TRAP-positive Multinucleated Cells Research Articles

Effects of local administration of tiludronic acid on experimental periodontitis in diabetic rats.

Tiludronic acid (TIL) presents antiresorptive and anti-inflammatory properties and has not been evaluated in the periodontitis-diabetes mellitus (DM) association to date, to the best knowledge of the authors. This study evaluates effects of local administration of TIL on experimental periodontitis (EP) in rats with streptozotocin-induced DM. Thirty-two animals (Rattus norvegicus albinus, Wistar) were divided into groups DM/C (Control), DM/EP, DM/EP/TIL1, and DM/EP/TIL3. In EP groups, a ligature was placed around mandibular first molars. In groups DM/EP/TIL1 and DM/EP/TIL3, TIL solutions (1 and 3mg/kg, respectively) were injected into the gingival tissue of mandibular molars every other day for 10 days, until euthanasia. Periodontal tissues were analyzed by microcomputed tomography (micro-CT), histomorphometry, immunohistochemistry (tartrate-resistant acid phosphatase [TRAP], receptor activator of nuclear factor-κB ligand [RANKL], osteoprotegerin, cleaved caspase 3), and quantitative reverse transcription-polymerase chain reaction (interleukin [IL]-1β, vascular endothelial growth factor [VEGF]). In micro-CT analyses, groups DM/EP/TIL1 and DM/EP/TIL3 presented reduced alveolar bone resorption (P<0.05). Group DM/EP/TIL3 presented decreased attachment loss (P<0.05). The amount of TRAP-positive multinucleated cells was decreased in TIL groups (P<0.05). Group DM/EP/TIL3 presented a lower immunolabeling pattern for RANKL (P<0.05). TIL treatment decreased genic expression of IL-1β, and in group DM/EP/TIL3, expression of VEGF was increased (P<0.05). Local administration of TIL promoted a protective effect against tissue destruction in EP in diabetic rats, and the dosage of 3mg/kg of TIL promoted the best results regarding its antiresorptive and anti-inflammatory effects.

Binding of PICK1 PDZ domain with calcineurin B regulates osteoclast differentiation

The calcineurin/nuclear factor of activated T cell (NFAT) signaling pathway plays a major role in osteoclast differentiation; however, the proteins that react with the calcineurin-NFAT complex in osteoclasts to regulate osteoclastogenesis remain unclear. Here, we present evidence that PICK1 also positively regulates calcineurin B in osteoclasts to activate NFAT to promote osteoclastogenesis. mRNA and protein expression of PICK1 in murine primary bone marrow macrophages (BMMs) was significantly increased during RANKL-induced osteoclast differentiation. The interaction of PICK1 with calcineurin B in BMMs was confirmed by co-immunoprecipitation. An inhibitor of the PICK1 PDZ domain significantly decreased osteoclastogenesis marker gene expression and the number of TRAP-positive multinucleated cells among RAW264.7 osteoclast progenitor cells. Overexpression of PICK1 in RAW264.7 cells significantly increased the number of TRAP-positive mature osteoclasts. Increased NFAT activation with transcriptional activation of PICK1 during RAW264.7 osteoclastogenesis was also confirmed in a tetracycline-controlled PICK1 expression system. These results suggest that the PDZ domain of PICK1 directly interacts with calcineurin B in osteoclast progenitor cells and promotes osteoclast differentiation through activation of calcineurin-NFAT signaling.

