RESUMO
PURPOSE: The purpose of this research is to demonstrate echinacoside promotes osteogenesis and angiogenesis and inhibits osteoclast formation. METHODS: We conducted a cell experiment in vitro to study how echinacoside affects angiogenesis, osteogenesis and osteoclast formation. We used polymerase chain reaction and Western blotting to detect the expression levels of proteins and genes related to angiogenesis, osteogenesis and osteoclast formation. We established a bone fracture model with rats to test angiogenesis, osteogenesis and osteoclast formation of echinacoside. We labelled osteogenic markers, blood vessels and osteoclastic markers in fracture sections of rats. RESULTS: The in vitro cell experiments showed echinacoside improved the osteogenic activity of mouse embryo osteoblast precursor cells and promoted the migration and tube formation of human umbilical vein endothelial cells. In addition, it inhibited differentiation of mouse leukaemia cells of monocyte macrophage. Echinacoside increased the expression of related proteins and genes and improved angiogenesis and osteogenesis while inhibiting osteoclast formation by repressing the expression of related proteins and genes. From in vivo experiments, the results of IHC and HE experiments demonstrated echinacoside significantly decreased the content of MMP-9 and improved the content of VEGF and OCN. The fluorescence immunoassay showed echinacoside promoted the activities of RUNX2 and VEGF and inhibited CTSK. Echinacoside reduced the content of TNF-α, IL-1ß and IL-6, thus demonstrating its anti-inflammatory activity. CONCLUSION: Echinacoside improved angiogenesis and osteogenesis and inhibited osteoclast formation to promote fracture healing.
Assuntos
Glicosídeos , Células Endoteliais da Veia Umbilical Humana , Metaloproteinase 9 da Matriz , Neovascularização Fisiológica , Osteoclastos , Osteogênese , Animais , Osteogênese/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Humanos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Ratos , Glicosídeos/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Masculino , Diferenciação Celular/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Ratos Sprague-Dawley , Movimento Celular/efeitos dos fármacos , Osteocalcina/metabolismo , Osteocalcina/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , AngiogêneseRESUMO
Vascular calcification (VC) is characterized by pathological depositions of calcium and phosphate in the arteries and veins via an active cell-regulated process, in which vascular smooth muscle cells (VSMCs) transform into osteoblast/chondrocyte-like cells as in bone formation. VC is associated with significant morbidity and mortality in chronic kidney disease (CKD) and cardiovascular disease, but the underlying mechanisms remain unclear. In this study we investigated the role of large-conductance calcium-activated potassium (BK) channels in 3 experimental VC models. VC was induced in vascular smooth muscle cells (VSMCs) by ß-glycerophosphate (ß-GP), or in rats by subtotal nephrectomy, or in mice by high-dosage vitamin D3. We showed that the expression of BK channels in the artery of CKD rats with VC and in ß-GP-treated VSMCs was significantly decreased, which was functionally confirmed by patch-clamp recording. In ß-GP-treated VSMCs, BK channel opener NS1619 (20 µM) significantly alleviated VC by decreasing calcium content and alkaline phosphatase activity. Furthermore, NS1619 decreased mRNA expression of ostoegenic genes OCN and OPN, as well as Runx2 (a key transcription factor involved in preosteoblast to osteoblast differentiation), and increased the expression of α-SMA protein, whereas BK channel inhibitor paxilline (10 µM) caused the opposite effects. In primary cultured VSMCs from BK-/- mice, BK deficiency aggravated calcification as did BK channel inhibitor in normal VSMCs. Moreover, calcification was more severe in thoracic aorta rings of BK-/- mice than in those of wild-type littermates. Administration of BK channel activator BMS191011 (10 mg· kg-1 ·d-1) in high-dosage vitamin D3-treated mice significantly ameliorated calcification. Finally, co-treatment with Akt inhibitor MK2206 (1 µM) or FoxO1 inhibitor AS1842856 (3 µM) in calcified VSMCs abrogated the effects of BK channel opener NS1619. Taken together, activation of BK channels ameliorates VC via Akt/FoxO1 signaling pathways. Strategies to activate BK channels and/or enhance BK channel expression may offer therapeutic avenues to control VC.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Calcificação Vascular/patologia , Fosfatase Alcalina/efeitos dos fármacos , Animais , Aorta Torácica/efeitos dos fármacos , Benzimidazóis/farmacologia , Colecalciferol/farmacologia , Modelos Animais de Doenças , Glicerofosfatos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nefrectomia , Osteocalcina/efeitos dos fármacos , Osteopontina/efeitos dos fármacos , Fragmentos de Peptídeos/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
