RESUMEN
The activation of NLRP3 results in the assembly of inflammasome that regulates caspase-1 activation and the subsequent secretion of bioactive interleukin (IL)-1ß. Excessive activation of the NLRP3 inflammasome is mechanistically linked to diverse pathophysiological conditions, including airway inflammation. Here, we discovered that Curcuma phaeocaulis can suppress caspase-1 activation and processing of pro-IL-1ß into mature cytokine in macrophages stimulated with NLRP3 inflammasome activators, such as SiO2 or TiO2 nanoparticles. Furthermore, in the bronchoalveolar lavage fluids of animals administered the nanoparticles, the in vitro effects of C. phaeocaulis translated into a decrease in IL-1ß levels and cell infiltration. Demethoxycurcumin (DMC) and curcumin were found to be responsible for the inflammasome inhibitory activity of C. phaeocaulis. Interestingly, in contrast to the previously reported higher antioxidant- and NFκB-inhibitory activities of curcumin, DMC exhibited approximately two-fold stronger potency than curcumin against nanoparticle induced activation of NLRP3 inflammasome. In the light of these results, both compounds seem to act independently of their antioxidant- and NFκB-inhibitory properties. Although how C. phaeocaulis inhibits nanoparticle-activated NLRP3 inflammasome remains to be elucidated, our results provide a basis for further research on C. phaeocaulis extract as an anti-inflammatory agent for the treatment of disorders associated with excessive activation of NLRP3 inflammasome.
Asunto(s)
Curcumina , Nanopartículas , Animales , Antioxidantes/farmacología , Caspasa 1 , Caspasas , Curcuma , Curcumina/farmacología , Inflamasomas , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Interleucina-1beta/farmacología , Macrófagos , Ratones , FN-kappa B/farmacología , Proteína con Dominio Pirina 3 de la Familia NLR , Dióxido de Silicio/farmacologíaRESUMEN
Neuropathic pain is associated with an increased sensitivity to painful stimuli or abnormal sensitivity to otherwise innocuous stimuli. However, in addition to adverse effects, currently available drugs have shown limited response in patients with neuropathic pain, which provides a rationale to explore new drug classes acting on novel targets and with better efficacy and safety profiles. Here, we found that saikosaponins potently inhibit agonist-induced activation of the transient receptor potential A1 (TRPA1) channel, which has been reported to mediate neuropathic pain by sensing a variety of chemical irritants. Molecular docking and site-directed mutagenesis analyses suggested that saikosaponins bind to the hydrophobic pocket in TRPA1 near the Asn855 residue, which, when mutated to Ser, was previously associated with enhanced pain perception in humans. In support of these findings, saikosaponin D significantly attenuated agonist-induced nociceptive responses and vincristine-induced mechanical hypersensitivity in mice. These results indicate that saikosaponins are TRPA1 antagonists and provide a basis for further elaboration of saikosaponin derivatives for the development of new therapeutics for neuropathic pain.
Asunto(s)
Ácido Oleanólico/análogos & derivados , Saponinas/farmacología , Canal Catiónico TRPA1/antagonistas & inhibidores , Animales , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Hiperalgesia/diagnóstico , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Simulación del Acoplamiento Molecular , Neuralgia/diagnóstico , Neuralgia/tratamiento farmacológico , Ácido Oleanólico/química , Ácido Oleanólico/aislamiento & purificación , Ácido Oleanólico/metabolismo , Ácido Oleanólico/farmacología , Dimensión del Dolor , Saponinas/química , Saponinas/aislamiento & purificación , Saponinas/metabolismo , Canal Catiónico TRPA1/química , Canal Catiónico TRPA1/metabolismoRESUMEN
Osteoporosis is a clinical condition characterized by low bone strength that leads to an increased risk of fracture. Strategies for the treatment of osteoporosis involve inhibition of bone resorption by osteoclasts and an increase of bone formation by osteoblasts. Here, we identified the extract derived from the stem part of Edgeworthia papyrifera that enhanced differentiation of MC3T3-E1 cells to osteoblast-like cells and inhibited osteoclast differentiation of RAW 264.7 cells in vitro. In support of our observation, rutin and daphnoretin, which were previously reported to inhibit osteoclast differentiation, were identified in E. papyrifera extract. In an animal model of osteoporosis, the ovariectomy-induced increases in bone resorption biomarkers such as pyridinoline and tartrate-resistant acid phosphatase were significantly reduced by E. papyrifera extract administration at 25.6 and 48.1%, respectively. Furthermore, the ovariectomy-induced bone loss in animal models of osteoporosis was significantly prevented by the administration of E. papyrifera in our study. Taking these observations into account, we suggest that E. papyrifera is an interesting candidate for further exploration as an anti-osteoporotic agent.
