RESUMEN
AIM: Vinpocetine (Vin) has long been used as a medicine to treat cerebrovascular disorders and as a dietary supplement to improve cognitive functions. Previous studies have revealed that the transcription factor nuclear factor kappa B (NF-κB) activity plays an important role in osteogenic differentiation of mesenchymal stem cells (MSC). Vin inhibits NF-κB-dependent inflammatory responses; however, the effect of Vin on the osteogenic differentiation of MSCs has not been reported. In this study, we aimed to the investigate effect of Vin on the osteogenic differentiation of rat bone marrow-derived MSCs (BMSCs). METHODS: We treated BMSCs with clinical plasma (0.17 µM) or higher concentrations (5 and 20 µM) of Vin with no significant effect on the cell viability. Alizarin Red S and alkaline phosphatase (ALP) stainings were used to evaluate mineralizations on days 14 and 21. Moreover, expressions of target genes were detected using qRT-PCR analysis. RESULTS: Osteogenic differentiation of BMSCs did not significantly change with Vin's clinical plasma concentration, but significantly decreased with higher concentrations. Calcium mineralization, ALP staining and mRNA gene expressions of Runx2 and ALP were decreased significantly with high concentrations of Vin, paticularly on day 21. CONCLUSION: Our in vitro findings suggest that clinically relevant concentration of Vin seems safe to use in elderly patients with respect to osteoporosis. On the other hand, Vin at high concentrations appears to be harmful to bone homeostasis.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Alcaloides de la Vinca/sangre , Alcaloides de la Vinca/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Células de la Médula Ósea/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Regulación de la Expresión Génica/efectos de los fármacos , FN-kappa B/efectos de los fármacos , RatasRESUMEN
OBJECTIVE: We examined the effects of photobiomodulation (PBM) on stereological parameters, and gene expression of Runt-related transcription factor 2 (RUNX2), osteocalcin, and receptor activator of nuclear factor kappa-B ligand (RANKL) in repairing tissue of tibial bone defect in streptozotocin (STZ)-induced type 1 diabetes mellitus (TIDM) in rats during catabolic response of fracture healing. BACKGROUND DATA: There were conflicting results regarding the efficacy of PBM on bone healing process in healthy and diabetic animals. MATERIALS AND METHODS: Forty-eight rats have been distributed into four groups: group 1 (healthy control, no TIDM and no PBM), group 2 (healthy test, no TIDM and PBM), group 3 (diabetic control, TIDM and no PBM), and group 4 (diabetic test, no TIDM and PBM). TIDM was induced in the groups 3 and 4. A partial bone defect in tibia was made in all groups. The bone defects of groups second and fourth were irradiated by a laser (890 nm, 80 Hz, 1.5 J/cm2). Thirty days after the surgery, all bone defects were extracted and were submitted to stereological examination and real-time polymerase chain reaction (RT-PCR). RESULTS: PBM significantly increased volumes of total callus, total bone, bone marrow, trabecular bone, and cortical bone, and the numbers of osteocytes and osteoblasts of callus in TIDM rats compared to those of callus in diabetic control. In addition, TIDM increased RUNX2, and osteocalcin in callus of tibial bone defect compared to healthy group. PBM significantly decreased osteocalcin gene expression in TIDM rats. CONCLUSIONS: PBM significantly increased many stereological parameters of bone repair in an STZ-induced TIDM during catabolic response of fracture healing. Further RT-PCR test demonstrated that bone repair was modulated in diabetic rats during catabolic response of fracture healing by significant increase in mRNA expression of RUNX2, and osteocalcin compared to healthy control rats. PBM also decreased osteocalcin mRNA expression in TIDM rats.
Asunto(s)
Curación de Fractura/efectos de la radiación , Terapia por Luz de Baja Intensidad , Osteotomía , Tibia/efectos de la radiación , Fracturas de la Tibia/radioterapia , Animales , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/complicaciones , Modelos Animales de Enfermedad , Femenino , Curación de Fractura/fisiología , Osteocalcina/biosíntesis , Ligando RANK/biosíntesis , Ratas , Ratas Wistar , Tibia/fisiopatología , Fracturas de la Tibia/complicaciones , Fracturas de la Tibia/fisiopatología , Fracturas de la Tibia/terapiaRESUMEN
Mesenchymal stem cell (MSC) dysfunction has been implicated in the pathogenesis of osteoporosis. MSCs derived from osteoporotic subjects demonstrate significant impairment in proliferation, adhesion and chemotaxis, and osteogenic differentiation, leading to reduced functional bone-forming osteoblasts and ultimately nett bone loss and osteoporosis. Epimedium herbs and its active compound Icaritin (ICT) have been used in Chinese ethnopharmacology for the treatment of metabolic bone diseases. Using an in-vitro cell culture model, we investigated the benefits of ICT treatment in enhancing MSC proliferation, migration and osteogenic differentiation, and provide novel data to describe its mechanism of action. ICT enhances MSC proliferation, chemotaxis to stromal cell-derived factor-1 (SDF-1) and osteogenic differentiation through the activation of signal transduction activator transcription factor 3 (STAT-3), with a consequential up-regulation in the expression and activity of cysteine (C)-X-C motif chemokine receptor 4 (CXCR4). These findings provide a strong basis for future clinical studies to confirm the therapeutic potential of ICT for the prevention and treatment of osteoporosis and fragility fractures.
