RESUMO
Osteoporosis, a disease defined by the primary bone strength due to a low bone mineral density, is a bone disorder associated with increased mortality in the older adult population. Osteoporosis is mainly treated via hormone replacement therapy, bisphosphates, and anti-bone resorption agents. However, these agents exert severe side effects, necessitating the development of novel therapeutic agents. Many studies are focusing on osteogenic agents as they increase the bone density, which is essential for osteoporosis treatment. Here, we aimed to investigate the effects of Diospyros lotus L. leaf extract (DLE) and its components on osteoporosis in MC3T3-E1 pre-osteoblasts and ovariectomized mice and to elucidate the underlying related pathways. DLE enhanced the differentiation of MC3T3-E1 pre-osteoblasts, with a 1.5-fold elevation in ALP activity, and increased the levels of osteogenic molecules, RUNX family transcription factor 2, and osterix. This alteration resulted from the activation of bone morphogenic protein 2/4 (BMP2/4) and transformation of growth factor ß (TGF ß) pathways. In ovariectomized mice, DLE suppressed the decrease in bone mineral density by 50% and improved the expression of other bone markers, which was confirmed by the 3~40-fold increase in osteogenic proteins and mRNA expression levels in bone marrow cells. The three major compounds identified in DLE exhibited osteogenic and estrogenic activities with their aglycones, as previously reported. Among the major compounds, myricitrin alone was not as strong as whole DLE with all its constituents. The osteogenic activity of DLE was partially suppressed by the inhibitor of estrogen signaling, indicating that the estrogenic activity of DLE participated in its osteogenic activity. Overall, DLE suppresses osteoporosis by inducing osteoblast differentiation.
Assuntos
Densidade Óssea , Diospyros , Osteoblastos , Osteogênese , Extratos Vegetais , Animais , Feminino , Camundongos , Densidade Óssea/efeitos dos fármacos , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/efeitos dos fármacos , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diospyros/química , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Osteoporose/prevenção & controle , Ovariectomia , Extratos Vegetais/farmacologia , Folhas de Planta/química , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Astragalus saponin IV(AS- IV) extracted from tranditional Chinese medicine Radix Astragali Mongolici, which had been reported to have medicinal properties in treating several types of diseases. This study aimed at investigating the biological functions of AS-IV on bone marrow mesenchymal cells(BMSCs) differentiation, therefore, seeking for a better application of AS-IV on fracture or other orthopedic disorders. METHODS: AS-IV was co-incubated with BMSCs in vitro to testify whether it can influence the proliferation and differentiation of BMSCs. Cell proliferation activity was detected by Cell Counting Kit-8 (CCK-8), while its differentiation promoting capibility was obtained by alkaline phosphatase (ALP) activity assay and Alizarin red S staining. Besides, differentiation protein markers of preosteoblast was detected by western blots. Neuron growth factor antagonists (NGFA) and microRNA-21 (miR-21) inhibitors were co incubated with AS-IV to search the regulatory pathways it activated in BMSCs. RESULTS: AS-IV incubation boosted the proliferation of BMSCs, and accelerated the differentiating direction into preosteoblasts. Runx2, OPN, BMP2, OCN proteins were up regulated after AS- IV treatment. MiR-21/NGF/BMP2/Runx2 pathway can participate the biological effects of AS- IV on BMSCs. CONCLUSION: AS- IV might be used as a therapeutic agent for bone fracture or other orthopedic disorders.
