RESUMEN
In the central nervous system, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential to maintain normal neuronal function. Recent studies have shown that HCN channels may be involved in the pathological process of ischemic brain injury, but the mechanisms remain unclear. Autophagy is activated in cerebral ischemia, but its role in cell death/survival remains controversial. In this study, our results showed that the HCN channel blocker ZD7288 remarkably decreased the percentage of apoptotic neurons and corrected the excessive autophagy induced by oxygen-glucose deprivation followed by reperfusion (OGD/R) in hippocampal HT22 neurons. Furthermore, in the OGD/R group, p-mTOR, p-ULK1 (Ser757), and p62 were significantly decreased, while p-ULK1 (Ser317), atg5, and beclin1 were remarkably increased. ZD7288 did not change the expression of p-ULK1 (Ser757), ULK1 (Ser317), p62, Beclin1, and atg5, which are involved in regulating autophagosome formation. Besides, we found that OGD/R induced a significant increase in Cathepsin D expression, but not LAMP-1. Treatment with ZD7288 at 10 µmol/L in the OGD/R group did not change the expression of cathepsin D and LAMP-1. However, chloroquine (CQ), which decreases autophagosome-lysosome fusion, eliminated the correction of excessive autophagy and neuroprotection by ZD7288. Besides, shRNA knockdown of HCN2 channels significantly reduced the accumulation of LC3-II and increased neuron survival in the OGD/R and transient global cerebral ischemia (TGCI) models, and CQ also eliminated the effects of HCN2-shRNA. Furthermore, we found that the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes decreased in Con-shRNA-transfected HT22 neurons exposed to OGD/R or CQ. In HCN2-shRNA-transfected HT22 neurons, the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes increased under OGD/R; however, the percentage was significantly decreased by the addition of CQ to HCN2-shRNA-transfected HT22 neurons. The present results demonstrated that blockade of HCN2 channels provides neuroprotection against OGD/R and TGCI by accelerating autophagic degradation attributable to the promotion of autophagosome and lysosome fusion.
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Autofagia , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/antagonistas & inhibidores , Neuronas , Neuroprotección , Daño por Reperfusión , Glucosa , Hipocampo/citología , Humanos , Canales de Potasio , PirimidinasRESUMEN
Anxiety is an affective disorder that is commonly observed after irreversible brain damage induced by cerebral ischemia and can delay the physical and cognitive recovery, which affects the quality of life of both the patient and family members. However, anxiety after ischemia has received less attention, and mechanisms underlying anxiety-like behaviours induced by chronic cerebral ischemia are under-investigated. In the present study, the chronic cerebral hypoperfusion model was established by the permanent occlusion of the bilateral common carotid arteries (two-vessel occlusion, 2VO) in rats, and anxiety-related behaviours were evaluated. Results indicated that 2VO induced obvious anxiety-like behaviours; the surface expressions of GABAB2 subunits were down-regulated; Brain derived neurotrophic factor (BDNF), tyrosine kinase B (TrkB) and neural cell adhesion molecule (NCAM) were reduced; Meanwhile, the surface expressions of G protein-activated inwardly rectifying potassium (GIRK, Kir3) channels were up-regulated in hippocampal CA1 in 2VO rats. Baclofen, a GABAB receptor agonist, significantly ameliorated the anxiety-like behaviours. It also improved the down-regulation of GABAB2 surface expressions, restored the levels of BDNF, TrkB and NCAM, and reversed the increased surface expressions of Kir3 in hippocampal CA1 in 2VO rats. However, the effects of baclofen were absent in shRNA-GABAB2 infected 2VO rats. These results suggested that activation of GABAB2 subunits could improve BDNF signalling and reverse Kir3 channel surface expressions in hippocampal CA1, which may alleviate the anxiety-like behaviours in rats with chronic cerebral hypoperfusion.
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Ansiedad/metabolismo , Enfermedades de las Arterias Carótidas/metabolismo , Receptores de GABA-B/metabolismo , Animales , Ansiedad/tratamiento farmacológico , Ansiedad/etiología , Baclofeno/farmacología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/metabolismo , Enfermedades de las Arterias Carótidas/tratamiento farmacológico , Enfermedades de las Arterias Carótidas/psicología , Arteria Carótida Común , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Agonistas de Receptores GABA-B/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Masculino , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Psicotrópicos/farmacología , Ratas Sprague-Dawley , Receptor trkB/metabolismo , Receptores de GABA-B/genética , Proteína de la Zonula Occludens-2/metabolismoRESUMEN
The selective hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288) blocks the induction of long-term potentiation in the perforant path-CA3 region in rat hippocampus in vivo. To explore the mechanisms underlying the action of ZD7288, we recorded excitatory postsynaptic potentials in perforant path-CA3 synapses in male Sprague-Dawley rats. We measured glutamate content in the hippocampus and in cultured hippocampal neurons using high performance liquid chromatography, and determined intracellular Ca(2+) concentration [Ca(2+)]i) using Fura-2. ZD7288 inhibited the induction and maintenance of long-term potentiation, and these effects were mirrored by the nonspecific HCN channel blocker cesium. ZD7288 also decreased glutamate release in hippocampal tissue and in cultured hippocampal neurons. Furthermore, ZD7288 attenuated glutamate-induced rises in [Ca(2+)]i in a concentration-dependent manner and reversed 8-Br-cAMP-mediated facilitation of these glutamate-induced [Ca(2+)]i rises. Our results suggest that ZD7288 inhibits hippocampal synaptic plasticity both glutamate release and resultant [Ca(2+)]i increases in rat hippocampal neurons.
