• BMB Reports
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• v.53 no.3
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• pp.154-159
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• 2020

We investigated the effects of physalin A, B, D, and F on osteoclastogenesis induced by receptor activator of nuclear factor κB ligand (RANKL). The biological functions of different physalins were first predicted using an in silico bioinformatic tool (BATMAN-TCM). Afterwards, we tested cell viability and cell apoptosis rate to analyze the cytotoxicity of different physalins. We analyzed the inhibitory effects of physalins on RANKL-induced osteoclastogenesis from mouse bone-marrow macrophages (BMMs) using a tartrate-resistant acid phosphatase (TRAP) stain. We found that physalin D has the best selectivity index (SI) among all analyzed physalins. We then confirmed the inhibitory effects of physalin D on osteoclast maturation and function by immunostaining of F-actin and a pit-formation assay. On the molecular level, physalin D attenuated RANKL-evoked intracellular calcium ([Ca(2+)](i)) oscillation by inhibiting phosphorylation of phospholipase Cγ2 (PLCγ2) and thus blocked the downstream activation of Ca2+/calmodulin-dependent protein kinases (CaMK)IV and cAMP-responsive element-binding protein (CREB). An animal study showed that physalin D treatment rescues bone microarchitecture, prevents bone loss, and restores bone strength in a model of rapid bone loss induced by soluble RANKL. Taken together, these results suggest that physalin D inhibits RANKL-induced osteoclastogenesis and bone loss via suppressing the PLCγ2-CaMK-CREB pathway.

• Archives of Pharmacal Research
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• v.26 no.11
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• pp.917-924
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• 2003

This research aims to test a new drug candidate based on a traditional medicinal herb, F1, an herbal extract obtained from Astragalus membranaceus and its main ingredient, 1-monolinolein that may have fewer side effects and less uterine hypertrophy. In vitro experiments, human osteoblast-like cell lines, MG-63 and Saos-2, were analyzed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and an alkaline phosphatase (ALP) assays. Mouse osteoclasts were induced through a calcium-deficient diet and inhibition effects were measured. In vivo experiments were done using ovariectomized (OVX) rats for 9 weeks. At necropsy, uterus weights were measured, trabecular bone area (TBA) of tibia and lumbar vertebra were measured bone histomorphology. In results, cell proliferation and ALP activity in Saos-2 by ether F1 or 1-monolinolein did not increased significantly compared to the control. The F1 inhibited osteoclast development ($IC_{25}=3.37{\times}10^{-5}$mg/mL) less than 17$\beta$-estradiol. The OVX rats administered F1 (2 mg/kg/day and 10 mg/kg/day) showed an increase in TBA of the tibia significantly (136.3${\pm}4.2% and 138.5{\pm}$10.3% of control). In conclusions, the herbal extract, F1 inhibited tibia and lumbar bone loss and did not cause uterine hypertrophy. However, 1-monolinolein, the main ingredient of the herbal extract, did not inhibit bone loss.

• Applied Microscopy
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• v.22 no.2
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• pp.118-126
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• 1992

