Effects of jsog-6 on protection against bone loss in ovariectomized mice through regulation of osteoblast differentiation and osteoclast formation

Chung et al. BMC Complementary and Alternative Medicine 2014, 14:184http://www.biomedcentral.com/1472-6882/14/184 Effects of JSOG-6 on protection against bone lossin ovariectomized mice through regulation ofosteoblast differentiation and osteoclast formation Hwa-Jin Chung1, Lan Cho1, Joon-Shik Shin2, Jinho Lee2, In-Hyuk Ha2, Hyen Joo Park1 and Sang Kook Lee1* Background: JSOG-6 is used as a traditional medicine to relieve the symptoms associated with inflammation,rheumatism, and osteoporosis in Korea. In the present study, we investigated the effects of JSOG-6 on bone lossprevention both in in vitro and in vivo as well as its underlying mechanism of action.
Methods: Protection against bone loss was assessed in an ovariectomized (OVX) mouse model. Bonemicroarchitecture was measured using a micro-computed tomography to detect the parameters of three-dimensionalstructure of a trabecular bone. Serum biomarkers were also evaluated in an OVX-induced model. Osteoclasts derivedfrom mouse bone marrow cells (BMCs) and osteoblastic MC3T3-E1 cells were also employed to investigate themechanism of action.
Results: Oral administration of JSOG-6 significantly increased the bone mineral density (BMD) of the femur in OVX micein vivo. Especially, the reduced Tb.No (trabecular bone number) in the OVX group was significantly recovered byJSOG-6 treatment. The serum levels of alkaline phosphatase (ALP), osteocalcin, C-terminal telopeptide, andtartrate-resistant acid phosphatase, biomarkers of bone resorption, were significantly elevated in OVX mice, butJSOG-6 effectively inhibited the increase in OVX mice. JSOG-6 was also found to enhance the osteoblasticdifferentiation and maturation with the increase of the density and ALP activity, a marker of osteoblasticdifferentiation, as well as calcium deposition, a marker of osteoblastic maturation in MC3T3-E1 cells. The effectsof JSOG-6 on osteoblastic differentiation were also associated in part with the increase of ALP and OPN mRNAexpressions and the decrease of RANKL mRNA expression in MC3T3-E1 cells.
Conclusions: The findings demonstrate that JSOG-6 induced protection against bone loss in OVX mice, and itsanti-osteoporotic property might be, in part, a function of the stimulation of osteoblast differentiation and theinhibition of osteoclast formation. These findings suggest that JSOG-6 might be an applicable therapeutictraditional medicine for the regulation of the osteoporotic response.
Keywords: JSOG-6, Ovariectomized mice, Bone loss, Osteoclast, Osteoblast altered. Bone mass is regulated by continuous bone re- Osteoporosis is an age-dependent metabolic bone disease modeling through bone resorption and bone formation by characterized by the decrease in bone mass, the deterior- osteoclasts and osteoblasts, respectively . Indeed, bone ation of bone tissue, and an increased risk of fractures homeostasis depends on maintaining a balance between []. In the process of osteoporosis, the bone mineral the activities of bone-forming osteoblasts and bone- density (BMD) is decreased, bone microarchitecture is de- resorbing osteoclasts , which is ultimately determined teriorated, and a variety of proteins in bone are also by the proliferation and differentiation of progenitors ofthese two bone-associated cells.
Osteoclasts are derived from hematopoietic cells within * Correspondence: 1 the monocyte/macrophage lineage [and regulated by the College of Pharmacy, Natural Products Research Institute, Seoul National University, San 56-1 Sillim-dong, Gwanak-gu, Seoul 151-742, KoreaFull list of author information is available at the end of the article 2014 Chung et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public DomainDedication waiver applies to the data made available in this article,unless otherwise stated.
