Assuntos
Anticorpos/química , Biologia Computacional/métodos , Gástrula/citologia , Análise de Célula Única/métodos , Software , Academias e Institutos/economia , Animais , Encéfalo/citologia , Encéfalo/virologia , Brasil , Núcleo Celular/metabolismo , Custos e Análise de Custo , Metilação de DNA , Gástrula/fisiologia , Humanos , Análise de Sequência de RNA , Análise de Célula Única/economia , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/metabolismo , Infecção por Zika virus/patologiaRESUMO
INTRODUCTION: This study investigated the mechanism of action of a synthetic tetrahydroisoquinoline alkaloid, MHTP, in an experimental model of acute lung injury (ALI) in two distinct moments: 72 h and 10 days. METHODOLOGY: To realize this study, 2.5 mg/kg of lipopolysaccharide (LPS) was intranasally administered in BALB/c mice, and nasal instillation of MHTP (1.25; 2.5; 5.0; 10 or 20 mg/kg) was administrated at 1, 24, and 48 h after LPS challenge. The data were statistically analyzed and p < 0.05 was considered statistically significant. RESULTS: MHTP treatment (2.5, 5.0, 10 or 20 mg/kg) significantly decreased neutrophil migration into the bronchoalveolar lavage fluid (BALF), tissue inflammatory cell infiltration, edema, and hemorrhage as well as collagen fiber deposition on the perialveolar regions at both moments. TNF-α and IL-6 levels were significantly diminished in the MHTP-treated animals at 72 h and maintained them, at a basal level, at 10-day observation. These effects of MHTP are due to downregulating p38MAPkinese/p65NFκB signaling pathway-TLR4 dependent. Also, the MHTP treatment promoted a survival rate at 100% and improved their body weights during the 10-day observation. Unlike, the LPS group (non-treated LPS challenged animals) presented less than 50% of surviving rate at 72 h and the animals that survived did not improve their physiological state at 10-day observation. CONCLUSIONS: These data showed for the first time the beneficial and effective activity of a nasal treatment with a synthetic tetrahydroisoquinoline alkaloid on an experimental model of ALI and pointed out the molecular mechanism related to it.
Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Tetra-Hidroisoquinolinas/uso terapêutico , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Administração Intranasal , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Interleucina-6/imunologia , Lipopolissacarídeos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos Endogâmicos BALB C , Tetra-Hidroisoquinolinas/farmacologia , Fator de Transcrição RelA/imunologia , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Mast cells (MCs) contribute to the control of local inflammatory reactions and become hyporesponsive after prolonged TLR4 activation by bacterial LPS. The molecular mechanisms involved in endotoxin tolerance (ET) induction in MCs are not fully understood. In this study, we demonstrate that the endocannabinoid 2-arachidonoylglycerol (2-AG) and its receptor, cannabinoid receptor 2 (CB2), play a role in the establishment of ET in bone marrow-derived MCs from C57BL/6J mice. We found that CB2 antagonism prevented the development of ET and that bone marrow-derived MCs produce 2-AG in a TLR4-dependent fashion. Exogenous 2-AG induced ET similarly to LPS, blocking the phosphorylation of IKK and the p65 subunit of NF-κB and inducing the synthesis of molecular markers of ET. LPS caused CB2 receptor trafficking in Rab11-, Rab7-, and Lamp2-positive vesicles, indicating recycling and degradation of the receptor. 2-AG also prevented LPS-induced TNF secretion in vivo, in a MC-dependent model of endotoxemia, demonstrating that TLR4 engagement leads to 2-AG secretion, which contributes to the negative control of MCs activation. Our study uncovers a functional role for the endocannabinoid system in the inhibition of MC-dependent innate immune responses in vivo.
Assuntos
Ácidos Araquidônicos/farmacologia , Endocanabinoides/farmacologia , Glicerídeos/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Mastócitos/imunologia , Receptor CB2 de Canabinoide/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Tolerância Imunológica/imunologia , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/imunologia , Camundongos , Camundongos Knockout , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Transporte Proteico/imunologia , Receptor CB2 de Canabinoide/genética , Receptor 4 Toll-Like/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/imunologia , proteínas de unión al GTP Rab7RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Senecio brasiliensis (Spreng) Less (S. brasiliensis), known as "Flor-das-almas", "Margaridinha" or "Maria mole", is used in folk medicine as an anti-inflammatory and to treat gastric ulcers and stomach pain. While the Senecio genus has been widely studied for its pharmacological activities to support its use in traditional medicine, few studies focus on the anti-inflammatory activities of the species. AIM OF THE STUDY: To investigate the anti-inflammatory activities of S. brasiliensis, a specie native to Brazil, using a murine model of pleurisy induced by carrageenan. MATERIAL AND METHODS: The flowers of S. brasiliensis were air-dried for 3 days and subjected to ethanol (96%) extraction for 7 days to obtain the crude extract (CE). The CE was subjected to acid-base extraction to obtain the alkaloid fraction (AF). The hexane (HEX), dichloromethane (DCM) and ethyl acetate (EtOAc) fractions were obtained by extracting from CE with different solvents. The alkaloids senecionine (Sen), integerrimine (Int) and senecionine N-oxide were obtained from AF by chromatographic fractionation and a mixture of 1,4-, 3,4-, 3,5- and 4,5-dicaffeoylquinic acids (DCQs) were obtained from the EtOAc fraction. The isolated alkaloids were identified through spectroscopic analysis of IR, NMR and LC-MS coupled with electrospray ionization mass spectrometry (ESI-MS), and the dicaffeoylquinic acids through the hierarchical key method. Swiss mice were used in the in vivo experiments. We evaluated the effect of the CE, its derived fractions (AF, HEX, DCM and EtOAc), and the isolated compounds (Sen, Int, N-oxide senecionine, and DCQs) on: leukocyte migration, exudate concentrations, myeloperoxidase (MPO) and adenosine-deaminase (ADA) activities, and tumor necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß) and interleukin 17A levels in the fluid leakage from the pleural cavity using a mouse model of pleurisy induced by carrageenan. The effects of the isolated compounds, Sen, Int, N-oxide senecionine and DCQs, were also analyzed for their ability to inhibit p65 phosphorylation (p-p65) in the nuclear factor-kappa B (NF-κB) pathway in the lung tissue. MPO and ADA were analyzed by colorimetric assays, and the cytokines and protein p65 levels were determined using an enzyme immunoassay (EIA). RESULTS: The CE, its EtOAc and AF fractions, and its isolated compounds (Sen, Int and DCQs), significantly reduced leukocyte migration (P < 0.05), MPO and ADA activities (P < 0.01), and TNF-α (P < 0.05), and IL-17A levels (P < 0.01). The CE, the EtOAc and AF fractions, and the DCQs also decreased IL-1ß levels (P < 0.01). The isolated compounds, Sen, Int and the DCQs, inhibited p65 phosphorylation (NF-κB) (P < 0.05). CONCLUSION: This study demonstrated that S. brasiliensis has important anti-inflammatory properties that are capable of inhibiting activated leukocytes by decreasing neutrophil migration. This effect may be attributed to the inhibition of pro-inflammatory cytokines and the reduction of the NF-κB pathway. The compounds Sen, Int, and DCQs may be responsible for the anti-inflammatory actions of S. brasiliensis.