The present study aimed to investigate the effect of ßreceptor blocker propranolol on early osseointegration of pure titanium implants and the underlying molecular regulatory mechanisms. An implant osseointegration model using the tibial metaphysis of New Zealand rabbits was established. The rabbits were divided into control and low, medium and highdose propranolol groups. The formation of implant osseointegration was detected by Xray scanning. Mesenchymal stem cells (MSCs) and osteoblasts (OBs) were isolated and cultured in vitro, isoproterenol was supplemented to simulate sympathetic action and propranolol was subsequently administrated. The effect of propranolol on cell proliferation and osteogenic differentiation were assessed by EdU, flow cytometry, alizarin red staining and alkaline phosphatase (ALP) detection. The expression levels of bone morphogenetic protein (BMP)2, RUNX family transcription factor (RunX)2, collagen (COL)1, osteocalcin (OCN) and ß2adrenergic receptor (AR) were detected by immunofluorescence, reverse transcriptionquantitative PCR and western blot assay. Propranolol effectively promoted implant osseointegration in vivo, facilitated proliferation of OBs, inhibited proliferation of MSCs and enhanced osteogenic differentiation of OBs and MSCs. The calcium content and ALP activity of cells treated with propranolol were markedly higher than in the control group. Propranolol also elevated mRNA and protein expression levels of BMP2, RunX2, COL1 and OCN in tissue and cells, and decreased the expression of ß2AR. The present study demonstrated that the ßreceptor blocker propranolol promoted osteogenic differentiation of OBs and MSCs and enhanced implant osseointegration. The present study provided a novel insight into the application and regulatory mechanisms of propranolol.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Osseointegração/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Propranolol/farmacologia , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/efeitos dos fármacos , Osteocalcina/metabolismo , Coelhos , Receptores Adrenérgicos beta 2/metabolismo , Titânio/farmacologiaRESUMO
OBJECTIVE: To investigate the therapeutic effects of PERK activator CCT020312 (CCT) on inflammation-mediated osteoporosis (IMO) in ovariectomized rats. METHODS: Rats were divided into Sham, IMO, IMO + 1 mg/kg CCT and IMO + 2 mg/kg CCT groups. IMO models were constructed by bilateral ovariectomy (OVX) on 1st day followed by injection with magnesium silicate (Talc) on the 59th day. Sham rats did not undergo OVX surgery and were injected with saline instead of Talc. From 60th to 79th day, rats were treated with DMSO (vehicle control) in the Sham and IMO groups, and 1 or 2 mg/kg CCT020312 in treatment groups. Osteopontin (OPN), osteocalcin (OCN), tartrate-resistant acid phosphatase (TRAP), C-terminal telopeptide of type I collagen (CTX-I), and pro-inflammatory factors were measured on the 80th day. ProdigyDEXA was used to evaluate bone mineral density and content (BMD/BMC). Bone volume/total volume (BV/TV), connectivity density (Conn.D), trabecular number (Tb.N), and trabecular separation (Tb.Sp) was assessed using 3D micro-CT scanner. RESULTS: CCT up-regulated Conn.D, BV/TV, and Tb.N, but down-regulated Tb.Sp in IMO rats. Besides, the declined femoral BMD and BMC in IMO rats were elevated after CCT treatment. Besides, IMO rats represented declined OPN and OCN, as well as increased TRAP, CTX-I, and pro-inflammatory factors, whereas those in the treatment groups were ameliorated regarding these indexes, with 2 mg/kg CCT showing better effect. CONCLUSION: PERK activator CCT020312 can be served as a new therapeutic option for the protection against bone loss in the OVX rat model associated with inflammation probably by manipulating inflammatory factors.
Assuntos
Densidade Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Ovariectomia , eIF-2 Quinase , Absorciometria de Fóton , Animais , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/metabolismo , Osso Esponjoso/diagnóstico por imagem , Osso Esponjoso/efeitos dos fármacos , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Fêmur/diagnóstico por imagem , Fêmur/efeitos dos fármacos , Fêmur/metabolismo , Humanos , Imageamento Tridimensional , Inflamação/metabolismo , Tamanho do Órgão , Osteocalcina/efeitos dos fármacos , Osteocalcina/metabolismo , Osteopontina/efeitos dos fármacos , Osteopontina/metabolismo , Osteoporose Pós-Menopausa/diagnóstico por imagem , Osteoporose Pós-Menopausa/metabolismo , Fragmentos de Peptídeos/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Ratos , Fosfatase Ácida Resistente a Tartarato/efeitos dos fármacos , Fosfatase Ácida Resistente a Tartarato/metabolismo , Microtomografia por Raio-XRESUMO
Osteogenic differentiation is critical to bone homeostasis, and its imbalance plays a key role in the progression of osteoporosis. Osteoblast cells are responsible for synthesizing new bone tissue, and understanding how to control osteoblastic differentiation is vital to the treatment of osteoporosis. Herein, we show that GPR173 signaling is involved in the regulation of osteoblastic differentiation in MC3T3-E1 cells. Our data reveals that GPR173 is abundantly expressed in MC3T3-E1 cells, and its expression is inducible upon the introduction of osteogenic media. The activation of GPR173 by its selective agonist phoenixin 20 induces the expression of several osteoblast signature genes including collagen type 1 alpha 1 (Col-I), osteocalcin (OCN), alkaline phosphatase (ALP) as well as increased matrix mineralization and ALP activity, suggesting that the activation of GPR173 promotes osteoblastic differentiation. Moreover, we show that the effect of phoenixin 20 is mediated by its induction on the key regulator runt-Related Transcription Factor 2 (Runx2). Mechanistically, we display that the action of phoenixin 20 requires the activation of MAPK kinase p38, and deactivation of p38 by its inhibitor SB203580 weakens the phoenixin 20-mediated induction of RUNX-2, ALP, and matrix mineralization. Silencing of GPR173 attenuates phoenixin 20-mediated osteoblastic differentiation, indicating its dependence on the receptor. Collectively, our study reveals a new role of GPR173 and its agonist phoenixin 20 in osteoblastic differentiation.
