RESUMO
Phytocannabinoids modulate inflammatory responses by regulating the production of cytokines in several experimental models of inflammation. Cannabinoid type-2 (CB2) receptor activation was shown to reduce the production of the monocyte chemotactic protein-2 (MCP-2) chemokine in polyinosinic-polycytidylic acid [poly-(I:C)]-stimulated human keratinocyte (HaCaT) cells, an in vitro model of allergic contact dermatitis (ACD). We investigated if nonpsychotropic cannabinoids, such as cannabidiol (CBD), produced similar effects in this experimental model of ACD. HaCaT cells were stimulated with poly-(I:C), and the release of chemokines and cytokines was measured in the presence of CBD or other phytocannabinoids (such as cannabidiol acid, cannabidivarin, cannabidivarinic acid, cannabichromene, cannabigerol, cannabigerolic acid, cannabigevarin, tetrahydrocannabivarin, and tetrahydrocannabivarinic acid) and antagonists of CB1, CB2, or transient receptor potential vanilloid type-1 (TRPV1) receptors. HaCaT cell viability following phytocannabinoid treatment was also measured. The cellular levels of endocannabinoids [anandamide (AEA), 2-arachidonoylglycerol] and related molecules (palmitoylethanolamide, oleoylethanolamide) were quantified in poly-(I:C)-stimulated HaCaT cells treated with CBD. We show that in poly-(I:C)-stimulated HaCaT cells, CBD elevates the levels of AEA and dose-dependently inhibits poly-(I:C)-induced release of MCP-2, interleukin-6 (IL-6), IL-8, and tumor necrosis factor-α in a manner reversed by CB2 and TRPV1 antagonists 6-iodopravadoline (AM630) and 5'-iodio-resiniferatoxin (I-RTX), respectively, with no cytotoxic effect. This is the first demonstration of the anti-inflammatory properties of CBD in an experimental model of ACD.
Assuntos
Anti-Inflamatórios/farmacologia , Canabidiol/farmacologia , Dermatite Alérgica de Contato/tratamento farmacológico , Anti-Inflamatórios/uso terapêutico , Ácidos Araquidônicos/metabolismo , Canabidiol/uso terapêutico , Linhagem Celular , Quimiocina CCL8/metabolismo , Dermatite Alérgica de Contato/metabolismo , Endocanabinoides/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Palmitoylethanolamide (PEA) is a nutraceutical compound that has been demonstrated to improve intestinal inflammation. We aimed at evaluating its antiproliferative and antiangiogenic effects in human colon adenocarcinoma Caco-2 cell line. Caco-2 cells were treated with increasing concentrations of PEA (0.001, 0.01 and 0.1 µM) in the presence of peroxisome proliferator-activated receptor-a (PPAR-α) or PPAR-γ antagonists. Cell proliferation was evaluated by performing a MTT assay. Vascular endothelial growth factor (VEGF) release was estimated by ELISA, while the expression of VEGF receptor and the activation of the Akt/mammalian target of rapamycin (mTOR) pathway were evaluated by western blot analysis. PEA caused a significant and concentration-dependent decrease of Caco-2 cell proliferation at 48 h. PEA administration significantly reduced in a concentration-dependent manner VEGF secretion and VEGF receptor expression. Inhibition of Akt phosphorylation and a downstream decrease of phospho-mTOR and of p-p70S6K were observed as compared with untreated cells. PPAR-α, but not PPAR-γ antagonist, reverted all effects of PEA. PEA is able to decrease cell proliferation and angiogenesis. The antiangiogenic effect of PEA depends on the specific inhibition of the AkT/mTOR axis, through the activation of PPAR-α pathway. If supported by in vivo models, our data pave the way to PEA co-administration to the current chemotherapeutic regimens for colon carcinoma. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Etanolaminas/química , PPAR alfa/metabolismo , Ácidos Palmíticos/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Amidas , Animais , Células CACO-2 , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo/efeitos dos fármacos , Etanolaminas/uso terapêutico , Humanos , Neovascularização Patológica , Ácidos Palmíticos/uso terapêutico , Transdução de SinaisRESUMO
This minireview highlights the importance of cannabidiol (CBD) as a promising drug for the therapy of inflammatory bowel diseases (IBD). Actual pharmacological treatments for IBD should be enlarged toward the search for low-toxicityand low-cost drugs that may be given alone or in combination with the conventional anti-IBD drugs to increase their efficacy in the therapy of relapsing forms of colitis. In the past, Cannabis preparations have been considered new promising pharmacological tools in view of their anti-inflammatory role in IBD as well as other gut disturbances. However, their use in the clinical therapy has been strongly limited by their psychotropic effects. CBD is a very promising compound since it shares the typical cannabinoid beneficial effects on gut lacking any psychotropic effects. For years, its activity has been enigmatic for gastroenterologists and pharmacologists, but now it is evident that this compound may interact at extra-cannabinoid system receptor sites, such as peroxisome proliferator-activated receptor-gamma. This strategic interaction makes CBD as a potential candidate for the development of a new class of anti-IBD drugs.
