Inhibition Of Osteoclastogenesis Research Articles

Echinacoside Inhibits Osteoclast Function by Down-Regulating PI3K/Akt/C-Fos to Alleviate Osteolysis Caused by Periprosthetic Joint Infection.

Infected osteolysis as a common secondary osteoporosis is associated with excessive osteoclastogenesis and bone resorption. The inhibition of osteoclastogenesis and bone resorption have been demonstrated an effective approach in the treatment of osteolytic diseases. Echinacoside (ECH) is a natural phenylethanoid glycoside with multiple biological functions, including anti-inflammatory, antioxidant, and osteoblast differentiation promotion. However, the effects of ECH on osteoclast differentiation and bone resorption function remain unknown. In vitro, we investigated the effects of ECH on osteoclast differentiation and bone resorption induced by RANKL and its potential mechanisms. In vivo, we established a periprosthetic joint infection (PJI) rat model and demonstrated the changes of infected osteolysis and osteoclasts activities in surgical sites. ECH (20 mg/kg) was injected intraperitoneally after debridement for 4 weeks. Radiological evaluation and bone histomorphometric analysis was performed to assess the efficacy of ECH. The results showed that ECH inhibited osteoclast differentiation, F-actin belts formation, bone resorption function and osteoclast-specific gene expression by preventing NFATc1 translocation, down-regulating its expression and affecting the PI3K/Akt/c-Fos pathway in vitro. ECH also alleviated in vivo PJI-induced osteolysis and maintained bone mass by inhibiting osteoclast activity. Our study indicated that ECH attenuated RANKL-induced osteoclastogenesis and PJI-induced bone loss and was shown as a potentially effective therapeutic agent for osteoclast-related bone diseases.

The New Face of a Well-Known Antibiotic: A Review of the Anticancer Activity of Enoxacin and Its Derivatives.

Simple SummaryEnoxacin is a second-generation quinolone with promising anticancer activity. In contrast to other members of the quinolone group, it exhibits an extraordinary cytotoxic mechanism of action. Enoxacin enhances RNA interference and promotes microRNA processing, as well as the production of free radicals. Interestingly, apart from its proapoptotic, cell cycle arresting and cytostatic effects, enoxacin manifests a limitation of cancer invasiveness. The underlying mechanisms are the competitive inhibition of vacuolar H+-ATPase subunits and c-Jun N-terminal kinase signaling pathway suppression. The newly synthesized enoxacin derivatives have shown a magnified cytotoxic effect with an emphasis on prooxidative, proapoptotic and microRNA interference actions. The mentioned mechanisms seem to contribute to a safer, more selective and more effective anticancer therapy. Enoxacin as a second-generation synthetic quinolone is known for its antibacterial action; however, in recent years there have been studies focusing on its anticancer potential. Interestingly, it turns out that compared to other fluoroquinolones, enoxacin exhibits uncommon cytotoxic properties. Besides its influence on apoptosis, the cell cycle and cell growth, it exhibits a regulatory action on microRNA biogenesis. It was revealed that the molecular targets of the enoxacin-mediated inhibition of osteoclastogenesis are vacuolar H+-ATPase subunits and the c-Jun N-terminal kinase signaling pathway, causing a decrease in cell invasiveness. Interestingly, the prooxidative nature of the subjected fluoroquinolone enhanced the cytotoxic effect. Crucial for the anticancer activity were the carboxyl group at the third carbon atom, fluorine at the seventh carbon atom and nitrogen at the eighth position of naphyridine. Modifications of the parent drug improved the induction of oxidative stress, cell cycle arrest and the dysregulation of microRNA. The inhibition of V-ATPase–microfilament binding was also observed. Enoxacin strongly affected various cancer but not normal cells, excluding keratinocytes, which suffered from phototoxicity. It seems to be an underestimated anticancer drug with pleiotropic action. Furthermore, its usage as a safe antibiotic with well-known pharmacokinetics and selectivity will enhance the development of anticancer treatment strategies. This review covers articles published within the years 2000–2021, with a strong focus on the recent years (2016–2021). However, some canonical papers published in twentieth century are also mentioned.

