Anti-osteoclastogenic Activity Research Articles

Napyradiomycin B4 Suppresses RANKL-Induced Osteoclastogenesis and Prevents Alveolar Bone Destruction in Experimental Periodontitis.

The unique structure and beneficial biological properties of marine natural products have drawn interest in drug development. Here, we examined the therapeutic potential of napyradiomycin B4 isolated from marine-derived Streptomyces species for osteoclast-related skeletal diseases. Bone marrow-derived macrophages were treated with napyradiomycin B4 in an osteoclast-inducing medium, and osteoclast formation, osteoclast-specific gene expression, and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) localization were evaluated using tartrate-resistant acid phosphatase staining, real-time PCR, and immunostaining, respectively. Phosphorylation levels of signaling proteins were assessed by immunoblot analysis to understand the molecular action of napyradiomycin B4. The in vivo efficacy of napyradiomycin B4 was examined under experimental periodontitis, and alveolar bone destruction was evaluated by microcomputed tomography (micro-CT) and histological analyses. Among the eight napyradiomycin derivatives screened, napyradiomycin B4 considerably inhibited osteoclastogenesis. Napyradiomycin B4 significantly suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation and disrupted the expression of NFATc1 and its target genes. Mitogen-activated extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK) phosphorylation levels were reduced by napyradiomycin B4 in response to RANKL. Under in vivo experimental periodontitis, napyradiomycin B4 significantly attenuated osteoclast formation and decreased the distance between the cementoenamel junction and alveolar bone crest. Our findings demonstrate the antiosteoclastogenic activity of napyradiomycin B4 by inhibiting the RANKL-induced MEK-ERK signaling pathway and its protective effect on alveolar bone destruction.

Ginseng (Panax ginseng) leaf extract modulates the expression of heme oxygenase-1 to attenuate osteoclast differentiation

Osteoporosis is an aging disease characterized by an imbalance between bone formation and resorption. However, drugs that inhibit bone resorption have various adverse effects. Ginseng (Panax ginseng), a prominent herbal medicine in East Asia for >2000 years, is renowned for its manifold beneficial properties, including antioxidant, anti-cancer, anti-diabetic, and anti-adipogenic activities. Despite its long history of use, the pharmacological functions of ginseng leaves are not yet fully comprehended. In this study, we evaluated the potential effects of ginseng leaf extract (GLE) on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation in RAW264.7 macrophage cells. Tartrate-resistant acid phosphatase (TRAP) staining revealed that GLE had significant anti-osteoclastogenic activity. GLE significantly reduced mRNA levels of osteoclast differentiation markers including TRAP, nuclear factor of activated T cell cytoplasmic 1, and cathepsin K. It also suppressed the production of reactive oxygen species (ROS) and secretion of high mobility group box-1 (HMGB1) in RANKL-treated RAW264.7 cells. In addition, GLE upregulated dose- and time-dependently the expression of heme oxygenase-1 (HO-1), eventually suppressing ROS production and HMGB1 secretion. This effects of GLE were significantly reversed by Tin Protoporphyrin IX dichloride, an inhibitor of HO-1, and HO-1 shRNA, indicating that HO-1 potently inhibits RANKL-induced osteoclast differentiation by inhibiting ROS production and HMGB1 secretion. Taken together, these observations suggest that GLE could have therapeutic potential as a natural product-derived medicine for the treatment of bone disorders.

Sucrosephenylpropanoid esters and isoflavonoids isolated from Belamcanda chinensis roots and their potential anti-osteoclastogenic activity

Repeated chromatography of the CH2Cl2 and EtOAc soluble fractions from the methanol extract of Belamcanda chinensis root yielded six new sucrosephenylpropanoid esters (1–6) and twenty-one known compounds (7–27). The structures of 1–6 were elucidated using diverse nuclear magnetic resonance (NMR) techniques and high-resolution mass spectrometry (HRMS) data analysis, together with chemical methods. All the twenty-seven isolated compounds were evaluated for their anti-osteoclastogenic activities. Preliminary screening results revealed that compounds 1 and 19 exhibited strong effects against RANKL-induced osteoclast formation in RAW264.7 cells. In addition, the treatment of mouse bone marrow macrophages (BMMs) with compounds 1 and 19 significantly decreased RANKL-induced TRAP-positive multinucleated osteoclast formation in a concentration-dependent manner without affecting cell viability. Further bioassay investigation showed that compounds 1 and 19 inhibited the expression of some osteoclast-specific marker genes and the transcription factor nuclear factor of activated T cells cytoplasmic 1 (NFATc1) in response to RANKL. To the best of our knowledge, this is the first investigation of anti-osteoclastogenic activity for compounds isolated from B. chinensis.