Asunto(s)
Flavonoides/farmacocinética , Células Madre Mesenquimatosas/efectos de los fármacos , Receptores CXCR4/fisiología , Factor de Transcripción STAT3/fisiología , Calcio/análisis , Adhesión Celular , Diferenciación Celular , División Celular , Movimiento Celular , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Evaluación Preclínica de Medicamentos , Humanos , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/citología , Osteoporosis/tratamiento farmacológico , Fosforilación , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptores CXCR4/biosíntesis , Receptores CXCR4/genética , Factor de Transcripción STAT3/antagonistas & inhibidores , Transcripción Genética , Regulación hacia ArribaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Aconiti Sinomontani Radix is frequently used in the treatment of Bi syndrome in traditional Chinese medicine. Several reports indicate that Aconiti Sinomontani Radix has therapeutic effects for rheumatoid arthritis (RA). However, the cellular mode of action is still unclear. To investigate the effect of alkaloid extracts of Aconiti Sinomontani Radix on proliferation and migration of human synovial sarcoma SW982 cells as well as the molecular mechanism underlying. MATERIALS AND METHODS: SW982 cells were examined for proliferation by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. Wound scratch assays were performed to assess the migrated rate of SW982 cells. Quantitative real-time PCR was used to measure the mRNA expression levels of Wnt5a, Runx2, MMP3, and Bmp2. Western blotting was used to measure the phosphorylated levels of JNK and NF-κB as well as the expression of MMP3. RESULTS: The alkaloid extract from Aconiti Sinomontani Radix (MQA) and MQB, which removed lappaconitine from MQA significantly inhibited the proliferation of SW982 in a dose-dependent manner. The proliferation inhibitory effect of MQB was more potent. Incubation with 10µg/ml MQB for 12, 24, and 36h inhibited the migration of SW982 cells by 83%, 58%, and 42%, respectively. Treatment with different concentrations of MQB for 24h inhibited mRNA expression of Wnt5a, Runx2, and MMP3, but Bmp2 mRNA expression was elevated by MQB. Further, MQB inhibited phosphorylation of JNK and NF-κB p65 as well as MMP3 expression by Western blotting analysis. CONCLUSION: The results showed that MQB inhibited proliferation and migration of SW982 cells possibly through suppressing Wnt5a-mediated JNK and NF-κB pathways. These results indicated that MQB might be an active extract of Aconiti Sinomontani Radix for targeting fibroblast-like synoviocytes (FLS) and be potential for RA therapy.
Asunto(s)
Aconitum/química , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Extractos Vegetales/farmacología , Sinoviocitos/citología , Sinoviocitos/efectos de los fármacos , Proteína Morfogenética Ósea 2/biosíntesis , Línea Celular , Ensayos de Migración Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Relación Dosis-Respuesta a Droga , Fibroblastos/citología , Expresión Génica/efectos de los fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Metaloproteinasa 3 de la Matriz/biosíntesis , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Proteína Wnt-5a/biosíntesisRESUMEN
BACKGROUND/AIMS: Mechanical loading plays an important role in the regulation of bone mass. However, bone cells are not always under physiological stress. In some cases, bone tissue is subjected to an overloaded mechanical environment. For example, a person who is weight training and a stevedore often experience bone pain, inflammation and other bone fatigue damage symptoms. Icariin is the major ingredient of Herba epimedii, which has been widely used for the treatment of bone injury in traditional Chinese medicine, but its mechanism remains unknown. The aim of this study was to probe the effect of icariin on the proliferation and differentiation of osteoblasts exposed to overload and to determine whether the Wnt/ß-catenin signalling pathway is involved in the drug response in osteoblasts. METHODS: Mouse MC3T3-E1 cells were exposed to mechanical tensile strain using a four- point bending device to create an overload damage model. An MTT assay was performed to determine the effects of icariin on MC3T3-E1 cell proliferation. The mRNA and protein levels of ALP, COL-I, OCN, RUNX2 and ß-catenin were assessed using RT-PCR and immunoblotting. The protein levels of ß-catenin in the MC3T3-E1 cells were also determined using fluorescence microscopy. The mineralization of osteoblasts was assessed using Alizarin Red S staining. RESULTS: We found that icariin enhanced the proliferation of osteoblasts exposed to overload and promoted MC3T3-E1 cell differentiation and mineralization. Furthermore, the gene and protein expression levels of ß-catenin and RUNX2 all increased with icariin treatment compared with those in the damage group. CONCLUSION: Our study suggested that icariin promotes proliferation and differentiation in osteoblasts exposed to overload. The effect of icariin on osteoblastic differentiation acted by activating the RUNX2 promoter and the Wnt/ß- catenin pathway.