The chemokine receptor type 4 antagonist, AMD3100, interrupts experimental tooth movement in rats

ObjectiveThe aim of this study was to clarify the role of the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis in osteoclast accumulation, and the influence of orthodontic tooth movement (OTM) under mechanical force application to periodontal tissues, by administration of the CXCR4 antagonist AMD3100. DesignThe upper right first molar (M1) of rats was moved mesially with a 10-g force titanium-nickel closed coil spring. Rats were treated with phosphate-buffered saline or AMD3100 (5mg/kg), which is a SDF-1 antagonist. After 0, 1, 3, and 7days, alveolar bones in all groups were examined at each time point by micro-computed tomography and histological analysis. ResultsTooth movement was decreased significantly in the AMD3100-treated group at 1, 3, and 7days after beginning OTM. The numbers of tartrate-resistant acid phosphatase-positive multinucleated cells in the periodontal ligament around the maxillary M1 were decreased significantly in the treated as compared to the control group on Days 1 and 3. ConclusionAdministration of AMD3100 decreases OTM and osteoclast accumulation in rat molars under orthodontic force application. These findings suggest that the SDF-1/CXCR4 axis plays an important role in alveolar bone metabolism during OTM.

Inorganic phosphate-induced impairment of osteoclast cell-cell fusion by the inhibition of AP-1-mediated DC-STAMP expression

Chronic kidney disease (CKD) causes hyperphosphatemia and secondary hyperparathyroidism, leading to several disorders of bone metabolism. Although high concentrations of extracellular inorganic phosphate (Pi) inhibit osteoclastogenesis, the molecular mechanism of this effect has not been fully understood. In the present study, therefore, we examined the effect of Pi on the differentiation of the osteoclast precursor RAW-D cells. Treatment with the receptor activator of nuclear factor-kappa B ligand induced the differentiation of RAW-D cells (osteoclastogenesis). However, Pi significantly weakened this effect, assessed by the tartrate-resistant acid phosphatase (TRAP) activity and the number of TRAP-positive multinucleated cells. Pi also reduced the expressions of nuclear factor of activated T-cell (NFAT) c1 and dendritic cell-specific transmembrane protein (DC-STAMP). Interestingly, the Pi-induced reduction of DC-STAMP gene promoter activity was lost when the activator protein 1 (AP-1) binding site was mutated. Since Pi strongly inhibited the expression of c-Fos which is the component of AP-1, the Pi-induced reduction of DC-STAMP expression was proposed to be mediated by the absence of c-Fos. These results suggested that hyperphosphatemia in the patients with CKD suppresses bone resorption by inhibiting osteoclastogenesis, and this impairs the regulation of bone metabolism.

Interleukin 17 regulates SHP-2 and IL-17RA/STAT-3 dependent Cyr61, IL-23 and GM-CSF expression and RANKL mediated osteoclastogenesis by fibroblast-like synoviocytes in rheumatoid arthritis

Interleukin (IL)-17 predominately produced by the Th17 cells, plays a crucial role in the fibroblast-like synoviocytes (FLS) mediated disease process of rheumatoid arthritis (RA). IL-17 exerts its pathogenic effects in RA-FLS by IL-17/IL-17RA/STAT-3 signaling. Recent studies have shown that RA-FLS produces SHP-2, Cyr61, IL-23, GM-CSF and RANKL which results in worsening of the disease. However, whether IL-17/IL-17RA/STAT-3 signaling regulates SHP-2, Cyr61, IL-23, GM-CSF and RANKL expressions in RA-FLS remains unknown. In this study, IL-17 treatment dramatically induced the production of Cyr61, IL-23 and GM-CSF in FLS isolated from adjuvant induced arthritis (AA) rats. Conversely, IL-17 mediated production of Cyr61, IL-23 and GM-CSF was abrogated by knockdown of IL-17RA using a small interfering RNA or blockade of STAT-3 activation with S3I-201 in AA-FLS. Interestingly, IL-17 treatment noticeably increased the expression of IL-17RA and SHP-2 in AA-FLS. However, silencing of IL-17RA reversed the effect of IL-17 on the expression of IL-17RA and SHP-2 in AA-FLS. In addition, an increased number of TRAP-positive multinucleated cells were observed in a coculture system consisting of IL-17 treated AA-FLS and rat bone marrow derived monocytes/macrophages. Further, mechanistically we found that IL-17 upregulated RANKL expression in AA-FLS that was dependent on the IL-17/IL-17RA/STAT-3 signaling cascade. Knockdown of IL-17RA or inhibition of STAT-3 activation decreased the IL- 17 induced RANKL expression by AA-FLS and their osteoclastogenic potential. Taken together, our findings demonstrate that IL-17 regulates SHP-2 expression and IL-17RA/STAT-3 dependent production of Cyr61, IL-23, GM-CSF and RANKL in AA-FLS and may reveal a new insight into the pathogenesis of RA.