Assuntos
Astrágalo/química , Desenvolvimento Ósseo/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Saponinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Feminino , Masculino , Camundongos , MicroRNAs/genética , Fator de Crescimento Neural/genética , Osteoporose/tratamento farmacológico , Ovariectomia , Ratos , Ratos Sprague-DawleyRESUMO
Exposure to environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes cleft palate at high rates, but little is known about the underlying biological mechanisms. In the present study, we cultured osteoblasts from human fetal palate mesenchymal cells (hFPMCs) to explore the effects of TCDD on osteogenic differentiation. The results showed that TCDD significantly decreased cell proliferation, alkaline phosphatase (ALP) activity and calcium deposition. RNA analyses and protein detection demonstrated that TCDD downregulated a wide array of pro-osteogenic biomarkers. Further investigation of the underlying molecular mechanisms revealed that exposure to TCDD activated aryl hydrocarbon receptor (AhR) signaling and inhibited BMP-2/TGF-ß1/Smad pathway molecules. The inactivation of AhR signaling using CRISPR/Cas9-mediated AhR deletion or by genetic siRNA knockdown significantly blocked the effects induced by TCDD, suggesting a critical role of AhR activation in the TCDD-mediated inhibition of hFPMC osteogenic differentiation. The cotreatment with TGF-ß1 or BMP-2 and TCDD significantly relieved the activation of AhR and rescued the impairment of osteogenesis caused by TCDD. Taken together, our findings indicated that TCDD inhibited the osteogenic differentiation of hFPMCs via crosstalk between AhR and BMP-2/TGF-ß1/Smad signaling pathway.
Assuntos
Poluentes Ambientais/toxicidade , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Palato/citologia , Dibenzodioxinas Policloradas/toxicidade , Transdução de Sinais/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Palato/efeitos dos fármacos , Palato/embriologia , Gravidez , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Proteínas Smad/efeitos dos fármacos , Fator de Crescimento Transformador beta/efeitos dos fármacosRESUMO
PURPOSE: Treatment of complex fractures in the elderly is a challenge for operative reconstruction due to degraded bone structure. Early peri-operative bone anabolic treatment could improve new bone formation, avoid implant loosening and accelerate fracture healing. METHODS: To compare the osteoanabolic potential of different drugs after distraction osteogenesis, 168 female Sprague-Dawley rats underwent lengthening of the right femur using a monolateral external fixator. Animals were randomly divided into six groups: vehicle-injected group, PTH(1-34), raloxifen, strontium ranelate, alendronate and simvastatin. Histomorphometry, CT-scanning, DEXA- and biomechanical analysis were performed to evaluate new bone formation, callus volume, mineralisation and biomechanical strength. Expression of bone metabolic mediators and differentiation indicators of distracted and intact bone were examined by RT-PCR and western blot. RESULTS: Histological analysis showed significant increase of the bone mass after treatment with PTH(1-34), raloxifen and strontium ranelate (p = 0.02). Raloxifen increased bone mineral content (BMC) of the whole distracted femur significantly (p = 0.007). Callus volume was significantly larger in the PTH(1-34), raloxifen and simvastatin groups (p = 0.001) compared to control. Ultimate load of distracted new formed bone was increased in PTH(1-34) and raloxifen groups. It seems that PTH(1-34) and raloxifen have a stronger effect on bone where a repair response is activated. Strontium ranelate demonstrates similar effects to PTH regarding new bone formation but shows low values for mineralisation and biomechanical strength. CONCLUSION: This study suggests that peri-operative treatment of complex and/or osteoporotic fractures with PTH(1-34) and raloxifen might be useful as a stimulator of bone formation and mineralisation to shorten the consolidation time in humans.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Densidade Óssea/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Fêmur/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Absorciometria de Fóton , Alendronato/farmacologia , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Western Blotting , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Proteína Morfogenética Óssea 2/genética , Calo Ósseo/diagnóstico por imagem , Calo Ósseo/metabolismo , Calo Ósseo/patologia , Hormônios e Agentes Reguladores de Cálcio/farmacologia , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Feminino , Fêmur/diagnóstico por imagem , Fêmur/patologia , Fêmur/cirurgia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Interleucina-6/genética , Fator Estimulador de Colônias de Macrófagos/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/genética , Osteocalcina/efeitos dos fármacos , Osteocalcina/genética , Osteogênese/genética , Osteogênese por Distração , Hormônio Paratireóideo/farmacologia , Ligante RANK/efeitos dos fármacos , Ligante RANK/genética , Cloridrato de Raloxifeno/farmacologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinvastatina/farmacologia , Tiofenos/farmacologia , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. OBJECTIVE: Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. MATERIAL AND METHODS: ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software. RESULTS: RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. CONCLUSIONS: In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.