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Flavonoides/farmacología , Modelos Biológicos , Osteoblastos/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Animales , Línea Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Ratones , Osteoblastos/citología , beta Catenina/biosíntesisRESUMEN
Cell-sheet tissue engineering retains the benefits of an intact extracellular matrix (ECM) and can be used to produce scaffold-free constructs. Adipose tissue-derived stem cells (ASCs) are multipotent and more easily obtainable than the commonly used bone marrow-derived stem cells (BMSCs). Although BMSC cell sheets have been previously reported to display multipotentiality, a detailed study of the development and multilineage potential of ASC cell sheets (ASC-CSs) is non-existent in the literature. The aims of this study were to temporally profile: (a) the effect of hyperconfluent culture duration on ASC-CSs development; and (b) the multipotentiality of ASC-CSs by differentiation into the osteogenic, adipogenic and chondrogenic lineages. Rabbit ASCs were first isolated and cultured until confluence (day 0). The confluent cells were then cultured in ascorbic acid-supplemented medium for 3 weeks to study cell metabolic activity, cell sheet thickness and early differentiation gene expressions at weekly time points. ASC-CSs and ASCs were then differentiated into the three lineages, using established protocols, and assessed by RT-PCR and histology at multiple time points. ASC-CSs remained healthy up to 3 weeks of hyperconfluent culture. One week-old cell sheets displayed upregulation of early differentiation gene markers (Runx2 and Sox9); however, subsequent differentiation results indicated that they did not necessarily translate to an improved phenotype. ASCs within the preformed cell sheet groups did not differentiate as efficiently as the non-hyperconfluent ASCs, which were directly differentiated. Although ASCs within the cell sheets retained their differentiation capacity and remained viable under prolonged hyperconfluent conditions, future applications of ASC-CSs in tissue engineering should be considered with care. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Tejido Adiposo/metabolismo , Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , Tejido Adiposo/citología , Animales , Antígenos de Diferenciación/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Células Madre Mesenquimatosas/citología , Conejos , Factor de Transcripción SOX9/biosíntesis , Regulación hacia ArribaRESUMEN
BACKGROUND: Vascular calcification (VC) is closely related to cardiovascular events in chronic kidney disease (CKD). Apelin has emerged as a potent regulator of cardiovascular function, but its role in VC during CKD remains unknown. We determined whether apelin plays a role in phosphate-induced mineralization of human aortic smooth muscle cells (HASMCs) and in adenine-induced CKD rats with aortic calcification. METHODS AND RESULTS: In vitro, apelin-13 was found to inhibit calcium deposition in HASMCs (Pi(+) Apelin(+) group vs Pi(+) Apelin(-) group: 50.1 ± 6.21 ug/mg vs 146.67 ± 10.02 ug/mg protein, p = 0.012) and to suppress the induction of the osteoblastic transformation genes BMP-2, osteoprotegerin (OPG) and Cbfa1. This effect was mediated by interference of the sodium-dependent phosphate cotransporter (Pit-1) expression and phosphate uptake. In vivo, decreased plasma apelin levels (adenine(+) apelin(-) vs vehicle: 0.37 ± 0.09 ng/ml vs 0.68 ± 0.16 ng/ml, p = 0.003) and downregulation of APJ in the aorta were found in adenine-induced CKD rats with hyperphosphatemia (adenine(+) apelin(-) vs vehicle: 6.91 ± 0.23 mmoL/L vs 2.3 ± 0.07 mmoL/L, p = 0.001) and aortic calcification. Exogenous supplementation of apelin-13 normalized the level of the apelin/APJ system and significantly ameliorated aortic calcification, as well as the suppression of Runx2, OPG and Pit-1 expression. CONCLUSIONS: Apelin ameliorates VC by suppressing osteoblastic differentiation of VSMCs through downregulation of Pit-1. These results suggest apelin may have potential therapeutic value for treatment of VC in CKD.
