RESUMO
Asiatic acid is a triterpenoid compound extracted from a medicinal plant Centella asiatica. It has been used as a highly efficient compound for the treatment of cancer and hyperlipidemia, as well as possessing potential antiinflammatory properties. However, its effects on bone metabolism and osteoporosis haven't been reported. The purpose of our research were to reveal the biomolecular effects of asiatic acid on osteoclasts, and its underlying molecular mechanisms regulating its effects on receptor activator of NF-κB ligand (RANKL)-induced signaling pathways. We found that asiatic acid inhibited multinucleated tartrate-resistant acid phosphatase (TRAcP)-positive osteoclast differentiation and osteoclast induced bone loss. Real time PCR showed that asiatic acid reduced the expression of down-cascade target genes including Ctsk, Nfatc1, Calcr, and Atp6v0d2. Western blot and luciferase reporter gene assays revealed that asiatic acid inhibits RANKL mediated NF-κB and NFATc1 signalings. Further, in vivo study demonstrated asiatic acid attenuates estrogen deficiency-induced bone loss in ovariectomized mice. MicroCT and histology analyses revealed that osteoclast numbers were significantly suppressed in asiatic acid treated groups. Furthermore, serum levels of TRAcP and CTX-1 were downregulated in treated groups. Taken together, our data show that asiatic acid can inhibit osteoclastic formation and reduce OVX-induced bone resorption through RANKL-activated NF-κB or NFATc1 signaling, suggesting that asiatic acid may be a potential and effective natural compound for the therapy of excessive RANKL-related osteolytic diseases.
RESUMO
Osteoporosis is the most common osteolytic disease characterized by excessive osteoclast formation and resultant bone loss, which afflicts millions of patients around the world. Astilbin, a traditional herb, is known to have anti-inflammatory, antioxidant and antihepatic properties, but its role in osteoporosis treatment has not yet been confirmed. In our study, astilbin was found to have an inhibitory effect on the RANKL-induced formation and function of OCs in a dose-dependent manner without cytotoxicity. These effects were attributed to its ability to suppress the activity of two transcription factors (NFATc1 and c-Fos) indispensable for osteoclast formation, followed by inhibition of the expression of bone resorption-related genes and proteins (Acp5/TRAcP, CTSK, V-ATPase-d2 and integrin ß3). Furthermore, we examined the underlying mechanisms and found that astilbin repressed osteoclastogenesis by blocking Ca2+ oscillations and the NF-κB and MAPK pathways. In addition, the therapeutic effect of MA on preventing bone loss in vivo was further confirmed in an ovariectomized mouse model. Therefore, considering its ability to inhibit RANKL-mediated osteoclastogenesis and the underlying mechanisms, astilbin might be a potential candidate for treating osteolytic bone diseases.
Assuntos
Reabsorção Óssea/prevenção & controle , Flavonóis/farmacologia , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Animais , Células Cultivadas , Medicamentos de Ervas Chinesas/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Integrina beta3/genética , Integrina beta3/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Ovariectomia , Fitoterapia/métodos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7 , Fosfatase Ácida Resistente a Tartarato/genética , Fosfatase Ácida Resistente a Tartarato/metabolismoRESUMO
Identification of natural compounds that inhibit osteoclastogenesis will facilitate the development of antiresorptive treatment of osteolytic bone diseases. Asiaticoside is a triterpenoid derivative isolated from Centella asiatica, which exhibits varying biological effects like angiogenesis, anti-inflammation, wound healing, and osteogenic differentiation. However, its role in osteoclastogenesis remains unknown. Here, we show that Asiaticoside can suppress RANKL-induced osteoclast formation and bone resorption in a dose-dependent manner. Asiaticoside attenuated the expression of osteoclast marker genes including Ctsk, Atp6v0d2, Nfatc1, Acp5, and Dc-stamp. Furthermore, Asiaticoside inhibited RANKL-mediated NF-κB and NFATc1 activities, and RANKL-induced calcium oscillation. Collectively, this study demonstrates that Asiaticoside inhibited osteoclast formation and function through attenuating RANKL-induced key signaling pathways, which may indicate that Asiaticoside is a potential antiresorptive agent against osteoclast-related osteolytic bone diseases.