Assuntos
Canabidiol/uso terapêutico , Fármacos Gastrointestinais/uso terapêutico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Humanos , Inflamação/tratamento farmacológico , Fitoterapia , Extratos Vegetais/uso terapêuticoRESUMO
Emerging evidence indicates that astrogliosis is involved in the pathogenesis of neurodegenerative disorders. Our previous findings suggested cannabinoids and Autacoid Local Injury Antagonism Amides (ALIAmides) attenuate glial response in models of neurodegeneration. The present study was aimed at exploring palmitoylethanolamide (PEA) ability to mitigate ß-amyloid (Aß)-induced astrogliosis. Experiments were carried out to investigate PEA's (10(-7) M) effects upon the expression and release of pro-inflammatory molecules in rat primary astrocytes activated by soluble Aß(1-42) (1 µg/ml) as well as to identify mechanisms responsible for such actions. The effects of Aß and exogenous PEA on the astrocyte levels of the endocannabinoidsand of endogenous ALIAmides were also studied. The peroxisome proliferator-activated receptor (PPAR)-α (MK886, 3 µM) or PPAR-γ (GW9662, 9 nM) antagonists were co-administered with PEA. Aß elevated endogenous PEA and d5-2-arachidonoylglycerol (2-AG) levels. Exogenous PEA blunted the Aß-induced expression of pro-inflammatory molecules. This effect was reduced by PPAR-α antagonist. Moreover, this ALIAmide, like Aß, increased 2-AG levels. These results indicate that PEA exhibits anti-inflammatory properties able to counteract Aß-induced astrogliosis, and suggest novel treatment for neuroinflammatory/ neurodegenerative processes.
Assuntos
Peptídeos beta-Amiloides/efeitos adversos , Anti-Inflamatórios não Esteroides/farmacologia , Astrócitos/efeitos dos fármacos , Gliose/tratamento farmacológico , Inflamação/tratamento farmacológico , Ácidos Palmíticos/farmacologia , Fragmentos de Peptídeos/efeitos adversos , Amidas , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/metabolismo , Western Blotting , Ensaio de Desvio de Mobilidade Eletroforética , Endocanabinoides , Etanolaminas , Imunofluorescência , Gliose/etiologia , Humanos , Inflamação/etiologia , PPAR alfa/antagonistas & inibidores , PPAR alfa/metabolismo , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The aim of this study was to obtain evidences of a possible analgesic role for palmitoylethanolamide (PEA) in chronic granulomatous inflammation sustained by mast cell (MC) activation in rats at 96 hours. PEA (200-400-800 µg/mL), locally administered at time 0, reduced in a concentration-dependent manner the expression and release of NGF in comparison with saline-treated controls. PEA prevented nerve formation and sprouting, as shown by histological analysis, reduced mechanical allodynia, evaluated by Von Frey filaments, and inhibited dorsal root ganglia activation. These results were supported by the evidence that MCs in granuloma were mainly degranulated and closely localized near nerve fibres and PEA significantly reduced MC degranulation and nerves fibre formation. These findings are the first evidence that PEA, by the modulation of MC activation, controls pain perception in an animal model of chronic inflammation, suggesting its potential use for the treatment of all those painful conditions in which MC activation is an initial key step.