Assuntos
Calcificação Fisiológica/genética , Diferenciação Celular/fisiologia , Osteoblastos/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/fisiologia , Diferenciação Celular/efeitos dos fármacos , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Imidazóis/farmacologia , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteocalcina/efeitos dos fármacos , Osteocalcina/genética , Hormônios Peptídicos/farmacologia , Piridinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiologia , Transcriptoma , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Recently, it has been suggested that glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), which play important roles in the homeostasis of glucose metabolism, could be involved in the regulation of bone metabolism. Inhibitors of dipeptidyl peptidase 4 (DPP-4), an enzyme that degrades GIP and GLP-1, are widely used clinically as a therapeutic agent for diabetes. However, the effects of DPP-4 inhibitors on bone metabolism remain unclear. In this study, we investigated the effects of linagliptin, a DPP-4 inhibitor, on bone fragility induced by type 2 diabetes mellitus (T2DM). Non-diabetic mice were used as controls, and T2DM mice were administered linagliptin orally on a daily basis for 12 weeks. In T2DM mice, decreased bone mineral density was observed in the lower limb bones along with low serum osteocalcin levels and high serum tartrate-resistant acid phosphatase-5b (TRAP) levels. In contrast, the decreased serum osteocalcin levels and increased serum TRAP levels observed in T2DM mice were significantly suppressed after the administration of linagliptin 30 mg/kg. Bone histomorphometric analysis revealed a reduced osteoid volume and osteoblast surface with an increase in the eroded surface and number of osteoclasts in T2DM mice. This decreased bone formation and increased bone resorption observed in the T2DM mice were suppressed and trabecular bone volume increased following the administration of 30 mg/kg linagliptin. Collectively, these findings suggest that linagliptin may improve the microstructure of trabecular bone by inhibiting both a decrease in bone formation and an increase in bone resorption induced by T2DM.
Assuntos
Osso e Ossos/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores da Dipeptidil Peptidase IV/farmacologia , Linagliptina/farmacologia , Administração Oral , Animais , Densidade Óssea/efeitos dos fármacos , Osso e Ossos/anormalidades , Osso e Ossos/metabolismo , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/complicações , Inibidores da Dipeptidil Peptidase IV/administração & dosagem , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Polipeptídeo Inibidor Gástrico/metabolismo , Polipeptídeo Inibidor Gástrico/farmacologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Linagliptina/administração & dosagem , Linagliptina/uso terapêutico , Masculino , Camundongos , Camundongos Obesos , Osteocalcina/sangue , Osteocalcina/efeitos dos fármacos , Fosfatase Ácida Resistente a Tartarato/sangue , Fosfatase Ácida Resistente a Tartarato/efeitos dos fármacosRESUMO
This study aimed to investigate the effects of resveratrol (RES) on bone metabolism and bone turnover related indexes in ovariectomized osteoporosis rats. 48 clean grade adult healthy unmated female SD rats were randomly divided into 6 groups, including normal control group (NCG), osteoporosis model group (OP MG), estrogen treatment group (17ß-E2 group), RES low dose group (RES-L), RES medium-dose group (RES-M) and RES high dose group (RES-H). The rats in NCG and OP MG were given distilled water once a day and the rats in the other two groups were given 17ß-E2 and resveratrol respectively. The levels of serum calcium (S-Ca), serum phosphorus (S-P), urinary calcium (U-Ca/Cr) and urinary phosphorus (U-P/Cr) were measured with an automatic biochemistry analyzer. The levels of serum osteocalcin (BGP), alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), type I amino front-end peptide (PINP), type I collagen strong carboxyl peptide (CTX-I), urine deoxypyridinoline (DPD) and serum estrogen were detected by enzyme-linked immunosorbent assay (ELISA). In the OP group, the serum estrogen levels, S-Ca and S-P decreased significantly and the expression of U-Ca/Cr and U-P/Cr increased significantly (P< 0.05). Compared with the OP group, the expression of S-Ca and S-P increased significantly and the expression of U-Ca/Cr and U-P/Cr decreased significantly (p< 0.05) after treatment. The levels of TRAP, BGP, DPD and CTX-I in the OP group increased significantly (P< 0.05). Compared with the OP group, the levels of TRAP decreased significantly (P< 0.05). The levels of PINP and ALP in OP MG increased significantly (P< 0.05). IP and ALP increased in the middle and lower levels (P< 0.05). The bone mineral density of the OP group decreased significantly (P< 0.05). Resveratrol can affect the changes in bone turnover in ovariectomized rats, promote bone formation in low estrogen state and inhibit bone resorption. Resveratrol may have a protective effect on the bone of ovariectomized rats.
