Trifolirhizin reduces osteoclast formation and prevents inflammatory osteolysis by inhibiting RANKL-induced activation of NF-κB and MAPK signaling pathways and ROS.

Inflammatory osteolysis is often caused by the excessive activation of osteoclasts stimulated by bacterial products such as lipopolysaccharide. The natural flavonoid trifolirhizin (TRI) has anti-inflammatory properties; however, its function in inflammatory bone lysis remains unclear. This study aimed to elucidate the potential regulatory mechanisms of TRI in osteoclasts.Tartrate-resistant acid phosphatase (TRAP) staining, acid secretion assays, podosomal actin belt fluorescence staining, and bone resorption assays were used to investigate the effects of TRI on osteoclast differentiation and bone resorption. A reactive oxygen species (ROS) measurement kit was used to detect the effect of TRI on ROS levels in osteoclasts. The effects of TRI on genes and signaling pathways related to osteoclast differentiation were determined by quantitative polymerase chain reaction (qPCR) and western blotting. A mouse model of lipopolysaccharide-mediated inflammatory osteolysis was established, and the effects of TRI treatment on bone mass were observed using micro-CT and histological examination. Mechanistically, TRI reduced ROS production by inhibiting receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, and by upregulating the expression levels of the anti-ROS enzymes heme oxygenase-1 (HO-1) and catalase (CAT), which contributed to the degradation of ROS, ultimately leading to a decrease in osteoclastogenesis. TRI inhibited osteoclast formation and ameliorated lipopolysaccharide (LPS)-mediated inflammatory osteolysis. Thus, TRI may be a candidate agent for anti-inflammatory osteolysis.

Regorafenib Attenuates Osteoclasts Differentiation by Inhibiting the NF-κB, NFAT, ERK, and p38 Signaling Pathways.

Osteolytic diseases such as osteoporosis and neoplastic bone metastases are caused by the excessive activation of osteoclasts. Inhibiting the excessive activation of osteoclasts is a crucial strategy for treating osteolytic diseases. This study investigated the roles and mechanisms of regorafenib, a tyrosine kinase inhibitor, on osteoclasts and osteolytic diseases. We first identified the potential targets and mechanisms of regorafenib on osteoclast-related osteolytic diseases using network pharmacological analysis and molecular docking techniques. Then, we verified its role and mechanism on osteoclasts via cellular and animal experiments. Network pharmacology analysis identified 89 common targets shared by regorafenib and osteoclast-related osteolytic diseases. Enrichment analysis suggested that regorafenib may act on osteoclast-related osteolytic diseases by modulating targets such as AKT1, CASP3, MMP9, and MAPK3, regulating biological processes such as cell proliferation, apoptosis, and phosphorylation regulation, and influencing signaling pathways such as MAPK, PI3K/AKT, and osteoclast differentiation. The molecular docking results indicated that regorafenib and AKT1, CASP3, MMP9, MAPK3, and MAPK14 were stably docked. Cell experiments demonstrated that regorafenib significantly inhibited osteoclast differentiation and bone resorption in RAW 264.7 cells and bone marrow macrophages in a dose-dependent manner, with up to 50% reduction at 800 nM concentration without exhibiting cytotoxic effects. Furthermore, Western blot and RT-qPCR results demonstrated that regorafenib inhibited osteoclast differentiation by blocking the transduction of RANKL-induced NF-κB, p38, ERK, and NFAT signaling pathways. In vivo studies using an ovariectomized mouse model showed that regorafenib significantly improved bone volume fraction (BV/TV), bone surface to total volume (BS/TV), and number of trabeculae (TB.N), as well as reduced trabecular separation (Tb.Sp) compared to the OVX groups (P < 0.05). TRAcP staining results revealed a reduction in the number of osteoclasts with regorafenib treatment (P < 0.01). These results indicate that regorafenib exerts its protective effects against osteoclast-related osteolytic disease by inhibiting the RANKL-induced NF-κB, NFAT, ERK, and p38 signaling pathways. This study proves that regorafenib may serve as a potential therapeutic agent for osteoclast-related osteolytic diseases.

