RESUMEN
This study aims to investigate the therapeutic effect of icariin(ICA) on thioacetamide(TAA)-induced femoral osteolysis in rats. RAW264.7 cells were treated with TAA and ICA. Cell counting kit-8(CCK-8) assay was used to detect cell proliferation, and tartrate-resistant acid phosphatase(TRAP) staining to examine the formation of osteoclasts. The expression of TRAP, cathepsin K, c-FOS, and NFATc1 in RAW264.7 cells was determined by Western blot and immunofluorescence method. Thirty-two SD rats were randomized into the control group, TAA group(intraperitoneal injection of TAA at 300 mg·kg~(-1)), ICA group(gavage of ICA at 600 mg·kg~(-1)) and TAA + ICA group(intraperitoneal injection of TAA at 300 mg·kg~(-1) and gavage of ICA at 600 mg·kg~(-1)). Administration was performed every other day for 6 weeks. Body weight and length of femur were recorded at execution. Pathological injury and osteoclast differentiation of femur were observed based on hematoxylin-eosin(HE) staining and TRAP staining, and the changes of bone metabolism-related indexes alkaline phosphatase(ALP), calcium(Ca), phosphorus(P), magnesium(Mg), and cross-linked N-telopeptide of type â collagen(NTX-â ) in serum were detected. Three-point bending test and micro-CT were applied to evaluate the quality of femur, and Western blot to detect the levels of osteoclast-related proteins TRAP, cathepsin K, RANK, RANKL, p38, p-p38, ERK, p-ERK, JNK, p-JNK, c-Fos, and NFATc1. The results showed ICA could inhibit TAA-induced production of TRAP-positive cells, the expression of osteoclast-related proteins, and nuclear translocation of NFATc1. ICA alleviated the weight loss, reduction of femur length, and growth inhibition induced by TAA in SD rats. ICA ameliorated the decline of femur elastic modulus caused by TAA and significantly restored trabecular bone mineral density(BMD), trabecular pattern factor(Tb.Pf), trabecular number(Tb.N), trabecular thickness(Tb.Th), and structure model index(SMI), thus improving bone structure. Western blot results showed ICA suppressed femoral osteoclast differentiation induced by TAA through RANKL-p38/ERK-NFATc1 signaling pathway. ICA inhibits osteoclast differentiation and prevents TAA-induced osteolysis by down-regulating RANKL-p38/ERK-NFAT signaling pathway.
Asunto(s)
Resorción Ósea , Osteólisis , Ratas , Animales , Osteoclastos , Catepsina K/genética , Catepsina K/metabolismo , Catepsina K/farmacología , Tioacetamida/metabolismo , Tioacetamida/farmacología , Resorción Ósea/metabolismo , Resorción Ósea/patología , Osteólisis/metabolismo , Osteólisis/patología , Diferenciación Celular , Ratas Sprague-Dawley , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismoRESUMEN
Mature osteoclasts express the vitamin D receptor (VDR) and are able to respond to active vitamin D (1α, 25-dihydroxyvitamin D3; 1,25(OH)2D3) by regulating cell maturation and activity. However, the in vivo consequences of vitamin D signalling directly within functionally mature osteoclasts is only partially understood. To investigate the in vivo role of VDR in mature osteoclasts, conditional deletion of the VDR under control of the cathepsin K promoter (CtskCre/Vdr-/-), was assessed in 6 and 12-week-old mice, either under normal dietary conditions (NormCaP) or when fed a low calcium (0.03 %), low phosphorous (0.08 %) diet (LowCaP). Splenocytes from CtskCre/Vdr-/- mice were co-cultured with MLO-Y4 osteocyte-like cells to assess the effect on osteoclastogenesis. Six-week-old CtskCre/Vdr-/- mice demonstrated a 10 % decrease in vertebral bone volume (p < 0.05), which was associated with increased osteoclast size (p < 0.05) when compared to Vdrfl/fl control mice. Control mice fed a LowCaP diet exhibited extensive trabecular bone loss associated with increased osteoclast surface, number and size (p < 0.0001). Interestingly, CtskCre/Vdr-/- mice fed a LowCaP diet showed exacerbated loss of bone volume fraction (BV/TV%) and trabecular number (Tb.N), by a further 22 % and 21 %, respectively (p < 0.05), suggesting increased osteoclastic bone resorption activity with the loss of VDR in mature osteoclasts under these conditions. Co-culture of CtskCre/Vdr-/- splenocytes with MLO-Y4 cells increased resulting osteoclast numbers 2.5-fold, which were greater in nuclei density and exhibited increased resorption of dentine compared to osteoclasts derived from Vdrfl/fl splenocyte cultures. These data suggest that in addition to RANKL-mediated osteoclastogenesis, intact VDR signalling is required for the direct regulation of the differentiation and activity of osteoclasts in both in vivo and ex vivo settings.
