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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.
Assuntos
Osteoclastos , Osteogênese , Osteoporose , Extratos Vegetais , Espécies Reativas de Oxigênio , Fator 6 Associado a Receptor de TNF , Animais , Fator 6 Associado a Receptor de TNF/metabolismo , Camundongos , Osteogênese/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Feminino , Osteoporose/tratamento farmacológico , Extratos Vegetais/farmacologia , Proteínas com Motivo Tripartido/metabolismo , Ligante RANK/metabolismo , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma/metabolismo , Ovariectomia , Reabsorção Óssea/tratamento farmacológico , Células RAW 264.7 , Diferenciação Celular/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , HumanosRESUMO
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.
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Lipopolissacarídeos , Fator 2 Relacionado a NF-E2 , Proteína 3 que Contém Domínio de Pirina da Família NLR , Osteoclastos , Osteólise , Piroptose , Espécies Reativas de Oxigênio , Animais , Masculino , Camundongos , Anti-Inflamatórios/farmacologia , Inflamassomos/metabolismo , Inflamassomos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/efeitos dos fármacos , Osteólise/induzido quimicamente , Osteólise/tratamento farmacológico , Osteólise/metabolismo , Osteólise/patologia , Piroptose/efeitos dos fármacos , Ligante RANK/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
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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.
Assuntos
Reabsorção Óssea , Ácidos Cafeicos , Osteoporose , Succinatos , Animais , Feminino , Humanos , Camundongos , Reabsorção Óssea/prevenção & controle , Diferenciação Celular , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Osteoclastos , Osteogênese , Osteoporose/patologia , Ovariectomia/efeitos adversos , Ligante RANK/metabolismoRESUMO
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.
Assuntos
Reabsorção Óssea , Sesquiterpenos , Feminino , Humanos , Osteogênese , Espécies Reativas de Oxigênio/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Osteoclastos , Sesquiterpenos/farmacologia , Sesquiterpenos/metabolismo , Reabsorção Óssea/metabolismo , Ligante RANK/farmacologia , Diferenciação Celular , NF-kappa B/metabolismoRESUMO
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.
Assuntos
Reabsorção Óssea , Osteoporose , Humanos , Feminino , Camundongos , Animais , Osteogênese , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Osteoclastos , Reabsorção Óssea/patologia , Osteoporose/tratamento farmacológico , Osteoporose/etiologia , Osteoporose/metabolismo , NF-kappa B/metabolismo , Diferenciação Celular , Ovariectomia , Ligante RANK/metabolismoRESUMO
Oxide supported noble metals are extensively investigated for ambient formaldehyde oxidation, and the Ag-CoOx complex is one promising combination in terms of cost and activity. Further, we previously observed that cooperating Ag with Li + greatly boosted formaldehyde degradation on CoOx. Yet, there is still room for improvement in removal efficiency, mineralization capacity and resistance to severe conditions. These objectives could be realized via strategically formulating the Li+ sites of Li-CoOx composite in this sister study. Three samples with Li + ---Co3+-O2- connections (L-CO), spinel Li+ (LCO-S) and layered Li+ (LCO-L) were obtained at low (300 °C), moderate (500 °C) and high (700 °C) temperatures, respectively. The specific Li+ positions and componential interaction were demonstrated by Hyperspectral imaging (HSI), XRD, SEM, TEM, HAADF mapping, UV-vis DRS and XPS. Moreover, the effect of reactive oxygen exposure on catalytic oxidation of formaldehyde (330-350 mg/m3) was disclosed through CO-TPR and O2-TPD. Compared with the LCO-S and LCO-L, L-CO exhibited dominant formaldehyde degradation due to the larger content of surface oxygen. After Ag decoration, the Li+---Co3+-O2- connections uniquely caused a strong binding of Ag species with catalyst host, which boosted the amount of reactive oxygen and finally resulted in an even higher elimination of â¼73% (CO2 yield = â¼21%), 47% higher than that of the L-CO (CO2 yield = â¼6%). But in contrast, the Ag@LCO-S only achieved â¼53% removal (CO2 yield = â¼9%) and Ag modification was powerless in altering the inertness of LCO-L, demonstrating that the chemical environment of alkali metal is crucial to effectively tuning the catalyst activity. The advantage of Ag@L-CO in formaldehyde depollution was further reflected from its much better resistance to moisture and aromatic compound omnipresent in indoor air. For the first time, this study extended the understanding of the alkali-metal-promoted formaldehyde oxidation reaction to an in-depth level.