Inhibition of osteoclastogenesis after bisphosphonate therapy discontinuation: an in vitro approach.

Osteoclasts are specialized cells that degrade and resorb bone. Bisphosphonates (BPs) are drugs with well-known capacity to inhibit the resorption of mineralized tissues. Nitrogen-containing BPs, like alendronate (ALN) and zoledronic acid (ZA), inactivate osteoclast activity mostly by alterations on the cytoskeleton architecture of the cell. In this study, we used an in vitro model to test the hypothesis that bisphosphonates may have inhibitory effects on the osteoclastogenesis and osteoclast activity after the therapy was discontinued. Primary osteoclasts were generated from mouse bone marrow in media supplemented with 1,25-dihydroxyvitamin D3 and cultivated over bones pre-treated with ALN and ZA. The pre-saturation of the bone slices with bisphosphonates did not affect cell viability. We found, however, that by disrupting the gene expression of RANKL and OPG the osteoclastogenesis and resorption activity of osteoclasts was significantly disturbed. These inhibitory effects were confirmed by scanning electron microscopy resorption assay, assessment of osteoclast ultrastructure, and by gene expression analysis of TRAP and Cathepsin K. In conclusion, ALN and ZA adhered to the bone matrix reduced the osteoclast activity in vitro.

Glucocorticoid-induced expansion of classical monocytes contributes to bone loss

Classical monocytes are commonly involved in the innate inflammatory response and are the progenitors of osteoclasts. Excess endogenous glucocorticoids (GCs) can increase the levels of classical monocytes in blood and bone marrow. The role of this cell population in high-dose exogenous GC-induced osteoporosis (GIOP) remains to be elucidated. In this study, GIOP was established in rats and mice by daily methylprednisolone injection, and monocyte subsets were analyzed by flow cytometry. We demonstrated that classical monocytes accumulate in bone marrow during GIOP. Similarly, the monocyte proportion among bone marrow nucleated cells was also increased in patients with steroid treatment history. We sorted classical monocytes and analyzed their transcriptional profile in response to GCs by RNA sequencing. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that classical monocytes isolated from GC-treated rats exhibited osteoclast differentiation potential. Deletion of classical monocytes by clodronate liposome treatment prevented GIOP via inhibition of osteoclastogenesis and restoration of CD31HiendomucinHi vessels. Regarding the molecular mechanism, classical monocytes express high levels of glucocorticoid receptors. In vitro treatment with GCs increased both the percentage and absolute number of monocytes and promoted their proliferation. In summary, classical monocytes mediated GC-induced bone loss and are a potential target for therapeutic intervention in GIOP treatment.

AB0078 NESFATIN-1 EXPRESSION ASSOCIATED WITH A MARKER FOR BONE MATRIX FORMATION P1NP IN RHEUMATOID ARTHRITIS

BackgroundCurrently, the role of tissue cytokines in the pathogenesis of various diseases is being actively studied [1,2]. Nesfatin-1 (NF-1) is an endogenous peptide with pleiotropic activity. Recently, there have been works on the involvement of NF-1 in osteogenesis. In the experiment, intravenous administration of NF-1 to ovariectomized rats (OVX) leads to an increase in bone mineral density (BMD). There are works showing inhibition of osteoclastogenesis in mice [3,4]. We assume that the level of NF-1 affects the processes of bone matrix formation.The aim of the study was to determine the relationship between the level of NF-1, bone mineral density, composite body composition and markers of bone metabolism in patients with rheumatoid arthritis (RA).ObjectivesThe study randomized 110 patients with RA according to the ACR/EULAR criteria, 2010 and 30 people in the control group.MethodsAll of them underwent osteodensitometry LUNAR DPX-Pro with the determination of composite body composition (BCT) and bone mineral density (BMD). NF-1 levels and bone turnover markers were determined using a commercial ELISA test.ResultsThe average concentration of NF-1 in patients with RA was 50.49±34.05 ng/ml, which is significantly higher than in healthy individuals. The average level of NF-1 in the blood serum of healthy individuals was 31.61±3.17 ng/ml (M±σ). The level of reference indicators of NF-1 in healthy individuals, defined as М±2σ, ranged from 25.27 to 37.95 ng/ml.According to the level of NF-1, all patients with RA were divided into 2 subgroups. The 1st group included patients (n=44) with normal serum NF-1 concentration (less than 37.95 ng/ml), the 2nd group (n=66) included patients with elevated NF-1 levels (more than ng/ml).During the analysis of the results of the study, we revealed a statistically significant correlation between NF-1 and the N-terminal propeptide of type I procollagen (P1NP)) (r = 0.218, p = 0.022). The level of CRP and the erythrocyte sedimentation rate positively correlated with the indicators of NF-1.However, we found no relationship between NF-1 levels and BMD; composite body composition; as well as the level of C-terminal type I collagen telopeptide (β-CTx).ConclusionThus, the relationship between nesfatin-1 and a marker of bone matrix formation (P1NP) was revealed. This may indicate a possible effect of nesfatin-1 on the differentiation and function of osteoblasts.