Active fraction of Polyrhachis vicina (Rogers) inhibits osteoclastogenesis by targeting Trim38 mediated proteasomal degradation of TRAF6.

BACKGROUND: Reactive Oxygen Species (ROS) is a key factor in the pathogenesis of osteoporosis (OP) primarily characterized by excessive osteoclast activity. Active fraction of Polyrhachis vicina Rogers (AFPR) exerts antioxidant effects and possesses extensive promising therapeutic effects in various conditions, however, its function in osteoclastogenesis and OP is unknown. PURPOSE: The aim of this study is to elucidate the cellular and molecular mechanisms of AFPR in OP. STUDY DESIGN AND METHODS: CCK8 assay was used to evaluate the cell viability under AFPR treatment. TRAcP staining, podosome belts staining and bone resorption were used to test the effect of AFPR on osteoclastogenesis. Immunofluorescence staining was used to observe the effect of AFPR on ROS production. si-RNA transfection, coimmunoprecipitation and Western-blot were used to clarify the underlying mechanisms. Further, an ovariectomy (OVX) -induced OP mice model was used to identify the effect of AFPR on bone loss using Micro-CT scanning and histological examination. RESULTS: In the present study, AFPR inhibited osteoclast differentiation and bone resorption induced by nuclear factor-κB receptor activator (NF-κB) ligand (RANKL) in dose-/ time-dependent with no cytotoxicity. Meanwhile, AFPR decreased RANKL-mediated ROS levels and enhanced ROS scavenging enzymes. Mechanistically, AFPR promoted proteasomal degradation of TRAF6 by significantly upregulating its K48-linked ubiquitination, subsequently inhibiting NFATc1 activity. We further observed that tripartite motif protein 38 (TRIM38) could mediate the ubiquitination of TRAF6 in response to RANKL. Moreover, TRIM38 could negatively regulate the RANKL pathway by binding to TRAF6 and promoting K48-linked polyubiquitination. In addition, TRIM38 deficiency rescued the inhibition of AFPR on ROS and NFATc1 activity and osteoclastogenesis. In line with these results, AFPR reduced OP caused by OVX through ameliorating osteoclastogenesis. CONCLUSION: AFPR alleviates ovariectomized-induced bone loss via suppressing ROS and NFATc1 by targeting Trim38 mediated proteasomal degradation of TRAF6. The research offers innovative perspectives on AFPR's suppressive impact in vivo OVX mouse model and in vitro, and clarifies the fundamental mechanism.

Hecogenin alleviates LPS-induced osteolysis via regulating pyroptosis and ROS involved Nrf2 activation.

Reactive oxidative species (ROS) generation triggers pyroptosis and induces development of inflammatory osteolysis. Hecogenin (HG) has anti-inflammatory and antioxidative property, but its effects on inflammatory osteolysis remains unclear. In our study, we investigated the mechanism of HG on pyroptosis and its effect on inflammatory osteolysis in vitro and in vivo. The impact of HG on osteoclastogenesis was evaluated using cytotoxicity, TRAcP staining and bone resorption assays. The RNA-sequencing was employed to identify potential signaling pathways, and then RT-qPCR, western blot, immunofluorescence, and ELISA were used to verify. To determine the protective effect of HG in vivo, Lipopolysaccharide (LPS)-induced animal models were utilized, along with micro-CT and histological examination. HG suppressed RANKL-induced osteoclast differentiation, bone resorption, NFATc1 activity and downstream factors. RNA-sequencing results showed that HG inhibited osteoclastogenesis by modulating the inflammatory response and macrophage polarization. Furthermore, HG inhibited the NF-κB pathway, and deactivated the NLRP3 inflammasome. HG activated the expression of nuclear factor E2-related factor 2 (Nrf2) to eliminate ROS generation. Importantly, the inhibitory effect of HG on NLRP3 inflammasome could be reversed by treatment with the Nrf2 inhibitor ML385. In vivo, HG prevented the mice against LPS-induced osteolysis by suppressing osteoclastogenesis and inflammatory factors. In conclusion, HG could activate Nrf2 to eliminate ROS generation, inactivate NLRP3 inflammasome and inhibit pyroptosis, thereby suppressing osteoclastogenesis in vitro and alleviating inflammatory osteolysis in vivo, which indicating that HG might be a promising candidate to treat inflammatory osteolysis.

