Assuntos
Aspirina/uso terapêutico , Osteoporose Pós-Menopausa/tratamento farmacológico , Osteoprotegerina/efeitos dos fármacos , Ligante RANK/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Densidade Óssea , Citocinas/sangue , Feminino , Humanos , Osteoporose Pós-Menopausa/diagnóstico por imagem , Osteoporose Pós-Menopausa/patologia , Ratos , Ratos Sprague-Dawley , Tomografia Computadorizada por Raios XRESUMO
Twelve new terpenoids (1-12) were isolated from the stems of Fissistigma polyanthoides, an anti-inflammatory medicinal plant traditionally used in Vietnam. Most of them (1-9) possess a sesquiterpenoid backbone (e.g., guaiane, germacrane, and cadinane) connected to a 2'-O-trans-cinnamoyl)-ß-d-glucopyranose moiety, which is rare in Nature. Among them, compounds 4 (5/8-fused ring) and 6 (spiran [4,5] ring) represent uncommonly rearranged sesquiterpenoids. Compounds 10-12 are a novel monoterpene and two megastigmane derivatives, respectively. The individual structures were elucidated by combining NMR and MS data, and their configuration was established in NOESY and ECD experiments. Compounds 1-9 were also examined for their potential to interact with nuclear factor-kappa B activator protein 1 (NF-κB/AP-1) signaling by using the myelomonocytic reporter cell line THP-1Blue-CD14. Compounds 1-5 showed dose-dependent inhibitory effects [IC50 13.7 µM (1) to 49.0 µM (5)] on lipopolysaccharide-stimulated cells. However, compounds 1 to 4 also negatively affected cell viability in the same concentration range, while compound 5 was less potently cytotoxic.
Assuntos
Annonaceae/química , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Caules de Planta/química , Terpenos/química , Terpenos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Lipopolissacarídeos/farmacologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Sesquiterpenos/química , Sesquiterpenos/farmacologia , VietnãRESUMO
This aim of this study was to assess the molecular mechanism of osteoporosis in schizophrenia patients with risperidone use. Here, we investigated the effects of risperidone on cellular proliferation and apoptosis of a preosteoblast cell line, MC3T3-E1. Cell viability and apoptotic rate of MC3T3-E1 were detected by cell counting kit-8 and flow cytometry at a serial dose of risperidone and at different time points, respectively. Bone transformation relevant gene serum osteocalcin (BGP), collagen 1, tumor necrosis factor-α (TNF-α), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) mRNA levels were determined by real-time PCR (qPCR). Their protein expression patterns were evaluated using western blot. The results revealed that risperidone dramatically inhibited MC3T3-E1 cell proliferation in a dose-dependent manner. It also significantly induced MC3T3-E1 cell apoptosis. TNF-α gene and protein levels were greatly enhanced after risperidone treatment. In contrast, BGP, collagen 1, OPG, and RANKL gene and protein levels were markedly downregulated. Our study indicated that risperidone suppressed MC3T3-E1 cell proliferation and induced apoptosis. It also regulated BGP gene and protein expression.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Risperidona/farmacologia , Animais , Linhagem Celular , Colágeno/efeitos dos fármacos , Citometria de Fluxo , Osteocalcina/efeitos dos fármacos , Osteoprotegerina/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
This aim of this study was to assess the molecular mechanism of osteoporosis in schizophrenia patients with risperidone use. Here, we investigated the effects of risperidone on cellular proliferation and apoptosis of a preosteoblast cell line, MC3T3-E1. Cell viability and apoptotic rate of MC3T3-E1 were detected by cell counting kit-8 and flow cytometry at a serial dose of risperidone and at different time points, respectively. Bone transformation relevant gene serum osteocalcin (BGP), collagen 1, tumor necrosis factor-α (TNF-α), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) mRNA levels were determined by real-time PCR (qPCR). Their protein expression patterns were evaluated using western blot. The results revealed that risperidone dramatically inhibited MC3T3-E1 cell proliferation in a dose-dependent manner. It also significantly induced MC3T3-E1 cell apoptosis. TNF-α gene and protein levels were greatly enhanced after risperidone treatment. In contrast, BGP, collagen 1, OPG, and RANKL gene and protein levels were markedly downregulated. Our study indicated that risperidone suppressed MC3T3-E1 cell proliferation and induced apoptosis. It also regulated BGP gene and protein expression.
