The aim of this study was to investigate the bone regenerative effects of fucosterol in estrogen-deficient ovariectomized (OVX) rats.
The aim of this study was to investigate the bone regenerative effects of fucosterol in estrogen-deficient ovariectomized (OVX) rats.
Bone regeneration was assessed in fucosterol-treated MG63 cells in vitro via assays for osteoblast proliferation, alkaline phosphatase, and osteoclast differentiation. Osteoblast proliferation rates, alkaline phosphatase activity, and mineralization were increased in the fucosterol-treated group. Moreover, differentiation of osteoclasts was decreased in the fucosterol-treated group. In the in vivo assay, female rats were OVX. Twelve weeks after ovariectomy, rats were divided into seven groups, each oral administrate everyday for 7 weeks. The bone mineral density of femoral bones was higher in fucosterol groups than in OVX control, and body weight was lower in fucosterol groups. Among bone-quality parameters, bone volume/total volume increased and trabecular separation decreased in fucosterol groups relative to the OVX control. Bone formation and resorption were evaluated using the serum biomarkers osteocalcin and CTx. Fucosterol tripled the level of serum osteocalcin relative to the OVX group and reduced the serum level of CTx.
These results suggest that fucosterol has the dual potentials to activate osteoblasts to stimulate bone formation and suppress differentiation of osteoclasts so as to reduce bone resorption.
bone mineral density
bone volume/total volume
C-terminal telopeptide of type 1 collagen
estrogen replacement therapy
macrophage colony-stimulating factor
micro computed tomography
receptor activator of nuclear factor-κB ligand
tartrate-resistant acid phosphatase
Osteoporosis, a prevalent metabolic bone disease, is characterized by low bone mass and deterioration of bone tissue. It mainly affects postmenopausal women and elderly people, and can result in hip and vertebral fracture .
Bone mass is maintained through repeated cycles of construction and destruction to maintain the balance between bone formation and resorption, which are controlled by osteoblasts and osteoclasts, respectively . The basic processes of bone formation are very similar to the development of other organs with the exception of a unique mineralization phase involving the deposition of calcium and phosphate to provide the structural integrity and framework of mature skeletal bone .
Estrogen replacement therapy (ERT) is effective for the treatment of postmenopausal related conditions such as bone loss . Specifically, several studies have shown that ERT prevents osteoclastogenesis, resulting in the maintenance of skeletal bone mass and a reduction in the risk of fracture in postmenopausal women . Some findings from a Women's Health Initiative trial reported that ERT is associated with several adverse side effects, including coronary events, pulmonary embolism, and breast cancer. [6, 7]. Also, it was reported that there were no ERT-associated adverse side effects for breast cancer from other Women's Health Initiative trials . Recently, attention has focused on phytoestrogens as possible alternatives to or adjuncts of ERT. Phytoestrogens such as isoflavones are plant-derived substances that structurally resemble estrogen and display hormonal activity [9, 10].
Fucosterol was first isolated in 1934 from brown algae. The structural backbone of fucosterol is similar to human estrogen . Fucosterol is abundant in brown algae, including sargassum species such as Hizikia fusiforme. A previous report showed that fucosterol has anti-inflammatory and anticancer effects and protects skin from ultraviolet-induced damage [12-14]. However, to the best of our knowledge, there has been no study of the bone regenerative effects of fucosterol. In the present study, we investigated the ability of fucosterol to counter bone loss in an ovariectomy-induced osteoporosis model in rats.