Periplogenin attenuates LPS-mediated inflammatory osteolysis through the suppression of osteoclastogenesis via reducing the NF-κB and MAPK signaling pathways.

The key target for treating inflammatory osteolysis is osteoclasts. In an inflammatory environment, osteoclast differentiation increases, and bone resorption is enhanced. Periplogenin (Ppg) is a traditional Chinese medicine. It has anti-inflammatory and antitumor effects, but its impact on inflammatory osteolysis is unknown. This study found that Ppg prevented LPS-induced skull osteolysis by inhibiting the expression of inflammatory cytokines and osteoclast production. In vitro, Ppg blocked the RANKL-induced generation of osteoclasts, the development of pseudopodia bands, and bone resorption. Ppg also attenuated the expression of NFATc1, c-Fos, CTSK, and Atp6v0d2 proteins by inhibiting the NFATc1 signaling pathway. In addition, Ppg inhibited the expression of osteoclast-specific genes, including NFATc1, c-Fos, CTSK, Atp6v0d2, and Mmp9. Moreover, Ppg also inhibited NF-κB and MAPK pathways. In vivo, Ppg reduced the number of osteoclasts on the surface of the bone and suppressed LPS-induced osteolysis of the skull. These outcomes suggest that Ppg can serve as a new alternative therapy for treating inflammatory osteolysis by inhibiting inflammation and osteoclasts.

Cichoric acid targets RANKL to inhibit osteoclastogenesis and prevent ovariectomy-induced bone loss.

BACKGROUND AND AIM: Osteoporosis, a systemic metabolic bone disease, is characterized by the decline of bone mass and quality due to excessive osteoclast activity. Currently, drug-targeting osteoclasts show promising therapy for osteoporosis. In this study, we investigated the effect of cichoric acid (CA) on receptor activator of nuclear kappa-B ligand (RANKL)-induced osteoclastogenesis and the bone loss induced by ovariectomy in mice. EXPERIMENTAL PROCEDURE: Molecular docking technologies were employed to examine the interaction between CA and RANKL. CCK8 assay was used to evaluate the cell viability under CA treatment. TRAcP staining, podosome belt staining, and bone resorption assays were used to test the effect of CA on osteoclastogenesis and osteoclast function. Further, an OVX-induced osteoporosis mice model was employed to identify the effect of CA on bone loss using micro-CT scanning and histological examination. To investigate underlying mechanisms, network pharmacology was applied to predict the downstream signaling pathways, which were verified by Western blot and immunofluorescence staining. KEY RESULTS: The molecular docking analysis revealed that CA exhibited a specific binding affinity to RANKL, engaging multiple binding sites. CA inhibited RANKL-induced osteoclastogenesis and bone resorption without cytotoxic effects. Mechanistically, CA suppressed RANKL-induced intracellular reactive oxygen species, nuclear factor-kappa B, and mitogen-activated protein kinase pathways, followed by abrogated nuclear factor activated T-cells 1 activity. Consistent with this finding, CA attenuated post-ovariectomy-induced osteoporosis by ameliorating osteoclastogenesis. CONCLUSIONS AND IMPLICATIONS: CA inhibited osteoclast activity and bone loss by targeting RANKL. CA might represent a promising candidate for treating osteoclast-related diseases, such as osteoporosis.