Assuntos
Animais , Osteoblastos/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Risperidona/farmacologia , Proliferação de Células/efeitos dos fármacos , Osteocalcina/efeitos dos fármacos , Linhagem Celular , Colágeno/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Osteoprotegerina/efeitos dos fármacos , Citometria de FluxoRESUMO
INTRODUCTION: To date, no study has investigated the antiresorptive property of lunasin. Hence, the present study aimed to assess the ability of lunasin to inhibit the osteoclast formation using RAW 264.7 cells. We hypothesized that lunasin is able to inhibit osteoclast formation. METHODS: In the present study, the murine monocytic cell line RAW 264.7 was induced to differentiate into mature osteoclasts in the presence of recombinant receptor activator of nuclear factor kappa-B ligand. Tartrate-resistant acid phosphatase, a marker of osteoclasts, was used to identify osteoclasts. Cell lines were divided into different groups and exposed to different concentrations of 50 µmol/L, 75 µmol/L, and 100 µmol/L active and inactive lunasin. The control group was RAW 264.7 cells with receptor activator of nuclear factor kappa-B ligand. Tartrate-resistant acid phosphatase-positive cells of 3 or more nuclei, indicative of mature osteoclasts, were counted by 3 observers. The mean number of the data collected was analyzed using 1-way analysis of variance and the multiple comparison post hoc Bonferroni correction. RESULTS: There was a significant difference in the reduction of osteoclast formation in all the active lunasin groups (P < .001) compared with the control group and the inactive lunasin group (P < .001). CONCLUSIONS: Considering the suppressive effect of lunasin on osteoclastogenesis, the use of lunasin as a potential antiresorptive agent can be evaluated in future studies.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Osteoclastos/efeitos dos fármacos , Células RAW 264.7/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Proteínas de Soja/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Camundongos , Osteoclastos/fisiologia , Células RAW 264.7/fisiologia , Receptor Ativador de Fator Nuclear kappa-B/fisiologiaRESUMO
OBJECTIVE: This study aimed to evaluate the prognostic implications of insulin-like growth factor-II mRNA binding protein-3 (IMP3) and podoplanin (PDPN) as therapeutic targets against oral squamous cell carcinoma (OSCC) with bone invasion. STUDY DESIGN: We elucidated the correlation of IMP3 and PDPN expression with bone invasion in 160 OSCC tissue specimens, and assessed a mouse calvarium xenograft model using an IMP3- and PDPN-depleted OSCC cell line. RESULTS: The retrospective analysis revealed that the expression of IMP3 and PDPN is significantly correlated with T stage, lymph node metastasis, and the overall survival of OSCC patients. In addition, the dual expression of IMP3 and PDPN but not the single expression of either IMP3 or PDPN was associated with bone invasion and the number of osteoclasts in patients with OSCC. In support of these findings, IMP3 or PDPN depletion inhibited the invasive capacity of OSCC cells in a three-dimensional culture system, tumorigenesis, and regional bone destruction in a xenograft mouse model. In addition, IMP3 or PDPN depletion inhibited the expression of interleukin (IL)-6 and IL-8 in OSCC cells, and decreased the expression of receptor activator of NF-κB ligand (RANKL) in xenograft tumor tissues of OSCC. CONCLUSIONS: These results suggest that IMP3 and PDPN may have strong influence on the pathogenesis of OSCC, especially in bone invasion, and may serve as novel therapeutic targets with prognostic implications for bone-invasive OSCC.
Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Glicoproteínas de Membrana/farmacologia , Neoplasias Bucais/metabolismo , Proteínas de Ligação a RNA/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Xenoenxertos , Humanos , Interleucina-11/biossíntese , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Metástase Linfática , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Osteoclastos/patologia , Prognóstico , Ligante RANK/biossíntese , Ligante RANK/efeitos dos fármacos , RNA Mensageiro/genética , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Receptor Ativador de Fator Nuclear kappa-B/biossíntese , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Estudos Retrospectivos , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
