ObjectiveThe purpose of this study was to reveal the genetic correlation of RANKL polymorphisms with bone metastasis in breast cancer patients.MethodsIn this study, 139 bone metastasis patients and 152 no metastasis were included as the case and control groups. Real-time polymerase chain reaction (PCR) and allelic discrimination method were respectively applied for the genotyping of rs7325635 and rs2277438. Polymorphism genotype and allele frequencies were compared by χ2 test between the two groups. The risk of bone metastasis development caused by RANKL genetic variants was evaluated by odds ratio (OR) with 95% confidence interval (95%CI). The linkage of two polymorphisms was examined by Haploview. Binary and multivariate logistic analyses were used to optimize the results.ResultsRs2277438 GG genotype and G allele frequencies were significantly higher in bone metastasis patients than that in no-metastasis patients (P < 0.05); they were significantly correlated with the increased risk of bone metastasis occurrence (GG: OR = 2.065, 95%CI=1.104-3.863; G: OR = 1.486, 95%CI=1.068-2.068). Compared with the G–G haplotype, the A–A haplotype was found to significantly reduce the risk of bone metastasis in breast cancer patients (OR = 0.647, 95%CI=0.436-0.959). The multivariate logistic analysis indicated that family history, Ki67, and rs2277438 were positively correlated with bone metastasis, but menopausal state, clinical staging, and rs7325635 were negatively correlated with bone metastasis in breast cancer.ConclusionThe RANKL rs2277438 variant may be a potential genetic susceptibility factor associated with bone metastasis risk in breast cancer patients, though further functional validation is warranted. Rs7325635 was not independently associated with bone metastasis. The linkage disequilibrium between these two polymorphisms and their combined haplotype effect may play a role in bone metastasis susceptibility.

Objective Diabetic osteoporosis is a secondary complication of diabetes mellitus, characterized by reduced bone mass, increased bone fragility, and impaired fracture healing. However, the mechanisms underlying diabetic bone loss remain to be fully elucidated. More importantly, there is an urgent need to identify therapeutic agents that not only lower blood glucose levels but also alleviate bone loss. Therefore, this study aims to investigate the mechanisms of diabetes-associated bone loss and to explore potential therapeutic agents. Methods We established a mouse model of type 2 diabetes (T2D) induced by streptozotocin and a high-fat diet (HFD). Bone mass and osteoclast numbers were assessed using micro-CT and TRAP staining. In vitro, the effects of MSDC-0160 (MSD) on osteoclast differentiation and function were evaluated through TRAP staining and bone resorption assays. To elucidate the molecular mechanisms underlying MSD-mediated inhibition of osteoclastogenesis, qPCR, Western blotting, and immunofluorescence staining were performed. Finally, micro-CT scanning and immunohistochemical staining were conducted to examine the effects of MSD on bone microstructure and the bone microenvironment in T2D mice, as well as to clarify specific mechanism of action. Results T2D mice exhibited significant bone loss and enhanced osteoclast activation. Moreover, mitochondrial pyruvate carrier (MPC) activity was elevated in osteoclasts of T2D mice. Given the potential for mitigating diabetic bone loss by inhibiting MPC activity, we selected MSD, a novel insulin sensitizer that also serves as an MPC inhibitor. Further detailed investigations revealed that MSD suppresses osteoclast differentiation and function by reducing the energy supply required for osteoclast maturation. This effect results from impaired mitochondrial oxidative phosphorylation (OXPHOS) and reduced mitochondrial biogenesis. In vivo administration of MSD significantly ameliorated bone loss and reduced osteoclast numbers in T2D mice. Conclusion Our findings indicate that the bone loss in T2D mice is associated with excessive osteoclast activation, where MPC playing a crucial role in osteoclast differentiation and maturation. MSD, a novel insulin sensitizer, mitigates diabetic bone loss by suppressing MPC activity in osteoclasts.

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.