5′-Methylthioadenosine strongly suppresses RANKL-induced osteoclast differentiation and function via inhibition of RANK-NFATc1 signalling pathways

Excessive osteoclast-mediated bone resorption is a critical cause of osteoporosis affecting many aging people worldwide. 5′-Methylthioadenosine (MTA) is a natural sulfur-containing nucleoside normally produced in prokaryotes, plants, yeast, and higher eukaryotes via polyamine metabolism. MTA affects various physiological responses particularly the inflammatory pathway in both normal and cancerous cells and modulates the activation of nuclear factor-κB involved in the osteoclastogenesis signalling process. While several studies have reported that natural products possess anti-osteoclastogenesis phenolics and flavonoids, the effect of nucleoside derivatives on osteoclastogenesis remains limited. Therefore, this study aimed to explore the molecular mechanisms by which MTA affects pre-osteoclastic RAW 264.7 cells as a potential alleviation compound for inflammation-mediated bone loss. Osteoclasts were established by incubating RAW264.7 macrophage cells with receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor, the vital cytokines for activation of osteoclast differentiation. Cell viability was measured using MTT assays at 24, 48, and 72 h. The suppressive effect of MTA on RANKL-induced osteoclast differentiation and function was assessed using tartrate-resistant acid phosphatase (TRAP) analysis, qRT-PCR, and pit formation, Western blot, and immunofluorescence assays. MTA showed dose-dependent anti-osteoclastogenic activity by inhibiting TRAP-positive cell and pit formation and reducing essential digestive enzymes, including TRAP, cathepsin K, and matrix metallopeptidase 9. MTA was observed to suppress the osteoclast transduction pathway through (RANKL)-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NFƘB); it attenuated NFƘB-P65 expression and down-regulated cFos proto-oncogene and nuclear factor of activated T cell c1 (NFATc1), the main regulators of osteoclasts. Moreover, the suppression of RANK (the initial receptor triggering several osteoclastogenic transduction pathways) was observed. Thus, this study highlights the potential of MTA as an effective therapeutic compound for restoring bone metabolic disease by inhibiting the RANK-NFATc1 signal pathway.

Isolation and Characterization of an Anti-Osteoporotic Compound from Melia toosendan Fructus.

Melia toosendan fructus, traditionally employed in traditional Chinese and Korean herbal medicine, exhibits diverse biological properties encompassing anti-tumor, anti-inflammatory, and anti-viral effects. However, its influence on bone metabolism remains largely unexplored. In this study, we investigated the impact of an ethanolic extract of Melia toosendan fructus (MTE) on osteoclast differentiation and characterized its principal active constituent in osteoclast differentiation and function, as well as its effects on bone protection. Our findings demonstrate that MTE effectively inhibits the differentiation of osteoclast precursors induced by receptor activator of nuclear factor κB ligand (RANKL). Utilizing a bioassay-guided fractionation approach coupled with UHPLC-MS/MS analysis, we isolated and identified the triterpenoid compound toosendanin (TSN) as the active constituent responsible for MTE's anti-osteoclastogenic activity. TSN treatment downregulated the expression of nuclear factor of activated T cells c1, a pivotal osteoclastogenic transcription factor, along with molecules implicated in osteoclast-mediated bone resorption, including tumor necrosis factor receptor-associated factor 6, carbonic anhydrase II, integrin beta-3, and cathepsin K. Furthermore, treatment of mature osteoclasts with TSN impaired actin ring formation, acidification, and resorptive function. Consistent with our in vitro findings, TSN administration mitigated trabecular bone loss and reduced serum levels of the bone resorption marker, C-terminal cross-linked telopeptides of type I collagen, in a mouse bone loss model induced by intraperitoneal injections of RANKL. These results suggest that TSN, as the principal active constituent of MTE with inhibitory effects on osteoclastogenesis, exhibits bone-protective properties by suppressing both osteoclast differentiation and function. These findings imply the potential utility of TSN in the treatment of diseases characterized by excessive bone resorption.

