RESUMEN
Cannabis sativa L. is increasingly emerging for its protective role in modulating neuroinflammation, a complex process orchestrated among others by microglia, the resident immune cells of the central nervous system. Phytocannabinoids, especially cannabidiol (CBD), terpenes, and other constituents trigger several upstream and downstream microglial intracellular pathways. Here, we investigated the molecular mechanisms of a CBD- and terpenes-enriched C. sativa extract (CSE) in an in vitro model of neuroinflammation. We evaluated the effect of CSE on the inflammatory response induced by exposure to lipopolysaccharide (LPS) in BV-2 microglial cells, compared with CBD and ß-caryophyllene (CAR), CB2 receptors (CB2r) inverse and full agonist, respectively. The LPS-induced upregulation of the pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α was significantly attenuated by CSE and only partially by CBD, whereas CAR was ineffective. In BV-2 cells, these anti-inflammatory effects exerted by CSE phytocomplex were only partially dependent on CB2r modulation and they were mediated by the regulation of enzymes responsible for the endocannabinoids metabolism, by the inhibition of reactive oxygen species release and the modulation of JNK/p38 cascade with consequent NF-κB p65 nuclear translocation suppression. Our data suggest that C. sativa phytocomplex and its multitarget mechanism could represent a novel therapeutic strategy for neuroinflammatory-related diseases.
Asunto(s)
Cannabidiol , Cannabis , Cannabidiol/farmacología , Cannabidiol/uso terapéutico , Citocinas/metabolismo , Endocannabinoides/farmacología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Microglía , FN-kappa B/metabolismo , Receptor Cannabinoide CB2/metabolismoRESUMEN
Epigenetic modifications of DNA and histone proteins are emerging as fundamental mechanisms by which neural cells adapt their transcriptional response to environmental cues, such as, immune stimuli or stress. In particular, histone H3 phospho(Ser10)-acetylation(Lys14) (H3S10phK14ac) has been linked to activation of specific gene expression. The purpose of this study was to investigate the role of H3S10phK14ac in a neuroinflammatory condition. Adult male rats received a intraperitoneal injection of lipopolysaccharide (LPS) (830⯵g/Kg/i.p., nâ¯=â¯6) or vehicle (saline 1â¯mL/kg/i.p., nâ¯=â¯6) and were sacrificed 2 or 6â¯h later. We showed marked region- and time-specific increases in H3S10phK14ac in the hypothalamus and hippocampus, two principal target regions of LPS. These changes were accompanied by a marked transcriptional activation of interleukin (IL) 1ß, IL-6, Tumour Necrosis Factor (TNF) α, the inducible nitric oxide synthase (iNOS) and the immediate early gene c-Fos. By means of chromatin immunoprecipitation, we demonstrated an increased region- and time-specific association of H3S10phK14ac with the promoters of IL-6, c-Fos and iNOS genes, suggesting that part of the LPS-induced transcriptional activation of these genes is regulated by H3S10phK14ac. Finally, by means of multiple immunofluorescence approach, we showed that increased H3S10phK14ac is cell type-specific, being neurons and reactive microglia, the principal histological types involved in this response. Present data point to H3S10phK14ac as a principal epigenetic regulator of neural cell response to systemic LPS and underline the importance of distinct time-, region- and cell-specific epigenetic mechanisms that regulate gene transcription to understand the mechanistic complexity of neuroinflammatory response to immune challenges.
Asunto(s)
Histonas/metabolismo , Neuroinmunomodulación/efectos de los fármacos , Acetilación/efectos de los fármacos , Animales , Encéfalo/metabolismo , Epigénesis Genética/fisiología , Expresión Génica/efectos de los fármacos , Hipocampo/metabolismo , Hipotálamo/metabolismo , Lipopolisacáridos/farmacología , Masculino , Microglía/metabolismo , Microglía/fisiología , Neuroinmunomodulación/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Sprague-Dawley , Activación Transcripcional/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Converging evidence points at hypothalamus-pituitary-adrenal (HPA) axis hyperactivity and neuroinflammation as important factors involved in the etiopathogenesis of major depressive disorder (MDD) and in therapeutic efficacy of antidepressants. In this study, we examined the molecular effects associated with a response to a week-long treatment with escitalopram in the chronic escape deficit (CED) model, a validated model of depression based on the induction of an escape deficit after exposure of rats to an unavoidable stress. We confirmed our previous result that a treatment with escitalopram (10mg/kg) was effective after 7days in reverting the stress-induced escape deficit in approximately 50% of the animals, separating responders from non-responders. Expression of markers of HPA axis functionality as well as several inflammatory mediators were evaluated in the hypothalamus, a key structure integrating signals from the neuro, immune, endocrine systems. In the hypothalamus of responder animals we observed a decrease in the expression of CRH and its receptors and an increase in GR protein in total and nuclear extracts; this effect was accompanied by a significant decrease in circulating corticosterone in the same cohort. Hypothalamic IL-1ß and TNFα expression were increased in stressed animals, while CXCL2, IL-6, and ADAM17 mRNA levels were decreased in escitalopram treated rats regardless of the treatment response. These data suggest that efficacy of a one week treatment with escitalopram may be partially mediated by a decrease HPA axis activity, while in the hypothalamus the drug-induced effects on the expression of immune modulators did not correlate with the behavioural outcome.
