RANKL-induced Reactive Oxygen Species Research Articles

Unique cartilage matrix-associated protein inhibits osteoclast differentiation by alleviating RANKL-induced reactive oxygen species

Unique cartilage matrix-associated protein (UCMA) is a γ-carboxyglutamic acid-rich secretory protein primarily expressed in adult cartilage. UCMA promotes osteoblast differentiation and reduces high glucose-induced reactive oxygen species (ROS) production in osteoblasts; however, its role in osteoclasts remains unclear. Since Ucma is not expressed in osteoclasts, treatment with recombinant UCMA protein (rUCMA) was employed to investigate the effect of UCMA on osteoclasts. The rUCMA-treated osteoclasts exhibited significantly reduced osteoclast differentiation, resorption activity, and osteoclast-specific gene expression. Moreover, rUCMA treatment reduced RANKL-induced ROS production and increased the expression of antioxidant genes in osteoclasts. This study demonstrates that UCMA effectively inhibits RANKL-stimulated osteoclast differentiation and oxidative stress.

Isobavachin attenuates osteoclastogenesis and periodontitis-induced bone loss by inhibiting cellular iron accumulation and mitochondrial biogenesis

As bone-resorbing cells rich in mitochondria, osteoclasts require high iron uptake to promote mitochondrial biogenesis and maintain a high-energy metabolic state for active bone resorption. Given that abnormal osteoclast formation and activation leads to imbalanced bone remodeling and osteolytic bone loss, osteoclasts may be crucial targets for treating osteolytic diseases such as periodontitis. Isobavachin (IBA), a natural flavonoid compound, has been confirmed to be an inhibitor of receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation from bone marrow-derived macrophages (BMMs). However, its effects on periodontitis-induced bone loss and the potential mechanism of its anti-osteoclastogenesis effect remain unclear. Our study demonstrated that IBA suppressed RANKL-induced osteoclastogenesis in BMMs and RAW264.7 cells and inhibited osteoclast-mediated bone resorption in vitro. Transcriptomic analysis indicated that iron homeostasis and reactive oxygen species (ROS) metabolic process were enriched among the differentially expressed genes following IBA treatment. IBA exerted its anti-osteoclastogenesis effect by inhibiting iron accumulation in osteoclasts. Mechanistically, IBA attenuated iron accumulation in RANKL-induced osteoclasts by inhibiting the mitogen-activated protein kinase (MAPK) pathway to upregulate ferroportin1 (Fpn1) expression and promote Fpn1-mediated intracellular iron efflux. We also found that IBA inhibited mitochondrial biogenesis and function, and reduced RANKL-induced ROS generation in osteoclasts. Furthermore, IBA attenuated periodontitis-induced bone loss by reducing osteoclastogenesis in vivo. Overall, these results suggest that IBA may serve as a promising therapeutic strategy for bone diseases characterized by osteoclastic bone resorption.

Isoquercitrin attenuates the osteoclast-mediated bone loss in rheumatoid arthritis via the Nrf2/ROS/NF-κB pathway

An excess of osteoclastogenesis significantly contributes to the development of rheumatoid arthritis (RA). Activation of the nuclear factor erythroid-2 related factor 2 (Nrf2) and nuclear factor kappa B (NF-κB) ligand (RANKL)-induced reactive oxygen species (ROS)-to-NF-κB signaling cascade are important mechanisms regulating osteoclastogenesis; however, whether Nrf2 is involved in RANKL-induced NF-κB activation is controversial. Isoquercitrin, a natural flavonoid compound, has been shown to have Nrf2-dependent antioxidant effects inprevious studies. We sought to verify whether isoquercitrin could modulate RANKL-induced NF-κB activation by activating Nrf2, thereby affecting osteoclastogenesis. Tartrate-resistant acid phosphatase staining, F-actin ring staining and resorption pit assay suggested that isoquercitrin significantly inhibited osteoclastogenesis and osteolytic function. Mitosox staining showed that RANKL-induced ROS generation was significantly inhibited by isoquercitrin from day 3 of the osteoclast differentiation cycle. Quantitative real-time PCR, Western blot, and immunofluorescence indicated that isoquercitrin activated the Nrf2 signaling pathway and inhibited NF-κB expression. And when we used the Nrf2-specific inhibitor ML385, the inhibition of NF-κB by isoquercitrin disappeared. Moreover, we found that Nrf2 is not uninvolved in RANKL-induced NF-κB activation and may be related to the timing of ROS regulation. When we limited isoquercitrin administration to 2 days, Nrf2 remained activated and the inhibition of NF-κB disappeared. In vivo experiments suggested that isoquercitrin attenuated RA modeling-induced bone loss. Overall, isoquercitrin-activated Nrf2 blocked the RANKL-induced ROS-to-NF-κB signaling cascade response, thereby inhibiting osteoclastogenesis and bone loss. These findings provide new ideas for the treatment of RA.