Chung et al. BMC Complementary and Alternative Medicine 2014, 14:184 combined action of the receptor activator of the NF-κB ligand (RANKL) However, osteoblasts induce the Female ICR mice (18–20 g, 8 weeks old) were purchased increase in bone mass by inhibiting the ability of osteo- from Central Laboratory Animal Inc. (Seoul, Korea). Ani- clasts. Osteoblasts also express RANKL and osteopro- mals were housed under standard laboratory conditions tegerin (OPG), a decoy receptor of RANKL, which are with free access to food and water. The temperature was essential factors regulating the formation and activation thermostatically regulated to 22°C ± 2°C, and a 12-hour of osteoclasts (osteoclastogenesis) Therefore, the light/dark schedule was maintained. Prior to their use, they balance of RANKL and OPG mainly contributes to bone were allowed one week for acclimatization within the work remodeling because RANKL stimulates osteoclastogen- area environment. All animal experiments were carried out esis, while OPG suppresses bone resorption [Yet, in accordance with the Institutional Animal Care and Use excessive osteoclastogenesis and defects in osteoblasto- Committee Guidelines of Ewha Womans University (per- genesis are associated with bone diseases such as osteo- mission number: EWHA2010-2-07).
porosis and rheumatoid arthritis [].
At 9 weeks of age, mice were bilaterally OVX, and 8 mice JSOG-6 (named Bogangyeongol-hwan) consists of a mix- were Sham-operated (Sham). After 1 week of recovery from ture of six crude drugs, and each drug has been widely used surgery, the OVX mice were randomly divided into 5 in traditional medicine to treat various bone disorders such groups of 8 mice per group (OVX control, 17β-estradiol as arthritis, degenerative disc disease and osteoporosis. Of (E2, 20 μg/kg), and JSOG-6 (50, 150, or 450 mg/kg)).
the six components of JSOG-6, Harpagophytum procum- JSOG-6 was orally administered in distilled water (0.3 mL) bens var. sublobatum (Engl.) Stapf, radix, called Devil's claw, for 12 weeks, and the same volume of distilled water was is an herbal substance commonly used by patients with used in the Sham- and OVX control groups. After 12 weeks osteoarthritis (OA). Anti-inflammatory activities of the of treatment, the animals were sacrificed, and blood sam- root extracts of H. procumbens var. sublobatum have ples were collected for serum isolation. The femur bones also been reported in various inflammation models were dissected and divested of soft tissue for analysis of tra- Another component of JSOG-6, Drynaria for- tunei (Kunze ex Mett.) J. Sm., rhizome also showed ananti-osteogenic effect in a bone-resorption model [ Analysis of serum bone biomarkers as well as anti-inflammatory properties [Poria cocos Serum calcium (Ca) and phosphorus (P) were measured F.A.Wolf, sclerotium and Rehmannia glutinosa as previously described ]. The serum concentrations (Gaertn.) DC., radix exhibited anti-inflammatory of osteocalcin (OCN) and alkaline phosphatase (ALP) effects. Furthermore, Panax ginseng C.A.Mey., radix activity were assayed using an ELISA kit (Biomedical showed an anti-osteoporotic effect in an OVX rat model Technologies Inc., Stoughton, MA, USA) and QuantiChrome These reported data suggest that JSOG-6 might ALP assay kit (DALP-250, BioAssay Systems, CA, USA) have the potential to alleviate the symptoms of bone according to the manufacturer's instructions, respectively.
The serum levels of C-terminal telopeptides (CTx), bone In the present study, we investigated the activities of resorption biomarkers that indicate osteoclastic activity, JSOG-6 in vitro and in in vivo bone-remodeling models were also analyzed using commercial ELISA kits (Serum and examined its underlying molecular mechanism.
CrossLaps, Nordic Bioscience, Herlev, Denmark). Thetartrate-resistant acid phosphatase (TRAP) concentrationwas determined by a mouse TRAP assay kit (Suomen Bioanalytikka Oy, Turku, Finland).
