RESUMO
Research on flavonoids from plant sources has recently sparked increasing interest because of their beneficial health properties. Different studies have shown that flavonoids change the intracellular Ca2+ homeostasis linked to alterations in the function of mitochondria, Ca2+ channels and Ca2+ pumps. These findings hint at plasma membrane Ca2+-ATPase (PMCA) involvement, as it transports Ca2+ actively to the extracellular medium coupled to ATP hydrolysis, thus maintaining ion cellular homeostasis. The present study aims to investigate the effect of several natural flavonoids on PMCA both in isolated protein systems and in living cells, and to establish the relationship between flavonoid structure and inhibitory activity on PMCA. Our results show that natural flavonoids inhibited purified and membranous PMCA with different effectiveness: quercetin and gossypin were the most potent and their inhibition mechanisms seem to be different, as quercetin does not prevent ATP binding whereas gossypin does. Moreover, PMCA activity was inhibited in human embryonic kidney cells which transiently overexpress PMCA, suggesting that the effects observed on isolated systems could occur in a complex structure like a living cell. In conclusion, this work reveals a novel molecular mechanism through which flavonoids inhibit PMCA, which leads to Ca2+ homeostasis and signaling alterations in the cell.
Assuntos
ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Células HEK293 , HumanosRESUMO
We examine the fracture mechanics of tearing graphene. We present a molecular dynamics simulation of the propagation of cracks in clamped, free-standing graphene as a function of the out-of-plane force. The geometry is motivated by experimental configurations that expose graphene sheets to out-of-plane forces, such as back-gate voltage. We establish the geometry and basic energetics of failure and obtain approximate analytical expressions for critical crack lengths and forces. We also propose a method to obtain graphene's toughness. We observe that the cracks' path and the edge structure produced are dependent on the initial crack length. This work may help avoid the tearing of graphene sheets and aid the production of samples with specific edge structures.
RESUMO
CNP::EGFP transgenic mice, genetically engineered to express the enhanced green fluorescent protein (EGFP) under the control of the 2-3-cyclic nucleotide 3-phosphodiesterase (CNPase) promoter in oligodendroglial and Schwann cells, constitute a very important and widely used tool for the study of oligodendrocyte (OLG) development and function in young mice. Our results showed that CNP::EGFP mice were significantly more susceptible to CPZ-induced demyelination, as evaluated by MBP immunostaining, oligodendroglial progenitor cell (OPC) recruitment and astroglial, microglial and nestin response. This enhanced vulnerability was a consequence of their hypomyelination. CNP::EGFP control mice also displayed a significant decrease in corpus callosum (CC) thickness and MBP immunoreactivity. Morphometric analysis further showed a significant decrease in the frequency of myelinated axons, myelin turns (lamellae) and g-ratio carried out in the optic nerve (ON) and CC of CNP::EGFP, as compared to WT mice. Moreover, our results showed a decrease in the number of axons of small caliber, concomitantly with an increase in the number of axons of bigger size with more and enlarged mitochondria, which suggests a high energy demand. These findings and those displaying that MBP+ cells and NF200 staining in the CNP::EGFP cortex were more sparsely distributed provide evidence of axonal loss, which was supported by a decreased number of NeuN+ cells in the CA3 fields of the hippocampus. Transgenic mice also showed an increase in microglial and astroglial activation, accompanied by enhanced lipid peroxidation and recruitment of morphologically altered OPC. Finally, CNPase protein levels proved to be lower than MBP in the CC, which might indicate an altered pattern in myelin proteins with a CNPase deficiency. Behavioral analysis of adult CNP::EGFP transgenic mice supported our results, as it revealed a decrease in locomotion, exploratory activity and motor impairment, as compared to their WT littermates. Our data highlight the relevance of confronting results obtained in adult CNP::EGFP mice with those observed in WT mice. According to our findings, CNP::EGFP hypomyelination might be triggered by the cellular stress induced by the high level of EGFP expression in mature OLG. Adult CNP::EGFP mice could be considered a useful tool to evaluate future therapies for demyelinating diseases such as multiple sclerosis (MS), since these animals present chronic demyelination with axonal degeneration, a characteristic of such pathologies.