IKK/NF-κB and ROS signal axes are involved in Tenacissoside H mediated inhibitory effects on LPS-induced inflammatory osteolysis.

Periodontal disease and arthroplasty prosthesis loosening and destabilization are both associated with osteolysis, which is predominantly caused by abnormal bone resorption triggered by pro-inflammatory cytokines. Osteoclasts (OCs) are critical players in the process. Concerns regarding the long-term efficacy and side effects of current frontline therapies, however, remain. Alternative therapies are still required. The aim of this work was to investigate the involvement of Tenacissoside H (TDH) in RANKL-mediated OC differentiation, as well as inflammatory osteolysis and associated processes. In vitro, bone marrow-derived macrophages (BMMs) cultured with RANKL and M-CSF were used to detect TDH in the differentiation and function of OCs. Real-time quantitative PCR was used to measure the expression of specific genes and inflammatory factors in OCs. Western blot was used to identify NFATc1, IKK, NF-κB, MAPK pathway, and oxidative stress-related components. Finally, an LPS-mediated calvarial osteolysis mouse model was employed to explore TDH's role in inflammatory osteolysis. The results showed that in vivo TDH inhibited the differentiation and resorption functions of OCs and down-regulated the transcription of osteoclast-specific genes, as well as Il-1ß, Il-6 and Tnf-α. In addition, TDH inhibited the IKK and NF-κB signalling pathways and down-regulated the level of ROS. In vivo studies revealed that TDH improves the bone loss caused by LPS. TDH may be a new candidate or treatment for osteoclast-associated inflammatory osteolytic disease.

Tussilagone inhibits osteoclastogenesis by modulating mitochondrial function and ROS production involved Nrf2 activation.

Reactive Oxygen Species (ROS) play an essential role in the pathogenesis of osteoporosis mainly characterized by excessive osteoclasts (OCs) activity. OCs are rich in mitochondria for energy support, which is a major source of total ROS. Tussilagone (TSG), a natural Sesquiterpenes from the flower of Tussilago farfara, has plentiful beneficial pharmacological characteristics with anti-inflammatory and anti-oxidative activity, but its effects and mechanism in osteopathology are still unclear. In our study, we investigated the regulation of ROS generated from the mitochondria in OCs. We found that TSG inhibited OCs differentiation and bone resorption without any cytotoxicity. Mechanistically, TSG reduced RANKL-mediated total ROS level by down-regulating intracellular ROS production and mitochondrial function, leading to the suppression of NFATc1 transcription. We also found that nuclear factor erythroid 2-related factor 2 (Nrf2) could enhance ROS scavenging enzymes in response to RANKL-induced oxidative stress. Furthermore, TSG up-regulated the expression of Nrf2 by inhibiting its proteosomal degradation. Interestingly, Nrf2 deficiency reversed the suppressive effect of TSG on mitochondrial activity and ROS signaling in OCs. Consistent with this finding, TSG attenuated post-ovariectomy (OVX)- and lipopolysaccharide (LPS) induced bone loss by ameliorating osteoclastogenesis. Taken together, TSG has an anti-bone resorptive effect by modulating mitochondrial function and ROS production involved Nrf2 activation.

Myrislignan targets extracellular signal-regulated kinase (ERK) and modulates mitochondrial function to dampen osteoclastogenesis and ovariectomy-induced osteoporosis.