Orientin suppresses osteoclastogenesis and ameliorates ovariectomy-induced osteoporosis via suppressing ROS production.

The aberrant differentiation of osteoclasts is a key feature of the pathogenesis of osteoporosis, which has a devastating impact on human health. While the effects of Orientin (Ori) on osteoporosis, particularly on RANKL-stimulated osteoclast production and activation, remain still unclear, Ori has been found to display several biological activities, including antioxidant and anti-inflammatory. In this work, we investigated the possible pathways through which Ori suppressed RANKL-induced osteoclast development and showed for the first time that it does so. The macrophages from the bone marrow (BMMs) were cultivated and then treated with Ori after being stimulated with RANKL. Then, TRAP-positive multinucleated cells were counted, and F-actin ring analysis was used to assess Ori's impact on mature osteoclast development. In addition, dihydroethidium (DHE) staining was used to evaluate the impact of Ori on RANKL-induced reactive oxygen species (ROS). In addition, we performed western blotting and quantitative RT-PCR analysis to investigate probable causes of these downregulation effects. We discovered that Ori inhibits the creation of osteoclasts, the gene and protein expressions unique to osteoclasts, and the ROS production. By activating Nrf2 and other ROS-scavenging enzymes, Ori reduces intracellular ROS levels. The expression of the main transcription factor of osteoclast development, c-Fos, was downregulated together with NFATc1, CTSK, and NFATc2, thanks to Ori's inhibition of RANKL-induced NF-κB. Consistent with its in vitro antiosteoclastogenic action, Ori therapy in the ovariectomized (OVX) rat model was also able to restore bone mass and improve microarchitecture in the distal femurs. Together, our results demonstrate that Ori is a flavonoid molecule with therapeutic promise for bone illnesses associated with osteoclasts, such as osteoporosis.

Corylifol A protects against ovariectomized-induced bone loss and attenuates RANKL‐induced osteoclastogenesis via ROS reduction, ERK inhibition, and NFATc1 activation

Osteoclast differentiation and function are critical targets for anti-osteoporosis treatment. Oxidative stress also plays an important regulatory role in the differentiation of osteoclasts. Corylifol A (CA) is a flavonoid extracted from the Psoralea fruit. It has anti-inflammatory and antioxidant properties despite its unknown effect on osteoporosis. This study found that CA prevented estrogen-deficiency-induced bone loss and suppressed osteoclastogenesis in ovariectomized (OVX) mice by inhibiting intracellular reactive oxygen species (ROS) levels. In vivo, CA effectively prevented trabecular bone loss and reduced osteoclasts' number on the bone surface in OVX mice, as demonstrated in micro-CT, osteometry, and immunohistochemical data. However, CA did not affect cortical bone. In vitro, CA inhibited RANKL-induced podosome belt formation, osteoclastogenesis, and bone resorption functions. CA suppressed RANKL-induced ROS by boosting antioxidant enzymes (Catalase and NQO1) and NFATc1 signaling pathway related protein expression, including integrin αvβ3, NFATc1 and CTSK. Moreover, CA inhibited osteoclast-specific genes, including Ctsk, Acp5, and Mmp9. CA also attenuated the MAPK/ERK pathway, but did not affect the NF-κB signaling pathway. In terms of osteogenesis, CA did not inhibit or promote osteogenic differentiation and mineralization in vitro. These results reveal that CA could be a new replacement therapy for treating estrogen-deficiency osteoporosis via suppressing osteoclastogenesis and intracellular ROS.

MCU Inhibitor Ruthenium Red Alleviates the Osteoclastogenesis and Ovariectomized Osteoporosis via Suppressing RANKL-Induced ROS Production and NFATc1 Activation through P38 MAPK Signaling Pathway.