Preparation of test samplesA mixture of six crude drugs (Harpagophytum procumbens Analysis of bone microarchitecture var. sublobatum (Engl.) Stapf, radix (120 g), Drynaria fortu- Bone microarchitecture of the femur was scanned using a nei (Kunze ex Mett.) J. Sm., rhizome (120 g), Equus asinus micro-computed tomography (μCT system, SkyScan 1076, L., gelatinized (120 g), Poria cocos F.A.Wolf, sclerotium Aart-selaar, Belgium) in the region of 0.6–2.1 mm from (120 g), Rehmannia glutinosa (Gaertn.) DC., radix (120 g), the growth plate. The X-ray source was set at a voltage of and Panax ginseng C.A.Mey., radix (120 g)) was boiled in 50 kV and a current of 200 μA and filtered with a 0.5 mm tap water (10 L) for 6 h, and the extract was freeze-dried to aluminum filter. The scanning angular rotation was 180° obtain the JSOG-6 extracts (259 g, 35.6%). The crude drugs with an angular step of 0.5°. The voxel size was fixed at were purchased from an herbal market in Seoul, Korea, 8.9 μm. The morphometric index of the bone region was and authenticated by Dr. S.H. Lee, Jaseng Hospital of determined from the microtomographic data using a 3D Korean Medicine in Seoul, Korea. Voucher specimens of image (SkyScan). The following measures characterizing the plants used in this study were deposited in the herbar- the three-dimensional structure of a trabecular bone were ium at Jaseng Hospital of Oriental Medicine.
determined: the ratio of bone components to volume of Chung et al. BMC Complementary and Alternative Medicine 2014, 14:184 interest (BV/TV,%), trabecular thickness (Tb.Th, mm), tra- (2 × 104 cells/mL) were incubated with or without vari- becular separation (Tb.Sp, mm), trabecular bone number ous concentrations of JSOG-6. After 4 days, the cells (Tb.N, mm-1), and structure model index (SMI).
were washed with PBS and fixed with 70% ethanol for BV/TV indicates the ratio which a trabecular bone is 5 min and then extracted into lysis solution (10 mM Tris, occupied with a given volume of interest, usually mea- 0.1% Triton X-100 buffer (pH 7.5). Enzyme activity was sured as a% value. Tb.Th and Tb.Sp define the shape of determined using p-nitrophenylphosphate (p-NPP) as a a trabecular bone, whereas Tb.N implies the number of substrate. The color change reflecting the conversion of p- traversals made per unit length on a random linear path NPP to p-nitrophenol was measured at 405 nm. The pro- across a trabecular bone through a given volume of inter- tein concentration of cell lysates was measured using the est. SMI quantifies the relative prevalence of rod-, plate-, Bradford assay.
or sphere-shapes in a trabecular bone structure.
Mineralization assay α-Modified minimal essential medium (α-MEM), fetal bo- The calcium deposition of MC3T3-E1 cells was deter- vine serum (FBS), sodium pyruvate, L-glutamine, antibiotic- mined by a previously reported method MC3T3- antimycotic solution, and trypsin-EDTA were purchased E1 cells (2 × 104 cells/mL) were incubated with or from Invitrogen Co. (Grand Island, NY, USA). RANKL without various concentrations of JSOG-6 for 14 days.
and macrophage-colony stimulating factor (M-CSF) were The cells were washed twice with PBS and fixed with purchased from R&D systems (Minneapolis, MN, USA). As- 70% ethanol for 30 min. The fixed MC3T3-E1 cells were stained with 2% Alizarin Red S solution (pH 4.0) 2,5-diphenyltetrazolium bromide (MTT), and other chemicals for 5 min. The plate was washed several times by dis- were obtained from Sigma (St. Louis, MO, USA) unless tilled water, and then the cells were observed under a Cell cultureMouse calvaria MC3T3-E1 cells, obtained from American Real-time reverse transcriptase-polymerase chain reaction Type Culture Collection (ATCC, Rockville, MD, USA), (Real-time RT-PCR) were cultured in α-MEM supplemented with 10% heat- Lipopolysaccharide (LPS), a cell component of Gram- inactivated FBS, 100 units/mL penicillin, 100 μg/mL negative bacteria, is an important mediator of pathological streptomycin, and 0.25 μg/mL amphotericin B. Cells bone destruction associated with inflammation. MC3T3- were incubated at 37°C and 5% CO2 in a humidified E1 cells were stimulated with 1 μg/mL LPS in the presence or absence of JSOG-6 for 48 h. Total cellular RNA wasextracted with TRI reagent (Sigma, St. Louis, MO, USA) Evaluation of growth inhibitory potential according to the manufacturer's recommended procedure.