Assuntos
Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Resveratrol/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Feminino , Osteocalcina/efeitos dos fármacos , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Osteoporose/metabolismo , Ovariectomia/métodos , Fósforo/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Treatments for infectious bone defects such as periodontitis require antibacterial and osteogenic differentiation capabilities. Nanotechnology has prompted the development of multifunctional material. In this research, we aim to synthesize a nanoparticle that can eliminate periodontal pathogenic microorganisms and simultaneously stimulate new bone tissue regeneration and mineralization. QAS-modified core-shell mesoporous silica containing Ag nanoparticles (Ag@QHMS) was successfully synthesized through the classic hydrothermal method and surface quaternary ammonium salt functionalization. The Ag@QHMS in vitro antibacterial activity was explored via coculture with Staphylococcus aureus, Escherichia coli, and Porphyromonas gingivalis biofilms. Bone mesenchymal stem cells (BMSCs) were selected for observing cytotoxicity, apoptosis, and osteogenic differentiation. Ag@QHMS showed a good sustained release profile of Ag+ and a QAS-grafted mesoporous structure. Compared with the single-contact antibacterial activity of QHMS, Ag@QHMS exhibited a more efficient and stable concentration-dependent antimicrobial efficacy; the minimum inhibitory concentration was within 100 µg/ml, which was below the BMSC biocompatibility concentration (200 µg/ml). Thus, apoptosis would not occur while promoting the increased expression of osteogenic-associated factors, such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and collagen type 1 (COL-1). A safe concentration of particles can stimulate cell alkaline phosphatase and matrix calcium salt deposition. The dual antibacterial effect from the direct contact killing of QAS and the sustained release of Ag nanoparticles, along with the Ag-promoted osteogenic differentiation, had been verified and utilized in Ag@QHMS. This system demonstrates the potential for utilizing pluripotent biomaterials to treat complex lesions.
Assuntos
Antibacterianos/farmacologia , Nanopartículas Metálicas/administração & dosagem , Osteogênese/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , Dióxido de Silício/química , Prata/química , Animais , Bactérias/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Mesenquimais/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Nanomedicina/métodos , Osteocalcina/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Engenharia Tecidual/métodosRESUMO
BACKGROUND: Statins are the most widely used drugs in elderly patients; the most common clinical application of statins is in aged hyperlipemia patients. There are few studies on the effects and mechanisms of statins on bone in elderly mice with hyperlipemia. The study is to examine the effects of atorvastatin on bone phenotypes and metabolism in aged apolipoprotein E-deficient (apoE-/-) mice, and the possible mechanisms involved in these changes. METHODS: Twenty-four 60-week-old apoE-/- mice were randomly allocated to two groups. Twelve mice were orally gavaged with atorvastatin (10 mg/kg body weight/day) for 12 weeks; the others served as the control group. Bone mass and skeletal microarchitecture were determined using micro-CT. Bone metabolism was assessed by serum analyses, qRT-PCR, and Western blot. Bone marrow-derived mesenchymal stem cells (BMSCs) from apoE-/- mice were differentiated into osteoblasts and treated with atorvastatin and silent information regulator 1 (Sirt1) inhibitor EX-527. RESULTS: The results showed that long-term administration of atorvastatin increases bone mass and improves bone microarchitecture in trabecular bone but not in cortical bone. Furthermore, the serum bone formation marker osteocalcin (OCN) was ameliorated by atorvastatin, whereas the bone resorption marker tartrate-resistant acid phosphatase 5b (Trap5b) did not appear obviously changes after the treatment of atorvastatin. The mRNA expression of Sirt1, runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and OCN in bone tissue were increased after atorvastatin administration. Western blot showed same trend in Sirt1 and Runx2. The in vitro study showed that when BMSCs from apoE-/- mice were pretreated with EX527, the higher expression of Runx2, ALP, and OCN activated by atorvastatin decreased significantly or showed no difference compared with the control. The protein expression of Runx2 showed same trend. CONCLUSIONS: Accordingly, the current study validates the hypothesis that atorvastatin can increase bone mass and promote osteogenesis in aged apoE-/- mice by regulating the Sirt1-Runx2 axis.
Assuntos
Atorvastatina/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteogênese/efeitos dos fármacos , Sirtuína 1/metabolismo , Administração Oral , Idoso , Fosfatase Alcalina/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/metabolismo , Densidade Óssea/efeitos dos fármacos , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso Esponjoso/efeitos dos fármacos , Osso Esponjoso/metabolismo , Carbazóis/metabolismo , Carbazóis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/efeitos dos fármacos , Osteocalcina/sangue , Osteocalcina/efeitos dos fármacos , Microtomografia por Raio-X/métodosRESUMO
The Indian hedgehog (IHH) signaling pathway is an important pathway for bone growth and development. The aim of the present study was to examine the role of the IHH signaling pathway in the development of the ossification of ligamentum flavum (OLF) at the cellular and tissue levels. The expression levels and localization of the osteogenic genes Runt-related transcription factor 2 (RUNX2), Osterix, alkaline phosphatase (ALP), osteocalcin (OCN) and IHH were evaluated in OLF tissues by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. Non-ossified ligamentum flavum (LF) sections were used as control samples. The tissue explant method was used to obtain cultured LF cells. In addition, OLF cells were subjected to cyclic stretch application for 0, 6, 12 or 24 h. The expression levels of osteogenic genes, and the IHH signaling pathway genes IHH, Smoothened (SMO), GLI family zinc finger 1 (GLI1), GLI2 and GLI3 were evaluated with RT-qPCR and western blotting. Osteogenic differentiation was further evaluated by assessing ALP activity and staining. Moreover, the effect of cyclopamine (Cpn), an IHH signaling inhibitor, on osteogenic differentiation was examined. The RT-qPCR and immunohistochemical results indicated that the mRNA and protein expression levels of RUNX2, Osterix, ALP, OCN and IHH were significantly higher in the OLF group compared with the LF group. Furthermore, application of cyclic stretch to OLF cells resulted in greater ALP activity, and significant increases in mRNA and protein expression levels of RUNX2, Osterix, ALP and OCN in a time-d00ependent manner. Cyclic stretch application also led to significant increases in IHH signaling pathway genes, including IHH, SMO, GLI1 and GLI2, while no significant effect was found on GLI3 expression level. In addition, it was found that Cpn significantly reversed the effect of cyclic stretch on the ALP activity, and the expression levels of RUNX2, Osterix, ALP, OCN, GLI1 and GLI2. Collectively, the present results suggested that the IHH signaling pathway may mediate the effect of cyclic stretch on the OLF cells.
