KR20170011403A - Medical compound comprising Harpagoside by inhibit Osteoclast Differentiation - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for inhibiting osteoclast differentiation and, more specifically, to a pharmaceutical composition for inhibiting osteoclast differentiation, comprising harpagoside as an active ingredient.

Description

TECHNICAL FIELD The present invention relates to an osteoclast differentiation agent for treating inflammation-related bone diseases through inhibition of osteoclast differentiation,

The present invention relates to an agent for the treatment of inflammation-related bone diseases through inhibition of osteoclast differentiation using an active ingredient of hapgocide, and more particularly to an osteoclast cell comprising an osteoclast as an active ingredient for treating inflammatory bone diseases such as inflammatory osteoporosis and rheumatoid arthritis And a therapeutic agent for inflammation-related bone diseases through inhibition of differentiation.

The global aging trend is expected to increase the number of people suffering from bone metabolic diseases such as rheumatoid arthritis, osteoporosis and Paget's disease, and related markets.

goal The highest risk associated with the disease is a high risk of fracture, especially as a primary diagnostic indicator of osteoporosis, which seriously lowers quality of life and increases the risk of death.

The osteoclasts were stimulated with a cytokine receptor activator of the nuclear factor (NF) -κB ligand (RANKL) and a macrophage colony stimulating factor (M-CSF), also known as CSF-1, Absorbing polynuclear giant cells. RANKL, a member of the tumor necrosis factor (TNF) family, is an osteoclast marker that accelerates cell differentiation. M-CSF is a major regulator of osteoclast precursor and macrophage proliferation and survival.

The binding of RANKL and RANK receptors is mediated by a combination of NF-κB, Akt, and mitogen-activated protein kinase (MAPK) including p38, extracellular signal-regulated kinase (ERK), and c-jun N-terminal kinase (JNK) Activates a number of downstream signaling pathways, leading to the activation of nuclear factors of the transcription factors c-Fos and NFATc1, which are required for osteoclast differentiation.

The RANKL-RANK axis also stimulates the Ca ²⁺ signaling pathway through the activation of phospholipase C (PLC) γ. During the osteoclast formation process, activated PLCγ hydrolyzes phosphatidylinositol-4,5-biphosphate (PIP2) with inositol-1,4,5-triphosphate (IP3) to up-regulate intracellular Ca ²⁺ And NFATc1 activation.

It has been reported that various medicinal compounds made from demon's toenail plant extracts that act on osteoclast differentiation and function have therapeutic benefits with few side effects in the treatment or prevention of bone disease, A pharmaceutical composition suitable for guided bone disease has not yet been reported.

Accordingly, a problem to be solved by the present invention is to provide a pharmaceutical composition suitable for inflammation-induced bone disease.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for inhibiting osteoclast differentiation, which comprises a lower granulocyte as an active ingredient.

In one embodiment of the present invention, the pharmaceutical composition for inhibiting osteoclast differentiation is a pharmaceutical composition for treating or preventing inflammation-induced bone disease, and the bone disease is not a pharmaceutical composition for treating or preventing osteoporosis induced by estrogen deficiency.

The pharmaceutical compositions according to the present invention are particularly suitable for inflammation-induced bone diseases and have different therapeutic effects from estrogen-depleted bone diseases.

FIG. 1 is a bottom view of the undergarment according to an embodiment of the present invention and experimental results thereof.
Figure 2 shows that RANKL-mediated osteoclastogenesis is inhibited by RANKL-induced c-Fos and NFATc1 expression in order to determine whether hypoglossacids according to one embodiment of the present invention modulate RANKL-mediated osteoclastogenesis by inhibiting c-Fos and NFATc1 activation. The results of the experiment are shown in Fig.
FIG. 3 shows the results of a hypogaside effect test for RANKL-induced early signaling in order to investigate the mechanisms underlying the suppression of down-chain-mediated inhibition of osteoclastogenesis.
Figure 4 shows the results of the test for inhibiting LPS-induced bone loss in the lower corpus.
Figure 5 is a test result for OVX-mediated bone loss that is not affected by the lower coronary.