BACKGROUND: Activated osteoclasts cause excessive bone resorption, and disrupt bone homeostasis, leading to osteoporosis. The extracellular signal-regulated kinase (ERK) signaling is the classical pathway related to osteoclast differentiation, and mitochondrial reactive oxygen species are closely associated with the differentiation of osteoclasts. Myrislignan (MRL), a natural product derived from nutmeg, has multiple pharmacological activities; however, its therapeutic effect on osteoporosis is unclear. Here, we investigated whether MRL could inhibit osteoclastogenesis and bone mass loss in an ovariectomy mouse model by suppressing mitochondrial function and ERK signaling. METHODS: Tartrate-resistant and phosphatase (TRAP) and bone resorption assays were performed to observe the effect of MRL on osteoclastogenesis of bone marrow macrophages. MitoSOX RED and tetramethyl rhodamine methyl ester (TMRM) staining was performed to evaluate the inhibitory effect of MRL on mitochondria. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was performed to detect whether MRL suppressed the expression of osteoclast-specific genes. The impact of MRL on the protein involved in the mitogen-activated protein kinase (MAPK) and nuclear factor-κB pathways was evaluated using western blotting. In addition, a specific ERK agonist LM22B-10, was used to revalidate the inhibitory effect of MRL on ERK. Finally, we established an ovariectomy mouse model to assess the therapeutic effect of MRL on osteoporosis in vivo. RESULTS: MRL inhibited osteoclast differentiation and the associated bone resorption, by significantly decreasing osteoclastic gene expression. Mechanistically, MRL inhibited the phosphorylation of ERK by suppressing the mitochondrial function, thereby downregulating the nuclear factor of activated T cells 1 (NFATc1) signaling. LM22B-10 treatment further verified the targeted inhibition effect of MRL on ERK. Microscopic computed tomographic and histologic analyses of the tibial tissue sections indicated that ovariectomized mice had lower bone mass and higher expression of ERK compared with normal controls. However, MRL treatment significantly reversed these effects, indicating the anti-osteoporosis effect of MRL. CONCLUSION: We report for the first time that MRL inhibits ERK signaling by suppressing mitochondrial function, thereby ameliorating ovariectomy-induced osteoporosis. Our findings can provide a basis for the development of a novel therapeutic strategy for osteoporosis.

Aminooxyacetic acid hemihydrochloride leads to decreased intracellular ATP levels and altered cell cycle of prostate cancer cells by suppressing energy metabolism.

The second most common cancer among men is prostate cancer, which is also the fifth leading reason for male cancer deaths worldwide. Bone metastases are the main factor affecting the prognosis of prostate cancer. Consequently, antitumor and anti-prostate cancer-induced bone destruction medicines are urgently needed. We previously discovered that aminooxyacetic acid hemihydrochloride (AOAA) suppressed bone resorption and osteoclast growth by decreasing adenosine triphosphate (ATP) production and limiting oxidative phosphorylation (OXPHOS). Here, we evaluated the impacts of AOAA on prostate cancer RM-1 cells in vitro. It's found that AOAA significantly inhibited cell proliferation, migration, and invasiveness, decreased ATP levels, increased ROS, halted the cell cycle phase, and triggered apoptosis. AOAA also decreased mitochondrial membrane potential and the ability to uptake glucose, suggesting that the antitumor effects of AOAA were expressed through the inhibition of OXPHOS and glycolysis. Furthermore, we assessed the effects of AOAA in vivo using a prostate cancer-induced bone osteolysis mice model. AOAA also delayed tumor growth and bone destruction in vivo. On the whole, our findings imply that AOAA may potentially have therapeutic effects on prostate cancer and prostate cancer-induced osteolysis.

CGK733 alleviates ovariectomy-induced bone loss through blocking RANKL-mediated Ca2+ oscillations and NF-κB/MAPK signaling pathways.

Osteoporosis is a prevalent systemic metabolic disease in modern society, in which patients often suffer from bone loss due to over-activation of osteoclasts. Currently, amelioration of bone loss through modulation of osteoclast activity is a major therapeutic strategy. Ataxia telangiectasia mutated (ATM) inhibitor CGK733 (CG) was reported to have a sensitizing impact in treating malignancies. However, its effect on osteoporosis remains unclear. In this study, we investigated the effects of CG on osteoclast differentiation and function, as well as the therapeutic effects of CG on osteoporosis. Our study found that CG inhibits osteoclast differentiation and function. We further found that CG inhibits the activation of NFATc1 and ultimately osteoclast formation by inhibiting RANKL-mediated Ca2+ oscillation and the NF-κB/MAPK signaling pathway. Next, we constructed an ovariectomized mouse model and demonstrated that CG improved bone loss in ovariectomized mice. Therefore, CG may be a potential drug for the prevention and treatment of osteoporosis.