Assuntos
Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Ligamento Amarelo/metabolismo , Ossificação Heterotópica/genética , Ossificação Heterotópica/metabolismo , Transdução de Sinais , Estresse Mecânico , Adulto , Idoso , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Diferenciação Celular , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Proteínas Hedgehog/antagonistas & inibidores , Humanos , Ligamento Amarelo/patologia , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Osteocalcina/efeitos dos fármacos , Osteocalcina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Fator de Transcrição Sp7/efeitos dos fármacos , Fator de Transcrição Sp7/metabolismo , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/metabolismo , Proteína Gli3 com Dedos de Zinco/metabolismoRESUMO
INTRODUCTION: Vitamin K (VK) is a co-factor in the post-translational gamma glutamic carboxylation of Gla-proteins. VK-dependent coagulation factors are carboxylated in the liver by VK1. Osteocalcin and Matrix-Gla protein (MGP) are carboxylated in extrahepatic tissues by VK2. A model of VK deficiency would be suitable for studying extrahepatic Gla-proteins provided that severe bleeding is prevented. AIM: The aim of this work was to adapt an established protocol of vascular calcification by warfarin-induced inactivation of MGP as a calcification inhibitor, in an attempt to create a broader state of subclinical VK deficiency and to verify its safety. MATERIALS AND METHODS: Two consecutive experiments, each lasting 4 weeks, were required to modify the dosing schedule of warfa-rin and VK1 and to adapt it to the Wistar rats used. The original high doses of warfarin used initially had to be halved and the protective dose of VK1 to be doubled, in order to avoid treatment-induced hemorrhagic deaths. The second experiment aimed to confirm the efficacy and safety of the modified doses. To verify the VK deficiency, blood vessels were examined histologically for calcium deposits and serum osteocalcin levels were mea-sured. RESULTS: The original dosing schedule induced VK deficiency, manifested by arterial calcifications and dramatic changes in carboxyl-ated and uncarboxylated osteocalcin. The modified dosing regimen caused similar vascular calcification and no bleeding. CONCLUSION: The modified protocol of carefully balanced warfarin and VK1 doses is an effective and safe way to induce subclinical VK deficiency that can be implemented to investigate VK-dependent proteins like osteocalcin.
Assuntos
Anticoagulantes/toxicidade , Antifibrinolíticos/farmacologia , Artérias/efeitos dos fármacos , Modelos Animais de Doenças , Osteocalcina/efeitos dos fármacos , Ratos , Vitamina K 1/farmacologia , Vitamina K 2/metabolismo , Deficiência de Vitamina K/metabolismo , Varfarina/toxicidade , Animais , Artérias/patologia , Doenças Assintomáticas , Proteínas de Ligação ao Cálcio/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Carbono-Carbono Ligases/metabolismo , Proteínas da Matriz Extracelular/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Osteocalcina/metabolismo , Calcificação Vascular/patologia , Deficiência de Vitamina K/induzido quimicamente , Proteína de Matriz GlaRESUMO
BACKGROUND: The effects of C-type natriuretic peptide (CNP) and fibroblast growth factor (FGF)-23 appear to oppose each other during the process of bone formation, whereas few studies exist on the interaction between CNP and FGF-23. The main objective of the present study is to probe whether CNP is directly responsible for the regulation of osteoblast or via antagonizing FGF-23. METHODS: Osteoblasts were cultured in the absence or presence of CNP (0, 10, and 100 pmol/L) for 24 h, 48 h and 72 h, respectively. RESULTS: The findings of the present study indicated that: (1) CNP significantly stimulated osteoblastic proliferation and collagen (Col)-X expression; (2) both osteoblastic (osteocalcin, procollagen type I carboxy-terminal propeptide, total alkaline phosphatase and bone-specific alkaline phosphatase) and osteolytic (tartrate-resistant acid phosphatase and cross-linked carboxyterminal telopeptide of type I collagen) bone turnover biomarkers were up-regulated by CNP in osteoblasts; (3) FGF-23 mRNA and protein were significantly down-regulated at 24 h by CNP in osteoblasts, but the expression of FGF receptor-1/Klotho had no significant change. CONCLUSIONS: CNP stimulates osteoblastic proliferation and Col-X expression via the down-regulation of FGF-23 possibly in vitro. However, the specific mechanisms of the interaction between CNP and FGF-23 in osteoblasts are still unclear according to our findings. A further study on osteoblasts cultured with CNP and FGF-23 inhibitor will be undertaken in our laboratory.