Hereinafter, the present invention will be described in detail with reference to the drawings. The following embodiments are provided by way of example so that those skilled in the art can fully understand the spirit of the present invention. Therefore, the present invention is not limited to the embodiments described below, but may be embodied in other forms. In the drawings, the width, length, thickness, etc. of components may be exaggerated for convenience. Like reference numerals designate like elements throughout the specification. In addition, abbreviations displayed throughout this specification should be interpreted to the extent that they are known and used in the art unless otherwise indicated herein.

To identify novel compounds capable of functioning as anti-resorptive agents, some natural compounds were screened by analyzing TRAP staining. HaPagoside (HAR, FIG. 1A) is an iridoglycoside isolated from Harpagophytum procumbens used in the treatment of pain, arthritis, fever, ulcers and boils in South Africa and has been used as an anti-inflammatory and analgesic.

The inventors have discovered that HAR inhibited RANKL-mediated osteoclast differentiation from bone marrow macrophages (BMM). Based on the association between chronic inflammation and bone disease, the pharmacological effects of HAR on RANKL-induced osteoclast differentiation and the bone destruction in lipopolyadergaside (LPS) -induced and ovariectomized (OVX) Were investigated in vitro.

HAR inhibits RANKL-induced osteoclast differentiation and function.

To assess the effect of HAR on osteoclast differentiation, mouse BMM treated with M-CSF and RANKL were cultured in the presence or absence of HAR. TRAP-positive osteoclasts were observed more in the control group and HAR treatment inhibited the formation of TARP-positive osteoclasts in a dose-dependent manner (Fig. 1B). XTT analysis was performed to evaluate whether the HAR inhibitory effect on osteoclast differentiation was due to cytotoxicity. The HAR concentration used in the experiment was not cytotoxic. Formation of the F-actin loop structure in osteoclasts is a crucial marker for bone resorption activity of osteoclasts. Thus, we tested whether HAR inhibited osteoclast function and bone resorption. HAR treatment destroyed F-actin ring formation in a dose-dependent manner (Figure 1C). Additionally, in the case of mature osteoclasts seeded on hydroxyapatite-coated plates, the bone resorption region was observed in the control group, but HAR inhibited reabsorption in a dose-dependent manner (Fig. 1D). These findings suggest that HAR weakens osteoclast formation and bone resorption activity.

HAR inhibits RANKL-induced c-Fos and NFATc1 and osteoclast marker gene expression.

To determine whether HAR regulates RANKL-mediated osteoclastogenesis by inhibiting c-Fos and NFATc1 activation, the effect of HAR on RANKL-induced c-Fos and NFATc1 expression was tested. For BMMs treated with RANKL for 12-48 h, the mRNA expression of c-Fos and NFATc1 proteins and mRNA was increased in the control group as determined by Western blotting and quantitative real-time PCR, respectively, and these were reduced by treatment with HAR 2A and 2B). We also examined whether HAR regulates mRNA expression of TRAP , OSCAR ,? 3-integrin , DC-STAMP , CTR , and cathepsin K , the transcription factor genes involved in osteoclastogenesis and function during RANKL-induced osteoclast differentiation . Each transcript level of these factors was significantly inhibited by HAR treatment (FIG. 2C).

It is well known that c-Fos and NFATc1 are required for the differentiation of osteoclast precursors into bone-resorbing osteoclasts. The c-Fos gene product is derived from the Fos family members c-Fos, FosB, Fos-related antigen (Fra) -1 and Fra-2 and the Jun proteins c-Jun, JunB and JunD -1 complex, which regulates target gene transcription. c-Fos contains a C-terminal transactivation domain that is critically involved in cancer formation and cell transformation. The c-Fos-deficient mice experience osteoclastogenesis due to osteoclast differentiation, and the damaged osteoclast formation in mouse BMM is completely ablated by ectopic c-Fos expression. NFATc1, which is strongly induced by RANKL, belongs to the NFAT transcription factor family first identified in T cells. NFATc1-deficient embryonic stem cells do not differentiate into osteoclasts, which can be induced in BMM by the ectopic expression of NFATc1 in the absence of RANKL. NFATc1-deficient mice are defective in osteoclast formation and exhibit symptoms of osteoporosis. NFATc1 modulation by c-Fos during osteoclast formation is required for the expression of osteoclast-specific genes such as TRAP , OSCAR ,? 3- integrin , DC-STAMP , CTR , and cathepsin K. Thus, these results suggest that HAR diminished mRNA and protein expression of c-Fos and NFATc1 leading to downregulation of various transcription factors associated with osteoclasts.

