scholarly journals Inhibitory Effects of Astaxanthin on CML-HSA-Induced Inflammatory and RANKL-Induced Osteoclastogenic Gene Expression in RAW 264.7 Cells

Biomedicines ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
A. N. M. Mamun-Or-Rashid ◽  
Tanzima Tarannum Lucy ◽  
Masayuki Yagi ◽  
Yoshikazu Yonei
Keyword(s):  
Gene Expression ◽  
Raw 264.7 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Marker Genes ◽  
Tartrate Resistant Acid Phosphatase ◽  
Raw 264.7 ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Inflammatory Gene Expression ◽  
Inflammatory Gene

Objective: Elevated levels of serum Nε-carboxymethyllysine (CML), a well-known advanced glycation end-product (AGE), were observed in patients with inflammation or osteoporosis. Astaxanthin was reported to possess anti-inflammatory and antioxidant effects. In the present study, we investigated the effects of commercially available dietary supplement AstaReal ACTR (ASR) capsule content as astaxanthin on CML-HSA-induced inflammatory and receptor activator of nuclear factor-kappa-Β ligand (RANKL)-induced osteoclastogenic gene expression. Methods: RAW 264.7 murine macrophage cells were stimulated with CML-HSA to trigger inflammatory gene expression and treated with either a vehicle control or varied concentrations of astaxanthin. Inflammatory gene expression was measured using an enzyme-linked immunosorbent assay (ELISA) or qPCR. We triggered osteoclastogenesis using RANKL, and osteoclastogenic gene expression was measured through tartrate-resistant acid phosphatase (TRAP) activity, staining, immunofluorescence, and qPCR analyses. Results: CML-HSA showed a stimulatory effect on inflammatory gene expression, and astaxanthin reduced the expression by at least two-fold. The levels of autoinflammatory gene expression were reduced by astaxanthin. The RANKL-induced osteoclastogenesis was significantly inhibited by astaxanthin, with reductions in the activation of nuclear factor-κB (NF-κB), the expression of NFATc1 (nuclear factor of activated T cells 1), multinucleated cell formation, and the expression of mature osteoclast marker genes. Conclusion: Astaxanthin has potential as a remedy for CML-HSA-induced inflammation and RANKL-induced excessive bone loss.

scholarly journals The inhibitory effect of melatonin on osteoclastogenesis of RAW 264.7 cells in low concentrations of RANKL and MCSF

2020 ◽  
Vol 44 (6) ◽  
pp. 427-436
Author(s):  
Hala JARRAR ◽  
Damla ÇETİN ALTINDAL ◽  
Menemşe GÜMÜŞDERELİOĞLU
Keyword(s):  
Cathepsin K ◽  
Suppressive Effect ◽  
Inhibitory Effect ◽  
Raw 264.7 Cells ◽  
Tartrate Resistant Acid Phosphatase ◽  
Raw 264.7 ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Repressive Effect ◽  
Activity Trap

RAW 264.7 cells are one of the most recommended cell lines for investigating the activity and differentiation of osteoclasts. These cells differentiate into osteoclasts in the presence of two critical components: receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (MCSF). Melatonin (MEL) hormone has recently become one of the small molecules used in the field of bone regeneration and bone disease treatment, as it has the ability to inhibit the differentiation of osteoclasts directly by suppression of the NF-κB signaling pathway. The main aim of the current study is to determine sufficient RANKL/MCSF concentrations for differentiation of the cells to osteoclasts and to describe the repressive effect of MEL on the osteoclastogenesis of these cells. In this regard, it was found that 10 ng/mL of RANKL- and MCSF-containing medium is suitable for inducing osteoclastogenesis of the cells. In addition, melatonin at doses in the range of 100–1000 μM does not have a cytotoxic effect. Subsequently, results of tartrate resistant acid phosphatase (TRAP) activity, TRAP staining, and relative expressions of cathepsin K, nuclear factor of activated T cells one (NFATC1), and TRAP genes showed a suppressive effect of MEL —especially 800 μM— on RANKL-induced osteoclastogenesis of these cells.

