Anti-Osteoporotic Effect of Morroniside on Osteoblast and Osteoclast Differentiation In Vitro and Ovariectomized Mice In Vivo

Bone remodeling is a continuous process of bone synthesis and destruction that is regulated by osteoblasts and osteoclasts. Here, we investigated the anti-osteoporotic effects of morroniside in mouse preosteoblast MC3T3-E1 cells and mouse primary cultured osteoblasts and osteoclasts in vitro and ovariectomy (OVX)-induced mouse osteoporosis in vivo. Morroniside treatment enhanced alkaline phosphatase activity and positively stained cells via upregulation of osteoblastogenesis-associated genes in MC3T3-E1 cell lines and primary cultured osteoblasts. However, morroniside inhibited tartrate-resistant acid phosphatase activity and TRAP-stained multinucleated positive cells via downregulation of osteoclast-mediated genes in primary cultured monocytes. In the osteoporotic animal model, ovariectomized (OVX) mice were administered morroniside (2 or 10 mg/kg/day) for 12 weeks. Morroniside prevented OVX-induced bone mineral density (BMD) loss and reduced bone structural compartment loss in the micro-CT images. Taken together, morroniside promoted increased osteoblast differentiation and decreased osteoclast differentiation in cells, and consequently inhibited OVX-induced osteoporotic pathogenesis in mice. This study suggests that morroniside may be a potent therapeutic single compound for the prevention of osteoporosis.

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Identification and Functional Characterization of Metabolites for Bone Mass in Peri-/Post-menopausal Chinese Women

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgab146 ◽

2021 ◽

Author(s):

Rui Gong ◽

Hong-Mei Xiao ◽

Yin-Hua Zhang ◽

Qi Zhao ◽

Kuan-Jui Su ◽

...

Keyword(s):

Fatty Acids ◽

Bone Loss ◽

Osteoclast Differentiation ◽

Dodecanoic Acid ◽

Mineral Density ◽

Ovariectomized Mice ◽

Post Menopausal ◽

Functional Mechanisms

Abstract Context Although metabolic profiles appear to play an important role in menopausal bone loss, the functional mechanisms by which metabolites influence bone mineral density (BMD) during menopause are largely unknown. Objective We aimed to systematically identify metabolites associated with BMD variation and their potential functional mechanisms in peri-/post-menopausal women. Design and Methods We performed serum metabolomic profiling and whole-genome sequencing for 517 perimenopausal (16%) and early postmenopausal (84%) women aged 41 to 64 years in this cross-sectional study. Partial least squares (PLS) regression and general linear regression analysis were applied to identify BMD-associated metabolites, and weighted gene co-expression network analysis was performed to construct co-functional metabolite modules. Furthermore, we performed Mendelian randomization analysis to identify causal relationships between BMD-associated metabolites and BMD variation. Finally, we explored the effects of a novel prominent BMD-associated metabolite on bone metabolism through both in vivo/in vitro experiments. Results Twenty metabolites and a co-functional metabolite module (consisting of fatty acids) were significantly associated with BMD variation. We found dodecanoic acid (DA), within the identified module, causally decreased total hip BMD. Subsequently, the in vivo experiments might support that dietary supplementation with DA could promote bone loss, as well as increase the osteoblast and osteoclast numbers in normal/ovariectomized mice. DA treatment differentially promoted osteoblast and osteoclast differentiation, especially for osteoclast differentiation at higher concentrations in vitro (e.g.,10, 100μM). Conclusions This study sheds light on metabolomic profiles associated with postmenopausal osteoporosis risk, highlighting the potential importance of fatty acids, as exemplified by DA, in regulating BMD.

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Anti-Osteoporotic Effects of Kukoamine B in Osteoblast and Osteoclast Cells and Ovariectomized Mice

10.20944/preprints201710.0196.v1 ◽

2017 ◽

Author(s):

Eunkuk Park ◽

Jeonghyun Kim ◽

Mun-Chang Kim ◽

Subin Yeo ◽

Jieun Kim ◽

...