RESUMEN
AIMS: Brain ischemia activates astrocytes in a process known as astrogliosis. Although this process has beneficial effects, excessive astrogliosis can impair neuronal recovery. Polyinosinic-polycytidylic acid (Poly IC) has shown neuroprotection against cerebral ischemia-reperfusion injury, but whether it regulates reactive astrogliosis and glial scar formation is not clear. METHODS: We exposed cultured astrocytes to oxygen-glucose deprivation/reoxygenation (OGD/R) and used a rat middle cerebral artery occlusion (MCAO)/reperfusion model to investigate the effects of Poly IC. Astrocyte proliferation and proliferation-related molecules were evaluated by immunostaining and Western blotting. Neurological deficit scores, infarct volumes and neuroplasticity were evaluated in rats after transient MCAO. RESULTS: In vitro, Poly IC inhibited astrocyte proliferation, upregulated Toll-like receptor 3 (TLR3) expression, upregulated interferon-ß, and downregulated interleukin-6 production. These changes were blocked by a neutralizing antibody against TLR3, suggesting that Poly IC function is TLR3-dependent. Moreover, in the MCAO model, Poly IC attenuated reactive astrogliosis, reduced brain infarction volume, and improved neurological function. In addition, Poly IC prevented MCAO-induced reductions in soma size, dendrite length, and number of dendritic bifurcations in cortical neurons of the infarct penumbra. CONCLUSIONS: By ameliorating astrogliosis-related damage, Poly IC is a potential therapeutic agent for attenuating neuronal damage and promoting recovery after brain ischemia.
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Gliosis/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Inductores de Interferón/uso terapéutico , Poli I-C/uso terapéutico , Recuperación de la Función/efectos de los fármacos , Receptor Toll-Like 3/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/ultraestructura , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Modelos Animales de Enfermedad , Glucosa/deficiencia , Hipoxia/tratamiento farmacológico , Inductores de Interferón/farmacología , Masculino , Poli I-C/farmacología , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión , Regulación hacia Arriba/efectos de los fármacosRESUMEN
GABA receptors play an important role in ischemic brain injury. Studies have indicated that autophagy is closely related to neurodegenerative diseases. However, during chronic cerebral hypoperfusion, the changes of autophagy in the hippocampal CA1 area, the correlation between GABA receptors and autophagy, and their influences on hippocampal neuronal apoptosis have not been well established. Here, we found that chronic cerebral hypoperfusion resulted in rat hippocampal atrophy, neuronal apoptosis, enhancement and redistribution of autophagy, down-regulation of Bcl-2/Bax ratio, elevation of cleaved caspase-3 levels, reduction of surface expression of GABAA receptor α1 subunit and an increase in surface and mitochondrial expression of connexin 43 (CX43) and CX36. Chronic administration of GABAB receptors agonist baclofen significantly alleviated neuronal damage. Meanwhile, baclofen could up-regulate the ratio of Bcl-2/Bax and increase the activation of Akt, GSK-3ß and ERK which suppressed cytodestructive autophagy. The study also provided evidence that baclofen could attenuate the decrease in surface expression of GABAA receptor α1 subunit, and down-regulate surface and mitochondrial expression of CX43 and CX36, which might enhance protective autophagy. The current findings suggested that, under chronic cerebral hypoperfusion, the effects of GABAB receptors activation on autophagy regulation could reverse neuronal damage.
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Baclofeno/farmacología , Isquemia Encefálica/metabolismo , Región CA1 Hipocampal/efectos de los fármacos , Neuroprotección/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Biomarcadores , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/genética , Caspasa 3/metabolismo , Membrana Celular/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Masculino , Mitocondrias/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Proteína delta-6 de Union ComunicanteRESUMEN
1-(2,6-Dimethylphenoxy)-2-(3,4-dimethoxyphenylethylamino)-propane hydrochloride (DDPH) is a novel antihypertensive agent based on structural characteristics of mexiletine and verapamine. We investigated the effect of DDPH on vasodilatation and neuroprotection in a rat model of cerebral ischemia in vivo, and a rabbit model of isolated basilar arteries in vitro. Our results show that DDPH (10 mg/kg) significantly increased hippocampal blood flow in vivo in cerebral ischemic rats, and exerted dose-dependent relaxation of isolated basilar arteries contracted by histamine or KCl in the in vitro rabbit model. DDPH (3 × 10(-5) M) also inhibited histamine-stimulated extracellular calcium influx and intracellular calcium release. Our findings suggest that DDPH has a vasodilative effect both in vivo and in vitro, which mediates a neuroprotective effect on ischemic nerve tissue.