Asunto(s)
Calcio de la Dieta/farmacología , Osteoclastos/fisiología , Osteoporosis/etiología , Receptores de Calcitriol/genética , Animales , Huesos/diagnóstico por imagen , Huesos/fisiopatología , Calcio/sangre , Catepsina K/genética , Catepsina K/metabolismo , Técnicas de Cocultivo , Masculino , Ratones Noqueados , Ratones Transgénicos , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteogénesis , Fósforo/metabolismo , Receptores de Calcitriol/metabolismo , Microtomografía por Rayos XRESUMEN
Panax ginseng has a wide range of activities including a neuroprotective effect, skin protective effects, enhanced DNA repairing, anti-diabetic activity, and protective effects against vascular inflammation. In the present study, we sought to discover the inhibitory effects of a mixture of natural products containing Panax ginseng, Ziziphus jujube, Rubi fructus, Artemisiae asiaticae and Scutellaria baicalensis (PZRAS) on osteoclastogenesis and bone remodeling, as neither the effects of a mixture containing Panax ginseng extract, nor its molecular mechanism on bone inflammation, have been clarified yet. PZRAS upregulated the levels of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSH-R) and glutathione peroxidase (GSH-Px) and reduced malondialdehyde (MDA) in LPS-treated RAW264.7 cells. Moreover, treatment with PZRAS decreased the production of IL-1ß and TNF-α. PZRAS also inhibited osteoclast differentiation through inhibiting osteoclastspecific genes like MMP-2, 9, cathepsin K, and TRAP in RANKL-treated RAW264.7 cells. Additionally, PZRAS has inhibitory functions on the RANKL-stimulated activation of ERK and JNK, which lead to a decrease in the expression of NFATc1 and c-Fos. In an in vivo study, bone resorption induced by LPS was recovered by treatment with PZRAS in bone volume per tissue volume (BV/TV) compared to control. Furthermore, the ratio of eroded bone surface of femurs was significantly increased in LPStreated mice compared to vehicle group, but this ratio was significantly reversed in PZRAS-treated mice. These results suggest that PZRAS could prevent or treat disorders with abnormal bone loss.
Asunto(s)
Resorción Ósea/prevención & control , Inflamación/prevención & control , Osteogénesis/efectos de los fármacos , Extractos Vegetales/farmacología , Animales , Catepsina K/genética , Catepsina K/metabolismo , Diferenciación Celular/efectos de los fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolisacáridos/toxicidad , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos ICR , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ligando RANK/genética , Ligando RANK/metabolismo , Células RAW 264.7 , Fosfatasa Ácida Tartratorresistente/genética , Fosfatasa Ácida Tartratorresistente/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The metabolicosteopathy known as postmenopausal osteoporosisiscaused by disruption of the balance between bone resorption and osteogenesis, processes that are mediated by osteoclasts and osteoblasts, respectively. The current therapeutic approaches to treating osteoporosis have several limitations. In this study, we demonstrated that the natural chemical compound isoalantolactone (IAL) could inhibit osteoclastogenesis, without affecting osteogenesis. This is the first study reporting a role of IAL in suppressing the receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast formation in a dose-dependent manner, and downregulating the expression of osteoclast-related marker genes. Furthermore, IAL abrogated the phosphorylation of c-Jun N-terminal kinase (JNK)/p38, NF-κB, and phosphatidylinositol 3-kinase (PI3K)-AKT, and also diminished the expression of osteoclastogenesis-related proteins. In conclusion, our results indicated that IAL has promise for the treatment of osteoporosis and other metabolicbone diseases.
Asunto(s)
Resorción Ósea/tratamiento farmacológico , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Sesquiterpenos/uso terapéutico , Actinas/metabolismo , Animales , Catepsina K/genética , Catepsina K/metabolismo , Células Cultivadas , Femenino , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Ovariectomía , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ligando RANK , Sesquiterpenos/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Cathepsin K is a protease known to be involved in not only bone remodeling and resorption, but also articular cartilage degradation that leads to osteoarthritis (OA). Hyaluronan (HA) plays a pivotal role in maintaining homeostasis within articular chondrocytes. Intra-articular supplementation of high molecular weight hyaluronan (HMW-HA) has been widely used in OA treatment. However, its prospective mechanism of action is still unclear. In this study, we examined the suppressive effect of HA on enhanced cathepsin K expression induced by mechanical stress loading. A human chondrocytic HCS-2/8 cells were cultured in silicon chambers and subjected to cyclic tensile stress (CTS) loading. CTS loading significantly increased messenger ribonucleic acid and protein expression of cathepsin K, which appeared to be suppressed by pre-treatment with HMW-HA. Activation of nuclear factor-kappa B (NF-κB) was induced by CTS loading, and suppressed by pre-treatment with HMW-HA. Helenalin, a chemical inhibitor of NF-κB, clearly suppressed the enhanced expression of cathepsin K, as well as NF-κB activation induced by CTS loading. The suppressive effect of HMW-HA on enhanced cathepsin K expression via NF-κB inhibition impacts the effectiveness of HMW-HA in OA treatment. Our findings provide new evidence supporting the biological effectiveness of intra-articular HMW-HA injections for treatment of OA.