Assuntos
Proliferação de Células/genética , Fatores de Crescimento de Fibroblastos/genética , Peptídeo Natriurético Tipo C/metabolismo , Osteoblastos/metabolismo , Fosfatase Alcalina/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/genética , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Colágeno Tipo X/efeitos dos fármacos , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Ensaio de Imunoadsorção Enzimática , Fatores de Crescimento de Fibroblastos/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/metabolismo , Imunofluorescência , Expressão Gênica , Regulação da Expressão Gênica , Glucuronidase/efeitos dos fármacos , Glucuronidase/genética , Glucuronidase/metabolismo , Técnicas In Vitro , Proteínas Klotho , Peptídeo Natriurético Tipo C/farmacologia , Osteoblastos/efeitos dos fármacos , Osteocalcina/efeitos dos fármacos , Osteocalcina/metabolismo , Osteogênese/genética , Fragmentos de Peptídeos/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Cultura Primária de Células , Pró-Colágeno/efeitos dos fármacos , Pró-Colágeno/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Fosfatase Ácida Resistente a Tartarato/efeitos dos fármacos , Fosfatase Ácida Resistente a Tartarato/metabolismoRESUMO
Objective: This study aims to investigate the beneficial effects of 17ß-estradiol supplementation on the function of osteoblastic cells through the Sirtuin-1/nuclear transcription factor-κB/matrix metalloproteinase-8 (Sirt1/NF-κB/MMP-8) pathway.Methods: Mouse primary osteoblasts were obtained from neonatal mouse calvaria, and the cells were treated with or without 17ß-estradiol. We first detected the effect of 17ß-estradiol on the function of osteoblastic cells. Then, the changes in estrogen receptor-α (ERα), Sirt1, NF-κB, and MMP-8 were determined after the osteoblasts were treated with 17ß-estradiol. During supplementation with 17ß-estradiol, knockdown of Sirt1 in osteoblasts was used to further measure the changes of NF-κB and MMP-8 and observe the cell function.Results: In primary osteoblastic cells, exposure to 17ß-estradiol improved cell viability and increased the levels of bone formation biomarkers, including osteocalcin, osteoprotegerin (OPG), procollagen type 1 N-terminal propeptide (P1NP), and alkaline phosphatase (ALP). In addition, 17ß-estradiol supplement activated ERα and Sirt1 expression and inhibited NF-κB and MMP-8 expression. Moreover, these effects induced by 17ß-estradiol were reversed by knockdown of Sirt1 in mouse primary osteoblasts.Conclusion: 17ß-Estradiol replacement therapy may treat postmenopausal osteoporosis by improving osteoblastic cell function via the Sirt1/NF-κB/MMP-8 pathway.
Assuntos
Estradiol/farmacologia , Metaloproteinase 8 da Matriz/metabolismo , NF-kappa B/metabolismo , Osteoblastos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismo , Fosfatase Alcalina/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Terapia de Reposição de Estrogênios/métodos , Feminino , Humanos , Camundongos , Modelos Animais , Osteoblastos/metabolismo , Osteocalcina/efeitos dos fármacos , Osteoporose Pós-Menopausa/tratamento farmacológico , Osteoprotegerina/efeitos dos fármacos , Fragmentos de Peptídeos/efeitos dos fármacos , Pró-Colágeno/efeitos dos fármacosRESUMO
Osteoporosis is a metabolic disease characterized by reduced osteoblast differentiation and proliferation. Oxidative stress plays a role in the pathogenesis of osteoporosis. Aucubin (AU), an iridoid glycoside, was previously shown to promote osteoblast differentiation. We investigated the effects of AU on MG63 human osteoblast-like cells treated with dexamethasone (Dex) or hydrogen peroxide (H2O2) to induce oxidative damage. AU protected MG63 cells against apoptosis, and promoted increased expression of cytokines associated with osteoblast differentiation, including collagen I, osteocalcin (OCN), osteopontin (OPN), and osterix. In Dex- and H2O2-treated MG63 cells, AU also enhanced the expression of anti-oxidative stress-associated factors in the nuclear respiratory factor 2 signaling pathway, including superoxide dismutases 1 and 2, heme oxygenases 1 and 2, and catalase. In vivo, using a Dex-induced mouse model of osteoporosis, AU promoted increased cortical bone thickness, increased bone density, and tighter trabecular bone. Additionally, it stimulated an increase in the expression of collagen I, OCN, OPN, osterix, and phosphorylated Akt and Smads in bone tissue. Finally, AU stimulated the expression of cytokines associated with osteoblast differentiation in bone tissue and serum. Our data indicate AU may have therapeutic efficacy in osteoporosis.