Asunto(s)
Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Osteoporosis/tratamiento farmacológico , Extractos Vegetales/farmacología , Thymelaeaceae/química , Fosfatasa Alcalina/metabolismo , Aminoácidos/orina , Animales , Biomarcadores/sangre , Biomarcadores/orina , Resorción Ósea/tratamiento farmacológico , Diferenciación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Femenino , Ratones , Ratones Endogámicos , Modelos Animales , Osteoporosis/etiología , Extractos Vegetales/análisis , Células RAW 264.7 , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Although the roles of p21-activated serine/threonine kinase 1 (PAK1) have been reported in some neurodegenerative diseases, details regarding neurodegeneration are still limited. Hence, we tried to determine the role of PAK1 and molecular mechanisms of neuronal death involved in neurodegeneration. RESULTS: Expression of a dominant-negative form of PAK1 (PAK1(H83,86L, K229R), PAK1-DN) decreased the cell viability and increased cell death induced by oxidative stress. Indeed, oxidative stress decreased the phosphorylation of PAK1 in neuroblastoma cells, cultured dopamine (DA) neurons, or rat midbrains. PAK1-DN reduced the level of Bcl-2 protein, through an ubiquitin/proteasome-dependent mechanism. The level of Bcl-2 may be regulated by PAK1-ERK signaling and/or PAK1, directly. Conversely, expression of an active form of PAK1 (PAK1(T423E), PAK1-CA) could recover both loss of DA neurons in the substantia nigra (SN) and behavioral defects in a 6-OHDA-induced hemiparkinsonian rat model. CONCLUSIONS: Our data suggest that the oxidative stress-induced down-regulation of PAK1 activity could be involved in the loss of mesencephalic DA neurons through modulation of neuronal death, suggesting a novel role of PAK1 as a molecular determinant and mechanisms in the pathogenesis of Parkinson's disease.
Asunto(s)
Neuronas Dopaminérgicas/enzimología , Neuronas Dopaminérgicas/patología , Regulación hacia Abajo , Mesencéfalo/patología , Quinasas p21 Activadas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Calcineurina/metabolismo , Inhibidores de la Calcineurina/farmacología , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Modelos Biológicos , Estrés Oxidativo/efectos de los fármacos , Oxidopamina , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas Sprague-DawleyRESUMEN
Axon regeneration after injury in the central nervous system is hampered in part because if an age-dependent decline in the intrinsic axon growth potential, and one of the strategies to stimulate axon growth in injured neurons involves pharmacological manipulation of implicated signaling pathways. Here we report phenotypic cell-based screen of chemical libraries and structure-activity-guided optimization that resulted in the identification of compound 7p which promotes neurite outgrowth of cultured primary neurons derived from the hippocampus, cerebral cortex, and retina. In an animal model of optic nerve injury, compound 7p was shown to induce growth of GAP-43 positive axons, indicating that the in vitro neurite outgrowth activity of compound 7p translates into stimulation of axon regeneration in vivo. Further optimization of compound 7p and elucidation of the mechanisms by which it elicits axon regeneration in vivo will provide a rational basis for future efforts to enhance treatment strategies.