Tereticornate A suppresses RANKL-induced osteoclastogenesis via the downregulation of c-Src and TRAF6 and the inhibition of RANK signaling pathways

Excessive osteoclast differentiation and activation are closely associated with the development and progression of osteoporosis. Natural plant-derived compounds that can inhibit osteoclastogenesis are an efficient strategy for the prevention and treatment of osteoporosis. Tereticornate A (TA) is a natural terpene ester compound extracted from the leaves and branches of Eucalyptus gracilis, with antiviral, antibacterial, and anti-inflammatory activities. However, the effect of TA on osteoclastogenesis and the underlying molecular mechanism remain unclear. Based on the key role of the NF-κB pathway in the regulation of osteoclastogenesis and the observation that TA exhibits an anti-inflammatory effect by inhibiting NF-κB activity, we speculated that TA could exert anti-osteoclastogenesis activity. Herein, TA could inhibit the RANKL-induced osteoclast differentiation and formation of F-actin rings in RAW 264.7 cells. Mechanistically, TA downregulated the expression of c-Src and TRAF6, and also suppressed the RANKL-stimulated canonical RANK signaling pathways, including AKT, MAPK (p38, JNK, and ERK), and NF-κB; ultimately, downregulating the expression of NFATc1 and c-Fos, the key transcriptional factors required for the expression of genes (e.g., TRAP, cathepsin K, β-Integrin, MMP-9, ATP6V0D2, and DC-STAMP) that govern osteoclastogenesis. Our findings demonstrated that TA could effectively inhibit RANKL-induced osteoclastogenesis via the downregulation of c-Src and TRAF6 and the inhibition of RANK signaling pathways. Thus, TA could serve as a novel osteoclastogenesis inhibitor and might have beneficial effects on bone health.

Wheel-Running Exercise Protects Ovariectomized Mice from Bone Loss via IFN-γ-Mediated Suppression of the NF-κB and MAPK Pathways.

Physical exercise is recommended as a preventative approach for osteoporosis; however, the effect of physical exercise on bone mass remains controversial. Additionally, the immune regulation of physical exercise on bone mass remains unclear. To determine whether wheel-running (WR) exercise contributes to improving bone mineral density (BMD) and investigate the involved immune mechanism, ovariectomized (OVX) and sham-operated mice were treated with 8 weeks of WR exercise. The distal femurs of the mice were sequentially scanned, reconstructed, and analyzed using microcomputed tomography and related software to assess BMD and bone microarchitecture. Flow cytometry assays were applied to investigate alterations in immune cells and inflammatory cytokines. In vitro, osteoclast differentiation was conducted to determine the effect of IFN-γ on osteoclastogenesis and the underlying mechanism. As a result, trabecular parameters were decreased in the OVX mice compared with the sham group. However, WR exercise significantly improved the deterioration in the bone microarchitecture of the OVX mice with an increase of 60.00% in BMD, 55.18% in bone volume, 66.67% in trabecular number, 32.52% in trabecular thickness, and a decrease of 19.44% in trabecular separation. Similarly, WR exercise increased the proportion of CD8+ T cells from 7.26 ± 1.71% to 10.23 ± 1.35% in the spleen and from 1.62 ± 0.54% to 2.38 ± 0.43% in the bone marrow of the OVX mice (P < 0.05). The expression of IFN-γ was also increased in the OVX + WR mice compared with the OVX mice (1.65 ± 0.45% vs. 2.26 ± 0.34%, P < 0.05). In vitro studies demonstrated an inhibitory effect of IFN-γ on osteoclastogenesis in a dose- and time-dependent manner. Meanwhile, the classical NF-κB and MAPK pathways were found to be critical in IFN-γ-mediated inhibition of osteoclast differentiation. In conclusion, our study discovered that WR exercise rescued bone loss in the OVX mice in an IFN-γ-mediated immunomodulatory manner. After WR exercise, IFN-γ expression was restored by activated CD8+ T cells, consequently leading to the inhibition of osteoclastogenesis and the recovery from bone loss through the NF-κB and MAPK pathways.