Asunto(s)
Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/farmacología , ARN/genética , Insuficiencia Renal Crónica/complicaciones , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Calcificación Vascular/prevención & control , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Aorta Torácica/patología , Western Blotting , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Inmunohistoquímica , Ligandos , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Osteoprotegerina/biosíntesis , Osteoprotegerina/efectos de los fármacos , Osteoprotegerina/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/biosíntesis , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/efectos de los fármacos , Calcificación Vascular/etiología , Calcificación Vascular/metabolismoRESUMEN
Osteoarthritis (OA) is the most common cause of joint chronic pain and involves the entire joints. Subchondral osteoarthritic osteoblasts present a mineralization defect and, to date, only a few molecules (Vitamin D3 and Bone Morphogenetic Protein2) could improve the mineralization potential of this cell type. In this context, we have tested for the first time the effect of nacre extract on the mineralization capacity of osteoblasts from OA patients. Nacre extract is known to contain osteogenic molecules which have demonstrated their activities notably on the MC3T3 pre-osteoblastic cell line. For this goal, molecules were extracted from nacre (ESM, Ethanol Soluble Matrix) and tested on osteoblasts of the subchondral bone from OA patients undergoing total knee replacement and on MC3T3 cells for comparison. We chose to investigate the mineralization with Alizarin Red staining and with the study of extracellular matrix (ECM) structure and composition. In a complementary way the structure of the ECM secreted during the mineralization phase was investigated using second harmonic generation (SHG). Nacre extract was able to induce the early presence (after 7 days) of precipitated calcium in cells. Raman spectroscopy and electron microscopy showed the presence of nanograins of an early crystalline form of calcium phosphate in OA osteoblasts ECM and hydroxyapatite in MC3T3 ECM. SHG collagen fibers signal was present in both cell types but lower for OA osteoblasts. In conclusion, nacre extract was able to rapidly restore the mineralization capacity of osteoarthritis osteoblasts, therefore confirming the potential of nacre as a source of osteogenic compounds.
Asunto(s)
Calcificación Fisiológica/efectos de los fármacos , Fosfatos de Calcio/metabolismo , Nácar/farmacología , Osteoartritis/metabolismo , Osteoblastos/metabolismo , Células 3T3 , Animales , Artroplastia de Reemplazo de Rodilla , Línea Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Durapatita/metabolismo , Matriz Extracelular/metabolismo , Humanos , Ratones , Microscopía Electrónica de Rastreo , Osteocalcina/biosíntesis , Osteopontina/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría RamanRESUMEN
Metastasis is the most insidious aspect of breast cancer, but effective strategies to control this malignant process are still lacking. In previous studies, we screened over 200 extracts from plants of genus Chloranthaceae by bioactivity-guided fractionation, and found that Codonolactone (CLT) exhibited potential antimetastatic properties in breast cancer cells. This sesquiterpene lactone was isolated from Chloranthus henryi Hemsl, and is also found in other medical herbs, such as Codonopsis pilosula, Atractylodes macrocephala Koidz and others. Here, we report that CLT inhibited the ability of invasion and migration in metastatic breast cancer cells. Furthermore, CLT exhibited significant suppression on formation of lung metastatic foci of breast cancer in vivo. We next investigated the mechanism of CLT-induced metastasis inhibitory effects in breast cancer cells. A significant inhibition on activity and expression of MMP-9 and MMP-13 was observed. Moreover, data from western blotting, Runx2 transcription factor assay and chromatin immunoprecipitation assay showed that binding ability of Runx2 to sequences of the mmp-13 promoter was inhibited by CLT. Collectively, these findings suggested that the antimetastatic properties of CLT in breast cancer were due to the inhibition of MMPs, which might be associated with a downregulation of Runx2 transcriptional activity.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Lactonas/administración & dosificación , Sesquiterpenos/administración & dosificación , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lactonas/química , Extractos Vegetales/administración & dosificación , Extractos Vegetales/química , Sesquiterpenos/química , Activación Transcripcional/efectos de los fármacos , Viridiplantae/químicaRESUMEN
The proliferation and osteogenic capacity of mesenchymal stem cells (MSCs) needs to be improved for their use in cell-based therapy for osteoporosis. (-)-Epigallocatechin-3-gallate (EGCG), one of the green tea catechins, has been widely investigated in studies of osteoblasts and osteoclasts. However, no consensus on its role as an osteogenic inducer has been reached, possibly because of the various types of cell lines examined and the range of concentrations of EGCG used. In this study, the osteogenic effects of EGCG are studied in primary human bone-marrow-derived MSCs (hBMSCs) by detecting cell proliferation, alkaline phosphatase (ALP) activity and the expression of relevant osteogenic markers. Our results show that EGCG has a strong stimulatory effect on hBMSCs developing towards the osteogenic lineage, especially at a concentration of 5 µM, as evidenced by an increased ALP activity, the up-regulated expression of osteogenic genes and the formation of bone-like nodules. Further exploration has indicated that EGCG directes osteogenic differentiation via the continuous up-regulation of Runx2. The underlying mechanism might involve EGCG affects on osteogenic differentiation through the modulation of bone morphogenetic protein-2 expression. EGCG has also been found to promote the proliferation of hBMSCs in a dose-dependent manner. This might be associated with its antioxidative effect leading to favorable amounts of reactive oxygen species in the cellular environment. Our study thus indicates that EGCG can be used as a pro-osteogenic agent for the stem-cell-based therapy of osteoporosis.
