RESUMO
Purinergic signaling modulates immune function and is involved in the immunopathogenesis of several viral infections. This study aimed to investigate alterations in purinergic pathways in coronavirus disease 2019 (COVID-19) patients. Mild and severe COVID-19 patients had lower extracellular adenosine triphosphate and adenosine levels, and higher cytokines than healthy controls. Mild COVID-19 patients presented lower frequencies of CD4+ CD25+ CD39+ (activated/memory regulatory T cell [mTreg]) and increased frequencies of high-differentiated (CD27- CD28- ) CD8+ T cells compared with healthy controls. Severe COVID-19 patients also showed higher frequencies of CD4+ CD39+ , CD4+ CD25- CD39+ (memory T effector cell), and high-differentiated CD8+ T cells (CD27- CD28- ), and diminished frequencies of CD4+ CD73+ , CD4+ CD25+ CD39+ mTreg cell, CD8+ CD73+ , and low-differentiated CD8+ T cells (CD27+ CD28+ ) in the blood in relation to mild COVID-19 patients and controls. Moreover, severe COVID-19 patients presented higher expression of PD-1 on low-differentiated CD8+ T cells. Both severe and mild COVID-19 patients presented higher frequencies of CD4+ Annexin-V+ and CD8+ Annexin-V+ T cells, indicating increased T-cell apoptosis. Plasma samples collected from severe COVID-19 patients were able to decrease the expression of CD73 on CD4+ and CD8+ T cells of a healthy donor. Interestingly, the in vitro incubation of peripheral blood mononuclear cell from severe COVID-19 patients with adenosine reduced the nuclear factor-κB activation in T cells and monocytes. Together, these data add new knowledge to the COVID-19 immunopathology through purinergic regulation.
Assuntos
5'-Nucleotidase , Apirase , COVID-19 , Linfócitos T , 5'-Nucleotidase/metabolismo , Adenosina/sangue , Trifosfato de Adenosina/sangue , Anexinas , Apirase/metabolismo , Antígenos CD28/metabolismo , COVID-19/imunologia , Citocinas/sangue , Proteínas Ligadas por GPI/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Receptores Purinérgicos , Transdução de Sinais , Linfócitos T/imunologiaRESUMO
The severity score of quinolinic acid (QA)-induced seizures was investigated after N-methyl-D-aspartate (NMDA) preconditioning associated with adenosine receptors. Also, the levels of adenosine A1 and A2A receptors and subunits of NMDA receptors in the hippocampi of mice were determined to define components of the resistance mechanism. Adult CF-1 mice were treated intraperitoneally with saline or NMDA (75 mg/kg), and some mice were treated intracerebroventricularly (i.c.v.) with 0.1 pmol of adenosine receptor antagonists 8-cyclopentyltheophylline (CPT; receptor A1) or ZM241385 (receptor A2A) 0, 1, or 6 h after NMDA administration. These adenosine receptor antagonists were administered to block NMDA's protective effect. Seizures and their severity scores were evaluated during convulsions induced by QA (36.8 nmol) that was administered i.c.v. 24 h after NMDA. The cell viability and content of subunits of the NMDA receptors were analyzed 24 h after QA administration. NMDA preconditioning reduced the maximal severity 6 displayed in QA-administered mice, inducing protection in 47.6% of mice after QA-induced seizures. CPT increased the latency of seizures when administered 0 or 6 h, and ZM241385 generated the same effect when administered 6 h after NMDA administration. The GluN1 content was lower in the hippocampi of the QA mice and the NMDA-preconditioned animals without seizures. GluN2A content was unaltered in all groups. The results demonstrated the components of resistance evoked by NMDA, in which adenosine receptors participate in a time-dependent mode. Similarly, the reduction on GluN1 expression in the hippocampus may contribute to this effect during the preconditioning period.