Assuntos
Osso e Ossos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Glucosídeos Iridoides/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoporose/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Linhagem Celular , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Dexametasona/efeitos adversos , Fator de Transcrição de Proteínas de Ligação GA/efeitos dos fármacos , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Glucocorticoides/efeitos adversos , Humanos , Peróxido de Hidrogênio/toxicidade , Camundongos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteocalcina/efeitos dos fármacos , Osteocalcina/metabolismo , Osteopontina/efeitos dos fármacos , Osteopontina/metabolismo , Osteoporose/induzido quimicamente , Osteoporose/patologia , Oxidantes/toxicidade , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas Smad/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Transcrição Sp7/efeitos dos fármacos , Fator de Transcrição Sp7/metabolismoRESUMO
OBJECTIVES: Alveolar bone osteoporosis has attracted more and more attention because of its profound impact on stomatognathic function and treatment, but current treatments have not been targeted to alveolar bone and might even cause severe side effects. Thus, identifying the effects of anti-osteoporosis agents on alveolar bone is essential. Icariin ameliorates metabolic dysfunction of long bones, but its effects on alveolar bone remain unclarified. MATERIALS AND METHODS: BMSCs were isolated from rat mandibles (mBMSCs). The osteogenic potential of mBMSCs and the signalling pathway involved under icariin treatment were measured by ALP and alizarin red staining, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence. Dual-luciferase assay, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation were used to investigate the molecular mechanism. Ovariectomized and sham-operated rats treated with or without icariin were analysed by micro-CT, TRAP staining and calcein double labelling. RESULTS: We found that icariin promoted osteoblast differentiation of mBMSCs. Furthermore, STAT3 was critical for icariin-promoted osteoblast differentiation, as indicated by increased phosphorylation levels in icariin-treated mBMSCs, while preventing STAT3 activation blocked icariin-induced osteoblast differentiation. Mechanistically, icariin-promoted transcription of the downstream osteogenic gene osteocalcin (Ocn) through STAT3 and STAT3 bound to the promoter of Ocn. Notably, icariin prevented the alveolar bone osteoporosis induced by oestrogen deficiency through promoting bone formation. CONCLUSIONS: For the first time, our work provides evidence supporting the potential application of icariin in promoting osteogenesis and treating alveolar bone osteoporosis.
Assuntos
Perda do Osso Alveolar/tratamento farmacológico , Estrogênios/metabolismo , Flavonoides/farmacologia , Osteogênese/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Perda do Osso Alveolar/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/efeitos dos fármacos , Osteocalcina/metabolismo , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/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
OBJECTIVE: Abnormal remodeling of subchondral bone (SB) induced by estrogen deficiency has been shown to be involved in osteoarthritis (OA). Raloxifene (RAL) is commonly used to treat postmenopausal osteoporosis (OP). However, little is known about its effects on OA combined with estrogen deficiency. This study was performed to evaluate the efficacy of RAL on patella baja-induced patellofemoral joint OA (PFJOA) in an ovariectomized rat model. DESIGN: Patellar ligament shortening (PLS) and ovariectomy (OVX) were performed simultaneously in 3-month-old female Sprague-Dawley rats, which were treated with RAL (10 mg/kg/day) or vehicle at 72 h postoperatively for 10 weeks. PFJOA was assessed by immunohistochemistry (IHC), real-time polymerase chain reaction (PCR), tartrate-resistant acid phosphatase (TRAP) staining, enzyme-linked immunosorbent assay (ELISA), micro-computed tomography (µCT), histomorphology and behavioral analyses. RESULTS: X-ray examinations showed that patella baja was successfully established by PLS. Histomorphological analysis revealed that PFJOA was significantly exacerbated by OVX and markedly alleviated by RAL. Moreover, RAL improved cartilage metabolism by decreasing MMP-13, ADAMTS-4, and caspase-3 and increasing Col-II and aggrecan at both the protein and mRNA levels. Furthermore, RAL markedly improved bone mass and SB microarchitecture and reduced osteoclast numbers and the serum osteocalcin and CTX-I levels. Although RAL showed a trend toward reducing pain sensitivity based on mechanical allodynia testing, this result was not statistically significant. CONCLUSION: These findings demonstrate that RAL treatment retards PFJOA progression in an ovariectomized rat model, suggesting that it may be a potential candidate for amelioration of the progression of PFJOA accompanied by postmenopausal OP.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Osteoartrite do Joelho/diagnóstico por imagem , Articulação Patelofemoral/efeitos dos fármacos , Cloridrato de Raloxifeno/farmacologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Proteína ADAMTS4/efeitos dos fármacos , Proteína ADAMTS4/genética , Proteína ADAMTS4/metabolismo , Agrecanas/efeitos dos fármacos , Agrecanas/genética , Agrecanas/metabolismo , Animais , Remodelação Óssea , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Caspase 3/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Contagem de Células , Colágeno Tipo I/sangue , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo II/efeitos dos fármacos , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Fêmur/diagnóstico por imagem , Fêmur/efeitos dos fármacos , Fêmur/metabolismo , Fêmur/patologia , Humanos , Imuno-Histoquímica , Metaloproteinase 13 da Matriz/efeitos dos fármacos , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Osteocalcina/sangue , Osteocalcina/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoporose Pós-Menopausa/tratamento farmacológico , Osteoporose Pós-Menopausa/metabolismo , Ovariectomia , Patela/diagnóstico por imagem , Patela/efeitos dos fármacos , Patela/metabolismo , Patela/patologia , Ligamento Patelar/cirurgia , Articulação Patelofemoral/diagnóstico por imagem , Articulação Patelofemoral/metabolismo , Articulação Patelofemoral/patologiaRESUMO
Osteoporosis is caused by disturbance in the dynamic balance of bone remodelling, a physiological process, vital for maintenance of healthy bone tissue in adult humans. In this process, a new bone is formed by osteoblasts and the pre-existing bone matrix is resorbed by osteoclasts. Imperatorin, a widely available and inexpensive plant extract with antioxidative and apoptotic effects, is reported to treat osteoporosis. However, the underlying mechanism and specific effects on bone metabolism have not been elucidated. In this study, we used rat bone marrow-derived mesenchymal stem cells and found that imperatorin can activate RUNX2, COL1A1 and osteocalcin by promoting the Ser9 phosphorylation of GSK3ß and entry of ß-catenin into the nucleus. Imperatorin also enhanced the production of phospho-AKT (Ser473), an upstream factor that promotes the Ser9 phosphorylation of GSK3ß. We used ipatasertib, a pan-AKT inhibitor, to inhibit the osteogenic effect of imperatorin, and found that imperatorin promotes osteogenesis via AKT/GSK3ß/ß-catenin pathway. Next, we used rat bone marrow-derived monocytes, to check whether imperatorin inhibits osteoclast differentiation via AKT/GSK3ß/ß-catenin pathway. Further, we removed the bilateral ovaries of rats to establish an osteoporotic model. Intragastric administration of imperatorin promoted osteogenesis and inhibited osteoclast in vivo. Our experiments showed that imperatorin is a potential drug for osteoporosis treatment.