scholarly journals Inhibition of Osteoclastogenesis by Thioredoxin-Interacting Protein-Derived Peptide (TN13)

2019 ◽  
Vol 8 (4) ◽  
pp. 431 ◽  
Author(s):  
Mi Kim ◽  
Won Kim ◽  
Jae-Eun Byun ◽  
Jung Choi ◽  
Suk Yoon ◽  
...  
Keyword(s):  
Bone Loss ◽  
Osteoclast Differentiation ◽  
Bone Diseases ◽  
Raw 264.7 Cells ◽  
Tartrate Resistant Acid Phosphatase ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Interacting Protein ◽  
Hematopoietic Stem ◽  
Thioredoxin Interacting Protein

Overactivated osteoclasts lead to many bone diseases, including osteoporosis and rheumatoid arthritis. The p38 MAPK (p38) is an essential regulator of the receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis and bone loss. We previously reported TAT conjugated thioredoxin-interacting protein-derived peptide (TAT-TN13) as an inhibitor of p38 in hematopoietic stem cells (HSCs). Here, we examined the role of TAT-TN13 in the differentiation and function of osteoclasts. TAT-TN13 significantly suppressed RANKL-mediated differentiation of RAW 264.7 cells and bone marrow macrophages (BMMs) into osteoclasts. TAT-TN13 also inhibited the RANKL-induced activation of NF-κB and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), leading to the decreased expression of osteoclast-specific genes, including tartrate-resistant acid phosphatase (TRAP) and Cathepsin K. Additionally, TAT-TN13 treatment protected bone loss in ovariectomized (OVX) mice. Taken together, these results suggest that TAT-TN13 inhibits osteoclast differentiation by regulating the p38 and NF-κB signaling pathway; thus, it may be a useful agent for preventing or treating osteoporosis.

scholarly journals Inhibition of osteoclastogenesis and inflammation by phosvitin and phosvitin hydrolysate via NF-κB and MAPK pathways in RAW 264.7 cells

2021 ◽  
Vol 13 ◽  
Author(s):  
Jiandong Ren ◽  
Subhadeep Chakrabarti ◽  
Jianping Wu
Keyword(s):  
Raw264.7 Cells ◽  
Raw 264.7 Cells ◽  
Tartrate Resistant Acid Phosphatase ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Necrosis Factor Alpha ◽  
Monocyte Chemoattractant Protein 1 ◽  
Tnf Α ◽  
Factor Alpha ◽  
Oxide Synthase

Phosvitin (PV) is an egg protein. Our recent study showed both phosvitin and phosvitin hydrolysate (PVH) could promote osteoblast differentiation in osteoblast cells. The objective of the study was to investigate the effects of PV and PVH on osteoclastogenesis and possible signalling pathways in RAW264.7 cells. Both PV and PVH inhibited osteoclastogenesis (fewer tartrate-resistant acid phosphatase (TRAP) positive cells and lower TRAP activity), reduced levels of transcription factors, c-Fos and NFATc1 (nuclear factor of activated T-cells, cytoplasmic 1), and suppressed inflammatory biomarkers TNF-α (tumor necrosis factor alpha), MCP-1 (monocyte chemoattractant protein 1), RANTES (regulated on activation, normal T cell expressed and secreted), and inducible nitric oxide synthase. The inhibitory effects of PV and PVH on RAW264.7 cells differentiation were likely mediated through p38, c-Jun N-terminal kinases (JNK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. These results indicated that PV and PVH might inhibit bone resorption activities.  

scholarly journals Anti-inflammatory effect of neo-lignan isoamericanin A via suppression of NF-κB in liposaccharide-stimulated RAW 264.7 cells

2020 ◽  
Vol 19 (9) ◽  
pp. 1857-1862
Author(s):  
Ji Yun Yeo ◽  
Kwang Woo Hwang ◽  
So-Young Park
Keyword(s):  
Nitric Oxide ◽  
Proinflammatory Cytokines ◽  
Seed Extract ◽  
Raw 264.7 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
No Production ◽  
Raw 264.7 ◽  
Nuclear Factor ◽  
Active Constituent ◽  
Anti Inflammatory