Assuntos
Dióxido de Carbono , Formaldeído , Oxirredução , Formaldeído/química , Óxidos/química , Oxigênio/químicaRESUMO
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.
Assuntos
Doenças Ósseas Metabólicas , Reabsorção Óssea , Osteoporose , Feminino , Animais , Camundongos , Humanos , Osteogênese , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Microtomografia por Raio-X/efeitos adversos , Transdução de Sinais , Osteoclastos/metabolismo , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/etiologia , Reabsorção Óssea/prevenção & controle , Diferenciação Celular , Osteoporose/tratamento farmacológico , Osteoporose/etiologia , Osteoporose/prevenção & controle , Doenças Ósseas Metabólicas/complicações , Doenças Ósseas Metabólicas/metabolismo , Doenças Ósseas Metabólicas/patologia , Ovariectomia/efeitos adversos , Ligante RANK/metabolismoRESUMO
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.
Assuntos
NF-kappa B , Ossificação Heterotópica , Camundongos , Animais , NF-kappa B/metabolismo , Osteogênese , Sirtuína 1 , Macrófagos/metabolismo , Citocinas/farmacologiaRESUMO
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.
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Osteoporose Pós-Menopausa , Animais , Feminino , Humanos , Camundongos , Biomarcadores , Metabolômica , Osteoporose Pós-Menopausa/tratamento farmacológico , Osteoporose Pós-Menopausa/metabolismo , Fenilalanina , Triptofano , Tirosina , Espectrometria de MassasRESUMO
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.
Assuntos
Reabsorção Óssea , Osteoporose , Animais , Camundongos , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Microtomografia por Raio-X , Diferenciação Celular , Osteoclastos , Reabsorção Óssea/metabolismo , Sistema de Sinalização das MAP Quinases , Osteogênese , Osteoporose/tratamento farmacológico , Osteoporose/patologia , Estrogênios/farmacologia , Ligante RANK/metabolismoRESUMO
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.
Assuntos
Fator 2 Relacionado a NF-E2 , Osteogênese , Animais , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Simulação de Acoplamento Molecular , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Osteoclastos , NF-kappa B/metabolismo , Ligante RANK/metabolismoRESUMO
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 κB 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-κB, MAPK, PI3K/AKT/GSK3-ß, 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-1ß, TNF-α 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.
Assuntos
Conservadores da Densidade Óssea , Osteólise , Animais , Conservadores da Densidade Óssea/farmacologia , Diferenciação Celular , Flavanonas , Quinase 3 da Glicogênio Sintase/metabolismo , Inflamação/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lipopolissacarídeos/farmacologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Fator Estimulador de Colônias de Macrófagos/uso terapêutico , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Osteoclastos , Osteólise/induzido quimicamente , Osteólise/tratamento farmacológico , Oxidantes/metabolismo , Oxidantes/farmacologia , Oxidantes/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante RANK/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
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The hyperactivity of osteoclasts caused by postmenopausal estrogen deficiency plays an imperative role in the progression of osteoporosis. Although osteoporosis-related drugs have been widely used to alleviate this disorder, there is an urgent need for drugs with fewer side effects. In this study, we found that epoxymicheliolide (EMCL), a derivative of parthenolide, has a high affinity to ERK1/2, but the treatment and mechanism of osteoporosis using EMCL have not been explored. Therefore, we intended to figure out the effects and potential mechanisms of EMCL on RANKL-stimulated osteoclast formation and function in vitro, construct an OVX murine model to simulate the therapeutic effects of EMCL on estrogen-deficient bone loss subsequently. EMCL restrained the phosphorylation of ERK1/2 in the RANKL-stimulated MAPK pathway, which in sequence inhibited the transcription and expression of the main osteoclast transcription factor NFATc1, resulting in the suppression of osteoclastogenesis and bone resorption. However, the same concentration of EMCL did not affect the proliferation and differentiation of osteoblasts. In vivo experiments showed that EMCL can significantly resist osteoporosis caused by estrogen deficiency, alleviate bone loss, and reduce the number of osteoclasts. These results suggest that EMCL can reduce osteoclast production and bone resorption by inhibiting ERK1/2 phosphorylation and NFATc1 entering the nucleus, and could be used in the treatment of osteoporosis caused by estrogen deficiency and hyperactivity of osteoclasts.
Assuntos
Reabsorção Óssea , Osteoporose , Animais , Reabsorção Óssea/etiologia , Diferenciação Celular , Estrogênios/farmacologia , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos , Osteogênese , Osteoporose/metabolismo , Ovariectomia/efeitos adversos , Ligante RANK/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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.