Assuntos
Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/genética , Inibidores da Monoaminoxidase/toxicidade , Proteínas da Mielina/metabolismo , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/genética , Aldeídos/metabolismo , Animais , Encéfalo/patologia , Encéfalo/ultraestrutura , Antígeno CD11b/metabolismo , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Ectodisplasinas/metabolismo , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Interleucina-1beta/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/patologia , Desempenho Psicomotor/efeitos dos fármacos , Desempenho Psicomotor/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco/efeitos dos fármacos , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Galectins control critical pathophysiological processes, including the progression and resolution of central nervous system (CNS) inflammation. In spite of considerable progress in dissecting their role within lymphoid organs, their functions within the inflamed CNS remain elusive. Here, we investigated the role of galectin-glycan interactions in the control of oligodendrocyte (OLG) differentiation, myelin integrity and function. Both galectin-1 and -3 were abundant in astrocytes and microglia. Although galectin-1 was abundant in immature but not in differentiated OLGs, galectin-3 was upregulated during OLG differentiation. Biochemical analysis revealed increased activity of metalloproteinases responsible for cleaving galectin-3 during OLG differentiation and modulating its biological activity. Exposure to galectin-3 promoted OLG differentiation in a dose- and carbohydrate-dependent fashion consistent with the 'glycosylation signature' of immature versus differentiated OLG. Accordingly, conditioned media from galectin-3-expressing, but not galectin-3-deficient (Lgals3(-/-)) microglia, successfully promoted OLG differentiation. Supporting these findings, morphometric analysis showed a significant decrease in the frequency of myelinated axons, myelin turns (lamellae) and g-ratio in the corpus callosum and striatum of Lgals3(-/-) compared with wild-type (WT) mice. Moreover, the myelin structure was loosely wrapped around the axons and less smooth in Lgals3(-/-) mice versus WT mice. Behavior analysis revealed decreased anxiety in Lgals3(-/-) mice similar to that observed during early demyelination induced by cuprizone intoxication. Finally, commitment toward the oligodendroglial fate was favored in neurospheres isolated from WT but not Lgals3(-/-) mice. Hence, glial-derived galectin-3, but not galectin-1, promotes OLG differentiation, thus contributing to myelin integrity and function with critical implications in the recovery of inflammatory demyelinating disorders.
Assuntos
Diferenciação Celular , Bainha de Mielina/fisiologia , Oligodendroglia/citologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Axônios/metabolismo , Comportamento Animal , Células Cultivadas , Cuprizona/toxicidade , Galectina 1/metabolismo , Galectina 3/deficiência , Galectina 3/genética , Galectina 3/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/metabolismo , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Polissacarídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Ratos WistarRESUMO
The aim of the present work is to demonstrate the putative sedative and anxiolytic-like effects of a hydro-ethanolic extract obtained from the aerial parts of Aloysia polystachya (Verbenaceae) in male mice using several behavioural assays. Groups of male mice orally treated with doses of 1.0, 10.0 and 100.0 mg/kg of the extract did not show any significant alteration of their locomotor activity, body temperature or motor coordination. The same treatment increased the duration of the sleeping time induced by 30.0 mg/kg i.p. of sodium pentobarbital. However, the sleeping time induced by ethyl ether was not modified by the oral administration of the extract, not confirming the putative sedative effect of the plant. The ethanolic extract also significantly increased the percentage of both entries (1.0 and 100.0 mg/kg) and the time spent (10.0 and 100.0 mg/kg) into the open arms of the elevated plus maze (EPM). Nevertheless, the binding of (3)H-flunitrazepam ((3)H-FNZ) to the benzodiazepine binding site (BDZ-bs), in washed crude synaptosomal membranes from rat cerebral cortex, was not affected by the semi-purified components from Aloysia polystachya. These results indicate an anxiolytic-like profile of action for the extract of Aloysia polystachya without sedative side effect, being this activity probably mediated by other mechanism than BDZ-bs modulation at the GABA(A) receptors.
