RESUMEN
Postmenopausal osteoporosis, a chronic condition that predominantly affects postmenopausal women, presents a significant impediment to their overall well-being. The condition arises from estrogen deficiency, leading to enhanced osteoclast activity. Salvia miltiorrhiza, a well-established Chinese herbal medicine with a history of clinical use for osteoporosis treatment, contains diverse active constituents that have shown inhibitory effects on osteoclast formation and bone loss. Dihydrotanshinone I (DTI), a phenanthrenonequinone compound derived from the root of Salvia miltiorrhiza, has been identified as a potential therapeutic agent, although its mechanism of action on osteoclasts remains elusive. In this study, we aimed to elucidate the inhibitory potential of DTI on RANKL-induced osteoclastogenesis. We observed the ability of DTI to effectively impede the expression of key osteoclast-specific genes and proteins, as assessed by Real-time PCR and Western Blotting analyses. Mechanistically, DTI exerted its inhibitory effects on osteoclast formation by modulating critical signaling pathways including NF-κB, ERK, and calcium ion signaling. Notably, DTI intervention disrupted the nuclear translocation and subsequent transcriptional activity of the NFATc1, thus providing mechanistic insights into its inhibitory role in osteoclastogenesis. To further assess the therapeutic potential of DTI, we employed an ovariectomized osteoporosis animal model to examine its impact on bone loss. Encouragingly, DTI demonstrated efficacy in mitigating bone loss induced by estrogen deficiency. In conclusion, our investigation elucidates the ability of DTI to regulate multiple signaling pathways activated by RANKL, leading to the inhibition of osteoclast formation and prevention of estrogen-deficiency osteoporosis. Consequently, DTI emerges as a promising candidate for the treatment of osteoporosis.
Asunto(s)
Resorción Ósea , Osteoporosis , Animales , Femenino , Humanos , Resorción Ósea/prevención & control , Diferenciación Celular , Estrógenos/deficiencia , Estrógenos/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos , Osteogénesis , Osteoporosis/metabolismo , Ligando RANK/metabolismo , Transducción de SeñalRESUMEN
Brevilin A (BA) is the primary component of Centipeda minima, which is widely used in Chinese traditional medicine. The anti-inflammatory and anti-tumor properties of BA have been established; however, its function in bone metabolism is not well understood. This study revealed that concentrations of BA below 1.0 µM did not inhibit the proliferation of bone marrow macrophages but did impede the differentiation and bone resorption activity of osteoclasts. Furthermore, BA suppressed the expression of osteoclast-specific genes Mmp9, Acp5, Dc-stamp, Ctsk, and Atp6v0d2. In addition, mTOR, ERK, and NFATc1 activation in bone marrow macrophages were suppressed by BA. As a whole, BA blocks the mTOR and ERK signaling pathways, which is responsible for the development and activity of osteoclasts, and the resorption of bone.
Asunto(s)
Resorción Ósea , Osteoclastos , Animales , Osteoclastos/metabolismo , Resorción Ósea/genética , Resorción Ósea/patología , Serina-Treonina Quinasas TOR/metabolismo , Ligando RANK/farmacología , Ligando RANK/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Diferenciación Celular/genética , Osteogénesis/genéticaRESUMEN
Acrylamide (ACR), a potential neurotoxin, is generated from the Maillard reaction between reducing sugars and free amino acids during food processing. Our work focuses on clarifying the role of the leucine-rich repeat kinase 2 (LRRK2) and nuclear factor of activated T cells, cytoplasmic 2 (NFATc2) in the polarization of BV2 cells to the M1 proinflammatory type induced by ACR. Specifically, ACR promoted the phosphorylation of LRRK2 and NFATc2 in BV2 microglia. Furthermore, selectively phosphorylated LRRK2 by ACR induced nuclear translocation of NFATc2 to trigger a neuroinflammatory cascade. Knock-down of LRRK2 by silencing significantly diminished ACR-induced microglial neurotoxic effect with the decline of IL-1ß, IL-6, and iNOS levels and the decrease of NFATc2 expression in BV2 cells. After pretreated with Toll-Like Receptor 2 (TLR2) and TLR4 inhibitors separately, both the activation of LRRK2 and the release of pro-inflammatory factors were inhibited in BV2 cells. Gallic acid (GA) is ubiquitous in most parts of the medicinal plant. GA alleviated the increased CD11b expression, IL-6 and iNOS levels induced by ACR in BV2 microglia. In conclusion, this study shows that ACR leads to the cascade activation of LRRK2-NFATc2 mediated by TLR2 and TLR4 to induce microglial toxicity.
