RESUMEN
INTRODUCTION: There is increasing evidence for the involvement of chronic inflammation and oxidative stress in the pathogenesis of Alzheimer's disease (AD). Nuclear factor erythroid 2-related factor 2 (Nrf2) is an anti-inflammatory transcription factor that regulates the oxidative stress defense. Our previous experiments demonstrated that kavalactones protect neuronal cells against Amyloid ß (Aß)-induced oxidative stress in vitro by Nrf2 pathway activation. Here, we tested an in vivo kavalactone treatment in a mouse model of AD. METHODS: The kavalactone methysticin was administered once a week for a period of 6 months to 6 month old transgenic APP/Psen1 mice by oral gavage. Nrf2 pathway activation was measured by methysticin treatment of ARE-luciferase mice, by qPCR of Nrf2-target genes and immunohistochemical detection of Nrf2. Aß burden was analyzed by CongoRed staining, immunofluorescent detection and ELISA. Neuroinflammation was assessed by immunohistochemical stainings for microglia and astrocytes. Pro-inflammatory cytokines in the hippocampus was determined by Luminex multi-plex assays. The hippocampal oxidative damage was detected by oxyblot technique and immunohistochemical staining against DT3 and 4-HNE. The cognitive ability of mice was evaluated using Morris water maze. RESULTS: Methysticin treatment activated the Nrf2 pathway in the hippocampus and cortex of mice. The Aß deposition in brains of methysticin-treated APP/Psen1 mice was not altered compared to untreated mice. However, methysticin treatment significantly reduced microgliosis, astrogliosis and secretion of the pro-inflammatory cytokines TNF-α and IL-17A. In addition, the oxidative damage of hippocampi from APP/Psen1 mice was reduced by methysticin treatment. Most importantly, methysticin treatment significantly attenuated the long-term memory decline of APP/Psen1 mice. CONCLUSION: In summary, these findings show that methysticin administration activates the Nrf2 pathway and reduces neuroinflammation, hippocampal oxidative damage and memory loss in a mouse model of AD. Therefore, kavalactones might be suitable candidates to serve as lead compounds for the development of a new class of neuroprotective drugs.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Trastornos de la Memoria/tratamiento farmacológico , Fármacos Neuroprotectores/administración & dosificación , Presenilina-1/genética , Piranos/administración & dosificación , Administración Oral , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Aprendizaje por Laberinto/efectos de los fármacos , Trastornos de la Memoria/genética , Trastornos de la Memoria/metabolismo , Ratones , Ratones Transgénicos , Factor 2 Relacionado con NF-E2/metabolismo , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Piranos/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Mechanical ventilation (MV) elicits complex and clinically relevant cellular responses in the lungs. The current study was designed to define the role of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), a major regulator of the cellular antioxidant defense system, in the pulmonary response to MV. Nrf2 activity was quantified in ventilated isolated perfused mouse lungs (IPL). Regulation of amphiregulin (AREG) was investigated in BEAS-2B cells with inactivated Nrf2 or Keap1, the inhibitor of Nrf2, using a luciferase vector with AREG promoter. AREG-dependent Nrf2 activity was examined in BEAS-2B cells, murine precision-cut lung slices (PCLS), and IPL. Finally, Nrf2 knockout and wild-type mice were ventilated to investigate the interplay between Nrf2 and AREG during MV in vivo. Lung functions and inflammatory parameters were measured. Nrf2 was activated in a ventilation-dependent manner. The knockdown of Nrf2 and Keap1 via short hairpin RNA in BEAS-2B cells and an EMSA with lung tissue revealed that AREG is regulated by Nrf2. Conversely, AREG application induced a significant Nrf2 activation in BEAS-2B cells, PCLS, and IPL. The signal transduction of ventilation-induced Nrf2 activation was shown to be p38 MAP kinase-dependent. In vivo ventilation experiments indicated that AREG is regulated by Nrf2 during MV. We conclude that Areg expression is regulated by Nrf2. During high-pressure ventilation, Nrf2 becomes activated and induces AREG, leading to a positive feedback loop between Nrf2 and AREG, which involves the p38 MAPK and results in the expression of cytoprotective genes.