Osteoporosis is a disorder of bone metabolism that is extremely common in elderly patients as well as in postmenopausal women. The main manifestation is that the bone resorption capacity is greater than the bone formation capacity, which eventually leads to a decrease in bone mass, increasing the risk of fracture. There is growing evidence that inhibiting osteoclast formation and resorption ability can be effective in treating and preventing the occurrence of osteoporosis. Our study is the first time to explore the role of the mitochondrial calcium uniporter (MCU) and its inhibitor ruthenium red (RR) in bone metabolism, clarifying the specific mechanism by which it inhibits osteoclast formation in vitro and plays a therapeutic role in osteoporosis in vivo. We verified the suppressive effects of RR on the receptor activator of nuclear factor-κB ligand (RANKL-)-induced differentiation and bone resorption function of osteoclasts in vitro. The reactive oxygen species (ROS) production stimulated by RANKL and the expression level of P38 MAPK/NFATc1 were also found to be inhibited by RR. Moreover, the promotion of RR on osteogenesis differentiation was investigated by alkaline phosphatase (ALP) and alizarin red S (ARS) staining and the detection of osteogenesis-specific gene expression levels by quantitative polymerase chain reaction (qPCR) and western blotting. Moreover, in ovariectomy (OVX-)-induced osteoporosis models, RR can downregulate the expression and function of the MCU, relieving bone loss and promoting osteogenesis to present a therapeutic effect on osteoporosis. This new finding will provide an important direction for the study of RR and MCU in the study of bone metabolism therapy targets.

Urolithin B suppresses osteoclastogenesis via inhibiting RANKL‐induced signalling pathways and attenuating ROS activities

Osteoporosis (OP) has severely affected human health, which is characterized by abnormal differentiation of osteoclasts. Urolithin B (UB), as a potential natural drug, has been reported to exhibit numerous biological activities including antioxidant and anti‐inflammatory but its effects on OP, especially on RANKL‐stimulated osteoclast formation and activation, are still understood. In our study, we have demonstrated for the first time that UB inhibits RANKL‐induced osteoclast differentiation and explored its potential mechanisms of action. The RAW264.7 cells were cultured and induced with RANKL followed by UB treatment. Then, the effects of UB on mature osteoclast differentiation were evaluated by counting tartrate‐resistant acid phosphatase (TRAP)‐positive multinucleated cells and F‐actin ring analysis. Moreover, the effects of UB on RANKL‐induced reactive oxygen species (ROS) were measured by 2′, 7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) staining. Further, we explored the potential mechanisms of these downregulation effects by performing Western blotting and quantitative RT‐PCR examination. We found that UB represses osteoclastogenesis, F‐actin belts formation, osteoclast‐specific gene expressions and ROS activity in a time‐ and concentration‐dependent manner. Mechanistically, UB attenuates intracellular ROS levels by upregulation of Nrf2 and other ROS scavenging enzymes activation. Furthermore, UB also inhibited RANKL‐induced NF‐κB, MAPK and Akt signalling pathway and suppressed expression of c‐Fos and nuclear factor of activated T cells 1 (NFATc1), which is the master transcription factor of osteoclast differentiation. Taken together, our findings confirm that UB is a polyphenolic compound that can be a potential therapeutic treatment for osteoclast‐related bone diseases such as osteoporosis.

Azilsartan Suppresses Osteoclastogenesis and Ameliorates Ovariectomy-Induced Osteoporosis by Inhibiting Reactive Oxygen Species Production and Activating Nrf2 Signaling.