MC3T3-E1 cells (1 × 104 cells/mL in 96-well plates) Total RNA (1 μg) was reverse-transcribed using oligo-(dT)15 were treated with various concentrations of JSOG-6 primers and avian myeloblastosis virus (AMV) reverse tran- and incubated at 37°C in a humidified atmosphere with scriptase (Promega, Madison, WI, USA).
5% CO2. After JSOG-6 treatment for 72 h, MTT solu- Real-time RT-PCR was conducted with a MiniOpti- tion (5 mg/mL in PBS) was added to the medium (final con system (Bio-Rad, Hercules, CA, USA) using 5 μL concentration 500 μg/mL) and further incubated for of the reverse-transcription product, iQ™ SYBR® Green 4 h. The medium was discarded and 200 μL of (100% or Supermix (Bio-Rad, Hercules, CA, USA) and primers 10%) DMSO was added to each well to dissolve the for- for a total volume of 20 μL. Standard thermal cycler mazan. The absorbance was measured at 570 nm. The conditions were employed (95°C for 20 s, 40 cycles of effect of JSOG-6 on cell growth was calculated as a per- 95°C for 20 s, 56°C for 20 s, and 72°C for 30 s, followed centage relative to solvent-treated control incubations, by 95°C for 1 min, and 55°C for 1 min). The threshold and the IC50 values were calculated using non-linear cycle (CT), the fractional cycle number at which the regression analysis (percent cell proliferation versus amount of amplified target gene reaches a fixed thresh- old, was determined by MJ Opticon Monitor software.
The mean threshold cycle (CT) value for each transcript Alkaline phosphatase (ALP) activity was normalized by dividing it by the mean CT value for The ALP activity was determined according to a previ- the β-actin transcript for that sample. Normalized tran- ously described method MC3T3-E1 cells were in- script levels were expressed relative to those obtained cubated in osteogenic medium containing 400 μM from the control. Real-time RT-PCR primer sequences ascorbic acid and 5 mM β-glycerophosphate. The cells are listed in Table

Chung et al. BMC Complementary and Alternative Medicine 2014, 14:184 Table 1 Sequences of target gene-specific primers used in real-time PCR Preparation of total cell lysates (pH 6.8), 4% SDS, 10% glycerol, 0.006% bromophenol MC3T3-E1 cells (5 × 105 cells/mL in 60 mm dish) blue, 50 mM sodium fluoride, 5 mM sodium orthova- were incubated with or without various concentra- nadate, and 2% β-mercaptoethanol). Cell lysates were tions of JSOG-6 for 48 h. To obtain total cell lysates, boiled for an additional 20 min and stored at −20°C.
the cells were washed with ice-cold PBS and lysed in The protein content of cell lysates was determined by boiling 2X sample loading buffer (250 mM Tris–HCl the BCA method.
Figure 1 Effect of JSOG-6 on bone loss in OVX mice. (A) Change in body weight 12 weeks after ovarietomy. (B) Effect of JSOG-6 on bone 3DmicroCT image of the distal femur in OVX mice. (C) Effect of JSOG-6 on the bone morphometric parameters BMD, BV/TV (%), and Tb.No (1/mm)as analyzed with micro-CT SkyScan CTAN software. Data represent the mean ± S.D. (n = 8). *P < 0.01 indicates statistically significant differencesfrom the OVX mice group.
Chung et al. BMC Complementary and Alternative Medicine 2014, 14:184 Table 2 Effect of JSOG-6 on the serum parameters in OVX mice Calcium concentration Potassium concentration Osteocalcin level The serum levels of calcium, potassium, ALP, CTx, OCN, and TARP were analyzed as described in the . Data represent the mean ± S.D. (n = 8). *P < 0.01indicates statistically significant differences from the OVX mice group.
Figure 2 Effect of JSOG-6 on ALP activity in MC3T3-E1 cells. (A) Cell viability was measured by the MTT method as described in the(B) MC3T3-E1 cells (2 × 104 cells/mL) were incubated with JSOG-6 in the presence of ascorbic acid and β-glycerophosphate for 4 days.
The ALP activity was corrected for the amount of protein. Data represent the mean ± S.D. (n = 3). *P < 0.05, **P < 0.01 indicates statisticallysignificant differences from the control group. N.C., negative control; P.C., positive control (ascorbic acid + β-glycerophosphate).