Assuntos
Ansiolíticos/farmacologia , Extratos Vegetais/farmacologia , Verbenaceae , Animais , Comportamento Animal/efeitos dos fármacos , Temperatura Corporal/efeitos dos fármacos , Flunitrazepam/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Atividade Motora/efeitos dos fármacos , Sono/efeitos dos fármacosRESUMO
A large number of structurally different classes of ligands, many of them sharing the main characteristics of the benzodiazepine (BDZ) nucleus, are active in the modulation of anxiety, sedation, convulsion, myorelaxation, hypnotic and amnesic states in mammals. These compounds have high affinity for the benzodiazepine binding site (BDZ-bs) of the GABA(A) receptor complex. Since 1989 onwards our laboratories established that some natural flavonoids were ligands for the BDZ-bs which exhibit medium to high affinity in vitro and anxiolytic activity in vivo. Further research resulted in the production of synthetic flavonoid derivatives with increased biochemical and pharmacological activities. The currently accepted receptor/pharmacophore model of the BDZ-bs (Zhang, W.; Koeler, K. F.; Zhang, P.; Cook, J. M. Drug Des. Dev. 1995, 12, 193) accounts for the general requirements that should be met by this receptor for ligand recognition. In this paper we present a model pharmacophore which defines the characteristics for a ligand to be able to interact and bind to a flavone site, in the GABA(A) receptor. closely related to the BDZ-bs. A model of a flavone binding site has already been described (Dekermendjian, K.; Kahnberg, P.; Witt, M. R.; Sterner, O.; Nielsen, M.; Liljerfors, T. J. Med. Chem. 1999, 42, 4343). However, this alternative model is based only on graphic superposition techniques using as template a non-BDZ agonist. In this investigation all the natural and synthetic flavonoids found to be ligands for the BDZ-bs have been compared with the classical BDZ diazepam. A QSAR regression analysis of the parameters that describe the interaction demonstrates the relevance of the electronic effects for the ligand binding, and shows that they are associated with the negatively charged oxygen atom of the carbonyl group of the flavonoids and with the nature of the substituent in position 3'.
Assuntos
Benzodiazepinas/metabolismo , Flavonoides/metabolismo , Receptores de GABA-A/metabolismo , Animais , Ansiolíticos/metabolismo , Sítios de Ligação , Diazepam/química , Interações Medicamentosas , Flunitrazepam/metabolismo , Ligantes , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Ensaio Radioligante , Ratos , Sinaptossomos/metabolismoRESUMO
6,3'-dibromoflavone and 6-nitro-3'-bromoflavone inhibited [(3)H]flunitrazepam binding to the benzodiazepine binding site of the gamma amino butyric acid receptor complex with K(i) values between 17 and 36 nM in different brain regions. Their gamma amino butyric acid ratio for [(3)H]flunitrazepam binding to cerebral cortex membranes indicated partial agonistic properties. Both compounds had similar pharmacological effects: they produced anxiolytic-like effects at low doses but did not alter locomotor activity or muscle tonicity; sedation was caused only at doses higher than 30 mg/kg in mice. These synthetic flavone derivatives join an existing family of 6,3'-disubstituted flavone compounds with high affinity for the benzodiazepine binding site and partial agonistic profiles.
Assuntos
Encéfalo/metabolismo , Flavonoides/metabolismo , Receptores de GABA-A/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Sítios de Ligação , Encéfalo/efeitos dos fármacos , Flavonoides/farmacologia , Técnicas In Vitro , Cinética , Ligantes , Masculino , Camundongos , Ratos , Ratos Wistar , Receptores de GABA-A/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
6-Chloro-3'-nitroflavone integrates a list of nearly 70 flavone derivatives synthesized in our laboratories. The effects of 6-chloro-3'-nitroflavone on the benzodiazepine binding sites (BDZ-BSs) of the GABA(A) receptor were examined in vitro and in vivo. 6-Chloro-3'-nitroflavone inhibited the [3H]flunitrazepam ([3H]FNZ) binding to rat cerebral cortex membranes with a Ki of 6.68 nM and the addition of GABA to extensively washed membranes did not modify its affinity for the BDZ-BSs (GABA-shift = 1.16+/-0.12). The binding assays performed in rat striatal and cerebellar brain membranes showed that this compound has similar affinity to different populations of BDZ-BSs. Electrophysiological experiments revealed that 6-chloro-3'-nitroflavone did not affect GABA(A)-receptors (GABA(A)-Rs) responses recorded in Xenopus oocytes expressing alpha1beta2gamma2s subunits, but blocked the potentiation exerted by diazepam (DZ) on GABA-activated chloride currents. In vivo experiments showed that 6-chloro-3'-nitroflavone did not possess anxiolytic, anticonvulsant, sedative, myorelaxant actions in mice or amnestic effects in rats; however, 6-chloro-3'-nitroflavone antagonized diazepam-induced antianxiety action, anticonvulsion, short-term, and long-term amnesia and motor incoordination. These biochemical, electrophysiological, and pharmacological results suggest that 6-chloro-3'-nitroflavone behaves as an antagonist of the BDZ-BSs.