Sec-O-Glucosylhamaudol Inhibits RANKL-Induced Osteoclastogenesis by Repressing 5-LO and AKT/GSK3β Signaling.

Sec-O-glucosylhamaudol (SOG), an active flavonoid compound derived from the root of Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk., exhibits analgesic, anti-inflammatory, and high 5-lipoxygenase (5-LO) inhibitory effects. However, its effect on osteoclastogenesis was unclear. We demonstrated that SOG markedly attenuated RANKL-induced osteoclast formation, F-actin ring formation, and mineral resorption by reducing the induction of key transcription factors NFATc1, c-Fos, and their target genes such as TRAP, CTSK, and DC-STAMP during osteoclastogenesis. Western blotting showed that SOG significantly inhibited the phosphorylation of AKT and GSK3β at the middle–late stage of osteoclastogenesis without altering calcineurin catalytic subunit protein phosphatase-2β-Aα expression. Moreover, GSK3β inhibitor SB415286 partially reversed SOG-induced inhibition of osteoclastogenesis, suggesting that SOG inhibits RANKL-induced osteoclastogenesis by activating GSK3β, at least in part. 5-LO gene silencing by small interfering RNA in mouse bone marrow macrophages markedly reduced RANKL-induced osteoclastogenesis by inhibiting NFATc1. However, it did not affect the phosphorylation of AKT or GSK3β, indicating that SOG exerts its inhibitory effects on osteoclastogenesis by suppressing both the independent 5-LO pathway and AKT-mediated GSK3β inactivation. In support of this, SOG significantly improved bone destruction in a lipopolysaccharide-induced mouse model of bone loss. Taken together, these results suggest a potential therapeutic effect for SOG on osteoclast-related bone lysis disease.

Pharmacological characterization of AS2690168, a novel small molecule RANKL signal transduction inhibitor

Pathological osteolysis is associated with excessive bone resorption by activated osteoclasts. Given that receptor activator of NF-kB and its ligand (RANKL) are key players in the differentiation and activation of osteoclasts, the RANKL/RANK signaling pathway is considered a promising target for the development of effective osteoclastogenesis inhibitors. We previously found that the orally available compound, AS2690168, suppresses RANKL-induced osteoclastogenesis of RAW264 cells. In this report, we further characterized the pharmacological profiles of AS2690168 in vitro and in vivo. AS2690168 suppressed soluble RANKL (sRANKL)-induced NFATc1 mRNA expression in RAW264 cells at 0.3 and 3.0 μM. It also suppressed calcium release from parathyroid hormone-stimulated mouse calvaria with an IC50 value of 0.46 μM. Oral administration of AS2690168 completely suppressed the decrease in femoral bone mineral content in an sRANKL-induced osteopenic mice model at 3.0 mg/kg. It also significantly suppressed the decrease in femoral bone mineral density and increase in serum tartrate-resistant acid phosphatase-5b levels in ovariectomized rats at doses of 0.3, 1 and 3 mg/kg. Finally, AS260168 suppressed the increase in urine deoxypyridinoline in a rat prednisolone-induced osteoporosis model at 10 mg/kg. These results suggest that AS2690168 is a promising treatment for bone disorders with excessive bone resorption.