Microwave-assisted synthesis of novel steroidal heterocyclic analogs as potent inhibitors of RANKL-induced osteoclastogenesis.

Osteoporosis is a significant public health issue in our aging population. It is an excessive bone resorption condition brought on by osteoclastogenesis, which makes bones more brittle. In the present work, a series of novel heterosteroidal derivatives have been synthesized using the microwave technique and were evaluated as antiosteoclastogenic agents. The structures of the newly synthesized compounds have been confirmed using analytical and spectral data. The antiosteoclastogenic activity of the newly synthesized compounds was estimated in vitro against osteoclast-differentiated cells from the RAW 264.7 cell line. The pregnenolone dimer 10, the pyridinotestosterone derivative 2, and the phenylnicotinonitrile pregnenolone derivative 8a attained the most promising antiosteoclastogenic activity displaying IC50 (the half maximal inhibitory concentration) values of 5.45 ± 5.30, 11.88 ± 2.09, and 13.40 ± 3.00 µM, respectively, in comparison with dimethyl itaconate (IC50 = 17.76 ± 3.20 µM) and alendronate (IC50 = 4.48 ± 1.89 µM) as reference compounds. Finally, an in silico ADME (Absorption, Distribution, Metabolism, and Excretion) study was conducted to evaluate the synthesized compounds' pharmacokinetic and drug-likeness properties. The results manifested that almost all the investigated compounds' properties were compatible with the specified optimal range, which indicates their reassuring pharmacokinetic properties.

Inhibition of Osteoclast Differentiation and Promotion of Osteogenic Formation by Wolfiporia extensa Mycelium.

Osteoporosis, Greek for "porous bone," is a bone disease characterized by a decrease in bone strength, microarchitectural changes in the bone tissues, and an increased risk of fracture. An imbalance of bone resorption and bone formation may lead to chronic metabolic diseases such as osteoporosis. Wolfiporia extensa, known as "Bokryung" in Korea, is a fungus belonging to the family Polyporaceae and has been used as a therapeutic food against various diseases. Medicinal mushrooms, mycelium and fungi, possess approximately 130 medicinal functions, including antitumor, immunomodulating, antibacterial, hepatoprotective, and antidiabetic effects, and are therefore used to improve human health. In this study, we used osteoclast and osteoblast cell cultures treated with Wolfiporia extensa mycelium water extract (WEMWE) and investigated the effect of the fungus on bone homeostasis. Subsequently, we assessed its capacity to modulate both osteoblast and osteoclast differentiation by performing osteogenic and anti-osteoclastogenic activity assays. We observed that WEMWE increased BMP-2-stimulated osteogenesis by inducing Smad-Runx2 signal pathway axis. In addition, we found that WEMWE decreased RANKL-induced osteoclastogenesis by blocking c-Fos/NFATc1 via the inhibition of ERK and JNK phosphorylation. Our results show that WEMWE can prevent and treat bone metabolic diseases, including osteoporosis, by a biphasic activity that sustains bone homeostasis. Therefore, we suggest that WEMWE can be used as a preventive and therapeutic drug.

Citropten Ameliorates Osteoclastogenesis Related to MAPK and PLCγ/Ca2+ Signaling Pathways through the Regulation of Amyloid Beta.

Osteoporosis and Alzheimer's disease are typical types of dementia in seniors, which share common risk factors. Previous studies have shown that citizens with osteoporosis are more likely than healthy individuals to be at risk of Alzheimer's disease. Citropten, found in Citrus aurantifolia, has been reported to have several pharmacological activities; however, its antiosteoclastogenic activity remains unknown. Here, receptor activator nuclear factor κB ligand (RANKL)-induced osteoclast differentiation, formation, and function in the presence of amyloid beta (Aβ) were attenuated by citropten in the RAW 264.7 cell line. The expression of osteoclast specific genes and proteins indicated that citropten pretreatment lowers the MAPK and PLCγ/Ca2+ signaling pathways. Molecular docking simulations revealed that citropten interacts with the active sites of proteins in the calcium signaling pathway, which have negative binding affinities. These findings indicate that, through Aβ regulation, the RANKL-induced osteoclast can be suppressed by citropten, suggesting that citropten is a potential candidate for treating osteoclastogenesis-related diseases.

