kamiya, T., Kobayashi, Y., Kanaoka, K., Nakashima, T., Kato, Y., Mizuno, A., Sakai, H., 1998. Fluorescence microscopic demonstration of cathepsin K activity as the major lysosomal cysteine proteinase in osteoclasts. J. Biochem. 123 (4), 752-759.
Kitaura, H., Sands, M.S., Aya, K., Zhou, P., Hirayama, T., Uthgenannt, B., Wei, S., Takeshita, S., Novack, D.V., Silva, M.J., Abu-Amer, Y., Ross, F.P., Teitelbaum, S.L., 2004. Marrow stromal cells and osteoclast precursors differentially contribute to TNF-alpha-induced osteoclastogenesis in vivo. J. Immunol. 173 (8), 4838-4846.
Lakkakorpi, P.T., Nakamura. L, Young, M., Lipfert, L., Rodan, GA, Duong, L.T., 2000. Abnormal localization and hyperclustering of avb3 integrins and associated proteins in Src-deficient or tyrosine A9-treated osteoclasts. J Cell Science 114, 149-160.
Lee, N.H., Lee, M.Y., Lee, J.A., Jung, D.Y., Seo, C.S., Kim, J.H., Shin, H.K., 2010. Anti-asthmatic effect of Sanguisorba officinalis L. and potential role of heme oxygenase-1 in an ovalbumin-induced murine asthma model. Int J Mol Med. 26 (2), 201-208.
Lee, N.K., Choi, Y.G., Baik, J.Y., Han, S.Y., Jeong, D.W., Bae, Y.S., Kim, N., Lee, S.Y., 2005. A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation. Blood 106 (3), 852-859.
Lee, S.E., Woo. K.M., Kim, S.Y., Kim, H.-M., Kwack, K., Lee, Z.H., Kim, H.-H., 2002. The phosphatidylinositol 3-kinase, p38, and extracellular signal-regulated kinase pathways are involved in osteoclast differentiation. Bone 30 (1), 71-77.
Lee, S.Y., Lee, K.S., Yi, S.H., Kook, S.H., Lee, J.C., 2013. Acteoside suppresses RANKL-mediated osteoclastogenesis by inhibiting c-Fos induction and NF-kappaB pathway and attenuating ROS production. PLoS One 8, e80873.
Matsumoto, M., Sudo, T., Saito, T., Osada, H., Tsujimoto, M., 2000. Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL). J. Biol. Chem. 275 (40), 31155-31161.
Matsuo, K., Galson, D.L, Zhao, C., Peng, L, Bachler, MA, Amano, H., Aburatani, H., Ishikawa, H.. Wagner, E.F., 2004. Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-Fos. J. Biol. Chem. 279 (25), 26475-26480.
Naidu, S., Viayan, V., Santoso, S., Kietzmann, T., Immenschuh, S., 2009. Inhibition and genetic deficiency of p38 MAPK up-regulates heme oxygenase-1 gene expression via Nrf2. J. Immunol. 182 (11), 7048-7057.
Poss, K.D., Tonegawa, S., 1997. Reduced stress defense in heme oxygenase 1-deficient cells. Proc. Natl. Acad. Sci. U. S. A. 94 (20), 10925-10930.
Ryter, S.W., Alam, J., Choi, A.M., 2006. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol. Rev. 86 (2), 583-650.
Saftig, P., Hunziker, E., Wehmeyer, O., Jones, S., Boyde, A., Rommerskirch, W., Moritz, J.D., Schu, P., von Figura, K., 1998. Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice. Proc Natl Acad Sci U S A. 95 (23), 13453-13458.
Sakai, E., Shimada-Sugawara, M., Nishishita, K., Fukuma, Y., Naito, M., Okamoto, K., Nakayama, K., Tsukuba, T., 2012. Suppression of RANKL-dependent heme oxygenase-1 is required for high mobility group box 1 release and osteoclastogenesis. J. Cell. Biochem. 113 (2), 486-498.