Long-term therapy with intravenous zoledronate increases the number of nonattached osteoclasts

The purpose of this study was to investigate the influence of long-term therapy with intravenous zoledronate (ZA) on the healing of extraction sockets in rats. Forty rats, divided into groups C (Control) and Z (Zoledronate), received intravenous injections of either saline solution or ZA for 24 weeks. Their right maxillary incisor was extracted. Euthanasia was performed at 7 or 28 days postoperative. Histomorphometric (Newly Formed Bone Area) and immunohistochemical (RANKL, OPG and TRAP) analyses were performed. Data were statistically analyzed (ANOVA, Tukey's test and Kruskal–Wallis, Dunn's Multiple Comparison test).Groups C and Z showed similar new bone area, RANKL and OPG immunolabeling. The number of TRAP-positive multinucleated cells was significantly higher in Group Z than in Group C at 28 days. A significantly higher proportion of nonattached osteoclasts were seen in Group Z than in Group C at both periods of analysis. Long-term therapy with intravenous ZA stimulated nonattached osteoclast formation in extraction sockets in rats, thus decreasing local bone resorption. However, it did not influence bone formation by osteoblasts.

Evidence for altered osteoclastogenesis in splenocyte cultures from VDR knockout mice

The indirect action of 1α,25(OH)2-vitamin-D3 (1,25D) on the osteoclast through stromal signalling is well established. The role of vitamin D in osteoclasts through direct 1,25D-VDR signalling is less well known. We showed previously that local 1,25D synthesis in osteoclasts modified osteoclastogenesis and osteoclastic resorptive activity. In this study, we hypothesised that osteoclasts lacking VDR expression would display an enhanced resorptive capacity due to the loss of 1,25D signalling. Splenocytes were cultured under osteoclast-differentiating conditions from mice with global deletion of the Vdr gene (VDRKO) and this was compared with age-matched wild-type littermate controls (WT). In VDRKO cultures, osteoclastogenesis was reduced, as indicated by fewer TRAP-positive multinucleated cells at all time points measured (p < 0.05) compared to WT levels. However, VDRKO osteoclasts demonstrated greater resorption on a per cell basis than their WT counterparts. VDRKO cultures expressed greatly increased c-Fos mRNA compared to WT. In addition, the ratio of expression of the pro-apoptotic gene Bax to the pro-survival gene Bcl-2 was decreased in VDRKO cultures, implying that these osteoclasts may survive longer than WT osteoclasts. Our data indicate abnormal osteoclastogenesis due to the absence of Vdr expression, consistent with direct effects of vitamin D signalling being important for regulating the maturation and resorptive activities of osteoclasts.

Histamine and Histamine H4 Receptor Promotes Osteoclastogenesis in Rheumatoid Arthritis

Histamine H4 receptor (H4R) has immune-modulatory and chemotaxic effects in various immune cells. This study aimed to determine the osteoclastogenic role of H4R in rheumatoid arthritis (RA). The concentration of histamine in synovial fluid (SF) and sera in patients with RA was measured using ELISA. After RA SF and peripheral blood (PB) CD14+ monocytes were treated with histamine, IL-17, IL-21 and IL-22, and a H4R antagonist (JNJ7777120), the gene expression H4R and RANKL was determined by real-time PCR. Osteoclastogenesis was assessed by counting TRAP–positive multinucleated cells in PB CD14+ monocytes cultured with histamine, Th17 cytokines and JNJ7777120. SF and serum concentration of histamine was higher in RA, compared with osteoarthritis and healthy controls. The expression of H4R was increased in PB monocytes in RA patients. Histamine, IL-6, IL-17, IL-21 and IL-22 induced the expression of H4R in monocytes. Histamine, IL-17, and IL-22 stimulated RANKL expression in RA monocytes and JNJ7777120 reduced the RANKL expression. Histamine and Th17 cytokines induced the osteoclast differentiation from monocytes and JNJ7777120 decreased the osteoclastogenesis. H4R mediates RANKL expression and osteoclast differentiation induced by histamine and Th17 cytokines. The blockage of H4R could be a new therapeutic modality for prevention of bone destruction in RA.