Osteoporosis is characterized by a decrease in bone mass and destruction of the bone microarchitecture, and it commonly occurs in postmenopausal women and the elderly. Overactivation of osteoclasts caused by the inflammatory response or oxidative stress leads to osteoporosis. An increasing number of studies have suggested that intracellular reactive oxygen species (ROS) are strongly associated with osteoclastogenesis. As a novel angiotensin (Ang) II receptor blocker (ARB), azilsartan was reported to be associated with the inhibition of intracellular oxidative stress processes. However, the relationship between azilsartan and osteoclastogenesis is still unknown. In this study, we explored the effect of azilsartan on ovariectomy-induced osteoporosis in mice. Azilsartan significantly inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis and downregulated the expression of osteoclast-associated markers (Nfatc1, c-Fos, and Ctsk) in vitro. Furthermore, azilsartan reduced RANKL-induced ROS production by increasing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2). Mechanistically, azilsartan inhibited the activation of MAPK/NF-κB signaling pathways, while Nrf2 silencing reversed the inhibitory effect of azilsartan on MAPK/NF-κB signaling pathways. Consistent with the in vitro data, azilsartan administration ameliorated ovariectomy (OVX)-induced osteoporosis, and decreased ROS levels in vivo. In conclusion, azilsartan inhibited oxidative stress and may be a novel treatment strategy for osteoporosis caused by osteoclast overactivation.

The Novel Antioxidant Compound JSH-23 Prevents Osteolysis by Scavenging ROS During Both Osteoclastogenesis and Osteoblastogenesis.

Inflammatory osteolysis is a pathological skeletal disease associated with not only the production of inflammatory cytokines but also local oxidative status. Excessive reactive oxygen species (ROS) promote bone resorption by osteoclasts and induce the apoptosis of osteoblasts. In consideration of the lack of effective preventive or treatments options against osteolysis, the exploitation of novel pharmacological compounds/agents is critically required. In our study, we found that a novel antioxidant compound, JSH-23, plays a role in restoring bone homeostasis by scavenging intracellular ROS during both osteoclastogenesis and osteoblastogenesis. Mechanically, JSH-23 suppressed RANKL-induced osteoclastogenesis, bone resorption and the expression of specific genes (including NFATc1, c-Fos, TRAP, CTSK and DC-STAMP) via inhibition of the NF-κB signaling pathway. Meanwhile, JSH-23 suppressed RANKL-induced ROS generation via the TRAF6/Rac1/NOX1 pathway and the enhanced expression of Nrf2/HO-1. In addition, JSH-23 attenuated H2O2-induced apoptosis and mineralization reduction in osteoblasts by reducing ROS production and enhancing Nrf2/HO-1 expression. Our in vivo results further revealed that JSH-23 exerts its protective effects on bone mass through its antioxidant activity. In conclusion, our results show that the application of JSH-23 might be a novel and plausible strategy for the treatment of osteolysis-related disease.

Oroxylin A reduces osteoclast formation and bone resorption via suppressing RANKL-induced ROS and NFATc1 activation

Excessive bone erosion by osteoclasts is associated with osteoporosis, rheumatoid arthritis, and periprosthetic osteolysis. Targeting osteoclasts may serve as an effective treatment for osteolytic diseases. Although drugs are currently available for the treatment of these diseases, exploring potential anti-osteoclast natural compounds with safe and effective treatment remains needed. Oroxylin A (OA), a natural flavonoid isolated from the root of Scutellaria baicalensis Georgi, has numerous beneficial pharmacological characteristics, including anti-inflammatory and antioxidant activity. However, its effects and mechanisms on osteoclast formation and bone resorption have not yet been clarified. Our research showed that OA attenuated the formation and function of osteoclast induced by RANKL in a time- and concentration-dependent manner without any cytotoxicity. Mechanistically, OA suppressed intracellular reactive oxygen species (ROS) levels through the Nrf2-mediated antioxidant response. Moreover, OA inhibited the activity of NFATc1, the master transcriptional regulator of RANKL-induced osteoclastogenesis. OA exhibited protective effects in mouse models of post-ovariectomy (OVX)- and lipopolysaccharide (LPS)‐induced bone loss, in accordance with its in vitro anti-osteoclastogenic effect. Collectively, our findings highlight the potential of OA as a pharmacological agent for the prevention of osteoclast-mediated osteolytic diseases.

Oxymatrine Attenuates Osteoclastogenesis via Modulation of ROS-Mediated SREBP2 Signaling and Counteracts Ovariectomy-Induced Osteoporosis.