OBJECTIVE: To determine whether mandibular condylar cartilage degradation induced by experimentally abnormal occlusion could be ameliorated via systemic administration of strontium or NBD peptide. METHODS: Six-week-old female C57BL/6J mice were used. From the seventh day after mock operation or unilateral anterior crossbite (UAC) treatment, the control and UAC mice were further respectively pharmacologically treated for 2 weeks or 4 weeks of saline (CON + Saline and UAC + Saline groups), SrCl2 (CON + SrCl2 and UAC + SrCl2 groups) or NBD peptide (CON + NBD peptide and UAC + NBD peptide groups). Changes in condylar cartilage and subchondral bone were assessed 21 and 35 days after mock operation or UAC procedure by histology and micro-CT. Real-time PCR and/or immunohistochemistry (IHC) were performed to evaluate changes in expression levels of col2a1, aggrecan, ADAMTS-5, tnf-α, il-1ß, nfkbia, nuclear factor-kappaB phospho-p65 in condylar cartilage, and rankl/rank/opg in both condylar cartilage and subchondral bone. RESULTS: Cartilage degradation with decreased col2a1 and aggrecan expression, and increased ADAMTS-5, tnf-α/il1-ß, nfkbia and NF-κB phospho-p65 was observed in UAC + Saline groups. Subchondral bone loss with increased osteoclast numbers and decreased opg/rankl ratio was found in UAC + Saline groups compared to age-match CON + Saline groups. Cartilage degradation and subchondral bone loss were reversed by treatment of SrCl2 or NBD peptide while the same dosage in control mice induced few changes in condylar cartilage and subchondral bone. CONCLUSIONS: The results demonstrate reverse effect of systemic administration of strontium or NBD peptide on UAC-induced condylar cartilage degradation and subchondral bone loss.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Má Oclusão , Côndilo Mandibular/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Peptídeos/farmacologia , RNA Mensageiro/efeitos dos fármacos , Estrôncio/farmacologia , Proteínas ADAM/efeitos dos fármacos , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS5 , Agrecanas/efeitos dos fármacos , Agrecanas/genética , Agrecanas/metabolismo , Animais , Cartilagem Articular/metabolismo , Colágeno Tipo II/efeitos dos fármacos , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Oclusão Dentária , Feminino , Proteínas I-kappa B/efeitos dos fármacos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Imuno-Histoquímica , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Côndilo Mandibular/metabolismo , Côndilo Mandibular/patologia , Camundongos , Camundongos Endogâmicos C57BL , Inibidor de NF-kappaB alfa , Osteoclastos/metabolismo , Osteoprotegerina/efeitos dos fármacos , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Proteoglicanas/efeitos dos fármacos , Proteoglicanas/metabolismo , Ligante RANK/efeitos dos fármacos , Ligante RANK/genética , Ligante RANK/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Fator de Transcrição RelA/efeitos dos fármacos , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Advanced glycation end products (AGEs) disturb bone remodeling during aging, and this process is accelerated in diabetes. However, their role in modulation of osteoclast-induced bone resorption is controversial, with some studies indicating that AGEs enhance bone resorption and others showing the opposite effect. We determined whether AGEs present at different stages of osteoclast differentiation affect bone resorption differently. Based on increased levels of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK), we identified day 4 of induction as the dividing time of cell fusion stage and mature stage in RAW264.7 cell-derived osteoclast-like cells (OCLs). AGE-modified BSA (50-400 µg/ml) or control BSA (100 µg/ml) was then added at the beginning of each stage. Results showed that the presence of AGEs at the cell fusion stage reduced pit numbers, resorption area, and CTSK expression. Moreover, expression of receptor activator of nuclear factor-κB (RANK) as well as the number of TRAP-positive cells, nuclei per OCL, actin rings, and podosomes also decreased. However, the presence of AGEs at the mature stage enlarged the resorption area markedly and increased pit numbers slightly. Intriguingly, only the number of nuclei per OCL and podosomes increased. These data indicate that AGEs biphasically modulate bone resorption activity of OCLs in a differentiation stage-dependent manner. AGEs at the cell fusion stage reduce bone resorption dramatically, mainly via suppression of RANK expression in osteoclast precursors, whereas AGEs at the mature stage enhance bone resorption slightly, most likely by increasing the number of podosomes in mature OCLs.
Assuntos
Remodelação Óssea/efeitos dos fármacos , Reabsorção Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Produtos Finais de Glicação Avançada/farmacologia , Osteoclastos/efeitos dos fármacos , Fosfatase Ácida/metabolismo , Actinas/efeitos dos fármacos , Actinas/metabolismo , Animais , Western Blotting , Catepsina K/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Imuno-Histoquímica , Isoenzimas/metabolismo , Camundongos , Osteoclastos/citologia , Osteoclastos/metabolismo , Podossomos/efeitos dos fármacos , Podossomos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Fosfatase Ácida Resistente a TartaratoRESUMO