In vitro biological evaluation of epigallocatechin gallate (EGCG) release from three-dimensional printed (3DP) calcium phosphate bone scaffolds.

Three-dimensional printed (3DP) tricalcium phosphate (TCP) scaffolds can guide bone regeneration, especially for patient-specific defect repair applications in low-load bearing sites. Epigallocatechin gallate (EGCG), a green tea compound, has gained attention as a safer alternative treatment for bone disorders. The 3DP TCP scaffold is designed for localized EGCG delivery, which can enhance in vitro osteogenic ability, anti-osteoclastogenic activity, vascularization formation, and chemoprevention. In the cocultures of human bone marrow-derived mesenchymal stem cells (hMSCs) and monocytes (THP-1), EGCG release enhances osteogenic differentiation of hMSCs at day 16 compared to the control; this is indicated by a 2.8- and 4.0-fold upregulation of Runt-related transcription factor 2 (Runx2) and bone gamma-carboxyglutamic acid-containing protein (BGLAP), the early and late osteoblast differentiation marker expressions. However, EGCG significantly downregulates the receptor activator of nuclear factor-κB ligand (RANKL) expression by 7.0-fold, indicating that EGCG suppresses RANKL-induced osteoclast maturation. EGCG also stimulates endothelial tube formation at as early as 3 hours when human umbilical vein endothelial cells (HUVECs) grow on Matrigel. It reduces human osteosarcoma MG-63 cell viability by 66% compared to the control at day 11. An in vitro release kinetics study demonstrates that EGCG shows a ∼64% release within a day followed by a sustained release in the physiological environment (pH 7.4) because its phenolic hydroxyl groups are easily deprotonated at physiological pH. These findings contribute to developing a multifunctional scaffold for the treatment of low load-bearing patient-specific bone defects after trauma and tumor excision.

The Structure, Function and Regulation of Protein Tyrosine Phosphatase Receptor Type J and Its Role in Diseases.

Protein tyrosine phosphatase receptor type J (PTPRJ), also known as DEP-1, HPTPη, or CD148, belongs to the R3 subfamily of receptor protein tyrosine phosphatases (RPTPs). It was first identified as an antioncogene due to its protein level being significantly downregulated in most epithelial tumors and cancer cell lines (e.g., colon, lung, thyroid, breast, and pancreas). PTPRJ regulates mouse optic nerve projection by inhibiting the phosphorylation of the erythropoietin-producing hepatocellular carcinoma (Eph) receptor and abelson murine leukemia viral oncogene homolog 1 (c-Abl). PTPRJ is crucial for metabolism. Recent studies have demonstrated that PTPRJ dephosphorylates JAK2 at positions Y813 and Y868 to inhibit leptin signaling. Akt is more phosphorylated at the Ser473 and Thr308 sites in Ptprj-/- mice, suggesting that PTPRJ may be a novel negative regulator of insulin signaling. PTPRJ also plays an important role in balancing the pro- and anti-osteoclastogenic activity of the M-CSF receptor (M-CSFR), and in maintaining NFATc1 expression during the late stages of osteoclastogenesis to promote bone-resorbing osteoclast (OCL) maturation. Furthermore, multiple receptor tyrosine kinases (RTKs) as substrates of PTPRJ are probably a potential therapeutic target for many types of diseases, such as cancer, neurodegenerative diseases, and metabolic diseases, by inhibiting their phosphorylation activity. In light of the important roles that PTPRJ plays in many diseases, this review summarizes the structural features of the protein, its expression pattern, and the physiological and pathological functions of PTPRJ, to provide new ideas for treating PTPRJ as a potential therapeutic target for related metabolic diseases and cancer.