Fucosterol extract, which is a concentrate of hiziki (H. fusiforme), was obtained from Wan-do Su-hyup (Wan-do, Korea). Hiziki was extracted for 4 h in ethanol at a dried material to solvent ratio of 1:10 w/v. The extract was collected by filtration (Whatman, USA), and the solvent was evaporated under vacuum. The extract was successively partitioned between hexane, chloroform, ethyl acetate, and 50% methanol. Removal of the solvents resulted in the productions of hexane, chloroform, ethyl acetate, and 50% methanol fractions. Chloroform fraction from silica gel column dissolved in dichloromethane and partitioned with 30% MeOH. Lower dichloromethane layer was collected using separation funnel and evaporated to dryness. Dichloromethane fraction was dissolved in methanol containing 2 M of NaOH and heated at 40°C for 30 min for hydrolysis of alkaline-sensitive compounds. Fucosterol from alkaline methanol was extracted with ethyl acetate after adding the same volume of water. Ethyl acetate phase containing crude fucosterol evaporated was to dryness and redissolved in cyclohexane. For additional purification, in cyclohexane, crude fucosterol was loaded to small dry-packed silica gel column (1 × 5 cm). The column was washed in cyclohexane and subsequently eluted with cyclohexane with increasing amount of ethyl acetate (indicated volume percentage of ethyl acetate): (1) 5%, (2) 30%, (3) 50%, and (4) 100%.
A fucosterol standard was purchased from Sigma-Aldrich (St. Louis, USA). Analysis of the fucosterol was performed using a Waters (USA) LC/ESI-MS system equipped with a 2795 Alliance HT separation module, 2996 PDA detector, and Acquity SQD mass analyzer in positive ion mode. Chromatographic separation was carried out on a Waters C18 (0.46 × 25 cm) column. The mobile phase consisted of (A) water containing 0.1% formic acid and (B) ACN containing 0.1% formic acid. Elution was performed as follows: 0–5 min, 80% B; 15 min, 100% B; 20 min, 80% B; 25 min, 80% B. The flow rate was 1 mL/min and the injection volume was 10 μL.
The LC/MS system employed for MassLynx software (Waters). The following conditions were used: ion source temperature 150°C, capillary voltage 3.5–4.5 kV, cone voltage 35–90 V (indicated for each experiment separately), dryer gas (N2) flow 500 L/h, and cone gas 50 L/h. Mass scanning ranged from 50 to 2000 kDa and scanning speed was 0.2 s/scan (Fig. 1) (Table 1).
|Compound||Regression equation||Correlation coefficient (R2)||Composition (μg/mg, extract)|
|Fucosterol||y = 16 796 809.4817x – 247 977 504.9130||0.9992||236.92 ± 5.74|
MG63 cell proliferation was evaluated using a cell-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded at 2 × 103 cells per well in 96-well plates. After 24 h of incubation, the cells were washed with PBS and cultured in DMEM containing the indicated concentrations of fucosterol and 17-β estradiol (0.1, 1, or 10 μg/mL final concentration). 17-β Estradiol was purchased from Sigma-Aldrich. After 72 h of incubation with fucosterol, MTT solution in PBS was added to a final concentration of 0.5 mg/mL, followed by incubation for 3 h at 37°C. At the end of 3 h of incubation, the supernatant medium was removed, and cells were suspended in 100 μL of DMSO for 10 min. Absorbance was measured at 520 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The cell proliferation rates were calculated from the OD readings and represented as percentages of the vehicle control.
MG63 cells were maintained in complete medium. Cells were seeded at 2 × 103 cells per well in 96-well plates. After 24 h of incubation, the medium was changed to a differentiation medium containing 10 mM glycerol 2-phosphate, 50 μg/mL ascorbic acid, and the indicated concentrations of fucosterol or 17-β estradiol (0.1, 1, 10 μg/mL final concentrations in culture medium). After 3 days, the cells were washed with PBS and 0.1% Triton X-100 was added, followed by 1 h of incubation. Supernatant was transferred to 96-well plates, and 0.1 M Tris buffer (pH 10), 5 mM MgSO4, and 6 mM p-nitrophenol phosphate were added, followed by incubation for 1 h at 37°C. The reaction was stopped by adding 1 M H2SO4, and the absorbance was measured at 405 nm. The ALP activity is represented as a specific activity, defined as unit activity per total protein content. Protein content was determined via the Bradford method. One unit of ALP activity represents the hydrolysis of 1 μM p-nitrophenol phosphate per minute at 37°C. The ALP-specific activity was presented in percentage value, which was compared with ALP-specific activity in control cells (cells cultured in standard growth medium) that establish basal activity (100%).