Asunto(s)
Acetamidas/farmacología , Axones/efectos de los fármacos , Descubrimiento de Drogas , Regeneración Nerviosa/efectos de los fármacos , Sulfonamidas/farmacología , Acetamidas/síntesis química , Acetamidas/química , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ratones , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
Asthma is a chronic inflammatory disease of lung airways, and pharmacological inhibitors of cyclic adenosine monophosphate-specific phosphodiesterase 4 (PDE4) have been considered as therapeutics for the treatment of asthma. However, development of PDE4 inhibitors in clinical trials has been hampered because of the severe side effects of non-selective PDE4 inhibitors. Here, screening of a plant extract library in conjunction with dereplication technology led to identification of baicalin as a new type of PDE4-selective inhibitor. We demonstrated that while rolipram inhibited the enzyme activity of a range of PDE4 subtypes in in vitro enzyme assays, baicalin selectively inhibited the enzyme activity of PDE4A and 4B. In addition, baicalin suppressed lipopolysaccharide-induced TNF-α expression in macrophage where PDE4B plays a key role in lipopolysaccharide-induced signaling. Furthermore, baicalin treatment in an animal model of allergic asthma reduced inflammatory cell infiltration and TNF-α levels in bronchoalveolar lavage fluids, indicating that the antiinflammatory effects of baicalin in vivo are attributable, in part, to its ability to inhibit PDE4.
Asunto(s)
Antiinflamatorios/farmacología , Asma/tratamiento farmacológico , Flavonoides/farmacología , Inhibidores de Fosfodiesterasa 4/farmacología , Extractos Vegetales/farmacología , Animales , Asma/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Modelos Animales de Enfermedad , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Células RAW 264.7 , Rolipram/farmacología , Rosaceae/química , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The SLC45A2 gene encodes a Membrane-Associated Transporter Protein (MATP). Mutations of this gene cause oculocutaneous albinism type 4 (OCA4). However, the molecular mechanism of its action in melanogenesis has not been elucidated. Here, we discuss the role of MATP in melanin production. The SLC45A2 gene is highly enriched in human melanocytes and melanoma cell lines, and its protein, MATP, is located in melanosomes. The knockdown of MATP using siRNAs reduced melanin content and tyrosinase activity without any morphological change in melanosomes or the expression of melanogenesis-related proteins. Interestingly, the knockdown of MATP significantly lowered the melanosomal pH, as verified through DAMP analysis, suggesting that MATP regulates melanosomal pH and therefore affects tyrosinase activity. Finally, we found that the reduction of tyrosinase activity associated with the knockdown of MATP was readily recovered by copper treatment in the in vitro L-DOPA oxidase activity assay of tyrosinase. Considering that copper is an important element for tyrosinase activity and that its binding to tyrosinase depends on melanosomal pH, MATP may play an important role in regulating tyrosinase activity via controlling melanosomal pH.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Melanosomas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Monofenol Monooxigenasa/metabolismo , Línea Celular Tumoral , Humanos , Concentración de Iones de Hidrógeno , Indoles/metabolismo , Melaninas/metabolismo , Melanocitos/metabolismo , Melanoma/metabolismo , Proteínas de la Membrana/metabolismo , Mutación/genética , Oxidación-ReducciónRESUMEN
The zinc transporter protein ZIP13 plays critical roles in bone, tooth, and connective tissue development, and its dysfunction is responsible for the spondylocheirodysplastic form of Ehlers-Danlos syndrome (SCD-EDS, OMIM 612350). Here, we report the molecular pathogenic mechanism of SCD-EDS caused by two different mutant ZIP13 proteins found in human patients: ZIP13(G64D), in which Gly at amino acid position 64 is replaced by Asp, and ZIP13(ΔFLA), which contains a deletion of Phe-Leu-Ala. We demonstrated that both the ZIP13(G64D) and ZIP13(ΔFLA) protein levels are decreased by degradation via the valosin-containing protein (VCP)-linked ubiquitin proteasome pathway. The inhibition of degradation pathways rescued the protein expression levels, resulting in improved intracellular Zn homeostasis. Our findings uncover the pathogenic mechanisms elicited by mutant ZIP13 proteins. Further elucidation of these degradation processes may lead to novel therapeutic targets for SCD-EDS.