Asunto(s)
Bronquios/fisiología , Familia de Proteínas EGF/metabolismo , Factor 2 Relacionado con NF-E2/genética , Respiración Artificial , Anfirregulina , Animales , Elementos de Respuesta Antioxidante/fisiología , Bronquios/citología , Células Cultivadas , Retroalimentación Fisiológica/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/metabolismo , Técnicas de Cultivo de Órganos , Regiones Promotoras Genéticas/fisiología , Mucosa Respiratoria/citología , Mucosa Respiratoria/fisiología , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
INTRODUCTION: Rheumatoid arthritis (RA) is characterized by progressive inflammation associated with rampantly proliferating synoviocytes and joint destruction due to oxidative stress. Recently, we described nuclear factor erythroid 2-related factor 2 (Nrf2) as a major requirement for limiting cartilage destruction. NF-κB and AP-1 are the main transcription factors triggering the inflammatory progression in RA. We used sulforaphane, an isothiocyanate, which is both an Nrf2 inducer and a NF-κB and AP-1 inhibitor. METHODS: Cultured synoviocytes were stimulated with sulforaphane (SFN) with or without TNF-α pre-treatment. NF-κB, AP-1, and Nrf2 activation was investigated via dual luciferase reporter gene assays. Matrix metalloproteinases (MMPs) were measured via zymography and luminex technique. Cytokine levels were detected using ELISA. Cell viability, apoptosis and caspase activity were studied. Cell proliferation was analysed by real-time cell analysis. RESULTS: SFN treatment decreased inflammation and proliferation dose-dependently in TNF-α-stimulated synoviocytes. SFN did not reduce MMP-3 and MMP-9 activity or expression significantly. Interestingly, we demonstrated that SFN has opposing effects on naïve and TNF-α-stimulated synoviocytes. In naïve cells, SFN activated the cytoprotective transcription factor Nrf2. In marked contrast to this, SFN induced apoptosis in TNF-α-pre-stimulated synoviocytes. CONCLUSIONS: We were able to show that SFN treatment acts contrary on naïve and inflammatory synoviocytes. SFN induces the cytoprotective transcription factor Nrf2 in naïve synoviocytes, whereas it induces apoptosis in inflamed synoviocytes. These findings indicate that the use of sulforaphane might be considered as an adjunctive therapeutic strategy to combat inflammation, pannus formation, and cartilage destruction in RA.
Asunto(s)
Isotiocianatos/farmacología , Factor 2 Relacionado con NF-E2/agonistas , FN-kappa B/antagonistas & inhibidores , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Sulfóxidos , Membrana Sinovial/citología , Factor de Transcripción AP-1/metabolismoRESUMEN
INTRODUCTION: The mechanisms of ventilator-induced lung injury (VILI), including the role of MAP kinases, are frequently studied in different mouse strains. A useful model for such studies is the isolated perfused mouse lung. As a further development we present the one-lung method that permits to continue perfusion and ventilation of the right lung after removal of the left lung. This method was used to compare the effect of high pressure ventilation (HPV) on pro-inflammatory signaling events in two widely used mouse strains (C57BL/6, BALB/c) and to further define the role of p38 in VILI. METHODS: Lungs were perfused and ventilated for 30 min under control conditions before they were randomized to low (8 cm H(2)O) or high (25 cm H(2)O) pressure ventilation (HPV) for 210 min, with the left lung being removed after 180 min. In the left lung we measured the phosphorylation of p38, JNK, ERK and Akt kinase, and in the right lung gene expression and protein concentrations of Il1b, Il6, Tnf, Cxcl1, Cxcl2, and Areg. RESULTS: Lung mechanics and kinase activation were similar in both mouse strains. HPV increased all genes (except Tnf in BALB/c) and all mediators in both strains. The gene expression of mRNA for Il1b, Il6, Cxcl1 and Cxcl2 was higher in BALB/c mice. Backward regression of the kinase data at tâ=â180 min with the gene and protein expression data at tâ=â240 min suggested that p38 controls HPV-induced gene expression, but not protein production. This hypothesis was confirmed in experiments with the p38-kinase inhibitor SB203580. CONCLUSIONS: The one-lung method is useful for mechanistic studies in the lungs. While C57BL/6 show diminished pro-inflammatory responses during HPV, lung mechanics and mechanotransduction processes appear to be similar in both mouse strains. Finally, the one-lung method allowed us to link p38 to gene expression during VILI.