Assuntos
Diazepam/farmacologia , Flavonoides/farmacologia , Flunitrazepam/metabolismo , Moduladores GABAérgicos/farmacologia , Aprendizagem/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacos , Animais , Anticonvulsivantes , Diazepam/uso terapêutico , Antagonistas de Receptores de GABA-A , Masculino , Camundongos , Ratos , Ratos Wistar , Convulsões/tratamento farmacológico , XenopusRESUMO
6-Methyl-3'-bromoflavone inhibited [(3)H]flunitrazepam binding to the benzodiazepine binding site of the GABA(A) receptor (BDZ-bs) with Ki values between 10 and 50 nM in different brain regions. The GABA ratio of 1.03 for [(3)H]flunitrazepam binding to cerebral cortex, 0.76 for cerebellum, 0.7 for hippocampus, 0.7 for striatum, and 0.8 for spinal cord indicated an antagonistic or weak inverse agonistic profile of 6-methyl-3'-bromoflavone on BDZ-bs. Unlike classical benzodiazepines, it had no anticonvulsant, anxiolytic, myorelaxant, sedative, amnestic or motor incoordination effects. However, it antagonized the muscle relaxant, the sedative effect, and the changes in locomotor activity induced by diazepam. Taken together, these findings suggest that 6-methyl-3'-bromoflavone has an antagonistic profile on the BDZ-bs.
Assuntos
Encéfalo/metabolismo , Flavonoides/farmacologia , Antagonistas GABAérgicos/farmacologia , Receptores de GABA-A/metabolismo , Medula Espinal/metabolismo , Sinaptossomos/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Flavonoides/síntese química , Flavonoides/química , Flunitrazepam/farmacocinética , Antagonistas de Receptores de GABA-A , Hipocampo/metabolismo , Membranas Intracelulares/metabolismo , Cinética , Ligantes , Masculino , CamundongosRESUMO
The list of activities of plant flavonoids did not include effects on the central nervous system (CNS) up to 1990, when our laboratory described the existence of natural anxiolytic flavonoids. The first of these was chrysin (5,7-dihydroxyflavone), followed by apigenin (5,7,4'-trihydroxyflavone) and flavone itself. Semisynthetic derivatives of flavone obtained by introducing halogens, nitro groups or both in its molecule, give rise to high affinity ligands for the benzodiazepine receptor, active in-vivo; 6,3'-dinitroflavone, for example, is an anxiolytic drug 30 times more potent than diazepam. The data collected in this paper make clear that some natural flavonoids are CNS-active molecules and that the chemical modification of the flavone nucleus dramatically increases their anxiolytic potency.
Assuntos
Ansiolíticos/farmacologia , Sistema Nervoso Central/efeitos dos fármacos , Flavonoides/farmacologia , Ansiolíticos/química , Camomila , Flavonoides/química , Humanos , Óleos Voláteis/química , Óleos Voláteis/farmacologia , Plantas MedicinaisRESUMO
6-Bromo-3'-nitroflavone is a synthetic flavone derivative that selectively recognizes benzodiazepine receptors and has potent anxiolytic-like effects. Here, we describe in detail its pharmacological characterization. When i.p. injected in mice, 6-bromo-3'-nitroflavone (0.01-0.3 mg/kg) had an anxiolytic-like effect in the elevated plus-maze test. This effect was blocked by the specific benzodiazepine receptor antagonist, flumazenil. In addition, it exhibited anxiolytic-like actions when given orally (1 mg/kg). 6-Bromo-3'-nitroflavone did not exhibit myorelaxant effects (up to 30 mg/kg, i.p.). Unlike diazepam, this flavonoid produced no anterograde amnesia in a one-trial inhibitory avoidance learning. On the other hand, 6-bromo-3'-nitroflavone possessed mild anticonvulsant activity (0.1 mg/kg, i.p.) and provoked sedative-depressant actions only at doses 100-1000 times higher than those producing anxiolytic-like effects. 6-Bromo-3'-nitroflavone (0.1-1 mM) produced a lower potentiation of gamma-amino-butyric acid (GABA)-stimulated 36Cl- influx (126-138%) in comparison to diazepam (0.1 mM: 166%) in cerebral cortical membrane vesicles. Taken together, these findings suggest that 6-bromo-3'-nitroflavone has anxiolytic-like action possibly behaving as a partial agonist of the benzodiazepine receptors.