Assuntos
Proteína Morfogenética Óssea 2/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Tretinoína/farmacologia , Fosfatase Alcalina/análise , Fosfatase Alcalina/efeitos dos fármacos , Análise de Variância , Western Blotting , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/efeitos dos fármacos , Osteocalcina/análise , Osteocalcina/efeitos dos fármacos , Osteogênese/fisiologia , Osteonectina/análise , Osteonectina/efeitos dos fármacos , Valores de Referência , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
AIMS: To evaluate the short term effect of zoledronic acid on bone remodeling in the streptozotocin induced diabetes rats. MATERIALS AND METHODS: Diabetes was induced by an injection of streptozotocin (60 mg/kg). The rats were treated with zoledronic acid (0.1 mg/kg) at the onset of diabetes (Z-I group) and 2 weeks later (Z-II group). Rats were sacrificed at the 1, 2, 3, 4 and 5 weeks after the onset of diabetes. Real-time PCR and western blot were performed to detect the expression of the following osteogenic gene mRNAs and their proteins: bone morphogenetic proteins 2 (BMP2), Runx2, Osterix and Noggin. The bone mineral density (BMD) and the mechanical resistance test was measured. RESULTS: BMP2, Runx2 and Osterix mRNA and protein expression in group D had regulated down, while Noggin expression increased. Z-I treatment could reverse the results. However group Z-II showed only a transient reversing effect. On the 5th week in group D, the BMD decreased, the bone trabecular distance increased, while the trabecular thickness and bone trabecular volume were reduced, the biomechanics index decreased significantly. Zoledronic acid treatment restored these alterations. CONCLUSIONS: Zoledronic acid administered in the early stage of the diabetes could prevent the osteopenia. The underlying mechanisms might be that zoledronic acid treatment reversed the effect of diabetes on the expression of osteoblast-regulating transcription factors: BMP2, Runx2 and Osterix.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Densidade Óssea , Doenças Ósseas Metabólicas/tratamento farmacológico , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Subunidades alfa de Fatores de Ligação ao Core/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Fatores de Transcrição/efeitos dos fármacos , Ácido Zoledrônico/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Conservadores da Densidade Óssea/administração & dosagem , Doenças Ósseas Metabólicas/etiologia , Doenças Ósseas Metabólicas/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Feminino , Ratos , Ratos Wistar , Fatores de Transcrição/metabolismo , Ácido Zoledrônico/administração & dosagemRESUMO
Abstract Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. Objective Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. Material and Methods ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software. Results RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. Conclusions In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.
Assuntos
Humanos , Osteogênese/efeitos dos fármacos , Tretinoína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteogênese/fisiologia , Valores de Referência , Fatores de Tempo , Osteocalcina/análise , Osteocalcina/efeitos dos fármacos , Osteonectina/análise , Osteonectina/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Western Blotting , Reprodutibilidade dos Testes , Análise de Variância , Fosfatase Alcalina/análise , Fosfatase Alcalina/efeitos adversos , Proteína Morfogenética Óssea 2/metabolismo , Células-Tronco Mesenquimais/metabolismoRESUMO
OBJECTIVE: To evaluate the dose-response relationship of exercise loading in the cartilage-subchondral bone (SB) unit in surgically-induced post-traumatic osteoarthritis (PTOA) of the knee. DESIGN: Destabilized medial meniscus (DMM) surgery was performed on the right knee of 12-week-old male Wistar rats, and sham surgery was performed on the contralateral knee. Four weeks after the surgery, the animals were subjected to moderate (12 m/min) or intense (21 m/min) treadmill exercises for 30 min/day, 5 days/week for 4 weeks. PTOA development in articular cartilage and SB was examined using histological and immunohistochemical analyses, micro-computed tomography (micro-CT) analysis, and biomechanical testing at 8 weeks after surgery. Gremlin-1 was injected to determine the role of bone morphogenetic protein (BMP) signaling on PTOA development following moderate exercise. RESULTS: Moderate exercise increased BMP-2, BMP-4, BMP-6, BMP receptor 2, pSmad-5, and inhibitor of DNA binding protein-1 expression in the superficial zone chondrocytes and suppressed cartilage degeneration, osteophyte growth, SB damage, and osteoclast-mediated SB resorption. However, intense exercise had little effect on BMP expression and even caused progression of these osteoarthritis (OA) changes. Gremlin-1 injection following moderate exercise caused progression of the PTOA development down to the level of the non-exercise DMM-operated knee. CONCLUSIONS: Exercise regulated cartilage-SB PTOA development in DMM-operated knees in a dose-dependent manner. Our findings shed light on the important role of BMP expression in superficial zone chondrocytes in attenuation of PTOA development following physiological exercise loading. Further studies to support a mechanism by which BMPs would be beneficial in preventing PTOA progression are warranted.