Assuntos
Anticonvulsivantes/uso terapêutico , N-Metilaspartato/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Purinérgicos P1/metabolismo , Convulsões/tratamento farmacológico , Animais , Anticonvulsivantes/administração & dosagem , Anticonvulsivantes/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Injeções Intraperitoneais , Masculino , Camundongos , N-Metilaspartato/administração & dosagem , N-Metilaspartato/farmacologia , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , Ácido Quinolínico/toxicidade , Convulsões/etiologiaRESUMO
The guanine-based purines (GBPs) have essential extracellular functions such as modulation of glutamatergic transmission and trophic effects on neurons and astrocytes. We previously showed that GBPs, such as guanosine-5'-monophosphate (GMP) or guanosine (GUO), promote the reorganization of extracellular matrix proteins in astrocytes, and increase the number of neurons in a neuron-astrocyte co-culture protocol. To delineate the molecular basis underlying these effects, we isolated cerebellar neurons in culture and treated them with a conditioned medium derived from astrocytes previously exposed to GUO or GMP (GBPs-ACM) or, directly, with GUO or GMP. Agreeing with the previous studies, there was an increase in the number of ß-tubulin III-positive neurons in both conditions, compared with controls. Interestingly, the increase in the number of neurons in the neuronal cultures treated directly with GUO or GMP was more prominent, suggesting a direct interaction of GBPs on cerebellar neurons. To investigate this issue, we assessed the role of adenosine and glutamate receptors and related intracellular signaling pathways after GUO or GMP treatment. We found an involvement of A2A adenosine receptors, ionotropic glutamate N-methyl-D-aspartate (NMDA), and non-NMDA receptors in the increased number of cerebellar neurons. The signaling pathways extracellular-regulated kinase (ERK), calcium-calmodulin-dependent kinase-II (CaMKII), protein kinase C (PKC), phosphatidilinositol-3'-kinase (PI3-K), and protein kinase A (PKA) are also potentially involved with GMP and GUO effect. Such results suggest that GMP and GUO, and molecules released in GBPs-ACM promote the survival or maturation of primary cerebellar neurons or both via interaction with adenosine and glutamate receptors.
Assuntos
Adenosina/metabolismo , Guanosina/metabolismo , Neurônios/metabolismo , Receptores de Glutamato/metabolismo , Animais , Astrócitos/metabolismo , Sistema Nervoso Central/metabolismo , Ácido Glutâmico/metabolismo , Guanosina Monofosfato/metabolismo , Receptores Purinérgicos P1/metabolismoRESUMO
Creatine acts intracellularly as energy buffer and storage, demonstrating protective effects in animal models of neurodegenerative diseases. However, its permeability throught blood-brain barrier (BBB) is reduced. The aim of the present study was developing a carrier to facilitate the delivery of creatine to the central nervous system. Creatine nanoliposomes were produced, characterized and assayed in models of toxicity in vitro and in vivo. Particles showed negative zeta potential (-12,5 mV), polydispersity index 0.237 and medium-size of 105 nm, which was confirmed by transmission electron microscopy (TEM) images. Toxicity assay in vitro was evaluated with blank liposomes (no drug) or creatine nanoliposomes at concentrations of 0.02 and 0.2 mg/mL, that did not influence the viability of Vero cells. The result. of the comet assay that the nanoliposomes are not genotoxic, togeher with cell viability demonstrated that the nanoliposomes are not toxic. Besides, in vivo assays not demonstrate toxicity in hematological and biochemical markers of young rats. Nevertheless, increase content of creatine in the cerebral cortex tissue after subchronic treatment was observed. Altogether, results indicate increase permeability of creatine to the BBB that could be used as assay for in vivo studies to confirm improved effect than free creatine.
Assuntos
Encéfalo/efeitos dos fármacos , Creatina/toxicidade , Lipossomos/toxicidade , Nanopartículas/toxicidade , Polissorbatos/toxicidade , Animais , Encéfalo/ultraestrutura , Chlorocebus aethiops , Microscopia Eletrônica de Transmissão , Modelos Animais , Ratos , Ratos Wistar , Células VeroRESUMO
N-methyl-d-aspartate (NMDA) preconditioning is induced by subtoxic doses of NMDA and it promotes a transient state of resistance against subsequent lethal insults. Interestingly, this mechanism of neuroprotection depends on adenosine A1 receptors (A1R), since blockade of A1R precludes this phenomenon. In this study we evaluated the consequences of NMDA preconditioning on the hippocampal A1R biology (i.e. expression, binding properties and functionality). Accordingly, we measured A1R expression in NMDA preconditioned mice (75mg/kg, i.p.; 24h) and showed that neither the total amount of receptor, nor the A1R levels in the synaptic fraction was altered. In addition, the A1R binding affinity to the antagonist [(3)H] DPCPX was slightly increased in total membrane extracts of hippocampus from preconditioned mice. Next, we evaluated the impact of NMDA preconditioning on A1R functioning by measuring the A1R-mediated regulation of glutamate uptake into hippocampal slices and on behavioral responses in the open field and hot plate tests. NMDA preconditioning increased glutamate uptake into hippocampal slices without altering the expression of glutamate transporter GLT-1. Interestingly, NMDA preconditioning also induced antinociception in the hot plate test and both effects were reversed by post-activation of A1R with the agonist CCPA (0.2mg/kg, i.p.). NMDA preconditioning or A1R modulation did not alter locomotor activity in the open field. Overall, the results described herein provide new evidence that post-activation of A1R modulates NMDA preconditioning-mediated responses, pointing to the importance of the cross-talk between glutamatergic and adenosinergic systems to neuroprotection.