Asunto(s)
Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/patología , Zinc/metabolismo , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Eliminación de Secuencia , Ubiquitina/metabolismoRESUMEN
The leptin receptor, OBR, is involved in the regulation of whole-body energy homeostasis. Most obese people are resistant to leptin and do not respond to the hormone. The prevention and reversal of leptin resistance is one of the major current goals of obesity research. We showed previously that increased OBR cell surface expression concomitantly increases cellular leptin signaling and prevents obesity development in mice. Improvement of OBR cell surface expression can thus be considered as an interesting anti-obesity therapeutic strategy. To identify compounds that increase the surface expression of OBR, we developed a cell-based, phenotypic assay to perform a high-content screen (HCS) against a library of 50,000 chemical compounds. We identified 67 compounds that increased OBR cell surface expression with AC50 values in the low micromolar range and no effect on total OBR expression and cellular toxicity. Compounds were classified into 16 chemical clusters, of which 4 potentiated leptin-promoted signaling through the JAK2/STAT3 pathway. In conclusion, development of a robust phenotypic screening approach resulted in the discovery of four new scaffolds that demonstrate the desired biological activity and could constitute an original therapeutic solution against obesity and associated disorders.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Obesidad/metabolismo , Fenotipo , Receptores de Leptina/metabolismo , Línea Celular , Descubrimiento de Drogas/métodos , Expresión Génica , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento , Humanos , Obesidad/tratamiento farmacológico , Obesidad/genética , Receptores de Leptina/genética , Proteínas Recombinantes de Fusión , Bibliotecas de Moléculas PequeñasRESUMEN
Kv4.2, a pore-forming α-subunit of voltage-gated A-type potassium channels, is expressed abundantly in the soma and dendrites of hippocampal neurons, and is responsible for somatodendritic I(A) current. Recent studies have suggested that changes in the surface levels of Kv4.2 potassium channels might be relevant to synaptic plasticity. Although the function and expression of Kv4.2 protein have been extensively studied, the dendritic localization of Kv4.2 mRNA is not well described. In this study, Kv4.2 mRNAs were shown to be localized in the dendrites near postsynaptic regions. The dendritic transport of Kv4.2 mRNAs were mediated by microtubule- based movement. The 500 nucleotides of specific regions within the 3'-untranslated region of Kv4.2 mRNA were found to be necessary and sufficient for its dendritic localization. Collectively, these results suggest that the dendritic localization of Kv4.2 mRNAs might regulate the dendritic surface level of Kv4.2 channels and synaptic plasticity.
Asunto(s)
Dendritas/metabolismo , ARN Mensajero/genética , Secuencias Reguladoras de Ácido Ribonucleico/fisiología , Canales de Potasio Shal/genética , Canales de Potasio Shal/metabolismo , Animales , Animales Recién Nacidos , Hipocampo/metabolismo , Modelos Biológicos , Plasticidad Neuronal/genética , Neuronas/citología , Neuronas/metabolismo , Transporte de Proteínas/genética , ARN Mensajero/química , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Secuencias Reguladoras de Ácido Ribonucleico/genética , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , Distribución TisularRESUMEN
Although the dendritic localization and translation of a subset of mRNAs plays a pivotal role in synaptic plasticity, the dendritic mRNAs and their functions have been only minimally characterized thus far. In this study, we isolated mRNAs from Staufen2-containing ribonucleoprotein complexes, which function as modules for the transport of mRNA to the dendrites, and then constructed a cDNA library. Apolipoprotein E gene (APOE) mRNA was isolated from the dendritic mRNA-specific cDNA library. The specific localization of APOE mRNA was evaluated via in situ hybridization. The specific regions involved in the dendritic transport of APOE mRNA were determined using a visualization system employing green fluorescent protein-tagged bacteriophage MS2 RNA-binding protein. As a result, the proximal N-terminal or C-terminal regions of the ApoE-coding sequences were determined to be sufficient for dendritic transport. The level of dendritic APOE mRNA was significantly increased by depolarization-induced neuronal activity, but was reduced in the cell body regions. We assessed the functions of neuronal ApoE. The reduction of ganglioside GM1 by cholesterol depletion was completely blocked by ApoE over-expression. In addition, ApoE over-expression increased the immunoreactivity of the post-synaptic density 95 kDa antibody in the dendrites. These findings indicate that neuronal ApoE may be relevant to lipid rafts or synaptic structural plasticity.