RESUMEN
The hyperpolarization-activated cyclic-nucleotide-gated non-selective cation (HCN) channels play a vital role in the neurological basis underlying nervous system diseases. However, the role of HCN channels in drug addiction is not fully understood. In the present study, we investigated the expression of HCN1 and HCN2 subunits in hippocampal CA1 and the potential molecular mechanisms underlying the modulation of HCN channels in rats with chronic morphine exposure with approaches of electrophysiology, water maze, and Western blotting. We found that chronic morphine exposure (5 mg/kg, sc, for 7 days) caused an inhibition of long-term potentiation (LTP) and impairment of spatial learning and memory, which is associated with a decrease in HCN1, and an increase in HCN2 on cell membrane of hippocampal CA1 area. Additional experiments showed that the imbalance of cell membrane HCN1 and HCN2 expression under chronic morphine exposure was related to an increase in expression of TPR containing Rab8b interacting protein (TRIP8b) (1a-4) and TRIP8b (1b-2), and phosphorylation of protein kinase A (PKA) and adaptor protein 2 µ2 (AP2 µ2). Our results demonstrate the novel information that drug addiction-induced impairment of learning and memory is involved in the imbalance of HCN1 and HCN2 subunits, which is mediated by activation of TRIP8b (1a-4), TRIP8b (1b-2), PKA and AP2 µ2.
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Región CA1 Hipocampal/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Trastornos de la Memoria/inducido químicamente , Morfina/toxicidad , Narcóticos/toxicidad , Aprendizaje Espacial/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Trastornos de la Memoria/patología , Actividad Motora/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Cerebral ischemic preconditioning (IPC) protects brain against ischemic injury. Activation of Toll-like receptor 3 (TLR3) signaling can induce neuroprotective mediators, but whether astrocytic TLR3 signaling is involved in IPC-induced ischemic tolerance is not known. METHODS: IPC was modeled in mice with three brief episodes of bilateral carotid occlusion. In vitro, IPC was modeled in astrocytes by 1-h oxygen-glucose deprivation (OGD). Injury and components of the TLR3 signaling pathway were measured after a subsequent protracted ischemic event. A neutralizing antibody against TLR3 was used to evaluate the role of TLR3 signaling in ischemic tolerance. RESULTS: IPC in vivo reduced brain damage from permanent middle cerebral artery occlusion in mice and increased expression of TLR3 in cortical astrocytes. IPC also reduced damage in isolated astrocytes after 12-h OGD. In astrocytes, IPC or 12-h OGD alone increased TLR3 expression, and 12-h OGD alone increased expression of phosphorylated NFκB (pNFκB). However, IPC or 12-h OGD alone did not alter the expression of Toll/interleukin receptor domain-containing adaptor-inducing IFNß (TRIF) or phosphorylated interferon regulatory factor 3 (pIRF3). Exposure to IPC before OGD increased TRIF and pIRF3 expression but decreased pNFκB expression. Analysis of cytokines showed that 12-h OGD alone increased IFNß and IL-6 secretion; 12-h OGD preceded by IPC further increased IFNß secretion but decreased IL-6 secretion. Preconditioning with TLR3 ligand Poly I:C increased pIRF3 expression and protected astrocytes against ischemic injury; however, cells treated with a neutralizing antibody against TLR3 lacked the IPC- and Poly I:C-induced ischemic protection and augmentation of IFNß. CONCLUSIONS: The results suggest that IPC-induced ischemic tolerance is mediated by astrocytic TLR3 signaling. This reprogramming of TLR3 signaling by IPC in astrocytes may play an important role in suppression of the post-ischemic inflammatory response and thereby protect against ischemic damage. The mechanism may be via activation of the TLR3/TRIF/IRF3 signaling pathway.