BACKGROUND: Portulaca oleracea (PO) has been widely used as traditional medicine because of its pharmacological activities. However, the effects of PO on osteoclasts that modulate bone homeostasis are still elusive. METHODS: In this study, we examined the effects of PO ethanol extract (POEE) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated Ca(2+) mobilization, nuclear factor of activated T-cell c1 (NFATc1) amplification, tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cell (MNC) formation, and cytotoxicity. RESULTS: Our results demonstrated that POEE suppressed RANKL-induced Ca(2+) oscillations by inhibition of Ca(2+) release from internal Ca(2+) stores, resulting in reduction of NFATc1 amplification. Notably, POEE attenuated RANKL-mediated cytotoxicity and cleavage of polyadenosine 5'-diphosphate-ribose polymerase (PARP), resulted in enhanced formation of TRAP+ MNCs. CONCLUSIONS: These results present in vitro effects of POEE on RANKL-mediated osteoclastogenesis and suggest the possible use of PO in treating bone disorders, such as osteopetrosis.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Extratos Vegetais , Portulaca/química , Receptor Ativador de Fator Nuclear kappa-B , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/toxicidade , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/metabolismoRESUMO
OBJECTIVE: Human bone marrow mesenchymal stromal cells (hBM-MSC) are being applied in tissue regeneration and treatment of autoimmune diseases (AD). Their cellular and immunophenotype depend on isolation and culture conditions which may influence their therapeutic application and reflect their in vivo biological functions. We have further characterised the phenotype induced by fibroblast growth factor 2 (FGF2) on healthy donor hBM-MSC focusing on the osteoimmunological markers osteoprotegerin (OPG), receptor activator of nuclear factor kB (RANK), RANK ligand (RANKL) and HLA-DR and their regulation of expression by the inflammatory cytokines IL1ß and IFNγ. METHODS: RANK, RANKL, OPG and HLA-DR expression in hBM-MSC expanded under specific culture conditions, were measured by RT-PCR and flow cytometry. MAPKs induction by FGF2, IL1ß and IFNγ in hBM-MSC was analysed by immunoblotting and RT-PCR. RESULTS: In hBM-MSC, OPG expression is constitutive and FGF2 independent. RANKL expression depends on FGF2 and ERK1/2 activation. IL1ß and IFNγ activate ERK1/2 but fail to induce RANKL. Only IL1ß induces P38MAPK. The previously described HLA-DR induced by FGF2 through ERK1/2 on hBM-MSC, is suppressed by IL1ß through inhibition of CIITA transcription. HLA-DR induced by IFNγ is not affected by IL1ß in hBM-MSC, but is suppressed in articular chondrocytes and lung fibroblasts. CONCLUSIONS: RANKL expression and IL1ß regulated MHC-class II, both induced via activation of the ERK1/2 signalling pathway, are specific for progenitor hBM-MSC expanded in the presence of FGF2. HLA-DR regulated by IL1ß and ERK1/2 is observed on hBM-MSC during early expansion without FGF2 suggesting previous in vivo acquisition. Stromal progenitor cells with this phenotype could have an osteoimmunological role during bone regeneration.
Assuntos
Células da Medula Óssea/metabolismo , Fator 2 de Crescimento de Fibroblastos/imunologia , Antígenos HLA-DR/genética , Interferon gama/imunologia , Interleucina-1beta/imunologia , Células-Tronco Mesenquimais/metabolismo , Osteoprotegerina/genética , Ligante RANK/genética , Receptor Ativador de Fator Nuclear kappa-B/genética , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Antígenos HLA-DR/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Interferon gama/farmacologia , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Osteoprotegerina/efeitos dos fármacos , Osteoprotegerina/metabolismo , Ligante RANK/efeitos dos fármacos , Ligante RANK/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
There is a well recognized link between the bone and the immune system and in recent years there has been a major effort to elucidate the multiple functions of the molecules expressed in both bone and immune cells. Several molecules that were initially identified and studied in the immune system have been shown to have essential functions also in the bone. An interdisciplinary field embracing immune and bone biology has been brought together and called "osteoimmunology". The co-regulation of the skeletal and immune systems strikingly exemplifies the extreme complexity of such an interaction. Their interdependency must be considered in designing therapeutic approaches for either of the two systems. In other words, it is necessary to think of the osteoimmune system as a complex physiological unit. Denosumab was originally introduced to specifically target bone resorption, but it is now under evaluation for its effect on the long term immune response. Similarly, our current and still growing knowledge of the intimate link between the immune system and bone will be beneficial for the safety of drugs targeting either of these integrated systems. Given the large number of molecules exerting functions on both the skeletal and immune systems, osteoimmunological understanding is becoming increasingly important. Both bone and immune systems are frequently disrupted in cancer; and they may be crucial in regulating tumor growth and progression. Some therapies - such as bisphosphonates and receptor activator of NF-κB ligand (RANKL) targeted drugs - that aim at reducing pathologic osteolysis in cancer may interact with the immune system, thus providing potential favorable effects on survival.