Assuntos
Centella/química , Macrófagos/efeitos dos fármacos , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Triterpenos/farmacologia , Animais , Sinalização do Cálcio , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/genética , Osteoclastos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7 , Triterpenos/isolamento & purificaçãoRESUMO
Osteoporosis is the most common osteolytic disease characterized by excessive osteoclast formation and resultant bone loss, which afflicts millions of patients around the world. Madecassoside (MA), isolated from Centella asiatica, was reported to have anti-inflammatory and antioxidant activities, but its role in osteoporosis treatment has not yet been confirmed. In our study, MA was found to have an inhibitory effect on the RANKL-induced formation and function of OCs in a dose-dependent manner without cytotoxicity. These effects were attributed to its ability to suppress the activity of two transcription factors (NFATc1 and c-Fos) indispensable for osteoclast formation, followed by inhibition of the expression of bone resorption-related genes and proteins (Acp5/TRAcP, CTSK, ATP6V0D2/V-ATPase-d2, and integrin ß3). Furthermore, we examined the underlying mechanisms and found that MA represses osteoclastogenesis by blocking Ca2+ oscillations and the NF-κB and MAPK pathways. In addition, the therapeutic effect of MA on preventing bone loss in vivo was further confirmed in an ovariectomized mouse model. Therefore, considering its ability to inhibit RANKL-mediated osteoclastogenesis and the underlying mechanisms, MA might be a potential candidate for treating osteolytic bone diseases.
Assuntos
Estrogênios/metabolismo , Osteogênese/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Ligante RANK/metabolismo , Triterpenos/farmacologia , Animais , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Linhagem Celular , Centella , Feminino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoporose/metabolismo , Extratos Vegetais , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Fosfatase Ácida Resistente a Tartarato/metabolismoRESUMO
Osteoclasts are responsible for bone resorption during the process of bone remodeling. Increased osteoclast numbers and bone resorption activity are the main factors contributing to bone loss-related diseases such as osteoporosis. Therefore, modulating the formation and function of osteoclasts is critical for the effective treatment of osteolysis and osteoporosis. Kavain is the active ingredient extracted from the root of the kava plant, which possesses known anti-inflammatory properties. However, the effects of kavain on osteoclastogenesis and bone resorption remain unclear. In this study, we found that kavain inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and fusion using tartrate-resistant acid phosphatase staining and immunofluorescence. Furthermore, kavain inhibited bone resorption performed by osteoclasts. Using reverse transcription-polymerase chain reaction and western blot analysis, we found that kavain downregulates the expression of osteoclast marker genes, such as nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1), v-atpase d2 (Atp6v0d2), dendrocyte expressed seven transmembrane protein (Dcstamp), matrix metallopeptidase 9 (Mmp9), cathepsin K (Ctsk), and Acp5. Additionally, kavain repressed RANKL-induced calcium oscillations, nuclear factor of activated T cells activation, and mitogen-activated protein kinase phosphorylation, while leaving NF-κB unaffected. We found no effects of kavain on either osteoblast proliferation or differentiation. Besides, kavain inhibited bone loss in ovariectomized mice by suppressing osteoclastogenesis. Collectively, these data suggest a potential use for kavain as a candidate drug for the treatment of osteolytic diseases.
Assuntos
Reabsorção Óssea/tratamento farmacológico , Proteína Quinase 1 Ativada por Mitógeno/genética , Fatores de Transcrição NFATC/genética , Osteogênese/efeitos dos fármacos , Pironas/farmacologia , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Sinalização do Cálcio/efeitos dos fármacos , Catepsina K/genética , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Kava/química , Metaloproteinase 9 da Matriz/genética , Camundongos , NF-kappa B/química , NF-kappa B/genética , Osteogênese/genética , Osteoporose , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Pironas/química , Ligante RANK/genética , Células RAW 264.7 , Fosfatase Ácida Resistente a Tartarato/genética , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Choline, a hydrophilic cation, has versatile physiological roles throughout the body, including cholinergic neurotransmission, memory consolidation and membrane biosynthesis and metabolism. Choline kinases possess enzyme activity that catalyses the conversion of choline to phosphocholine, which is further converted to cytidine diphosphate-coline (CDP-choline) in the biosynthesis of phosphatidylcholine (PC). PC is a major constituent of the phospholipid bilayer which constitutes the eukaryotic cell membrane, and regulates cell signal transduction. Choline Kinase consists of three isoforms, CHKα1, CHKα2 and CHKß, encoded by two separate genes (CHKA(Human)/Chka(Mouse) and CHKB(Human)/Chkb(Mouse)). Both isoforms have similar structures and enzyme activity, but display some distinct molecular structural domains and differential tissue expression patterns. Whilst Choline Kinase was discovered in early 1950, its pivotal role in the development of muscular dystrophy, bone deformities, and cancer has only recently been identified. CHKα has been proposed as a cancer biomarker and its inhibition as an anti-cancer therapy. In contrast, restoration of CHKß deficiency through CDP-choline supplements like citicoline may be beneficial for the treatment of muscular dystrophy, bone metabolic diseases, and cognitive conditions. The molecular structure and expression pattern of Choline Kinase, the differential roles of Choline Kinase isoforms and their potential as novel therapeutic targets for muscular dystrophy, bone deformities, cognitive conditions and cancer are discussed.