Assuntos
Granuloma/complicações , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Mastócitos/patologia , Ácidos Palmíticos/uso terapêutico , Amidas , Animais , Carragenina , Endocanabinoides , Etanolaminas , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Granuloma/patologia , Hiperalgesia/patologia , Masculino , Mastócitos/efeitos dos fármacos , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/patologia , Fatores de Crescimento Neural/metabolismo , Neurogênese/efeitos dos fármacos , Ácidos Palmíticos/administração & dosagem , Ácidos Palmíticos/farmacologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Down syndrome (DS) is caused by trisomy of chromosome 21 and is characterized by mental retardation, seizures and premature Alzheimer's disease. To examine neuropathological mechanisms giving rise to this disorder, we generated multiple human DS neural progenitor cell (NPC) lines from the 19-21 week frontal cortex and characterized their genomic and functional properties. Microarray profiling of DS progenitors indicated that increased levels of gene expression were not limited to chromosome 21, suggesting that increased expression of genes on chromosome 21 altered transcriptional regulation of a subset of genes throughout the entire genome. Moreover, many transcriptionally dysregulated genes were involved in cell death and oxidative stress. Network analyses suggested that upregulated expression of chromosome 21 genes such as S100B and amyloid precursor protein activated the stress response kinase pathways, and furthermore, could be linked to upregulation of the water channel aquaporin 4 (AQP4). We further demonstrate in DS NPCs that S100B is constitutively overexpressed, that overexpression leads to increased reactive oxygen species (ROS) formation and activation of stress response kinases, and that activation of this pathway results in compensatory AQP4 expression. In addition, AQP4 expression could be induced by direct exposure to ROS, and siRNA inhibition of AQP4 resulted in elevated levels of ROS following S100B exposure. Finally, elevated levels of S100B-induced ROS and loss of AQP4 expression led to increased programmed cell death. These findings suggest that dysregulation of chromosome 21 genes in DS neural progenitors leads to increased ROS and thereby alters transcriptional regulation of cytoprotective, non-chromosome 21 genes in response to ongoing cellular insults.
Assuntos
Aquaporina 4/genética , Aquaporina 4/metabolismo , Síndrome de Down/genética , Síndrome de Down/metabolismo , Células-Tronco Embrionárias/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Proteínas S100/genética , Proteínas S100/metabolismo , Apoptose , Sequência de Bases , Estudos de Casos e Controles , Linhagem Celular , Cromossomos Humanos Par 21/genética , Primers do DNA/genética , Síndrome de Down/embriologia , Síndrome de Down/patologia , Células-Tronco Embrionárias/patologia , Perfilação da Expressão Gênica , Humanos , Neurônios/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100 , TransfecçãoRESUMO
Palmitoylethanolamide (PEA) and some of its analogues have shown great efficacy in the treatment of pain and inflammation. Adelmidrol - the International Nonproprietary Name (INN) of the di-amide derivative of azelaic acid - is one of these analogues. The anti-inflammatory and analgesic effects of PEA and adelmidrol are hypothesized to be mediated, at least in part, by mast cell down-modulation. Mast cell mediators released at early stage of the inflammatory process drive the inflammatory reaction to chronicity as it happens in X-carrageenin-induced granulomatous tissue formation. In the present study, the choice of testing adelmidrol depends upon the physicochemical properties of the compound, i.e. the amphipatic feature, that make it more easily soluble than PEA. In this study, we investigated the effect of adelmidrol on granuloma formation induced by lambda-carrageenin-soaked sponge implant in rats. Our results show that the local administration of the compound under study significantly decreases weight and neo-angiogenesis in granulomatous tissue. The anti-inflammatory effect was due to the modulation of mast cells degranulation, as shown by histological analysis and by the inhibition of the release of several pro-inflammatory and pro-angiogenic enzymes (e.g. iNOS, chymase and metalloproteinase MMP-9), and mediators (e.g. nitric oxide and TNF-alpha). The results indicate that adelmidrol, given locally, may represent a potential therapeutic tool in controlling chronic inflammation.
Assuntos
Ácidos Dicarboxílicos/farmacologia , Granuloma/prevenção & controle , Inflamação/prevenção & controle , Ácidos Palmíticos/farmacologia , Amidas , Animais , Western Blotting , Carragenina , Contagem de Células , Doença Crônica , Ácidos Dicarboxílicos/química , Relação Dose-Resposta a Droga , Endocanabinoides , Etanolaminas , Granuloma/induzido quimicamente , Granuloma/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/patologia , Neovascularização Patológica/prevenção & controle , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Ácidos Palmíticos/química , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of several genes involved in the immune and inflammatory process. Previously, we demonstrated that NF-kappaB activation can be significantly inhibited by a double stranded oligodeoxynucleotide (ODN). Nevertheless, the therapeutic use of ODN requires a delivery system able to improve poor crossing of cell membranes and rapid in vivo enzymatic degradation. Poly(D,L-lactide-co-glycolide) (PLGA) microspheres can increase ODN stability in biological environment and release the encapsulated drug in long time frames. Here, we used a decoy ODN against NF-kappaB and we investigated its effect, when administered in naked form or when delivered by PLGA microspheres, in a rat model of chronic inflammation. The subcutaneous implant of lambda-carrageenin-soaked sponges caused leukocyte infiltration and formation of granulation tissue which were inhibited up to 15 days by co-injection of microspheres releasing decoy ODN whereas naked decoy ODN showed this effect only up to 5 days. Molecular analysis performed on granulation tissue demonstrated an inhibition of NF-kappaB activation correlated to a decrease of tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) expression. Our results suggest that microspheres could be an useful tool to improve pharmacokinetics of decoy ODN and may represent a strategy to inhibit NF-kappaB activation in chronic inflammation.