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Astrocitos/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/prevención & control , Precondicionamiento Isquémico , Receptor Toll-Like 3/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Astrocitos/patología , Encéfalo/patología , Isquemia Encefálica/complicaciones , Isquemia Encefálica/patología , Células Cultivadas , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Proteína Ácida Fibrilar de la Glía/metabolismo , Glucosa/deficiencia , Infarto de la Arteria Cerebral Media/patología , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/metabolismo , Interleucina-6/metabolismo , Masculino , Ratones , Oxígeno , Fosforilación , Poli I-C/farmacología , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismoRESUMEN
Hyperpolarization-activated cyclic-nucleotide-gated cation nonselective (HCN) channels are involved in the pathology of nervous system diseases. HCN channels and γ-aminobutyric acid (GABA) receptors can mutually co-regulate the function of neurons in many brain areas. However, little is known about the co-regulation of HCN channels and GABA receptors in the chronic ischemic rats with possible features of vascular dementia. Protein kinase A (PKA) and TPR containing Rab8b interacting protein (TRIP8b) can modulate GABAB receptors cell surface stability and HCN channel trafficking, respectively, and adaptor-associated kinase 1 (AAK1) inhibits the function of the major TRIP8b-interacting protein adaptor protein 2 (AP2) via phosphorylating the AP2 µ2 subunit. Until now, the role of these regulatory factors in chronic cerebral hypoperfusion is unclear. In the present study, we evaluated whether and how HCN channels and GABAB receptors were pathologically altered and investigated neuroprotective effects of GABAB receptors activation and cross-talk networks between GABAB receptors and HCN channels in the hippocampal CA1 area in chronic cerebral hypoperfusion rat model. We found that cerebral hypoperfusion for 5 weeks by permanent occlusion of bilateral common carotid arteries (two-vessel occlusion, 2VO) induced marked spatial and nonspatial learning and memory deficits, significant neuronal loss and decrease in dendritic spine density, impairment of long-term potentiation (LTP) at the Schaffer collateral-CA1 synapses, and reduction of surface expression of GABAB R1, GABAB R2, and HCN1, but increase in HCN2 surface expression. Meanwhile, the protein expression of TRIP8b (1a-4), TRIP8b (1b-2), and AAK1 was significantly decreased. Baclofen, a GABAB receptor agonist, markedly improved the memory impairment and alleviated neuronal damage. Besides, baclofen attenuated the decrease of surface expression of GABAB R1, GABAB R2, and HCN1, but downregulated HCN2 surface expression. Furthermore, baclofen could restore expression of AAK1 protein and significantly increase p-PKA, TRIP8b (1a-4), TRIP8b (1b-2), and p-AP2 µ2 expression. Those findings suggested that, under chronic cerebral hypoperfusion, activation of PKA could attenuate baclofen-induced decrease in surface expression of GABAB R1 and GABAB R2, and activation of GABAB receptors not only increased the expression of TRIP8b (1a-4) and TRIP8b (1b-2) but also regulated the function of TRIP8b via AAK1 and p-AP2 µ2, which restored the balance of HCN1/HCN2 surface expression in rat hippocampal CA1 area, and thus ameliorated cognitive impairment.
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Región CA1 Hipocampal/metabolismo , Trastornos del Conocimiento/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales de Potasio/metabolismo , Receptores de GABA-B/metabolismo , Animales , Potenciación a Largo Plazo , Masculino , Neuronas/metabolismo , Transporte de Proteínas/fisiología , Ratas Sprague-Dawley , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The recording of hippocampal and cortical long-term potentiation (LTP) in rats in vivo is an appropriate and commonly used method to describe changes in cellular mechanisms underlying synaptic plasticity. Recently, we introduced a method for the simultaneous recording of LTP in bilateral CA1 regions and parietal association cortex (PtA), and observed differences between the Schaffer collateral-CA1 pathway (SC), Schaffer collateral/associational commissural pathway (SAC) and Schaffer collateral/associational commissural-cortex pathway (SACC). In this study, we found that (1) synaptic transmission of the SAC and SACC pathways depended on hippocampal commissural fibers [dorsal and ventral hippocampal commissural fibers, the medial septum (MS) and hippocampal CA3 commissural fibers], (2) nerve conduction velocity of the SACC pathway might be higher than that of the SAC pathway, (3) the input/output (I/O) curve of the SC pathway was shifted to the left side, compared to that of the SAC and SACC pathways, (4) all three pathways could induce stable LTP; however, LTP of the SAC and SACC pathways was much stronger than that of the SC pathway, (5) the degree of paired-pulse facilitation (PPF) was weaker in the SC pathway than that in the SAC and SACC pathways, (6) after cutting off the corpus callosum and commissural fibers, spatial learning and memory were impaired, and the ability to explore the novel environment and spontaneous locomotor activity were weakened. Taken together, our results suggested that hippocampal commissural fibers were very important for exchanging information between hemispheres, and basic differences in electrophysiological properties of hippocampal-cortical neural networks play a vital role in the processes of learning and memory.