Assuntos
Comportamento Animal/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/efeitos dos fármacos , N-Metilaspartato/farmacologia , Receptor A1 de Adenosina/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Antagonistas do Receptor A1 de Adenosina/farmacologia , Animais , Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Atividade Motora/efeitos dos fármacos , Agonistas do Receptor Purinérgico P1/farmacologia , Xantinas/farmacologiaRESUMO
OBJECTIVE: Methylphenidate (MPD) is a drug prescribed for the treatment of attention deficit/hyperactivity disorder and its therapeutic effect is attributed to the inhibition of dopamine. METHODS: Young male Wistar rats were administered MPD (1, 2, 5, or 10 mg/kg) once a day or an intraperitoneal injection of saline for 28 days (chronic treatment) or for 1 day (acute treatment). Two hours after the last administration the animals were decapitated and their striatum was dissected. RESULTS: In this work, we show that continued treatment with MPD is capable of modifying the levels of phosphorylation of proteins JNK1/2 (c-Jun amino-terminal kinases 1 and 2) and ERK1/2 (extracellular signal-regulated kinases 1 and 2). Whereas the level of phosphorylation of protein ERK increased significantly, that of proteins JNK1/2 diminished. CONCLUSION: The alteration in the level of activation of mitogen-activated protein kinases can be a molecular mechanism through which MPD exerts its therapeutic effect.
RESUMO
Recent studies have focused on the role of N-methyl-d-aspartate (NMDA) in brain injury. The present study is aimed at verifying memory, anxiety/depression parameters, and cellular viability in the brain of mice preconditioned with NMDA and subjected to the model of mild traumatic brain injury. For this purpose, male albino CF-1 mice were pre-treated with NMDA (75 mg/kg) and subjected to brain trauma, and after 24h submitted to memory tasks and anxiety and depression-like behavioral tests. The memory tests were evaluated at 1.5h, 24h, and 7 days after the training. In addition, the cellular viability was evaluated in the cerebral cortex and hippocampus 96 h after the trauma. It was observed that the cellular viability was reduced in the hippocampus of the mice subjected to trauma and the preconditioning with NMDA was able to protect this damage. All mice learnt the task in the habituation test, but in the object recognition task the mice preconditioned with NMDA were protected against impairment induced by TBI in both short and long-term memory. On the other hand, in the step-down inhibitory avoidance test, only the mice treated with NMDA showed impairment of long-term memory (7 days after training session). The evaluation of anxiety/depression behavior showed no changes after TBI. In conclusion, NMDA preconditioning induced impairment of the long-term memory; however, it was able to protect against the novel recognition memory impairment and increase the cellular survival in the hippocampus of mice exposed to traumatic brain injury.
Assuntos
Lesões Encefálicas/complicações , Sobrevivência Celular/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Transtornos da Memória/prevenção & controle , N-Metilaspartato/farmacologia , Reconhecimento Psicológico/efeitos dos fármacos , Animais , Ansiedade/metabolismo , Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Lesões Encefálicas/metabolismo , Depressão/metabolismo , Hipocampo/metabolismo , Masculino , Transtornos da Memória/etiologia , Transtornos da Memória/metabolismo , Camundongos , Atividade Motora/efeitos dos fármacos , Permeabilidade , Percepção Visual/efeitos dos fármacosRESUMO
Nevirapine (NVP) and efavirenz (EFV) belong to the class of anti-HIV drugs called non-nucleoside reverse transcriptase inhibitors (NNRTIs), commonly used as part of highly active antiretroviral therapy (HAART). Although the HAART is able to bring down viral load to undetectable levels and restore immune function, their prolonged use causes several adverse effects. It has been demonstrated that both NVP and EFV are able to cross the blood-brain barrier, causing important central nervous system-related side effects. Thus, this study investigated the effects of chronic administration of EFV (10 mg/kg) and NVP (3.3 mg/kg) in mice submitted to two distinct series of experiments, which aimed to evaluate: (1) the emotional behavior (elevated plus-maze, forced swimming, and open-field test) and (2) the cognitive performance (object recognition and inhibitory avoidance test) of mice. Our results demonstrated that EFV, but not NVP, reduced the exploration to open arms in the elevated plus-maze test. Neither NVP nor EFV altered mouse behavior in the forced swimming and open-field tests. Both drugs reduced the recognition index in the object recognition test, but only EFV significantly impaired the aversive memory assessed in the inhibitory avoidance test 24 h after training. In conclusion, our findings point to a genuine anxiogenic-like effect to EFV, since it reduced exploration to open arms of elevated plus-maze test without affecting spontaneous locomotion. Additionally, both drugs impaired recognition memory, while only the treatment with EFV impaired significantly aversive memory.
