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Patulin (PAT) is a fungi-derived natural product with immunosuppressive properties, but the signaling pathways involved in the effect of PAT remain undefined. This work aimed to evaluate the effect of PAT in lipopolysaccharide (LPS)-activated murine peritoneal macrophages. For this purpose, murine peritoneal macrophages were stimulated with LPS and treated with PAT at different concentrations (62.5-4,000 ng/ml). Cell viability assay showed that PAT at concentrations up to 250 ng/ml did not affect macrophage viability. Then, nitric oxide response was evaluated using the nontoxic PAT concentrations (62.5; 125; and 250 ng/ml). As a result, PAT, at concentration of 250 ng/ml, significantly reduced nitric oxide production (by 98.4%), inducible nitric oxide synthase (iNOS) expression (by 83.5%), and iNOS messenger ribonucleic acid expression (by 100.0%) in the cells stimulated with LPS. Next, we investigated whether PAT interferes with interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor (TNF)-α release. PAT significantly reduced LPS-induced IL-1β release (by 80.6%), without interfering with the production of IL-6, IL-10, and TNF-α. Moreover, PAT significantly reduced cluster of differentiation (CD) 69 (by 63.1%) and Toll-like receptor (TLR) 4 (by 91.9%) protein expression in the presence of LPS, without altering the gene expression of these proteins. Finally, to investigate PAT mechanism of action, mitogen-activated protein (MAP) kinase phosphorylation analysis was performed. PAT significantly reduced LPS-triggered phosphorylation of all MAP kinases assessed: extracellular signal-regulated kinase (ERK; by 89.5%), c-Jun N-terminal kinase (JNK; by 77.5%), and p38 (by 72.3%). Taken together, these data suggest that PAT downregulates acute inflammatory response, inhibiting nitric oxide production by suppressing CD69-TLR4/ERK-JNK-p38 MAP kinases/Nos2/iNOS signaling pathway in murine peritoneal macrophages. Thus, PAT could become a promising pharmacological tool for the control of inflammation, capable of completely inhibiting the nitric oxide pathway. Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES A patulina (PAT) é um produto natural derivado de fungos com propriedades imunossupressoras. No entanto, as vias de sinalização envolvidas no efeito da PAT permanecem indefinidas. Este trabalho teve como objetivo avaliar o efeito da PAT em macrófagos peritoneais murinos ativados com lipopolissacarídeo (LPS). Para isso, macrófagos peritoneais murinos foram estimulados com LPS e tratados com a PAT em diferentes concentrações (62,5-4.000 ng/ml). O ensaio de viabilidade celular mostrou que a PAT em concentrações de até 250 ng/ml não afetou a viabilidade dos macrófagos. Então, foi avaliada a produção de óxido nítrico usando as concentrações não tóxicas de PAT (62,5; 125 e 250 ng/ml). Como resultado, a PAT, na concentração de 250 ng/ml, reduziu significativamente a produção do óxido nítrico (98,4%), a expressão da óxido nítrico sintase induzida (iNOS; 83,5%) e a expressão do ácido ribonucleico mensageiro da iNOS (100,0%) nas células estimuladas com o LPS. Em seguida, foi investigado se a PAT interfere na produção de interleucina (IL) 1β, IL-6, IL-10 e fator de necrose tumoral (TNF)-α. A PAT reduziu significativamente a produção da IL-1β (80,6%) induzida pelo LPS, sem interferir na produção da IL-6, IL-10 e TNF-α. Além disso, a PAT reduziu significativamente a expressão das proteínas grupamento de diferenciação (CD) 69 (63,1%) e receptor do tipo Toll (TLR) 4 (91,9%) na presença do LPS, sem alterar a expressão gênica dessas proteínas. Por fim, para investigar o mecanismo de ação da PAT, foi realizada a análise da fosforilação das proteínas ativadas por mitógenos (MAP) quinases. A PAT reduziu significativamente a fosforilação induzida pelo LPS de todas as MAP quinases avaliadas: quinase regulada por sinal extracelular (ERK; 89,5%), quinase N-terminal c-Jun (JNK; 77,5%) e p38 (72,3%). Em conjunto, esses dados inéditos sugerem que a PAT regula negativamente a resposta inflamatória aguda, inibindo a produção de óxido nítrico ao suprimir a via de sinalização CD69-TLR4/MAP quinases ERK-JNK-p38/Nos2/iNOS em macrófagos peritoneais murinos. Assim, a PAT pode se tornar uma ferramenta farmacológica promissora para o controle da inflamação, capaz de inibir completamente a via do óxido nítrico.

Inflammation is an immune response that aims to establish a tissue homeostasis during an infection or injury. Recognized as a beneficial process, an inflammation can become harmful when in excess. Thus, therapeutic therapies that are applied to the resolution of the inflammation are studied and developed, with natural products and their derivatives playing a prominent role in the discoveries of new anti-inflammatory molecules. In this context, the objective of this study was to evaluate the antiinflammatory potential of milonine, a Cissampelos sympodialis Eichl alkaloid, in vivo, against the release of mediators by using the murine model of acute inflammation and pain. Swiss mice pretreated with milonine were submitted to protocols of: paw edema induced by LPS, prostaglandin (PGE2), bradykinin (BK) and serotonin (5-HT) to evaluate the anti-edematogenic activity of the alkaloid; microvascular permeability induced by acetic acid to obtain a total protein concentration and histological analysis of the peritoneum; the models of carrageenan-induced peritonitis to evaluate the effect of milonine on the migration of the total and differential cells; and the formalin test to evaluate its nociceptive activity. The results show that milonine was capable of significantly reduce (p