Arteperoxides A–C, tris-normonoterpene–sesquiterpene conjugates with peroxide-bridges from Artemisia judaica exhibiting antiosteoclastogenic activity

Antiosteoclastogenic-guided screening was conducted with 120 extracts of the medicinal plants collected in Egypt that led to the selection of Artemisia judaica L. (Asteraceae). Three undescribed davanone-related terpenoids, arteperoxides A–C, were isolated from the extract with two known derivatives, hydroxydavanone and davana acid. Structural analysis revealed that arteperoxides A–C were tris-normonoterpene–sesquiterpene conjugates with peroxide bridges. Although davanone derivatives with peroxides, such as a hydroperoxyl and peroxyhemiketal groups, have been isolated from Artemisia species, arteperoxides A–C are the first variations observed to contain peroxide bridges between two terpene-derived units. The absolute configurations of arteperoxides A and B were studied based on their spectroscopic data compared with those of the semisynthetic analogs that have ether linkages. The natural and synthetic compounds were tested for the antiosteoclastogenic activity, and arteperoxide C and hydroxydavanone were more potent than other compounds at 20 μM.

Pterostilbene-isothiocyanate impedes RANK/TRAF6 interaction to inhibit osteoclastogenesis, promoting osteogenesis in vitro and alleviating glucocorticoid induced osteoporosis in rats

Prolonged glucocorticoid treatment often leads to glucocorticoid-induced osteoporosis (GIOP), a common iatrogenic complication. This study has explored the anti-osteoporotic potential of semi-synthetic compound, pterostilbene isothiocyanate (PTER-ITC) in GIOP rat model and bone formation potential in vitro. Dysregulated bone-remodelling leads to osteoporosis. PTER-ITC has shown anti-osteoclastogenic activity in vitro. However, its molecular target remains unidentified, which has been explored in this study through in silico and experimental approaches. Alizarin Red S and von-Kossa staining, and alkaline phosphatase (ALP) activity showed the osteogenic differentiation potential of PTER-ITC in pre-osteoblastic mouse MC3T3-E1 and human hFOB 1.19 cells, further, confirmed through the expressions of osteogenic markers at transcriptional (RT-qPCR) and translational (immunoblotting) levels. The anti-osteoclastogenic property of PTER-ITC was confirmed through inhibition of actin ring formation in mouse RAW 264.7 and human THP-1 macrophagic cells. Molecular docking and molecular dynamic simulation showed that PTER-ITC inhibited the crucial osteoclastogenic RANK/TRAF6 interaction, which was further confirmed biochemically through co-immunoprecipitation assay. Osteoporotic bone architecture [validated through scanning electron microscopy (SEM), X-ray radiography, and micro-computed tomography (µ-CT)], physiology (confirmed through compression testing, Young’s modulus and stress versus strain output) and histology (verified through hematoxylin-eosin, Alizarin Red S, von-Kossa and Masson-trichrome staining) of PTER-ITC-treated GIOP female Wistar rats were assuaged. Osteoporotic amelioration through PTER-ITC treatment was further substantiated through serum biomarkers, like, parathyroid hormone (PTH), ALP, calcium (Ca2+), Procollagen type I N-terminal propeptide (P1NP), and 25-hydroxy vitamin D. In conclusion, this study identifies the molecular target of PTER-ITC in impeding osteoclastogenesis and facilitating osteogenesis to ameliorate osteoporosis.

Structures and antiosteoclastogenic activity of compounds isolated from edible lotus (Nelumbo nucifera Gaertn.) leaves and stems