Osteoporosis, mainly caused by osteoclast-induced bone resorption, has become a major health problem in post-menopausal women and the elderly. Growing evidence indicates that inhibiting osteoclastogenesis is an efficient approach to develop alternative therapeutic agents for treating osteoporosis. In this study, we identified the potential regulating role of Oxymatrine (OMT), a quinazine alkaloid extracted from Sophora flavescens with various therapeutic effects in many diseases, on osteoclastogenesis for the first time. We found that OMT attenuated RANKL-induced osteoclast formation in both time- and dose-dependent manners. Further, OMT significantly suppressed RANKL-induced sterol regulatory element-binding protein 2 (SREBP2) activation and the expression of the nuclear factor of activated T cells 1 (NFATc1). Moreover, OMT inhibited the generation of RANKL-induced reactive oxygen species (ROS), and the upregulation of ROS could rescue the inhibition of SREBP2 by OMT. More importantly, ovariectomy (OVX) mouse model showed that OMT could effectively improve ovariectomy (OVX)-induced osteopenia by inhibiting osteoclastogenesis in vivo. In conclusion, our data demonstrated that OMT impaired ROS mediated SREBP2 activity and downstream NFATc1 expression during osteoclastogenesis, suppressed OVX-induced osteopenia in vivo, which suggested that OMT could be a promising compound for medical treatment against osteoporosis.

Deficiency of optineurin enhances osteoclast differentiation by attenuating the NRF2-mediated antioxidant response

Abnormally increased resorption contributes to bone degenerative diseases such as Paget’s disease of bone (PDB) through unclear mechanisms. Recently, the optineurin (OPTN) gene has been implicated in PDB, and global OPTN knockout mice (Optn−/−) were shown to exhibit increased formation of osteoclasts (osteoclastogenesis). Growing evidence, including our own, has demonstrated that intracellular reactive oxygen species (ROS) stimulated by receptor activator of nuclear factor kappa-B ligand (RANKL) can act as signaling molecules to promote osteoclastogenesis. Here, we report that OPTN interacts with nuclear factor erythroid-derived factor 2-related factor 2 (NRF2), the master regulator of the antioxidant response, defining a pathway through which RANKL-induced ROS could be regulated for osteoclastogenesis. In this study, monocytes from Optn−/− and wild-type (Optn+/+) mice were utilized to differentiate into osteoclasts, and both qRT-PCR and tartrate-resistant acid phosphatase (TRAP) staining showed that the Optn−/− monocytes exhibited enhanced osteoclastogenesis compared to the Optn+/+ cells. CellROX® staining, qRT-PCR, and Western blotting indicated that OPTN deficiency reduced the basal expression of Nrf2, inhibited the expression of NRF2-responsive antioxidants, and increased basal and RANKL-induced intracellular ROS levels, leading to enhanced osteoclastogenesis. Coimmunoprecipitation (co-IP) showed direct interaction, and immunofluorescence staining showed perinuclear colocalization of the OPTN-NRF2 granular structures during differentiation. Finally, curcumin and the other NRF2 activators attenuated the hyperactive osteoclastogenesis induced by OPTN deficiency. Collectively, our findings reveal a novel OPTN-mediated mechanism for regulating the NRF2-mediated antioxidant response in osteoclasts and extend the therapeutic potential of OPTN in the aging process resulting from ROS-triggered oxidative stress, which is associated with PDB and many other degenerative diseases.

Inhibitory Effect of a Rosmarinic Acid-Enriched Fraction Prepared from Nga-Mon (Perilla frutescens) Seed Meal on Osteoclastogenesis through the RANK Signaling Pathway.

The aim of this study is to determine antioxidant and anti-inflammatory activities relating to the antiosteoporosis effects of various perilla seed meal (PSM) fractions. The remaining waste of perilla seed obtained from cold oil compression was extracted with 70% ethanol and sequentially fractionated according to solvent polarity with hexane, dichloromethane, ethyl acetate, and water. The results indicated that the seed-meal ethyl acetate fraction (SMEF) exhibited the highest antioxidant and anti-inflammatory activities, and rosmarinic acid (RA) content. The signaling pathways induced by the receptor activator of the nuclear factor kappa B (NF-κB) ligand (RANKL) that trigger reactive oxygen species (ROS) and several transcription factors, leading to the induction of osteoclastogenesis, were also investigated. The SMEF clearly showed attenuated RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts and TRAP activity. A Western blot analysis showed that the SMEF significantly downregulated RANKL-induced NF-κB, AP-1 activation, and the nuclear factor of activated T-cell 1 (NFATc1) expression. SMEF also suppressed RANKL-induced osteoclast-specific marker gene-like MMP-9 using zymography. Furthermore, the SMEF showed inhibition of RANKL-induced ROS production in RAW 264.7 cells. The results suggest that the SMEF, which contained high quantities of RA, could be developed as a natural active pharmaceutical ingredient for osteoclastogenic protection and health promotion.