Asunto(s)
Microglía , Receptor Toll-Like 2 , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Acrilamida/metabolismo , Receptor Toll-Like 4/metabolismo , Interleucina-6/metabolismo , Línea Celular , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , FN-kappa B/metabolismoRESUMEN
Osteoporosis, an immune disease characterized by bone mass loss and microstructure destruction, is often seen in postmenopausal women. Isoimperatorin (ISO), a bioactive, natural furanocoumarin isolated from many traditional Chinese herbal medicines, has therapeutic effects against various diseases; however, its effect on bone homeostasis remains unclear. In this study, we investigated the effect of ISO on the differentiation and activation of osteoclast and its molecular mechanism in vitro, and evaluated the effect of ISO on bone metabolism by ovariectomized (OVX) rat model. In vitro experiments showed that ISO affected RANKL-induced MAPK, NFAT, NFATc1 trafficking and expression, osteoclast F-actin banding, osteoclast-characteristic gene expression, ROS inhibitory activity, and calcium oscillations, NF-κB signaling pathway. In vivo experiments showed that oral administration of ISO effectively reduced bone loss caused by ovariectomy and retained bone mass.Collectively, ISO inhibits RANK/RANKL binding, thereby reducing the activity of NFATc1, calcium, and ROS and inhibiting osteoclast generation. In addition, ISO protects bone mass by slowing osteoclast production and downregulating NFATc1 gene and protein expression in the bone tissue microenvironment and inhibits OVX-induced bone loss in vivo.
Asunto(s)
Resorción Ósea , Furocumarinas , Animales , Femenino , Humanos , Ratas , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/prevención & control , Resorción Ósea/metabolismo , Diferenciación Celular , Furocumarinas/farmacología , FN-kappa B/metabolismo , Factores de Transcripción NFATC/genética , Osteoclastos , Osteogénesis , Ovariectomía , Ligando RANK/farmacología , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción , Receptor Activador del Factor Nuclear kappa-B/metabolismoRESUMEN
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
Diabetes osteoporosis is a chronic complication of diabetes mellitus (DM) and is associated with osteoclast formation and enhanced bone resorption. Specnuezhenide (SPN) is an active compound with anti-inflammatory and immunomodulatory properties. However, the roles of SPN in diabetic osteoporosis remain unknown. In this study, primary bone marrow macrophages (BMMs) were pretreated with SPN and were stimulated with receptor activator of nuclear factor kappa B ligand (RANKL; 50â ng/mL) to induce osteoclastogenesis. The number of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining. The protein levels of cellular oncogene fos/nuclear factor of activated T cells c1 (c-Fos/NFATc1), nuclear factor kappa-B (NF-κB), and mitogen-activated protein kinases (MAPKs) were evaluated by western blot analysis. NF-κB luciferase assays were used to examine the role of SPN in NF-κB activation. The DM model group received a high-glucose, high-fat diet and was then intraperitoneally injected with streptozotocin (STZ). Micro-CT scanning, serum biochemical analysis, histological analysis were used to assess bone loss. We found that SPN suppressed RANKL-induced osteoclast formation and that SPN inhibited the expression of osteoclast-related genes and c-Fos/ NFATc1. SPN inhibited RANKL-induced activation of NF-κB and MAPKs. In vivo experiments revealed that SPN suppressed diabetes-induced bone loss and the number of osteoclasts. Furthermore, SPN decreased the levels of bone turnover markers and increased the levels of runt-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), calcium (Ca) and phosphorus (P). SPN also regulated diabetes-related markers. This study suggests that SPN suppresses diabetes-induced bone loss by inhibiting RANKL-induced osteoclastogenesis, and provides an experimental basis for the treatment of diabetic osteoporosis.