The pathway and time course of fucose-containing glycoprotein synthesis and intracellular translocation in osteoclasts of the mice maxillary alveolar bone were investigated by electron microscopic radioautography. Male Balb-C mice weighing 17gm were anesthetized with Nembutal and injected via the external jugular vein with 2.5 mCi of $L-[6-^{3}H]-fucose$ (specific activity 16.8 mCi/mmol) in 0.1 ml of sterile saline solution. At 5, 10, 20, 35 minutes and 8 hours after administration of the $^{3}H-fucose$, animals were killed by intracardiac perfusion of 30ml of 2% glutaraldehyde in a modified Tyroid solution, pH 7.4. The maxillae were then removed and further fixed in Karnovsky fixative for an additional 3-4 hours. After rinsing in 0.1M cacodylate buffer for 10 minutes, the maxillae were demineralized for 2 weeks at $4^{\circ}C$ in ethylene diamine tetra acetate containing 2% glutaraldehyde. The first interdental areas were mesiodistally sectioned into slices of 1mm thickness and postfixed in osmium tetroxide. Tissues were then dehydrated and embedded in Poly Bed. To prepare electron microscopic radioautography, the dipping method of Kopriwa (1973) was employed. Thin sections were coated with a crystalline monolayer of ILford $L_4$ photographic emulsion. After exposure for 4 months at $4^{\circ}C$, the sections were developed Kodak Microdol-X and Phenidon (for compact grains), fixed in 30% sodium thiosulfate, stained with uranyl acetate and lead citrate and examined in the electron microscope (JEOL 1200 EX). At 5, 10 and 20 minutes after injection, $^{3}H-fucose$ was concentrated in Golgi cisternae of the osteoblasts. By 35 minutes the labels were observed over small vesicles in the suprannclear area of osteoclasts. At 8 hours, numerous silver grains were located on the ruffled border and cell membrane of osteoclasts. These results indicate that fucose molecules are added in the Golgi apparatus and small vesicles appear to be responsible for translocation of the glycoproteins to the marginal portion of osteoblasts. The glycoproteins are distributed on the osteoclast cell surface and especially over the ruffled border.

• Experimental and Molecular Medicine
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• v.50 no.11
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• pp.2.1-2.16
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• 2018

Supplementation of mesenchymal stem cells (MSCs) at sites of bone resorption is required for bone homeostasis because of the non-proliferation and short lifespan properties of the osteoblasts. Calcium ions ($Ca^{2+}$) are released from the bone surfaces during osteoclast-mediated bone resorption. However, how elevated extracellular $Ca^{2+}$ concentrations would alter MSCs behavior in the proximal sites of bone resorption is largely unknown. In this study, we investigated the effect of extracellular $Ca^{2+}$ on MSCs phenotype depending on $Ca^{2+}$ concentrations. We found that the elevated extracellular $Ca^{2+}$ promoted cell proliferation and matrix mineralization of MSCs. In addition, MSCs induced the expression and secretion of osteopontin (OPN), which enhanced MSCs migration under the elevated extracellular $Ca^{2+}$ conditions. We developed in vitro osteoclast-mediated bone resorption conditions using mouse calvaria bone slices and demonstrated $Ca^{2+}$ is released from bone resorption surfaces. We also showed that the MSCs phenotype, including cell proliferation and migration, changed when the cells were treated with a bone resorption-conditioned medium. These findings suggest that the dynamic changes in $Ca^{2+}$ concentrations in the microenvironments of bone remodeling surfaces modulate MSCs phenotype and thereby contribute to bone regeneration.

• Journal of Acupuncture Research
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• v.32 no.3
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• pp.69-81
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• 2015

Purpose : The first purpose of this study is to find out whether the water extract of Rehmanniae Radix Preparat(RRP), Cuscutae Semen(CS) and their combination(Ssangbohwan, SBH) have the effect of suppressing Receptor activator of nuclear factor kappa-B ligand(RANKL)-induced osteoclast differentiation. The second purpose of this study is to find out whether the water extract of RRP, CS and SBH have the effect of inhibiting osteoporosis in an osteoporosis model induced by lipopolysaccharide(LPS). Methods : After promoting differentiation of osteoclasts by treating the RANKL, we observed the effect by the administration of RRP, CS and SBH. In addition, by means of Reverse transcription polymerase chain reaction(RT-PCR), we assayed mRNA expression levels of NFATc1, c-Fos, TRAP and GAPDHS(Glyceraldehyde-3-phosphate dehydrogenase, spermatogeni) from bone marrow macrophages(BMMs). Similarly, the protein expression levels of NFATc1 (Nuclear factor of activated T-cells, cytoplasmic1), C-Fos, MAPKs(Mitogen-activated protein kinases) and ${\beta}$-actin in cell lysates were analyzed by means of Western Blotting. Finally, we determined the anti-osteoporotic effects of RRP, CS and SBH, through the use of Lipopolysaccharide-induced bone-loss mouse. Results : RRP, CS and SBH showed remarkable inhibitive effect on RANKL-treated osteoclast differentiation without cytotoxicity. SBH inhibited the phosphorylation of p38, Jun N-terminal kinases(JNK), and I-${\kappa}B$ and down-regulated the induction of c-Fos and NFATc1 by RANKL. RRP, CS suppressed degradation of I-${\kappa}B$, but it did not affect c-Fos and NFATc1 by RANKL. Lastly, in vivo data showed that RRP and SBH prevented bone erosion by LPS treatment. Conclusions : These results demonstrate SBH can be effective remedy for bone-loss diseases such as osteoporosis.