Keyword(s):

Osteoblast Differentiation ◽

Osteoclast Differentiation ◽

Osteoblastic Differentiation ◽

Nodule Formation ◽

Mouse Bone Marrow ◽

Mineral Density ◽

Bone Mineral Density Loss ◽

Ovariectomized Mice

Osteoporosis is an abnormal bone remodeling condition characterized by decreased bone density, which leads to high risks of broken bones. Previous studies have demonstrated that Lycii Radicis Cortex (LRC) extract inhibits bone loss in ovariectomized (OVX) mice by enhancing the osteoblast differentiation. A bioactive compound, Kukoamine B (KB), was identified from a fractionation of LRC extract as a candidate component responsible for an anti-osteoporotic effect. This study investigated the anti-osteoporotic effects of KB using in vitro and in vivo osteoporosis models. KB treatment significantly increased the osteoblastic differentiation and mineralized nodule formation of osteoblastic MC3T3-E1 cells, while it significantly decreased the osteoclast differentiation of primary-cultured monocytes derived from mouse bone marrow. The effects of KB on osteoblastic and osteoclastic differentiations under more physiological conditions were also examined. In the co-culture of MC3T3-E1 cells and monocytes, KB promoted osteoblast differentiation but did not affect osteoclast differentiation. For the in vivo experiments, KB significantly inhibited OVX-induced bone mineral density loss and restored the impaired bone structural properties in osteoporosis model mice. These results suggest that KB may be a potential therapeutic candidate for the treatment of osteoporosis.

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Circaea Mollis Siebold & Zucc. Prevents Bone Loss in a Mouse Postmenopausal Osteoporosis Model by Promoting of Osteoblast Differentiation (P06-130-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz031.p06-130-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Gyhye Yoo ◽

Ji-Hye Park ◽

Yang-Ju Son ◽

Chang Ho Lee ◽

Chu Won Nho

Keyword(s):

Breast Cancer ◽

Side Effects ◽

Bone Loss ◽

Postmenopausal Osteoporosis ◽

Osteoclast Differentiation ◽

Mineral Density ◽

Promising Alternative ◽

Ovariectomized Mice

Abstract Objectives Postmenopausal osteoporosis, a condition of low bone density consequent to decreased estrogen levels after menopause in women, is generally treated with hormone replacement therapy. However, long-term hormone use may cause critical side effects including breast cancer. Alternatively, phytoestrogens, which have similar structures to steroid hormones, are reported to cure postmenopausal symptoms with fewer side effects. Here, we investigated the effects of EtOH extract of Circaea mollis Siebold & Zucc. (EECM), a traditional herbal medicine in Asia that exhibits anti-arthritic activities, on postmenopausal osteoporosis. Methods In vitro model: MCF7 breast cancer cells and MC3T3-E1 pre-osteoblast cells were utilized to estimate estrogenic and osteogenic activity. Osteoblastic markers were measured by western blot and real-time PCR. In vivo model: Female mature C57BL/6 mice were ovariectomized and oral administrated with 10 mg/kg and 40 mg/kg of EECM respectively. Results EECM increased alkaline phosphatase activity and osteoblastic markers including osteoprotegerin at day 6 during mouse preosteoblast differentiation. EECM inhibited osteoclast differentiation and bone resorption in an osteoblast-osteoclast primary co-culture system via osteoprotegerin-mediated RANK/RANKL signaling. In ovariectomized mice, EECM prevented bone mineral density decrease and recovered osteoblastic molecules. Conclusions EECM enhanced the differentiation of osteoblasts via osteogenic markers and modulated RANK/RANKL signaling via an elevation of OPG from osteoblasts in vitro and in vivo. Therefore, EECM may be effective in preventing bone loss and offers a promising alternative for the nutritional management of postmenopausal osteoporosis. Funding Sources This work was supported by the Center Project for the Korea-Mongolia Science and Technology Cooperation (2U06170). Supporting Tables, Images and/or Graphs

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Melatonin inhibits osteoclastogenesis and bone loss in ovariectomized mice by regulating PRMT1-mediated signaling

Endocrinology ◽

10.1210/endocr/bqab057 ◽

2021 ◽

Author(s):

Joo-Hee Choi ◽

Ah-Ra Jang ◽

Min-Jung Park ◽

Dong-il Kim ◽

Jong-Hwan Park

Keyword(s):