Asunto(s)
Catepsina K/metabolismo , Condrocitos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/farmacología , FN-kappa B/metabolismo , Estrés Mecánico , Adyuvantes Inmunológicos/farmacología , Cartílago Articular/citología , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Catepsina K/genética , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Humanos , Receptores de Hialuranos/genética , FN-kappa B/genética , Transducción de SeñalRESUMEN
Osteoclasts are responsible for bone resorption during the process of bone remodeling. Increased osteoclast numbers and bone resorption activity are the main factors contributing to bone loss-related diseases such as osteoporosis. Therefore, modulating the formation and function of osteoclasts is critical for the effective treatment of osteolysis and osteoporosis. Kavain is the active ingredient extracted from the root of the kava plant, which possesses known anti-inflammatory properties. However, the effects of kavain on osteoclastogenesis and bone resorption remain unclear. In this study, we found that kavain inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and fusion using tartrate-resistant acid phosphatase staining and immunofluorescence. Furthermore, kavain inhibited bone resorption performed by osteoclasts. Using reverse transcription-polymerase chain reaction and western blot analysis, we found that kavain downregulates the expression of osteoclast marker genes, such as nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1), v-atpase d2 (Atp6v0d2), dendrocyte expressed seven transmembrane protein (Dcstamp), matrix metallopeptidase 9 (Mmp9), cathepsin K (Ctsk), and Acp5. Additionally, kavain repressed RANKL-induced calcium oscillations, nuclear factor of activated T cells activation, and mitogen-activated protein kinase phosphorylation, while leaving NF-κB unaffected. We found no effects of kavain on either osteoblast proliferation or differentiation. Besides, kavain inhibited bone loss in ovariectomized mice by suppressing osteoclastogenesis. Collectively, these data suggest a potential use for kavain as a candidate drug for the treatment of osteolytic diseases.
Asunto(s)
Resorción Ósea/tratamiento farmacológico , Proteína Quinasa 1 Activada por Mitógenos/genética , Factores de Transcripción NFATC/genética , Osteogénesis/efectos de los fármacos , Pironas/farmacología , Animales , Resorción Ósea/genética , Resorción Ósea/patología , Señalización del Calcio/efectos de los fármacos , Catepsina K/genética , Diferenciación Celular/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Kava/química , Metaloproteinasa 9 de la Matriz/genética , Ratones , FN-kappa B/química , FN-kappa B/genética , Osteogénesis/genética , Osteoporosis , Extractos Vegetales/química , Extractos Vegetales/farmacología , Raíces de Plantas/química , Pironas/química , Ligando RANK/genética , Células RAW 264.7 , Fosfatasa Ácida Tartratorresistente/genética , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
Pro-domain of a cysteine cathepsin contains a highly conserved Ex2Rx2Fx2Nx3Ix3N (ERFNIN) motif. The zymogen structure of cathepsins revealed that the Arg(R) residue of the motif is a central residue of a salt-bridge/H-bond network, stabilizing the scaffold of the pro-domain. Importance of the arginine is also demonstrated in studies where a single mutation (Arg â Trp) in human lysosomal cathepsin K (hCTSK) is linked to a bone-related genetic disorder "Pycnodysostosis". In the present study, we have characterized in vitro Arg â Trp mutant of hCTSK and the same mutant of hCTSL. The R â W mutant of hCTSK revealed that this mutation leads to an unstable zymogen that is spontaneously activated and auto-proteolytically degraded rapidly. In contrast, the same mutant of hCTSL is sufficiently stable and has proteolytic activity almost like its wild-type counterpart; however it shows an altered zymogen activation condition in terms of pH, temperature and time. Far and near UV circular dichroism and intrinsic tryptophan fluorescence experiments have revealed that the mutation has minimal effect on structure of the protease hCTSL. Molecular modeling studies shows that the mutated Trp31 in hCTSL forms an aromatic cluster with Tyr23 and Trp30 leading to a local stabilization of pro-domain and supplements the loss of salt-bridge interaction mediated by Arg31 in wild-type. In hCTSK-R31W mutant, due to presence of a non-aromatic Ser30 residue such interaction is not possible and may be responsible for local instability. These differences may cause detrimental effects of R31W mutation on the regulation of hCTSK auto-activation process compared to altered activation process in hCTSL.
Asunto(s)
Arginina/metabolismo , Catepsina K/metabolismo , Catepsina L/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arginina/química , Catepsina K/química , Catepsina K/genética , Catepsina L/química , Catepsina L/genética , Dicroismo Circular , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , Especificidad por SustratoRESUMEN
The present study aimed to investigate bone deterioration in glucocorticoidinduced osteoporosis (GIOP) mice, and the antiosteoporosis effect and underlying molecular mechanism of icariin. Dexamethasone (DSM) treatment was demonstrated to facilitate the induction of hypercalciuria in GIOP mice. Icariin treatment reversed the dexamethasone (DXM)induced disequilibrium of calcium homeostasis and bone resorption, and increased serum alkaline phosphatase, tartrate resistant acid phosphatase, osteocalcin and deoxypyridinoline. Haematoxylin and eosin staining revealed an increase in disconnections and separation in the trabecular bone network of the tibial proximal metaphysis, in the GIOP group. Icariin treatment reversed the DXMinduced trabecular deleterious effects, and stimulated bone remodeling in GIOP mice. Furthermore, the results demonstrated that the mRNA and protein expression of cathepsin K were significantly increased in GIOP mice, compared with the control group. Icariin treatment may suppress the expression of cathepsin K in the tibia of GIOP mice. The levels of microRNA (miR)186 were markedly reduced in the tibia of GIOP mice compared with control group; however, this was inhibited by icariin treatment. Bioinformatics analysis demonstrated that miR186 regulates cathepsin K via binding to the upstream 3'untranslated region. Furthermore, transfection with miR186 mimics resulted in inhibition of cathepsin K expression, whereas miR186 inhibitors facilitated cathepsin K expression in osteoclasts. In conclusion, the present study demonstrated the protective effects of icariin against bone deteriorations in the experimental GIOP mice, and the underlying mechanism was mediated, at least partially, via activation of miR186mediated suppression of cathepsin K. These results provide evidence to support the use of icariin as a therapeutic approach in the management of glucocorticoidinduced bone loss, and the disequilibrium of calcium homeostasis.