Dual Oxidase Maturation Factor 1 Positively Regulates RANKL-Induced Osteoclastogenesis via Activating Reactive Oxygen Species and TRAF6-Mediated Signaling.

Receptor activator of NF-κB ligand (RANKL) induces generation of intracellular reactive oxygen species (ROS), which act as second messengers in RANKL-mediated osteoclastogenesis. Dual oxidase maturation factor 1 (Duoxa1) has been associated with the maturation of ROS-generating enzymes including dual oxidases (Duox1 and Duox2). In the progression of osteoclast differentiation, we identified that only Duoxa1 showed an effective change upon RANKL stimulation, but not Duox1, Duox2, and Duoxa2. Therefore, we hypothesized that Duoxa1 could independently act as a second messenger for RANKL stimulation and regulate ROS production during osteoclastogenesis. Duoxa1 gradually increased during RANKL-induced osteoclastogenesis. Using siRNA or retrovirus transduction, we found that Duoxa1 regulated RANKL-stimulated osteoclast formation and bone resorption positively. Furthermore, knockdown of Duoxa1 decreased the RANKL-induced ROS production. During Duoxa1-related control of osteoclastogenesis, activation of tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated early signaling molecules including MAPKs, Akt, IκB, Btk, Src and PLCγ2 was affected, which sequentially modified the mRNA or protein expression levels of key transcription factors in osteoclast differentiation, such as c-Fos and NFATc1, as well as mRNA expression of osteoclast-specific markers. Overall, our data indicate that Duoxa1 plays a crucial role in osteoclastogenesis via regulating RANKL-induced intracellular ROS production and activating TRAF6-mediated signaling.

Schisandrin A restrains osteoclastogenesis by inhibiting reactive oxygen species and activating Nrf2 signalling.

ObjectivesIntracellular reactive oxygen species (ROS) induced by receptor activator of NF‐kB ligand (RANKL) has been proven to be a critical factor in the development of osteoclasts. This study aimed to prove that schisandrin A (Sch), a novel anti‐oxidant compound, is able to suppress osteoclastogenesis and prevent bone loss in ovariectomized (OVX) mice by suppressing ROS via nuclear factor erythroid 2‐related factor (Nrf2).Material and MethodsMicro‐CT was used to detect bone formation. The effects of Sch on receptor activator of nuclear factor‐κB (NF‐κB) ligand (RANKL)‐induced reactive oxygen species (ROS) were measured by dihydroethidium (DHE) staining in vivo and 2',7'‐dichlorodihydrofluorescein diacetate (DCFH‐DA) staining in vitro. Immunofluorescence staining was used to detect the expression of Nrf2 in vivo. siRNA was used to evaluate the effect of Nrf2 in osteoclastogenesis.ResultsSch suppresses RANKL‐induced ROS production by regulating nuclear factor erythroid 2‐related factor (Nrf2) in vitro and vivo. Mechanistically, Sch enhances the expression of Nrf2 by regulating the degradation of Nrf2. Further, Sch suppresses phosphorylation of P65 and its nuclear translocation, as well as the degradation of IκBα. Collectively, our findings reveal that Sch protects against OVX‐induced bone loss by suppressing ROS via Nrf2.ConclusionsOur results showed the potential of anti‐oxidant compound schisandrin A in the treatment of osteoporosis, highlighting Nrf2 as a novel promising target in osteoclast‐related disease.