AG490 suppresses interleukin-34-mediated osteoclastogenesis in mice bone marrow macrophages.

Interleukin-34 (IL-34) has been recently identified as a novel cytokine, substituting for the function of macrophage colony-stimulating factor (M-CSF), a pivotal osteoclastogenic factor involved in bone-related diseases (e.g., osteomyelitis of the jaws). However, the molecular mechanisms are not fully understood. This study aimed to explore the potential mechanism of IL-34 in receptor activator of NF-kB ligand (RANKL)-induced osteoclast formation. We found that IL-34 alone significantly maintained the survival of bone marrow macrophages (BMMs) and enhanced the expression of the osteoclast-related genes TRAP, Ctsk, and NFATc1, as well as TRAP-positive multinucleated cells combined with RANKL, which can be reversed by AG490. Conversely, AG490 did not affect the M-CSF-mediated osteoclastogenesis in the presence of RANKL. The protein expression of p-STAT3 in BMMs was enhanced by IL-34 combined with RANKL compared with RANKL alone, and AG490 inhibited the expression of p-SATA3 at protein level in the IL-34 plus RANKL group, resulting in significantly increased Smad7 expression. This study demonstrated for the first time that IL-34 may play a crucial role in RANKL-induced osteoclastogenesis by promoting the proliferation and differentiation of BMMs, stimulating p-STAT3 expression, and inhibiting the expression of Smad7 in the absence of M-CSF.

Atorvastatin inhibits osteoclast differentiation by suppressing NF-κB and MAPK signaling during IL-1β-induced osteoclastogenesis.

Background/AimsTo define the effect of statins on interleukin 1β (IL-1β)-induced osteoclastogenesis and elucidate the underlying mechanisms.MethodsBone marrow cells were obtained from 5-week-old male ICR (Institute for Cancer Research) mice, and they were cultured to differentiate them into osteoclasts with macrophage colony-stimulating factor and the receptor activator of nuclear factor (NF)-κB ligand in the presence or absence of IL-1β or atorvastatin. The formation of osteoclasts was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and resorption pit assay with dentine slice. The molecular mechanisms of the effects of atorvastatin on osteoclastogenesis were investigated using reverse transcription polymerase chain reaction and immunoblotting for osteoclast specific molecules.ResultsAtorvastatin significantly reduced the number of TRAP-positive multinucleated cells as well as the bone resorption area. Atorvastatin also downregulated the expression of the NF of activated T-cell c1 messenger RNA and inhibited the expression of osteoclast-specific genes. A possible underlying mechanism may be that atorvastatin suppresses the degradation of the inhibitors of NF-κB and blocks the activation of the c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38; thus, implicating the NF-κB and mitogen-activated protein kinases pathway in this process.ConclusionsAtorvastatin is a strong inhibitor of inflammation-induced osteoclastogenesis in inflammatory joint diseases.

Excavatolide B Attenuates Rheumatoid Arthritis through the Inhibition of Osteoclastogenesis.