MG63 cells (1 × 104 cells/well in 24-well plates) were maintained in complete medium as described previously. When the cells reached confluence, the medium was changed to a differentiation medium containing 10 mM glycerol 2-phosphate and 50 μg/mL ascorbic acid, which was changed every 3 days. After an additional 21 days, the cells were stained using alizarin red S solution (40 mM; Sigma, St. Louis, USA) for 10 min to evaluate the level of mineralization. After washing with distilled water, the stain was eluted with cetylpyridinium chloride (10%, w/v). The absorbance at 562 nm was measured with a plate reader (Multiskan Spectrum, Thermo, Japan).
RAW264.7 cells were seeded into 24-well plates at a density of 1 × 104 cells per 1 mL of DMEM containing 10% FBS. Inducer of osteoclast in macrophage colony-stimulating factor (M-CSF; 30 ng/mL final) and receptor activator of nuclear factor-κB ligand (RANKL; 100 ng/mL final) were added 1 day after treatment with various concentrations of fucosterol and 17-β estradiol (Sigma-Aldrich). After 3 days, the cells were fixed with fixative solution (25 mL citrate solution, 65 mL acetone, 8 mL 37% formaldehyde) and stained for tartrate-resistant acid phosphatase (TRAP) using a TRAP kit (Sigma) according to the manufacturer's directions. TRAP-positive multinucleated cells containing three or more nuclei were counted as osteoclasts.
RAW264.7 cells were incubated with E2 and fucosterol for 24 h and were stimulated with 200 ng/mL RANKL for 30 min. Cell lysate was prepared by lysing the cells in buffer containing 50 mM Tris–HCl, 137 mM NaCl, 1% NP-40, 10% glycerol, 1 mM sodium fluoride supplemented with protease and phosphatase inhibitors. The lysates were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After blocking with 5% skimmed milk, the membrane was probed with primary antibody (RANK). The same membranes were stripped and reprobed with β-actin. Blots were developed using HRP-conjugated secondary antibody and visualized using ECL (Amersham).
Female Sprague–Dawley rats (6 weeks old) were obtained from Dae-han Bio (Um sung, Korea). Rats were anesthetized with ketamine/Rompun combination (4:1, 10 mg/kg, i.p.) and underwent bilateral ovariectomy at 7 weeks of age. Small incisions were made bilaterally on the sides of the back to expose the ovaries retroperitoneally. The ovaries were clamped and removed, and the uterine tubes were ligated. The muscle and skin were then sutured. The sham procedure consisted of anesthesia, visualization of the ovaries via incisions into the abdominal cavity, and closure of the wounds. Body weight was measured once a week during the experimental period.
The rats were randomly divided into seven groups: (A) normal, (B) sham, (C) ovariectomized (OVX), (D) estradiol 10 mg/kg, (E) fucosterol 25 mg/kg, (F) fucosterol 50 mg/kg, (G) fucosterol 100 mg/kg for 7 weeks beginning 12 weeks postoperation (each n = 8). This study has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Kyung Hee University. IACUC number is KHUASP(SU)-10–17.
Architectural changes in the trabecular bone of the rat femur bone were analyzed with microcomputed tomography (μCT; Skyscan model 1076, Skyscan, Kontich, Belgium). Exposures were carried out at 60 kVp and 400 μA for 400 s. The CT data were obtained and analyzed using software (software CT Analyzer V 18.104.22.168, Skyscan) and the 3D, 2D application program. From among approximately 250 3D images and 400 2D images, five each were selected based on image clarity and angle by observation with the naked eye.
We obtained bone morphometric parameters, including bone volume over total volume (BV/TV), and trabecular number, separation, and thickness by analyzing volume of interest. The scan analysis operator was unaware of the treatments associated with each specimen.
Bone mineral density (BMD) of whole femurs was measured with Lunar DPX-IQ dual-energy X-ray absorptiometry (Lunar Corporation, Madison, WI, USA), and analyzed with PlXlmus2 software.