Assuntos
Antineoplásicos/farmacologia , Conservadores da Densidade Óssea/farmacologia , Neoplasias Ósseas/imunologia , Remodelação Óssea/imunologia , Reabsorção Óssea/imunologia , Osso e Ossos/metabolismo , Difosfonatos/farmacologia , Osteoclastos/efeitos dos fármacos , Ligante RANK/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/uso terapêutico , Doenças Ósseas/imunologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Osso e Ossos/imunologia , Ácido Clodrônico/farmacologia , Denosumab , Difosfonatos/uso terapêutico , Humanos , Ácido Ibandrônico , Imidazóis/farmacologia , Sistema Imunitário , Osteoclastos/imunologia , Osteoprotegerina/efeitos dos fármacos , Osteoprotegerina/metabolismo , Pamidronato , Ligante RANK/antagonistas & inibidores , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Ácido ZoledrônicoRESUMO
The creation of the eruption pathway requires the resorption of the occlusal alveolar bone by osteoclasts and signaling events between bone and dental follicle are necessary. The aim of the present study has been to evaluate the effect of alendronate on osteoclastogenesis and the expression of the regulator proteins of osteoclast activation, namely RANK, RANKL and OPG, in the bone that covers the first molar germ. Newborn Wistar rats were treated daily with 2.5 mg/kg alendronate for 4, 8, 14, 21 and 28 days, whereas controls received sterile saline solution. At the time points cited, maxillae were fixed, decalcified and processed for light and electron microscopic analysis. TRAP histochemistry was performed on semi-serial sections and the osteoclasts in the occlusal half of the bony crypt surface were counted. TUNEL analysis was carried out on paraffin sections. The occlusal bone that covers the upper first molar was removed in additional 4- and 8-day-old alendronate-treated and control rats in which the expression of RANK, RANKL and OPG was analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. TRAP-positive osteoclasts were more numerous in the alendronate group at all time points, despite their unactivated phenotype and the presence of apoptotic cells. RANKL expression in the alendronate specimens was inhibited at all time points, unlike in controls. Our findings indicate that the expression of RANKL in the occlusal portion of the bony crypt is unrelated to osteoclast recruitment and differentiation but is crucial to their activation during the creation of the eruption pathway.
Assuntos
Alendronato/farmacologia , Conservadores da Densidade Óssea/farmacologia , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Erupção Dentária/efeitos dos fármacos , Fosfatase Ácida/metabolismo , Animais , Reabsorção Óssea/metabolismo , Diferenciação Celular , Saco Dentário/citologia , Saco Dentário/metabolismo , Isoenzimas/metabolismo , Osteoprotegerina/biossíntese , Osteoprotegerina/efeitos dos fármacos , Osteoprotegerina/metabolismo , Ligante RANK/biossíntese , Ligante RANK/efeitos dos fármacos , Ratos , Ratos Wistar , Receptor Ativador de Fator Nuclear kappa-B/biossíntese , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Fosfatase Ácida Resistente a TartaratoRESUMO
BACKGROUND AND OBJECTIVE: The interleukin (IL)-1 receptor antagonist (Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is unclear whether the IL-1Ra plays a protective role in periodontal disease. The purpose of this study was to compare IL-1Ra knockout (KO) and wild-type (WT) mice in regard to proinflammatory cytokine production, osteoclast formation and bone resorption in response to periodontal bacterial lipopolysaccharide (LPS). MATERIAL AND METHODS: Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10 µg/mL of Aggregatibacter actinomycetemcomitans LPS for 24 h. The levels of IL-1alpha (IL-1α), IL-1beta (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-6 were measured in periotoneal Mφs supernatant fluid (PM-SF) using an ELISA. Bone marrow cells were obtained from the mice and stimulated with PM-SF for 9 d, then stained with TRAP. The frequency of TRAP-positive multinucleated giant cell formation was calculated based on a fusion index. PM-SF-stimulated calvarial bone resorption was analyzed using micro-computed tomography, and calvarial histological analysis was performed using hematoxylin and eosin and TRAP staining. The expression of cyclooxygenase-2 (Cox2), prostanoid receptor EP4 (Ep4) and Rank mRNAs in bone marrow cells were measured using real-time quantitative PCR, while prostaglandin E2 (PGE2 ) production was determined by ELISA. RESULTS: The levels of IL-1α, IL-1ß, TNF-α and IL-6 in IL-1Ra KO mice PM-SF stimulated with A. actinomycetemcomitans LPS were significantly increased by approximately 4- (p < 0.05), 5- (p < 0.05), 1.3- (p < 0.05) and 6- (p < 0.05) fold, respectively, compared with the levels in WT mice. Moreover, osteoclast formation, expression of Rank, Ep4 and Cox2 mRNAs and production of PGE2 were significantly increased by approximately 2- (p < 0.05), 1.6- (p < 0.05), 2.5- (p < 0.05), 1.6- (p < 0.05) and 1.9- (p < 0.05) fold, respectively, in IL-1Ra KO mice stimulated with A. actinomycetemcomitans LPS compared with WT mice. CONCLUSION: IL-1Ra regulates IL-1 activity and appears to reduce the levels of other inflammatory cytokines, including TNF-α and IL-6, while it also reduces expression of the EP4 receptor related to prostanoid sensitivity and osteoclast formation. These results suggest that IL-1Ra is an important molecule for inhibition of inflammatory periodontal bone resorption.