Purpose: To investigate the potential anti-inflammatory effects of the seeds of Opuntina humifusa and its active constituents.Methods: The extract of O. humifusa seeds was tested for the inhibition of nitric oxide (NO) production in liposaccharide (LPS)-stimulated RAW 264.7 cells using Griess reagent. The active constituents were isolated using bioassay-guided isolation methods. The effects of the active constituent on NO, proinflammatory cytokines, nuclear factor-kappa B (NF-κB) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) were evaluated by enzyme-linked immunosorbent assay (ELISA) and western blot analysis.Results: The seed extract of O. humifusa significantly attenuated LPS-induced NO production in RAW 264.7 cells (p < 0.05). Bioassay-guided fractionation resulted in the isolation of isoamericanin A as an active constituent. Isoamericanin A reduced LPS-induced production of NO, iNOS, and proinflammatory cytokines (TNF-α and IL-6) in a concentration-dependent manner (p < 0.05). Furthermore, the effect was accompanied by decreased translocation of NF-κB from the cytosol to the nucleus and the decreased phosphorylation of IκB in the cytosol induced by LPS (p < 0.05).Conclusion: The seed extract of O. humifusa and its active constituent, isoamericanin A, have antiinflammatory effects in LPS-stimulated RAW 264.7 cells, suggesting that they have potentials as antiinflammatory agents. Keywords: Opuntia humifusa seeds, Isoamericanin A, Nitric oxide, RAW 264.7 cells, NF-kappa B

scholarly journals Fermented Sea Tangle (Laminaria japonica Aresch) Suppresses RANKL-Induced Osteoclastogenesis by Scavenging ROS in RAW 264.7 Cells

Foods ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 290
Author(s):  
Jeong ◽  
Ji ◽  
Lee ◽  
Hong ◽  
Kim ◽  
...  
Keyword(s):  
Osteoclast Differentiation ◽  
Laminaria Japonica ◽  
Asia Pacific ◽  
Raw 264.7 Cells ◽  
Marker Genes ◽  
Raw 264.7 ◽  
Related Factor ◽  
Nuclear Factor ◽  
Food Ingredient ◽  
Laminaria Japonica Aresch

Sea tangle (Laminaria japonica Aresch), a brown alga, has been used for many years as a functional food ingredient in the Asia-Pacific region. In the present study, we investigated the effects of fermented sea tangle extract (FST) on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-stimulated osteoclast differentiation, using RAW 264.7 mouse macrophage cells. FST was found to inhibit the RANKL-stimulated activation of tartrate-resistance acid phosphatase (TRAP) and F-actin ring structure formation. FST also down-regulated the expression of osteoclast marker genes like TRAP, matrix metalloproteinase-9, cathepsin K and osteoclast-associated receptor by blocking RANKL-induced activation of NF-κB and expression of nuclear factor of activated T cells c1 (NFATc1), a master transcription factor. In addition, FST significantly abolished RANKL-induced generation of reactive oxygen species (ROS) by activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and its transcriptional targets. Hence, it seems likely that FST may have anti-osteoclastogenic potential as a result of its ability to inactivate the NF-κB-mediated NFATc1 signaling pathway and by reducing ROS production through activation of the Nrf2 pathway. Although further studies are needed to inquire its efficacy in vivo, FST appears to have potential use as an adjunctive or as a prophylactic treatment for osteoclastic bone disease.

scholarly journals Effects of the Lysine Methyltransferase Inhibitor AZ505 on Bone Metabolism

2021 ◽  
Vol 28 (4) ◽  
pp. 297-305
Author(s):  
Min-Kyoung Song ◽  
Suhan Jung ◽  
Seojin Hong ◽  
Jun-Oh Kwon ◽  
Min Kyung Kim ◽  
...  
Keyword(s):  
Bone Metabolism ◽  
Transmembrane Protein ◽  
Osteoclast Differentiation ◽  
Marker Genes ◽  
Tartrate Resistant Acid Phosphatase ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Alizarin Red ◽  
Trap Staining