Serum osteocalcin as an indicator of bone protection, and C-terminal telopeptide of type 1 collagen (CTx) as an indicator of bone resorption were measured after the treatment period using an ELISA reader (VERSAmax, Sunnyvale, USA), Rat-MIDTM Osteocalcin EIA Kit and RatLapsTM EIA Kit (IDS Ltd., UK).
The results were determined using the Statistical Analysis System (PRISM 5, Graph Pad). Analysis of variance was used to identify statistically significant differences among the groups (p < 0.05). Tukey's multiple comparison test was used for post hoc test (p < 0.05). The values were expressed as mean values ± SD.
The proliferation of MG63 cells was examined to investigate osteogenic effects. The proliferation rate of MG63 in the fucosterol-treated group increased to 15% (at 10 μg/mL) compared to the control group. The effect of fucosterol on proliferation activity elevation was dependent on dose. Estradiol-treated group was increased 7% (at 0.1 μg/mL), but high dose of estradiol-treated group did not show an effect on proliferation. The effect of estradiol on proliferation activity elevation was not shown in a dose-dependent manner (Fig. 2).
To evaluate the effect of fucosterol on the differentiation of osteoblastic MG63 cells, the activity of ALP was examined. The ALP activities of MG63 cells in the fucosterol-treated group increased to 55% (at 10 μg/mL) compared to the control group. The effect of fucosterol on ALP activity elevation was dependent on dose. While estradiol-treated group was increased to 36% (at 0.1 μg/mL) compared to control group (Fig. 3).
In addition to proliferation and differentiation of osteoblasts, extracellular matrix mineralization is also a crucial part of bone formation. It was examined whether fucosterol could stimulate the matrix mineralization of MG63 cells, which increases the anabolic activity in bone metabolism. Calcium deposits, which are responsible for mineralized nodule formation in extracellular matrix, were stained with alizarin red S dye that was eluted and measured by spectrophotometer. The administration of fucosterol extract promoted the extracellular matrix mineralization of osteoblasts increased to 37% (at 100 μg/mL) compared to the control group. Estradiol-treated group was increased to 23% (at 100 μg/mL) compared to the control group (Fig. 4).
Homeostasis of bone is maintained by the balance between bone formation and resorption. Osteoclasts are responsible for bone resorption and formed by fusion of monocyte-macrophage precursors . The murine macrophage/osteoclast precursor cell line RAW264.7 is a widely used preosteoclast model that differentiates into osteoclasts when stimulated with M-CSF and RANKL . Osteoclast differentiation was estimated by measuring multinucleated TRAP-positive cell formation in RAW264.7 cells following addition of M-CSF and RANKL. It was observed that the intensity of multinucleated TRAP-positive staining cells was increased in the control group, while there were almost no multinucleated TRAP-positive staining cells in the fucosterol-treated group. Analyses of these images were performed using an image analyzer to quantify the extent of differentiation. The fucosterol-treated group was decreased with the formation of RANKL-stimulated osteoclast to 40% (at 1 μg/mL; inhibition rate 60%) and 34% (at 10 μg/mL; inhibition rate 66%) compared to control group, while the estradiol-treated group was decreased to 73% (at 10 μg/mL; inhibition rate 27%) compared to control group (Fig. 5).
With a view to find out whether fucosterol caused the suppression of RANK signaling due to altering RANK expression or not, Western blot analysis of RANK was performed.
The RANK expression results showed that the fucosterol decreased to 40% (at 10 μg/mL; inhibition rate 60%) compared to control group, while estradiol was decreased to 54% (at 10 μg/mL; inhibition rate 46%) compared to control group (Fig. 6).