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Articulação do Joelho/metabolismo , Osteoartrite do Joelho/metabolismo , Condicionamento Físico Animal , Suporte de Carga , Animais , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/efeitos dos fármacos , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 6/efeitos dos fármacos , Proteína Morfogenética Óssea 6/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/efeitos dos fármacos , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proteínas Morfogenéticas Ósseas/efeitos dos fármacos , Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Citocinas , Proteína 1 Inibidora de Diferenciação , Articulação do Joelho/efeitos dos fármacos , Masculino , Osteoartrite do Joelho/etiologia , Proteínas/farmacologia , Ratos , Ratos Wistar , Proteína Smad5/efeitos dos fármacos , Proteína Smad5/metabolismo , Lesões do Menisco Tibial/complicações , Lesões do Menisco Tibial/metabolismoRESUMO
Daidzein, the most widely studied soy phytoestrogen, is not only a potential antiosteoporosis agent owing to its possible osteogenic activity, but also shows anticancer activity. However, the mechanisms through which daidzein affects osteoblast function have not been well understood. Here, we show that daidzein stimulated cell proliferation and differentiation of osteoblasts, demonstrated by upregulation of XTT activity, enhancement of alkaline phosphatase (ALP) activity, and upregulation of osteoblast-specific marker genes, including Runt-related transcription factor 2 (Runx2) and Smad1, as well as up-regulation of Runx2 and Smad1 protein expression. To determine the mechanisms underlying daidzein's effects on osteoblast differentiation, we first tested the role of daidzein in bone morphogenetic protein (BMP)-2 gene expression in OCT1 cells, and found that it significantly upregulated the expression of BMP-2. Furthermore, it significantly enhanced the phosphorylated protein level of Smad1/5/8and protein expression of Osterix (Osx, a direct target gene of BMP signaling) and increased the activity of BMP signaling reporter (12xSBE-OC-Luc). Finally, we demonstrated that daidzein stimulated Col I, Runx2, and ALP expression, while these effects were significantly blocked by the BMP signaling inhibitor noggin. Thus, our data indicate that daidzein acts through stimulating the activation of BMP-2/Smads pathway to promote osteoblast proliferation and differentiation.
Assuntos
Proteína Morfogenética Óssea 2/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Isoflavonas/farmacologia , Osteoblastos/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Osteoblastos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosAssuntos
Proteína Morfogenética Óssea 2/efeitos dos fármacos , Calciofilaxia/tratamento farmacológico , Calciofilaxia/metabolismo , Proteínas de Ligação ao Cálcio/efeitos dos fármacos , Proteínas da Matriz Extracelular/efeitos dos fármacos , Vitamina K/metabolismo , Varfarina/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Biópsia por Agulha , Proteína Morfogenética Óssea 2/metabolismo , Calciofilaxia/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Razão de Chances , Resultado do Tratamento , Varfarina/uso terapêutico , Proteína de Matriz GlaRESUMO
The most significant increase in metabolic syndrome over the previous decade occurred in women of reproductive age, which is alarming given that metabolic syndrome is associated with reproductive problems including subfertility and early pregnancy loss. Individuals with metabolic syndrome often consume excess fructose, and several studies have concluded that excess fructose intake contributes to metabolic syndrome development. Here, we examined the effects of increased fructose consumption on pregnancy outcomes in mice. Female mice fed a high-fructose diet (HFrD) for 6 weeks developed glucose intolerance and mild fatty liver but did not develop other prominent features of metabolic syndrome such as weight gain, hyperglycemia, and hyperinsulinemia. Upon mating, HFrD-exposed mice had lower pregnancy rates and smaller litters at midgestation than chow-fed controls. To explain this phenomenon, we performed artificial decidualization experiments and found that HFrD consumption impaired decidualization. This appeared to be due to decreased circulating progesterone as exogenous progesterone administration rescued decidualization. Furthermore, HFrD intake was associated with decreased bone morphogenetic protein 2 expression and signaling, both of which were restored by exogenous progesterone. Finally, expression of forkhead box O1 and superoxide dismutase 2 [Mn] proteins were decreased in the uteri of HFrD-fed mice, suggesting that HFrD consumption promotes a prooxidative environment in the endometrium. In summary, these data suggest that excess fructose consumption impairs murine fertility by decreasing steroid hormone synthesis and promoting an adverse uterine environment.