One new 1,4-bis-phenyl-1,4-butanedione glycoside (14), one new eudesmane-type sesquiterpenoid (16), and 16 known compounds were isolated from the leaves and stems of Nelumbo nucifera Gaertn. The structures of the isolated compounds were elucidated by interpretation of their 1D and 2D NMR spectroscopic and HRESIMS data. Time-dependent density functional theory calculations and Electronic Circular Dichroism (ECD) spectroscopy was used to determine absolute configurations of the new eudesmane-type sesquiterpenoid (16). All the isolated compounds were examined for their antiosteoclastogenic activity. Preliminarily results of the TRAP staining on RAW 264.7 cells indicated that compounds 1 and 11 possess potential inhibitory effects on RANKL-induced osteoclast formation. Further bioassay investigation was carried out to reveal that compounds 1 and 11 suppressed RANKL-induced osteoclast formation in a concentration-dependent manner with the inhibition up to 55% and 78% at the concentration of 10 μM, respectively. In addition, the structure-activity relationship analysis showed that the 1,3-dioxole substitute and the double bond at C-6a/C-7 in the aporphine skeleton may be responsible for the antiosteoclastogenic activity. The findings provided valuable insights for the discovery and structural modification of aporphine alkaloids as the antiosteoclastogenic lead compounds.

Flavonoids from Dalbergia cochinchinensis: Impact on osteoclastogenesis

Background/purposeDalbergia cochinchinensi has been widely used in traditional medicine because of its flavonoids. This study examined which components in D. cochinchinensis were capable of reducing or even stimulating the formation of bone-resorbing osteoclasts. Materials and methodsWe have isolated subfamilies of chalcones (isoliquiritigenin, butein), flavones (7-hydroxy-6-methoxyflavone) and neoflavanoids (5-methoxylatifolin), and performed an in vitro bioassay on osteoclastogenesis. The flavonoids were tested for their potential to change the expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK) in murine bone marrow cultures being exposed to RANKL, M-CSF and TGF-β1 using RT-PCR, histochemistry and immunoassay. ResultsWe could confirm that isoliquiritigenin and butein significantly lower the expression of TRAP and CTSK in this setting. Moreover, histochemistry supported the decrease of TRAP by the chalcones. We further observed a trend towards an increase of osteoclastogenesis in the presence of 5-methoxylatifolin and 7-hydroxy-6-methoxyflavone, particular in bone marrow cultures being exposed to RANKL and M-CSF. Consistently, the anti-inflammatory activity was restricted to isoliquiritigenin and butein in murine RAW 264.7 inflammatory macrophages stimulated by lipopolysaccharide (LPS). With respect to osteoblastogenesis, neither of the flavonoids but butyrate, a short chain fatty acid, increased the osteogenic differentiation marker alkaline phosphatase activity in ST2 murine mesenchymal cells. ConclusionWe have identified two flavonoids from D. cochinchinensis with a potential pro-osteoclastogenic activity and confirm the anti-osteoclastogenic activity of isoliquiritigenin and butein.

Anti-Osteoporosis Effect of Perilla frutescens Leaf Hexane Fraction through Regulating Osteoclast and Osteoblast Differentiation.

Osteoporosis is the result of an imbalance in the bone-remodeling process via an increase in osteoclastic activity and a decrease in osteoblastic activity. Our previous studies have shown that Perilla frutescens seed meal has anti-osteoclastogenic activity. However, the role of perilla leaf hexane fraction (PLH) in osteoporosis has not yet been investigated and reported. In this study, we aimed to investigate the effects of PLH in osteoclast differentiation and osteogenic potential using cell-based experiments in vitro. From HPLC analysis, we found that PLH contained high luteolin and baicalein. PLH was shown to inhibit RANKL-induced ROS production and tartrate-resistant acid phosphatase (TRAP)-positive multi-nucleated osteoclasts. Moreover, PLH significantly downregulated the RANKL-induced MAPK and NF-κB signaling pathways, leading to the attenuation of NFATc1 and MMP-9 expression. In contrast, PLH enhanced osteoblast function by regulating alkaline phosphatase (ALP) and restoring TNF-α-suppressed osteoblast proliferation and osteogenic potential. Thus, luteolin and baicalein-rich PLH inhibits osteoclast differentiation but promotes the function of osteoblasts. Collectively, our data provide new evidence that suggests that PLH may be a valuable anti-osteoporosis agent.