Chung et al. BMC Complementary and Alternative Medicine 2014, 14:184 Western blot analysis M-CSF. The medium was replaced with 50% volume Equal amounts of cell lysates (40 50 μg) were subjected with and without JSOG-6 every 2 days, and the culture to 8% and 10% SDS-PAGE and electrotransferred onto was terminated after 5 days.
polyvinylidene difluoride (PVDF) membranes (Millipore,MA, USA). Membranes were blocked in PBST (PBS with0.1% Tween-20) containing 5% non-fat dry milk for 1 h at Tartrate-resistant acid phosphatase (TRAP) staining room temperature. After washing 3 times with PBST, Osteoclast differentiation was assessed by TRAP (Sigma- membranes were incubated with primary antibodies Aldrich, St. Louis, MO, USA) activity. 5 days after stimulat- against OPG, RANKL, c-Fos, and TRAF6 (Santa Cruz ing the cells with M-CSF and RANKL (100 and 30 ng/mL, Biotechnology, Santa Cruz, CA, USA), p-ERK, ERK (Cell respectively), the cells were washed with PBS and fixed with Signaling, Danvers, MA, USA), and β-actin (Sigma) for 4% paraformaldehyde for 5 min. The cells were rinsed in 3 h at room temperature or overnight at 4°C. Membranes de-ionized water and incubated in tartrate-staining solution were washed 3 times with PBST and incubated with the in the dark for 1 h at 37°C. The cells were rinsed in de- corresponding secondary antibodies (Santa Cruz) for ionized water and allowed to air dry. TRAP-positive multi- 90 min at room temperature. The blots were washed 3 nucleated cells containing 3 or more nuclei were counted times with PBST and visualized using an enhanced as osteoclasts.
chemiluminescence (ECL) Western blotting detectionsystem (Lab Frontier, Suwon, Korea).
Murine bone marrow-derived osteoclasts All experiments were repeated at least 3 times. Data BMCs were isolated from 4-week-old mice as previously are presented as the mean ± SD for the indicated num- described [The cells were plated into 96-well plates ber of independently performed experiments. The statis- in 30 ng/mL of M-CSF for 24 h. Next, the cells were tical significance within a parameter was evaluated by treated with the indicated concentrations of JSOG-6 in one-way analysis of variation (ANOVA) coupled with the presence of 100 ng/mL of RANKL and 30 ng/mL of Dunnett's t-test.
Figure 3 Effect of JSOG-6 on the mineralization of MC3T3-E1 cells. (A) MC3T3-E1 cells (2 × 104 cells/mL) were incubated with JSOG-6 inthe presence of ascorbic acid and β-glycerophosphate for 14 days. Mineralized nodule formation was assessed by Alizarin red S staining.
Data represent the mean ± S.D. (n = 3). *P < 0.01 indicates statistically significant differences from the control group. (B) Representativemicroscopic observation of JSOG-6 on the formation of calcification nodules with staining Alizarin red S. N.C., negative control; P.C., positivecontrol (ascorbic acid + β-glycerophosphate).
