RESUMO
Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder characterized by alpha-L-iduronidase (IDUA) deficiency, an enzyme responsible for glycosaminoglycan degradation. Musculoskeletal impairment is an important component of the morbidity related to the disease, as it has a major impact on patients' quality of life. To understand how this disease affects bone structure, morphological, biomechanical and histological analyses of femurs from 3- and 6-month-old wild type (Idua +/+) and MPS I knockout mice (Idua -/-) were performed. Femurs from 3-month-old Idua -/- mice were found to be smaller and less resistant to fracture when compared to their age matched controls. In addition, at this age, the femurs presented important alterations in articular cartilage, trabecular bone architecture, and deposition of type I and III collagen. At 6 months of age, femurs from Idua -/- mice were more resistant to fracture than those from Idua +/+. Our results suggest that the abnormalities observed in bone matrix and articular cartilage in 3-month-old Idua -/- animals caused bone tissue to be less flexible and more likely to fracture, whereas in 6-month-old Idua -/- group the ability to withstand more load before fracturing than wild type animals is possibly due to changes in the bone matrix.
Assuntos
Iduronidase/metabolismo , Mucopolissacaridose I/metabolismo , Mucopolissacaridose I/patologia , Animais , Fenômenos Biomecânicos/fisiologia , Colágeno/metabolismo , Modelos Animais de Doenças , Feminino , Fêmur/enzimologia , Fêmur/metabolismo , Fêmur/patologia , Iduronidase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridose I/enzimologiaAssuntos
Fraturas do Fêmur/enzimologia , Fêmur/enzimologia , Consolidação da Fratura , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Modelos Animais de Doenças , Fraturas do Fêmur/genética , Fraturas do Fêmur/fisiopatologia , Fêmur/lesões , Fêmur/fisiopatologia , Masculino , MicroRNAs/genética , Fosforilação , RNA Interferente Pequeno , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Supraphysiological levels of the osteoblast-enriched mineralization regulator ectonucleotide pyrophosphatase or phosphodiesterase-1 (NPP1) is associated with type 2 diabetes mellitus. We determined the impact of osteoblast-specific Enpp1 ablation on skeletal structure and metabolic phenotype in mice. Female, but not male, 6-week-old mice lacking osteoblast NPP1 expression (osteoblast-specific knockout [KO]) exhibited increased femoral bone volume or total volume (17.50% vs. 11.67%; p < .01), and reduced trabecular spacing (0.187 vs. 0.157 mm; p < .01) compared with floxed (control) mice. Furthermore, an enhanced ability of isolated osteoblasts from the osteoblast-specific KO to calcify their matrix in vitro compared to fl/fl osteoblasts was observed (p < .05). Male osteoblast-specific KO and fl/fl mice showed comparable glucose and insulin tolerance despite increased levels of insulin-sensitizing under-carboxylated osteocalcin (195% increase; p < .05). However, following high-fat-diet challenge, osteoblast-specific KO mice showed impaired glucose and insulin tolerance compared with fl/fl mice. These data highlight a crucial local role for osteoblast NPP1 in skeletal development and a secondary metabolic impact that predominantly maintains insulin sensitivity.
Assuntos
Osso e Ossos/enzimologia , Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina , Osteoblastos/enzimologia , Osteogênese , Diester Fosfórico Hidrolases/deficiência , Pirofosfatases/deficiência , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Osso e Ossos/patologia , Osso Esponjoso/enzimologia , Osso Esponjoso/patologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fêmur/enzimologia , Fêmur/patologia , Insulina/sangue , Masculino , Camundongos Knockout , Osteoblastos/patologia , Osteocalcina/sangue , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Fatores Sexuais , Crânio/enzimologia , Crânio/patologia , Tíbia/enzimologia , Tíbia/patologiaRESUMO
INTRODUCTION: Resveratrol (RES) exhibits estrogen-like effects and has potential applications to treatment of osteoporosis caused by estrogen deficiency; however, the specific mechanism of action of RES remains unclear. Here, we examined the therapeutic effects of RES on ovariectomized (OVX) rats with osteoporosis and determined the underlying mechanism. METHODS: We established an OVX rat model to study osteoporosis caused by estrogen deficiency. The treatment groups were given orally with RES (50, 100, and 200 mg/day), the estrogen group received 0.8 mg/kg E2 daily via oral route, and the sham-operated and control groups received an equivalent dose of sodium carboxymethylcellulose orally. After 12 weeks of treatment, we used real-time quantitative polymerase chain reaction (PCR) and Western blot analysis to measure the gene and protein expression of miR-92b-3p, Nox4, NF-κBp65, IκB, BMP2, Smad7, and RUNX-2 in bone tissues. Right femur structural parameters were evaluated by micro-CT. Dual-energy X-ray 4500 W was used to determine systemic bone mineral density (BMD). Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the serum levels of bone alkaline phosphatase (BALP), osteoprotegerin (OPG), anti-tartrate acid phosphatase-5b (PTRA5b), and carboxylated terminal peptide (CTX-I). The rat femoral bone specimens were stained using hematoxylin and eosin for pathological examination. RESULTS: We observed increased levels of serum estrogen in both ovaries, elevated miR-92b-3p levels in bone tissues, reduced levels of Nox4, NF-κBp65, p-IκB-a, and cathepsin K, and elevated gene and protein expression of BMP2, Smad7, and RUNX-2 in the OVX rat model of osteoporosis after treatment with RES. Elevated levels of BALP, OPG, ALP, and BMD along with reduced levels of TRAP-5b and CTX-I were also observed. The structural model index (SMI) and the trabecular space (Tb. Sp) decreased, while the trabecular thickness (Tb. Th), bone volume fraction (BV/TV), trabecular number (Tb.N), and tissue bone density (Conn.D) increased, thereby improving osteoporosis induced by estrogen deficiency in both ovaries. CONCLUSION: Cathepsin K expression and Nox4/NF-κB signaling pathway were suppressed by the elevated expression of miR-92b-3p. This inhibition was pivotal in the protective effect of RES against osteoporosis induced by estrogen deficiency in both ovaries. Thus, RES efficiently alleviated osteoporosis induced by estrogen deficiency in rats.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Remodelação Óssea/efeitos dos fármacos , Estrogênios/deficiência , Fêmur/efeitos dos fármacos , MicroRNAs/metabolismo , NADPH Oxidase 4/metabolismo , NF-kappa B/metabolismo , Osteoporose Pós-Menopausa/prevenção & controle , Resveratrol/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Catepsina K/genética , Catepsina K/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fêmur/enzimologia , Fêmur/patologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , MicroRNAs/genética , NADPH Oxidase 4/genética , NF-kappa B/genética , Osteoporose Pós-Menopausa/enzimologia , Osteoporose Pós-Menopausa/genética , Osteoporose Pós-Menopausa/patologia , Ovariectomia , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Osteoporosis is characterized by a bone loss associated to an increased bone marrow adiposity; however, it is still unclear what kind of lipids are involved. Therefore, the main purpose of this study was to see if there is any local bone lipid changes related to osteoporosis, by using the ovariectomy-induced osteoporosis (OVX) rat model. Female SD rats (operated at 6 months of age for skeletal maturity) were divided in control SHAM and OVX groups (n = 6/group) and maintained for 9 month post-surgery. Lipids were analyzed in two compartments of femoral diaphyses: bone marrow (BM) and mineralized tissue (MT), by chromatographic methods. As expected, osteoporotic femurs had a larger BM mass associated with a two-fold increase of lipid content. The MT had a similar lipid enrichment, indicating that adiposity affected the mineral part as well. The main lipids concerned were triglycerides, sphingomyelin, phosphatidylcholine and phosphatidylserine in BM, and triglycerides and cholesterol esters in MT. The increase of both energy-storage and membrane-associated lipids in BM suggested that cell number and/or size was enhanced to allow more triglyceride storage. Interestingly, in MT of osteoporotic femurs, sphingomyelin was decreased, suggesting that its catabolism could be linked to osteoporosis. In both femoral compartments, fatty acid profiles were enriched in 14:0 and 16:1, lowered in 18:0 and 20:4 n-6, and two-fold higher stearoyl-CoA desaturase indexes (16:1/16:0 and 18:1/18:0 ratios), suggesting an increased de novo lipogenesis in osteoporotic femurs. Thus, the present study is first to report local changes of individual lipids in rat osteoporotic femurs and suggests that osteoporosis is a pathologic condition associated with an enhanced de novo lipogenesis. Further studies will be needed to better understand the consequences of these lipid changes in osteoporotic bones.
Assuntos
Adiposidade , Fêmur/metabolismo , Osteoporose/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Animais , Ácidos Graxos/metabolismo , Feminino , Fêmur/enzimologia , Metabolismo dos Lipídeos , Lipogênese , Osteoporose/enzimologia , Osteoporose/etiologia , Ovariectomia , Ratos Sprague-DawleyRESUMO
Glucocorticoid-induced osteoporosis (GIO) is a secondary osteoporosis with extensive use of glucocorticoids (GCs). GCs can increase bone fragility and fracture via inhibiting osteoblastic proliferation and differentiation. Luteolin (LUT), a kind of plant flavonoid, has been reported to exhibit the antioxidant activity, but the effects of LUT on GIO still remain unclear. This study aimed to investigate the effects of LUT on GIO both in vivo and in vitro and elaborate the potential molecular mechanisms. LUT increased the superoxide dismutase activity, glutathione level and decreased reactive oxygen species (ROS) level and lactate dehydrogenase release in GIO. Meanwhile, LUT decreased caspase-3, caspase-9, and Bax protein expressions and increased Bcl-2 protein expression in GIO. LUT increased the ratio of osteoprotegerin (OPG)/receptor activator of nuclear factor-κB Ligand (RANKL) messenger RNA (mRNA) expression and mRNA expression levels of osteogenic markers, including runt-related transcription factor 2, osterix, collagen type I, and osteocalcin. LUT also enhanced the extracellular signal-regulated kinases (ERK) phosphorylation, glycogen synthase kinase 3ß (GSK-3ß) phosphorylation, mRNA expression levels of lipoprotein-receptor-related protein 5 (Lrp-5) and ß-catenin. Further study revealed that Lrp-5 small interfering RNA (siRNA )and ERK-siRNA reduced the effects of LUT on GSK-3ß phosphorylation, alkaline phosphatase (ALP) activity and the ratio of OPG/RANKL mRNA expression. Moreover, ERK-siRNA decreased Lrp-5 mRNA expression in vitro. These results indicated that LUT promoted proliferation by attenuating oxidative stress and promoted osteoblastic differentiation by regulating the ERK/Lrp-5/GSK-3ß pathway in GIO. This study may bring to light the possible mechanisms involved in the action of LUT in GIO treatment, and benefit for further research on GIO.