Bone Loss ◽

Osteoclast Differentiation ◽

Tumor Necrosis Factor Receptor ◽

Estrogen Deficiency ◽

Melatonin Receptors ◽

Mineral Density ◽

Melatonin Treatment ◽

Ovariectomized Mice

Abstract Melatonin, a pineal gland hormone, has been suggested to treat postmenopausal osteoporosis due to its inhibitory effect on osteoclast differentiation. We previously reported that protein arginine methyltransferase 1 (PRMT1) was an important mediator of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. However, the relationship between melatonin and PRMT1 in osteoclast differentiation and estrogen deficiency-induced osteoporosis is unclear. In this study, we investigated the inhibitory mechanisms of melatonin in vitro and in vivo by focusing on PRMT1. Melatonin treatment effectively blocked RANKL-induced osteoclastogenesis by inhibiting PRMT1 and asymmetric dimethylarginine (ADMA) expression. RANKL-induced tumor necrosis factor receptor-associated factor 6 (TRAF6) and the phosphorylation of JNK were also suppressed by melatonin, and TRAF6 siRNA attenuated RANKL-induced p-JNK and PRMT1 production. Melatonin inhibited the transcriptional activity of NF-κB by interfering with the binding of PRMT1 and NF-κB subunit p65 in RANKL-treated BMDMs. Our results also revealed that melatonin inhibits RANKL-induced PRMT1 expression through receptors-independent pathway. Thus, the anti-osteoclastogenic effect of melatonin was mediated by a cascade of inhibition of RANKL-induced TRAF6, JNK, PRMT1, and NF-κB signaling in melatonin receptors-independent pathway. In vivo, ovariectomy caused significant decreases in bone mineral density, but melatonin treatment alleviated the ovariectomized (OVX)-induced bone loss by inhibiting bone resorption. Furthermore, the expression PRMT1 and TRAP mRNA was upregulated in OVX-femurs, but effectively suppressed by melatonin injection. These findings suggest that melatonin inhibited osteoclast differentiation and estrogen deficiency-induced osteoporosis by suppressing RANKL-induced TRAF6, JNK, PRMT1, and NF-κB signaling cascades in melatonin receptors-independent pathway.

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Osteoprotective Effects of Loganic Acid on Osteoblastic and Osteoclastic Cells and Osteoporosis-Induced Mice

International Journal of Molecular Sciences ◽

10.3390/ijms22010233 ◽

2020 ◽

Vol 22 (1) ◽

pp. 233

Author(s):

Eunkuk Park ◽

Chang Gun Lee ◽

Eunguk Lim ◽

Seokjin Hwang ◽

Seung Hee Yun ◽

...

Keyword(s):

Osteoclast Differentiation ◽

Osteoblastic Differentiation ◽

Common Disease ◽

Root Extract ◽

Mouse Bone Marrow ◽

Mineral Density ◽

Bone Mineral Density Loss ◽

In Vivo Experiments

Osteoporosis is a common disease caused by an imbalance of processes between bone resorption by osteoclasts and bone formation by osteoblasts in postmenopausal women. The roots of Gentiana lutea L. (GL) are reported to have beneficial effects on various human diseases related to liver functions and gastrointestinal motility, as well as on arthritis. Here, we fractionated and isolated bioactive constituent(s) responsible for anti-osteoporotic effects of GL root extract. A single phytochemical compound, loganic acid, was identified as a candidate osteoprotective agent. Its anti-osteoporotic effects were examined in vitro and in vivo. Treatment with loganic acid significantly increased osteoblastic differentiation in preosteoblast MC3T3-E1 cells by promoting alkaline phosphatase activity and increasing mRNA expression levels of bone metabolic markers such as Alpl, Bglap, and Sp7. However, loganic acid inhibited osteoclast differentiation of primary-cultured monocytes derived from mouse bone marrow. For in vivo experiments, the effect of loganic acid on ovariectomized (OVX) mice was examined for 12 weeks. Loganic acid prevented OVX-induced bone mineral density loss and improved bone structural properties in osteoporotic model mice. These results suggest that loganic acid may be a potential therapeutic candidate for treatment of osteoporosis.

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Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL

International Journal of Molecular Sciences ◽

10.3390/ijms22010434 ◽

2021 ◽

Vol 22 (1) ◽

pp. 434

Author(s):

Yuria Jang ◽

Hong Moon Sohn ◽

Young Jong Ko ◽

Hoon Hyun ◽

Wonbong Lim

Keyword(s):

Nuclear Translocation ◽

Critical Role ◽

Osteoclast Differentiation ◽

Point Mutations ◽

Tartrate Resistant Acid Phosphatase ◽

Mice Model ◽

Gsk 3Β ◽

Rank Signaling

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.