Assuntos
Portadores de Fármacos/química , Inflamação/tratamento farmacológico , Ácido Láctico/química , NF-kappa B/metabolismo , Oligonucleotídeos Fosforotioatos/uso terapêutico , Ácido Poliglicólico/química , Animais , Western Blotting , Doença Crônica , Modelos Animais de Doenças , Ensaio de Desvio de Mobilidade Eletroforética , Tecido de Granulação/efeitos dos fármacos , Tecido de Granulação/metabolismo , Inflamação/metabolismo , Masculino , Microesferas , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Oligonucleotídeos Fosforotioatos/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Cannabidiol (CBD) is the main non-psychotropic component of the glandular hairs of Cannabis sativa. It displays a plethora of actions including anticonvulsive, sedative, hypnotic, antipsychotic, antiinflammatory and neuroprotective properties. However, it is well established that CBD produces its biological effects without exerting significant intrinsic activity upon cannabinoid receptors. For this reason, CBD lacks the unwanted psychotropic effects characteristic of marijuana derivatives, so representing one of the bioactive constituents of Cannabis sativa with the highest potential for therapeutic use.The present review reports the pharmacological profile of CBD and summarizes results from preclinical and clinical studies utilizing CBD, alone or in combination with other phytocannabinoids, for the treatment of a number of CNS disorders.
Assuntos
Canabidiol/farmacologia , Cannabis/química , Doenças do Sistema Nervoso Central/tratamento farmacológico , Animais , Canabidiol/uso terapêutico , Canabidiol/toxicidade , HumanosRESUMO
Activated microglia and astrocytes produce a large number of inflammatory and neurotoxic substances in various brain pathologies, above all during neurodegenerative disorders. In the search for new neuroprotective compounds, interest has turned to marijuana derivatives, since in several in vitro, in vivo, and clinical studies, they have shown a great ability to control neuroinflammation. Despite the emerging evidence regarding pharmacological activities of cannabinoids, their effective introduction into clinical therapy still remains controversial and strongly limited by their unavoidable psychotropicity. Since the psychotropic effect of cannabinoids is generally linked to the activation of the CB1 receptor on neurons, the aim of our review is to clarify the function of the two cannabinoid receptors on glial cells and the differential role played by them, highlighting the emerging evidence of a CB2-mediated control of neuroinflammation that could liberate cannabinoids from the slavery of their central side effects. Despite the emerging evidence regarding pharmacological activities of cannabinoids, however their effective introduction in the clinical therapy remains still controversial and strongly limited by their unavoidable psychotropicity. Since the psychotropic effect of cannabinoids is generally linked to the activation of CB1 receptor on neurons, aim of our review is to clarify the functioning of the two cannabinoid receptors on glial cells and the differential role played by them, highlighting the emerging evidence of a CB2-mediated control of neuro-inflammation that could liberate cannabinoids from the slavery of the central side effects.