Osteoclasts are multinucleated giant cells of macrophage/monocyte lineage, and cell differentiation with the upregulation of osteoclast-related proteins is believed to play a major role in the destruction of the joints in the course of rheumatoid arthritis (RA). Pro-inflammatory cytokines, such as interleukin-17A (IL-17A) and macrophage colony-stimulating factor (M-CSF), can be overexpressed in RA and lead to osteoclastogenesis. In a previous study, we found that cultured-type soft coral-derived excavatolide B (Exc-B) exhibited anti-inflammatory properties. In the present study, we thus aimed to evaluate the anti-arthritic activity of Exc-B in in vitro and in vivo models. The results demonstrated that Exc-B inhibits LPS-induced multinucleated cell and actin ring formation, as well as TRAP, MMP-9, and cathepsin K expression. Additionally, Exc-B significantly attenuated the characteristics of RA in adjuvant (AIA) and type II collagen-induced arthritis (CIA) in rats. Moreover, Exc-B improved histopathological features, and reduced the number of TRAP-positive multinucleated cells in the in vivo AIA and CIA models. Immunohistochemical analysis showed that Exc-B attenuated the protein expression of cathepsin K, MMP-2, MMP-9, CD11b, and NFATc1 in ankle tissues of AIA and CIA rats. Level of interleukin-17A and macrophage colony-stimulating factor were also decreased by Exc-B. These findings strongly suggest that Exc-B could be of potential use as a therapeutic agent by inhibiting osteoclast differentiation in arthritis. Moreover, this study also illustrates the use of the anti-inflammatory marine compound, Exc-B, as a potential therapeutic strategy for RA.

Mixed effects of caffeic acid phenethyl ester (CAPE) on joint inflammation, bone loss and gastrointestinal inflammation in a murine model of collagen antibody-induced arthritis.

To investigate the effect of caffeic acid phenethyl ester (CAPE) on local and systemic inflammation and bone loss in collagen antibody-induced arthritis (CAIA) mice. Four groups of mice (n=8 per group) were allocated; control, CAPE (1mg/kg), CAIA and CAIA+CAPE (1mg/kg). Local inflammation and bone loss were evaluated using clinical paw scores, in vivo micro-computed tomography (micro-CT), histological assessment and tartrate-resistant acid phosphatase (TRAP) staining. Serum levels of C-reactive protein (CRP) and C-terminal telopeptide (CTX-1) were measured by ELISA. Jejunum and colon sections were evaluated histopathologically for damage and toxicity. Greater paw scores and percentage change in paw volume were observed in CAIA+CAPE compared to the control groups (p<0.05). Bone volume over time remained unchanged (p=0.94) and the number of multinucleated TRAP-positive cells was greatest in CAIA+CAPE mice (p<0.05). CRP and CTX-1 levels did not differ between groups. CAIA+CAPE mice exhibited lower colon toxicity scores and a reduced percentage of cavitated goblet cells in the colon crypts compared with CAIA mice (p=0.026 and p=0.003, respectively). Histopathology in the jejunum was not altered. CAPE did not reduce paw inflammation or bone loss in CAIA mice. CAPE reduced histopathological changes in the colon of CAIA mice.

IL-6 Enhances Osteocyte-Mediated Osteoclastogenesis by Promoting JAK2 and RANKL Activity In Vitro