Chung et al. BMC Complementary and Alternative Medicine 2014, 14:184 demonstrated by treatment with E2, a positive control, Effect of JSOG-6 on bone loss in an OVX-induced in the same experimental condition. In addition, the microstructural index parameters were observed in the The anti-osteoporotic activity of JSOG-6 was primarily per- OVX-induced bone loss model (Figure C). The BMD formed in an in vivo experiment employing an OVX- of the OVX group was markedly reduced by 38.7% in induced bone loss mouse model. Body weight change was comparison with the Sham group. The BMD of the tra- monitored during the administration of JSOG-6. As shown becular bone of the femur in mice treated with JSOG-6 in Figure , the increase in body weight in the OVX (50 mg/kg) was shown to be 83.0% higher than that in group was significantly higher than that in the Sham- the OVX group and was not significantly different from operation group (P < 0.01). However, mice given an oral ad- that in the Sham control group (P < 0.01). E2 also in- ministration of JSOG-6 for 12 weeks after OVX showed a creased the BMD by 80.9% in the OVX-induced bone relatively slow increase in body weight compared to the loss mouse model. The BV/TV was also markedly re- OVX group. The estradiol (E2, 10 μg/kg)-treated group duced in the OVX group (64.7%) compared with the Sham showed a similar result. The destruction of the trabecular group, but the treatment of OVX mice with JSOG-6 or E2 bone of the femur was also observed by 3D-μCT. As illus- resulted in a significant increase in the BV/TV in the trated in Figure OVX caused a significant deterioration OVX-induced bone loss model (P < 0.01). The Tb.No in of trabecular bone architecture compared with the Sham- the OVX group was 63.8% lower than that of the Sham control group (P < 0.01). However, treatment with JSOG-6 group, but JSOG-6 treatment (50, 150, and 450 mg/kg) retarded or recovered the destruction of the trabecular significantly recovered the Tb.No value to 113.3, 145.6, bone of the femur in a dose-dependent manner in the and 192.2%, respectively, in a dose-dependent manner OVX-induced bone loss model. The protective effect on than the OVX group. The E2-treated group also showed a trabecular bone microarchitecture was also clearly 150% recovery of Tb.No compared to the OVX group.
1 10 25 100
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JSOG-6 ( g/ml)
JSOG-6 ( g/ml)
JSOG-6 ( g/ml)
1 10 25 100
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Figure 4 Effect of JSOG-6 on osteoblastic gene expression. MC3T3-E1 cells (2 × 104 cells/mL) were treated with the indicated concentrationsof JSOG-6 for 48 h, and the mRNA levels of osteoblastic genes were examined using real-time PCR. The results are presented as a relative expressionlevel compared to unstimulated cells and were normalized to β-actin. Data represent the mean ± S.D. (n = 3). *P < 0.05, **P < 0.01 indicates statisticallysignificant differences from the control group.

Chung et al. BMC Complementary and Alternative Medicine 2014, 14:184 Effect of JSOG-6 on serum biochemical markers in the RANKL play important roles in the maintenance of bone mass and the regulation of bone remodeling. The OPG/ The effects of JSOG-6 on the bone metabolic bio- RANKL ratio is the major index of osteoclastogenic stimu- markers were also determined with the serum of blood lation. JSOG-6 treatment induced the expression of OPG samples collected in OVX mice. The serum levels of cal- mRNA in a concentration-dependent manner, whereas cium and potassium were not significantly different in JSOG-6 significantly suppressed the expression of RANKL the Sham, JSOG-6 treatment, and OVX groups (P < mRNA (*P < 0.05, **P < 0.01). In addition, JSOG-6 signifi- 0.01) (Table The serum levels of the bone formation cantly increased the OPG/RANKL ratio in MC3T3-E1 cells markers ALP and OCN were significantly increased in (*P < 0.01) (Figure ). These findings suggest that JSOG-6 the OVX group compared to the Sham group. However, might modulate the process of osteoclastogenesis via its ef- JSOG-6 treatment decreased the elevated serum levels of fect on the OPG/RANKL gene expressions.
ALP and OCN in the OVX mice (P < 0.01). In addition, theserum level of TRAP, which is responsible for enhanced os-teoclastogenesis and activation of mature osteoclasts for Effect of JSOG-6 on the protein levels of OPG, RANKL, and bone resorption, was increased in the OVX group, but ERK in MC3T3-E1 cells JSOG-6 treatment significantly reduced TRAP activity in a The effect of JSOG-6 on the protein expressions of OPG, dose-dependent manner (P < 0.01). The increase in an add- RANKL, and ERK in MC3T3-E1 cells was evaluated to con- itional bone resorption marker, CTx, in the OVX group firm the activity of JSOG-6 on osteogenic differentiation. As was also inhibited by JSOG-6 treatment (P < 0.01) (Table shown in Figure , JSOG-6 increased the protein expres-sion of OPG in a concentration-dependent manner, but Effect of JSOG-6 on the ALP activity of MC3T3-E1 cells the protein expression of RANKL was suppressed. In The ALP staining activity, a marker of osteoblastic differ- addition, JSOG-6 treatment induced ERK phosphorylation entiation , was evaluated in osteoblastic MC3T3-El cells. There was no effect on MC3T3-El cell viability withJSOG-6 treatment up to 100 μg/mL as determined by theMTT assay (>95% cell viability) (Figure A). Therefore,the cells were treated with up to 100 μg/mL JSOG-6 tounderstand the biological effects of JSOG-6 without caus-ing cytotoxicity. The MC3T3-El cells were differentiatedin the presence of ascorbic acid and β-glycerophosphate inthe cell culture medium. After 4 days of differentiation,JSOG-6 was found to enhance the density and ALP activ-ity in a concentration-dependent manner (Figure ).