Assuntos
Canabinoides/farmacologia , Gliose/metabolismo , Microglia/fisiologia , Fármacos Neuroprotetores/farmacologia , Receptor CB1 de Canabinoide/efeitos dos fármacos , Receptor CB2 de Canabinoide/efeitos dos fármacos , Animais , Canabinoides/uso terapêutico , Expressão Gênica , Gliose/tratamento farmacológico , Humanos , Microglia/efeitos dos fármacos , Receptor CB1 de Canabinoide/fisiologia , Receptor CB2 de Canabinoide/fisiologiaRESUMO
In this study, we investigated the mechanism of S100B neurotoxicity and the effect of cannabinoids, in C6 cells treated with 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP) and co-cultured with differentiated PC12 cells. MPTP concentration- and time-dependently increased S100B density in C6 cells. This effect was followed by increased C6 cell proliferation and decreased cell viability of co-cultured PC12 cells. An antibody against S100B, given to PC12 cells before co-culture, led to their survival. Treatment with arachidonyl-2-chloroethylamide, a CB1 agonist, significantly inhibited MPTP-induced S100B density in C6 cells and protected co-cultured PC12 cells from cell death. Because MPTP selectively increased the levels of anandamide in C6 cells, the involvement of the endocannabinoid system was investigated by using selective inhibitors of endocannabinoid inactivation (cellular re-uptake or enzymatic hydrolysis) and selective cannabinoid CB1 and CB2 receptor antagonists and by silencing the CB1 receptor. Our data suggest that selective activation of CB1 receptors by either exogenous or endogenous cannabinoids might afford neuroprotection in MPTP-induced neurotoxicity also by controlling S100B up-regulation in activated glial cells.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/efeitos adversos , Ácidos Araquidônicos/farmacologia , Intoxicação por MPTP/metabolismo , Fatores de Crescimento Neural/metabolismo , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptor CB1 de Canabinoide/efeitos dos fármacos , Proteínas S100/metabolismo , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Animais , Anticorpos , Apoptose/efeitos dos fármacos , Ácidos Araquidônicos/metabolismo , Cálcio/metabolismo , Caspase 3/metabolismo , Comunicação Celular/efeitos dos fármacos , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Relação Dose-Resposta a Droga , Endocanabinoides , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Intoxicação por MPTP/patologia , Fatores de Crescimento Neural/imunologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/enzimologia , Neurônios/metabolismo , Neurônios/patologia , Células PC12 , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/metabolismo , Pirazóis/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/efeitos dos fármacos , Receptor CB2 de Canabinoide/metabolismo , Rimonabanto , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/imunologia , Serotonina/análogos & derivados , Serotonina/farmacologia , Fatores de Tempo , Regulação para CimaRESUMO
Chronic inflammation is often associated with granuloma formation that is a hallmark of many human diseases. The transcription factor nuclear factor-kappa B (NF-kappaB) plays a central role in this process by regulating the expression of several pro-inflammatory genes. Cannabinoids (CBs) from Cannabis sativa L. exert a large number of biological effects including anti-inflammatory and anti-angiogenic effects. In this study, we investigated the role of CBs on granuloma formation induced by lambda-carrageenin-soaked sponge implant in rat. Our results show that local administration of WIN 55,212-2, a CB(1)/CB(2) agonist, given daily or at time of implantation significantly decreased weight and neo-angiogenesis in granuloma tissue and inhibited nuclear factor-kappa B (NF-kappaB)/DNA binding that was associated with a reduced inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), tumor necrosis factor alpha (TNF-alpha), and vascular endothelial growth factor (VEGF) messenger RNA (mRNA) and protein expression. Also, arachidonyl-2-chloroethylamide (ACEA), a CB(1) selective agonist, and JWH-015, a CB(2) selective agonist, exhibited the same effects that were reversed by SR141716-A and SR144528, respectively, CB(1) and CB(2) selective antagonists. These results indicate that CBs given locally may represent a potential therapeutic tool in controlling chronic inflammation avoiding psychotropic effects.
Assuntos
Analgésicos/administração & dosagem , Analgésicos/farmacologia , Benzoxazinas/administração & dosagem , Benzoxazinas/farmacologia , Granuloma/patologia , Morfolinas/administração & dosagem , Morfolinas/farmacologia , NF-kappa B/metabolismo , Naftalenos/administração & dosagem , Naftalenos/farmacologia , Neovascularização Patológica , Animais , Agonistas de Receptores de Canabinoides , Antagonistas de Receptores de Canabinoides , Carragenina/farmacologia , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Masculino , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Inducible nitric oxide synthase (iNOS) is an homodimeric enzyme which produces large amounts of nitric oxide (NO) in response to inflammatory stimuli. Several factors affect the synthesis and catalytic activity of iNOS. Particularly, dimerization of NOS monomers is promoted by heme, whereas an intracellular depletion of heme and/or L-arginine considerably decreases NOS resistance to proteolysis. In this study, we found that oxalomalate (OMA, oxalomalic acid, alpha-hydroxy-beta-oxalosuccinic acid), an inhibitor of both aconitase and NADP-dependent isocitrate dehydrogenase, inhibited nitrite production and iNOS protein expression in lipopolysaccharide (LPS)-activated J774 macrophages, without affecting iNOS mRNA content. Furthermore, injection of OMA precursors to LPS-stimulated rats also decreased nitrite production and iNOS expression in isolated peritoneal macrophages. Interestingly, alpha-ketoglutarate or succinyl-CoA administration reversed OMA effect on NO production, thus correlating NO biosynthesis with the anabolic capacity of Krebs cycle. When protein synthesis was blocked by cycloheximide in LPS-activated J774 cells treated with OMA, iNOS protein levels, evaluated by Western blot analysis and (35)S-metabolic labelling, were decreased, suggesting that OMA reduces iNOS biosynthesis and induces an increase in the degradation rate of iNOS protein. Moreover, we showed that OMA inhibits the activity of the iNOS from lung of LPS-treated rats by enzymatic assay. Our results, demonstrating that OMA acts regulating synthesis, catalytic activity and degradation of iNOS, suggest that this compound might have a potential role in reducing the NO overproduction occurring in some pathological conditions.