Inflammatory osteolysis is frequently associated with excessive osteoclast (OC) activation triggered by bacterial components such as lipopolysaccharide (LPS), accompanied by bone destruction and resorption. Receptor activator of nuclear factor kappa B ligand (RANKL) is a pivotal cytokine primarily secreted by osteoblasts, bone marrow stromal cells, and activated T lymphocytes. Wogonoside (WG), a flavonoid extracted from the root of Scutellaria baicalensis, possesses anti-inflammatory properties; however, its role in inflammatory osteolysis remains unclear. The aim of this study was to investigate the regulatory mechanism of WG in RANKL- and LPS-mediated OC differentiation and its effects on a mouse model of inflammatory osteolysis. Tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assays were used to evaluate the impact of WG on OC differentiation and function. Intracellular reactive oxygen species (ROS), Fe2+, and lipid peroxidation levels during OC differentiation were measured using 2 ',7 '-dichlorodihydrofluorescein diacetate (H2DCFDA), FerroOrange, and BODIPY 581/591 C11 fluorescent probes, respectively. The effects of WG on OC differentiation-related genes and downstream proteins and signaling pathways were examined by qPCR and Western blotting. In an LPS-induced inflammatory osteolysis model, micro-CT and histological staining (TRAP and HE) were employed to assess bone mass. Enzyme-linked immunosorbent (ELISA) and immunohistochemistry were applied to detect inflammatory cytokines in serum and the expression of antioxidant and inflammatory factors in bone tissue. Mechanistically, WG activated nuclear factor erythroid-2 related factor 2 (Nrf2) and its downstream SLC40A1, suppressed ROS-related iron metabolism dysregulation, and inhibited the mitogen-activated protein kinase (MAPK) signaling pathway and nuclear factor of activated T cells 1 (NFATc1), ultimately attenuating osteoclastogenesis. In vivo, WG ameliorated LPS-mediated inflammatory osteolysis. Thus, WG may represent a novel candidate drug for the treatment of OC-related inflammatory osteolytic diseases.

Osteoporosis is a skeletal condition caused by an excess of osteoclasts, resulting in an imbalance in bone metabolism. Sinensetin (SIN), one of the main ingredients in citrus fruits, provides a variety of pharmacological properties, like antioxidant, but its effects on osteoporosis remains unknown. Herein, we explored at how SIN affected RANKL-induced osteoclastogenesis and ovariectomy (OVX)-induced osteoporotic mice. Our research found that SIN, without compromising cell viability, inhibited RANKL-mediated osteoclastogenesis and the NFATc1 signaling pathway in a concentration-dependent manner. Further, RNA sequencing analysis suggested that the molecular mechanism of SIN inhibitory effect on osteoclasts is related to the cytoskeleton reorganization. The results indicated that SIN prevents the cytoskeleton reorganization of preosteoclasts via the c-Src-mediated PI3K/PAK4/AKT signaling axis. Meanwhile, SIN enhanced the expression of phosphorylation and activity of AMP-activated protein kinase (AMPK) in response to RANKL. Further, SIN targets AMPK to reduce intracellular Reactive oxygen species (ROS) levels, thereby blocking c-Src activation. Finally, we verified that SIN inhibits osteoclast activity, thus preventing OVX-induced bone loss. These findings suggest that SIN serves as an AMPK activator that abrogates RANKL-induced osteoclastogenesis and OVX-induced bone loss via hindering cytoskeleton reorganization.