Assuntos
Aggregatibacter actinomycetemcomitans/fisiologia , Citocinas/efeitos dos fármacos , Dinoprostona/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Lipopolissacarídeos/farmacologia , Osteoclastos/efeitos dos fármacos , Regulação para Cima , Fosfatase Ácida/análise , Animais , Células da Medula Óssea/efeitos dos fármacos , Reabsorção Óssea/imunologia , Técnicas de Cultura de Células , Ciclo-Oxigenase 2/efeitos dos fármacos , Células Gigantes/efeitos dos fármacos , Proteína Antagonista do Receptor de Interleucina 1/genética , Interleucina-1alfa/análise , Interleucina-1beta/efeitos dos fármacos , Interleucina-6/análise , Isoenzimas/análise , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Camundongos Knockout , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP4/efeitos dos fármacos , Crânio/imunologia , Fosfatase Ácida Resistente a Tartarato , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
BACKGROUND: Simvastatin is a cholesterol-lowering drug whose pleiotropic effects may have a therapeutic impact on bone. This study evaluates the effect of simvastatin on rats subjected to experimental periodontal disease. METHODS: Periodontitis was induced by ligature placement around the maxillary left second molar of rats for 11 days. Groups of six animals received oral saline or simvastatin (3, 10, and 30 mg/kg/day) until sacrifice on day 11. Alveolar bone loss was determined by macroscopic and histologic examination. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total alkaline phosphatase (TAP) were evaluated. Gingival myeloperoxidase activity and gingival levels of interleukin-1ß (IL-1ß), tumor necrosis factor-α, IL-10, reduced glutathione, malonaldehyde, and nitrate/nitrite were analyzed to investigate oxidative stress and inflammation. Expression of inducible nitric oxide synthase (iNOS), matrix metalloproteinases 1 and 8 (MMP-1 and -8), bone morphogenetic protein-2 (BMP-2), receptor activator of nuclear factor κB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) were also investigated by immunohistochemistry to assess bone turnover and metabolism. Immunofluorescence microscopy was used to confirm the expression of RANKL in rats' maxillae. RESULTS: Treatment with simvastatin improved alveolar bone loss within all of the parameters studied, thus demonstrating anti-inflammatory and antioxidant activity. Simvastatin reduced expression of iNOS, MMP-1 and -8, RANK, and RANKL and increased BMP-2 and OPG levels in the periodontal tissue. Simvastatin (30 mg/kg) increased TAP activity on day 11 compared with the saline group. No differences were found in the levels of AST and ALT in any of the groups studied. CONCLUSION: The present data suggest that simvastatin prevents inflammatory bone resorption in experimental periodontitis, which may be mediated by its anti-inflammatory and antioxidant properties.
Assuntos
Anti-Inflamatórios/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Periodontite/prevenção & controle , Sinvastatina/uso terapêutico , Alanina Transaminase/efeitos dos fármacos , Fosfatase Alcalina/efeitos dos fármacos , Perda do Osso Alveolar/prevenção & controle , Animais , Antioxidantes/uso terapêutico , Aspartato Aminotransferases/efeitos dos fármacos , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Feminino , Gengiva/efeitos dos fármacos , Glutationa/efeitos dos fármacos , Interleucina-10/análise , Interleucina-1beta/efeitos dos fármacos , Malondialdeído/análise , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Metaloproteinase 8 da Matriz/efeitos dos fármacos , Nitratos/análise , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Nitritos/análise , Osteoprotegerina/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/efeitos dos fármacos , Ligante RANK/efeitos dos fármacos , Ratos , Ratos Wistar , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
INTRODUCTION: Porphyromonas endodontalis lipopolysaccharide (LPS) has been shown to have a high positive rate in infected root canals and symptomatic apical periodontitis. It may play an integral role as a potent stimulator of inflammatory cytokines involved in apical lesions. The receptor activator of nuclear factor-κB ligand (RANKL) has been proven to be the key regulator of bone remodeling. This study investigated P. endodontalis LPS-induced RANKL production and LPS signaling in mouse osteoblasts. METHODS: LPS-induced RANKL production in mouse osteoblast MC3T3-E1 cells was measured by Western blot and real-time polymerase chain reaction, and the Toll-like receptors (TLRs) were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. RESULTS: Both of the anti-TLR2 and anti-TLR4 antibodies significantly (P < .05) inhibited the expression of RANKL from osteoblasts stimulated with P. endodontalis LPS; only anti-TLR2 antibody had a significant (P < .05) inhibitory effect on E. coli LPS signaling. SP600125 (c-Jun N-terminal kinase [JNK] inhibitor) prevented the up-regulation of RANKL expression in P. endodontalis LPS-infected osteoblasts (P < .05). The inhibitory effect of wortmannin (phosphatidylinositol 3-kinase inhibitor) and PD98059 (mitogen-activated protein kinase [MAPK]/extracellular signal-regulated kinase [ERK] kinase-1/2 [MEK 1/2] inhibitor) were observed in E. coli LPS-treated mouse osteoblasts (P < .05). CONCLUSIONS: Results from this study showed that P. endodontalis LPS has the ability to promote the expression of RANKL in mouse osteoblasts, and this induction was mainly through the TLR2/4-JNK signaling pathway, a situation quite different from that of typical bacterial endotoxin (E. coli LPS).