Assuntos
Ansiolíticos/farmacologia , Flavonoides/farmacologia , Agonistas de Receptores de GABA-A , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Aprendizagem da Esquiva/fisiologia , Flavonoides/uso terapêutico , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Pentobarbital/farmacologia , Pentilenotetrazol/farmacologia , Desempenho Psicomotor/efeitos dos fármacos , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Sono/efeitos dos fármacos , Sono/fisiologiaRESUMO
Solution phase combinatorial synthesis of flavone derivatives and evaluation of their affinity for the central benzodiazepine receptors is described. The libraries preparation is simple and provides a convenient method for rapid compound generation and screening. Thirty one new compounds were obtained of which the most promising, as high affinity benzodiazepine receptor ligands, were 6-bromo-3'-fluoroflavone; 6,3'-dichloroflavone; 6-bromo-3'-chloroflavone and 6-chloro-3'-bromoflavone.
Assuntos
Flavonoides/síntese química , Flavonoides/metabolismo , Receptores de GABA-A/metabolismo , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Cromatografia Líquida de Alta Pressão , Flavonoides/química , Flunitrazepam/metabolismo , Camundongos , Estrutura Molecular , Ratos , Soluções , Ácido gama-Aminobutírico/metabolismoRESUMO
Benzodiazepines (BDZs) are the most widely prescribed class of psychoactive drugs in current therapeutic use, despite the important unwanted side-effects that they produce such as sedation, myorelaxation, ataxia, amnesia, ethanol and barbiturate potentiation and tolerance. Searching for safer BDZ-receptor (BDZ-R) ligands we have recently demonstrated the existence of a new family of ligands which have a flavonoid structure. First isolated from plants used as tranquilizers in folkloric medicine, some natural flavonoids have shown to possess a selective and relatively mild affinity for BDZ-Rs and a pharmacological profile compatible with a partial agonistic action. In a logical extension of this discovery various synthetic derivatives of those compounds, such as 6,3'-dinitroflavone were found to have a very potent anxiolytic effect not associated with myorelaxant, amnestic or sedative actions. This dinitro compound, in particular, exhibits a high affinity for the BDZ-Rs (Ki = 12-30 nM). Due to their selective pharmacological profile and low intrinsic efficacy at the BDZ-Rs, flavonoid derivatives, such as those described, could represent an improved therapeutic tool in the treatment of anxiety. In addition, several flavone derivatives may provide important leads for the development of potent and selective BDZ-Rs ligands.
Assuntos
Flavonoides/metabolismo , Receptores de GABA-A/metabolismo , Animais , Flavonoides/química , Flavonoides/farmacologia , Ligantes , Estrutura MolecularRESUMO
6,3'-Dintroflavone is a synthetic flavone derivative with high affinity for central benzodiazepine receptors that has anxiolytic effects. Here, we describe its biochemical and pharmacological characterization. 6,3'-Dinitroflavone inhibited differentially [3H]flunitrazepam binding to central benzodiazepine receptors in several brain regions, showing a lower Ki value in the cerebellum (central benzodiazepine receptor type I-enriched area), and a higher Ki value in the spinal cord and in the dentate gyrus (central benzodiazepine receptor type II-enriched area). When i.p. injected in mice, 6,3'-dinitroflavone had a potent anxiolytic effect in the elevated plus maze test. This effect was blocked by the specific central benzodiazepine receptor antagonist, Ro 15-1788. 6,3'-Dinitroflavone did not exhibit anticonvulsant or myorelaxant effects in mice or amnestic effects in rats. Moreover, it abolished the myorelaxant effect of diazepam. On the other hand, 6,3'-dinitroflavone possessed a mild sedative action only at doses 100-300-fold greater than the anxiolytic one. Based on these findings, we suggest that 6,3'-dinitroflavone has a benzodiazepine partial agonist profile, with low selectivity for central benzodiazepine receptor types I and II.
Assuntos
Ansiolíticos/farmacologia , Comportamento Animal/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Flavonoides/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Animais , Autorradiografia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Sistema Nervoso Central/metabolismo , Flunitrazepam/farmacologia , Ligantes , Masculino , Camundongos , Ratos , Ratos Wistar , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
6-Bromoflavone, obtained by bromination of flavanone, binds to central benzodiazepine receptors with a Ki=70 nM and has a clear anxiolytic activity in mice, at 0.5 mg/kg i.p. A survey of the structure/affinity relationship for those receptors in a series of natural and synthetic flavonoids is presented.