Ovariectomy significantly increased the final body weights in the OVX control (389.56 ± 27.01 g) after 15 weeks (p < 0.01), compared with the normal (331.92 ± 24.63 g) and sham groups (309.49 ± 23.99 g). The oral administrations of estradiol and fucosterol group weight gain were significantly decreased to 29.1%, 25.5% compared to OVX group (Table 2), which is similar to [17, 18] data; both estradiol and fucosterol had preventing activity on ovariectomy-induced increasing of body weight [17, 18].
|Measure body weight||Normal||Sham||OVX||Estradiol||Fucosterol 25 mg/kg||Fucosterol 50 mg/kg||Fucosterol 100 mg/kg|
|Initial (g)||204 ± 9.2||202 ± 11.7||203 ± 10.8||202 ± 11.1||200 ± 14.1||201 ± 11.9||203 ± 10.7|
|Final (g)||331 ± 24.6**||309 ± 24.0**||389 ± 27.1||335 ± 18.8**||379 ± 23.5||365 ± 17.9*||341 ± 24.2*|
The mineral density of the femurs of the rats is shown in Fig. 7. The BMD in the OVX group (187.6 ± 2.14 mg/cm2) was significantly decreased compared to the sham group (207.6 ± 5.82 mg/cm2). The oral administrations of estradiol and fucosterol were increased to 7%, 9% of the BMD compared to the OVX group (Fig. 7). The results meant that estradiol and fucosterol treatment significantly ameliorated the loss of bone density induced by the ovariectomy, which is similar to the findings of other phytoestrogen studies [19, 20].
μCT images showed that the microarchitecture of bone was notably improved 7 weeks after treatment with fucosterol compared to the control group (Fig. 8). Bone morphometric parameters were worsened by ovariectomy. The μCT observations showed that oral administration of fucosterol increased the ratio of trabecular BV/TV by 45% for the OVX control, and decreased trabecular separation to 87% for the OVX control (Table 3) .
|Normal||Sham||OVX||Estradiol||Fuco 25||Fuco 50||Fuco 100|
|BV/TV, %||21.40 ± 1.06***||21.32 ± 1.86***||6.62 ± 0.29||8.91 ± 0.37**||7.49 ± 2.44*||8.01 ± 0.37*||9.60 ± 0.55**|
|Tb.Th, mm||0.09 ± 0.00||0.09 ± 0.00||0.10 ± 0.00||0.10 ± 0.00||0.10 ± 0.00||0.10 ± 0.00||0.10 ± 0.00|
|Tb.N, mm−1||2.35 ± 0.15***||2.37 ± 0.26***||0.66 ± 0.01||0.82 ± 0.04**||0.75 ± 0.25||0.77 ± 0.04*||0.97 ± 0.06***|
|Tb.Sp, mm||0.64 ± 0.00***||0.62 ± 0.03***||1.06 ± 0.06||0.93 ± 0.07*||1.01 ± 0.12||1.03 ± 0.07||0.89 ± 0.06**|
The level of serum osteocalcin, a bone formation biomarker [22, 23], was reduced after ovariectomy in the OVX group (272.59 ± 63.51 ng/mL), compared to the sham group (565.59 ± 90.46 ng/mL). The fucosterol-treated group was increased to 201% (822.61 ± 137.84 ng/mL at 100 mg/kg) compared to OVX group. In contrast, the estradiol-treated group was increased to 67% (457.81 ± 131.10 ng/mL at 10 mg/kg) compared to OVX group (Fig. 9A).
Ovariectomy increased the serum level of CTx in the OVX group (118.88 ± 21.51 ng/mL). The fucosterol-treated group was decreased to 60% (47.26 ± 6.53 ng/mL at 100 mg/kg) compared to OVX group. The estradiol-treated group was also decreased to 63% (44.26 ± 8.52 ng/mL at 10 mg/kg) compared to OVX group (Fig. 9B).
Remodeling in bone is a balance between bone formation and resorption driven by osteoblastic and osteoclastic activities, respectively. To study effects on osteoblastogenesis, parameters of bone formation such as proliferation, differentiation, and extracellular matrix mineralization of a human osteoblastic cell line (MG63) were used. The presence of mature osteoblasts during the proliferation phase is indicated by the production of ALP, processing of procollagen to collagen, and the deposition of extracellular matrix containing additional proteins that can later be mineralized . ALP synthesis and ALP activity are increased as osteoblasts differentiate, especially in the early stage of osteogenesis .
