RESUMEN
Icariin, a prenylated flavonol glycoside isolated from the herb Epimedium, has been considered as a potential alternative therapy for osteoporosis. Previous research has shown that, unlike other flavonoids, icariin is unlikely to act via the estrogen receptor, but its exact mechanism of action is unknown. In this study, using rat calvarial osteoblast culture and rat bone growth models, we demonstrated that icariin promotes bone formation by activating the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway requiring functional primary cilia of osteoblasts. We found that icariin increases the peak bone mass attained by young rats and promotes the maturation and mineralization of rat calvarial osteoblasts. Icariin activated cAMP/PKA/CREB signaling of the osteoblasts by increasing intracellular cAMP levels and facilitating phosphorylation of both PKA and CREB. Blocking cAMP/PKA/CREB signaling with inhibitors of the cAMP-synthesizing adenylyl cyclase (AC) and PKA inhibitors significantly inhibited the osteogenic effect of icariin in the osteoblasts. Icariin-activated cAMP/PKA/CREB signaling was localized to primary cilia, as indicated by localization of soluble AC and phosphorylated PKA. Furthermore, blocking ciliogenesis via siRNA knockdown of a cilium assembly protein, IFT88, inhibited icariin-induced PKA and CREB phosphorylation and also abolished icariin's osteogenic effect. Finally, several of these outcomes were validated in icariin-treated rats. Together, these results provide new insights into icariin function and its mechanisms of action and strengthen existing ties between cAMP-mediated signaling and osteogenesis.
Asunto(s)
Flavonoides/farmacología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Densidad Ósea/efectos de los fármacos , Desarrollo Óseo/efectos de los fármacos , Cilios/efectos de los fármacos , Cilios/metabolismo , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Medicamentos Herbarios Chinos/farmacología , Epimedium/química , Femenino , Osteogénesis/genética , Osteogénesis/fisiología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Effects of sinusoidal electromagnetic fields (SEMFs) on bone metabolism have not yet been well defined. The present study investigated SEMF effects on bone formation and resorption in rat femur bone tissues in vitro. Cultured femur diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues were treated with 50 Hz 1.8 mT SEMFs 1.5 h per day for up to 12 days and treatment effects on bone formation and resorption markers and associated gene expression were examined. Treatment with SEMFs caused a significant increase in alkaline phosphatase (ALP) activity and inhibited the tartrate-resistant acid phosphatase (TRACP) activity in the femoral diaphyseal or metaphyseal tissues. SEMFs also significantly increased levels of mRNA expression of osterix (OSX), insulin-like growth factor (IGF-1) and ALP in the bone tissues. SEMF treatment decreased glucose content and increased lactic acid contents in the culture conditioned medium. In addition, treatment with SEMFs decreased mRNA expression levels of bone resorption-related genes TRACP, macrophage colony stimulating factor (M-CSF) and cathepsin K (CTSK) in the cultured bone tissues. In conclusion, the current study demonstrated that treatment with 1.8 mT SEMFs at 1.5 h per day promoted bone formation, increased metabolism and inhibited resorption in both metaphyseal and diaphyseal bone tissues in vitro.
Asunto(s)
Resorción Ósea/terapia , Campos Electromagnéticos , Fémur/efectos de la radiación , Magnetoterapia , Osteogénesis/efectos de la radiación , Fosfatasa Ácida/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/fisiopatología , Fémur/metabolismo , Fémur/fisiología , Fémur/fisiopatología , Regulación de la Expresión Génica/efectos de la radiación , Glucosa/metabolismo , Isoenzimas/metabolismo , Ácido Láctico/metabolismo , Masculino , Osteogénesis/genética , Ratas , Ratas Wistar , Fosfatasa Ácida TartratorresistenteRESUMEN
Although pulsed electromagnetic fields (PEMFs) have been approved as a therapy for osteoporosis, action mechanisms and optimal parameters are elusive. To determine the optimal intensity, exposure effects of 50 Hz PEMFs of 0.6-3.6 mT (0.6 interval at 90 min/day) were investigated on proliferation and osteogenic differentiation of cultured calvarial osteoblasts. All intensity groups stimulated proliferation significantly with the highest effect at 0.6 mT. The 0.6 mT group also obtained the optimal osteogenic effect as demonstrated by the highest ALP activity, ALP(+) CFU-f colony formation, nodule mineralization, and expression of COL-1 and BMP-2. To verify our hypothesis that the primary cilia are the cellular sensors for PEMFs, osteoblasts were also transfected with IFT88 siRNA or scrambled control, and osteogenesis-promoting effects of 0.6 mT PEMFs were found abrogated when primary cilia were inhibited by IFT88 siRNA. Thus primary cilia of osteoblasts play an indispensable role in mediating PEMF osteogenic effect in vitro.