GSH attenuates RANKL-induced osteoclast formation in vitro and LPS-induced bone loss in vivo

Osteoclasts are capable of adhering the bone matrix, then secrete acid and lytic enzymes to resorb it. Reactive oxygen species (ROS), as a signaling messenger, plays an important role in the receptor activator nuclear factor κB ligand (RANKL) signal pathway during osteoclast differentiation. Glutathione (GSH) is known to be a powerful antioxidant which can scavenge intracellular ROS. This study aimed to investigate whether GSH can as a protective agent against the RANKL-stimulated osteoclastogenesis by suppressing intracellular ROS. Here, we showed that GSH markedly restricted RNAKL-induced differentiation of bone marrow-derived macrophages (BMMs) to form osteoclasts. GSH suppressed RANKL-induced ROS generation and subsequent ROS-induced NF-κB signaling pathways within BMMs during osteoclastogenesis. Further, GSH acted to significantly downregulate the osteoclastogenic genes expression of nuclear factor in activated T cells, cytoplasmic1 (NFATc1), C-fos, the tartrate-resistant acid phosphatase (TRAP), and osteoclast-associated immunoglobulin-like receptor (OSCAR). Our results suggested that GSH inhibits intracellular ROS-mediated NF-κB signal pathway involved in osteoclast differentiation. These findings might form the basis of a new strategy for treating bone disease associated with excessive bone resorption.

The Roles of ROS Generation in RANKL-Induced Osteoclastogenesis: Suppressive Effects of Febuxostat.

Receptor activator of NF-κB ligand (RANKL), a critical mediator of osteoclastogenesis, is upregulated in multiple myeloma (MM). The xanthine oxidase inhibitor febuxostat, clinically used for prevention of tumor lysis syndrome, has been demonstrated to effectively inhibit not only the generation of uric acid but also the formation of reactive oxygen species (ROS). ROS has been demonstrated to mediate RANKL-mediated osteoclastogenesis. In the present study, we therefore explored the role of cancer-treatment-induced ROS in RANKL-mediated osteoclastogenesis and the suppressive effects of febuxostat on ROS generation and osteoclastogenesis. RANKL dose-dependently induced ROS production in RAW264.7 preosteoclastic cells; however, febuxostat inhibited the RANKL-induced ROS production and osteoclast (OC) formation. Interestingly, doxorubicin (Dox) further enhanced RANKL-induced osteoclastogenesis through upregulation of ROS production, which was mostly abolished by addition of febuxostat. Febuxostat also inhibited osteoclastogenesis enhanced in cocultures of bone marrow cells with MM cells. Importantly, febuxostat rather suppressed MM cell viability and did not compromise Dox’s anti-MM activity. In addition, febuxostat was able to alleviate pathological osteoclastic activity and bone loss in ovariectomized mice. Collectively, these results suggest that excessive ROS production by aberrant RANKL overexpression and/or anticancer treatment disadvantageously impacts bone, and that febuxostat can prevent the ROS-mediated osteoclastic bone damage.

The Nrf2 activator RTA-408 attenuates osteoclastogenesis by inhibiting STING dependent NF-κb signaling

The dysregulation of ROS production and osteoclastogenesis is involved in the progress of osteoporosis. To identify novel and effective targets to treat this disease, it is important to explore the underlying mechanisms. In our study, we firstly tested the effect of the Nrf2 activator RTA-408, a novel synthetic triterpenoid under clinical investigation for many diseases, on osteoclastogenesis. We found that it could inhibit osteoclast differentiation and bone resorption in a time- and dose-dependent manner. Further, RTA-408 enhanced the expression and activity of Nrf2 and significantly suppressed RANKL-induced reactive oxygen species (ROS) production. Nrf2 regulates the STING expression and STING induces the production of IFN-β. Here, we found that RTA-408 could suppress STING expression, but that it does not affect Ifnb1 expression. RANKL-induced degradation of IκBα and the nuclear translocation of P65 was suppressed by RTA-408. Although this compound was not found to influence STING–IFN-β signaling, it suppressed the RANKL-induced K63-ubiquitination of STING via inhibiting the interaction between STING and the E3 ubiquitin ligase TRAF6. Further, adenovirus-mediated STING overexpression rescued the suppressive effect of RTA-408 on NF-κB signaling and osteoclastogenesis. In vivo experiments showed that this compound could effectively attenuate ovariectomy (OVX)-induced bone loss in C57BL/6 mice by inhibiting osteoclastogenesis. Collectively, we show that RTA-408 inhibits NF-κB signaling by suppressing the recruitment of TRAF6 to STING, in addition to attenuating osteoclastogenesis and OVX-induced bone loss in vivo, suggesting that it could be a promising candidate for treating osteoporosis in the future.