Niloticin inhibits osteoclastogenesis by blocking RANKL–RANK interaction and suppressing the AKT, MAPK, and NF-κB signaling pathways

Dysregulation of osteoclasts or excessive osteoclastogenesis significantly –contributes to the occurrence and development of osteolytic diseases, including osteoporosis, inflammatory bone erosion, and tumor-induced osteolysis. The protein-protein interaction between the receptor activator of nuclear factor (NF)-κB (RANK) and its ligand (RANKL) mediates the differentiation and activation of osteoclasts, making it a key therapeutic target for osteoclastogenesis inhibition. However, very few natural compounds exerting anti-osteoclastogenesis activity by inhibiting the RANKL-RANK interaction have been found. Niloticin is a natural tetracyclic triterpenoid compound with anti-viral, antioxidative, and mosquitocidal activities. However, its role in osteoclastogenesis remains unknown. The present study found that niloticin directly binds to RANK with an equilibrium dissociation constant of 5.8 μM, blocking RANKL-RANK interaction, thereby inhibiting RANKL-induced AKT, MAPK (p38, JNK, and ERK1/2), and NF-κB (IKKα/β, IκBα, and p65) pathways activation, and reducing the expression of key osteoclast differentiation-related regulatory factors (NFATc1, c-Fos, TRAP, c-Src, β3-Integrin, and cathepsin K) in osteoclast precursors, ultimately negatively regulating osteoclastogenesis. These findings suggest that niloticin could serve as a novel osteoclastogenesis inhibitor and might have beneficial effects on bone health.

Stimulation of Osteoclast Formation by Oncostatin M and the Role of WNT16 as a Negative Feedback Regulator.

Oncostatin M (OSM), which belongs to the IL-6 family of cytokines, is the most potent and effective stimulator of osteoclast formation in this family, as assessed by different in vitro assays. Osteoclastogenesis induced by the IL-6 type of cytokines is mediated by the induction and paracrine stimulation of the osteoclastogenic cytokine receptor activator of nuclear factor κ-B ligand (RANKL), expressed on osteoblast cell membranes and targeting the receptor activator of nuclear factor κ-B (RANK) on osteoclast progenitor cells. The potent effect of OSM on osteoclastogenesis is due to an unusually robust induction of RANKL in osteoblasts through the OSM receptor (OSMR), mediated by a JAK–STAT/MAPK signaling pathway and by unique recruitment of the adapter protein Shc1 to the OSMR. Gene deletion of Osmr in mice results in decreased numbers of osteoclasts and enhanced trabecular bone caused by increased trabecular thickness, indicating that OSM may play a role in physiological regulation of bone remodeling. However, increased amounts of OSM, either through administration of recombinant protein or of adenoviral vectors expressing Osm, results in enhanced bone mass due to increased bone formation without any clear sign of increased osteoclast numbers, a finding which can be reconciled by cell culture experiments demonstrating that OSM can induce osteoblast differentiation and stimulate mineralization of bone nodules in such cultures. Thus, in vitro studies and gene deletion experiments show that OSM is a stimulator of osteoclast formation, whereas administration of OSM to mice shows that OSM is not a strong stimulator of osteoclastogenesis in vivo when administered to adult animals. These observations could be explained by our recent finding showing that OSM is a potent stimulator of the osteoclastogenesis inhibitor WNT16, acting in a negative feedback loop to reduce OSM-induced osteoclast formation.

Anti-Osteoporosis Potencies of Zingiber officinale Rosc. Rhizome Water Extract and DFA III Produced from Dahlia spp. L.: in vivo and in vitro Studies

