RESUMEN
Resveratrol is a dietary polyphenol that displays neuroprotective properties in several in vivo and in vitro experimental models, by modulating oxidative and inflammatory responses. Glutathione (GSH) is a key antioxidant in the central nervous system (CNS) that modulates several cellular processes, and its depletion is associated with oxidative stress and inflammation. Therefore, this study sought to investigate the protective effects of resveratrol against GSH depletion pharmacologically induced by buthionine sulfoximine (BSO) in C6 astroglial cells, as well as its underlying cellular mechanisms. BSO exposure resulted in several detrimental effects, decreasing glutamate-cysteine ligase (GCL) activity, cystine uptake, GSH intracellular content and the activities of the antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR). Moreover, BSO increased reactive oxygen/nitrogen species (ROS/RNS) levels and pro-inflammatory cytokine release. Resveratrol prevented these effects by protecting astroglial cells against BSO-induced cytotoxicity, by modulating oxidative and inflammatory responses. Additionally, we observed that pharmacological inhibition of heme oxygenase 1 (HO-1), an essential cellular defense against oxidative and inflammatory injuries, abolished all the protective effects of resveratrol. These observations suggest HO-1 pathway as a cellular effector in the mechanism by which resveratrol protects astroglial cells against GSH depletion, a condition that may be associated to neurodegenerative diseases.
Asunto(s)
Astrocitos/enzimología , Butionina Sulfoximina/efectos adversos , Glutatión/metabolismo , Hemo-Oxigenasa 1/metabolismo , Estilbenos/farmacología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/inmunología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Masculino , Estrés Oxidativo/efectos de los fármacos , Ratas , Resveratrol , Transducción de Señal/efectos de los fármacosRESUMEN
Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na(+)/K(+)-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate-glutamine cycle, as well as glutamate and D-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems.
Asunto(s)
Acetilcolinesterasa/análisis , Aminoácidos/análisis , Astrocitos/química , Corteza Cerebral/química , ATPasa Intercambiadora de Sodio-Potasio/análisis , Acetilcolinesterasa/metabolismo , Factores de Edad , Aminoácidos/metabolismo , Animales , Astrocitos/metabolismo , Células Cultivadas , Corteza Cerebral/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Proteínas Transportadoras de GABA en la Membrana Plasmática/análisis , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Masculino , Ratas , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Astrocytes are important brain targets of ammonia, a neurotoxin implicated in the development of hepatic encephalopathy. During hyperammonemia, the pivotal role of astrocytes in brain function and homeostasis is impaired. These cells are abundantly interconnected by gap junctions (GJ), which are intercellular channels that allow the exchange of signaling molecules and metabolites. This communication may also increase cellular vulnerability during injuries, while GJ uncoupling could limit the extension of a lesion. Therefore, the current study was performed to investigate whether astrocyte coupling through GJ contributes to ammonia-induced cytotoxicity. We found that carbenoxolone (CBX), an effective GJ blocker, prevented the following effects induced by ammonia in astrocyte primary cultures: (1) decrease in cell viability and membrane integrity; (2) increase in reactive oxygen species production; (3) decrease in GSH intracellular levels; (4) GS activity; (5) pro-inflammatory cytokine release. On the other hand, CBX had no effect on C6 astroglial cells, which are poorly coupled via GJ. To our knowledge, this study provides the first evidence that GJ play a role in ammonia-induced cytotoxicity. Although more studies in vivo are required to confirm our hypothesis, our data suggest that GJ communication between astrocytes may transmit damage signals and excitotoxic components from unhealthy to normal cells, thereby contributing to the propagation of the neurotoxicity of ammonia.
Asunto(s)
Amoníaco/toxicidad , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Animales , Carbenoxolona/farmacología , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Glutatión/metabolismo , Mediadores de Inflamación/metabolismo , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Hyperammonemia induces significant changes in the central nervous system (CNS) in direct association with astroglial functions, such as oxidative damage, glutamatergic excitotoxicity, and impaired glutamine synthetase (GS) activity and pro-inflammatory cytokine release. Classically, lipoic acid (LA) and N-acetylcysteine (NAC) exhibit antioxidant and anti-inflammatory activities by increasing glutathione (GSH) biosynthesis and decreasing pro-inflammatory mediator levels in glial cells. Thus, we evaluated the protective effects of LA and NAC against ammonia cytotoxicity in C6 astroglial cells. Ammonia decreased GSH levels and increased cytokine release and NFκB transcriptional activation. LA and NAC prevented these effects by the modulation of ERK and HO1 pathways. Taken together, these observations show that LA and NAC prevent the ammonia-induced inflammatory response.