Assuntos
Deciduoma/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Frutose/toxicidade , Tamanho da Ninhada de Vivíparos/efeitos dos fármacos , Taxa de Gravidez , Progesterona/metabolismo , Edulcorantes/toxicidade , Aborto Espontâneo , Animais , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Decídua/efeitos dos fármacos , Decídua/metabolismo , Decídua/patologia , Deciduoma/metabolismo , Deciduoma/patologia , Técnicas de Cultura Embrionária , Transferência Embrionária , Endométrio/metabolismo , Endométrio/patologia , Fígado Gorduroso , Feminino , Fertilização in vitro , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Intolerância à Glucose , Imuno-Histoquímica , Síndrome Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Gravidez , Superovulação , Superóxido Dismutase/efeitos dos fármacos , Superóxido Dismutase/metabolismoRESUMO
PURPOSE: Antiseptic solutions are commonly used in dentistry for a number of sterilization procedures, including harvesting of bone chips, irrigation of extraction sockets, and sterilization of osteonecrotic bone. Despite its widespread use, little information is available regarding the effects of various antiseptic solutions on bone cell viability, morphology, and the release of growth factors. MATERIALS AND METHODS: The antiseptic solutions included 1) 0.5% povidone iodine (PI), 2) 0.2% chlorhexidine diguluconate (CHX), 3) 1% hydrogen peroxide (H2O2), and 4) 0.25% sodium hypochlorite (HYP). Bone samples collected from porcine mandibular cortical bone were rinsed in the antiseptic solutions for 10 minutes and assessed for cell viability using an MTS assay and protein release of transforming growth factor (TGF-ß1), bone morphogenetic protein 2 (BMP2), vascular endothelial growth factor (VEGF), interleukin (IL)-1ß, and receptor activator of nuclear factor κB ligand (RANKL) using an enzyme-linked immunosorbent assay at 15 minutes and 4 hours after rinsing. RESULTS: After antiseptic rinsing, changes to the surface protein content showed marked alterations, with an abundant protein layer remaining on CHX-rinsed bone samples. The amount of surface protein content gradually decreased in the following order: CHX, H2O2, PI, and HYP. A similar trend was also observed for the relative cell viability from within bone samples after rinsing, with up to 6 times more viable cells found in the CHX-rinsed bone samples than in the HYP- and PI-rinsed samples. An analysis of the growth factors found that both HYP and PI had significantly lower VEGF and TGF-ß1 protein release from bone samples at 15 minutes and 4 hours after rinsing compared with CHX and H2O2. A similar trend was observed for RANKL and IL-1ß protein release, although no change was observed for BMP2. CONCLUSIONS: The results from the present study have demonstrated that antiseptic solutions present with very different effects on bone samples after 10 minutes of rinsing. Rinsing with CHX maintained significantly higher cell viability and protein release of growth factors potent to the bone remodeling cycle.