Assuntos
Dexametasona , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fêmur/efeitos dos fármacos , Glucocorticoides , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Luteolina/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoporose/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Osso Esponjoso/efeitos dos fármacos , Osso Esponjoso/enzimologia , Osso Esponjoso/patologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Fêmur/enzimologia , Fêmur/patologia , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Osteoblastos/enzimologia , Osteoblastos/patologia , Osteogênese/efeitos dos fármacos , Osteoporose/induzido quimicamente , Osteoporose/enzimologia , Osteoporose/patologia , Estresse Oxidativo/efeitos dos fármacos , FosforilaçãoRESUMO
Down syndrome (DS) is caused by three copies of human chromosome 21 (Hsa21) and results in phenotypes including intellectual disability and skeletal deficits. Ts65Dn mice have three copies of ~50% of the genes homologous to Hsa21 and display phenotypes associated with DS, including cognitive deficits and skeletal abnormalities. DYRK1A is found in three copies in humans with Trisomy 21 and in Ts65Dn mice, and is involved in a number of critical pathways including neurological development and osteoclastogenesis. Epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, inhibits Dyrk1a activity. We have previously shown that EGCG treatment (~10mg/kg/day) improves skeletal abnormalities in Ts65Dn mice, yet the same dose, as well as ~20mg/kg/day did not rescue deficits in the Morris water maze spatial learning task (MWM), novel object recognition (NOR) or balance beam task (BB). In contrast, a recent study reported that an EGCG-containing supplement with a dose of 2-3mg per day (~40-60mg/kg/day) improved hippocampal-dependent task deficits in Ts65Dn mice. The current study investigated if an EGCG dosage similar to that study would yield similar improvements in either cognitive or skeletal deficits. Ts65Dn mice and euploid littermates were given EGCG [0.4mg/mL] or a water control, with treatments yielding average daily intakes of ~50mg/kg/day EGCG, and tested on the multivariate concentric square field (MCSF)-which assesses activity, exploratory behavior, risk assessment, risk taking, and shelter seeking-and NOR, BB, and MWM. EGCG treatment failed to improve cognitive deficits; EGCG also produced several detrimental effects on skeleton in both genotypes. In a refined HPLC-based assay, its first application in Ts65Dn mice, EGCG treatment significantly reduced kinase activity in femora but not in the cerebral cortex, cerebellum, or hippocampus. Counter to expectation, 9-week-old Ts65Dn mice exhibited a decrease in Dyrk1a protein levels in Western blot analysis in the cerebellum. The lack of beneficial therapeutic behavioral effects and potentially detrimental skeletal effects of EGCG found in Ts65Dn mice emphasize the importance of identifying dosages of EGCG that reliably improve DS phenotypes and linking those effects to actions of EGCG (or EGCG-containing supplements) in specific targets in brain and bone.
Assuntos
Catequina/análogos & derivados , Cognição/efeitos dos fármacos , Síndrome de Down/tratamento farmacológico , Síndrome de Down/patologia , Fêmur/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Administração Oral , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Catequina/farmacologia , Cognição/fisiologia , Modelos Animais de Doenças , Síndrome de Down/enzimologia , Síndrome de Down/psicologia , Fêmur/diagnóstico por imagem , Fêmur/enzimologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Fenótipo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Distribuição Aleatória , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologia , Falha de Tratamento , Quinases DyrkRESUMO
In the phase 3 Study of Eliglustat Tartrate (Genz-112638) in Patients With Gaucher Disease Who Have Reached Therapeutic Goals With Enzyme Replacement Therapy (ENCORE), at 1 year, eliglustat was noninferior to imiglucerase enzyme therapy in maintaining stable platelet counts, hemoglobin concentrations, and spleen and liver volumes. After this primary analysis period, patients entered a long-term extension phase in which all received eliglustat. Duration on eliglustat ranged from 2 to 5 years, depending on timing of enrollment (which spanned 2 years), treatment group to which patients were randomized, and whether they lived in the United States when commercial eliglustat became available. Here we report long-term safety and efficacy of eliglustat for 157 patients who received eliglustat in the ENCORE trial; data are available for 46 patients who received eliglustat for 4 years. Mean hemoglobin concentration, platelet count, and spleen and liver volumes remained stable for up to 4 years. Year to year, all 