Assuntos
Inibidores Enzimáticos/farmacologia , Macrófagos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Oxalatos/farmacologia , Acil Coenzima A/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Antagonismo de Drogas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Ácidos Cetoglutáricos/farmacologia , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/enzimologia , Masculino , Óxido Nítrico Sintase Tipo II/genética , Nitritos/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder. A massive accumulation of beta-amyloid (Abeta) peptide aggregates has been proposed as pivotal event in AD. Abeta-induced toxicity is accompanied by a variegated combination of events including oxidative stress. The Wnt pathway has multiple actions in the cascade of events triggered by Abeta, and drugs that rescue Wnt activity may be considered as novel therapeutics for AD treatment. Cannabidiol, a non-psychoactive marijuana component, has been recently proposed as an antioxidant neuroprotective agent in neurodegenerative diseases. Moreover, it has been shown to rescue PC12 cells from toxicity induced by Abeta peptide. However, the molecular mechanism of cannabidiol-induced neuroprotective effect is still unknown. Here, we report that cannabidiol inhibits hyperphosphorylation of tau protein in Abeta-stimulated PC12 neuronal cells, which is one of the most representative hallmarks in AD. The effect of cannabidiol is mediated through the Wnt/beta-catenin pathway rescue in Abeta-stimulated PC12 cells. These results provide new molecular insight regarding the neuroprotective effect of cannabidiol and suggest its possible role in the pharmacological management of AD, especially in view of its low toxicity in humans.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Canabidiol/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Ácido Ascórbico/farmacologia , Cannabis/química , Quinase 3 da Glicogênio Sintase/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Proteínas do Tecido Nervoso/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Fragmentos de Peptídeos/farmacologia , Fosforilação , Ratos , Transdução de Sinais , Tiadiazóis/farmacologia , Proteínas Wnt/efeitos dos fármacos , beta Catenina/efeitos dos fármacos , Proteínas tauRESUMO
In view of the pro-inflammatory scenario observed in Alzheimer's disease, in the recent years anti-inflammatory drugs have been proposed as potential therapeutic agents. We have previously shown that cannabidiol, the main non-psychotropic component from Cannabis sativa, possess a variegate combination of anti-oxidant and anti-apoptotic effects that protect PC12 cells from Abeta toxicity. In parallel, cannabidiol has been described to have anti-inflammatory properties in acute models of inflammation but the possible inhibitory effect of cannabidiol on iNOS protein expression and nitrite production in the nitrosative stress induced by Abeta in neuronal cell-line is un-investigated. Stimulation of differentiated PC12 cells with Abeta (1-42) (1 microg/mL) for 36 h caused a significant increase of nitrite production, compared to un-stimulated cells, that was inhibited in a concentration-dependent manner by both the non-selective iNOS inhibitor, L-NAME (0.3-30 microM), and, at higher extent, by the selective iNOS inhibitor SMT (0.3-30 microM). CBD (10(-6) to 10(-4) M) inhibited both nitrite production and iNOS protein expression induced by Abeta (1-42). Cannabidiol effect was mediated through the inhibition of phosphorylated form of p38 MAP kinase and transcription factor nuclear factor-kappaB activation in a concentration-dependent manner. The here reported data increases our knowledge about the possible neuroprotective mechanism of cannabidiol, highlighting the importance of this compound to inhibit beta-amyloid induced neurodegeneration, in view of its low toxicity in humans.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Anti-Inflamatórios/farmacologia , Canabidiol/farmacologia , NF-kappa B/fisiologia , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Humanos , Isotiurônio/análogos & derivados , Isotiurônio/farmacologia , NF-kappa B/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Células PC12 , Fosforilação , RatosRESUMO
Among the wide range of neuro-inflammatory signalling molecules released by beta-amyloid-stimulated astroglial cells, nitric oxide (NO) plays a fundamental role in AD aethiopathogenesis since it directly promotes neuronal tau protein hyperphosphorylation leading to neurofibrillary tangle formation. Synthetic cannabinoids (CBs), via a selective CB1 receptor activation, negatively modulates both iNOS protein expression and NO production induced by pro-inflammatory stimuli. In this study we investigated the role of both the non-selective WIN 55,212-2 and the selective CB1 receptor agonist, ACEA, on: (i) NO production, (ii) iNOS protein expression in (1-42) beta-amyloid peptide (Abeta)-stimulated C6 rat glioma cells and (iii) tau protein hyperphosphorylation in co-cultured differentiated PC12 neurons. Our results demonstrated that synthetic CBs, by a selective CB1 effect, down-regulate iNOS protein expression and NO production in Abeta-stimulated C6 cells. This effect leads, in turn, to a significant and concentration-dependent inhibition of NO-dependent tau protein hyperphosphorylation in co-cultured PC12 neurons. The results of the present study extend our knowledge about the neuroprotective actions of synthetic CBs on Abeta-dependent neurotoxicity in vitro. Furthermore, our study allows us to identify, in the CB1-mediated inhibition of astroglial-derived NO, a new potential target to blunt tau hyperphosphorylation and the consequent related tauopathy in AD.