HAR contains ERK, JNK, and Syk-Btk-PLC [gamma] 2-Ca ²⁺ It regulates osteoclast formation through signaling.

In order to investigate the mechanism underlying the HAR-mediated inhibition of osteoclast formation, the effect of HAR on the RANKL-induced early signaling pathway was tested. The phosphorylation of ERK and JNK induced by RANKL stimulation was slightly down-regulated by HAR treatment (Fig. 3A). Furthermore, phosphorylation of Syk, Btk, and PLCγ2 was also inhibited by HAR (FIG. 3B). Based on these observations, the effect of HAR on the RANKL-induced Ca ²⁺ response was evaluated. HAR treatment reduced the amplitude and frequency of Ca ²⁺ oscillations induced by RANKL (Figure 3C).

Upper signaling leading to the induction of c-Fos and NFATc1 involves RANKL-dependent phosphorylation of several signaling pathways in MAPK and PLCγ2-Ca2 ⁺ pathways critical for osteoclastogenesis. SB203580, a p38 inhibitor, directly destroys osteoclast formation in co-cultures of mouse osteoblasts and BMC by directly targeting osteoclast precursors.

The JNK inhibitor SP600125 stimulated the apoptotic effects of the RANKL / RANK / tumor necrosis factor receptor-related factor on the osteoclasts and the inhibition of ERK by the overexpression of dominant-negative Ras resulted in apoptosis of osteoclast- .

Ca ²⁺ signaling is important for osteoclast differentiation. RANKL triggers PLCγ activation and leads to Ca ²⁺ mobilization. PLCγ family member of PLCγ1 with PLCγ2 is requires the phosphorylation of these tyrosine residues for the catalytic activity, which regulates the intracellular Ca ^{2 +} levels in protein kinase C activation and hematopoietic cells. The PLC and catalyzes the conversion of PIP2 IP3, which play a role in up-regulation of Ca ^{2 +} levels within cells, and diacylglycerol. Although PLCγ1 is not, phosphorylation of PLCγ2 is required for RANKL-mediated Ca ²⁺ signaling in osteoclast differentiation. PLCγ2 complexes with adapter molecules that are important for the differentiation of the scaffold protein GRB-associated binding protein 2 precursor cells into osteoclasts, leading to its phosphorylation and recruitment. PLCγ2 phosphorylation is directly related to Ca ²⁺ oscillation and Ca ²⁺ -dependent translocation of NFATc1 induced by RANKL. The PLCγ2-Ca2 ⁺ -NFATc1 signaling sequence is downstream of the activation of the tyrosine kinases Btk and Tec, which are responsible for the interaction between RANK and immunoreceptor tyrosine-based activation motif (ITAM) signaling and within the receptor- It regulates osteoclast formation through phosphorylation of ITAM, which leads to the recruitment of Syk tyrosine kinase. In this study, HAR inhibited Ca ²⁺ oscillation through inactivation of Syk, Btk, and PLCγ2, resulting in inhibition of RANKL-induced osteoclast differentiation (FIG. 3). These results demonstrate the relevance of ERK and JNK as well as Syk-Btk-PLCγ2-Ca ²⁺ signaling in HAR-mediated inhibition of osteoclastogenesis.

HAR inhibits LPS-induced bone loss.

The major cell wall components of LPS-gram-negative bacteria trigger release of cytokines, chemokines, metalloproteinases, and other agents that induce bone resorption, and are involved in inflammatory-mediated bone loss by macrophages. LPS is a TNF-α, interleukin -1 (IL-1), prostaglandin E ₂ and adjusting the production of RANKL derived from osteoblasts and pre-bone by reducing the osteoclasts and interacts with RANK and expression of M-CSF Receptor Increases the rate of reabsorption. LPS also stimulates osteoclast differentiation and activation through RANKL-induced MAPK signaling.