Asunto(s)
Diabetes Mellitus , Osteoporosis , Células de la Médula Ósea/metabolismo , Calcio/metabolismo , Diferenciación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Diabetes Mellitus/metabolismo , Glucosa/metabolismo , Glucósidos , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Osteogénesis , Osteoporosis/tratamiento farmacológico , Osteoporosis/etiología , Osteoporosis/metabolismo , Osteoprotegerina/metabolismo , Fósforo/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Piranos , Ligando RANK/farmacología , Transducción de Señal , Estreptozocina , Fosfatasa Ácida Tartratorresistente/metabolismoRESUMEN
A number of studies demonstrated that some tea extracts exert inhibitory effects on osteoclastogenesis induced by receptor activator of nuclear factor κB ligand (RANKL). However, the effect of purple tea, a famous tea in China, on osteoclastogenesis remains unclear. In this study, a water-based purple tea extract (PTE) was found to suppress osteoclast formation, osteoclastic resorption pit area formation, and F-actin ring formation within RANKL-stimulated bone marrow macrophages (BMMs). Furthermore, our results demonstrated that PTE could inhibit expression of master transcription factors NFATc1 and c-Fos and their target genes DC-STAMP, Ctsk, and Atp6v0d2. Western blot analysis revealed that PTE treatment led to reduced RANKL-induced phosphorylation of Akt and GSK3ß without altering transient activation of NF-κB and MAPKs (p38, JNK, ERK1/2) signaling. In addition, the results demonstrated that PTE treatment of RANKL-stimulated BMMs could down-regulate Blimp1 expression and up-regulate Irf8 expression. In summary, these results suggest that PTE treatment of RANKL-stimulated BMMs inhibited osteoclast differentiation via modulation of Blimp1-Irf8 and Akt/GSK3ß signaling pathways. Aligning with our in vitro results, in vivo PTE administration ameliorated bone loss in LPS-treated mice. Taken together, the results presented in this work suggest that PTE treatment possesses anti-osteolytic activity.
Asunto(s)
Resorción Ósea , Ligando RANK , Animales , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/genética , Resorción Ósea/metabolismo , Diferenciación Celular , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos , Osteogénesis , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ligando RANK/metabolismo , Té/metabolismo , Agua/metabolismoRESUMEN
Osteoporosis is characterized by reduction in bone mass and microarchitectural deterioration of the bone, which causes bone fragility and fracture susceptibility. Ishige sinicola, a brown alga, reportedly affects osteoblast differentiation. However, its protective effect on estrogen deficiency-induced bone loss has not been elucidated. This study aimed to investigate the effect of I. sinicola extract (ISE) on ovariectomy (OVX)-induced bone loss in vivo and osteoclastogenesis in vitro. Female Sprague-Dawley rats were randomly assigned to the sham-operated (SHAM) group and four OVX subgroups: SHAM, OVX, ISE20 (20 mg/kg), ISE200 (200 mg/kg), and estradiol (10 µg/kg). After 6 weeks of treatment, the bone mineral density (BMD), femur indices, and serum biomarker levels were measured. Furthermore, the effects of ISE on osteoclastogenesis and the expression of osteoclast-specific markers were measured. ISE administration improved the trabecular bone structure, bone biomechanical properties, BMD, and bone mineralization degree. In addition, the levels of serum bone turnover markers were decreased in the ISE group compared with those in the OVX group. Moreover, ISE inhibited osteoclast formation by downregulating NFATc1, TRAP, c-Src, c-Fos, and cathepsin K without any cytotoxic effects on RANKL-induced osteoclast formation. Therefore, we suggest that ISE has therapeutic potential in postmenopausal osteoporosis.