Assuntos
Colina Quinase/química , Colina Quinase/metabolismo , Sistema Musculoesquelético/enzimologia , Neoplasias/enzimologia , Animais , Biomarcadores Tumorais , Colina Quinase/deficiência , Colina Quinase/genética , Humanos , Doenças Metabólicas/tratamento farmacológico , Camundongos , Estrutura Molecular , Distrofias Musculares/tratamento farmacológico , Sistema Musculoesquelético/fisiopatologia , Neoplasias/fisiopatologiaRESUMO
Receptor activator of nuclear factor kappa-B ligand (RANKL) induces differentiation and function of osteoclasts through triggering multiple signaling cascades, including NF-κB, MAPK, and Ca(2+) -dependent signals, which induce and activate critical transcription factor NFATc1. Targeting these signaling cascades may serve as an effective therapy against osteoclast-related diseases. Here, by screening a panel of natural plant extracts with known anti-inflammatory, anti-tumor, or anti-oxidant properties for possible anti-osteoclastogenic activities we identified Eriodictyol. This flavanone potently suppressed RANKL-induced osteoclastogenesis and bone resorption in a dose-dependent manner without detectable cytotoxicity, suppressing RANKL-induced NF-κB, MAPK, and Ca(2+) signaling pathways. Eriodictyol also strongly inhibited RANKL-induction of c-Fos levels (a critical component of AP-1 transcription factor required by osteoclasts) and subsequent activation of NFATc1, concomitant with reduced expression of osteoclast specific genes including cathepsin K (Ctsk), V-ATPase-d2 subunit, and tartrate resistant acid phosphatase (TRAcP/Acp5). Taken together, these data provide evidence that Eriodictyol could be useful for the prevention and treatment of osteolytic disorders associated with abnormally increased osteoclast formation and function. J. Cell. Physiol. 231: 1983-1993, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Flavanonas/farmacologia , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Animais , Células da Medula Óssea/citologia , Reabsorção Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Flavanonas/metabolismo , Camundongos , NF-kappa B/metabolismo , Osteoclastos/citologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The RANKL-induced NF-κB signaling pathway is required for osteoclast formation and function. By screening for compounds that inhibit RANKL-induced NF-κB activation using a luciferase reporter gene assay in RAW264.7 cells, we identified triptolide (PG490), as a candidate compound targeting osteoclast differentiation and osteoclast-mediated osteolysis. Triptolide (PG490) is an active compound of the medicinal herb Tripterygium wilfordii Hook F (TWHF) or Lei Gong Teng with known anti-inflammatory properties. We found that triptolide inhibited osteoclastogenesis and bone resorption, as well as RANKL-induced NF-ÒB activities as monitored by luciferase reporter gene assays and the nuclear translocation of p65. In vivo studies showed that triptolide attenuates titanium-induced osteolysis and osteoclast formation in a mouse calvarial model. Considering that drugs which protect against localized bone loss are critically needed for the effective treatment of particle-induced osteolysis, our data suggest that triptolide might have therapeutic potential for the treatment of bone lytic diseases caused by prosthetic wear particles.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Diterpenos/farmacologia , Osteoclastos/metabolismo , Osteólise/tratamento farmacológico , Fenantrenos/farmacologia , Ligante RANK/metabolismo , Titânio/toxicidade , Fator de Transcrição RelA/metabolismo , Animais , Linhagem Celular , Compostos de Epóxi/farmacologia , Camundongos , Osteoclastos/patologia , Osteólise/induzido quimicamente , Osteólise/metabolismo , Osteólise/patologiaRESUMO
The maintenance of bone homeostasis requires tight coupling between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the precise molecular mechanism(s) underlying the differentiation and activities of these specialized cells are still largely unknown. Here, we identify choline kinase ß (CHKB), a kinase involved in the biosynthesis of phosphatidylcholine, as a novel regulator of bone homeostasis. Choline kinase ß mutant mice (flp/flp) exhibit a systemic low bone mass phenotype. Consistently, osteoclast numbers and activity are elevated in flp/flp mice. Interestingly, osteoclasts derived from flp/flp mice exhibit reduced sensitivity to excessive levels of extracellular calcium, which could account for the increased bone resorption. Conversely, supplementation of cytidine 5'-diphosphocholine in vivo and in vitro, a regimen that bypasses CHKB deficiency, restores osteoclast numbers to physiological levels. Finally, we demonstrate that, in addition to modulating osteoclast formation and function, loss of CHKB corresponds with a reduction in bone formation by osteoblasts. Taken together, these data posit CHKB as a new modulator of bone homeostasis.