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Corteza Cerebral/citología , Hipocampo/citología , Aprendizaje por Laberinto/fisiología , Memoria/fisiología , Vías Nerviosas/fisiología , Neuronas/fisiología , Potenciales de Acción/fisiología , Animales , Corteza Cerebral/fisiología , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/fisiología , Conducta Exploratoria/fisiología , Hipocampo/fisiología , Masculino , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/fisiología , VigiliaRESUMEN
Cullin-RING ubiquitin ligases (CRLs) are the largest family of E3 ligases and require cullin neddylation for their activation. The NEDD8-activating enzyme inhibitor MLN4924 reportedly blocked cullin neddylation and inactivated CRLs, which resulted in apoptosis induction and tumor suppression. However, CRL roles in ovarian cancer cell survival and the ovarian tumor repressing effects of MLN4924 are unknown. We show here that CRL4 components are highly expressed in human epithelial ovarian cancer tissues. MLN4924-induced DNA damage, cell cycle arrest, and apoptosis in ovarian cancer cells in a time- and dose-dependent manner. In addition, MLN4924 sensitized ovarian cancer cells to other chemotherapeutic drug treatments. Depletion of CRL4 components Roc1/2, Cul4a, and DDB1 had inhibitory effects on ovarian cancer cells similar to MLN4924 treatment, which suggested that CRL4 inhibition contributed to the chemotherapeutic effect of MLN4924 in ovarian cancers. We also investigated for key CRL4 substrate adaptors required for ovarian cancer cells. Depleting Vprbp/Dcaf1 did not significantly affect ovarian cancer cell growth, even though it was expressed by ovarian cancer tissues. However, depleting Cdt2/Dcaf2 mimicked the pharmacological effects of MLN4924 and caused the accumulation of its substrate, CDT1, both in vitro and in vivo. MLN4924-induced DNA damage and apoptosis were partially rescued by Cdt1 depletion, suggesting that CRL4(CDT2) repression and CDT1 accumulation were key biochemical events contributing to the genotoxic effects of MLN4924 in ovarian cancer cells. Taken together, these results indicate that CRL4(CDT2) is a potential drug target in ovarian cancers and that MLN4924 may be an effective anticancer agent for targeted ovarian cancer therapy.
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Ciclopentanos/farmacología , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Proteínas Nucleares/genética , Neoplasias Ováricas/tratamiento farmacológico , Pirimidinas/farmacología , Ubiquitina-Proteína Ligasas/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Carcinoma Epitelial de Ovario , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN , Relación Dosis-Respuesta a Droga , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Proteínas Nucleares/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Activation of alpha2-adrenoceptors inhibits long-term potentiation and long-term depression in many brain regions. However, effectiveness and mechanism of alpha2-adrenoceptors for synaptic plasticity at the Schaffer collateral-CA1 synapses in rat in vivo is unclear. In the present study, we investigated the effects of alpha2-adrenoceptors agonist clonidine on high-frequency stimulation (HFS)-induced long-term potentiation (LTP) and paired-pulse facilitation (PPF) at the Schaffer collateral-CA1 synapse of rat hippocampus in vivo. Clonidine (0.05, 0.1 mg/kg, ip) inhibited synaptic plasticity in a dose-dependent manner, accompanying with the decreasing of aortic pressure and heart rate (HR) in anesthetized rats. Clonidine (1.25, 2.5 µg/kg, icv, 10 min before HFS) also dose-dependently inhibited synaptic plasticity, which had no remarkable effect on HR and aortic pressure. But, 20 min after HFS, administration of clonidine (2.5 µg/kg) had no effect on LTP. The inhibitory effect of clonidine (2.5 µg/kg) on LTP was completely reversed by yohimbine (18 µg/kg, icv) and ZD7288 (5 µg/kg, icv). Moreover, the inhibition was accompanied by a significant increase of the normalized PPF ratio. Furthermore, clonidine at 1 and 10 µM significantly decreased glutamate (Glu) content in the culture supernatants of hippocampal neurons, and yohimbine at 1 and 10 µM had no effect on Glu release, while it could reverse the inhibition of clonidine (1 and 10 µM) on Glu release. In conclusion, clonidine can suppress the induction of LTP at the Schaffer collateral-CA1 synapse, and the possible mechanism is that activation of presynaptic alpha2-adrenoceptors reduces the Glu release by inhibiting HCN channels.