Asunto(s)
Células de la Médula Ósea/citología , Catequina/análogos & derivados , Células Madre Mesenquimatosas/citología , Osteogénesis/efectos de los fármacos , Fosfatasa Alcalina/biosíntesis , Antioxidantes/farmacología , Proteína Morfogenética Ósea 2/biosíntesis , Catequina/farmacología , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Proliferación Celular/efectos de los fármacos , Tratamiento Basado en Trasplante de Células y Tejidos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Humanos , Osteogénesis/genética , Osteoporosis/terapia , Té/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidoresRESUMEN
AIMS: High cardiovascular mortality in patients with end-stage renal disease is closely associated with arterial medial calcification (AMC) caused by hyperphosphatemia, the mechanism of which associated hormones (FGF-23, klotho) and osteochondrogenic events is unclear. We examined the effect of Lanthanum carbonate on AMC via regulating the abnormalities in phosphorus metabolism of uremic rats. MAIN METHODS: 45 healthy SD rats were randomly divided into 3 groups: Normal group (n=15), CRF group (n=15), CRF diet supplemented with 2% La (n=15). AMC in great arteries were evaluated by VonKossa. Osteochondrogenic specific genes were analyzed by Immunohistochemistry and qRT-PCR. Serum FGF-23 and klotho levels were detected by ELISA kit. KEY FINDINGS: Serum phosphate was markedly increased in CRF group (6.94 ± 0.97 mmol/L) and 2%La group (5.12 ± 0.84 mmol/L) at week 4, while the latter became hypophosphatemic (2.92 ± 0.73 mmol/L vs CRF group, p<0.01) at week 10. Inhibitory effect of 2%La on development of AMC was reflected by downregulated Runx2, Osterix, BSP, Osteocalcin and collagenII and a reduction of FGF-23 at week 4(vs CRF group, p<0.01) but not week 10. SIGNIFICANCE: Beneficial effects of Lanthanum carbonate on progression of AMC in CRF could be mainly due to the decreased phosphate retention and FGF-23 in early stage and likewise a reduction of bone-associated proteins via osteochondrogenic pathway. Lanthanum carbonate has no effect on soluble klotho and serum FGF-23 in late stage of CRF.
Asunto(s)
Calcinosis/prevención & control , Lantano/uso terapéutico , Fósforo Dietético/efectos adversos , Uremia/tratamiento farmacológico , Animales , Calcinosis/sangre , Calcinosis/complicaciones , Colágeno Tipo II/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Regulación hacia Abajo/efectos de los fármacos , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/biosíntesis , Hiperfosfatemia/sangre , Hiperfosfatemia/tratamiento farmacológico , Hiperfosfatemia/etiología , Sialoproteína de Unión a Integrina/biosíntesis , Masculino , Osteocalcina/biosíntesis , Fosfatos/sangre , Ratas , Factores de Transcripción/biosíntesis , Túnica Media/patología , Uremia/sangre , Uremia/complicaciones , Uremia/patologíaRESUMEN
BACKGROUND AND AIMS: The therapeutic effects of pulsed electromagnetic fields (PEMFs) on osteoporosis have been documented. However, the precise mechanisms by which PEMFs elicit these favorable biological responses are still not fully understood. This study aimed to systematically investigate the effects of PEMFs on bone mass and Wnt/ß-catenin signaling pathway in ovariectomized rats. METHODS: Thirty 3-month-old female Sprague Dawley rats were randomly assigned to one of three groups: sham-operated control (sham), ovariectomy (OVX), and ovariectomy with PEMFs treatment (PEMFs). One week following ovariectomy surgery, rats in the PEMFs group were exposed to PEMFs for 40 min/day, 5 days/week, for 12 weeks. RESULTS: After 12-week interventions, serum 17ß-estradiol and bone-specific alkaline phosphatase levels increased in the PEMFs group. Bone mineral density of the femur and the fifth lumbar vertebral body also increased in the PEMFs group. Histomorphometrical studies showed that PEMFs improved trabecular area, trabecular width, and trabecular number by 77.50%, 17.38% and 51.06%, respectively, and reduced trabecular separation by 44.28% compared with the OVX group. Biomechanical studies showed that PEMFs increased maximum load and energy to failure in the fifth lumbar vertebral body. Quantitative real-time RT-PCR analysis showed that PEMFs increased the mRNA expressions of Wnt3a, low-density lipoprotein receptor-related protein 5(LRP5), ß-catenin, c-myc and runt-related gene 2 (Runx2), and reduced dickkopf1 (DKK1) in ovariectomized rats. However, mRNA expression of Axin2 was not affected by PEMFs. CONCLUSIONS: PEMFs can prevent ovariectomy-induced bone loss and deterioration of bone microarchitecture and strength, at least partly, through activation of Wnt/ß-catenin signaling pathway.