BACKGROUND: Zingiber officinale Rosc. is estrogenic and thus can be developed as an anti-osteoporosis. Difructose anhydride III (DFA III), possesses anti-osteoporosis potencies. This study aimed to investigate the anti-osteoporosis activity of ginger rhizome water extract (GE) and DFA III from dahlia tubers in ovariectomized (OVX) rat models and to determine their anti-osteoclastogenic effect in vitro.METHODS: This study was conducted using 25 female rats. Blood sampling was carried out at the beginning and end of treatments. Femur bones were isolated after daily 14-day treatments, measured for density, and processed for histological staining. RAW 264.7 cells were induced by osteoclast differentiation factor. A cell viability assay was employed to determine the cytotoxicity of DFA III and GE. The inhibition of osteoclastogenesis was investigated by tartrate-resistant acid phosphatase staining.RESULTS: All groups showed no difference in body weight elevation and serum lipid profiles. The GE and DFA III caused no effect on bone density. However, the GE or DFA III groups showed higher osteoblast numbers compared with the control groups. A significantly less osteoclast was found in the GE+DFA III group. The GE and DFA III showed no toxicity on RAW 264.7 cells. GE showed strong inhibitory effects on the post stimulation osteoclastogenesis model. The combination of GE and DFA III was synergistic in reducing the osteoclastogenesis confluency in RAW 264.7 cells.CONCLUSION: The data support our hypothesis that GE and DFA III can decrease the risk of osteoporosis by osteoclastogenesis inhibition.KEYWORDS: Dahlia spp., estrogenic, ginger, osteoclast, osteoporosis, ovariectomy, RAW 264.7 cell

ThPOK Inhibits Osteoclast Formation Via NFATc1 Transcription and Function.

ABSTRACTBoth LRF (Zbtb7a) and ThPOK (Zbtb7b) belong to the POK (BTB/POZ and Kruppel) family of transcription repressors that participate in development, differentiation, and oncogenesis. Although LRF mediates osteoclast differentiation by regulating NFATc1 expression, the principal established function of ThPOK is transcriptional control of T‐cell lineage commitment. Whether ThPOK affects osteoclast formation or function is not known. We find that marrow macrophage ThPOK expression diminishes with exposure to receptor activator of NF‐kB ligand (RANKL), but ThPOK deficiency does not affect osteoclast differentiation. On the other hand, enhanced ThPOK, in macrophages, substantially impairs osteoclastogenesis. Excess ThPOK binds the NFATc1 promoter and suppresses its transcription, suggesting a mechanism for its osteoclast inhibitory effect. Despite suppression of osteoclastogenesis by excess ThPOK being associated with diminished NFATc1, osteoclast formation is not rescued by NFATc1 overexpression. Thus, ThPOK appears to inhibit NFATc1 transcription and its osteoclastogenic capacity, while its depletion has no effect on the bone‐resorptive cell. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

ThPOK inhibits osteoclast formation via NFATc1 transcription and function

Both LRF (Zbtb7a) and ThPOK (Zbtb7b) belong to the POK (BTB/POZ and Kruppel) family of transcription repressors which participate in development, differentiation and oncogenesis. While LRF mediates osteoclast differentiation by regulating NFATc1 expression, the principal established function of ThPOK is transcriptional control of T-cell lineage commitment. Whether ThPOK affects osteoclast formation or function, is not known.We find that marrow macrophage ThPOK expression diminishes with exposure to RANKL but ThPOK deficiency does not affect osteoclast differentiation. On the other hand, enhanced ThPOK, in macrophages, substantially impairs osteoclastogenesis. Excess ThPOK binds the NFATc1 promoter and suppresses its transcription suggesting a mechanism for its osteoclast inhibitory effect. Despite suppression of osteoclastogenesis by excess ThPOK being associated with diminished NFATc1, osteoclast formation is not rescued by NFATc1 overexpression. Thus, ThPOK appears to inhibit NFATc1 transcription and its osteoclastogenic capacity while its depletion has no effect on the bone resorptive cell. This article is protected by copyright. All rights reserved.

Epoxymicheliolide inhibits osteoclastogenesis and resists OVX-induced osteoporosis by suppressing ERK1/2 and NFATc1 signaling

The hyperactivity of osteoclasts caused by postmenopausal estrogen deficiency plays an imperative role in the progression of osteoporosis. Although osteoporosis-related drugs have been widely used to alleviate this disorder, there is an urgent need for drugs with fewer side effects. In this study, we found that epoxymicheliolide (EMCL), a derivative of parthenolide, has a high affinity to ERK1/2, but the treatment and mechanism of osteoporosis using EMCL have not been explored. Therefore, we intended to figure out the effects and potential mechanisms of EMCL on RANKL-stimulated osteoclast formation and function in vitro, construct an OVX murine model to simulate the therapeutic effects of EMCL on estrogen-deficient bone loss subsequently. EMCL restrained the phosphorylation of ERK1/2 in the RANKL-stimulated MAPK pathway, which in sequence inhibited the transcription and expression of the main osteoclast transcription factor NFATc1, resulting in the suppression of osteoclastogenesis and bone resorption. However, the same concentration of EMCL did not affect the proliferation and differentiation of osteoblasts. In vivo experiments showed that EMCL can significantly resist osteoporosis caused by estrogen deficiency, alleviate bone loss, and reduce the number of osteoclasts. These results suggest that EMCL can reduce osteoclast production and bone resorption by inhibiting ERK1/2 phosphorylation and NFATc1 entering the nucleus, and could be used in the treatment of osteoporosis caused by estrogen deficiency and hyperactivity of osteoclasts.