Isorhamnetin attenuates osteoarthritis by inhibiting osteoclastogenesis and protecting chondrocytes through modulating reactive oxygen species homeostasis.

Increasing evidence indicates that osteoarthritis (OA) is a musculoskeletal disease affecting the whole joint, including both cartilage and subchondral bone. Reactive oxygen species (ROS) have been demonstrated to be one of the important destructive factors during early‐stage OA development. The objective of this study was to investigate isorhamnetin (Iso) treatment on osteoclast formation and chondrocyte protection to attenuate OA by modulating ROS. Receptor activator of nuclear factor‐kappa B ligand (RANKL) was used to establish the osteoclast differentiation model in bone marrow macrophages (BMMs) in vivo. H2O2 was used to induce ROS, which could further cause chondrocyte apoptosis. We demonstrated that Iso suppressed RANKL‐induced ROS generation, which could mediate osteoclastogenesis. Moreover, we found that Iso inhibited osteoclast formation and function by suppressing the expression of osteoclastogenesis‐related genes and proteins. We proved that Iso inhibited RANKL‐induced activation of mitogen‐activated protein kinase activation of mitogen‐activated protein kinase (MAPK), nuclear factor‐kappa B (NF‐κB) and AKT signalling pathways in BMMs. In addition, Iso inhibited ROS‐induced chondrocyte apoptosis by regulating apoptosis‐related proteins. Moreover, Iso was administered to an anterior cruciate ligament transection (ACLT)‐induced OA mouse model. The results indicated that Iso exerted beneficial effects on inhibiting excessive osteoclast activity and chondrocyte apoptosis, which further remedied cartilage damage. Overall, our data showed that Iso is an effective candidate for treating OA.

Pseurotin A Inhibits Osteoclastogenesis and Prevents Ovariectomized-Induced Bone Loss by Suppressing Reactive Oxygen Species.

Rationale: Growing evidence indicates that intracellular reactive oxygen species (ROS) accumulation is a critical factor in the development of osteoporosis by triggering osteoclast formation and function. Pseurotin A (Pse) is a secondary metabolite isolated from Aspergillus fumigatus with antioxidant properties, recently shown to exhibit a wide range of potential therapeutic applications. However, its effects on osteoporosis remain unknown. This study aimed to explore whether Pse, by suppressing ROS level, is able to inhibit osteoclastogenesis and prevent the bone loss induced by estrogen-deficiency in ovariectomized (OVX) mice.Methods: The effects of Pse on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis and bone resorptive function were examined by tartrate resistant acid phosphatase (TRAcP) staining and hydroxyapatite resorption assay. 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) was used to detect intracellular ROS production in vitro. Western blot assay was used to identify proteins associated with ROS generation and scavenging as well as ROS-mediated signaling cascades including mitogen-activated protein kinases (MAPKs), NF-κB pathways, and nuclear factor of activated T cells 1 (NFATc1) signaling. The expression of osteoclast-specific genes was assessed by qPCR. The in vivo potential of Pse was determined using an OVX mouse model administered with Pse or vehicle for 6 weeks. In vivo ROS production was assessed by intravenous injection of dihydroethidium (DHE) into OVX mice 24h prior to killing. After sacrifice, the bone samples were analyzed using micro-CT and histomorphometry to determine bone volume, osteoclast activity, and ROS level ex vivo.Results: Pse was demonstrated to inhibit osteoclastogenesis and bone resorptive function in vitro, as well as the downregulation of osteoclast-specific genes including Acp5 (encoding TRAcP), Ctsk (encoding cathepsin K), and Mmp9 (encoding matrix metalloproteinase 9). Mechanistically, Pse suppressed intracellular ROS level by inhibiting RANKL-induced ROS production and enhancing ROS scavenging enzymes, subsequently suppressing MAPK pathway (ERK, P38, and JNK) and NF-κB pathways, leading to the inhibition of NFATc1 signaling. Micro-CT and histological data indicated that OVX procedure resulted in a significant bone loss, with dramatically increased the number of osteoclasts on the bone surface as well as increased ROS level in the bone marrow microenvironment; whereas Pse supplementation was capable of effectively preventing these OVX-induced changes.Conclusion: Pse was demonstrated for the first time as a novel alternative therapy for osteoclast-related bone diseases such as osteoporosis through suppressing ROS level.