Asunto(s)
Citalopram/metabolismo , Citalopram/farmacología , Depresión/tratamiento farmacológico , Hormona Adrenocorticotrópica/metabolismo , Animales , Antidepresivos/uso terapéutico , Corticosterona/análisis , Corticosterona/sangre , Corticosterona/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Depresión/metabolismo , Trastorno Depresivo Mayor/metabolismo , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Hipotálamo/metabolismo , Masculino , Sistema Hipófiso-Suprarrenal/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Estrés Psicológico/complicacionesRESUMEN
To gain insight into the possible immune targets of antidepressant, we evaluated the expression of several inflammatory mediators in the hypothalamus of rats chronically (28 days) treated with the serotonin selective reuptake inhibitor fluoxetine (5mg/kg, i.p.) or the tricyclic compound imipramine (15 mg/kg, i.p.). We focused our attention on the hypothalamus as it plays a key role in determining many of the somatic symptoms experienced by depressed patients. This brain region, critical also for expression of motivated behaviours, participates in the control of the hypothalamic-pituitary-adrenal axis activity and in stress response as well as coordinates physiological functions such as sleep and food intake that have been found altered in a high percentage of depressed patients. Notably, hypothalamus is a key structure for brain cytokine expression and function as it integrates signals from the neuro, immune, endocrine systems. By means of quantitative Real Time PCR experiments we demonstrated that a chronic treatment with either fluoxetine or imipramine resulted in a reduction of IL-6 and IFN-γ mRNAs and increased IL-4 mRNA expression in the rat hypothalamus. Moreover, we demonstrated that hypothalamic expression of members of IL-18 system was differentially affected by chronic antidepressant treatments. Chronically administered fluoxetine decreased IL-8 and CX3CL1 hypothalamic expression, while a chronic treatment with imipramine decreased p11 mRNA. Our data suggest that a shift in the balance of the inflammation toward an anti-inflammatory state in the hypothalamus may represent a common mechanism of action of both the chronic treatments with fluoxetine and imipramine.
Asunto(s)
Antidepresivos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Animales , Anexina A2/genética , Citocinas/genética , Regulación de la Expresión Génica/inmunología , Inflamación/genética , Inflamación/inmunología , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Interleucina-18/genética , Proteínas S100/genética , Factores de TiempoRESUMEN
The cytokine IL-18 acts on the CNS both in physiological and pathological conditions. Its action occurs through the heterodimeric receptor IL-18Ralpha\beta. To better understand IL-18 central effects, we investigated in the mouse brain the distribution of two IL-18Ralpha transcripts, a full length and an isoform lacking the intracellular domain hypothesized to be a decoy receptor. Both isoforms were expressed in neurons throughout the brain primarily with overlapping distribution but also with some unique pattern. These data suggest that IL-18 may modulate neuronal functions and that its action may be regulated through expression of a decoy receptor.
Asunto(s)
Encéfalo/metabolismo , Subunidad alfa del Receptor de Interleucina-18/metabolismo , Interleucina-18/metabolismo , Empalme Alternativo , Animales , Encéfalo/inmunología , Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Exones , Hipocampo/metabolismo , Hipotálamo/metabolismo , Inmunohistoquímica , Hibridación in Situ , Interleucina-18/inmunología , Subunidad alfa del Receptor de Interleucina-18/inmunología , Subunidad beta del Receptor de Interleucina-18/metabolismo , Intrones , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
The Ca(2+)/calmodulin-dependent protein kinase kinases alpha and beta (CaMKKs alpha and beta) are novel members of the CaM kinase family. The CaMKKbeta was cloned from mouse brain. The deduced amino acid sequence shared 96.43% homology with the rat CaMKKbeta. Both the alpha and beta isoforms were widely distributed throughout the adult mouse brain. Additionally, all peripheral tissues examined displayed CaMKK alpha and beta expression.