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Envejecimiento/fisiología , Anestesia , Región CA1 Hipocampal/fisiología , Clonidina/farmacología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Potenciación a Largo Plazo/efectos de los fármacos , Sinapsis/fisiología , Animales , Presión Sanguínea/efectos de los fármacos , Región CA1 Hipocampal/efectos de los fármacos , Células Cultivadas , Clonidina/administración & dosificación , Glutamatos/metabolismo , Frecuencia Cardíaca/efectos de los fármacos , Inyecciones Intraperitoneales , Inyecciones Intraventriculares , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Yohimbina/administración & dosificación , Yohimbina/farmacologíaRESUMEN
BACKGROUND: The role of DAXX in ovarian cancer development and metastasis has not been investigated before now. RESULTS: Overexpression of DAXX enhanced ovarian cancer cell proliferation, colony formation, and migration, whereas Daxx depletion had the opposite effects. CONCLUSION: DAXX promotes ovarian cancer cell proliferation and chemoresistance. SIGNIFICANCE: ModulatingDAXXmay be an effective strategy for preventing the recurrence and chemoresistance of ovarian cancers. Understanding the genes involved in apoptosis and DNA damage responses may improve therapeutic strategies for ovarian cancer. The death domain-associated protein DAXX can be either a pro-apoptotic or an anti-apoptotic factor, depending on the cell type and context. In this study, we found that DAXX was highly expressed in human ovarian surface epithelial tumors but not in granulosa cell tumors. In cultured ovarian cancer cells, DAXX interacted with promyelocytic leukemia protein (PML) and localized to subnuclear domains (so-called PML nuclear bodies). A role for DAXX in ovarian cancer cell proliferation, metastasis, and radio/chemoresistance was examined. Overexpression of DAXX enhanced multiple ovarian cancer cell lines' proliferation, colony formation, and migration, whereas Daxx depletion by RNA interference had the opposite effects. When transplanted into nude mice, ovarian cancer cells that overexpressed DAXX displayed enhanced tumorigenesis capability in vivo, whereas Daxx depletion inhibited tumor development. Importantly, Daxx induced tumorigenic transformation of normal ovarian surface epithelial cells. Daxx also protected ovarian cancer cells against x-irradiation- and chemotherapy-induced DNA damage by interacting with PML. Taken together, our results suggest that DAXX is a novel ovarian cancer oncogene that promotes ovarian cancer cell proliferation and chemoresistance in ovarian cancer cells. Thus, modulating DAXX-PML nuclear body activity may be an effective strategy for preventing the recurrence and chemoresistance of ovarian cancers.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cistadenoma Seroso/metabolismo , Resistencia a Antineoplásicos , Proteínas Nucleares/metabolismo , Neoplasias Ováricas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Transformación Celular Neoplásica/metabolismo , Proteínas Co-Represoras , Cistadenoma Seroso/tratamiento farmacológico , Cistadenoma Seroso/secundario , Daño del ADN , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Expresión Génica , Humanos , Ratones , Ratones Desnudos , Chaperonas Moleculares , Proteínas Nucleares/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Ovario/metabolismo , Ovario/patología , Proteína de la Leucemia Promielocítica , Tolerancia a Radiación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AIM: To examine the neuroprotective effects of the Toll-like receptor 3 (TLR3) agonist Poly I:C in acute ischemic models in vitro and in vivo. METHODS: Primary astrocyte cultures subjected to oxygen-glucose deprivation (OGD) were used as an in vitro simulated ischemic model. Poly I:C was administrated 2 h before OGD. Cell toxicity was measured using MTT assay and LDH leakage assay. The levels of TNFα, IL-6 and interferon-ß (IFNß) in the media were measured using ELISA. Toll/interleukin receptor domain-containing adaptor-inducing IFNß (TRIF) protein levels were detected using Western blot analysis. A mouse middle cerebral artery occlusion (MCAO) model was u sed for in vivo study. The animals were administered Poly I:C (0.3 mg/kg, im) 2 h before MCAO, and examined with neurological deficit scoring and TTC staining. The levels of TNFα and IL-6 in ischemic brain were measured using ELISA. RESULTS: Pretreatment with Poly I:C (10 and 20 µg/mL) markedly attenuated OGD-induced astrocyte injury, and significantly raised the cell viability and reduced the LDH leakage. Poly I:C significantly upregulated TRIF expression accompanied by increased downstream IFNß production. Moreover, Poly I:C significantly suppressed the pro-inflammatory cytokines TNFα and IL-6 production. In mice subjected to MCAO, administration of Poly I:C significantly attenuated the neurological deficits, reduced infarction volume, and suppressed the increased levels of TNFα and IL-6 in the ischemic striatum and cortex. CONCLUSION: Poly I:C pretreatment exerts neuroprotective and anti-inflammatory effects in the simulated cerebral ischemia models, and the neuroprotection is at least in part due to the activation of the TLR3-TRIF pathway.
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Astrocitos/efectos de los fármacos , Ataque Isquémico Transitorio/prevención & control , Fármacos Neuroprotectores/uso terapéutico , Poli I-C/uso terapéutico , Receptor Toll-Like 3/agonistas , Animales , Astrocitos/metabolismo , Western Blotting , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Glucosa/metabolismo , Ataque Isquémico Transitorio/metabolismo , Ratones , Fármacos Neuroprotectores/farmacología , Oxígeno/metabolismo , Poli I-C/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Menispermum dauricum rhizome has been widely used in China to treat various cardiovascular and thrombosis disorders. Some studies have reported that the phenolic alkaloids of Menispermum dauricum rhizome (PAM) have protective effects against brain ischemia injury, but the mechanism of this action remains to be clarified. In the present study, we investigated the possible mechanisms of action of PAM on experimental brain ischemia injury. Oxygen and glucose deprivation (OGD) in rat primary cortical cultures and middle cerebral artery occlusion in rats were used to mimic ischemia-reperfusion injury, respectively. The results suggested that PAM protected rat primary cortical cultures against OGD-reoxygenation induced cytotoxicity. PAM decreased extracellular glutamate content and markedly prevented the effects induced by OGD on protein level of GLT-1 and EAAC1 glutamate transporters. In addition, it reduced intracellular ROS generation. In vivo, PAM significantly reduced cerebral infarct area and ameliorated neurological functional deficits at different time points. Our findings revealed that the possible mechanism of action of PAM protected against brain ischemia injury involves regulation of GLT-1, EAAC1 and ROS generation.