Background: An enhanced number of osteoclasts accompanied by abnormal bone resorption activity are major contributing factors to systemic bone diseases such as osteoporosis. Directing the suppression of osteoclastogenesis is a crucial approach to combating osteoporosis. Nevertheless, the effects of Schisandrol B (SolB) from Schisandra chinensis lignan on osteoclasts and osteoporosis remain unclear.Purpose: This study aims to uncover how SolB inhibits osteoclasts and prevents estrogen deficiency-induced osteoporosis.Methods: The impacts of SolB osteoclast production, F-actin ring formation, bone resorption, intracellular ROS, and antioxidant enzymes were examined in vitro. Transcriptome analysis was used to analyze the effects of SolB on RANKL-mediated signaling pathways. Molecular docking, dynamics, and surface plasmon resonance (SPR) were used for binding confirmation of SolB and the target proteins. The OVX model was used to examine SolB's protective effect on bone mass in mice.Results: In vitro experiments demonstrated that SolB inhibited osteoclast production and bone resorption by enhancing antioxidant enzymes to reduce intracellular ROS and decrease intracellular Ca2+ and NFATc1 expression. SolB suppressed TRAF6-NF-κB and MAPK signaling pathways activation stimulated by RANKL. SPR results revealed that SolB could bind to TRAF6 and NOX1. SolB effectively protected against bone loss in OVX mice by lowering NOX1 expression and enhancing antioxidant enzyme expression.Conclusion: Our results suggest that SolB is a potentially effective drug for preventing and treating postmenopausal osteoporosis.

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.

Rationale: Osteoclasts are giant bone-resorbing cells that need vigorous mitochondrial respiration to support their activation. Rc3h1, an RNA-binding protein, precisely governs the homeostasis of mRNA. However, the precise role of Rc3h1 in regulating iron metabolism and mitochondrial respiration in osteoclasts is not yet understood. Methods: We generated Rc3h1-deficient mice in osteoclast precursors and mature osteoclasts. The bone mass and osteoclast activity in bone tissues were evaluated. Moreover, we assessed the differentiation, bone resorption, iron content, and mitochondrial function of osteoclasts in vitro. In the end, the target gene of Rc3h1 and its role in mediating the effect of Rc3h1 on mitochondrial respiration in osteoclasts were further investigated. Results: Mice lacking Rc3h1 exhibit low bone mass. In addition, Rc3h1 deletion in osteoclasts significantly promotes osteoclast activation. Mechanistically, Rc3h1 post-transcriptionally represses the expression of transferrin receptor 1 (Tfr1), restricting iron absorption and mitochondrial respiration in osteoclasts. Inhibition of Tfr1 in Rc3h1-deficient osteoclasts diminishes excessive osteoclast formation and mitochondrial respiration. Conclusion: These findings suggest that Rc3h1 has a negative effect on osteoclast activation via limiting iron resorption and mitochondrial respiration. Finally, targeting the Rc3h1/Tfr1 axis might represent a potential therapeutic approach for bone-loss diseases.

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.

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.

目的:探究常春藤皂苷元(Hed)对胶质母细胞瘤细胞系U87 MG生长增殖以及运动能力的影响.方法:将Hed分为0μmol/L组、20μmol/L组、40μmol/L组、60μmol/L组以及80μmol/L组,采用CCK-8法检测Hed处理48 h的细胞活力以确定给药浓度;平板克隆实验检测细胞生长状况;EdU染色实验检测细胞DNA复制情况;流式细胞术检测细胞周期分布;伤口愈合实验和Transwell实验检测细胞迁移和侵袭;蛋白免疫印迹实验检测细胞中Cyclin D1、CDK2、CDK4以及MMP2蛋白的表达.结果:Hed处理U87 MG细胞48 h后,40μmol/L组的U87 MG细胞活力被显著抑制(P<0.05).与0μmol/L组相比,20μmol/L组和40μmol/L组的U87 MG细胞的克隆形成率显著下降,DNA复制被显著抑制,G1期细胞所占比例显著减少,伤口愈合率显著下降,迁移和侵袭能力显著减弱(均P<0.05),且抑制作用呈剂量依赖性.Cyclin D1、CDK2、CDK4以及MMP2蛋白的相对表达量均显著减少(均P<0.05).结论:Hed可抑制U87 MG胶质母细胞瘤的生长增殖和运动能力,其机制可能与抑制细胞周期相关蛋白以及MMP2有关.

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.

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.

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.

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.

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-kappa B phosphorylation, and alleviating osteoporosis in ovariectomized (OVX) mice. Taken together, 4 -MC reduces ROS by inhibiting Keap1, thereby preventing OVX-induced bone loss.