Asunto(s)
Osteogénesis , Phaeophyceae , Animales , Densidad Ósea , Regulación hacia Abajo , Femenino , Humanos , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos , Ovariectomía/efectos adversos , Extractos Vegetales/metabolismo , Extractos Vegetales/farmacología , Ligando RANK/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Cannabis sativa L. extracts (CSE) are used for treating inflammatory conditions, but little is known about their immunomodulatory effects. We investigated a novel CSE with high (14%) CBD and low (0.2%) THC concentration in comparison with pure CBD on primary human lymphocytes. METHODS: Proliferation, cell cycle distribution, apoptosis/necrosis and viability were analysed with standard methods. Genotoxicity was evaluated with the comet-assay. The effect on T lymphocyte activation was evaluated via CD25/CD69 marker expression, degranulation assays and the production of cytokines. The influence on the transcription factors was analysed using Jurkat reporter cell lines. Specific CB2 receptor antagonist SR144528 and TRPV1 receptor antagonist A78416B were used to study the involvement of CB2 or TRPV1 receptors. RESULTS: CSE inhibited the proliferation of activated T lymphocytes in a dose-dependent manner without inducing apoptosis, necrosis, or affecting cell viability and DNA integrity. The inhibitory effect was mediated via the suppression of T lymphocytes activation, particularly by the suppression of CD25 surface marker expression. Furthermore, CSE interferes with the functionality of the T lymphocytes, as indicated by inhibition of degranulation, IL-2, and IFN-γ production. AP-1-and-NFAT-reporter activation was reduced implicating an AP-1-and-NFAT-mediated mode of action. The effects were in part reversed by SR144528 and A78416B, showing that the effects were mainly mediated by CB2 and TRPV1 receptors. CONCLUSION: CSE and CBD have immunomodulatory effects and interfere with the activation and functionality of T lymphocytes. A comparison between CSE and CBD suggests that the immunosuppressive effect of CSE is mostly due to the effect of CBD.
Asunto(s)
Inmunosupresores/metabolismo , Extractos Vegetales/metabolismo , Linfocitos T/inmunología , Apoptosis , Cannabis/inmunología , Degranulación de la Célula , Proliferación Celular , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Extractos Vegetales/inmunología , Psicotrópicos , Receptor Cannabinoide CB2/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Osteoporosis is a chronic disease that has become a serious public health problem due to the associated reduction in quality of life and its increasing financial burden. It is known that inhibiting osteoclast differentiation and promoting osteoblast formation prevents osteoporosis. As there is no drug with this dual activity without clinical side effects, new alternatives are needed. Here, we demonstrate that austalide K, isolated from the marine fungus Penicillium rudallenes, has dual activities in bone remodeling. Austalide K inhibits the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and improves bone morphogenetic protein (BMP)-2-mediated osteoblast differentiation in vitro without cytotoxicity. The nuclear factor of activated T cells c1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), dendritic cell-specific transmembrane protein (DC-STAMP), and cathepsin K (CTSK) osteoclast-formation-related genes were reduced and alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and osteopontin (OPN) (osteoblast activation-related genes) were simultaneously upregulated by treatment with austalide K. Furthermore, austalide K showed good efficacy in an LPS-induced bone loss in vivo model. Bone volume, trabecular separation, trabecular thickness, and bone mineral density were recovered by austalide K. On the basis of these results, austalide K may lead to new drug treatments for bone diseases such as osteoporosis.
Asunto(s)
Conservadores de la Densidad Ósea/uso terapéutico , Resorción Ósea/prevención & control , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Penicillium/química , Xantenos/uso terapéutico , Animales , Conservadores de la Densidad Ósea/aislamiento & purificación , Conservadores de la Densidad Ósea/farmacología , Resorción Ósea/inducido químicamente , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Sedimentos Geológicos/microbiología , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos ICR , Estructura Molecular , Factores de Transcripción NFATC/biosíntesis , Factores de Transcripción NFATC/genética , Osteoporosis , Penicillium/aislamiento & purificación , Ligando RANK/farmacología , Fosfatasa Ácida Tartratorresistente/antagonistas & inhibidores , Xantenos/aislamiento & purificación , Xantenos/farmacologíaRESUMEN
Osteoporosis is a systemic skeletal disease characterized by reduced bone mineral density (BMD), which results in an increased risk of fracture. Melandrium firmum (Siebold & Zucc.) Rohrbach (MFR), 'Wangbulryuhaeng' in Korean, is the dried aerial portion of Melandrii Herba Rohrbach, which is a member of the Caryophyllaceae family and has been used to treat several gynecological conditions as a traditional medicine. However, to the best of our knowledge, the effect of MFR on osteoclast differentiation and osteoporosis has not been assessed. To evaluate the effects of MFR on osteoclast differentiation, tartrateresistant acid phosphatase staining, actin ring formation and bone resorption assays were used. Additionally, receptor activator of nuclear factorκB ligandinduced expression of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) and cFos were measured using western blotting and reverse transcriptionPCR. The expression levels of osteoclastrelated genes were also examined. To further investigate the antiosteoporotic effects of MFR in vivo, an ovariectomized (OVX) rat model of menopausal osteoporosis was established. Subsequently, the femoral head was scanned using microcomputed tomography. The results revealed that MFR suppressed osteoclast differentiation, formation and function. Specifically, MFR reduced the expression levels of osteoclastrelated genes by downregulating transcription factors, such as NFATc1 and cFos. Consistent with the in vitro results, administration of MFR water extract to OVX rats reduced BMD loss, and reduced the expression levels of NFATc1 and cathepsin K in the femoral head. In conclusion, MFR may contribute to alleviate osteoporosislike symptoms. These results suggested that MFR may exhibit potential for the prevention and treatment of postmenopausal osteoporosis.