Asunto(s)
Densidad Ósea/efectos de la radiación , Terapia por Estimulación Eléctrica , Magnetoterapia , Transducción de Señal/efectos de la radiación , Vía de Señalización Wnt/efectos de la radiación , Absorciometría de Fotón , Fosfatasa Alcalina/sangre , Animales , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Modelos Animales de Enfermedad , Estradiol/sangre , Femenino , Fémur/efectos de la radiación , Fémur/ultraestructura , Regulación de la Expresión Génica/efectos de la radiación , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/biosíntesis , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Vértebras Lumbares/efectos de la radiación , Vértebras Lumbares/ultraestructura , Ovariectomía , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Proteína Wnt3A/biosíntesis , Proteína Wnt3A/genéticaRESUMEN
Osteogenic differentiation of vascular smooth muscle cells (VSMCs) results in medial artery calcification, which is common in diabetes, but the pathogenesis is poorly understood. We aimed to explore the pathophysiological roles of insulin resistance (IR) on medial artery calcification in rats with 10% fructose in drinking water. After 12 weeks of fructose feeding, rats showed severe IR, with increased levels of fasting blood glucose, serum insulin and oral glucose tolerance test (OGTT). Fructose-fed rats showed aortic calcification, increased aortic calcium deposition and irregular elastic fibers in the medial layer of the vessel wall. Moreover, plasma phosphorus concentration, calcium × phosphorus product and alkaline phosphatase (ALP) activity, and aortic calcium content and ALP activity were significantly increased. Fructose feeding increased mRNA levels of osteopontin, type III sodium-dependent phosphate co-transporter, bone morphogenetic protein-2 and the key transcription factor core binding factor alpha 1 in aortic tissue and downregulated mRNA levels of osteoprotegerin and matrix γ-carboxyglutamic acid protein. Fructose feeding decreased protein levels of smooth-muscle lineage markers and induced severe lipid peroxidation injury. IR induced by high fructose feeding could evoke osteogenic transdifferentiation of VSMCs and promote vascular calcification.
Asunto(s)
Aorta Torácica/patología , Carbohidratos de la Dieta/administración & dosificación , Fructosa/administración & dosificación , Resistencia a la Insulina , Músculo Liso Vascular/patología , Calcificación Vascular/patología , Calcificación Vascular/fisiopatología , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/metabolismo , Animales , Glucemia/metabolismo , Proteínas Morfogenéticas Óseas/biosíntesis , Proteínas Morfogenéticas Óseas/genética , Calcio/análisis , Proteínas de Unión al Calcio/biosíntesis , Diferenciación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Proteínas de la Matriz Extracelular/biosíntesis , Prueba de Tolerancia a la Glucosa , Insulina/sangre , Peroxidación de Lípido , Masculino , Músculo Liso Vascular/metabolismo , Osteopontina/biosíntesis , Osteopontina/genética , Osteoprotegerina/biosíntesis , Fósforo/sangre , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/biosíntesis , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Túnica Media/patología , Proteína Gla de la MatrizRESUMEN
Astragaloside IV (ASI), a pure compound derived from Radix Astragali, is commonly used in degenerative bone diseases such as osteoporosis. Our previous study identified in vivo the osteogenetic effect of Fu Fang Qi She Pills (FFQSP), a Chinese herbal formula containing Radix Astragali from which ASI was extracted. In this study, we investigated the osteogenetic effects of ASI under the conditions of centrifugating pressure on OCT-1 cells. These preosteoblasts were grown in 3D-culture, and treated with ASI at 50 micromol/l with centrifugation at 200 rpm, 500 rpm for 3 and 5 days. Morphocytological examination, morphometry of alkaline phosphatases (ALP) staining was performed. Expression of type I collagen (Col I) was detected by immunocytochemistry assays. ALP, Col1a2, Osteocalcin (OC), and runt-related transcription factor-2 (Runx2) mRNA expression were determined via real-time PCR. The results showed ASI plus 500 rpm for 3 days and ASI plus 200 rpm for 5 days significantly induced osteogenesis related protein and gene expression. We concluded that ASI would promote osteogenesis when cells of preosteoblast OCT-1 were subjected to proper centrifugating pressure and a pertinent period of time.