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Alcaloides/farmacología , Isquemia Encefálica/tratamiento farmacológico , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 3 de Aminoácidos Excitadores/metabolismo , Menispermum/química , Fármacos Neuroprotectores/farmacología , Fenoles/farmacología , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/prevención & control , Alcaloides/aislamiento & purificación , Alcaloides/uso terapéutico , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Hipoxia de la Célula , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 3 de Aminoácidos Excitadores/genética , Líquido Extracelular/química , Líquido Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Lactato Deshidrogenasas/metabolismo , Masculino , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/fisiología , Fármacos Neuroprotectores/aislamiento & purificación , Fármacos Neuroprotectores/uso terapéutico , Fenoles/aislamiento & purificación , Fenoles/uso terapéutico , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Rizoma/químicaRESUMEN
The function of Epigallocatechin gallate (EGCG), a main component of green tea, has been widely investigated, amelioration of synaptic transmission and neuroprotective effects against ischemia-induced brain damage among others. However, the mechanism underlying is still unveiled. We investigated the effects of EGCG on high frequency stimulation-induced long-term potentiation (LTP) in the Schaffer collateral-CA1 synapse with or without cerebral ischemia injury induced by middle cerebral artery occlusion (MCAO) in vivo to examine the possible relations between EGCG and synaptic transmission. Application of EGCG modulated synaptic transmission and produced a dose-dependent improvement of the induction of LTP. However, relative high-dose EGCG can block the induction of LTP at the Schaffer collateral-CA1 synapse in normal rat in vivo. In addition, the effects of EGCG were observed on the infarct volume and neurological deficit in rats subjected to MCAO; furthermore, the cell viability of primary cultured rat hippocampal and cortical neurons suffered from oxygen-glucose deprivation were evaluated with MTT and LDH assay, which showed significant neuroprotective properties in vitro. Surprisingly, the contents of the glutamate (Glu), glycine (Gly), and gamma-aminobutyric acid amino acids were totally disequilibrated before and after cerebral ischemia injury and could be rebalanced to original level by application of EGCG. Our results suggest that EGCG is able to improve the efficiency of synaptic transmission in cerebral ischemia injury with attenuated effect related to the neuroprotection of EGCG through regulating excitatory and inhibitory amino acid balance.
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Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/fisiopatología , Región CA1 Hipocampal/fisiopatología , Catequina/análogos & derivados , Potenciación a Largo Plazo/efectos de los fármacos , Sinapsis/efectos de los fármacos , Aminoácidos/metabolismo , Animales , Infarto Encefálico/complicaciones , Infarto Encefálico/patología , Infarto Encefálico/fisiopatología , Isquemia Encefálica/patología , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/patología , Catequina/farmacología , Catequina/uso terapéutico , Forma de la Célula/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/patología , Corteza Cerebral/fisiopatología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Glucosa/deficiencia , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Masculino , Neuronas/efectos de los fármacos , Neuronas/patología , Oxígeno , Ratas , Ratas Sprague-Dawley , Sinapsis/metabolismoRESUMEN
We previously reported that inhibition of Rho-kinase (ROCK) by hydroxyl fasudil improves cognitive deficit and neuronal damage in rats with chronic cerebral ischemia (Huang et al., Cell Mol Neurobiol 28:757-768, 2008). In this study, fasudil mesylate (FM) was investigated for its neuroprotective potential in rats with ischemia following middle cerebral artery occlusion (MCAO) and reperfusion. The effect of fasudil mesylate was also studied in rat brain cortical and hippocampal slices treated with oxygen-glucose deprivation (OGD) injury. Gross anatomy showed that cerebral infarct size, measured with 2,3,5-triphenyltetrazolium chloride (TTC) staining, was significantly smaller in the FM-treated than in the non-FM-treated ischemic rats. In the brain regions vulnerable to ischemia of ischemic rats, fasudil mesylate was also found to significantly restore the enzyme protein expression level of endothelial nitric oxide synthase (eNOS), which was decreased in ischemia. However, it remarkably reduced the protein synthesis of inducible nitric oxide synthase (iNOS) that was induced by ischemia and reperfusion. In rat brain slices treated with OGD injury, fasudil mesylate increased the neuronal cell viability by 40% for cortex and by 61% for hippocampus, respectively. Finally, in the presence of OGD and fasudil mesylate, superoxide dismutase (SOD) activity was increased by 50% for cortex and by 58% for hippocampus, compared to OGD only group. In conclusion, our in vivo study showed that fasudil mesylate not only decreased neurological deficit but also reduced cerebral infarct size, possibly and at least partially by augmenting eNOS protein expression and inhibiting iNOS protein expression after ischemia-reperfusion.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Daño por Reperfusión/tratamiento farmacológico , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Animales , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Modelos Animales de Enfermedad , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatología , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/fisiopatología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/fisiopatología , Masculino , Fármacos Neuroprotectores/uso terapéutico , Óxido Nítrico Sintasa de Tipo II/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , Superóxido Dismutasa/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Sales de Tetrazolio , Resultado del TratamientoRESUMEN