Asunto(s)
Conservadores de la Densidad Ósea/farmacología , Conservadores de la Densidad Ósea/uso terapéutico , Osteoclastos/efectos de los fármacos , Osteoporosis Posmenopáusica/tratamiento farmacológico , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Silene/química , Actinas/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Conservadores de la Densidad Ósea/química , Conservadores de la Densidad Ósea/toxicidad , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/metabolismo , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoclastos/patología , Osteoporosis Posmenopáusica/diagnóstico por imagen , Osteoporosis Posmenopáusica/etiología , Osteoporosis Posmenopáusica/patología , Ovariectomía/efectos adversos , Extractos Vegetales/química , Extractos Vegetales/toxicidad , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ligando RANK/toxicidad , Ratas Sprague-Dawley , Factor 6 Asociado a Receptor de TNF/metabolismo , Fosfatasa Ácida Tartratorresistente/metabolismoRESUMEN
Exposure through arsenic-contaminated air and food caused by the burning of coal is a major environmental public health concern in Guizhou Province of China. Previous studies have shown that immunological dysfunction is involved in the pathogenesis and carcinogenesis of arsenic; however, knowledge regarding effective prevention measures have not been fully examined. The effect of Ginkgo biloba extract (EGb761) on arsenic-induced skin damage of human immortalized keratinocyte cells (HaCaT) was first evaluated in this study. The results showed that 200 µg/mL EGb761 can reduce the expression of miR-155-5p, and the indicators reflecting arsenic-induced skin damage (Krt1, Krt6c and Krt10) in arsenic-exposed cells (P < 0.05), the expression levels of NF-AT1; the indicators reflecting arsenic-induced immunological dysfunction (IL-2, IFN-γ) in cells; and the levels of secreted IL-2 and IFN-γ in cell supernatants were significantly increased (P < 0.05). Further randomized controlled double-blind experiments showed that compared to the placebo control group, the expression level of miR-155-5p in the plasma of the Ginkgo biloba intervention group, the indicators in the serum reflecting arsenic-induced skin damage (Krt1, Krt6c, and Krt10) and the epithelial-mesenchymal transformation (EMT) vimentin were significantly reduced (P < 0.05), but the levels of NF-AT1 and the indicators reflecting arsenic-induced immunological dysfunction (IL-2, IFN-γ) and EMT (E-cadherin) in serum were significantly increased (P < 0.05). Our study provides some limited evidence that Ginkgo biloba L. can increase the expression of NF-AT1 by downregulating the level of miR-155-5p, alleviating immunological dysfunction, and decreasing the expression of EMT biomarkers, thus indirectly improving arsenic-induced skin damage.