Assuntos
Anti-Infecciosos Locais/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/análise , Mandíbula/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Técnicas de Cultura de Células , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Clorexidina/análogos & derivados , Clorexidina/farmacologia , Peróxido de Hidrogênio/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucina-1beta/efeitos dos fármacos , Mandíbula/citologia , Mandíbula/metabolismo , Proteínas de Membrana/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Povidona-Iodo/farmacologia , Ligante RANK/efeitos dos fármacos , Hipoclorito de Sódio/farmacologia , Suínos , Fatores de Tempo , Fator de Crescimento Transformador beta1/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacosRESUMO
Tumor necrosis factor-alpha (TNF-α) has been demonstrated to have a close relationship with inflammation in the body. Although most researchers confirmed that TNF-α can have an effect on the expression of osteoblast specific genes, they had not elucidated the regulation of inflammatory factors in osteogenic gene expression during the process of bone marrow mesenchymal stem cells (BMMSCs) differentiating to osteoblast. The aim of this study was to investigate the effect of TNF-α at different concentrations on osteogenetic differentiation of BMMSCs. In this study, BMMSCs proliferation was analyzed by using cell counting kit-8 assay, cell osteogenic differentiation was evaluated by means of alkaline phosphatase activity assay and Von Koaas staining and the messenger RNA (mRNA) expression of bone morphogenetic proteins-2 (BMP-2) and drosophila mothers against decapentaplegic protein 1 (Smad1) was measured through real-time polymerase chain reaction. The results indicated that a low concentration of TNF-α at short-term promotes the osteogenetic differentiation of BMMSCs and increases the mRNA expression of BMP-2 and Smad1, but inhibits the osteogenetic differentiation of BMMSCs and the expression of BMP-2 and Smad1 at long term. In addition, regardless of a short or long time, a high concentration of TNF-α inhibits the osteogenetic differentiation of BMMSCs and the expression of Smad1, but results in a high expression of BMP-2.
Assuntos
Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fator de Necrose Tumoral alfa/administração & dosagem , Fosfatase Alcalina/análise , Animais , Antraquinonas , Proteína Morfogenética Óssea 2/análise , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Contagem de Células , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Corantes , Feminino , Masculino , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/análise , Proteína Smad1/efeitos dos fármacos , Fatores de TempoRESUMO
BACKGROUND: Vascular calcification (VC) is highly prevalent in patients with chronic kidney disease (CKD). Low magnesium levels are associated with VC, and recent in vitro studies confirm a protective role of magnesium, which is mediated by its entry into the VSMCs through the Transient Receptor Potential Melastatin 7 (TRPM7) channel. The role of Angiotensin II (Ang II) on VC is still unclear. As Ang II is able to stimulate TRPM7 activity, we hypothesize that it might prevent VC. Thus, the aim of this study was to dissect the direct effect of Ang II on VC. MATERIALS AND METHODS: We worked with a model of high phosphate (HP)-induced calcification in human aortic smooth muscle cells, which resembles the CKD-related VC. RESULTS: Addition of Ang II to cells growing in HP decreased calcification, which was associated with the upregulation of the osteogenic factors BMP2, Runx2/Cbfa1, Osterix and ALP. A reduction of magnesium entry into the HP-calcifying cells was found. The treatment with Ang II avoided this reduction, which was reversed by the cotreatment with the TRPM7-inhibitor 2-APB. The protective effect of Ang II was related to AT1R-induced ERK1/2 MAPKinase activation. HP-induced calcification was also associated with the upregulation of the canonical Wnt/beta-catenin pathway, while its downregulation was related to attenuation of calcification by Ang II. CONCLUSION: As hypothesized, Ang II prevented phosphate-induced calcification in VSMCs, which appears mediated by the increase of magnesium influx and by the activation of the ERK1/2 and the inhibition of the canonical Wnt/beta-catenin signalling pathways.
Assuntos
Angiotensina II/farmacologia , Magnésio/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Canais de Cátion TRPM/efeitos dos fármacos , Calcificação Vascular/metabolismo , Vasoconstritores/farmacologia , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Compostos de Boro/farmacologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição Sp7 , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Regulação para Cima , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
BACKGROUND: Cigarette smoking inhibits bone-healing and leads to increased rates of pseudarthrosis. However, the mechanisms behind these effects are controversial. Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin)--a cigarette smoke constituent and potent activator of the aryl hydrocarbon receptor (Ahr)--negatively impacts bone quality and osteoblast differentiation. We hypothesized that activation of the Ahr by dioxin would inhibit bone morphogenetic protein (BMP)-2-mediated spinal fusion in a rat arthrodesis model. METHODS: Female Long-Evans rats were pretreated with dioxin or vehicle in six weekly doses, followed by bilateral posterior lumbar spinal fusion across the L4-L5 transverse processes using recombinant human BMP (rhBMP)-2. Treatments continued until sacrifice at four weeks postoperatively. A third group was treated with dioxin for six weeks, followed by a recovery period of four elimination half-lives to assess the reversible effects of dioxin exposure on spinal fusion capacity. Bone formation and fusion capacity were evaluated using fusion scoring, radiography, micro-computed tomography, and histologic analysis. RESULTS: Fusion scores for dioxin-treated and dioxin-recovery rats were significantly lower than those for controls. Although fusion rates were also significantly reduced in dioxin-treated animals relative to controls (50% versus 100%, respectively), rates were not significantly reduced in dioxin-recovery animals (80%). CONCLUSIONS: Dioxin treatment significantly inhibited spinal fusion in a rat arthrodesis model, and a prolonged cessation of dioxin exposure facilitated only a partial recovery of bone-healing capacity. This finding indicates that, although the effects of dioxin are persistent, an extended recovery from exposure could potentially restore bone regeneration in vivo. CLINICAL RELEVANCE: Development of a pharmacologic agent that reduces the adverse effects of cigarette smoke on bone-healing could prove useful to orthopaedic surgeons. Since dioxin and other similar cigarette smoke toxins exert their effects through Ahr pathway activation, the receptor represents a potential therapeutic target to improve spinal fusion rates in patients who smoke.