Guattegaumerine is a natural product with characteristics of being lipophilic and reaching high concentration in the brain, but its function in the central nervous system has not yet been observed. This study was designed to evaluate the neuroprotective effects of guattegaumerine on rat primary cultured cortical neurons. Following a 24-h exposure of the cells to combined serum-starvation and hydrogen peroxide, a significant augment in neuron damage as determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release were observed. Preincubation of guattegaumerine dramatically improved the cell viability and inhibited LDH release. Preincubation of guattegaumerine also dramatically inhibited malondialhehyde (MDA) production and elevated the decreased total antioxidative capacity in cells caused by the combined injury. Results of flow cytometry and immunohistochemistry showed that pre-addition of guattegaumerine interrupted the apoptosis of the neurons, reversed the up regulation of the pro-apoptotic gene (Bax) and the down regulation of the anti-apoptotic gene (Bcl-2). Furthermore, guattegaumerine suppressed the increase of intracellular calcium ([Ca(2+)](i)) stimulated by either H(2)O(2) or KCl in Ca(2+)-containing extracellular solutions, and high concentration of 2.5 microM guattegaumerine also suppressed the increase of [Ca(2+)](i) induced by H(2)O(2) in Ca(2+)-free solution. These observations suggested that guattegaumerine may possess potential protection against oxidative stress injury, which might be beneficial for neurons.
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Corteza Cerebral/citología , Citoprotección/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Isoquinolinas/farmacología , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Suero , Animales , Animales Recién Nacidos , Antioxidantes/metabolismo , Apoptosis/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Isoquinolinas/química , L-Lactato Deshidrogenasa/metabolismo , Malondialdehído/metabolismo , Neuronas/citología , Cloruro de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Piracetam is the derivate of gamma-aminobutyric acid, which improves the cognition,memory,consciousness, and is widely applied in the clinical treatment of brain dysfunction. In the present experiments, we study the effects of piracetam on chronic cerebral hypoperfused rats and observe its influence on amino acids, synaptic plasticity in the Perforant path-CA3 pathway and apoptosis in vivo. Cerebral hypoperfusion for 30 days by occlusion of bilateral common carotid arteries induced marked amnesic effects along with neuron damage, including: (1) spatial learning and memory deficits shown by longer escape latency and shorter time spent in the target quadrant; (2) significant neuronal loss and nuclei condensation in the cortex and hippocampus especially in CA1 region; (3) lower induction rate of long term potentiation, overexpression of BAX and P53 protein, and lower content of excitatory and inhibitory amino acids in hippocampus. Oral administration of piracetam (600 mg/kg, once per day for 30 days) markedly improved the memory impairment, increased the amino acid content in hippocampus, and attenuated neuronal damage. The ability of piracetam to attenuate memory deficits and neuronal damage after hypoperfusion may be beneficial in cerebrovascular type dementia.
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Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/etiología , Hipoxia-Isquemia Encefálica/complicaciones , Piracetam/uso terapéutico , Animales , Evaluación Preclínica de Medicamentos , Electrofisiología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Ratas , Ratas Sprague-Dawley , Natación , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
AIM: To study the effect of ZD7288 on synaptic transmission in the pathway from perforant pathway (PP) fibers to CA3 region in rat hippocampus. METHODS: The extracellular recording technique in vivo was used to record the CA3 region field potentials. High-performance liquid chromatography (HPLC) with fluorescence detection was applied to measure the content of amino acids in hippocampal tissues. The effect of ZD7288 and CsCl on the amplitudes of population spike (PS) in CA3 region evoked by stimulation (0.5 Hz) of the perforant pathway (PP) fibers, and the content of amino acids in hippocampal tissue were observed. RESULTS: Microinjection of ZD7288 (20, 100 and 200 nmol) and CsCl (1, 5 and 10 micromol) into CA3 region decreased the population spike (PS) amplitudes in a dose-dependent manner. The inhibitory effects appeared at 5 min after microinjection and lasted at least 90 min. In those rats treated with ZD7288 (100 nmol), the contents of glutamate, aspartate, glycine and GABA decreased significantly as compared to those of saline control (all P < 0.01, except P < 0.05 for that of glycine). A similar decrease in the contents of amino acids was observed when the rats were microinjected with CsCl (5 micromol). CONCLUSION; ZD7288 could obviously inhibit synaptic transmission in the pathway from PP fibers to CA3 region in rat hippocampus, and this action of ZD7288 may be associated with altered contents of amino acids.