Asunto(s)
Osteogénesis/efectos de los fármacos , Saponinas/farmacología , Triterpenos/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Línea Celular , Centrifugación , Colágeno Tipo I/biosíntesis , Colorantes , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Técnica del Anticuerpo Fluorescente , Osteocalcina/biosíntesis , Postura , ARN/biosíntesis , ARN/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sales de Tetrazolio , TiazolesRESUMEN
There has been a strong interest in searching for natural therapies for osteoporosis. Genistein, an isoflavone abundant in soy, and icariin, a prenylated flavonol glycoside isolated from Epimedium Herb, have both been identified to exert beneficial effects in preventing postmenopausal bone loss. However, the relative potency in osteogenesis between the individual phytoestrogen flavonoids remains unknown. The present study compared ability of genistein and icariin in enhancing differentiation and mineralization of cultured rat calvarial osteoblasts in vitro. Dose-dependent studies in osteoblast differentiation measuring alkaline phosphatase (ALP) activity revealed optimal concentrations of genistein and icarrin for stimulating osteogenesis to be both at 10(-5) M. Time course studies comparing the two compounds both at 10(-5) M demonstrated that icariin treatment always produced higher ALP activity, more and larger areas of CFU-F(ALP) colonies and mineralized nodules, more osteocalcin secretion, and calcium deposition, and a higher level of mRNA expression of osteogenesis-related genes COL1α2, BMP-2, OSX, and RUNX-2. However, they inhibited the proliferation of osteoblasts to a similar degree. In conclusion, although future in vivo studies are required to investigate whether icariin is more efficient in improving bone mass and/or preventing bone loss, our in vitro studies have demonstrated that icariin has a stronger osteogenic activity than genistein. In addition, while the prenyl group on C-8 of icariin could be the active group that takes part in osteoblastic differentiation and explains its greater potency in osteogenesis, mechanisms of action, and reasons for the relative potency of icariin versus genistein need to be further studied.
Asunto(s)
Calcificación Fisiológica/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Flavonoides/farmacología , Genisteína/farmacología , Osteoblastos/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Proteína Morfogenética Ósea 2/biosíntesis , Proteína Morfogenética Ósea 2/genética , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colágeno/biosíntesis , Colágeno/genética , Colágeno Tipo I , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Pruebas de Enzimas , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Cráneo/citología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Osteoarthritis (OA) is characterised by cartilage degradation and bone lesions. Subchondral bone may be involved in the pathogenesis of cartilage matrix breakdown. OBJECTIVE: To assess the role of bone remodelling in OA by studying the effect of bisphosphonate on OA development in mice with high bone remodelling. METHODS: Mice overexpressing Runx2 (Runx2-Tg) under the control of collagen type I that displayed high bone remodelling were used. Joint instability was performed by partial medial meniscectomy to induce OA. RESULTS: Six weeks after surgery, tibial cartilage of Runx2-Tg mice displayed an increased number of ADAMTS-4- and ADAMTS-5-expressing chondrocytes compared with controls (p<0.05). This increase was higher in Runx2-Tg mice than in wild-type mice, although their OA score did not differ (2.5+/-0.6 vs 2.4+/-0.2, P=NS). Pamidronate reduced the OA score in Runx2-Tg mice but not in wild-type littermates (1.2+/-0.5 vs 2.7+/-0.4; p<0.05) despite the reduction of bone resorption and of the expression of cartilage proteases in both genotypes. CONCLUSIONS: These findings support the hypothesis that the level of bone resorption influences cartilage metabolism and that inhibition might prevent the progression of OA. Targeting bone resorption might therefore provide an approach to the treatment of high bone resorbing forms of OA.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Conservadores de la Densidad Ósea/uso terapéutico , Remodelación Ósea/efectos de los fármacos , Resorción Ósea/prevención & control , Osteoartritis/tratamiento farmacológico , Proteínas ADAM/metabolismo , Animales , Artritis Experimental/etiología , Artritis Experimental/metabolismo , Artritis Experimental/fisiopatología , Conservadores de la Densidad Ósea/farmacología , Remodelación Ósea/fisiología , Resorción Ósea/complicaciones , Resorción Ósea/metabolismo , Resorción Ósea/fisiopatología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Difosfonatos/farmacología , Difosfonatos/uso terapéutico , Progresión de la Enfermedad , Evaluación Preclínica de Medicamentos , Epífisis/efectos de los fármacos , Epífisis/fisiopatología , Masculino , Meniscos Tibiales/cirugía , Ratones , Ratones Transgénicos , Osteoartritis/etiología , Osteoartritis/metabolismo , Osteoartritis/fisiopatología , Pamidronato , Proteoglicanos/metabolismoRESUMEN
The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.