• Journal of Acupuncture Research
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• v.34 no.2
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• pp.1-18
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• 2017

Objectives : The purpose of this study was to evaluate the effects of water extracts of Eucommiae cortex (EC), Psoraleae semen (PS), and their combination on receptor activator of nuclear factor-kappa-B ligand (RANKL)-induced osteoclast differentiation. Methods : We assayed the protein expression levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), c-Fos, mitogen-activated protein kinases (MAPKs), and ${\beta}-actin$ in cell lysates using western blotting. Similarly, mRNA expression levels of NFATc1, c-Fos, tartrateresistant acid phosphate (TRAP), and glyceraldehyde-3-phosphate dehydrogenase, spermatogeni (GAPDHS) from bone marrow macrophages (BMMs) were analyzed using reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, we determined the anti-osteoporotic effects of the water extracts of EC, PS, and their combination in a lipopolysaccharide (LPS)-induced bone-loss mouse model. Results : The in vitro data revealed showed that the combination of EC and PS extract showed a more remarkable inhibition of osteoclast differentiation than each herb did alone. The combination downregulated the induction of c-Fos, NFATc1, and TRAP by suppressing the phosphorylation of p38 and c-Jun N-terminal kinases (JNKs) and inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells ($NF-{\kappa}B$). Lastly, the in vivo data showed that PS reduced the LPS-induced bone erosion. Conclusion : The result of this study suggests that EC and PS could be potential therapeutic agents for bone loss diseases such as osteoporosis.

• International Journal of Oral Biology
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• v.31 no.2
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• pp.53-65
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• 2006

Calcium concentration in the bone resorption lacunae is high and is in the mM concentration range. Both osteoblast and osteoclast have calcium sensing receptor in the cell surface, suggesting the regulatory role of high extracellular calcium in bone metabolism. In vitro, high extracellular calcium stimulated osteoclastogenesis in coculture of mouse osteoblasts and bone marrow cells. Therefore we examined the genes that were commonly regulated by both high extracellular calcium and $1,25(OH)_2vitaminD_3(VD3)$ by using mouse oligo 11 K gene chip. In the presence of 10 mM $[Ca^{2+}]e$ or 10 nM VD3, mouse calvarial osteoblasts and bone marrow cells were co-cultured for 4 days when tartrate resistant acid phosphatase-positive multinucleated cells start to appear. Of 11,000 genes examined, the genes commonly regulated both by high extracellular calcium and by VD3 were as follows; 1) the expression of genes which were osteoclast differentiation markers or were associated with osteoclastogenesis were up-regulated both by high extracellular calcium and by VD3; trap, mmp9, car2, ctsk, ckb, atp6b2, tm7sf4, rab7, 2) several chemokine and chemokine receptor genes such as sdf1, scya2, scyb5, scya6, scya8, scya9, and ccr1 were up-regulated both by high extracellular calcium and by VD3, 3) the genes such as mmp1b, mmp3 and c3 which possibly stimulate bone resorption by osteoclast, were commonly up-regulated, 4) the gene such as c1q and msr2 which were related with macrophage function, were commonly down-regulated, 5) the genes which possibly stimulate osteoblast differentiation and/or mineralization of extracellular matrix, were commonly down-regulated; slc8a1, admr, plod2, lox, fosb, 6) the genes which possibly suppress osteoblast differentiation and/or mineralization of extracellular matrix, were commonly up-regulated; s100a4, npr3, mme, 7) the genes such as calponin 1 and tgfbi which possibly suppress osteoblast differentiation and/or mineralization of extracellular matrix, were up-regulated by high extracellular calcium but were down-regulated by VD3. These results suggest that in coculture condition, both high extracellular calcium and VD3 commonly induce osteoclastogenesis but suppress osteoblast differentiation/mineralization by regulating the expression of related genes.