Asunto(s)
Conservadores de la Densidad Ósea/uso terapéutico , Huesos/efectos de los fármacos , Catepsina K/antagonistas & inhibidores , Medicamentos Herbarios Chinos/uso terapéutico , Flavonoides/uso terapéutico , MicroARNs/metabolismo , Osteoporosis/tratamiento farmacológico , Animales , Huesos/metabolismo , Huesos/patología , Catepsina K/genética , Catepsina K/metabolismo , Dexametasona , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Osteoporosis/inducido químicamente , Osteoporosis/metabolismo , Osteoporosis/patología , Células RAW 264.7RESUMEN
This study was designed to investigate the effect of 10-hydroxycamptothecin (10-HCPT) on osteoclast formation. RAW264.7 cells were cultured in vitro with 100 ng/ml receptor activator for nuclear factor-κ B ligand (RANKL) and 30 ng/ml recombinant macrophage colony stimulating factor (M-CSF), and 10-HCPT with different solubilities were added. After five-day cultivation, tartrate-resistant acid phosphatase (TRAP) staining was used to observe the number of osteoclasts. mRNA expression of osteoclast-specific genes, such as TRAP, cathepsin K (CTSK) and matrix metalloproteinase protease 9 (MMP-9), was detected by real-time Polymerase Chain Reaction (PCR). The effect of 10-HCPT on the proliferation activity of RAW264.7 cells was detected using Cell Counting Kit-8 (CCK-8). CCK-8 detection showed that 10-HCPT with a certain concentration (1 ng/ml to 5 ng/ml) had no effect on cell proliferation (P>0.05); 10-HCPT could inhibit the generation of osteoclasts. With the increase of the concentration of 10-HCPT, the number of osteoclasts generated from cells cultured with 10-HCPT [1 ng/ml (86±11.14), 2 ng/ml (66.67±7.51), 5ng/ml (27.67±6.51)] was much lower than that of the control group (145±8.19), and the difference was statistically significant (all P=0, P less than 0.05); mRNA expression of osteoclast-specific gene TRAP [1 ng/ml (24.38±0.68), 2 ng/ml (20.09±1.86), 5 ng/ml (6.23±0.53)], CTSK [1 ng/ml (10.08±0.81), 2 ng/ml (7.30±0.30), 5 ng/ml (3.20±0.56)] and MMP-9 [1 ng/ml (43.54±6.96), 2 ng/ml (28.28±5.83), 5 ng/ml (11.07±2.53)] was much lower than that of the groups added with RANKL and M-CSF only (all P=0, P less than 0.05), and with the increase of concentration of 10-HCPT, the expression of osteoclast-specific genes showed a decreasing tendency. All the findings suggest that 10-HCPT can inhibit the formation of osteoclasts by reducing the expression of osteoclast-specific genes such as TRAP, CTSK and MMP-9.
Asunto(s)
Antirreumáticos/farmacología , Camptotecina/análogos & derivados , Osteoclastos/citología , Células RAW 264.7/efectos de los fármacos , Inhibidores de Topoisomerasa I/farmacología , Animales , Artritis Reumatoide/tratamiento farmacológico , Camptotecina/farmacología , Catepsina K/biosíntesis , Catepsina K/genética , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/farmacología , Metaloproteinasa 9 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ligando RANK/farmacología , Células RAW 264.7/citología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Fosfatasa Ácida Tartratorresistente/biosíntesis , Fosfatasa Ácida Tartratorresistente/genéticaRESUMEN
Cathepsin K (CatK) is essential for osteoclast-mediated bone resorption. CatK expression is also detected in breast cancer cells that metastasize to bone. Here, the CatK inhibitor L-235 dosed in prevention (10, 30, and 100 mg/kg, p.o., b.i.d.) or treatment regimen (30 mg/kg) was compared with the bisphosphonate zoledronic acid (ZOL, 7.5 µg/kg/wk, s.c.) in the intratibial injection model of MDA-MB-231 breast carcinoma in nude rats. Progression of osteolysis, skeletal tumor burden, and local metastasis was evaluated by radiography through 42 days and ex vivo µCT and histology. IHC and RT-PCR confirmed the increases in CatK protein and mRNA levels in human breast cancer primary and metastatic tumors. In the experimental model of breast cancer bone metastasis, L-235 dosed in preventive mode resulted in a dose-related reduction of osteolysis of 72%, 75%, and 87% respectively, compared with ZOL by 86% versus intact. Similarly, L-235 significantly reduced intratibial tumor volume by 29%, 40%, and 63%, respectively, compared with 56% by ZOL versus vehicle. Efficacy of L-235 and ZOL on reduction of osteolytic lesions and tumor burden was comparable in treatment versus preventive regimens. All L-235 doses inhibited cortical disruption and extraskeletal tumor growth to a level comparable with ZOL. Assessment of local metastasis demonstrated that treatment with the CatK inhibitor was more effective than ZOL in reducing breast cancer invasion. These data support the role of CatK in breast cancer skeletal growth and metastasis and CatK inhibitors may represent a novel oral therapy for treatment of metastatic breast cancer.