Therefore, in order to test the effect of HAR on LPS-induced bone loss in vivo, mice were treated with LPS with or without HAR, and after 9 days, micro-computed tomography (micro-CT) Was examined. Three-dimensional analysis revealed loss of soju bone after LPS treatment, which was not present in the HAR treatment group (Figure 4A): Morphometric analysis showed that HAR / BV, TV, Tb.Sp, and Tb.N recover But not Tb.Th (FIG. 4B). Serum levels of these proteins were measured by enzyme-linked immunosorbant assay (ELISA) to determine whether HAR contributed to RANKL and osteo-rotrogenin (OPG) expression. HAR treatment led to downregulation of RANKL and upregulation of OPG, thereby reducing the RANKL / OPG ratio compared to the LPS-only group (FIG. 4C). Histological analysis showed that the number of osteoclasts as well as LPS-induced osteoclast formation and bone loss (Figure 4D) (Figure 4E) were inhibited in the femur of mice treated with HAR and LPS. Finally, the serum C-terminal telopeptide type I collagen (CTX-1) concentration, a bone resorption marker, was substantially higher in the LPS-treated group. Moreover, the expression of CTX-1 in the serum of LPS + HAR was more effectively reduced than the LPS group (Fig. 4F).

OVX-mediated bone loss is not affected by HAR.

OVX mice were used as preclinical models for the study of osteoporosis and to investigate the effect of HAR on estrogen deficiency-induced bone loss. Three-dimensional visualization of the femur region by micro-CT showed a loss of bone mineral density (Fig. 5A), a reduction in BV / TV, Tb.Sp, and Tb.N induced by OVX (Fig. 5B) I did not let it. Histological examination also showed that osteoclastogenesis, bone loss and number of osteoclasts present in the femur were not restored in HAR-treated OVX mice (Fig. 5C and 5D). In addition, compared to the Sham (SH) group, serum levels of CTX-1 were increased in the OVX group. However, this level did not differ between OVX and HAR-treated OVX mice (Fig. 5E). These results demonstrate that HAR restores LPS-induced bone loss but not OVX-mediated bone loss.

Unlike the relationship between LPS and bone environment, estrogen regulates osteoclast formation and activity by promoting osteoclast apoptosis induced by transformation of growth factor beta 1, and estrogen agonist activates osteoclastogenesis through estrogen receptor-alpha activation , Which inhibits RANKL activity. Estrogen deficiency, a consequence of OVX, induces upregulation of osteoclast formation by promoting T-cell TNF-a production. OVX also induces differentiation of Th17 cells that secrete IL-7, promotes osteoclastogenesis and inhibits osteoclast differentiation, thereby promoting bone loss. In this study, micro-CT and histological analyzes were performed on mice treated with LPS and HAR simultaneously to evaluate the preventive effect of HAR on bone loss, and to evaluate the therapeutic effect of HAR, OVX- It was also performed on mice treated with HAR after induction of bone loss. HAR restored bone density in the LPS-induced bone loss model, but not in the OVX, demonstrating the protective effect of HAR on bone erosion. If the mechanism of bone loss due to LPS-induced inflammation is dependent on the modulation of PLCγ2-Ca ²⁺ signaling, unlike the case of OVX-induced bone loss, these findings suggest that HAR controls its effect on PLCγ2 activation . The main reason for this result is the lack of association with Syk-Btk-PLCγ2-Ca ²⁺ signaling in the OVX model. Previously PLCγ2-deficient (PLCγ2 ^{- / -} ) mice had considerably higher soymmetric bone than wild-type mice, but PLCγ2 ^{- / -} mice demonstrated that OVX-induced bone erosion was similar to wild-type mice. These results imply the presence of a PLCγ2-independent mechanism that leads to estrogen deficiency-induced osteoclastogenesis. Thus, we conclude that OVX-induced osteolysis with a PLCγ2-independent mechanism has not been restored by HAR, which primarily regulates osteoclast formation through a PLCγ2-dependent signaling pathway.