4 measures remained collectively stable (composite end point relative to baseline values) in ≥85% of patients as well as individually in ≥92%. Mean bone mineral density z scores (lumbar spine and femur) remained stable and were maintained in the healthy reference range throughout. Eliglustat was well tolerated over 4 years; 4 (2.5%) patients withdrew because of adverse events that were considered related to the study drug. No new or long-term safety concerns were identified. Clinical stability assessed by composite and individual measures was maintained in adults with Gaucher disease type 1 treated with eliglustat who remained in the ENCORE trial for up to 4 years. This trial was registered at www.clinicaltrials.gov as #NCT00943111.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Terapia de Reposição de Enzimas , Doença de Gaucher/tratamento farmacológico , Glucosilceramidase/uso terapêutico , Pirrolidinas/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Administração Oral , Adulto , Densidade Óssea/efeitos dos fármacos , Feminino , Fêmur/efeitos dos fármacos , Fêmur/enzimologia , Doença de Gaucher/enzimologia , Doença de Gaucher/fisiopatologia , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/enzimologia , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Baço/efeitos dos fármacos , Baço/enzimologiaRESUMO
The AKR1A1 protein is a member of the aldo-keto reductase superfamily that is responsible for the conversion of D-glucuronate to L-gulonate in the ascorbic acid (vitamin C) synthesis pathway. In a pCAG-eGFP transgenic mouse line that was produced by pronuclear microinjection, the integration of the transgene resulted in a 30-kb genomic DNA deletion, including the Akr1A1 gene, and thus caused the knockout (KO) of the Akr1A1 gene and targeting of the eGFP gene. The Akr1A1 KO mice (Akr1A1eGFP/eGFP) exhibited insufficient serum ascorbic acid levels, abnormal bone development and osteoporosis. Using micro-CT analysis, the results showed that the microarchitecture of the 12-week-old Akr1A1eGFP/eGFP mouse femur was shorter in length and exhibited less cortical bone thickness, enlargement of the bone marrow cavity and a complete loss of the trabecular bone in the distal femur. The femoral head and neck of the proximal femur also showed a severe loss of bone mass. Based on the decreased levels of serum osteocalcin and osteoblast activity in the Akr1A1eGFP/eGFP mice, the osteoporosis might be caused by impaired bone formation. In addition, administration of ascorbic acid to the Akr1A1eGFP/eGFP mice significantly prevented the condition of osteoporotic femurs and increased bone formation. Therefore, through ascorbic acid administration, the Akr1A1 KO mice exhibited controllable osteoporosis and may serve as a novel model for osteoporotic research.
Assuntos
Aldeído Redutase/genética , Deficiência de Ácido Ascórbico/genética , Fêmur/patologia , Técnicas de Inativação de Genes , Osteogênese , Osteoporose/genética , Aldeído Redutase/deficiência , Animais , Ácido Ascórbico/administração & dosagem , Ácido Ascórbico/sangue , Deficiência de Ácido Ascórbico/enzimologia , Deficiência de Ácido Ascórbico/patologia , Deficiência de Ácido Ascórbico/prevenção & controle , Modelos Animais de Doenças , Feminino , Fêmur/diagnóstico por imagem , Fêmur/enzimologia , Predisposição Genética para Doença , Camundongos Knockout , Osteoblastos/enzimologia , Osteoblastos/patologia , Osteocalcina/sangue , Osteoporose/enzimologia , Osteoporose/patologia , Osteoporose/prevenção & controle , Fenótipo , Fatores de Tempo , Microtomografia por Raio-XRESUMO
UNLABELLED: The increasing evidence suggesting the role of free radicals in bone resorption and bone loss prompted us to explore whether the consumption of antioxidant rich medicinal plant C. quadrangularis modifies antioxidant status in ovariectomized rats. METHODS: Twenty four female adult rats, 90days old showing regular estrous cycles were used for the present study. The animals were divided into two groups. The Group-1 rats (n=6) were sham operated and Group-II rats were bilaterally ovariectomized (n=18) and treated with C. quadrangularis for sixty days (100mg/kg body weight and 250mg/kg body weight). After sixty days, the rats were killed, femora were dissected out, minced and homogenized in Tris-HCl buffer (pH 7.4) and the supernatant was collected and used for biochemical assays. RESULTS: Ovariectomy registered a decrease (p<0.05) in the activities of SOD, GPx, GST, ALP, collagen content and increased (p<0.05) the activities of TRAP and lipid peroxidation. Simultaneous administration of C. quadrangularis maintained the enzyme activities in ovariectomized rats. CONCLUSION: C. quadrangularis, a natural herb may be used to treat the estrogen deficiency/menopause onset and ovariectomy induced oxidative stress.