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Antineoplásicos/farmacología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Catepsina K/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Animales , Antineoplásicos/administración & dosificación , Neoplasias Óseas/tratamiento farmacológico , Catepsina K/genética , Catepsina K/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/administración & dosificación , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Clasificación del Tumor , Metástasis de la Neoplasia , Osteólisis , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The pathogenesis of bone resorption in ß-thalassemia major is multifactorial and our understanding of the underlying molecular and cellular mechanisms remains incomplete. Considering the emerging importance of the endocannabinoid/endovanilloid system in bone metabolism, it may be instructive to examine a potential role for this system in the development of osteoporosis in patients with ß-thalassemia major and its relationship with iron overload and iron chelation therapy. This study demonstrates that, in thalassemic-derived osteoclasts, tartrate-resistant acid phosphatase expression inversely correlates with femoral and lumbar bone mineral density, and directly correlates with ferritin levels and liver iron concentration. The vanilloid agonist resiniferatoxin dramatically reduces cathepsin K levels and osteoclast numbers in vitro, without affecting tartrate-resistant acid phosphatase expression. The iron chelators deferoxamine, deferiprone and deferasirox decrease both tartrate-resistant acid phosphatase and cathepsin K expression, as well as osteoclast activity. Taken together, these data show that transient receptor potential vanilloid type 1 activation/desensitization influences tartrate-resistant acid phosphatase expression and activity, and this effect is dependent on iron, suggesting a pivotal role for iron overload in the dysregulation of bone metabolism in patients with thalassemia major. Our applied pharmacology provides evidence for the potential of iron chelators to abrogate these effects by reducing osteoclast activity. Whether iron chelation therapy is capable of restoring bone health in humans requires further study, but the potential to provide dual benefits for patients with ß-thalassemia major -preventing iron-overload and alleviating associated osteoporotic changes - is exciting.
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Sobrecarga de Hierro/fisiopatología , Osteoporosis/etiología , Osteoporosis/metabolismo , Canales Catiónicos TRPV/metabolismo , Talasemia beta/complicaciones , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Adulto , Biomarcadores/metabolismo , Western Blotting , Estudios de Casos y Controles , Catepsina K/genética , Catepsina K/metabolismo , Células Cultivadas , Femenino , Técnica del Anticuerpo Fluorescente , Estudios de Seguimiento , Humanos , Quelantes del Hierro/farmacología , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoporosis/tratamiento farmacológico , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/genética , Receptor Cannabinoide CB2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales Catiónicos TRPV/genética , Fosfatasa Ácida Tartratorresistente , Talasemia beta/metabolismoRESUMEN
OBJECTIVE: To characterise the effects of early and late electroacupuncture (EA) treatment on serum 17ß-oestradiol (E2), C-terminal cross-linking telopeptide of type I collagen (CTX-I), bone mineral density (BMD), biomechanical bone strength and mRNA expression of cathepsin K in ovariectomised (OVX) rats. METHODS: Sixty Sprague-Dawley rats underwent ovariectomy (n=40) or sham surgery (n=20) and were randomly divided into two batches. Batch 1 (n=30) consisted of 10 sham-operated rats (Sham-0 group) and 20 OVX rats: half commenced EA immediately (early EA group, n=10) and half were left untreated (OVX-0 group, n=10). Batch 2 (n=30) consisted of 10 sham-operated rats (Sham-12 group) and 20 OVX rats: half commenced EA treatment 12â weeks after ovariectomy (late EA group, n=10) and half were left untreated (OVX-12 group, n=10). Rats in batches 1 and 2 were killed after 12 and 24â weeks, respectively. Serum E2, CTX-I, BMD, bone strength and cathepsin K expression were determined by ELISA, dual energy X-ray absorptiometry, biomechanical assessment and qRT-PCR, respectively. RESULTS: Both early and late EA treatment increased serum E2 levels, reduced serum CTX- I levels and increased BMD and bone strength of the L5 vertebral body in OVX rats. Although early EA treatment similarly increased BMD and bone strength of the femur, late EA treatment did not. However, both early and late EA treatment reduced mRNA expression of cathepsin K in OVX rats. CONCLUSIONS: Early EA completely prevented and late EA partially prevented bone loss and deterioration of bone strength in OVX rats. The timing of initiation of EA treatment may be an important consideration for optimisation of effects. The influence of EA on bone strength appears to be at least partially mediated through regulation of the expression of cathepsin K.