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Cells in regenerating deer antler cartilage provide a microenvironment that supports osteoclast differentiation

Journal of Experimental Biology ◽

10.1242/jeb.204.3.443 ◽

2001 ◽

Vol 204 (3) ◽

pp. 443-455

Author(s):

C. Faucheux ◽

S. Nesbitt ◽

M. Horton ◽

J. Price

Keyword(s):

Type I Collagen ◽

Mononuclear Cells ◽

Osteoclast Differentiation ◽

Cathepsin K ◽

Collagen Matrix ◽

Tartrate Resistant Acid Phosphatase ◽

Type I ◽

Deer Antler

Deer antlers are a rare example of mammalian epimorphic regeneration. Each year, the antlers re-grow by a modified endochondral ossification process that involves extensive remodelling of cartilage by osteoclasts. This study identified regenerating antler cartilage as a site of osteoclastogenesis in vivo. An in vitro model was then developed to study antler osteoclast differentiation. Cultured as a high-density micromass, cells from non-mineralised cartilage supported the differentiation of large numbers of osteoclast-like multinucleated cells (MNCs) in the absence of factors normally required for osteoclastogenesis. After 48 h of culture, tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells (osteoclast precursors) were visible, and by day 14 a large number of TRAP-positive MNCs had formed (783+/−200 per well, mean +/− s.e.m., N=4). Reverse transcriptase/polymerase chain reaction (RT-PCR) showed that receptor activator of NF κ B ligand (RANKL) and macrophage colony stimulating factor (M-CSF) mRNAs were expressed in micromass cultures. Antler MNCs have the phenotype of osteoclasts from mammalian bone; they expressed TRAP, vitronectin and calcitonin receptors and, when cultured on dentine, formed F-actin rings and large resorption pits. When cultured on glass, antler MNCs appeared to digest the matrix of the micromass and endocytose type I collagen. Matrix metalloproteinase-9 (MMP-9) may play a role in the resorption of this non-mineralised matrix since it is highly expressed in 100 % of MNCs. In contrast, cathepsin K, another enzyme expressed in osteoclasts from bone, is only highly expressed in resorbing MNCs cultured on dentine. This study identifies the deer antler as a valuable model that can be used to study the differentiation and function of osteoclasts in adult regenerating mineralised tissues.

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Crataegus pinnatifida Bunge Inhibits RANKL-Induced Osteoclast Differentiation in RAW 264.7 Cells and Prevents Bone Loss in an Ovariectomized Rat Model

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/5521562 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Minsun Kim ◽

MinBeom Kim ◽

Jae-Hyun Kim ◽

SooYeon Hong ◽

Dong Hee Kim ◽

...

Keyword(s):

Bone Loss ◽

Rat Model ◽

Osteoclast Differentiation ◽

Ovariectomized Rat ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Mineral Density ◽

Crataegus Pinnatifida

Osteoporosis is characterized by a decrease in bone microarchitecture with an increased risk of fracture. Long-term use of primary treatments, such as bisphosphonates and selective estrogen receptor modulators, results in various side effects. Therefore, it is necessary to develop alternative therapeutics derived from natural products. Crataegus pinnatifida Bunge (CPB) is a dried fruit used to treat diet-induced indigestion, loss of appetite, and diarrhea. However, research into the effects of CPB on osteoclast differentiation and osteoporosis is still limited. In vitro experiments were conducted to examine the effects of CPB on RANKL-induced osteoclast differentiation in RAW 264.7 cells. Moreover, we investigated the effects of CPB on bone loss in the femoral head in an ovariectomized rat model using microcomputed tomography. In vitro, tartrate-resistant acid phosphatase (TRAP) staining results showed the number of TRAP-positive cells, and TRAP activity significantly decreased following CPB treatment. CPB also significantly decreased pit formation. Furthermore, CPB inhibited osteoclast differentiation by suppressing NFATc1, and c-Fos expression. Moreover, CPB treatment inhibited osteoclast-related genes, such as Nfatc1, Ca2, Acp5, mmp9, CtsK, Oscar, and Atp6v0d2. In vivo, bone mineral density and structure model index were improved by administration of CPB. In conclusion, CPB prevented osteoclast differentiation in vitro and prevented bone loss in vivo. Therefore, CPB could be a potential alternative medicine for bone diseases, such as osteoporosis.