Assuntos
Peptídeos beta-Amiloides/fisiologia , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Fragmentos de Peptídeos/fisiologia , Receptor CB1 de Canabinoide/agonistas , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Glioma , Humanos , Neurônios/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Fragmentos de Peptídeos/farmacologia , Feocromocitoma , Fosforilação , RatosRESUMO
S100beta is an astroglial-derived Ca2+ -binding protein having neurotrophic role on neurons and glial cells. An aberrant S100beta production has been observed in neurodegenerative disease, as Alzheimer's disease and Down syndrome. S100beta is responsible to start up a gliotic reaction by the release of pro-inflammatory mediators, including nitric oxide (NO) and cytokines from microglia and astrocytes, which are, in turn, deleterious for neurons. Interestingly, pro-inflammatory effect of S100beta seems not be restricted into the brain. Macrophages play a pivotal role in inflammatory diseases, occurring both in the brain and in the periphery. In this study, we tested the hypothesis that S100beta may affect macrophage functions, amplifying thus the inflammatory process. Our results demonstrate that S100beta stimulates both NO production and iNOS protein transcription and expression in J774 and rat peritoneal macrophages. NO production was concentration and time-dependently inhibited by two iNOS inhibitors, L-NAME and SMT. We also demonstrated that S100beta induced oxidative stress by increasing H2O2 production and lipid peroxidation of cell membrane in both macrophage types. The pro-oxidant potential of S100beta activates p38 MAP kinase (MAPK), which has been described to directly activate NF-kappaB. In our study, SB203580, a p38 MAPK inhibitor, and two NF-kappaB inhibitors, TLCK and BAY 11-7082, decreased both NO production and iNOS protein transcription and expression in S100beta-stimulated J774 and peritoneal rat macrophages. Moreover, additional studies demonstrated that S100beta affected also TNF-alpha protein expression in J774 macrophages. In conclusion, our results highlight the potential role of S100beta during an inflammatory scenario identifying macrophages as a novel S100beta-responsive cell-type.
Assuntos
Astrócitos/metabolismo , Autoantígenos/farmacologia , Fatores de Crescimento Neural/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas S100/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Animais , Biomarcadores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Isotiurônio/análogos & derivados , Isotiurônio/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Macrófagos Peritoneais , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Subunidade beta da Proteína Ligante de Cálcio S100RESUMO
The effects of ultramicronized palmitoylethanolamide were evaluated on pain behaviours and markers of mast cell (MC) activity in a rat model of endometriosis plus ureteral calculosis (ENDO+STONE)-induced viscerovisceral hyperalgesia (VVH). Female Sprague-Dawley rats that underwent surgical induction of endometriosis were randomly assigned to receive active (ultramicronized palmitoylethanolamide 10 mg·kg(-1)·d(-1), orally) or placebo treatment for 25 days. At day 21, they underwent ureteral stone formation and were video-recorded till day 25 to evaluate ureteral and uterine pain behaviours. At autopsy (day 25), ureteral condition and number and diameter of endometrial cysts were evaluated. The following were then measured: number and percentage of degranulating MCs, number of vessels, chymase, nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and Flk-1 (VEGF receptor) in cysts, and NGF in dorsal root ganglia (DRG). Ultramicronized palmitoylethanolamide-treated vs placebo-treated rats showed significantly lower number, duration and complexity of ureteral crises, shorter duration of uterine pain, and smaller cyst diameter (0.0001 < P < 0.004); a significantly higher percentage of expelled stones (P < 0.0001); significantly lower MC number (P < 0.01), vessel number (P < 0.01), chymase (P < 0.05), NGF (P < 0.05), VEGF (P < 0.01), and Flk-1 (P < 0.01) expression in cysts and NGF expression in DRG (P < 0.01). In all animals, the global duration of ureteral crises correlated linearly and directly with cyst diameter, MC number and chymase in cysts, and NGF in cysts and DRG (0.02 < P < 0.0002). Ultramicronized palmitoylethanolamide significantly reduces VVH from ENDO+STONE, probably by modulating MC expression/activity in cysts, thus reducing central sensitization due to noxious signals from endometriotic lesions. The results suggest potential utility of the compound for VVH in clinics.