Assuntos
Cissus/química , Fêmur/efeitos dos fármacos , Fêmur/patologia , Ovariectomia/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Envelhecimento , Fosfatase Alcalina/metabolismo , Animais , Colágeno/metabolismo , Feminino , Fêmur/enzimologia , Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Ratos Wistar , Superóxido Dismutase/metabolismo , Fosfatase Ácida Resistente a Tartarato/metabolismoRESUMO
Parathyroid hormone (PTH) is the only current anabolic treatment for osteoporosis in the United States. PTH stimulates expression of matrix metalloproteinase 13 (MMP13) in bone. Sirtuin 1 (SIRT1), an NAD-dependent deacetylase, participates in a variety of human diseases. Here we identify a role for SIRT1 in the action of PTH in osteoblasts. We observed increased Mmp13 mRNA expression and protein levels in bone from Sirt1 knock-out mice compared with wild type mice. PTH-induced Mmp13 expression was significantly blocked by the SIRT1 activator, resveratrol, in osteoblastic UMR 106-01 cells. In contrast, the SIRT1 inhibitor, EX527, significantly enhanced PTH-induced Mmp13 expression. Two h of PTH treatment augmented SIRT1 association with c-Jun, a component of the transcription factor complex, activator protein 1 (AP-1), and promoted SIRT1 association with the AP-1 site of the Mmp13 promoter. This binding was further increased by resveratrol, implicating SIRT1 as a feedback inhibitor regulating Mmp13 transcription. The AP-1 site of the Mmp13 promoter is required for PTH stimulation of Mmp13 transcriptional activity. When the AP-1 site was mutated, EX527 was unable to increase PTH-stimulated Mmp13 promoter activity, indicating a role for the AP-1 site in SIRT1 inhibition. We further showed that SIRT1 deacetylates c-Jun and that the cAMP pathway participates in this deacetylation process. These data indicate that SIRT1 is a negative regulator of MMP13 expression, SIRT1 activation inhibits PTH stimulation of Mmp13 expression, and this regulation is mediated by SIRT1 association with c-Jun at the AP-1 site of the Mmp13 promoter.
Assuntos
Metaloproteinase 13 da Matriz/metabolismo , Osteoblastos/enzimologia , Hormônio Paratireóideo/fisiologia , Sirtuína 1/fisiologia , Acetilação , Animais , Sítios de Ligação , Indução Enzimática , Feminino , Fêmur/citologia , Fêmur/enzimologia , Expressão Gênica , Masculino , Metaloproteinase 13 da Matriz/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
While it has been well-documented that excessive fluoride exposure caused the skeletal disease and osteoblasts played a critical role in the advanced skeletal fluorosis, the underlying mechanism that mediated these effects remain poorly understood. The present study was undertaken to examine the effect of fluoride on bone of rats and MC3T3-E1 cells in vitro. Herein we found pathological features of high bone turnover in fluoride-treated rats, which was supported by an increase of osteogenic and osteoclastogenic genes expression in different stages of fluoride exposure. The skeletal toxicity of fluoride was accompanied by activation of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR). A novel finding of this study was that expression of PKR-like endoplasmic reticulum kinase (PERK) was the same trend with receptor activator for nuclear factor-κ B ligand (RANKL), and NF-E2 p45-related factor 2 (Nrf2) was the same trend with Runt-related transcription factor 2 (Runx2) in bones of rats exposed to varied fluoride condition. Based on these data, we hypothesized that up-regulation of PERK probably played a role in mediating bone turnover induced by fluoride. Action of fluoride on MC3T3-E1 cells differentiation was demonstrated through analysis of alkaline phosphatase (ALP) activity and mineralized nodules formation. Meantime, an increase of binding immunoglobulin protein (BiP) expression indicated the active ER stress in cells exposed to various dose of fluoride. Blocking PERK expression using siRNA showed the obvious decrease of osteogenic and osteoclastogenic factors expression in MC3T3-E1 cells exposed to certain dose of fluoride that could positively stimulate osteoblastic viability. In conclusion these findings underscore the importance of PERK in modulating fluoride induced bone formation and bone resorption. Understanding the link between PERK and bone turnover could probe into the mechanism underlying different bone lesion of skeletal fluorosis.
Assuntos
Remodelação Óssea/efeitos dos fármacos , Fêmur/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Crânio/efeitos dos fármacos , Fluoreto de Sódio/toxicidade , eIF-2 Quinase/metabolismo , Animais , Densidade Óssea/efeitos dos fármacos , Remodelação Óssea/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fêmur/enzimologia , Fêmur/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Marcadores Genéticos , Masculino , Camundongos Endogâmicos C57BL , Osteoblastos/enzimologia , Osteoblastos/patologia , Osteoclastos/enzimologia , Osteoclastos/patologia , Interferência de RNA , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Crânio/enzimologia , Crânio/patologia , Fatores de Tempo , Transfecção , eIF-2 Quinase/genéticaRESUMO
Skeletal integrity is dependent on the coordinated actions of bone-forming osteoblasts and bone-resorbing osteoclasts, which recognize and respond to multiple environmental inputs. Here we have studied the roles in bone development and growth of Akt1 and Akt2, two closely related signaling proteins, by evaluating mice lacking either of these enzymes. Global deficiency of Akt1 but not Akt2 caused a reduction in whole body and femoral bone mineral density, in femoral cortical thickness and volume, and in trabecular thickness in both males and females when measured at 20-weeks of age, which was reflected in diminished femoral resistance to fracture. Haplo-deficiency of Akt1 in male mice also decreased femoral cortical and trabecular skeletal parameters, and reduced bone strength. Cell-based studies showed that genetic Akt1 deficiency diminished the rate of proliferation of osteoblast progenitors and impaired osteoclast differentiation in primary culture but that loss of Akt2 did not. Our results demonstrate differential effects of Akt1 and Akt2 on skeletal maturation and architecture through actions on both osteoblast and osteoclast precursors.