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Anti–IL-20 monoclonal antibody inhibits the differentiation of osteoclasts and protects against osteoporotic bone loss

Journal of Experimental Medicine ◽

10.1084/jem.20102234 ◽

2011 ◽

Vol 208 (9) ◽

pp. 1849-1861 ◽

Cited By ~ 47

Author(s):

Yu-Hsiang Hsu ◽

Wei-Yu Chen ◽

Chien-Hui Chan ◽

Chih-Hsing Wu ◽

Zih-Jie Sun ◽

...

Keyword(s):

Monoclonal Antibody ◽

Bone Loss ◽

Therapeutic Potential ◽

Osteoclast Differentiation ◽

Bone Destruction ◽

Osteoporotic Bone ◽

Mineral Density

IL-20 is a proinflammatory cytokine of the IL-10 family that is involved in psoriasis, rheumatoid arthritis, atherosclerosis, and stroke. However, little is known about the role of IL-20 in bone destruction. We explored the function of IL-20 in osteoclastogenesis and the therapeutic potential of anti–IL-20 monoclonal antibody 7E for treating osteoporosis. Higher serum IL-20 levels were detected in patients with osteopenia and osteoporosis and in ovariectomized (OVX) mice. IL-20 mediates osteoclastogenesis by up-regulating the receptor activator of NF-κB (RANK) expression in osteoclast precursor cells and RANK ligand (RANKL) in osteoblasts. 7E treatment completely inhibited osteoclast differentiation induced by macrophage colony-stimulating factor (M-CSF) and RANKL in vitro and protected mice from OVX-induced bone loss in vivo. Furthermore, IL-20R1–deficient mice had significantly higher bone mineral density (BMD) than did wild-type controls. IL-20R1 deficiency also abolished IL-20–induced osteoclastogenesis and increased BMD in OVX mice. We have identified a pivotal role of IL-20 in osteoclast differentiation, and we conclude that anti–IL-20 monoclonal antibody is a potential therapeutic for protecting against osteoporotic bone loss.

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Chemokine and chemokine receptor expression during colony stimulating factor-1–induced osteoclast differentiation in the toothless osteopetrotic rat: a key role for CCL9 (MIP-1γ) in osteoclastogenesis in vivo and in vitro

Blood ◽

10.1182/blood-2005-08-3365 ◽

2006 ◽

Vol 107 (6) ◽

pp. 2262-2270 ◽

Cited By ~ 57

Author(s):

Meiheng Yang ◽

Geneviève Mailhot ◽

Carole A. MacKay ◽

April Mason-Savas ◽

Justin Aubin ◽

...

Keyword(s):

Osteoclast Differentiation ◽

The Body ◽

Receptor Expression ◽

Long Bone ◽

Tartrate Resistant Acid Phosphatase ◽

Colony Stimulating Factor ◽

Cell Counts ◽

Stimulating Factor

AbstractOsteoclasts differentiate from hematopoietic precursors under systemic and local controls. Chemokines and receptors direct leukocyte traffic throughout the body and may help regulate site-specific bone resorption. We investigated bone gene expression in vivo during rapid osteoclast differentiation induced by colony-stimulating factor 1 (CSF-1) in Csf1-null toothless (tl/tl) rats. Long-bone RNA from CSF-1–treated tl/tl rats was analyzed by high-density microarray over a time course. TRAP (tartrate-resistant acid phosphatase)–positive osteoclasts appeared on day 2, peaked on day 4, and decreased slightly on day 6, as marrow space was expanding. TRAP and cathepsin K mRNA paralleled the cell counts. We examined all chemokine and receptor mRNAs on the arrays. CCL9 was strongly induced and peaked on day 2, as did its receptor, CCR1, and regulatory receptors c-Fms (CSF-1 receptor) and RANK (receptor activator of nuclear factor κB). Other chemokines and receptors showed little or no significant changes. In situ hybridization and immunohistochemistry revealed CCL9 in small, immature osteoclasts on day 2 and in mature cells at later times. Anti-CCL9 antibody inhibited osteoclast differentiation in culture and significantly suppressed the osteoclast response in CSF-1–treated tl/tl rats. While various chemokines have been implicated in osteoclastogenesis in vitro, this first systematic analysis of chemokines and receptors during osteoclast differentiation in vivo highlights the key role of CCL9 in this process.

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