Asunto(s)
Intoxicación por Arsénico/tratamiento farmacológico , Queratinocitos/efectos de los fármacos , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Enfermedades de la Piel/tratamiento farmacológico , Adulto , Anciano , Intoxicación por Arsénico/sangre , Intoxicación por Arsénico/complicaciones , Intoxicación por Arsénico/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Método Doble Ciego , Femenino , Ginkgo biloba , Humanos , Interferón gamma/sangre , Interferón gamma/genética , Interleucina-2/sangre , Interleucina-2/genética , Queratinocitos/metabolismo , Masculino , MicroARNs/sangre , Persona de Mediana Edad , Factores de Transcripción NFATC/sangre , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Enfermedades de la Piel/sangre , Enfermedades de la Piel/inducido químicamente , Enfermedades de la Piel/genéticaRESUMEN
The hypothalamic neuroendocrine system is strongly implicated in body energy homeostasis. In particular, the degree of production and release of arginine vasopressin (AVP) in the hypothalamus is affected by plasma osmolality, and that hypothalamic AVP is responsible for thirst and osmolality-dependent water and metabolic balance. However, the osmolality-responsive intracellular mechanism within AVP cells that regulates AVP synthesis is not clearly understood. Here, we report a role for tonicity-responsive enhancer binding protein (TonEBP), a transcription factor sensitive to cellular tonicity, in regulating osmosensitive hypothalamic AVP gene transcription. Our immunohistochemical work shows that hypothalamic AVP cellular activity, as recognized by c-fos, was enhanced in parallel with an elevation in TonEBP expression within AVP cells following water deprivation. Interestingly, our in vitro investigations found a synchronized pattern of TonEBP and AVP gene expression in response to osmotic stress. Those results indicate a positive correlation between hypothalamic TonEBP and AVP production during dehydration. Promoter and chromatin immunoprecipitation assays confirmed that TonEBP can bind directly to conserved binding motifs in the 5'-flanking promoter regions of the AVP gene. Furthermore, dehydration- and TonEBP-mediated hypothalamic AVP gene activation was reduced in TonEBP haploinsufficiency mice, compared with wild TonEBP homozygote animals. Therefore, our result support the idea that TonEBP is directly necessary, at least in part, for the elevation of AVP transcription in dehydration conditions. Additionally, dehydration-induced reductions in body weight were rescued in TonEBP haploinsufficiency mice. Altogether, our results demonstrate an intracellular machinery within hypothalamic AVP cells that is responsible for dehydration-induced AVP synthesis.
Asunto(s)
Arginina Vasopresina/metabolismo , Regulación de la Expresión Génica , Hipotálamo/metabolismo , Factores de Transcripción NFATC/metabolismo , Neuronas/metabolismo , Animales , Arginina Vasopresina/genética , Haploinsuficiencia , Ratones , Factores de Transcripción NFATC/genética , Concentración Osmolar , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Privación de AguaRESUMEN
Fritillariae thunbergii bulbus has been widely used to treat symptoms of coughs and airway congestion in the chest due to pathological colds and damp phlegm in traditional Chinese medicine. Despite its long history of traditional use, its pharmacological activities on osteoclastogenesis and osteoporosis have not been evaluated. This study investigated the effects of the water extract of Fritillariae thunbergii bulbus (WEFT) on osteoclast differentiation in bone marrow-derived macrophage cells and on ovariectomy (OVX)-induced osteoporosis in mice. We found that WEFT significantly inhibited osteoclastogenesis by downregulating the receptor activator of the NF-κB ligand (RANKL) signaling-induced nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expression. In an OVX-induced osteoporosis model, WEFT significantly prevented the OVX-induced trabecular loss of femurs, accompanied by a reduction in fat accumulation in the bone marrow and liver. In addition, WEFT significantly prevented weight gain and gonadal fat gain without recovering uterine atrophy. Using ultrahigh-performance liquid chromatography-tandem mass spectrometry, seven alkaloids (peimisine glucoside, yibeissine, peiminoside, sipeimine-glucoside, peimisine, peimine, and peiminine) were identified in WEFT. The results of this study suggest that WEFT can be a potential pharmacological candidate to reduce menopausal osteoporosis and menopause-related symptoms, such as fat accumulation.
