Patulina inibe a produção de óxido nítrico induzida por LPS ao suprimir a via de sinalização das MAP quinases

Abstract

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.