Effect of JSOG-6 on the mineralization of MC3T3-E1 cellsExtracellular matrix mineralization is one of the markers ofosteoblastic maturation. Alizarins red S staining was usedto evaluate extracellular matrix calcium deposition. As illus-trated in Figure when the cells were simultaneouslytreated with JSOG-6 for 14 days, the calcification nodules,represented in red, in MC3T3-E1 cells were increased in aconcentration-dependent manner. These results indicatethat the effect of JSOG-6 on the ALP activity and calciumdeposition stimulated osteoblast differentiation.
Effect of JSOG-6 on osteoblastic gene expressionThe effects of JSOG-6 on osteoblast differentiation was fur-ther elucidated using the analysis of expression of osteo- Figure 5 Effect of JSOG-6 on the protein levels of OPG, RANKL, genic differentiation mediators mRNA by real-time RT- and ERK in MC3T3-E1 cells. (A, B) MC3T3-E1 cells (2× 104 cells/mL) PCR. After 2 days of differentiation, JSOG-6 (100 μg/mL) were incubated for 48 h and then treated with JSOG-6 for 48 h. After caused a significant (P < 0.01) increase in the expressions of incubation, total cell extracts were obtained and subjected to Western ALP and OPN mRNA, whereas the expression of OCN blot analysis as described in the Data were representative ofthree separate experiments. β-Actin was used as an internal standard.
mRNA was decreased by JSOG-6 treatment. OPG and Chung et al. BMC Complementary and Alternative Medicine 2014, 14:184 Figure 6 Effect of JSOG-6 on RANKL-induced osteoclast differentiation. (A) Cell viability was measured by the MTT method as described inthe (B) Bone marrow cells (1 × 104 cells/mL) were incubated with JSOG-6 in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL)for 5 days. Osteoclastogenesis was confirmed by TRAP staining. Data represent the mean ± S.D. (n = 3). **P < 0.01 indicates statistically significantdifferences from the control group. (C) The expression of TRAP was determined by Western blot analysis as described in the Data arerepresentative of three separated experiments. β-Actin was used as an internal standard.
Effect of JSOG-6 on osteoclast differentiation JSOG-6 is an herbal preparation derived from six trad- TRAP staining was performed to evaluate the effect of itional medicines that have been clinically used to treat JSOG-6 on RANKL-induced osteoclast differentiation. As inflammation-associated diseases in oriental Korean medi- depicted in Figure , no cytotoxic effect on BMCs was ob- cine. However, the exact pharmacological effect and its served at a test concentration up to 100 μg/mL JSOG-6 as underlying mechanisms of action remain to be elucidated.
determined by the MTT assay (>85% cell survival rate).
Therefore, the present study was performed to investigate BMCs were allowed to differentiate into osteoclasts in the the anti-osteoporotic activity of JSOG-6 in an OVX mouse presence of RANKL and M-CSF for 5 days. JSOG-6 model and to further elucidate its underlying mechanisms inhibited the formation of TRAP-positive cells during of actions both in vitro and in vivo.
RANKL-induced osteoclast differentiation in a concentration- The OVX animal model has been widely used to study dependent manner (Figure Western blot analysis showed postmenopausal osteoporosis mimicked by estrogen in- that JSOG-6 also suppressed the protein expression of sufficiency Deterioration of trabecular 3D micro- TRAF6 in RANKL-stimulated cells (Figure ).
architecture is apparent in the OVX mouse model We found that JSOG-6 prevented the deterioration of microstructural parameters in the distal femur in mice.