Asunto(s)
Hueso Esponjoso/efectos de los fármacos , Hueso Esponjoso/metabolismo , Fritillaria/química , Osteogénesis/efectos de los fármacos , Osteoporosis Posmenopáusica/metabolismo , Extractos Vegetales/farmacología , Ligando RANK/metabolismo , Animales , Hueso Esponjoso/patología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos/efectos de los fármacos , Osteogénesis/genética , Osteoporosis Posmenopáusica/tratamiento farmacológico , Osteoporosis Posmenopáusica/etiología , Ovariectomía , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Ligando RANK/genética , Espectrometría de Masas en TándemRESUMEN
The 5-year survival rate of diffuse large B-cell lymphoma (DLBCL) can reach 60%. However, nearly half of patients undergo relapse/refractory issues with a survival period of less than 2 years. New therapeutic approaches are therefore needed to improve chemotherapy efficacy and patient survival. Bufalin (BF), isolated from the traditional Chinese medicine Chansu, has been reported to play an anticancer role in multiple cancer cell types. However, there are few reports of the effects of BF on the growth of DLBCL. In the present study, we demonstrated that BF exerts antitumor activity in DLBCL cells, both in vitro and in vivo. Treatment of DLBCL cells with BF resulted in increased proliferation and apoptosis in a dose- and time-dependent manner. Daily intraperitoneal injection of 1.5 mg/kg BF significantly delayed DLBCL xenograft growth in NOD/SCID mice without affecting body weight. Bioinformatics analysis showed that BF may regulate NFATC1 protein and affect expression of its downstream gene, cMYC. Our results suggest that BF can attenuate NFATC1 translocation by reducing the intracellular calcium concentration; BF may also have a low synergistic effect with cyclosporin A. In conclusion, we demonstrated that BF exerts antitumor activity that is mediated at least in part by the Ca2+/NFATC1/cMYC pathway. Our findings suggest that BF can be effectively applied as a novel potential therapeutic agent for DLBCL.
Asunto(s)
Antineoplásicos/farmacología , Bufanólidos/farmacología , Señalización del Calcio/efectos de los fármacos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Bufanólidos/uso terapéutico , Señalización del Calcio/genética , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Linfoma de Células B Grandes Difuso/patología , Masculino , Ratones , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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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
Calcineurin is a Ca2+ -dependent serine/threonine phosphatase that dephosphorylates nuclear factor of activated T cells (NFAT), allowing for NFAT entry into the nucleus. In skeletal muscle, calcineurin signaling and NFAT activation increases the expression of proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) and slow myosin heavy chain (MHC) I ultimately promoting fatigue resistance. Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that antagonizes calcineurin by re-phosphorylating NFAT preventing its entry into the nucleus. Here, we tested whether GSK3 inhibition in vivo with low dose lithium chloride (LiCl) supplementation (10 mg kg-1 day-1 for 6 weeks) in male C57BL/6J mice would enhance muscle fatigue resistance in soleus and extensor digitorum longus (EDL) muscles by activating NFAT and augmenting PGC-1α and MHC I expression. LiCl treatment inhibited GSK3 by elevating Ser9 phosphorylation in soleus (+1.8-fold, p = .007) and EDL (+1.3-fold p = .04) muscles. This was associated with a significant reduction in NFAT phosphorylation (-50%, p = .04) and a significant increase in PGC-1α (+1.5-fold, p = .05) in the soleus but not the EDL. MHC isoform analyses in the soleus also revealed a 1.2-fold increase in MHC I (p = .04) with no change in MHC IIa. In turn, a significant enhancement in soleus muscle fatigue (p = .04), but not EDL (p = .26) was found with LiCl supplementation. Lastly, LiCl enhanced specific force production in both soleus (p < .0001) and EDL (p = .002) muscles. Altogether, our findings show the skleletal muscle contractile benefits of LiCl-mediated GSK3 inhibition in mice.
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Suplementos Dietéticos , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Compuestos de Litio/administración & dosificación , Fatiga Muscular/efectos de los fármacos , Alimentación Animal/análisis , Animales , Calcineurina/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Contracción Muscular/efectos de los fármacos , Cadenas Pesadas de Miosina/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/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.