Assuntos
Escherichia coli/fisiologia , Lipopolissacarídeos/farmacologia , Osteoblastos/efeitos dos fármacos , Porphyromonas endodontalis/fisiologia , Ligante RANK/efeitos dos fármacos , Células 3T3/efeitos dos fármacos , Androstadienos/farmacologia , Animais , Antracenos/farmacologia , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Camundongos , Osteoprotegerina/análise , Inibidores de Fosfoinositídeo-3 Quinase , Reação em Cadeia da Polimerase em Tempo Real , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Receptor 2 Toll-Like , Receptor 4 Toll-Like , WortmaninaRESUMO
Osteoarthritis (OA) is characterized by qualitative and quantitative changes in the architecture and composition of all the joint structures. Glucosamine (GlcN) has been used to treat OA in humans, because GlcN is present in the cartilage tissues as a component of glycosaminoglycans, and exhibits the symptom-modifying effect on OA by normalizing cartilage metabolism. On the other hand, the pathological change of subchondral bone is associated with the initiation and progression of cartilage damage in OA. However, the effect of GlcN on bone metabolism remains unsolved. In the present study, we determined the effect of GlcN on bone metabolism (osteoblastic cell differentiation) using mouse osteoblastic MC3T3-E1 cells by evaluating the expression of early (type I collagen and alkaline phosphatase), middle (osteopontin) and late (osteocalcin and mineralization) stage differentiation markers, and further compared its effects to those of N-acetyl-D-glucosamine (GlcNAc), a derivative of GlcN. The results indicated that the mineralization of mature osteoblasts was increased by treatment with GlcN and GlcNAc. Furthermore, reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that GlcN and GlcNAc substantially increased the expression of a middle stage marker and a late stage marker, although they did not essentially affect the expression of early stage markers. In addition, GlcN and GlcNAc suppressed the expression of receptor activator of NF-κB ligand (RANKL), a key factor involved in the osteoclastic cell differentiation and activation. Together these observations suggest that both GlcN and GlcNAc may have a potential not only to induce osteoblastic cell differentiation especially at middle-late stages, but also to suppress the osteoclastic cell differentiation, thereby possibly increasing bone matrix deposition and decreasing bone resorption, and eventually modulating bone metabolism in OA.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Glucosamina/farmacologia , Osteoartrite/tratamento farmacológico , Osteoblastos/efeitos dos fármacos , Acetilglucosamina/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Colágeno Tipo I/metabolismo , Glicosaminoglicanos/metabolismo , Camundongos , Osteoartrite/patologia , Osteoblastos/citologia , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/antagonistas & inibidores , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND AND PURPOSE: Homocysteine has been linked to increased risk of ischemic stroke and other cardiovascular events. Matrix degradation and inflammation play an important role in these disorders, and we have demonstrated increased levels of matrix-degrading enzymes and inflammatory cytokines in hyperhomocysteinemic individuals. Recent studies suggest that RANK ligand (RANKL) through interaction with its receptor RANK can modulate matrix degradation and inflammation. The present study aimed to examine the role of the RANKL/RANK axis in hyperhomocystinemia. METHODS: RANKL/RANK was measured on protein or mRNA level before and after B-vitamin supplementation in hyperhomocysteinemic individuals. We also examined the in vitro effects of soluble RANKL in peripheral blood mononuclear cells from hyperhomocysteinemic individuals. RESULTS: Our main findings were: (1) compared to peripheral blood mononuclear cells from controls, cells from hyperhomocysteinemic individuals had significantly higher gene expression of RANKL and RANK; (2) folic acid treatment for 6 weeks in an open, uncontrolled study significantly reduced gene expression of RANKL/RANK in peripheral blood mononuclear cells from these individuals; (3) compared to placebo, treatment with folic acid, vitamin B(12), and vitamin B(6) for 3 months in a randomized, double-blind trial significantly lowered serum levels of soluble RANKL in hyperhomocysteinemic individuals; and (4) in vitro, soluble RANKL markedly increased the release of matrix metalloproteinase-9 and inflammatory cytokines from peripheral blood mononuclear cells in hyperhomocysteinemic subjects. CONCLUSIONS: Our findings suggest a dysregulated RANKL/RANK axis in hyperhomocysteinemic subjects. Based on their role in atherogenesis, this enhanced expression of RANKL and RANK could contribute to the increased risk of cardiovascular disease in hyperhomocystinemia. Moreover, treatment with B-vitamins may have beneficial implications for plaque stability in these individuals.
