This study investigated the regulatory potential of salidroside (SAL), a primary active compound in Rhodiola rosea L., on osteoclast differentiation by modulating the hypoxia-inducible factor 1-alpha (HIF-1a) pathway in osteoblasts. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were employed to validate whether the receptor activator of nuclear factor-?B ligand (RANKL) is the downstream target gene of HIF-1a in osteoblasts. The study also utilized lipopolysaccharide (LPS)-induced mouse osteolysis to examine the impact of SAL on osteolysis in vivo. Furthermore, conditioned medium (CM) from SAL-pretreated osteoblasts was used to investigate the paracrine effects on osteoclastogenesis through the HIF-1a pathway. Hypoxic condition-induced overexpression of HIF-1a upregulated RANKL levels by binding to the RANKL promoter and enhancing transcription in osteoblastic cells. In vivo, SAL significantly alleviated bone tissue hypoxia and decreased the expression of HIF-1a by downregulating the expression of RANKL, vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), and angiopoietin-like 4 (ANGPTL4). In the paracrine experiment, conditioned media from SAL-pretreated osteoblasts inhibited differentiation through the HIF-1a/RANKL, VEGF, IL-6, and ANGPTL4 pathways. RANKL emerges as the downstream target gene regulated by HIF-1a in osteoblasts. SAL significantly alleviates bone tissue hypoxia and bone loss in LPS-induced osteolysis through the HIF-1a/RANKL, VEGF, IL-6, and ANGPTL4 pathways. SAL inhibits osteoclast differentiation by regulating osteoblast paracrine secretion.
Animals ; Osteoblasts/cytology* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Glucosides/administration & dosage* ; Cell Differentiation/drug effects* ; Phenols/administration & dosage* ; Mice ; Osteoclasts/metabolism* ; RANK Ligand/genetics* ; Rhodiola/chemistry* ; Osteogenesis/drug effects* ; Signal Transduction/drug effects* ; Interleukin-6/genetics* ; Male ; RAW 264.7 Cells ; Osteolysis/genetics* ; Humans ; Mice, Inbred C57BL

Osteoclast-like cells are known to inhibit arterial calcification. Receptor activator of NF-κB ligand (RANKL) is likely to act as an inducer of osteoclast-like cell differentiation. However, several studies have shown that RANKL promotes arterial calcification rather than inhibiting arterial calcification. The present study was conducted in order to investigate and elucidate this paradox. Firstly, RANKL was added into the media, and the monocyte precursor cells were cultured. Morphological observation and Tartrate resistant acid phosphatase (TRAP) staining were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells. During arterial calcification, in vivo and in vitro expression of RANKL and its inhibitor, osteoprotegerin (OPG), was detected by real-time PCR. The extent of osteoclast-like cell differentiation was also assessed. It was found RANKL could induce osteoclast-like cell differentiation. There was no in vivo or in vitro expression of osteoclast-like cells in the early stage of calcification. At that time, the ratio of RANKL to OPG was very low. In the late stage of calcification, a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG. According to the results, the ratio of RANKL to OPG was very low during most of the arterial calcification period. This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation. This likely explains why RANKL had the ability to induce osteoclast-like cell differentiation but acted as a promoter of calcification instead.
Acid Phosphatase ; genetics ; metabolism ; Animals ; Aorta ; drug effects ; metabolism ; pathology ; Cell Differentiation ; Coculture Techniques ; Gene Expression Regulation ; Isoenzymes ; genetics ; metabolism ; Male ; Monocytes ; cytology ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; pathology ; Osteoclasts ; drug effects ; metabolism ; pathology ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tartrate-Resistant Acid Phosphatase ; Vascular Calcification ; genetics ; metabolism ; pathology

Eupatilin is the main active component of DA-9601, an extract from Artemisia. Recently, eupatilin was reported to have anti-inflammatory properties. We investigated the anti-arthritic effect of eupatilin in a murine arthritis model and human rheumatoid synoviocytes. DA-9601 was injected into collagen-induced arthritis (CIA) mice. Arthritis score was regularly evaluated. Mouse monocytes were differentiated into osteoclasts when eupatilin was added simultaneously. Osteoclasts were stained with tartrate-resistant acid phosphatase and then manually counted. Rheumatoid synoviocytes were stimulated with TNF-alpha and then treated with eupatilin, and the levels of IL-6 and IL-1beta mRNA expression in synoviocytes were measured by RT-PCR. Intraperitoneal injection of DA-9601 reduced arthritis scores in CIA mice. TNF-alpha treatment of synoviocytes increased the expression of IL-6 and IL-1beta mRNAs, which was inhibited by eupatilin. Eupatilin decreased the number of osteoclasts in a concentration dependent manner. These findings, showing that eupatilin and DA-9601 inhibited the expression of inflammatory cytokines and the differentiation of osteoclasts, suggest that eupatilin and DA-9601 is a candidate anti-inflammatory agent.
Animals ; Anti-Inflammatory Agents/pharmacology/*therapeutic use ; Arthritis, Experimental/chemically induced/*drug therapy ; Arthritis, Rheumatoid/drug therapy/pathology ; Cell Differentiation/*drug effects ; Cells, Cultured ; Collagen Type II ; Cytokines/biosynthesis ; Disease Models, Animal ; Drugs, Chinese Herbal/therapeutic use ; Female ; Flavonoids/pharmacology/*therapeutic use ; Humans ; Inflammation/drug therapy/immunology ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Lymph Nodes/cytology ; Mice ; Mice, Inbred DBA ; Monocytes/cytology ; Osteoclasts/*cytology ; Plant Extracts/pharmacology ; RNA, Messenger/biosynthesis ; Synovial Membrane/cytology ; T-Lymphocytes, Regulatory/cytology/immunology ; Tumor Necrosis Factor-alpha/pharmacology

