Efeito do flavonoide agatisflavona na modulação do NLRP3-inflamossoma, micro-RNAs e citocinas pró-inflamatórias associados a resposta microglial a estímulos inflamatórios e peptídeos aβ-amiloide

Abstract

Microglial activation plays a crucial role in the pathogenesis of neurodegenerative diseases (NDD), and the control of this activation has been the subject of investigations as a strategy for the development of new therapeutic approaches for NDD. Agatisflavone, purified from Cenostigma pyramidale (Tul.), has been shown to be neuroprotective in in vitro models of glutamate-induced excitotoxicity and inflammatory damage. However, the potential role of microglial regulation by agathisflavone in these neuroprotective effects remains unclear. In this work, we investigated in microglial cells from the cortex of rats and in microglial cells of human lineage induced to inflammatory damage, the effects of agatisflavone in modulating the inflammatory response in order to elucidate mechanisms of neuroprotection. For this purpose, cultures of human microglia of the C20 lineage and microglia isolated from the cortex of newborn Wistar rats were exposed to β-amyloid peptide oligomers (500nM) for 4h or Escherichia coli lipopolysaccharide (LPS, 1µg/mL) for 24h and then treated or not with agathisflavone (1µM) for 24h. Cultures of PC12 neuronal cells were exposed to rat microglia conditioned medium (MCM) treated or not with agathisflavone. In a first study, we observed that LPS induced microglia to assume an activated inflammatory state (increased CD68, with a more rounded/amoeboid phenotype). However, most microglia exposed to LPS and agathisflavone showed an anti-inflammatory profile (increased CD206 and branched phenotype), associated with reduced expression of NO, GSH, NLRP3 inflammasome, IL1-β, IL-6, IL-18, TNF, CCL5 and CCL2. Molecular docking also showed that agathisflavone bound to the NLRP3 NACTH inhibitory domain. Furthermore, in cultures of PC12 cells exposed to MCM previously treated with the flavonoid, most cells preserved their neurites and increased expression of β-tubulin III. In the second study, we observed that β-amyloid and LPS induced microglia cultures to assume an activated inflammatory state, with increased expression of miR-146a and miR-155 and inflammatory mediators IL1-β, IL-6 and NOS2. However, in cells exposed to inflammatory damage and treated with agathisflavone, we observed a significant reduction in the concentration of miR146a and miR-155, as well as the inflammatory cytokines evaluated. We also observed in cells stimulated only with β-amyloid, an increase in the ratio of p-STAT3/STAT3 signaling proteins, and in cells stimulated with β-amyloid and treated with flavonoid, a reduction in this ratio. Furthermore, we showed that agatisflavone downregulated microglial cytosolic calcium concentration, suggesting its antagonistic effect on purinergic receptor. Thus, these data reinforce the anti-inflammatory activity and the neuroprotective effect of agathisflavone, associated with the control of the NLRP3 inflammasome and inflammatory mediators, also highlighting its potential in the regulation of miRNAs associated with neuroinflammation. These results strengthen the potential of this flavonoid as a promising molecule for the treatment or prevention of neurodegenerative diseases.