Assuntos
Proteína Morfogenética Óssea 2/efeitos dos fármacos , Proteína Morfogenética Óssea 2/fisiologia , Dioxinas/efeitos adversos , Fusão Vertebral , Animais , Regeneração Óssea/efeitos dos fármacos , Feminino , Modelos Animais , Ratos , Ratos Long-EvansRESUMO
GH stimulates growth plate chondrogenesis and longitudinal bone growth directly at the growth plate. However, it is not clear yet whether these effects are entirely mediated by the local expression and action of IGF-1 and IGF-2. To determine whether GH has any IGF-independent growth-promoting effects, we generated (TamCart)Igf1r(flox/flox) mice. The systemic injection of tamoxifen in these mice postnatally resulted in the excision of the IGF-1 receptor (Igf1r) gene exclusively in the growth plate. (TamCart)Igf1r(flox/flox) tamoxifen-treated mice [knockout (KO) mice] and their Igf1r(flox/flox) control littermates (C mice) were injected for 4 weeks with GH. At the end of the 4-week period, the tibial growth and growth plate height of GH-treated KO mice were greater than those of untreated C or untreated KO mice. The systemic injection of GH increased the phosphorylation of Janus kinase 2 and signal transducer and activator of transcription 5B in the tibial growth plate of the C and KO mice. In addition, GH increased the mRNA expression of bone morphogenetic protein-2 and the mRNA expression and protein phosphorylation of nuclear factor-κB p65 in both C and KO mice. In cultured chondrocytes transfected with Igf1r small interfering RNA, the addition of GH in the culture medium significantly induced thymidine incorporation and collagen X mRNA expression. In conclusion, our findings demonstrate that GH can promote growth plate chondrogenesis and longitudinal bone growth directly at the growth plate, even when the local effects of IGF-1 and IGF-2 are prevented. Further studies are warranted to elucidate the intracellular molecular mechanisms mediating the IGF-independent, growth-promoting GH effects.
Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Lâmina de Crescimento/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Proteína Morfogenética Óssea 2/genética , Células Cultivadas , Condrócitos/efeitos dos fármacos , Colágeno Tipo X/efeitos dos fármacos , Colágeno Tipo X/genética , Lâmina de Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Janus Quinase 2/efeitos dos fármacos , Janus Quinase 2/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/genética , Fator de Transcrição RelA/efeitos dos fármacos , Fator de Transcrição RelA/genéticaRESUMO
BACKGROUND AND OBJECTIVE: The proteasome inhibitor, bortezomib, is known to induce osteoblastic differentiation in a number of cell lines, such as mesenchymal stem cells and osteoblastic precursor cells. As periodontal ligament (PDL) cells are multipotent, we examined whether bortezomib may induce the differentiation of PDL cells into hard-tissue-forming cells. MATERIAL AND METHODS: A mouse PDL clone cell line, MPDL22 cells, was cultured in mineralization medium in the presence or absence of bortezomib. Expression of calcification-related genes and calcified-nodule formation were evaluated by real-time PCR and Alizarin Red staining, respectively. RESULTS: Bortezomib increased the expression of calcification-related mRNAs, such as tissue nonspecific alkaline phosphatase isoenzyme (ALPase), bone sialoprotein (Bsp), runt-related transcription factor 2 (Runx2) and osteopontin, and calcified-nodule formation in MPDL22 cells. These effects were induced, in part, by increasing the cytosolic accumulation and nuclear translocation of ß-catenin, leading to an increase in expression of bone morphogenetic protein (Bmp)-2, -4 and -6 mRNAs. In addition, bortezomib enhanced BMP-2-induced expression of Bsp and osteopontin mRNAs and increased calcified-nodule formation in MPDL22 cells. CONCLUSION: Bortezomib induced cytodifferentiation and mineralization of PDL cells by enhancing the accumulation of ß-catenin within the cytosol and the nucleus and increasing the expression of Bmp-2, -4 and -6 mRNAs. Moreover, bortezomib enhanced the BMP-2-induced cytodifferentiation and mineralization of PDL cells, suggesting that bortezomib may be efficacious for use in periodontal regeneration therapy.