Asunto(s)
Densidad Ósea , Huesos/metabolismo , Catepsina K/metabolismo , Electroacupuntura , Osteoporosis/prevención & control , Animales , Catepsina K/genética , Colágeno Tipo I/sangre , Estradiol/sangre , Femenino , Fémur/metabolismo , Vértebras Lumbares/metabolismo , Osteoporosis/sangre , Osteoporosis/metabolismo , Ovariectomía , Péptidos/sangre , ARN Mensajero/metabolismo , Ratas Sprague-DawleyRESUMEN
This study investigated the effects of Drynaria total flavonoids on cathepsin K serum concentrations and gene expression, biomechanics and bone mineral density (BMD) of the tibial shaft in ovariectomized rat models of osteoporosis, and mechanism in the prevention and cure of osteoporosis. Seventy-two female Sprague-Dawley rats were divided into six groups. The rats in each group were subjected to gastric lavage after the model was established. The tibial shaft of the right hindlimb was obtained to measure the BMD. Serum cathepsin K concentrations were determined. The cathepsin K mRNA expression was also determined using fluorescent quantitative polymerase chain reaction. The three-point bending method was performed to measure the maximum bending load of the tibial shaft. The total flavonoid and normal groups had significant differences in serum cathepsin K concentrations compared with that in the estrogen group (P<0.05). The total flavonoid and sham-operated groups also showed significant differences in cathepsin K mRNA expression compared with that in the normal group (P<0.01). The maximum bending load of the rats in the total flavonoid group was significantly different from that in the estrogen group (P<0.05) and the sham-operated and normal groups (P<0.01). The high-dose total flavonoid group elicited a better effect on BMD than that by the medium- and low-dose groups (P<0.05). Thus, Drynaria total flavonoids inhibited the serum cathepsin K concentration and increased the maximum bending load of the tibial shaft in ovariectomized rats.
Asunto(s)
Catepsina K/genética , Catepsina K/metabolismo , Flavonoides/administración & dosificación , Osteoporosis/tratamiento farmacológico , Polypodiaceae/química , Animales , Densidad Ósea/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Flavonoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Osteoporosis/cirugía , Ovariectomía , Ratas , Ratas Sprague-Dawley , Tibia/efectos de los fármacos , Tibia/fisiopatologíaRESUMEN
Syzygium tetragonum Wall is a Chinese folk medicine for the treatment of rheumatism, joint swelling and pain. By High Content Screening (HCS), 8 compounds (1-8) from Syzygium tetragonum Wall were evaluated for their inhibitory activity on the nuclear translocation of NFATc1 in EGFP-NFATc1 U2OS cells. Among them, 6-[10'(Z)-heptadecenyl] salicylic acid (8) exhibited a significant inhibitory activity. In RAW 264.7 cells, it could dose-dependently prevent nuclear NFATc1 translocation induced by receptor activator of nuclear factor κB ligand (RANKL). The differentiation of osteoclasts from bone marrow derived macrophages (BMMs) was significantly inhibited by 8 in a dose-dependent manner. The mRNA expression of TRAP, CtsK, and MMP9, key enzymes for the bone resorption secreted by osteoclasts, were also significantly down-regulated; and MMP9 activity was also obviously decreased. More importantly, the bone resorption activity of osteoclasts was dose-dependently suppressed by compound 8. Our results suggest that compound 8 can effectively inhibit osteoclastogenesis and bone erosion via preventing NFATc1 nuclear translocation and might be a promising drug candidate for relevant diseases.
Asunto(s)
Factores de Transcripción NFATC/metabolismo , Osteoclastos/efectos de los fármacos , Salicilatos/farmacología , Syzygium/química , Fosfatasa Ácida/genética , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Resorción Ósea/genética , Catepsina K/genética , Línea Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Isoenzimas/genética , Macrófagos/efectos de los fármacos , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Medicina Tradicional China , Ratones Endogámicos BALB C , Estructura Molecular , Osteoclastos/metabolismo , Extractos Vegetales/análisis , Extractos Vegetales/química , Ligando RANK/metabolismo , Ligando RANK/farmacología , Salicilatos/aislamiento & purificación , Fosfatasa Ácida TartratorresistenteRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Postmenopausal osteoporosis is one of the most common disorders in women after menopause, which is linked to an estrogen deficiency and characterized by an excessive loss of trabecular bone. Rubus coreanus and Astragalus membranaceus have been used for their various pharmacological properties in Asia as a traditional medicine. The present study evaluated the anti-osteoporotic effects of the optimal combination of Rubus coreanus and Astragalus membranaceus in 7:3 mixture (RAM) in ovariectomized (OVX) mice by investigating bone biomechanical properties and the serum levels of TNF-α, osteocalcin, RANKL, OPG, and RANK-RANKL signal-related osteoclast differentiation markers. MATERIALS AND METHODS: A total of 36 mature female outbred ICR (Institute of cancer research) strain mice (7 weeks) were divided into 6 groups with 7 mice in each group as follows: (1) Sham-operated control mice (Sham) received daily oral phosphate-buffered-saline (PBS) of equal volumes through gavage. (2) OVX mice received a daily oral gavage of PBS (OVX). (3) OVX mice were treated daily with 50mg/kgb.w./day of RAM (4) with 100mg/kgb.w./day of RAM or (5) with 200mg/kgb.w./day of RAM via oral gavage. (6) OVX mice received i.p. injections of 17ß-estradiol (E2) (0.1mg/kgb.w./day) three times per week for 12 weeks. RESULTS: Micro-CT images showed that oral administration of RAM to OVX mice prevented tibial bone loss, preserved trabecular bone microarchitecture, and improved bone biomechanical properties. RAM administration also showed recovery effects on the levels of TNF-α, OPG and RANKL concentration in OVX-states. Additionally, we found that the mechanism by which RAM elicited anti-osteoporotic effects was by down-regulating the expression of TRAF6 and NFATc1 in RANKL-RANK pathway, a route of osteoclast differentiation, followed by reducing the production of osteoclast differentiation factors, calcitonin receptors and cathepsin K. CONCLUSIONS: Our research strongly suggests that RAM can be clinically used in the prevention and treatment of postmenopausal osteoporosis.