• The Journal of Korean Academy of Prosthodontics
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• v.57 no.2
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• pp.142-149
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• 2019

Purpose: The purpose of this study is to investigate the changes of osteoclast differentiation inhibition according to the period of precipitation when titanium disks were immersed in Modified simulated body fluid (mSBF). Materials and methods: Titanium alloy (Ti grade III) disks with machined surfaces and anodized surfaces were immersed in distilled water and mSBF, respectively. The immersion periods were 7 days, 14 days, 21 days and 28 days, and the control group was immersed in distilled water for each period. RAW 264.7 cells capable of differentiating into osteoclasts were used to measure the number of adherent cells, the measurement of TRAP activity, and the expression pattern of NFATc1 by western blotting. Results: The degree of inhibition of osteoclast differentiation was found to be statistically significant when the disks were immersed in mSBF for more than 14 days on both machined surfaces and anodized surfaces. There was no correlation between immersion time and cell attachment. When the disks were immersed for more than 14 days, TRAP activity was decreased and NFATc1 expression was inhibited. Futhermore, the decrease in TRAP activity and the inhibition of NFATc1 expression remained unchanged. Conclusion: Immersion of titanium disks in mSBF for more than 14 days can prevent RAW 264.7 cells from differentiating into osteoclasts. Inhibition activity does not change even if the immersion period is for more than 14 days.

• Journal of Life Science
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• v.21 no.8
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• pp.1199-1203
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• 2011

The present study evaluated the beneficial effects of polycalcium (a mixture of Polycan and calcium lactate-gluconate 1:9 [g/g]) on osteoporosis using in vitro assays. Cell proliferation and alkaline phosphatase activities of osteoblasts (human primary osteoblasts) and osteoclast differentiation of RAW264.7 cells (murine osteoclast progenitor cells) treated with different concentrations of polycalcium for various periods were assessed. Osteoblast proliferation was stimulated and prevented RANKL-induced osteoclast differentiation of RAW264.7 cells. These results support the development of ideal anti-osteoporotic agents, such as polycalcium, that exhibit properties that accelerate bone formation and inhibit bone resorption.

• Microbiology and Biotechnology Letters
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• v.43 no.4
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• pp.322-329
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• 2015

The effects of water extracts of Gallus gallus var. domesticus (Yeonsan ogolgye, GD) on the activities of osteoblast differentiation and the restraint of osteoclast formation were investigated. The water extract of GD in the human osteoblast "MG-63" cell, was examined in relation to alkaline phosphatase (ALP) activity and alizarin red stains. In order to observe the effects of osteoclasts formation, we analyzed RAW 264.7 cell tartrate-resistant acid phosphatase (TRAP) activity and TRAP stains. The ALP activity of the water extract of hen and cock flesh (3 years) were 133.8% and 129.6%, respectively. The ALP activity of flesh extracts was also higher than that of the skin extracts. Concerning the effects of age, the 3 years old flesh extracts had a higher activity than that of the one year old extracts. However the activity of the 3 years old skin extracts was lower than that of the one year old extracts. For gender conditions, the ALP activity of the hen extract was higher than that of the cock. The degree bone mineralization in the three years old hen flesh exhibited the highest rate, at 124.3%, amongst all the groups. The TRAP activity of the flesh extracts of the three years old cock revealed the lowest rate, at 31.8%, compared to the control. Our results demonstrate that the water extract of GD increases bone mineralization and osteoblast differentiation activity in MG-63 cells and enhances the inhibitory activity of bone-resorption in RAW 264.7 cells. In conclusion, the water extracts of GD seem to be effective in the prevention and treatment of bone related disorders.