Assuntos
Densidade Óssea/fisiologia , Fêmur/enzimologia , Osteoblastos/enzimologia , Osteogênese/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/enzimologia , Animais , Diferenciação Celular/fisiologia , Feminino , Fêmur/citologia , Masculino , Camundongos , Camundongos Mutantes , Osteoblastos/citologia , Proteínas Proto-Oncogênicas c-akt/genética , Células-Tronco/citologiaRESUMO
Although their primary therapeutic indications are different, aminobisphosphonates and statins target enzymes in the mevalonate pathway, which is critical for bone homeostasis. Previous studies have shown that some polymorphisms of the gene encoding farnesyl diphosphate synthase (FDPS), the main target of aminobisphosphonates, modulate the response to these drugs. In this study, we explored whether those single nucleotide polymorphisms (SNPs) also influence the changes in bone mineral density (BMD) following therapy with statins. Sixty-six patients with coronary heart disease were studied at baseline and after 1-year therapy with atorvastatin. BMD was measured by DXA. Three SNPs of the FDPS gene (rs2297480, rs11264359 and rs17367421) were analyzed by using Taqman assays. The results showed that there was no association between the SNPs and basal BMD. However, rs2297480 and rs11264359 alleles, which are in linkage disequilibrium, were associated with changes in hip BMD following atorvastatin therapy. Thus, patients with AA genotype at the rs2297480 locus had a 0.8 ± 0.8 % increase in BMD at the femoral neck, whereas in patients with AC/CC genotypes, BMD showed a 2.3 ± 0.8 % decrease (p = 0.02). Similar results were obtained regarding changes of BMD at the femoral trochanter and when alleles at the rs11264359 locus were analyzed. However, there was no association between BMD and rs17367421 alleles. In conclusion, these results suggest that polymorphisms of the FDPS gene may influence the bone response to various drugs targeting the mevalonate pathway, including not only aminobisphosphonates but also statins.
Assuntos
Densidade Óssea/efeitos dos fármacos , Doença das Coronárias/tratamento farmacológico , Fêmur/efeitos dos fármacos , Geraniltranstransferase/genética , Ácidos Heptanoicos/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Polimorfismo de Nucleotídeo Único , Pirróis/uso terapêutico , Absorciometria de Fóton , Idoso , Atorvastatina , Doença das Coronárias/diagnóstico , Feminino , Fêmur/diagnóstico por imagem , Fêmur/enzimologia , Frequência do Gene , Genótipo , Geraniltranstransferase/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Farmacogenética , Fenótipo , Fatores de Tempo , Resultado do TratamentoRESUMO
Bone integrity abnormality and imbalance between bone formation by osteoblasts and bone resorption by osteoclasts are known to result in metabolic bone diseases such as osteoporosis. Silymarin-rich milk thistle extract (MTE) and its component silibinin enhanced alkaline phosphatase activity of osteoblasts but reduced tartrate-resistant acid phosphatase (TRAP) activity of osteoclasts. The osteoprotective effects of MTE were comparable to those of estrogenic isoflavone. Low-dose combination of MTE and isoflavone had a pharmacological synergy that may be useful for osteogenic activity. This study attempted to reveal the suppressive effects of MTE on bone loss. C57BL/6 female mice were ovariectomized (OVX) as a model for postmenopausal osteopenia and orally administered 10 mg/kg MTE or silibinin for 8 weeks. The sham-operated mice served as estrogen controls. The treatment of ovariectomized mice with nontoxic MTE and silibinin improved femoral bone mineral density and serum receptor activator of nuclear factor- κB ligand/osteoprotegerin ratio, an index of osteoclastogenic stimulus. In addition, the administration of MTE or silibinin inhibited femoral bone loss induced by ovariectomy and suppressed femoral TRAP activity and cathepsin K induction responsible for osteoclastogenesis and bone resorption. Collectively, oral dosage of MTE containing silibinin in the preclinical setting is effective in preventing estrogen deficiency-induced bone loss.