Asunto(s)
Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Dendritas/fisiología , Plasticidad Neuronal/genética , ARN Mensajero/metabolismo , Sinapsis/genética , Animales , Animales Recién Nacidos , Apolipoproteínas E/fisiología , Células Cultivadas , Dendritas/ultraestructura , Hipocampo/citología , Hipocampo/fisiología , Microdominios de Membrana/genética , Microdominios de Membrana/metabolismo , Biosíntesis de Proteínas , Transporte de Proteínas/genética , ARN Mensajero/fisiología , Ratas , Sinapsis/ultraestructuraRESUMEN
Although transport and subsequent translation of dendritic mRNA play an important role in neuronal synaptic plasticity, the underlying mechanisms for modulating dendritic mRNA transport are almost completely unknown. In this study, we identified and characterized an interaction between Staufen2 and mitogen-activated protein kinase (MAPK) with co-immunoprecipitation assays. Staufen2 utilized a docking (D) site to interact with ERK1/2; deleting the D-site decreased colocalization of Staufen2 with immunoreactive ERK1/2 in the cell body regions of cultured hippocampal neurons, and it reduced the amount of Staufen2-containing RNP complexes in the distal dendrites. In addition, the deletion completely abolished the depolarization-induced increase of Staufen2-containing RNP complexes. These results suggest that the MAPK pathway could modulate dendritic mRNA transport through its interaction with Staufen2.
Asunto(s)
Dendritas/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Línea Celular , Humanos , Inmunoprecipitación , Ratones , Datos de Secuencia Molecular , Plasticidad Neuronal , Proteínas de Unión al ARN/genética , Ratas , Eliminación de SecuenciaRESUMEN
There is increasing evidence showing that mRNA is transported to the neuronal dendrites in ribonucleoprotein (RNP) complexes or RNA granules, which are aggregates of mRNA, rRNA, ribosomal proteins, and RNA-binding proteins. In these RNP complexes, Staufen, a double-stranded RNA-binding protein, is believed to be a core component that plays a key role in the dendritic mRNA transport. This study investigated the molecular mechanisms of the dendritic mRNA transport using green fluorescent protein-tagged Staufen2 produced employing a Sindbis viral expression system. The kinesin heavy chain was found to be associated with Staufen2. The inhibition of kinesin resulted in a significant decrease in the level of dendritic transport of the Staufen2-containing RNP complexes in neurons under non-stimulating or stimulating conditions. This suggests that the dendritic transport of the Staufen2-containing RNP complexes use kinesin as a motor protein. A mitogen-activated protein kinase inhibitor, PD98059, inhibited the activity-induced increase in the amount of both the Staufen2-containing RNP complexes and Ca(2+)/calmodulin-dependent protein kinase II alpha-subunit mRNA in the distal dendrites of cultured hippocampal neurons. Overall, these results suggest that dendritic mRNA transport is mediated via the Staufen2 and kinesin motor proteins and might be modulated by the neuronal activity and mitogen-activated protein kinase pathway.