In conclusion, the results of this study demonstrate for the first time that HAR inhibits RANKL-induced osteoclast differentiation through ERK, JNK, and Syk-Btk-PLCγ2-Ca ²⁺ signaling and down-signal activation of NFATc1 and target gene expression do. Furthermore, HAR restored bone density in the LPS-induced bone loss model, but not in the OVX-induced bone loss model. These findings suggest that HAR is a promising therapeutic for the treatment of inflammatory-related bone disease, such as inflammatory osteoporosis and rheumatoid arthritis, except postmenopausal osteoporosis. Further studies will use in vivo models of LPS- and OVX-induced bone loss to elucidate the detailed mechanism underlying HAR function.

Experimental Section

Test compounds and reagents.

HAR was purchased from Sigma-Aldrich (St. Louis, MO, USA) and diluted by dissolving in dimethylsulfoxide (DMSO). The purity of HAR (> 95%) was determined by high performance liquid chromatography (HPLC) analysis. 1,25-dihydroxyvitamin D ₃ (VitD ₃ ), prostaglandin E ₂ (PGE ₂ ), LPS, and monoclonal β-actin antibodies were also purchased from Sigma-Aldrich. Recombinant soluble human RANKL and M-CSF were obtained from PeproTech EC Ltd. (London, UK). Penicillin / streptomycin antibody, a-minimal essential medium (? -MEM), and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). (sc-7202), NFATc1 (sc-7294), IκB (sc-371), spleen tyrosine kinase (Syk) (sc-1077), phospho- 5283) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-p38 MAPK 9211, p38 MAPK 9212, phospho-ERK 1/2 9101, ERK 1/2 9102, phospho-JNK 9251, JNK 9252, Antibodies to Akt (9271), Akt (9272), phospho-inhibiting kappa B (IkB) (2859), Btk (3533), and phospho-Skk (2710) were purchased from Cell Signaling Technology Inc. USA). Antibodies to the phospho-Bruton's tyrosine kinase (Btk) (GTX61791) were purchased from GeneTex (Irvine, CA, USA). All other chemicals were of analytical grade or in accordance with the criteria required for cell culture experiments.

Declaration of ethics.

Experimental procedures were carried out in accordance with the recommendations of the National Institutes of Health's Laboratory Handling and Usage Guide (WKU-14-45). Surgery was performed under sodium pentobarbital anesthesia and all efforts were made to minimize pain. Animals were monitored daily to check for health conditions.

An experimental animal.

Male ICR mice (5 weeks) or female C57BL / 6J mice (10 weeks) were purchased from Damul Science (Daejeon, Korea) or Central Lab Animal Inc. (Seoul, Korea). The mice were housed under controlled temperature (22 ° C - 24 ° C) and humidity (55% 60%), 12: 12h person / cancer cycle.

Mouse BMM production and osteoclast differentiation.

Bone marrow cells were obtained by flushing the femur and tibia of 5-week-old ICR mice and resuspended in α-MEM supplemented with 10% FBS and 1% penicillin / streptomycin. Non-adherent cells were harvested and cultured for 3 days in the presence of M-CSF (30 ng / mL). The floating cells were discarded and the cells attached to the bottom of the culture dish were classified as BMM. Cells were seeded at 3.5 × 10 ⁴ / well and cultured for 4 days in the presence of M-CSF (30 ng / mL) and RANKL (100 ng / mL) with or without HAR, and then fixed at 3.7% formalin for 15 minutes. Permeabilized with 0.1% Triton X-100, and stained for TRAP. TARP-positive multinuclear cells (NMC) with more than 5 nuclei were counted as osteoclasts.

Cytotoxicity analysis.