Assuntos
Arterite/sangue , Matriz Extracelular/efeitos dos fármacos , Hiper-Homocisteinemia/tratamento farmacológico , Ligante RANK/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Complexo Vitamínico B/farmacologia , Adulto , Arterite/etiologia , Arterite/fisiopatologia , Células Cultivadas , Citocinas/metabolismo , Método Duplo-Cego , Matriz Extracelular/metabolismo , Feminino , Ácido Fólico/farmacologia , Humanos , Hiper-Homocisteinemia/complicações , Hiper-Homocisteinemia/fisiopatologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Placebos , Ligante RANK/metabolismo , Ligante RANK/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Vitamina B 12/farmacologia , Vitamina B 6/farmacologiaAssuntos
Anticorpos Monoclonais/uso terapêutico , Inibidores da Aromatase/efeitos adversos , Neoplasias da Mama/tratamento farmacológico , Osteoporose/induzido quimicamente , Osteoporose/prevenção & controle , Ligante RANK/uso terapêutico , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Adulto , Anticorpos Monoclonais Humanizados , Antineoplásicos Hormonais/efeitos adversos , Densidade Óssea/efeitos dos fármacos , Denosumab , Feminino , HumanosRESUMO
BACKGROUND AND AIM: NFkappaB plays a major role in the immune and inflammation responses of pancreatitis. Recently, there is increasing evidence that the expression and activity of PPARgamma may participate in the activity of NFkappaB. Therefore, we investigated a putative relationship of the two transcription factors in cerulein-treated pancreatic acinar AR42J cells. METHOD: AR42J were stimulated by cerulein with or without the presence of a PPARgamma activator pioglitazone or a PPARgamma antagonist GW9662. RESULTS: Treatment of AR42J cells with pioglitazone attenuated cerulein induced p50 and p65 NFkappaB dimer activity in the nucleus as measured by transcription factor assay. Cytosolic expression of IkappaBalpha protein was reduced by cerulein, basal signalling was not influenced by the PPARgamma inhibitor GW9662 and pioglitazone. Adversely, the inhibitory effect of pioglitazone on NFkappaB activity induced by cerulein was almost reversed by GW9662. CONCLUSION: These findings provide evidence for the involvement of the nuclear hormone receptors PPARgamma in the activity of NFkappaB in cerulein-treated AR42J cells.
Assuntos
Ceruletídeo/farmacologia , Hipoglicemiantes/farmacologia , Quinase I-kappa B/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , PPAR gama/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Animais , Western Blotting , Linhagem Celular , Citoplasma/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Imunoprecipitação , Ligantes , Subunidade p50 de NF-kappa B/biossíntese , Subunidade p50 de NF-kappa B/genética , Proteínas Nucleares/biossíntese , Fosforilação , Pioglitazona , Ratos , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Fator de Transcrição RelA/biossíntese , Fator de Transcrição RelA/genéticaRESUMO
Several epidemiological studies have reported that temporomandibular disorder is more prevalent in women, which suggests the involvement of sex hormones, such as estrogen, in the pathogenesis of this disease. PCR amplification and Western blotting were employed to target the expression of estrogen receptors (ERs) in human fibroblast-like synovial and ATDC5 cells. The effect of estrogen was investigated through the expression of RANKL, osteoprotegerin (OPG), M-CSF/CSF-1 and c-fms. We showed expression of M-CSF/ CSF-1 and c-fms, with time-dependent increase in both after the addition of estrogen. Based on previous studies reporting that M-CSF/CSF-1 regulates the proliferation and differentiation of hemopoietic progenitor cells into mature macrophages, we put forward a new hypothesis based on the increased inflammation and tendency of females to suffer more from temporomandibular disorder (TMD) in the presence of external exacerbating factors. Detection of RANKL and OPG in ATDC5 and expression of both in HFLS was confirmed with complete disappearance of the RANKL band, and marked increase in the expression of OPG after 1 h from the addition of estrogen.