Asunto(s)
Calcificación Fisiológica , Magnetoterapia/instrumentación , Magnetoterapia/métodos , Osteoblastos/fisiología , Osteogénesis , Cráneo/citología , Fosfatasa Alcalina , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Cilios , Campos Electromagnéticos , Técnicas In Vitro , Osteoblastos/citología , Ratas , Proteínas Supresoras de Tumor/genéticaRESUMEN
OBJECTIVE: To investigate the estrogenic activity of icariin and genistein with estrogen-dependent human breast cancer (MCF-7) cells. METHOD: MCF-7 cells were incubated with media containing 5% charcoal dextran-treated FBS in phenol red-free media for 48 h. CCK-8 kit was used to study the impact of defferent concentration of icariin and genistein on MCF-7 proliferation in vitro. Optimal concentration icariin and genistein were added into medium and total RNA was isolated after 12, 24, 36, 48 h. The gene expression of ERalpha, ERbeta, PS2, and PR were investigated by Real-time RT-PCR Total protein was also isolated and secretion of ERalpha, ERbeta, PS2, and PR were examined by Western blot. RESULT: 10 micromol x L(-1) icariin and genistein could promote the proliferation of MCF-7 evidently. However, the ability of genistein to promote the proliferation was better than icariin. With the concentration of 10 micromol x L(-1), genistein group had a stronger expression of ERa, PS2 and PR mRNA levels than icariin while ERbetaexpression had no significant difference in two group. The same effects were detected by western blotting. CONCLUSION: Both genistein and icariin have a strong estrogen-like effect, but the estrogenic activity of genistein is stronger than icariin. It showed that the activity of icariin is stron-ger than genistein to promote ROB maturation. So it must be that icariin promotes the maturation of osteoblasts in vitro by a estogen-independent mechanism.
Asunto(s)
Flavonoides/farmacología , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Estrógenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genisteína/farmacología , Humanos , Células MCF-7 , Osteoblastos/metabolismo , Presenilina-2/metabolismoRESUMEN
OBJECTIVE: To compare the effect of icariin and genistein in the osteogenic differentiation of rat bone marrow stromal cells (rBMSC). METHOD: Rat marrow stromal cells were seperated in vitro, and the optimal concentration of genisten and icriin were screened. Genistein and icariin with the concentration of 1 x 10(-5) mol x L(-1) were adopted to intereven rBMSCs cultured in vitro. Alkaline phosphatase (ALP) was determined at 3, 6, 9, 12,15 d after intervention; calcified nodule was detected with alizarin red staining at 12 d; OXS, Runx-2, bone morphogenetic protein (BMP-2) and Collagen-I mRNA expression were observed with Real-time RT-PCR at 12, 24, 48, 72, 96 h. RESULT: Genistein and icariin with the concentration of 1 x 10(-5) mol x L(-1) could increase the activity of ALP and the content of Ca, regulate OXS, BMP-2, Runx-2 and Collagen-I mRNA expression. CONCLUSION: Icariin showed a stronger effect in improving the osteogenic differentiation of rat bone marrow stromal cells than genistein.