BMM was seeded in 96-well plates at a density of 1 10 ⁴ / well. Cells were treated with M-CSF (30 ng / mL) and increasing concentrations of HAR. After 3 days, 50 μl of sodium 3'- [1- (phenyl-aminocarbonyl) -3,4-tetrazolium] -bis (4-methoxy-6-nitro) (XTT reagent), benzenesulfonic acid hydrate, And N-methyldibenzopyrazine methyl sulfate were added to each well, followed by incubation for 4 h. Optical density at 450 nm was measured in a microplate reader (Molecular Devices, Sunnyvale, Calif., USA).

F-actin ring staining.

BMM were incubated with M-CSF (30 ng / mL) and RANKL (100 ng / mL) for 3 days in the presence or absence of HAR. After 3 days, the cells were fixed in phosphate-buffered saline (PBS) containing 3.7% formalin for 20 minutes and permeabilized in PBS containing 0.1% Triton X-100 for 15 minutes. Cells were blocked with 2.5% bovine serum albumin for 30 minutes and then incubated with 4 ', 6-diamidino-2'-phenylindole (Sigma) at room temperature with Molecular Probes / Life Technologies, Carlsbad, Calif. For 30 minutes. Images were acquired using a fluorescence microscope (DMLB, Leica, Germany).

Resorption Pitz analysis.

Primary osteoblasts and BMC were co-cultured in collagen gel-coated 90-mm dishes in the presence of VitD ₃ and PGE ₂ for 6 days. The co-cultured cells were treated with 0.1% collagenase at 37 ° C for 10 minutes to desorb and then replated to hydroxyapatite-coated plates with or without HAR for 24 h. Cells were then removed and analyzed for resorption of absorbed Fitz using Image Pro-Plus version 4.0 (Media Cybernetics, Rockville, Md., USA).

Quantitative real-time RT-PCR analysis.

Total RNA was isolated using QIAzol reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. RNA (1 μg) was reverse-transcribed using oligo dT primer (10 μg) and dNTP (10 mM). The mixture was incubated for 5 min at 65 ° C and incubated for 50 min at 42 ° C with first strand buffer (50 mM Tris-HCl, pH 8.3; 75 mM KCl; 3 mM MgCl ₂ ), 100 mM dithiothreitol, CDNA was obtained by incubation with Super-script II reverse transcriptase (Invitrogen, Carlsbad, Calif., USA).

Real-time RT-PCR was performed in a 20 μl reaction mixture containing 10 μl SYBR Green Premix (Bioneer Co.), 10 pmol each forward and reverse primer, and 1 μg cDNA in an Exicycler 96 Real-Time Quantitative Thermal Block (Bioneer Co., Daejeon, Korea) . Amplification conditions were 95 ° C for 5 minutes, then 95 ° C for 1 minute, 60 ° C for 30 seconds and 72 ° C for 40 cycles. Fluorescence resulting from the incorporation of the SYBR Green dye into the double-stranded DNA was quantified using the threshold cycle (C _t ) value. The relative levels of c- Fos , NFATc1 , TRAP , OSCAR ,? 3 - integrin , DC-STAMP , CTR and cathepsin K were standardized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. The primers used for real-time RT-PCR are summarized in Table 1.

Western blot analysis.

Cells were dissolved in a buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1% deoxycholate, and protease inhibitor did. The cell lysate was centrifuged at 14,000 ㅧ g for 20 minutes and the protein concentration of the supernatant was determined. The sample was digested with 8% 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, Mass., USA) and blocked with 5% , The primary antibody (1: 1000), and then probed with a suitable secondary antibody conjugated with horseradish peroxidase (1: 3000, Enzo). Immunoreactivity was visualized using a chemiluminescence detection system (Millipore).

Ca ²⁺  Measure.

Separate BMMs were seeded on 22 ㅧ 22 mm cover slips (5 ㅧ 10 ⁴ cells each) and cultured for 24 h in the presence of M-CSF (30 ng / mL). If necessary, cells were directly flushed or pretreated with RANKL for 24 h with 100 μM HAR for the indicated time. The cells were then loaded with 5 μM Fura-2 / AM for 50 minutes at room temperature and washed with a bath solution (140 mM NaCl; 5 mM KCl; 1 mM MgCl ₂ ; 10 mM HEPES; 1 mM CaCl ₂ ; 10 mM glucose; and 310 mOsm, pH 7.4) The unloading dye was removed and then continuously perfused with prewarmed (37 ° C) bath solution. To measure the intensity of Fura-2 / AM fluorescence, cells were continuously excited at 340 and 380 nm and fluorescence emitted at 510 nm (non-F340 / F380) was captured using a charge-coupled device camera. The images were digitized and analyzed with MetaFluor software (Universal Imaging, Bedford Hills, NY, USA).