Icariin inhibits RANKL-induced osteoclastogenesis in RAW264.7 cells via inhibition of reactive oxygen species production by reducing the expression of NOX1 and NOX4

Icariin (ICA), isolated from Herba Epimedii, is a natural flavonoid glycoside that possesses antioxidant properties and inhibits osteoclastogenesis. However, the mechanism underlying osteoclastogenesis inhibition by ICA remains unclear. Here, we investigated the effects of ICA on receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells. ICA inhibited the expression of osteoclastogenesis-related genes in RAW264.7 cells induced by RANKL. ICA could inhibit osteoclastogenesis without inhibiting the viability of RAW264.7 cells. In addition, ICA inhibited reactive oxygen species production in RANKL-induced RAW264.7 cells. ICA reduced the expression of nuclear factor in activated T cells, cytoplasmic 1, and tartrate-resistant acid phosphatase, which are osteoclast-related molecules. Moreover, ICA decreased the expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX), specifically NOX1 and NOX4, in RANKL-induced RAW264.7 cells. Our findings suggest that ICA can be used as a potential therapeutic agent for osteolytic diseases such as osteoporosis.

Anti-Osteoporotic Mechanisms of Polyphenols Elucidated Based on In Vivo Studies Using Ovariectomized Animals.

Polyphenols are widely known for their antioxidant activity, i.e., they have the ability to suppress oxidative stress, and this behavior is mediated by the autoxidation of their phenolic hydroxyl groups. Postmenopausal osteoporosis is a common health problem that is associated with estrogen deficiency. Since oxidative stress is thought to play a key role in the onset and progression of osteoporosis, it is expected that polyphenols can serve as a safe and suitable treatment in this regard. Therefore, in this review, we aimed to elucidate the anti-osteoporotic mechanisms of polyphenols reported by in vivo studies involving the use of ovariectomized animals. We categorized the polyphenols as resveratrol, purified polyphenols other than resveratrol, or polyphenol-rich substances or extracts. Literature data indicated that resveratrol activates sirtuin 1, and thereafter, suppresses osteoclastogenic pathways, such as the receptor activator of the nuclear factor kappa B (RANK) ligand (RANKL) pathway, and promotes osteoblastogenic pathways, such as the wingless-related MMTV integration site pathway. Further, we noted that purified polyphenols and polyphenol-rich substances or extracts exert anti-inflammatory and/or antioxidative effects, which inhibit RANKL/RANK binding via the NF-κB pathway, resulting in the suppression of osteoclastogenesis. In conclusion, antioxidative and anti-inflammatory polyphenols, including resveratrol, can be safe and effective for the treatment of postmenopausal osteoporosis based on their ability to regulate the imbalance between bone formation and resorption.

A novel adiponectin receptor agonist (AdipoAI) ameliorates type 2 diabetes-associated periodontitis by enhancing autophagy in osteoclasts.