Assuntos
Estrogênios/deficiência , Osteoclastos/patologia , Osteoporose/tratamento farmacológico , Osteoporose/patologia , Ovariectomia , Extratos Vegetais/uso terapêutico , Silybum marianum/química , Fosfatase Ácida/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Catepsina K/metabolismo , Cromatografia Líquida de Alta Pressão , Estradiol/sangue , Feminino , Fêmur/efeitos dos fármacos , Fêmur/enzimologia , Fêmur/patologia , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/efeitos dos fármacos , Osteoblastos/enzimologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/enzimologia , Osteogênese/efeitos dos fármacos , Osteoporose/sangue , Fitoterapia , Extratos Vegetais/farmacologia , Silibina , Silimarina/farmacologia , Silimarina/uso terapêutico , Glycine max/química , Fosfatase Ácida Resistente a Tartarato , Útero/efeitos dos fármacos , Útero/patologiaRESUMO
SUMMARY: The present study shows no adverse effects of the anti-diabetic drug metformin on bone mass and fracture healing in rodents but demonstrates that metformin is not osteogenic in vivo, as previously proposed. INTRODUCTION: In view of the increased incidence of fractures in patients with type 2 diabetes mellitus (T2DM), we investigated the effects of metformin, a widely used T2DM therapy, on bone mass and fracture healing in vivo using two different rodent models and modes of metformin administration. METHODS: We first subjected 12-week-old female C57BL/6 mice to ovariectomy (OVX). Four weeks after OVX, mice received either saline or metformin administered by gavage (100 mg/kg/daily). After 4 weeks of treatment, bone micro-architecture and cellular activity were determined in tibia by micro-CT and bone histomorphometry. In another experiment, female Wistar rats aged 3 months were given only water or metformin for 8 weeks via the drinking water (2 mg/ml). After 4 weeks of treatment, a mid-diaphyseal osteotomy was performed in the left femur. Rats were sacrificed 4 weeks after osteotomy and bone architecture analysed by micro-CT in the right tibia while fracture healing and callus volume were determined in the left femur by X-ray analysis and micro-CT, respectively. RESULTS: In both models, our results show no significant differences in cortical and trabecular bone architecture in metformin-treated rodents compared to saline. Metformin had no effect on bone resorption but reduced bone formation rate in trabecular bone. Mean X-ray scores assessed on control and metformin fractures showed no significant differences of healing between the groups. Fracture callus volume and mineral content after 4 weeks were similar in both groups. CONCLUSIONS: Our results indicate that metformin has no effect on bone mass in vivo or fracture healing in rodents.
Assuntos
Densidade Óssea/efeitos dos fármacos , Consolidação da Fratura/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Densidade Óssea/fisiologia , Remodelação Óssea/efeitos dos fármacos , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/patologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Fraturas do Fêmur/fisiopatologia , Fêmur/enzimologia , Consolidação da Fratura/fisiologia , Hipoglicemiantes/sangue , Metformina/sangue , Camundongos , Camundongos Endogâmicos C57BL , Osteoporose/fisiopatologia , Ovariectomia , Ratos , Ratos Wistar , Tíbia/diagnóstico por imagem , Tíbia/efeitos dos fármacos , Tíbia/patologia , Tíbia/fisiopatologia , Microtomografia por Raio-X/métodosRESUMO
BACKGROUND: Bone microvascular remodeling is the primary predictor of bone structure and function. Remodeling by its very nature implies synthesis and degradation of the extracellular matrix. Normally, 50% of total protein in the vessel wall is elastin. During remodeling, elastin is degraded by specialized matrix metalloproteinases (MMPs). Because the turnover of elastin is 1000-fold slower than that of collagen, most of the elastin is replaced by stiffer collagen. Stiffer vessels impose pressure on the aortic valve, causing regurgitation and increased pulse pressure. On the other hand, high MMP activity will cause vascular dilatation, leading to aneurysm. Therefore, balanced constitutive remodeling is necessary for adequate bone structure and function. Interestingly, collagen-degrading MMPs are involved in various pathological conditions, including osteoporosis, osteoarthritis, and cardiovascular disease. Sodium nitroprusside is a nitric oxide donor that could potentially alter MMP activity via vasodilation in vivo, but can also produce peroxynitrite, which activates MMPs by combining with superoxide. Moreover, hydrogen sulfide is a known antioxidant as well as a vasodilator, and is also speculated to contribute directly to MMP activity. We hypothesized that hydrogen sulfide reduced activity of MMP in ex vivo bone tissue homogenates and that sodium nitroprusside would increase MMP activity in vitro. METHODS: We surgically removed the tibia and femur from anesthetized mice, and prepared bone tissue homogenates using a mortar and pestle, measured the protein concentration with a spectrophotometer, and detected MMP activity using gelatin gel zymography. RESULTS: Our data showed increased MMP activity at a sodium nitroprusside concentration of 1 µM, and MMP activity increased exponentially. There was a decrease in MMP activity with increasing hydrogen sulfide, beginning at 16 µM (P < 0.01) and continuing to 40 µM. Moreover, sodium nitroprusside 3 µM was able to overcome the decrease in MMP activity that occurred with hydrogen sulfide 40 µM; this resulted in a more pronounced exponential increase in MMP activity. CONCLUSION: There are several substances that can potentially be used to decrease MMP activity and to alleviate pathological remodeling by MMPs.
Assuntos
Remodelação Óssea/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Fêmur/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloproteinases da Matriz/metabolismo , Nitroprussiato/farmacologia , Tíbia/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Ativação Enzimática , Fêmur/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Espectrofotometria , Tíbia/enzimologiaRESUMO
CONTEXT: Cadmium (Cd) is a widespread environmental pollutant that is associated with increased risk of osteoporosis. It has been proposed that Cd's toxic effect on bone is exerted via impaired activation of vitamin D, secondary to the kidney effects. OBJECTIVE: The present study was designed to investigate the damaging impact of Cd in drinking water on bone from biochemical and histopathological point of view. MATERIALS AND METHODS: This study was conducted on 30, 3-months-old female Sprague Dawley rats exposed to cadmium chloride in a dose of 50 mg Cd/L in drinking water for 3 months. Serum was taken for determination of calcium, phosphorous levels, parathyroid hormone, 1,25 dihydroxy vitamin D(3), osteocalcin (OC) and bone specific alkaline phosphatase (BALP) activity. RESULTS: The result revealed that Cd administration induces significant increase in serum calcium (Ca), phosphorous (P) and parathyroid hormone (PTH) levels in concomitant with significant reduction in serum vitamin D(3), osteocalcin (OC) levels and bone specific alkaline phosphatase (BALP) activity. CONCLUSION: The present study provided clear evidence that long-term exposure to cadmium chloride produced marked abnormalities in bone biomarkers and increasing risk of fracture.