LPS-induced bone resorption mouse model.

To test the effect of HAR on LPS-induced bone loss, male ICR 5-week-old mice were randomly divided into 4 groups of 5 mice each. Mice were treated with PBS (control), LPS, HAR, or LPS and HAR (LPS + HAR) on the first day. HAR (10 mg / kg) and PBS were orally administered the day before the first LPS injection (5 mg / kg) and then every other day for 8 days. LPS was injected intraperitoneally on days 2 and 6. All mice were killed on the 10th day.

OVX-mediated bone loss mouse model.

Female C57BL / 6 8-week-old mice were randomly divided into 3 groups of 5 mice: sham-operated (SH), OVX control (OVX), HAR OVX with treatment (OVX + HAR). SH mice were incised and sutured without ovariectomy. Mice in the OVX and OVX + HAR groups were bilaterally ovariectomized. Four weeks after surgery, HAR (10 mg / kg) or PBS (control) was orally administered every other day for 28 days and all mice were killed on day 28.

Micro-CT and histological analysis.

The uninjured left femoral metapedic region of each mouse was examined by high resolution micro-CT (NFR-Polaris-S160; Nanofocus Ray, Iksan, Korea) analysis at 45 kVp supply voltage, 90 μA current, and 7 μm isotropic resolution. Femoral scans were performed at 2-mm intervals from the growth plate and a total of 350 sections were obtained per scan. After 3D reconstruction, the bone volume per tissue volume (BV / TV), shoal bone separation (Tb.Sp), shoal bone thickness (Tb.Th), and shochu bone number (Tb.N) were measured using INFINITT-Xelis software (INFINITT Healthcare, Seoul, Korea) for quantitative analysis. The femur was fixed in 4% neutral-buffer paraformaldehyde (Sigma) for 1 day, and calcium was removed in 12% EDTA for 3 weeks and embedded in paraffin. 5 mm thick sections were cut using RM2145 microtome (Leica Microsystems, Bannockburn, Ill., USA). The sections were stained with hematoxylin and eosin for histological examination, and the other sections were stained for TRAP. The parameters for bone resorption, including the number of osteoclasts per tissue field, were quantified using Image Pro-Plus software version 4.0 (Media Cybernetics, Silver Spring, MD, USA). The nomenclature, symbols, and units used in this study are those recommended by the American Society of Bone and Mineral Resources (ASBMR) Nomenclature Committee.

Measurement of RANKL, OPG, and CTX-I levels.

Serum RANKL and OPG levels were detected using a commercial ELISA kit according to the manufacturer's protocol (R & D Systems, Minneapolis, MN, USA). Serum CTX-I levels, a specific marker of bone resorption, were determined using a mouse-specific ELISA assay according to the manufacturer's protocol (Nordic Bioscience Diagnostics, Herlev, Denmark).

Statistical analysis.

Each experiment was performed at least three times, and all quantitative data are expressed as mean SD. Statistical analysis was performed using SPSS (Korean version 14.0; SPSS Inc., Chicago, IL, USA). Variance analysis and subsequent Tukey post-hoc tests were used to compare variables among the three groups. P < 0.05 was considered statistically significant.

Claims (3)

A pharmaceutical composition for inhibiting osteoclast differentiation,
A pharmaceutical composition for inhibiting the differentiation of osteoclasts, comprising a lower liposide as an active ingredient. The method according to claim 1,
Wherein the pharmaceutical composition for inhibiting osteoclast differentiation is a pharmaceutical composition for the treatment or prevention of an inflammatory bone disease. 3. The method of claim 2,
Wherein said bone disease is not a pharmaceutical composition for treating or preventing osteoporosis induced by estrogen deficiency.

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