Background: Protein methylation has important role in regulating diverse cellular responses, including differentiation, by affecting protein activity, stability, and interactions. AZ505 is an inhibitor of the SET and MYND domain-containing protein 2 lysine methylase. In this study, we investigated the effect of AZ505 on osteoblast and osteoclast differentiation in vitro and evaluated the effect of AZ505 in vivo on the long bones in mice.Methods: Osteoblast differentiation was assessed by alkaline phosphatase (ALP) and Alizarin red staining after culturing calvarial preosteoblasts in an osteogenic medium. Osteoclast differentiation was analyzed by tartrate-resistant acid phosphatase (TRAP) staining in bone marrow-derived macrophages cultured with macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). For in vivo experiments, mice were intraperitoneally injected with AZ505 and femurs were examined by micro-computed tomography.Results: AZ505 increased ALP and Alizarin red staining in cultured osteoblasts and the expression of osteoblast marker genes, including Runx2 and osteocalcin. AZ505 resulted in decreased TRAP-staining of osteoclasts and expression of c-Fos and nuclear factor of activated T cells transcription factors and osteoclast marker genes, including cathepsin K and dendritic cell-specific transmembrane protein. Unexpectedly, in vivo administration of AZ505 markedly decreased the trabecular bone mass of femurs. In support of this catabolic result, AZ505 strongly upregulated RANKL expression in osteoblasts.Conclusions: The results indicate that AZ505 has a catabolic effect on bone metabolism in vivo despite its anabolic effect in bone cell cultures. The findings indicate that cell culture data should be extrapolated cautiously to in vivo outcomes for studying bone metabolism.

scholarly journals Protective Effects of Fermented Oyster Extract against RANKL-Induced Osteoclastogenesis through Scavenging ROS Generation in RAW 264.7 Cells

2019 ◽  
Vol 20 (6) ◽  
pp. 1439 ◽  
Author(s):  
Jin-Woo Jeong ◽  
Sung Choi ◽  
Min Han ◽  
Gi-Young Kim ◽  
Cheol Park ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Osteoclast Differentiation ◽  
Bone Diseases ◽  
Raw 264.7 Cells ◽  
Ros Generation ◽  
Tartrate Resistant Acid Phosphatase ◽  
Raw 264.7 ◽  
Nuclear Factor ◽  
Protective Effects ◽  
Induced Expression

Excessive bone resorption by osteoclasts causes bone loss-related diseases and reactive oxygen species (ROS) act as second messengers in intercellular signaling pathways during osteoclast differentiation. In this study, we explored the protective effects of fermented oyster extract (FO) against receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation in murine monocyte/macrophage RAW 264.7 cells. Our results showed that FO markedly inhibited RANKL-induced activation of tartrate-resistant acid phosphatase and formation of F-actin ring structure. Mechanistically, FO has been shown to down-regulate RANKL-induced expression of osteoclast-specific markers by blocking the nuclear translocation of NF-κB and the transcriptional activation of nuclear factor of activated T cells c1 (NFATc1) and c-Fos. Furthermore, FO markedly diminished ROS production by RANKL stimulation, which was associated with blocking the expression of nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) and its regulatory subunit Rac-1. However, a small interfering RNA (siRNA) targeting NOX1 suppressed RANKL-induced expression of osteoclast-specific markers and production of ROS and attenuated osteoclast differentiation as in the FO treatment group. Collectively, our findings suggest that FO has anti-osteoclastogenic potential by inactivating the NF-κB-mediated NFATc1 and c-Fos signaling pathways and inhibiting ROS generation, followed by suppression of osteoclast-specific genes. Although further studies are needed to demonstrate efficacy in in vivo animal models, FO may be used as an effective alternative agent for the prevention and treatment of osteoclastogenic bone diseases.

scholarly journals 3-Hydroxyolean-12-en-27-oic Acids Inhibit RANKL-Induced Osteoclastogenesis in Vitro and Inflammation-Induced Bone Loss in Vivo