Sakai, E., Shimada-Sugawara, M., Yamaguchi, Y., Sakamoto, H., Fumimoto, R., Fukuma, Y., Nishishita, K., Okamoto, K., Tsukkuba, T., 2013. Fisetin inhibits osteoclastogenesis through prevention of RANKL-induced ROS production by Nrf2-mediated up-regulation of phase II antioxidant enzymes. J. Pharmacol. Sci. 121 (4), 288-298.
Schreck, R., Meier, B., Mannel, D.N., Droge, W., Baeuerle, PA, 1992. Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells. J. Exp. Med. 175 (5), 1181-1194.
Sharma, S.M., Bronisz, A., Hu, R., Patel, K., Mansky, K.C., Sif, S., 2007. MITF and PU.1 recruit p38 MAPK and NFATc1 to target genes during osteoclast differentiation. J. Biol. Chem. 282 (21), 15921-15929.
Song, I., Kim, J.H., Kim, K., Jin, H.M., Youn, B.U.. Kim, N., 2009. Regulatory mechanism of NFATc1 in RANKL-induced osteoclast activation. FEBS Lett. 583 (14). 2435-2440.
Soriano, P., Montgomery. C., Geske. R., Bradley, A., 1991. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64 (4), 693-702.
Takayanagi, H., Kim, T., Koga, T., Nishina, H., Isshiki, M., Yoshida, H., Saiura, A., Isobe. M., Yokochi, T., Inoue, J., Wagner, E.F., Mak, T.W., Kodama, T., Taniguchi, T., 2002. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev. Cell 3 (6), 889-901.
Tanaka, T., Nonaka, G.t Nihioka, I., 1985. Tannins and related compounds. Part 28. Revision of the structures of Sanguiin H-6, H-2, and H-3, and isolation and characterization of Sanguiin H-ll, a novel tetrameric hydrolysable tannin, and seven related tannins, from Sanguisorba officinalis. J Chem Research 1985, 176-177.
Watanabe, T., Kukita, T., Kukita, A., Wada, N., Toh, K., Nagata, K., Nomiyama, H., Iijima, T., 2004. Direct stimulation of osteodastogenesis by MIP-1[alpha]: evidence obtained from studies using RAW264 cell clone highly responsive to RANKL. J. Endocrinol. 180, 193-201.
Yokozawa, T., Chen, C.P., Tanaka, T., Kitani, K., 2002, Effects of sanguiin H-6, a component of Sanguisorbae Radix, on lipopolysaccharide-stimulated nitric oxide production. Biochem. Pharmacol. 63 (5), 853-858.
Eiko Sakai (a),  *, Yuri Aoki (a), Masako Yoshimatsu (b), Kazuhisa Nishishita (a), Mayumi Iwatake (a), Yutaka Fukuma (a), Kuniaki Okamoto (a), Takashi Tanaka (c), Takayuki Tsukuba (a)
(a) Division of Dental Pharmacology, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan
(b) Division of Orthodontics and Dentofacial Orthopedics, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan
(c) Division of Natural Product Chemistry, Department of Molecular Medicinal Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-813J, Japan
 Present address: Division of Applied Prosthodontics, Department of Developmental and Reconstructive Medicine, Nagasaki-University Graduate School of Biochemical Sciences. Nagasaki 852-8588, Japan.
Abbreviations: NF-[kappa]b, nuclear factor-[kappa]b; RANKL, receptor activator of nuclear factor-[kappa]b ligand; TNF-[alpha], tumor necrosis factor-alpha; M-CSF, macrophage colonystimulating factor; MAPK, mitogen-activated protein kinase; NFATc1, nuclear factor of activated T-cells cytoplasmic 1; TRAP, tartrate-resistant acid phosphatase; c-Src, cellular tyrosine protein kinase; ROS, reactive oxygen species; HO-1, heme oxygenase1; NO, nitric oxide; bmms, bone marrow macrophages; RT-PCR, real-time polymerase chain reaction; NAC, N-acetylcysteine; SD, standard deviation.
* Corresponding author at: Division of Dental Pharmacology, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan. Tel.: +81 95 819 7654; fax: +81 95 819 7655.
E-mail address: email@example.com (E. Sakai).
Please note: Some tables or figures were omitted from this article.