Casticin suppresses RANKL­induced osteoclastogenesis and prevents ovariectomy­induced bone loss by regulating the AKT/ERK and NF­κB signaling pathways.

Postmenopausal osteoporosis is a systemic metabolic disease that chronically endangers public health and is typically characterized by low bone mineral density and marked bone fragility. The excessive bone resorption activity of osteoclasts is a major factor in the pathogenesis of osteoporosis; therefore, strategies aimed at inhibiting osteoclast activity may prevent bone decline and attenuate the process of osteoporosis. Casticin (Cas), a natural compound, has anti­inflammatory and antitumor properties. However, the role of Cas in bone metabolism remains largely unclear. The present study found that the receptor activator of nuclear factor­κΒ (NF­κB) ligand­induced osteoclast activation and differentiation were inhibited by Cas. Tartrate­resistant acid phosphatase staining revealed that Cas inhibited osteoclast differentiation, and bone resorption pit assays demonstrated that Cas affected the function of osteoclasts. Cas significantly reduced the expression of osteoclast­specific genes and related proteins, such as nuclear factor of activated T cells, cytoplasmic 1 and c­Fos at the mRNA and protein level in a concentration­dependent manner. Cas inhibited osteoclast formation by blocking the AKT/ERK and NF­κB signaling pathways, according to the intracellular signaling analysis. The microcomputed tomography and tissue staining of tibiae from ovariectomized mice revealed that Cas prevented the bone loss induced by estrogen deficiency and reduced osteoclast activity in vivo. Collectively, these findings indicated that Cas may be used to prevent osteoporosis.

Ethyl caffeate inhibits macrophage polarization via SIRT1/NF-κB to attenuate traumatic heterotopic ossification in mice.

Heterotopic ossification (HO) denotes the presence of mature bone tissue in soft tissues or around joints. Inflammation is a key driver of traumatic HO, and macrophages play an important role in this process. Ethyl caffeate (ECF), a critical active compound found in Petunia, exerts significant anti-inflammatory effects. Herein, we established a mouse model of HO by transection of the Achilles tendon and back burn and found abundant macrophage infiltration in the early stage of HO, which decreased with time. In vitro and in vivo experiments indicated that ECF inhibited macrophage polarization, and mechanistic studies showed that it inhibited the SIRT1/NF-κB signalling pathway, thereby suppressing the release of downstream inflammatory cytokines. ECF reduced HO in mice, and its effect was comparable to indomethacin (INDO). In vitro studies revealed that ECF did not directly affect the mineralization of mesenchymal stem cells (MSCs) or osteogenic differentiation but inhibited these processes by reducing the level of inflammatory cytokines in the conditioned medium (CM). Thus, M1 macrophages may play a crucial role in the pathogenesis of HO, and ECF is a prospective candidate for the prevention of trauma-induced HO. DATA AVAILABILITY: Data will be made available on request.

UPLC/Q-TOF-MS-based metabolomics evaluate the efficacy of oroxylin A against postmenopausal osteoporosis.

Post-menopausal osteoporosis (PMOP) is a common metabolic bone malady characterized by bone mass loss and bone microarchitectural deterioration; however, there is currently no effective drug for its management. According to our previous study, oroxylin A (OA) could effectively protect ovariectomized (OVX)-osteoporotic mice from bone loss; however, its therapeutic targets are still unclear. From a metabolomic perspective, we studied serum metabolic profiles to discover potential biomarkers and OVX-related metabolic networks, which could assist us to comprehend the impact of OA on OVX. Five metabolites were identified as biomarkers associated with 10 related metabolic pathways, including phenylalanine, tyrosine and tryptophan biosynthesis, and phenylalanine, tryptophan and glycerophospholipid metabolism. After OA treatment, the expression of multiple biomarkers changed, with lysophosphatidylcholine (18:2) being a major significantly regulated biomarker. Our study demonstrated that OA's effects on OVX are probably related to the regulation of phenylalanine, tyrosine and tryptophan biosynthesis. Our findings explain the role of OA against PMOP in terms of metabolism and pharmacology and provide a pharmacological foundation for OA treatment of PMOP.