Background/Aims: Evidence suggests that IL-6 affects bone mass by modulating osteocyte communication towards osteoclasts. However, the mechanism by which IL-6 enhances osteocyte-mediated osteoclastogenesis is unclear. We aimed to investigate the inflammatory factors in serum after orthodontic surgery and their relationship between osteocytes and osteoclasts. Methods: Serum was obtained from 10 orthognathic surgery patients, and inflammatory factors were detected by ELISA. We treated the osteocyte-like cell line MLO-Y4 with recombinant mouse IL-6 and IL-6 receptor (IL-6R), and used quantitative RT-PCR and Western blotting to explore Receptor activator of nuclear factor-κB ligand (RANKL) expression at both the mRNA and protein level. MLO-Y4 cells were co-cultured with osteoclast precursor cells, and the formation of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining. To explore the role of JAK2 in the osteocyte-mediated osteoclastogenesis, AG490, a JAK2 inhibitor, was used to inhibit the JAK2-STAT3 pathway in osteocytes. Results: In our study, we found that IL-6 and RANKL were stimulated in serum 3-7 days after orthognathic surgery. Therefore, IL-6 and IL-6 receptor enhanced the expression of RANKL at both the mRNA and protein level in MLO-Y4. Furthermore, when MLO-Y4 cells were co-cultured with osteoclast precursor cells, it significantly stimulated osteoclastogenesis. Our study indicated that osteocytes could promote osteoclastic differentiation and the formation of TRAP-positive multinucleated cells after stimulation with IL-6 and IL-6R. Our results also indicated that treatment with IL-6 and IL-6R increased RANKL mRNA expression and the RANKL/OPG expression ratio. Meanwhile, the phosphorylation of Janus kinase 2 (JAK2) and Signal transducer and activator of transcription (STAT3) also correlated with RANKL levels. Furthermore, we investigated the effects of a specific JAK2 inhibitor, AG490, on the expression of RANKL in osteocyte-like MLO-Y4 cells and osteocyte-mediated osteoclastogenesis. The results showed that AG490 inhibited (p)-JAK2 and RANKL expression. Osteoclastic differentiation was decreased after pretreatment in MLO-Y4 with mouse IL-6/IL-6R and AG490; therefore, we concluded that IL-6 increased osteocyte-mediated osteoclastic differentiation by activating JAK2 and RANKL. Conclusion: The effects of IL-6/il-6R and AG490 on osteocyte-mediated osteoclastogenesis contribute to our understanding of the role of inflammatory factors in the interaction between osteocytes and osteoclast precursors. IL-6 and RANKL are key factors for bone remodelling after the orthodontic surgery, and their roles in bone remodelling may be fundamental mechanisms accelerating tooth movement by orthodontic surgery.

Inhibitory Effect of Myricetin on Matrix Metalloproteinase Expression and Activity in Periodontal Inflammation

Flavonoid myricetin, usually found in tea and medicinal plants, has antioxidant and anti-inflammatory effects. Our objectives in this study were to verify the effects of myricetin on periodontal ligament fibroblasts (PDLFs) under inflammatory conditions and to observe its effects on osteoclast generation and on cytokine expression in RAW264.7 cells. To determine the effects of myricetin on PDLFs, we examined the expression and activity of proteolytic enzymes, including MMP-1, MMP-2, and MMP-8, which all play an important role in chronic periodontitis. We observed the effects of myricetin on intracellular signal transduction to verify the molecular mechanism involved. By measuring the formation of TRAP–positive multinucleated cells and the expression and activity of MMP-8, we were able to assess the effects of myricetin on osteoclast generation. In addition, by measuring the secretion of IL-6 and NO, we could evaluate the effects of myricetin on inflammatory mediators. We found that Myricetin had no effect on the viability of the PDLFs in the presence of inflammation, but it did decrease both the expression of MMP-1 and MMP-8 and the enzyme activity of MMP-2 and MMP-8 in these fibroblasts. Myricetin also decreased the lipopolysaccharide-stimulated phosphorylation of JNK, p38 signaling, IKKB, AKT, and p65RelA in the PDLFs. In the RAW264.7 cells, myricetin inhibited both the expression and the activity of MMP-8. Furthermore, Myricetin not only suppressed the generation of LPS-stimulated osteoclasts, but it also slightly inhibited LPS-stimulated degradation of IkB and decreased the release of LPS-induced IL-6 and NO. These findings suggest that myricetin alleviates the tissue-destructive processes that occur during periodontal inflammation.

Roles of PKR in differentiation and apoptosis of bone-related cells

Double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine protein kinase which is activated by double-stranded RNAs and related to several signal transduction pathways. To examine the effects of PKR on bone metabolism, we established PKR-K/R mutant cells in which amino acid lysine at 296 is substituted with arginine. PKR regulated apoptosis in osteoblastic cells via nuclear factor kappa-B (NF-κB) cascade. MC3T3-E1 cells cultured with osteoblast differentiation medium differentiated into osteoblasts, while the mutant cells did not differentiate into osteoblasts. RAW246.7 cells triggered with receptor activator of NF-κB ligand (RANKL) formed tartrate-resistant acid phosphatase-positive multinucleated giant cells, whereas PKR-K/R mutant RAW cells did not. Differentiation of osteoblasts and osteoclasts was caused by NF-κB activation and signal transducer and activator of transcription1 (STAT1) ubiquitination and degradation. We also demonstrated involvement of PKR in chondrocyte differentiation. PKR prevented tumor necrosis factor-α- and interleukin 1α-induced bone resorption in calvaria and artificially induced periodontal disease in rat. Our findings indicate that PKR regulates bone metabolism invitro and invivo.