Our results showed that fucosterol stimulates the proliferation of MG63 cells 15% while estradiol does 7% above controls. It means fucosterol takes a role about showing no cell cytotoxicity as well as promoting cell proliferation effectively. ALP activity was also elevated in a fucosterol-treated group by 55% while in an estradiol-treated group by 7% above the control group, and mineralization activity was 37% higher in fucosterol-treated group while 23% in estradiol-treated group compared to that in control. These results suggest that fucosterol can increase osteogenesis by showing higher activities such as increasing cell proliferation (8%), activating ALP (19%), and differentiating osteoblasts (14%) compared to those activities of estradiol .
It is known that osteoclast differentiation and activation can be evaluated by the analysis of tumor necrosis factor receptor/TNF-like proteins (e.g. osteoprotegerin), RANK, and RANKL, which together regulates osteoclast function . With respect to resorption of bone, our data showed that RANKL-induced differentiation of the murine macrophage/osteoclast precursor cell line RAW264.7 into osteoclasts was inhibited by the addition of fucosterol and estradiol. Our data showed fucosterol had more increase on osteoblast differentiation and ALP than estradiol, but fucosterol has more inhibition rate on osteoclast differentiation related in vitro assay, RANK expression (33–39%), and TRAP assay (14%) than those inhibition of estradiol, respectively.
Ovariectomy-induced bone loss in rats is a well-accepted model for investigating agents that could help to prevent osteoporosis in postmenopausal women. Ovariectomy induced the standard phenotypes of menopause including gain of weight, loss of BMD, and degradation of microarchitecture of trabecular bone. Our results showed that the BMD of femoral bones and body weight were higher in fucosterol-treated groups than in OVX control groups. These results indicated that the oral administration of fucosterol prevents menopausal events induced by ovariectomy . Bone-quality parameters such as bone size, thickness, trabecular number, and degree of mineralization were highly correlated with BV/TV, as observed by correlation coefficients in this experiment and others . Our results demonstrating increased BV/TV indicate that fucosterol has positive effects on the recovery of microarchitecture of bone.
Biomarkers for bone formation and resorption, osteocalcin and CTx, were evaluated in the serum of rats . Osteocalcin, a biomarker for bone formation, was lowered by ovariectomy, while oral administration of fucosterol dramatically elevated 134% higher level of serum osteocalcin than that of estradiol. CTx, however, indicates the status of bone resorption, and its levels were elevated by ovariectomy . Fucosterol reduced the level of serum CTx. Interestingly, our data showed fucosterol had more activity on related in vivo assay, BMD, and bone-quality parameters including BV/TV and osteocalcin, than estradiol.
The current drug discovery about bone regenerative effects was focused on inhibiting bone resorption and preventing overproduction of cytokines, which is involved in osteoclastogenesis [31-33]. And there were many studies about the activity of estradiol on inhibiting osteoclast differentiation but it is rare to study about increasing osteoblast drug .
However, our results collectively suggest that fucosterol has the dual potentials to activate osteoblasts to stimulate bone formation and suppress differentiation of osteoclasts so as to reduce bone resorption. These dual effects may be necessary for treatment of postmenopausal osteoporosis, as opposed to using a purely antiresorptive approach in order to inhibit bone remodeling. Also, fucosterol may be effective in the treatment of postmenopausal syndrome because it reduced the body weight of OVX rats, but further studies are needed.
In summary, fucosterol stimulated bone regeneration and activated bone formation in ovariectomy-induced osteoporotic rats. Through these biological activities, fucosterol may play an important role in preventing osteoporosis and might be useful as a supplement for postmenopausal women. Further clinical studies will be required to define the precise role of fucosterol in the mechanism of bone metabolism and evaluate its estrogen-like behavior in postmenopausal women.
The authors have declared no conflict of interest.