Anti-osteoclastogenic cycloartane saponins from Mussaenda pubescens

The methanolic extract of Mussaenda pubescens Dryand leaves exhibited significant anti-osteoclastogenic activity. Chemical investigation of M. pubescens led to the isolation of one new cycloartane saponin, mussaendoside X (1) along with eight known compounds: heinsiagenin A 3-O-[α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→2)]-O-β-D-glucopyranoside (2), mussaendoside O (3), heinsiagenin A 3-O-[α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→2)]-O-β-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside (4), mussaendoside G (5), mussaendoside U (6), shanzhiside methyl ester (7), barlerin (8) and musaenoside (9). Their structures were elucidated by extensive spectroscopic methods including 1D- and 2D-NMR as well as MS analysis and comparison with the literature. Cycloartane saponins 1–6 positively suppressed osteoclast formation in an anti-osteoclastogenic screening assay. Consequently, treatment of RANKL-stimulated RAW 264.7 cells with compounds 1–4 significantly decreased the number of osteoclasts in a concentration-dependent manner. Six compounds from M. pubescens, with the new cycloartane, mussaendoside X, were shown for the first time as potential effective inhibitors of osteoclastogenesis.

Anti-osteoclastogenic activity of metabolites isolated from Viburnum lutescens Blume

• Two new compounds were isolated from Viburnum lutescens Blume. • The structures were successfully determined by spectroscopic evidence. • Compounds were evaluated anti-osteoclastogenic activity. • Triterpenoids significantly inhibited anti-osteoclastogenic activity. One new oleanane triterpenoid, 2-oxo-3 β ,23-dihydroxyolean-12-en-28-oic acid ( 1 ), one new megastigmane, (1 S ,3 S ,9 R )-megastigman-5-en-3,9,11-triol 11- O - β -D-glucopyranoside ( 6 ) and nine known compounds, 23-hydroxyursolic acid ( 2 ), 6 α -hydroxybetulic acid ( 3 ), alphitolic acid ( 4 ), 3-oxo-6 α -hydroxylup-20(29)-en-28-oic acid ( 5 ), (6 R ,9 R )-3-oxo- α -ionol 9- O - β -D-glucopyranoside ( 7 ), byzantionoside B ( 8 ), dihydrovomifoliol O- β -D-glucopyranoside ( 9 ), 1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-rhamnopyranoxypropyl)-methoxyphenoxy]-1,3-propanediol ( 10 ), and sweroside ( 11 ) were isolated from the methanol extract of the Viburnum lutescens Blume leaves. Their chemical structures were elucidated by 1D- and 2D-NMR and MS data and comparing their NMR data with those reported in the literature. In addition, the anti-osteoclastogenic activities of all compounds were also evaluated. The TRAP staining on RAW 264.7 cells indicated that compounds 1 , 2 , and 5 potently inhibited the osteoclastogenesis in RAW 264.7 cells in a concentration dependent manner.

Non-polar lipid from greenshell mussel (Perna canaliculus) inhibits osteoclast differentiation

The osteoclast-dependent bone resorption process is a crucial part of the bone regulatory system. The excessive function of osteoclasts can cause diseases of bone, joint, and other tissues such as osteoporosis and osteoarthritis. Greenshell mussel oil (GSM), a good source of long chain omega-3 polyunsaturated fatty acids (LCn-3PUFAs), was fractionated into total lipid, polar lipid, and non-polar lipid components and their anti-osteoclastogenic activity tested in RAW 264.7 cell cultures. Osteoclast differentiation process was achieved after 5 days of incubation with RANKL in 24-well culture plates. Introducing the non-polar lipid fraction into the culture caused a lack of cell differentiation, and a reduction in tartrate-resistant acid phosphatase (TRAP) activity and TRAP cell numbers in a dose-dependent manner (50% reduction at the concentration of 20 μg/mL, p < 0.001). Moreover, actin ring formation was significantly diminished by non-polar lipids at 10–20 μg/mL. The bone digestive enzymes released by osteoclasts into the pit formation were also compromised by downregulating gene expression of cathepsin K, carbonic anhydrase II (CA II), matrix metalloproteinase 9 (MMP-9), and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1). This study revealed that the non-polar lipid fraction of GSM oil contains bioactive substances which possess potent anti-osteoclastogenic activity.