Nicorandil inhibits osteoclast differentiation in vitro

Nicorandil is a hybrid angina therapeutic agent that has nitric oxide (NO) action and the ability to open ATP-sensitive K+ channels (KATP channels). A transient increase in NO and intracellular Ca2+ has been demonstrated to be highly involved in the differentiation and activation of osteoclasts. The objective of this study was to verify that the pharmacological effect of nicorandil suppresses the differentiation process of osteoclasts in vitro.Although little authentic NO production was detected in the culture medium in osteoclast formation assays, NO production increased only in the presence of nicorandil. The number of osteoclasts decreased markedly at late time-points after nicorandil addition compared with the number at early time-points. Both the number of TRAP-positive multinucleated cells and the number of cells that obtained F-actin rings decreased in the presence of nicorandil in a concentration-dependent manner. The osteo assay showed that the bone resorption area was also reduced with nicorandil in a concentration-dependent manner. An inhibition recovery experiment was conducted by adding a soluble guanylyl cyclase (sGC) inhibitor (ODQ) and a KATP channel-opening inhibitor (glibenclamide) during the osteoclast formation process. In the inhibition recovery experiment, the inhibitory effect of nicorandil on osteoclastogenesis was blocked by the addition of ODQ and glibenclamide. These results suggest that both the NO and KATP channel-opening activity of nicorandil inhibit osteoclast differentiation. Further study of nicorandil may lead to the development of drugs for osteoporosis treatment.

Interleukin 26 suppresses receptor activator of nuclear factor κB ligand induced osteoclastogenesis via down-regulation of nuclear factor of activated T-cells, cytoplasmic 1 and nuclear factor κB activity.

IL-26 has been shown to have high expression in RA. However, the effects of IL-26 on bone destruction in RA have not been evaluated. The aim of this study was to investigate the effects and mechanisms of IL-26 on RANK ligand (RANKL)-induced osteoclastogenesis. We treated cells with IL-26 in RANKL-induced oseteoclastogenesis to monitor osteoclast formation by tartrate-resistant acid phosphatase (TRAP) staining. Osteoclast activity was assessed by pit formation assay and F-actin ring formation. The mechanism of the inhibition was studied by biochemical analyses such as RT-PCR, immunofluorescence staining and immunoblotting. In addition, cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. IL-26 inhibited RANKL-induced TRAP-positive multinucleated cells and inhibited RANKL-induced nuclear factor κB (NF-κB) activation and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) nuclear translocation in RAW264.7 cells. Also, IL-26 significantly inhibited the bone-resorbing activity and F-actin ring formation ability of mature osteoclasts. Moreover, IL-26 suppressed RANKL-induced mitogen-activated protein kinase activation and NFATc1 downstream gene expression. We suggest that the inhibitory activity of IL-26 on osteoclastogenesis is via down-regulation of RANKL-induced NF-κB and NFATc1 expression. Our results suggest IL-26 as a possible new remedy against osteolytic bone destruction.