2020 ◽  
Vol 21 (15) ◽  
pp. 5240
Author(s):  
Wonyoung Seo ◽  
Suhyun Lee ◽  
Phuong Thao Tran ◽  
Thi Quynh-Mai Ngo ◽  
Okwha Kim ◽  
...  
Keyword(s):  
Bone Destruction ◽  
Osteoclast Formation ◽  
Bone Diseases ◽  
Marker Genes ◽  
Tartrate Resistant Acid Phosphatase ◽  
Specific Marker ◽  
Nuclear Factor ◽  
Activated T Cells

Olean-12-en-27-oic acids possess a variety of pharmacological effects. However, their effects and underlying mechanisms on osteoclastogenesis remain unclear. This study aimed to investigate the anti-osteoclastogenic effects of five olean-12-en-27-oic acid derivatives including 3α,23-isopropylidenedioxyolean-12-en-27-oic acid (AR-1), 3-oxoolean-12-en-27-oic acid (AR-2), 3α-hydroxyolean-12-en-27-oic acid (AR-3), 23-hydroxy-3-oxoolean-12-en-27-oic acid (AR-4), and aceriphyllic acid A (AR-5). Among the five olean-12-en-27-oic acid derivatives, 3-hydroxyolean-12-en-27-oic acid derivatives, AR-3 and AR-5, significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced mature osteoclast formation by reducing the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, F–actin ring formation, and mineral resorption activity. AR-3 and AR-5 decreased RANKL-induced expression levels of osteoclast-specific marker genes such as c-Src, TRAP, and cathepsin K (CtsK) as well as c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Mice treated with either AR-3 or AR-5 showed significant protection of the mice from lipopolysaccharide (LPS)-induced bone destruction and osteoclast formation. In particular, AR-5 suppressed RANKL-induced phosphorylation of JNK and ERK mitogen-activated protein kinases (MAPKs). The results suggest that AR-3 and AR-5 attenuate osteoclast formation in vitro and in vivo by suppressing RANKL-mediated MAPKs and NFATc1 signaling pathways and could potentially be lead compounds for the prevention or treatment of osteolytic bone diseases.

scholarly journals Osteoclasts Differentiation from Murine RAW 264.7 Cells Stimulated by RANKL: Timing and Behavior

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 117
Author(s):  
Nadia Lampiasi ◽  
Roberta Russo ◽  
Igor Kireev ◽  
Olga Strelkova ◽  
Oxana Zhironkina ◽  
...  
Keyword(s):  
Gene Expression ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Nuclear Factor ◽  
Protein Distribution ◽  
Cytoskeletal Organization ◽  
Receptor Activator ◽  
And Behavior ◽  
Nuclear Factor Κ B ◽  
Rankl Stimulation

The development of multi-nucleated cells is critical for osteoclasts (OCs) maturation and function. Our objective was to extend knowledge on osteoclastogenesis, focusing on pre-OC fusion timing and behavior. RAW 264.7 cells, which is a murine monocyte-macrophage cell line, provide a valuable and widely used tool for in vitro studies on osteoclastogenesis mechanisms. Cells were treated with the receptor activator of nuclear factor κ-B ligand (RANKL) for 1–4 days and effects on cell morphology, cytoskeletal organization, protein distribution, and OC-specific gene expression examined by TEM, immunofluorescence, and qPCR. Multinucleated cells began to appear at two days of Receptor Activator of Nuclear factor κ-B Ligand (RANKL) stimulation, increasing in number and size in the following days, associated with morphological and cytoskeletal organization changes. Interesting cellular extensions were observed in three days within cells labeled with wheat germ agglutinin (WGA)-Fluorescein isothiocyanate (FITC). The membrane, cytoplasmic, or nuclear distribution of RANK, TRAF6, p-p38, pERK1/2, and NFATc1, respectively, was related to OCs maturation timing. The gene expression for transcription factors regulating osteoclastogenesis (NFATc1, c-fos, RelA, MITF), molecules involved in RANKL-signaling transduction (TRAF6), cytoskeleton regulation (RhoA), fusion (DC-STAMP), migration (MMP9), and OC-specific enzymes (TRAP, CtsK), showed different trends related to OC differentiation timing. Our findings provide an integrated view on the morphological and molecular changes occurring during RANKL stimulation of RAW 264.7 cells, which are important to better understand the OCs’ maturation processes.

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