Assuntos
Fármacos Anti-HIV/farmacologia , Ansiedade/induzido quimicamente , Benzoxazinas/farmacologia , Cognição/efeitos dos fármacos , Nevirapina/farmacologia , Alcinos , Animais , Fármacos Anti-HIV/toxicidade , Ansiedade/fisiopatologia , Benzoxazinas/toxicidade , Cognição/fisiologia , Ciclopropanos , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Masculino , Camundongos , Nevirapina/toxicidadeRESUMO
Traumatic brain injury (TBI) causes impairment of fine motor functions in humans and nonhuman mammals that often persists for months after the injury occurs. Neuroprotective strategies for prevention of the sequelae of TBI and understanding the molecular mechanisms and cellular pathways are related to the glutamatergic system. It has been suggested that cellular damage subsequent to TBI is mediated by the excitatory neurotransmitters, glutamate and aspartate, through the excessive activation of the N-methyl-D-aspartate (NMDA) receptors. Thus, preconditioning with a low dose of NMDA was used as a strategy for protection against locomotor deficits observed after TBI in mice. Male adult mice CF-1 were preconditioned with NMDA (75 mg/kg) 24 hr before the TBI induction. Under anesthesia with O(2)/N(2)O (33%: 66%) inhalation, the animals were subjected to the experimental model of trauma that occurs by the impact of a 25 g weight on the skull. Sensorimotor gating was evaluated at 1.5, 6, or 24 hr after TBI induction by using footprint and rotarod tests. Cellular damage also was assessed 24 hr after occurrence of cortical trauma. Mice preconditioned with NMDA were protected against all motor deficits revealed by footprint tests, but not those observed in rotarod tasks. Although mice showed motor deficits after TBI, no cellular damage was observed. These data corroborate the hypothesis that glutamatergic excitotoxicity, especially via NMDA receptors, contributes to severity of trauma. They also point to a putative neuroprotective mechanism induced by a sublethal dose of NMDA to improve motor behavioral deficits after TBI.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Discinesias/tratamento farmacológico , N-Metilaspartato/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Animais , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Contagem de Células , Cerebelo/efeitos dos fármacos , Cerebelo/patologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Fragmentação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Discinesias/etiologia , Discinesias/patologia , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , N-Metilaspartato/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Equilíbrio Postural/efeitos dos fármacos , Índice de Gravidade de Doença , Fatores de Tempo , Resultado do TratamentoRESUMO
Neuropeptide S (NPS) is the endogenous ligand of a G-protein-coupled receptor named as NPSR. Behavioral effects have been recently attributed to NPS, i.e. hyperlocomotion, anxiolysis, and wakefulness. However, little is known about the mechanisms by which NPS evokes such biological actions. The present study aimed to investigate the role played by the adenosine A(2A) and A(1) receptors in hyperlocomotion induced by NPS. Spontaneous locomotion was assessed in an activity cage for 30 min in mice acutely treated with caffeine (a nonselective adenosine receptor antagonist), ZM241385 (a selective A(2A) receptor antagonist), or CPT (a selective A(1) receptor antagonist) before NPS challenge (0.1 nmol, i.c.v.), which induce hyperlocomotion in mice. The pretreatment with caffeine (3 mg/kg, i.p.), in an inactive dose per se, prevented the increase in locomotion evoked by NPS. The co-administration of NPS (0.1 nmol, i.c.v.) and ZM241385 (0.1 pmol, i.c.v.) counteracted hyperlocomotion evoked by NPS. The co-administration of NPS and CPT (0.1 pmol, i.c.v.) slightly facilitated the increase in locomotion evoked by NPS alone. In summary, the pharmacological blockade of A(2A) receptors significantly attenuated the stimulatory effects of NPS. By contrast, the antagonism of A(1) receptors facilitated NPS-induced hyperlocomotion in mice, but we cannot rule out a merely additive effect of two stimulatory systems in the brain. Altogether, this is the first evidence of a putative role played by A(2A) and A(1) receptors in modulating hyperlocomotion induced by NPS.