Isosinensetin alleviates estrogen deficiency-induced osteoporosis via suppressing ROS-mediated NF-κB/MAPK signaling pathways.

The formation of osteoclasts and their hyperactive bone resorption are related to the aggregation of intracellular reactive oxygen species (ROS). Flavonoids, derived from plant active ingredients, can alleviate the symptoms of osteoporosis (OP). Isosinensetin (Iss) is a flavonoid with antioxidant effects obtained mainly from citrus fruits, and its effect on osteoclastogenesis has not been reported. In this study, we investigated the antioxidant activity of Iss on osteoclast differentiation and function, as well as the therapeutic impact of Iss on OP. We found that Iss inhibited osteoclastogenesis and suppressed the bone resorption function of osteoclasts. Additionally, Iss reduced receptor activator of nuclear factor-κB ligand (RANKL)-induced intracellular ROS. Using quantitative real-time polymerase chain reaction and western blot, we further found that Iss inhibited osteoclast-specific genes and related proteins, while promoting the expression of antioxidant enzyme-related genes and proteins. Mechanistically, Iss reduces intracellular ROS by activating nuclear factor-erythroid 2-related factor 2 (Nrf2) and its related antioxidant enzymes and inhibits the downstream nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways of ROS, which in turn inhibits nuclear factor of activated T cells 1 (NFATc1), and ultimately inhibits osteoclastogenesis. In vivo, by micro-computed tomography (Micro-CT) assay and histological analyses, we found that Iss could reduce bone loss in ovariectomized (OVX) mice. Therefore, Iss has the potential as an OP preventative and therapeutic drug option.

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 αvß3, 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.

Pharmacology-based molecular docking of 4-methylcatechol and its role in RANKL-mediated ROS/Keap1/Nrf2 signalling axis and osteoclastogenesis.

4-Methylcatechol (4-MC) is an agonist of various neurotrophic factors, which can upregulate the expression of Heme oxygenase 1 (HO-1) protein by activating nuclear factor erythroid 2-related factor 2 (Nrf2), thereby inhibiting oxidative stress-induced neural stem cell death. During RANKL-stimulated osteoclast differentiation, intracellular reactive oxygen species (ROS) levels were increased. Nonetheless, the effect of 4-MC on osteoclast formation and bone resorption function has not been researched. In this study, we investigated the effect of HO-1 upregulation by 4-MC on RANKL-induced osteoclastogenesis and explored the molecular mechanism of HO-1 upregulation by 4-MC. We found that the small molecule compound 4-MC could bind to Keap1 amino acid residue of glycine GLY 367, isoleucine ILE 559 and valine VAL 606, with a predicted binding energy of -4.99 kcal/mol. 4-MC was found to inhibit osteoclast differentiation in vitro by activating Nrf2 to scavenge ROS, inhibiting NF-κB phosphorylation, and alleviating osteoporosis in ovariectomized (OVX) mice. Taken together, 4-MC reduces ROS by inhibiting Keap1, thereby preventing OVX-induced bone loss.

AKT/GSK3ß/NFATc1 and ROS signal axes are involved in AZD1390-mediated inhibitory effects on osteoclast and OVX-induced osteoporosis.