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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
BACKGROUND: Areca nut has anti-inflammatory, antiparasitic, antihypertensive, and antidepressant properties. The pathological hallmarks of inflammatory joint diseases are an increased number of osteoclasts and impaired differentiation of osteoblasts, which may disrupt the bone remodeling balance and eventually lead to bone loss. PURPOSE: The present study assessed the effects of arecoline, the main alkaloid found in areca nut, on osteoclast and osteoblast differentiation and function. METHOD: M-CSF/RANKL-stimulated murine bone marrow-derived macrophages (BMMs) were incubated with several concentrations of arecoline, and TRAP staining and pit formation were assessed to monitor osteoclast formation. Quantitative real-time RT-PCR and western blot analyses were used to analyze the expression of osteoclast-associated genes and signaling pathways. The effects of arecoline on bone were investigated in an in vivo mouse model of lipopolysaccharide (LPS)-induced trabecular bone loss after oral administration of arecoline. Alizarin red S staining and assays to measure ALP activity and the transcription level of osteoblast-related genes were used to evaluate the effects of arecoline on osteoblast differentiation and bone mineralization. RESULTS: In a dose-dependent manner, arecoline at concentrations of 50-100 µM reduced both the development of TRAP-positive multinucleated osteoclasts and the formation of resorption pits in M-CSF/RANKL-stimulated BMMs. In M-CSF/RANKL-stimulated BMMs, arecoline also suppressed the expression and translocation of c-Fos and NFATcl, and osteoclast differentiated-related genes via interference with the AKT, MAPK, and NF-kB activation pathways. Femur bone loss and microcomputed tomography parameters were recovered by oral administration of arecoline in the mouse LPS-induced bone loss model. Lastly, arecoline increased ALP activity, bone mineralization, and the expression of osteoblast differentiation-related genes, such as ALP and Runx2, in MC3T3-E1 cells. CONCLUSION: Our data suggest that arecoline may attenuate or prevent bone loss by suppressing osteoclastogenesis and promoting osteoblastogenesis. These findings provide evidence supporting arecoline's use as a potential therapeutic agent in bone-loss disorders and diseases.
Asunto(s)
Arecolina/farmacología , Resorción Ósea/tratamiento farmacológico , Osteoclastos/efectos de los fármacos , Animales , Resorción Ósea/metabolismo , Resorción Ósea/patología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Genes fos , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Masculino , Ratones Endogámicos DBA , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Osteoclastos/citología , Osteoclastos/fisiología , Osteogénesis/efectos de los fármacos , Osteoporosis/inducido químicamente , Osteoporosis/tratamiento farmacológico , Ligando RANK/metabolismo , Ligando RANK/farmacología , Microtomografía por Rayos XRESUMEN
Osteoporosis is a disease that leads to reduced bone mineral density. The increase in patient and medical costs because of global aging is recognized as a problem. Decreased bone mass is a common symptom of bone diseases such as Paget's disease, rheumatoid arthritis, and multiple myeloma. Osteoclasts, which directly affect bone mass, show a marked increase in differentiation and activation in the aforementioned diseases. Moreover, these multinucleated cells made from monocytes/macrophages under the influence of RANKL and M-CSF, are the only cells capable of resorbing bones. In this study, we found that the water extracts of Boseokchal (BSC-W) inhibited osteoclast differentiation in vitro and investigated its inhibitory mechanism. BSC-W was obtained by extracting flour of Boseokchal using hexane and water. To osteoclast differentiation, bone marrow-derived macrophage cells (BMMs) were cultured with the vehicle (0.1% DMSO) or BSC-W in the presence of M-CSF and RANKL for 4 days. Cytotoxicity was measured by CCK-8. Gene expression of cells was confirmed by real-time PCR. Protein expression of cells was observed by western blot assay. Bone resorption activity of osteoclast evaluated by bone pit formation assay using an Osteo Assay Plate. BSC-W inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner without exerting a cytotoxic effect on BMMs. BSC-W decreased the transcriptional and translational expression of c-Fos and NFATc1, which are regulators of osteoclastogenesis and reduced the mRNA expression level of TRAP, DC-STAMP, and cathepsin K, which are osteoclast differentiation marker. Furthermore, BSC-W reduced the resorption activity of osteoclasts. Taken together, our results indicate that BSC-W is a useful candidate for health functional foods or therapeutic agents that can help treat bone diseases such as osteoporosis.