Asunto(s)
Matriz Ósea/crecimiento & desarrollo , Expresión Génica/efectos de la radiación , Terapia por Luz de Baja Intensidad , Oseointegración/efectos de la radiación , Osteoblastos/efectos de la radiación , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/genética , Análisis de Varianza , Proteína Morfogenética Ósea 7/biosíntesis , Proteína Morfogenética Ósea 7/genética , Células Cultivadas/efectos de la radiación , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Humanos , Sialoproteína de Unión a Integrina/biosíntesis , Sialoproteína de Unión a Integrina/genética , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Láseres de Semiconductores/uso terapéutico , Osteoblastos/metabolismo , Osteocalcina/biosíntesis , Osteocalcina/genética , Osteopontina/biosíntesis , Osteopontina/genética , Osteoprotegerina/biosíntesis , Osteoprotegerina/genética , Ligando RANK/biosíntesis , Ligando RANK/genética , Estadísticas no Paramétricas , TitanioRESUMEN
The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.
Este estudo teve como objetivo investigar o efeito do laser diodo de gálio-alumÃnio-arsênio (GaAlAs) em células osteoblásticas humanas cultivadas sobre discos de Ti. Para tanto, células osteoblásticas foram obtidas por digestão enzimática de osso alveolar humano e cultivadas sobre discos de Ti por 17 dias. As células foram submetidas à irradiação no 3º e 7º dias na dose de 3 J/cm2 e comprimento de onda de 780 nm e células não irradiadas foram usadas como controle. A irradiação não alterou a proliferação celular, atividade de ALP e formação de matriz mineralizada. Microscopia por epifluorescência indicou que após 24 h da aplicação do laser, as culturas irradiadas apresentaram áreas sem células, que mais tarde foram repovoadas por células em fase de proliferação e menos diferenciadas. O laser aumentou a expressão gênica relativa da ALP, OC, BSP e BMP-7 e reduziu a de RUNX2, OPN e OPG. Os resultados indicam que a terapia com laser modula de forma complexa as respostas celulares, estimulando a diferenciação osteoblástica. Assim, é possÃvel sugerir possÃveis benefÃcios do laser na osseointegração de implantes de Ti apesar do efeito deletério à s células imediatamente após a irradiação.
Asunto(s)
Humanos , Matriz Ósea/crecimiento & desarrollo , Expresión Génica/efectos de la radiación , Terapia por Luz de Baja Intensidad , Oseointegración/efectos de la radiación , Osteoblastos/efectos de la radiación , Análisis de Varianza , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/genética , /biosíntesis , /genética , Células Cultivadas/efectos de la radiación , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Sialoproteína de Unión a Integrina/biosíntesis , Sialoproteína de Unión a Integrina/genética , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Láseres de Semiconductores/uso terapéutico , Osteoblastos/metabolismo , Osteocalcina/biosíntesis , Osteocalcina/genética , Osteopontina/biosíntesis , Osteopontina/genética , Osteoprotegerina/biosíntesis , Osteoprotegerina/genética , Ligando RANK/biosíntesis , Ligando RANK/genética , Estadísticas no Paramétricas , TitanioRESUMEN
Aceaea racemosa (formerly Cimicifuga racemosa, black cohosh, AR) extracts have been widely used as an alternative to hormonal replacement therapy for menopausal symptoms. Recent evidences suggest AR extracts are also effective in protecting against postmenopausal bone loss. To determine whether AR has any direct anabolic effect on osteoblasts, we investigated the ethanolic extract of AR on bone nodule formation in mouse MC3T3-E1 preosteoblast cells. AR did not stimulate osteoblast proliferation. Rather, at high doses of 1000 ng/mL for 48 h, AR suppressed (7.2+/-0.9% vs. control) osteoblast proliferation. At 500 ng/mL, a significant increase in bone nodule formation was seen with Von Kossa staining. Using quantitative PCR analysis, AR was shown to enhance the gene expression of runx2 and osteocalcin. Co-treatment with ICI 182,780, the selective estrogen receptor antagonist, abolished the stimulatory effect of AR on runx2 and osteocalcin gene induction, as well as on bone nodule formation in MC3T3-E1 cells. This is a first report of the direct effect of AR on enhancement of bone nodule formation in osteoblasts, and this action was mediated via an estrogen receptor-dependent mechanism. The results provide a scientific rationale at the molecular level for the claim that AR can offer effective prevention of postmenopausal bone loss.