Assuntos
Bortezomib/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Fosfatase Alcalina/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Proteína Morfogenética Óssea 4/efeitos dos fármacos , Proteína Morfogenética Óssea 6/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Citosol/efeitos dos fármacos , Isoenzimas/efeitos dos fármacos , Camundongos , Osteopontina/efeitos dos fármacos , Ligamento Periodontal/citologia , beta Catenina/efeitos dos fármacosRESUMO
Hedgehog (Hh) family members are involved in multiple cellular processes including proliferation, migration, differentiation, and cell fate determination. Recently, the novel Hh agonists Hh-Ag 1.3 and 1.7 were identified in a high-throughput screening of small molecule compounds that activate the expression of Gli1, a target of Hh signaling. This study demonstrates that Hh-Ag 1.3 and 1.7 strongly activate the expression of endogenous Gli1 and promote osteoblast differentiation in the mesenchymal stem cell line C3H10T1/2. Both compounds stimulated alkaline phosphatase activity in a dose-dependent manner, and induced osteoblast marker gene expression in C3H10T1/2 cells, which indicated that they had acquired an osteoblast identity. Of the markers, the expression of osterix/Sp7, a downstream target of runt-related transcription factor (Runx)2, was induced by Hh-Ag 1.7, which also rescued the osteoblast differentiation defect of RD-127, a mesenchymal cell line from Runx2-deficient mice. Hh-Ags also activated canonical Wnt signaling and synergized with low doses of BMP-2 to enhance osteoblastic potential. Thus, Hh-Ag 1.7 could be useful for bone healing in individuals with abnormalities in osteogenesis, such as osteoporosis patients and the elderly, and can contribute to the development of novel therapeutics for the treatment of bone fractures and defects.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proteínas Hedgehog/agonistas , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2/efeitos dos fármacos , Células Cultivadas , Células-Tronco Mesenquimais/citologia , Camundongos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Receptores de Superfície Celular/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacosRESUMO
INTRODUCTION: Prostacyclin (PGI2), a member of the prostaglandin family, can promote angiogenesis and cell proliferation. METHODS: In this study, the effect of the application of a PGI2 analog (iloprost) on dentin repair was examined in vitro and in vivo. RESULTS: Iloprost significantly stimulated the expression of vascular endothelial growth factor and osteo-/odontogenic marker messenger RNA in human dental pulp cells (HDPCs) under osteoinductive conditions in vitro. In addition, iloprost enhanced HDPC alkaline phosphatase enzymatic activity and mineral deposition. An in vivo study was performed using a rat molar mechanical pulp exposure model. After 30 days, histologic analysis revealed that there was a dramatic tertiary dentin formation in the iloprost-treated group compared with the calcium hydroxide and the untreated control groups. Furthermore, vascular endothelial growth factor protein expression in dental pulp tissue was increased in the iloprost-treated group as determined by immunohistochemical staining. CONCLUSIONS: Taken together, the present study, for the first time, shows that iloprost induces the expression of osteo-/odontogenic markers in vitro and promotes angiogenic factor expression and enhances tertiary dentin formation in vivo. This implies the potential clinical usefulness of iloprost in vital pulp therapy.