Synthesis and biological evaluation of rhein amides as inhibitors of osteoclast differentiation and bone resorption

Approaches of targeting excessive activation and differentiation of osteoclasts were considered as an effective treatment option for osteoporosis or osteopenia. In the present work, a series of rhein derivatives were synthesized and employed for their cytotoxicity screening against bone marrow-derived macrophages cells (BMMs) and their inhibition effects on osteoclasts activation and differentiation in vitro using an MTT assay and a TRAP activity assay respectively. Two rhein derivatives d6 and d11 inhibited BMMs activation and differentiation with 98% and 85% inhibitory activity respectively, without showing any cytotoxicity on BMMs. Subsequently, the most potent compound d6 was further validated for its inhibitory effects on the formation of TRAP-positive multinucleated cells and bone resorption as evaluated by TRAP staining and bone resorption assay. The regulation by d6 of osteoclast marker genes assay revealed that treatment of BMMs with M-CSF and RANKL resulted in the stimulation of mRNA expressions of NFATc1, c-fos, TRAP, MMP-9 and cathepsin K which were highly related with osteoclast activation and differentiation, while d6 decreased mRNA expressions of these genes. It was indicated that d6 might regulate osteoclasts activity through RANKL/RANK/NFATc1 pathway. Thus our current work is expected to provide a highly promising approach for the development of a new type of anti-osteoporosis agent.

Combined Effects of Soy Isoflavones and Docosahexaenoic Acid on Osteoclast Formation

Soy isoflavones and docosahexaenoic acid (DHA) are effective for maintaining bone health. This study investigated the combined effects of soy isoflavones and DHA on osteoclast formation. Mouse bone marrow cells were pre-cultured with macrophage colony-stimulating factor (M-CSF) for 3 days and then cultured with M-CSF and receptor activator of nuclear factor κB ligand (RANKL) for 6 days. RAW 264.7 cells were cultured with RANKL for 5 days. In mouse bone marrow cells, daidzein, genistein, and DHA significantly decreased the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP(+)MNCs), and the combination of soy isoflavones and DHA further decreased the number of TRAP(+)MNCs. Nuclear factor of activated T-cells c1 (NFATc1) mRNA expression tended to be decreased by daidzein, and was significantly decreased by genistein and DHA. Furthermore, the combination of daidzein and DHA caused significant reduction in NFATc1 mRNA expression compared to the control. In RAW 264.7 cells, daidzein tended to decrease and genistein significantly decreased the number of TRAP(+)MNCs, however, the combination of daidzein and DHA significantly decreased the number of TRAP(+)MNCs compared to the control. These results demonstrated that the combination of soy isoflavones and DHA decreased in osteoclast formation significantly, possibly by modulating the expression of specific genes.

Dioscin reduces ovariectomy-induced bone loss by enhancing osteoblastogenesis and inhibiting osteoclastogenesis

Our previous studies showed that dioscin can promote osteoblasts proliferation and differentiation in vitro, but its anti-osteoporosis effect in vivo and the underlying mechanisms remain unclear. In the present work, the results showed that dioscin significantly increased the viability of MC3T3-E1 cells, ALP level and alizarin red S staining area, markedly decreased the numbers of RANKL-induced TRAP-positive multinucleated cells and bone resorption pits formation, enhanced the levels of some osteogenic markers including COL1A2, ALP and OC, which suggested that dioscin clearly promoted osteoblasts proliferation and suppressed osteoclasts formation. In vivo experiments demonstrated that dioscin obviously reduced OVX-induced body weight increase, and improved the biochemical indexes including ALP, StrACP, OC, DPD/Cr, HOP/Cr, BMD, biomechanics and microarchitecture. Moreover, H&E, TB, TRAP staining, and fluorescent double labeling tests indicated that dioscin enhanced osteoblastogenesis and inhibited osteoclastogenesis. Further researches demonstrated that dioscin promoted osteoblastogenesis through up-regulating OPG/RANKL ratio, and inhibited osteoclastogenesis through down-regulating the levels of RANKL induced TRAF6 and the downstream signal molecules including MAPKs, Akt, NF-κB, AP-1, cathepsin K and NFATc1. In addition, dioscin also inhibited TLR4/MyD88 pathway to decrease the levels of TRAF6 and the related proteins. These findings provide new insights to elucidate the effects of dioscin against OVX-induced bone loss, which should be developed as a potential candidate for treating postmenopausal osteoporosis in the future.