Asunto(s)
Astragalus propinquus , Osteoporosis Posmenopáusica/metabolismo , Osteoporosis Posmenopáusica/prevención & control , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Rosaceae , Animales , Catepsina K/genética , Quimioterapia Combinada , Femenino , Frutas , Humanos , Ratones , Ratones Endogámicos ICR , Factores de Transcripción NFATC/genética , Osteocalcina/sangre , Osteoprotegerina/sangre , Ovariectomía , Fitoterapia , Raíces de Plantas , Ligando RANK/sangre , Radiografía , Receptores de Calcitonina/genética , Factor 6 Asociado a Receptor de TNF/genética , Tibia/diagnóstico por imagen , Tibia/efectos de los fármacos , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Yukmijihwang-tang (YMT) is a traditional herbal medicine known to enhance memory in brain injury models. The aims of this study were to evaluate the inhibitory effect of YMT on osteoclast differentiation and to determine its molecular mechanism of action. YMT dose-dependently inhibited receptor activator for nuclear factor-κB (NF-κB) ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP) activity and the formation of multinucleated osteoclasts in RAW264.7 cells. In addition, quantitative reverse transcription-polymerase chain reaction showed that YMT significantly decreased RANKL-induced expression of osteoclast differentiation-specific genes (TRAP, matrix metalloproteinase-9, cathepsin K, and the d2 isoform of vacuolar ATPase V(0) domain). Furthermore, YMT inhibited RANKL-induced phosphorylation of mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38), phosphorylation of I-κBα, phosphorylation of NF-κB p65, and the expression of transcription factors Fra-2 and nuclear factor of activated T-cells, cytoplasmic 1. Furthermore, YMT inhibited the bone-resorptive activity of differentiated osteoclasts, suggesting that YMT inhibits osteoclast differentiation by suppressing RANKL-induced signaling molecules and transcription factors that affect the regulation of genes for osteoclast differentiation. As such, YMT may have therapeutic potential in bone diseases.
Asunto(s)
Medicamentos Herbarios Chinos/farmacología , FN-kappa B/metabolismo , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Animales , Resorción Ósea/tratamiento farmacológico , Catepsina K/genética , Catepsina K/metabolismo , Diferenciación Celular , Línea Celular , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ligandos , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Inhibidor NF-kappaB alfa , Osteoclastos/metabolismo , Fosforilación , Ligando RANK/metabolismo , Transducción de Señal , Fosfatasa Ácida Tartratorresistente , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Maintenance of healthy bone requires the balanced activities of osteoclasts (OCs), which resorb bone, and osteoblasts, which build bone. Disproportionate action of OCs is responsible for the bone loss associated with postmenopausal osteoporosis and rheumatoid arthritis. NF-κB inducing kinase (NIK) controls activation of the alternative NF-κB pathway, a critical pathway for OC differentiation. Under basal conditions, TRAF3-mediated NIK degradation prevents downstream signaling, and disruption of the NIK:TRAF3 interaction stabilizes NIK leading to constitutive activation of the alternative NF-κB pathway. METHODOLOGY/PRINCIPAL FINDINGS: Using transgenic mice with OC-lineage expression of NIK lacking its TRAF3 binding domain (NT3), we now find that alternative NF-κB activation enhances not only OC differentiation but also OC function. Activating NT3 with either lysozyme M Cre or cathepsinK Cre causes high turnover osteoporosis with increased activity of OCs and osteoblasts. In vitro, NT3-expressing precursors form OCs more quickly and at lower doses of RANKL. When cultured on bone, they exhibit larger actin rings and increased resorptive activity. OC-specific NT3 transgenic mice also have an exaggerated osteolytic response to the serum transfer model of arthritis. CONCLUSIONS: Constitutive activation of NIK drives enhanced osteoclastogenesis and bone resorption, both in basal conditions and in response to inflammatory stimuli.