The purpose of this study was to determine whether the Ca2+ signaling pathway is involved in the ability of osteoprotegerin (OPG) to inhibit osteoclast differentiation and maturation. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-kappaB ligand (RANKL) to stimulate osteoclastogenesis and then treated with different concentrations of OPG, an inhibitor of osteoclast differentiation. The intracellular Ca2+ concentration [Ca2+]i and phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the different treatment groups were measured by flow cytometry and Western blotting, respectively. The results confirmed that M-CSF + RANKL significantly increased [Ca2+]i and CaMKII phosphorylation in osteoclasts (p < 0.01), and that these effects were subsequently decreased by OPG treatment. Exposure to specific inhibitors of the Ca2+ signaling pathway revealed that these changes varied between the different OPG treatment groups. Findings from the present study indicated that the Ca2+ signaling pathway is involved in both the regulation of osteoclastogenesis as well as inhibition of osteoclast differentiation and activation by OPG.
Animals ; Calcium/*metabolism ; *Calcium Signaling ; *Cell Differentiation/drug effects ; Cell Line ; Cell Survival/drug effects ; Gene Expression Regulation/drug effects ; Macrophage Colony-Stimulating Factor/metabolism ; Mice ; Osteoclasts/*cytology/*drug effects/*metabolism ; Osteoprotegerin/*pharmacology ; RANK Ligand/metabolism

Through morphological observation, HE staining, TRAP staining and toluidine blue staining of bone resorption pits to identify osteoclasts which obtained by 1α, 25-(OH)2 VitD3 inducing rabbit bone marrow cells. Three indicators-TRAP staining, TRAP enzyme activity detecting and the number and area of bone resorption pits were adapted to detect the effect of Sargentodoxae caulis on the activity of osteoclasts. Culturing MC3T3-E1 Subclong 14 cells and detecting the effect of S. caulis on differentiation and proliferation of them by MTT and detecting the alkaline phosphatase in cells. The results show that all of the low, middle and high doses of water and alcohol extracts of S. caulis have significant inhibition on osteoclast differentiation and bone resorption ability in a dose-dependent manner. The low and middle doses of water and alcohol extracts of S. caulis can stimulate differentiation and proliferation of MC3T3-ElSubclone 14 cells, which indicates S. caulis can prevent osteoporosis and the function could be achieved by inhibiting osteoclast activity and promoting the proliferation and differentiation of osteoblasts.
Animals ; Bone Resorption ; drug therapy ; physiopathology ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Mice ; Osteoclasts ; cytology ; drug effects ; Rabbits

OBJECTIVETo study the mechanism underlying the inhibitory effect of Qingluo Tongbi Granule (, QTG) on osteoclast differentiation in rheumatoid arthritis in rats.

METHODSFibroblast and monocyte co-culture were used to induce osteoclast differentiation in adjuvant-induced arthritic (AIA) rats. Serum containing QTG was prepared and added to the osteoclasts, and activation of the tumor necrosis factor receptor-associated factor 6/mitogen-activated protein kinase/nuclear factor of activated T cells, cytoplasmic1 (TRAF6/MAPK/NFATc1) pathways was examined.

RESULTSThe induced osteoclasts were multinucleated and stained positive for tartrate-resistant acid phosphatase (TRAP) staining. Serum containing QTG at 14.4, 7.2 or 3.6 g/kg inhibited the activation of TRAF6, extracellular regulated protein kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 and decreased the percentage of cells with nuclear NFATc1 in a dose-dependent manner, the high and middle doses exhibited clear inhibitory activity (P<0.01 and P<0.05, respectively). After the addition of MAPK inhibitors, the NFATc1 expression showed no significant difference compared with the control group (P>0.05).

CONCLUSIONSSerum containing QTG could generally inhibit the TRAF6/MAPK pathways and possibly inhibit the NFATc1 pathway. In addition, QTG may regulate other signaling pathways that are related to osteoclast differentiation and maturation.

Adjuvants, Immunologic ; adverse effects ; Animals ; Arthritis, Experimental ; pathology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Fibroblasts ; pathology ; Male ; Monocytes ; pathology ; Osteoclasts ; cytology ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley ; Synovial Membrane ; pathology

OBJECTIVETo discuss the effect of Drynariae Rhizoma's naringin on osteoclasts induced by mouse monocyte RAW264.7.