Assuntos
Endometriose/complicações , Etanolaminas/uso terapêutico , Hiperalgesia/tratamento farmacológico , Mastócitos/efeitos dos fármacos , Ácidos Palmíticos/uso terapêutico , Cálculos Ureterais/complicações , Amidas , Animais , Quimases/metabolismo , Modelos Animais de Doenças , Endometriose/metabolismo , Etanolaminas/farmacologia , Feminino , Hiperalgesia/complicações , Hiperalgesia/metabolismo , Mastócitos/metabolismo , Fator de Crescimento Neural/metabolismo , Ácidos Palmíticos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Cálculos Ureterais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Extracellular adenosine formation from ATP is controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5'-nucleotidase (e-5NT/CD73); the latter converts AMP to adenosine and inorganic phosphate, representing the rate limiting step controlling the ratio between extracellular ATP and adenosine. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has identified this pathway as an endogenous modulator in several diseases and was shown to be involved in the molecular mechanism of drugs, such as methotrexate, salicylates , interferon-ß. We evaluated whether CD73/adenosine/A2A signalling pathway is involved in nimesulide anti-inflammatory effect, in vivo and in vitro. We found that the adenosine A2A agonist, 4-[2-[[6-amino-9-(N-ethyl-ß-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680, 2mg/kg ip.), inhibited carrageenan-induced rat paw oedema and the effect was reversed by co-administration of the A2A antagonist -(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385; 3mg/kg i.p.). Nimesulide (5mg/kg i.p.) anti-inflammatory effect was inhibited by pre-treatment with ZM241385 (3mg/kg i.p.) and by local administration of the CD73 inhibitor, adenosine 5'-(α,ß-methylene)diphosphate (APCP; 400µg/paw). Furthermore, we found increased activity of 5'-nucleotidase/CD73 in paws and plasma of nimesulide treated rats, 4h following oedema induction. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin E2 production by lipopolysaccharide-activated J774 cell line was reversed by ZM241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by lipopolysaccharide-activated SiRNA CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide in part is mediated by CD73-derived adenosine acting on A2A receptors.
Assuntos
Adenosina/metabolismo , Anti-Inflamatórios/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , 5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/sangue , 5'-Nucleotidase/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Linhagem Celular , Inibidores de Ciclo-Oxigenase 2/administração & dosagem , Dinoprostona/sangue , Edema/tratamento farmacológico , Edema/imunologia , Edema/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Ratos Wistar , Receptor A2A de Adenosina/metabolismo , Sulfonamidas/administração & dosagemRESUMO
Nuclear Factor-κB (NF-κB) is a transcription factor regulating several genes involved in important physiological and pathological processes. NF-κB has been found constitutively activated in many inflammatory/immune diseases. In addition, a positive correlation between persistent activation of NF-κB and tumor promotion has been demonstrated. Since the IKK (IκB kinase) activation is an indispensable component of all pro-inflammatory signaling pathways leading to NF-κB activation, considerable efforts have been done in order to develop novel anti-inflammatory therapeutics targeting IKK. Association of the IKK complex relies on critical interactions between the C-terminus NBD (NEMO binding domain) of the catalytic subunits IKKα and IKKß, and the regulatory subunit NEMO (NF-κB Essential Modulator). Thus, this IKK/NEMO interacting region provides an attractive target to prevent the IKK complex formation and NF-κB activation. In this regard, we have identified non-peptide small molecule disruptors of IKKß/NEMO complex through a structure-based virtual screening (SBVS) of the NCI chemical library. Phenothiazine 22 and its close analogues (22.2, 22.4 and 22.10) were able to reduce nitrite production and iNOS mRNA expression in J774 murine macrophages stimulated with LPS for 24h. These effects were associated with a reduced NF-κB/DNA binding activity as well as a decreased expression of phosphorylated IKKß, IκBα and NF-κB/p65 in these cells. These observations suggest that compound 22 and its three structural analogues by inhibiting IKKß/NEMO association mediate the blockage of NF-κB signaling pathway and may prove effective in treatment of diseases in which the IKK/NF-κB pathway is dysregulated.