Assuntos
Furocumarinas/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Colágeno Tipo I , Cadeia alfa 1 do Colágeno Tipo I , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/efeitos dos fármacos , Osteocalcina/metabolismo , Osteoclastos/metabolismo , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Via de Sinalização Wnt/fisiologiaRESUMO
Osteonecrosis of the femoral head (ONFH) is usually caused by chronic and excessive alcohol dependency, and this condition largely suppresses the osteogenic differentiation of bone mesenchymal stem cells (BMSCs). As a trimethyl derivative of glycine, betaine is an important human nutrient that regulates a series of vital biological processes, including oxidative stress, inflammatory responses, osteoblast differentiation and cellular apoptosis. However, no study has investigated the role of betaine in alcohol-induced ONFH. In this study, we hypothesized that betaine might have protective effects on ethanol-treated BMSCs and decrease the morbidity of alcohol-induced ONFH in a rat model. In vitro, we found that ethanol significantly downregulated the expression of osteocalcin (OCN), collagen 1 (COL1) and RUNX2 via activating the mammalian target of rapamycin (mTOR) signaling cascade. However, the inhibitory effects were rescued by betaine co-treatment at concentrations of 1 mM and 10 mM. In vivo, the typical ONFH pathological changes in a rat model of alcohol-induced ONFH were investigated by using multiple methods, including hematoxylin-eosin staining, micro-CT scans, TdT-mediated dUTP nick end labeling (TUNEL) assays and immunohistochemical staining for OCN and COL1. Osteonecrotic lesions of the femoral head could be alleviated by betaine as evidenced by significant histological and radiological improvements. Collectively, betaine plays a protective role against ethanol-induced suppression of osteogenesis and mineralization of hBMSCs and is thus a potential pharmacotherapy for alcohol-induced ONFH in vivo.
Assuntos
Betaína/farmacologia , Etanol/farmacologia , Cabeça do Fêmur/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Cabeça do Fêmur/metabolismo , Necrose da Cabeça do Fêmur/tratamento farmacológico , Necrose da Cabeça do Fêmur/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/efeitos dos fármacos , Osteocalcina/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Genetic studies have revealed a critical role of Distal-homeobox (Dlx) genes in bone formation, and our previous study showed that Dlx2 overexpressing in neural crest cells leads to profound abnormalities of the craniofacial tissues. The aim of this study was to investigate the role and the underlying molecular mechanisms of Dlx2 in osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) and pre-osteoblast MC3T3-E1 cells. Initially, we observed upregulation of Dlx2 during the early osteogenesis in BMSCs and MC3T3-E1 cells. Moreover, Dlx2 overexpression enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization in BMSCs and MC3T3-E1 cell line. In addition, micro-CT of implanted tissues in nude mice confirmed that Dlx2 overexpression in BMSCs promoted bone formation in vivo. Unexpectedly, Dlx2 overexpression had little impact on the expression level of the pivotal osteogenic transcription factors Runx2, Dlx5, Msx2, and Osterix, but led to upregulation of Alp and Osteocalcin (OCN), both of which play critical roles in promoting osteoblast maturation. Importantly, luciferase analysis showed that Dlx2 overexpression stimulated both OCN and Alp promoter activity. Through chromatin-immunoprecipitation assay and site-directed mutagenesis analysis, we provide molecular evidence that Dlx2 transactivates OCN and Alp expression by directly binding to the Dlx2-response cis-acting elements in the promoter of the two genes. Based on these findings, we demonstrate that Dlx2 overexpression enhances osteogenic differentiation in vitro and accelerates bone formation in vivo via direct upregulation of the OCN and Alp gene, suggesting that Dlx2 plays a crucial role in osteogenic differentiation and bone formation.