Asunto(s)
Pulmón/metabolismo , Sistema de Señalización de MAP Quinasas , Respiración con Presión Positiva/efectos adversos , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Citocinas/biosíntesis , Inhibidores Enzimáticos/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Imidazoles/farmacología , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Pulmón/patología , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/farmacología , Especificidad de la Especie , Lesión Pulmonar Inducida por Ventilación Mecánica/patología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Corticotropin releasing factor (CRF) is a major mediator of central and peripheral responses to environmental stressors, and antagonism of its receptors (CRF-R1, -R2) is an active area of pharmacotherapeutic research for stress-related disorders. Stress responses include CRF activation of the hypothalamus-pituitary-adrenal axis and behavioural inhibition. Valid in vivo models for the study of these neuro-endocrine and -behavioural CRF pathways and their central-peripheral antagonism are important. The aims of this study in C57BL/6 mice were to describe the acute effects of intracerebroventricular (ICV) CRF using plasma ACTH-CORT titres and locomotor activity as readouts, and to study the impact on these readouts of central versus peripheral pre-treatment with the CRF-R1/2 antagonist, astressin. The following experiments were performed: Effects of (i) serial blood sampling (SBS) per se, (ii) physical confinement+SBS, (iii) ICV saline infusion+SBS, on plasma titres of ACTH-CORT. (iv) Effects of ICV or IP CRF infusion on plasma ACTH-CORT. (v) Effects of ICV CRF on plasma CRF. (vi) Effects of ICV or IP astressin on ICV or IP CRF-stimulated plasma CORT. (vii) Effects of ICV or IP astressin on ICV CRF-induced locomotor inactivity. Main findings were: (i)-(ii) Serial blood sampling per se and physical confinement+SBS led to similar, mild increases in plasma ACTH-CORT. (iii) ICV saline infusion led to a marked increase in plasma ACTH, possibly due to assay crossreactivity with "washed out" pituitary peptides, and a mild increase in plasma CORT. (iv) ICV CRF (0.001-1µg) induced no further increase in plasma ACTH versus vehicle, and induced dose-dependent increased plasma CORT. 1µg ICV CRF also reduced locomotor activity. (v) ICV CRF-induced dose-dependent increased plasma CRF. (vi) ICV astressin failed to block ICV CRF-induced increased plasma CORT, whereas IP astressin did do so. (vii) ICV CRF-induced locomotor inactivity was blocked by ICV astressin, but not by IP astressin. Therefore, ICV CRF-induced a dose-dependent increase in plasma CORT via a peripheral pathway and a reduction in locomotion via a central pathway, indicated by the double dissociation in the ability of astressin to antagonize these effects relative to its route of administration, IP or ICV, respectively. The preparation described here could be readily used to provide initial indications on the central and peripheral activity of CRF-R antagonists, including pharmacokinetics following peripheral administration.
Asunto(s)
Hormona Adrenocorticotrópica/metabolismo , Sistema Nervioso Central/metabolismo , Corticosterona/sangre , Hormona Liberadora de Corticotropina/administración & dosificación , Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Actividad Motora/fisiología , Animales , Sistema Nervioso Central/efectos de los fármacos , Hormona Liberadora de Corticotropina/farmacología , Relación Dosis-Respuesta a Droga , Infusiones Intraventriculares , Masculino , Ratones , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Estrés Psicológico/sangre , Factores de TiempoRESUMEN
The metabotropic glutamate receptor subtype 7 (mGluR7) is presynaptically located and modulates transmitter release. An earlier study from our group demonstrated that systemic administration of N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082), a selective allosteric mGluR7 agonist, attenuates the acquisition of conditioned fear measured by fear-potentiated startle. Aim of this study was to explore whether this effect is mediated by the basolateral amygdala, a crucial brain structure for acquisition of conditioned fear. Therefore, AMN082 was locally injected into the basolateral amygdala of rats and the effects of these injections on the acquisition of conditioned fear was measured. Our data clearly show that intra-amygdala injection of AMN082 impairs fear acquisition. This finding demonstrates that amygdaloid mGluR7 controls the learning of conditioned fear.
Asunto(s)
Amígdala del Cerebelo/efectos de los fármacos , Compuestos de Bencidrilo/farmacología , Condicionamiento Clásico/efectos de los fármacos , Receptores de Glutamato Metabotrópico/fisiología , Estimulación Acústica/métodos , Amígdala del Cerebelo/anatomía & histología , Amígdala del Cerebelo/fisiología , Animales , Compuestos de Bencidrilo/administración & dosificación , Condicionamiento Clásico/fisiología , Electrochoque/métodos , Extinción Psicológica/efectos de los fármacos , Extinción Psicológica/fisiología , Miedo/efectos de los fármacos , Miedo/fisiología , Miedo/psicología , Inyecciones/métodos , Aprendizaje/efectos de los fármacos , Aprendizaje/fisiología , Masculino , Memoria/efectos de los fármacos , Memoria/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/agonistas , Reflejo de Sobresalto/efectos de los fármacos , Reflejo de Sobresalto/fisiologíaRESUMEN
The bed nucleus of the stria terminalis (BNST) is involved in the mediation of fear behavior in rats. A previous study of our laboratory demonstrated that temporary inactivation of the BNST blocks fear behavior induced by exposure to trimethylthiazoline (TMT), a component of fox odor. The present study investigates whether noradrenaline release within the BNST is critical for TMT-induced fear behavior. First, we confirmed previous studies showing that the ventral BNST is the part of the BNST that receives the densest noradrenaline innervation. Second, using in vivo microdialysis, we showed that noradrenaline release within the BNST is strongly increased during TMT exposure, and that this increase can be blocked by local infusions of the alpha2-receptor blocker clonidine. Third, using intracerebral injections, we showed that clonidine injections into the ventral BNST, but not into neighboring brain sites, completely blocked TMT-induced potentiation of freezing behavior. The present data clearly show that the noradrenergic innervation of the ventral BNST is important for the full expression of behavioral signs of fear to the predator odor TMT.