Asunto(s)
Osteoclastos/metabolismo , Osteólisis/metabolismo , Osteoporosis/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Artritis/genética , Artritis/metabolismo , Sitios de Unión/genética , Western Blotting , Densidad Ósea , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Catepsina K/genética , Catepsina K/metabolismo , Células Cultivadas , Femenino , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Transgénicos , Muramidasa/genética , Muramidasa/metabolismo , FN-kappa B/metabolismo , Osteocalcina/sangre , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteólisis/genética , Osteoporosis/genética , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Ligando RANK/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor 3 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/metabolismo , Quinasa de Factor Nuclear kappa BRESUMEN
Our previous study showed that Erythrina variegata L. (EV) inhibited bone loss and improved bone properties in ovariectomised rats. The purpose of the present study is to investigate the potential mechanism involved in mediating the osteoprotective actions of EV. Female Sprague-Dawley rats were fed a phyto-oestrogen-free diet and subjected to either ovariectomy or a sham operation. Ovariectomised rats were treated with genistein (40 mg/kg) as well as low (200 mg/kg), medium (500 mg/kg) or high (1000 mg/kg) doses of EV extract. Bone properties and mRNA expressions were evaluated by micro-computed tomography and quantitative RT-PCR, respectively. Osteoclast differentiation in RAW 264.7 cells was studied by tartrate-resistant acid phosphatase (TRAP) staining. High doses of EV could decrease urinary Ca and P excretion, maintain serum Ca and P level, and exert beneficial effects on the micro-structure and morphology of trabecular bone and cortical bone in ovariectomised rats. EV suppressed the up-regulation of cathepsin K mRNA and the down-regulation of osteoprotegrin mRNA in the tibia of ovariectomised rats. TRAP-positive cell numbers were significantly decreased in receptor activator of nuclear factor-κB ligand (RANKL)-induced RAW 264.7 cells when co-cultured with EV extracts. The present study indicated that the protective effects of EV on bone properties in ovariectomised rats are likely to be mediated by its inhibitory actions on the process of bone resorption via the suppression of osteoclast differentiation and maturation.
Asunto(s)
Conservadores de la Densidad Ósea/farmacología , Resorción Ósea/tratamiento farmacológico , Erythrina , Osteoclastos/efectos de los fármacos , Osteoporosis/prevención & control , Fitoterapia , Extractos Vegetales/farmacología , Fosfatasa Ácida , Animales , Conservadores de la Densidad Ósea/uso terapéutico , Resorción Ósea/metabolismo , Calcio/metabolismo , Catepsina K/genética , Catepsina K/metabolismo , Diferenciación Celular/efectos de los fármacos , Femenino , Genisteína/farmacología , Genisteína/uso terapéutico , Isoenzimas , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Osteoclastos/metabolismo , Osteoporosis/metabolismo , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ovariectomía , Fósforo/metabolismo , Extractos Vegetales/uso terapéutico , Ligando RANK , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fosfatasa Ácida TartratorresistenteRESUMEN
Osteoporosis and obesity remain a major public health concern through its associated fragility and fractures. Several animal models for the study of osteoporotic bone loss, such as ovariectomy (OVX) and denervation, require unique surgical skills and expensive set up. The challenging aspect of these age-associated diseases is that no single animal model exactly mimics the progression of these human-specific chronic conditions. Accordingly, to develop a simple and novel model of post menopausal bone loss with obesity, we fed either a high fat diet containing 10% corn oil (CO) or standard rodent lab chow (LC) to 12-month-old female C57Bl/6J mice for 6 months. As a result, CO fed mice exhibited increased body weight, total body fat mass, abdominal fat mass and reduced bone mineral density (BMD) in different skeletal sites measured by dual energy X-ray absorptiometry. We also observed that decreased BMD with age in CO fed obese mice was accompanied by increased bone marrow adiposity, up-regulation of peroxisome proliferator-activated receptor γ, cathepsin k and increased proinflammatory cytokines (interleukin 6 and tumor necrosis factor α) in bone marrow and splenocytes, when compared to that of LC fed mice. Therefore, this appears to be a simple, novel and convenient age-associated model of post menopausal bone loss, in conjunction with obesity, which can be used in pre-clinical drug discovery to screen new therapeutic drugs or dietary interventions for the treatment of obesity and osteoporosis in the human population.
Asunto(s)
Adipocitos/metabolismo , Dieta , Grasas de la Dieta/metabolismo , Obesidad/metabolismo , Osteoporosis/fisiopatología , Animales , Peso Corporal , Densidad Ósea , Catepsina K/genética , Catepsina K/metabolismo , Células Cultivadas , Aceite de Maíz/metabolismo , Modelos Animales de Enfermedad , Femenino , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , PPAR gamma/genética , PPAR gamma/metabolismo , Bazo/citología , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
Soy isoflavone preparations, such as purified genistein and a soy extract (Novasoy), were reported previously to exert beneficial effects on bones. Our purpose in this study was to compare the effects of genistein and Novasoy on 3-dimensional trabecular bone parameters and the expression of bone-specific genes in ovariectomized (OVX) mice. The sham-operated mice were fed the control diet and OVX mice were fed diets containing genistein or Novasoy or the control diet, with or without 17beta-estradiol treatment, for 5 wk. Trabecular bone parameters of tibias were measured by microcomputed tomography and gene expression was assayed by real-time PCR. Consumption of diets containing genistein or Novasoy partially prevented the ovariectomy-induced increase in body weight but did not alter the uterus weight of the OVX mice. Novasoy, but not purified genistein, significantly preserved trabecular bone mass, bone volume, and trabecular bone separation in the proximal tibial metaphysis. Purified genistein decreased mRNA expression of receptor activator of nuclear factor-kappaB ligand (RANKL), carbonic anhydrase II, and cathepsin K and enhanced the ratio of osteoprotegrin:RANKL mRNA expression in the tibial head of the OVX mice. In contrast, the diet containing Novasoy suppressed the OVX-induced increase in serum alkaline phosphatase but did not alter bone-specific gene expression of tibia. Our study demonstrated that a soy extract containing a similar level of genistein in the form of Novasoy is more effective than purified genistein in improving tibial trabecular bone quality in OVX mice, but the mechanism of action might be distinct from that of genistein.