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 anti-inflammatory and antitumor properties. However, the role of Cas in bone metabolism remains largely unclear. The present study found that the receptor activator of nuclear factor-?B (NF-?B) ligand-induced osteoclast activation and differentiation were inhibited by Cas. Tartrate-resistant 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 osteoclast-specific genes and related proteins, such as nuclear factor of activated T cells, cytoplasmic 1 and c-Fos at the mRNA and protein level in a concentration-dependent manner. Cas inhibited osteoclast formation by blocking the AKT/ERK and NF-?B 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.

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.

Wear debris after total joint arthroplasty can attract the recruitment of macrophages, which release pro-inflammatory substances, triggering the activation of osteoclasts, thereby leading to periprosthetic osteolysis (PPOL) and aseptic loosening. However, the development of pharmacological strategies targeting osteoclasts to prevent periprosthetic osteolysis has not been fruitful. In this study, we worked toward researching the effects and mechanisms of a farnesyltransferase (FTase) inhibitor Lonafarnib (Lon) on receptor activator of nuclear factor κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis and bone resorption, as well as the impacts of Lon on titanium particle-induced osteolysis. To investigate the impacts of Lon on bone resorption and osteoclastogenesis in vitro, bone marrow macrophages were incubated and stimulated with RANKL and macrophage colony-stimulating factor (M-CSF). The influence of Lon on osteolysis prevention in vivo was examined utilizing a titanium particle-induced mouse calvarial osteolysis model. The osteoclast-relevant genes expression was explored by real-time quantitative PCR. Immunofluorescence was used to detect intracellular localization of nuclear factor of activated T cells 1 (NFATc1). SiRNA silence assay was applied to examine the influence of FTase on osteoclasts activation. Related signaling pathways, including NFATc1 signaling, NF-κB, mitogen-activated protein kinases pathways were identified by western blot assay. Lon was illustrated to suppress bone resorptive function and osteoclastogenesis in vitro, and it also reduced the production of pro-inflammatory substances and prevented titanium particle-induced osteolysis in vivo. Lon decreased the expression of osteoclast-relevant genes and suppressed NFATc1 nuclear translocation and auto-amplification. Mechanistically, Lon dampened FTase, and inhibition of FTase reduced osteoclast formation by suppressing ERK signaling. Lon is a promising treatment option for osteoclast-related osteolysis diseases including periprosthetic osteolysis by targeted inhibition of FTase through suppressing ERK signaling.

Background and objective: Bone loss occurs in several inflammatory diseases because of chronic persistent inflammation that activates osteoclasts (OCs) to increase bone resorption. Currently available antiresorptive drugs have severe side effects or contraindications. Herein, we explored the effects and mechanism of Alpinetin (Alp) on receptor activator of nuclear factor kappa B ligand (RANKL)-mediated OCs differentiation, function, and in inflammatory osteolysis of mice. Method: Primary mouse bone marrow-derived macrophages (BMMs) induced by RANKL and macrophage colony-stimulating factor (M-CSF) were utilized to test the impact of Alp on OCs differentiation, function, and intracellular reactive oxygen species (ROS) production, respectively. Expression of oxidant stress relevant factors and OCs specific genes were assessed via real-time quantitative PCR. Further, oxidative stress-related factors, NF-kappa B, MAPK, PI3K/AKT/GSK3-beta, and NFATc1 pathways were examined via Western blot. Finally, LPS-induced mouse calvarial osteolysis was used to investigate the effect of Alp on inflammatory osteolysis in vivo. Result: Alp suppressed OCs differentiation and resorption function, and down-regulated the ROS production. Alp inhibited IL-1 beta, TNF-alpha and osteoclast-specific gene transcription. It also blocked the gene and protein expression of Nox1 and Keap1, but enhanced Nrf2, CAT, and HO-1 protein levels. Additionally, Alp suppressed the phosphorylation of PI3K and P38, and restrained the expression of osteoclast-specific gene Nfatc1 and its auto-amplification, hence minimizing LPS-induced osteolysis in mice. Conclusion: Alp is a novel candidate or therapeutics for the osteoclast-associated inflammatory osteolytic ailment.