Osteoporosis is an age-dependent skeletal metabolic dis- Oral administration of JSOG-6 restored bone loss back ease in which patients suffer from lower bone density in OVX mice (Figure These results indicated that and lower bone mass compared to healthy individuals JSOG-6 was effective not only in preserving bone mass The disease is also associated with a homeostatic but also in rescuing the deterioration of bone microarch- imbalance between bone modeling and bone resorption.
itecture associated with OVX mice.
Although the main cause of the disease is not clear, en- Biochemical markers of bone turnover have been deter- docrinologic, nutritional, and genetic factors are thought mined previously, allowing the evaluation of the status of to be highly involved in osteoporosis.
bone remodeling . Analyses of the serum levels of ALP Chung et al. BMC Complementary and Alternative Medicine 2014, 14:184 and OCN, typical biomarkers of osteoblastic activity, and M-CSF and RANKL are important cytokines that CTx and TRAP, biomarkers of bone resorption, showed cause osteoclast precursors to differentiate into activated that the serum concentrations of ALP, OCN, CTx and osteoclasts . The present study showed that JSOG-6 TRAP in OVX mice were significantly higher than those suppressed RANKL-induced differentiation of osteoclasts in the Sham group. However, the levels of ALP and by down-regulating the protein expression of TRAF6 in TRAP in the JSOG-6-treated group were equivalent to BMCs (Figure These in vitro findings were well corre- the levels in the Sham group (Table These data sug- lated with the in vivo anti-osteoporotic effects of JSOG-6, gest that JSOG-6 most likely prevents bone loss through and thus JSOG-6 might be applicable to the clinic for im- decreased bone turnover.
proving age-dependent bone destruction disease.
In addition, the mechanisms underlying the cellular effects of JSOG-6 were investigated in osteoblasts and oste- oclasts. Osteoblasts, bone-forming cells, synthesize and The present study provides evidence that JSOG-6 has a po- regulate the deposition and mineralization of the extracellu- tential anti-osteoporotic activity both in vitro and in vivo.
lar matrix of bone [In this study, JSOG-6 treatment The underlying mechanisms of action of JSOG-6 are corre- was found to increase the ALP activity in a concentration- lated with the induction of ALP activation in osteoblasts, dependent manner in MC3T3-E1 cells (Figure ). JSOG-6 the increase in calcified bone matrix, and the reduction of also led to an increase of calcium deposition in MC3T3- E1 osteoclast formation. These data provide a pharmaco- logical basis for the use of JSOG-6 as a potential thera- To further explore the mechanism responsible for the ef- peutic strategy for protecting against osteoporotic bone fect of JSOG-6 on the regulation of osteoblasts, the markers loss in clinic.
of bone formation in MC3T3-E1 cells were detected byreal-time RT-PCR. LPS leads to the intracellular induction Competing interestsThe authors declare that they have no competing interest.
of p38, JNK phosphorylation, and NFκB in macrophagesand monocytes, and promotes the differentiation and sur- Authors' contributions vival of osteoclasts through the production of several fac- Conceived and designed the experiments: HJC, LC, and SKL. Performed theexperiments HJC and LC. Analyzed the data: HJC, LC, and SKL. Contributed tors such as PGE2, interleukin 1, RANKL, and TNF reagents, materials and analysis tools: HJC, LC, JSS, JL, IHH, and SKL. Wrote The levels of the bone formation biomarkers ALP, OPN, the paper: HJC, HJP, and SKL. Editing the paper: HJC, HJP, and SKL. All and OPG/RANKL mRNAs were up-regulated by JSOG-6 authors read and approved the final manuscript.
treatment (Figure RANKL drives osteoclastogenesis by providing an This work was supported by a grant from the JASENG Hospital of Korean important signal to osteoclast progenitors through the membrane-anchored receptor RANK in osteoclasts. Oste- oblasts also synthesize and secrete OPG, a decoy receptor 1College of Pharmacy, Natural Products Research Institute, Seoul National of RANKL, which blocks the interaction between RANKL University, San 56-1 Sillim-dong, Gwanak-gu, Seoul 151-742, Korea. 2JasengSpine and Joint Research Institute, Jaseng Medical Foundation, Jaseng and RANK. Therefore, the expression of OPG/RANKL Hospital of Korean Medicine, Seoul 135-896, Korea.
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