Fengshi Qutong capsule ameliorates bone destruction of experimental rheumatoid arthritis by inhibiting osteoclastogenesis

Ethnopharmacological relevanceBone destruction plays a key role in damaging the joint function of rheumatoid arthritis (RA). Fengshi Qutong capsule (FSQTC) consisting of 19 traditional Chinese medicines has been used for treating RA in China for many years. Preliminary studies show that FSQTC has analgesic activity and inhibits synovial angiogenesis of collagen-induced arthritis (CIA), but its role on bone destruction of RA is still unclear. Aim of the studyTo explore the effect of FSQTC on bone destruction of RA and the possible mechanism of osteoclastogenesis in vivo and in vitro. Materials and methodsLC-MS system was used to detect the quality control components of FSQTC. The anti-arthritic effect of FSQTC on CIA rats was evaluated by arthritis score, arthritis incidence and histopathology evaluation of inflamed joints. The effect of treatment with FSQTC on bone destruction of joint tissues was determined with X-ray and micro-CT quantification, and on bone resorption marker CTX-I and formation marker osteocalcin in sera were detected by ELISA. Then, osteoclast differentiation and mature were evaluated by TRAP staining, actin ring immunofluorescence and bone resorption assay both in joints and RANKL-induced RAW264.7 cells. In addition, RANKL, OPG, IL-1β and TNFα in sera were evaluated by ELISA. The molecular mechanisms of the inhibitions were elucidated by analyzing the protein and gene expression of osteoclastic markers CTSK, MMP-9 and β3-Integrin, transcriptional factors c-Fos and NFATc1, as well as phosphorylation of ERK1/2, JNK and P38 in joints and in RANKL-induced RAW264.7 cells using western blot and/or qPCR. ResultsIn this study, 12 major quality control components were identified. Our data showed that FSQTC significantly increased bone mineral density, volume fraction, trabecular thickness, and decreased trabecular separation of inflamed joints both at periarticular and extra-articular locations in CIA rats. FSQTC also diminished the level of CTX-I and simultaneously increased osteocalcin in sera of CIA rats. The effects were accompanied by reductions of osteoclast differentiation, bone resorption, and expression of osteoclastic markers (CTSK, MMP-9 and β3-Integrin) in joints. Interestingly, FSQTC treatment could reduce the protein level of RANKL, increase the expression of OPG, and decrease the ratio of RANKL to OPG in inflamed joints and sera of CIA rats. In addition, FSQTC inhibited the levels of pro-inflammatory cytokines implicated in bone resorption, such as IL-1β and TNFα in sera. When RAW264.7 cells were treated with RANKL, FSQTC inhibited the formation of TRAP + multinucleated cells, actin ring and the bone-resorbing activity in dose-dependent manners. Furthermore, FSQTC reduced the RANKL-induced expression of osteoclastic genes and proteins and transcriptional factors (c-Fos and NFATc1), as well as phosphorylation of mitogen-activated protein kinases (MAPKs). ConclusionFSQTC may inhibit bone destruction of RA by its anti-osteoclastogenic activity both in vivo and in vitro.

Increased frequency of Th17 cells and IL-17 levels are associated with low bone mineral density in postmenopausal women

Osteoporosis is one of the chronic and often neglected bone diseases in aging postmenopausal women that affect the quality of life. Studies on ovariectomized mice models indicated the reciprocal role of Th17 cells and Treg cells in the aetiology of osteoporosis. While Th17 cells promote osteoclastogenesis, Treg cells exhibit anti-osteoclastogenic activity. This exploratory study aimed to determine the difference in the frequency of these T-cell subtypes in pre-and postmenopausal women and to examine their association with BMD. In our study, the frequency of Treg cells, analyzed by flow cytometry, did not differ between pre-and postmenopausal women. However, plasma levels of IL-10 along with IL-10+CD4+T cells were higher in post- compared to premenopausal women. The frequency of Th17 cells was higher in postmenopausal women irrespective of their BMD, however, only postmenopausal women with low BMD had elevated IL-17 levels and their T-scores were associated with Th17 frequency. Collectively, the results suggest that estrogen insufficiency in postmenopausal women may lead to increased Th17 cell frequency and elevated IL-17 levels which are associated with low BMD. This study highlights, Th17 cells and IL-17 as key players in the pathogenesis of osteoporosis and they can be the potential targets for immunotherapy in the treatment of osteoporosis.