Asunto(s)
Flavonoides/farmacología , Genisteína/farmacología , Células Madre Mesenquimatosas/citología , Osteogénesis/efectos de los fármacos , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Expresión Génica/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
An effective method for preventing bone loss is by promoting osteoblast differentiation and bone formation. While dexamethasone has been routinely used as a classical inducer for osteoblast differentiation, limitations have been observed with its usage, including its varied effects on expression of osteoblast genes in different species and its potentials in suppressing osteoblastic differentiation and mineralization. In this study, we assessed the ability of flavonoid icariin in enhancing differentiation and mineralization of cultured rat primary osteoblasts in the absence of dexamethasone. It was found that, compared to the non-stimulated control, icariin at 10(-5) M produced a higher alkaline phosphatase activity, more and larger areas of alkaline phosphatase-positive colonies (CFU-FALP) and mineralized nodules, more osteocalcin secretion and calcium deposition, higher levels of mRNA expression of alkaline phosphatase, osteoblastic transcription factors osterix and runt-related transcription factor 2, and collagen 1α, higher levels of protein expression of collagen 1α, alkaline phosphatese, osterix, and runt-related transcription factor 2. In addition, icariin at 10(-5) M was always more potent than dexamethasone at its optimal concentration of 10(-8) M on the above osteoblast differentiation and mineralization markers. Taken together, our studies demonstrated that icariin has a pronounced ability in promoting osteoblast differentiation in vitro in the absence of dexamethasone.
Asunto(s)
Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Dexametasona/farmacología , Flavonoides/farmacología , Osteoblastos/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dexametasona/efectos adversos , Osteoblastos/citología , Osteogénesis/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
This study is to investigate effects of genistein on rat femoral bone metabolic in vitro. Rat femoral tissues was isolated and randomly divided into two groups including control group and genistein (1 x 10(-5) mol x(-1)) group. Determinations of alkaline phosphatase (ALP) activity, calcium content and osteoprotegerin (OPG), type I-collagen (Collagen-I), RANKL, Runx-2 and bone morphogenetic protein (BMP-2) mRNA expression were done by real-time PCR. The results showed that 1 x 10(-5) mol x L(-1) genistein could increase the activity of ALP and contents of Ca, regulate bone metabolism activity of OPG, RANKL, BMP-2, Collagen-I and Runx-2 mRNA expression level. Genistein can significantly modulate bone metabolism related gene expression level of rat femoral tissue in vitro, and can increase calcium content and the activity of ALP.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Calcio/metabolismo , Fémur/metabolismo , Genisteína/farmacología , Fitoestrógenos/farmacología , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligando RANK/genética , Ligando RANK/metabolismo , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Previous studies have found that 8-prenylflavonoids have a higher osteogenic activity than do flavonoids, which suggested that the 8-prenyl group may play an active role in bone-protective properties. To address this hypothesis, activities of 8-prenylnaringenin (PNG) and naringenin (NG) in osteoblast and osteoclast differentiation and function were compared in vitro. PNG was found to have a stronger ability than NG to improve osteoblast differentiation and osteogenic function in cultured rat calvarial osteoblasts, as demonstrated by levels of alkaline phosphatase activity, osteocalcin, calcium deposition, and the number and area of mineralized bone nodules, as well as mRNA expression of osteogenesis-related genes Bmp-2, OSX, and Runx-2. In addition, although expression of osteoclastogenic inducer receptor activator of nuclear factor kappa-B ligand (RANKL) was not affected, that of osteoclastogenesis inhibitor osteoprotegerin (OPG) and consequently the OPG/RANKL ratio were increased, more potently by PNG than NG. PNG was also found to have a higher potency than NG in inhibiting the osteoclast formation in rabbit bone marrow cells and their resorptive activity, as revealed by lower numbers of osteoclasts formed, lower numbers and areas of bone resorption pits, and lower mRNA expression levels of tartrate-resistant acid phosphatase and cathepsin K. Furthermore, PNG induced apoptosis of mature osteoclasts at a higher degree and at an earlier time than did NG. These results indicate that the 8-prenyl group plays an important role and contributes to the higher bone-protective activity of PNG in comparison with NG.