Assuntos
Cloreto de Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Fêmur/efeitos dos fármacos , Osteoporose/induzido quimicamente , Fosfatase Alcalina/metabolismo , Animais , Calcitriol/sangue , Cálcio/sangue , Cálcio/deficiência , Dieta , Feminino , Fêmur/enzimologia , Fêmur/metabolismo , Fêmur/patologia , Osteocalcina/sangue , Osteoporose/enzimologia , Osteoporose/metabolismo , Osteoporose/patologia , Hormônio Paratireóideo/sangue , Fósforo/sangue , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Tartrate resistant acid phosphatase (TRAP) was shown to be critical for skeleton development, and TRAP deficiency leads to a reduced resorptive activity during endochondral ossification resulting in an osteopetrotic phenotype and shortened long bones in adult mice. A proper longitudinal growth depends on a timely, well-coordinated vascularization and formation of the secondary ossification center (SOC) of the long bones epiphysis. Our results demonstrate that TRAP is not essential for the formation of the epiphyseal vascular network. Therefore, in wild type (Wt) controls as well as TRAP deficient (TRAP(-/-)) mutants vascularised cartilage canals are present from postnatal day (P) five. However, in the epiphysis of the TRAP(-/-) mice cartilage mineralization, formation of the marrow cavity and the SOC occur prematurely compared with the controls. In the mutant mice the entire growth plate is widened due to an expansion of the hypertrophic zone. This is not seen in younger animals but first detected at week (W) three and during further development. Moreover, an enhanced number of thickened trabeculae, indicative of the osteopetrotic phenotype, are observed in the metaphysis beginning with W three. Epiphyseal excavation was proposed as an important function of TRAP, and we examined whether TRAP deficiency affects this process. We therefore evaluated the marrow cavity volume (MCV) and the epiphyseal volume (EV) and computed the MCV to EV ratio (MCV/EV). We investigated developmental stages until W 12. Our results indicate that both epiphyseal excavation and establishment of the SOC are hardly impaired in the knockouts. Furthermore, no differences in the morphology of the epiphyseal bone trabeculae and remodeling of the articular cartilage layers are noted between Wt and TRAP(-/-) mice. We conclude that in long bones, TRAP is critical for the development of the growth plate and the metaphysis but apparently not for the epiphyseal vascularization, excavation, and establishment of the SOC.
Assuntos
Fosfatase Ácida/metabolismo , Desenvolvimento Ósseo , Epífises/enzimologia , Epífises/crescimento & desenvolvimento , Isoenzimas/metabolismo , Animais , Biomarcadores/metabolismo , Medula Óssea/crescimento & desenvolvimento , Matriz Óssea/citologia , Matriz Óssea/enzimologia , Calcificação Fisiológica , Cartilagem Articular/citologia , Cartilagem Articular/crescimento & desenvolvimento , Epífises/citologia , Fêmur/citologia , Fêmur/enzimologia , Fêmur/crescimento & desenvolvimento , Lâmina de Crescimento/citologia , Imuno-Histoquímica , Camundongos , Tamanho do Órgão , Osteoblastos/citologia , Osteoblastos/enzimologia , Osteogênese , Fosfatase Ácida Resistente a TartaratoRESUMO
Large conventional bone allografts are susceptible to fracture and nonunion due to incomplete revascularization and insufficient bone remodeling. We aim to improve bone blood flow and bone remodeling using surgical angiogenesis combined with delivery of fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF). Frozen femoral allografts were heterotopically transplanted in a rat model. The saphenous arteriovenous bundle was implanted within the graft medullary canal. Simultaneously, biodegradable microspheres containing phosphate buffered saline (control), FGF-2, VEGF, or FGF-2 + VEGF were placed within the graft. Rats were sacrificed at 4 and 18 weeks. Angiogenesis was determined by quantifying bone capillary density and measuring cortical bone blood flow. Bone remodeling was assessed by histology, histomorphometry, and alkaline phosphatase activity. VEGF significantly increased angiogenesis and bone remodeling at 4 and 18 weeks. FGF-2 did not elicit a strong angiogenic or osteogenic response. No synergistic effect of FGF-2 + VEGF was observed. VEGF delivered in microspheres had superior long-term effect on angiogenesis and osteogenesis in surgically revascularized frozen bone structural allografts as compared to FGF-2 or FGF-2 + VEGF. Continuous and localized delivery of VEGF by microencapsulation has promising clinical potential by inducing a durable angiogenic and osteogenic response in frozen allografts.