Type 2 diabetes (T2D)-associated periodontitis is severe and refractory in many cases. Considered an inflammatory disease, T2D predisposes to periodontitis by increasing whole-body inflammation. One mechanism of increased inflammation is thatT2D is mediated by loss of production or function of the anti-inflammatory hormone adiponectin. In our previous report, AdipoRon, an adiponectin receptor agonist, and AdipoAI, a newly discovered, more specific agonist, attenuated T2D-associated inflammation by inhibiting osteoclastogenesis and LPS-induced endotoxemia. Autophagy plays an important role during osteoclast differentiation and function. The impact of AdipoAI on osteoclast function and autophagy involved in osteoclastogenesis is not known. Here, we compare AdipoRon and AdipoAI potency, side effects and mechanism of action in T2D-associated periodontitis. The RAW 264.7 cell line was used for in vitro studies. We analyzed the potential cytotoxicity of AdipoAI using the CCK-8 assay. The anti-osteoclastogenic potential of AdipoAI was studied by real-time qPCR and tartrate-resistant acid phosphatase staining. The actions of AdipoAI involved in autophagy were tested by real-time qPCR, western blot and immunofluorescence staining. In the diet-induced obesity model of T2D, we investigated the impact of AdipoAI on fasting blood glucose, alveolar bone loss, and gingival inflammation in mice with experimental periodontitis. AdipoRon inhibited osteoclastogenesis and AdipoAI inhibited osteoclastogenesis at lower doses than AdipoRon without any cytotoxicity. In DIO mice with experimental periodontitis, AdipoAI reduced mouse body weight in 14days, reducing fasting glucose levels, alveolar bone destruction, osteoclast number along the alveolar bone surface, and decreased the expression of pro-inflammatory factors in periodontal tissues. AdipoAI and AdipoRon also enhanced LC3A/B expression when cultured with RANKL.3-Methyladenine, a known autophagy inhibitor, decreased LC3A/B expression and reversed the inhibition of osteoclastogenesis during AdipoAI treatment. Our results demonstrate that AdipoAI ameliorates the severity of T2D-associated periodontitis by enhancing autophagy in osteoclasts at lower doses than AdipoRon without demonstrable side effects. Thus, AdipoAI has pharmaceutical potential for treating diabetes-associated periodontal disease.

1,3-Dibenzyl-5-Fluorouracil Prevents Ovariectomy-Induced Bone Loss by Suppressing Osteoclast Differentiation.

Osteoclasts (OCs) are clinically important cells that resorb bone matrix. Accelerated bone destruction by OCs is closely linked to the development of metabolic bone diseases. In this study, we screened novel chemical inhibitors targeting OC differentiation to identify drug candidates for metabolic bone diseases. We identified that 1,3-dibenzyl-5-fluorouracil, also named OCI-101, is a novel inhibitor of osteoclastogenesis. The formation of multinucleated OCs is reduced by treatment with OCI-101 in a dose-dependent manner. OCI-101 inhibited the expression of OC markers via downregulation of receptor activator of NF-κB ligand and M-CSF signaling pathways. Finally, we showed that OCI-101 prevents ovariectomy-induced bone loss by suppressing OC differentiation in mice. Hence, these results demonstrated that OCI-101 is a good drug candidate for treating metabolic bone diseases.

Physicochemical properties and osteoclastogenesis for three premixed calcium silicate-based sealers post set.

Solubility, pH, ion release, cytotoxicity, and osteoclastogenesis inhibition in bone marrow-derived monocyte macrophages (BMMs) were evaluated in EndoSequence BC Sealer (END), Bio-C Sealer (BC), and Sealer Plus BC (SPBC). pH was determined after immersion of the sealers in deionized water (DW) and Minimum Essential Medium Alpha (α-MEM). Solubility was obtained by mass loss. Ion release was measured by using X-ray fluorescence spectroscopy (XRF). Cytotoxicity was evaluated by MTT assay. Inhibition of osteoclastogenesis was evaluated by tartrate-resistant acid phosphatase (TRAP). Data were analyzed using the t-test, ANOVA and Tukey/Dunnett's post-hoc tests (α = 0.05). END had the highest pH in DW (p < 0.05), and BC, in α-MEM (p < 0.05). Solubility in DW was the lowest for SPBC (p < 0.005). The highest calcium release was observed for BC in DW at 12 h (p < 0.05), and in α-MEM at 12 and 24 h (p < 0.05). The lowest toxicity was detected for END (p < 0.05). BC had the highest inhibitory effect on osteoclasts (p < 0.05). Overall, the highest solubility and pH values were found in DW. However, the calcium silicate-based sealer showed higher solubility than the ISO standards. Calcium release was the highest for BC. END showed the highest cell viability, and BC, the highest osteoclast inhibition.