As a common disease in modern society, osteoporosis is caused by osteoclast hyperactivation, leading to enhanced bone resorption. Reactive oxygen species (ROS) metobolism and nuclear factor-activated T cells 1 (NFATc1) activities are two crucial processes during osteoclastogenesis. AZD1390 (AZD), an inhibitor of ataxia telangiectasia mutated (ATM), has been reported for antitumor effects, but little is known about how it plays a function in metabolic bone disease. Here, we found that AZD inhibitsthe generation, function and ROS-scavenging enzyme activity of mature osteoclast induced by RANKL stimulation, in a dose-dependent manner.Mechanistic analysis shows thatAZD affects osteoclast function and differentiation by inhibiting RANKL-induced NFATc1 signaling pathway and by increasing ROS-scavenging enzymes production in oxidative stress pathways. Preclinical studies have shown that AZD protects against bone loss in an ovariectomy (OVX) mouse model. Finally, our data confirm that AZD may prevent OVX-induced bone loss by abrogating RANKL-induced AKT/GSK3ß/NFATc1 signaling pathways, and by promoting the expression of ROS scavenging enzymes in oxidative stress pathways.Collectively, our research shows that AZD has the potential as a new therapeutic agent for osteoporosis.

Aminooxyacetic acid hemihydrochloride inhibits osteoclast differentiation and bone resorption by attenuating oxidative phosphorylation.

Osteoclasts undergo active metabolic reprogramming to acquire the energy needed during differentiation and bone resorption. Compared with immature osteoclasts, mature osteoclasts comprise higher levels of electron transport chain enzymes and more metabolically active mitochondria. Of all energy metabolism pathways, oxidative phosphorylation is considered to be the most efficient in supplying energy to osteoclasts. We found that the malate-aspartate shuttle inhibitor aminooxyacetic acid hemihydrochloride inhibits osteoclastogenesis and bone resorption by inhibiting exchange of reducing equivalents between the cytosol and the mitochondrial matrix and attenuating mitochondrial oxidative phosphorylation in vitro. The weakening of the oxidative phosphorylation pathway resulted in reduced mitochondrial function and inadequate energy supply along with reduced reactive oxygen species production. Furthermore, treatment with aminooxyacetic acid hemihydrochloride helped recover bone loss in ovariectomized mice. Our findings highlight the potential of interfering with the osteoclast intrinsic energy metabolism pathway as a treatment for osteoclast-mediated osteolytic diseases.

Anti-Osteoclast Effect of Exportin-1 Inhibitor Eltanexor on Osteoporosis Depends on Nuclear Accumulation of IκBα-NF-κB p65 Complex.

Osteoporosis affects around 200 million people globally, with menopausal women accounting for the bulk of cases. In the occurrence and development of osteoporosis, a key role is played by osteoclasts. Excessive osteoclast-mediated bone resorption activity reduces bone mass and increases bone fragility, resulting in osteoporosis. Thus, considerable demand exists for designing effective osteoporosis treatments based on targeting osteoclasts. Eltanexor (Elt; KPT-8602) is a selective nuclear-export inhibitor that covalently binds to and blocks the function of the nuclear-export protein exportin-1 (XPO1), which controls the nucleus-to-cytoplasm transfer of certain critical proteins related to growth regulation and tumor suppression, such as p53, IκBα [nuclear factor-κB (NF-κB) inhibitor α] and FOXO1; among these proteins, IκBα, a critical component of the NF-κB signaling pathway that primarily governs NF-κB activation and transcription. How Elt treatment affects osteoclasts remains poorly elucidated. Elt inhibited the growth and activity of RANKL-induced osteoclasts in vitro in a dose-dependent manner, and Elt exerted no cell-killing effect within the effective inhibitory concentration. Mechanistically, Elt was found to trap IκBα in the nucleus and thus protect IκBα from proteasome degradation, which resulted in the blocking of the translocation of IκBα and NF-κB p65 and the consequent inhibition of NF-κB activity. The suppression of NF-κB activity, in turn, inhibited the activity of two transcription factors (NFATc1 and c-Fos) essential for osteoclast formation and led to the downregulation of genes and proteins related to bone resorption. Our study thus provides a newly identified mechanism for targeting in the treatment of osteoporosis.