METHODRAW264.7 cells were induced by 100 μg x L(-1) nuclear factor-κB receptor activator ligand (RANKL) and became mature osteoclasts, which were identified through TRAP specific staining and bone resorption. MTT method was sued to screen and inhibit and the highest concentration of osteoclasts. After being cultured with the screened medium containing naringin for 5 days, positive TRAP cell counting and bone absorption area analysis were adopted to observe the effect of naringin on the formation of osteoclast sells and the bone absorption function. The osteoclast proliferation was measured by flow cytometry. The effects of RANK, TRAP, MMP-9, NFATc1 and C-fos mRNA expressions on nuclear factor-κB were detected by RT-PCR.

RESULTNaringin could inhibit osteoclast differentiation, bone absorption function and proliferation activity of osteoclasts, significantly down-regulate RANK, TRAP, MMP-9 and NFATc1 mRNA expressions in the osteoclast differentiation process, and up-regulate the C-fos mRNA expression.

CONCLUSIONNaringin could inhibit osteoclast differentiation, proliferation and bone absorption function. Its mechanism may be achieved by inhibiting the specific gene expression during the osteoclast differentiation process.

Acid Phosphatase ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Flavanones ; pharmacology ; Isoenzymes ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; Mice ; NFATC Transcription Factors ; genetics ; Osteoclasts ; cytology ; drug effects ; Tartrate-Resistant Acid Phosphatase

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase (MAPK) signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1β (IL-1β)-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1β induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1β restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-1β restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.
3T3 Cells ; Animals ; Cartilage, Articular ; cytology ; Cell Survival ; physiology ; Cells, Cultured ; Chondrocytes ; drug effects ; enzymology ; Coculture Techniques ; Culture Media, Conditioned ; Gelatinases ; drug effects ; Interleukin-1beta ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; MAP Kinase Signaling System ; physiology ; Matrix Metalloproteinase 2 ; drug effects ; Matrix Metalloproteinase 9 ; drug effects ; Mice ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; drug effects ; Monocytes ; cytology ; NF-kappa B ; antagonists & inhibitors ; Osteoclasts ; physiology ; Protease Inhibitors ; analysis ; Tissue Inhibitor of Metalloproteinase-1 ; drug effects ; Tissue Inhibitor of Metalloproteinase-2 ; drug effects ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

To investigate 1alpha,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1alpha,25-(OH)2D3 during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1alpha,25-(OH)2D3 inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1alpha,25-(OH)2D3 administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.
Acid Phosphatase/metabolism ; Animals ; Blotting, Western ; Calcitriol/*pharmacology ; Calcium Channel Agonists/pharmacology ; *Cell Differentiation ; Cell Line ; Cell Proliferation ; Gene Expression Regulation, Enzymologic/*drug effects ; Isoenzymes/metabolism ; Matrix Metalloproteinase 9/*genetics/metabolism ; Mice ; Osteoclasts/*cytology/*enzymology ; Tetrazolium Salts ; Thiazoles

This study is to investigate the effect of 8-prenylnaringenin (8-PNG) on osteoclastogensis of bone marrow cells and bone resorption activity of osteoclasts. Osteoclasts were separated from long bone marrow of newborn rabbits and cultured in alpha-MEM containing 10% FBS. 8-PNG was added into culture media at 1 x 10(-7), 1 x 10(-6), 1 x 10(-5) mol xL(-1), separately. 17beta-Estradiol (E2, 1 x 10(-7) mol x L(-7)) was used as positive control. T RAP staining and TRAP activity measurement were performed after 5 days, and the bone resorption pits were analyzed after 7 days. Annexin V staining for the detection of apoptotic osteoclasts was performed after 2, 4, 8, 12, 24, 36 and 48 h separately. The mRNA expression level of TRAP and cathepsin K (CTSK) was measured by real-time RT-PCR. 8-PNG significantly reduced the number of osteoclasts which was TRAP staining positive and with more than three nucleus, the area and number of bone resorption pits decreased obviously in 8-PNG-supplemented groups. The apoptosis rate peaked earlier in the 8-PNG-supplemented groups and the mRNA expression level of TRAP and CTSK decreased significantly. All these inhibitory effects were in a dose dependent manner, the highest effect was obtained by 1 x 10(-5) mol x L(-1) 8-PNG. 8-PNG inhibits bone resorption activity of osteoclasts by inducing osteoclast apoptosis and inhibiting the gene expression and enzyme activity including TRAP and CTSK, and restrains bone marrow cells to osteoclast differentiation.
Acid Phosphatase ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; Bone Marrow Cells ; cytology ; Bone Resorption ; Cathepsin K ; genetics ; metabolism ; Cells, Cultured ; Dose-Response Relationship, Drug ; Flavanones ; administration & dosage ; pharmacology ; Isoenzymes ; genetics ; metabolism ; Osteoclasts ; cytology ; metabolism ; RNA, Messenger ; metabolism ; Rabbits ; Tartrate-Resistant Acid Phosphatase