Polissacarídeos sulfatados da alga Ulva lactuca L.: efeitos na modulação de fatores proinflamatórios em modelos experimentais e na translocação do Fator Nuclear NF - kB em RAW264.7

Abstract

Nowadays, have been studying the biological activity of different seaweed species. The specie Ulva lactuca,popularlk known as sea´nlettuce, is a green algae rich in ulvanes, a sulfate polysaccharide that has been relacionated with diverse biological activities. The objective of the work was the extraction of sulfate polysaccharides of Ulva lactuca L. and evaluation of its anti-inflammatory activity in macrophages stimulated with LPS. The seaweed dried was rasureted, delipidated and submitted to proteolysis. The crude polysaccharides was submitted to precipitation by increase amounts of acetone, obtaining four fractions denominated U30, U50, U70 and U100. These compounds were caracterizated by chemical dosages, molecular weigth estimation by agarose gel, gel filtration chromatography, fluorophore assisted carbohydrate electrophoresis (FACE) for monosaccharide composition analyses and nuclear magnetic ressonance (NMR). The capacity of reduce MTT was evaluate by MTT assay in macrophage cell line RAW 264.7. Also investigated the fractions anti-inflammatory activity in macrophages RAW 264.7 and peritoneal macrophage in mice C57BL/6 through production of nitric oxide by Griess method, MMP-2 and 9 activity by zimogram and expression by western blot, production of pro-inflammatory cytokines TNF-TNF-a e IL-6 to ELISA assay, the translocation of nuclear factor kB, the number of lipid droplets in cells and lipid peroxidation in macrophages. The fraction showed sugar content between 4,15 and 6,96 mg/mL, sulfate between 2,50 and 3,40 mg/mL and uronic between 0,74 e 2,86 mg/mL. Phenolic or protein content were not observed. The fractions were composed by rhamnose, glucose, galactose and uronic acid monosaccharides. For NMR analyzes, the fractions U50 e U70 are formed by ulvanobiuronic acids. No one fractions reduce the capacity of macrophages RAW 264.7 in reduce MTT. In the anti-inflammatory assay, the fraction U50 reduced in 17% and 21% the NO production in 12,5 and 25 µg/mL, still U70 fraction reduced the NO production in 58 and 70% in 12,5 e 25 µg/mL concentration. The fraction U70 decreased the activity of MMP-9 in 69% in 25 µg/mL concentration and the fraction U50 in 40 and 33% in 12,5 e 100 µg/mL concentrations. Both fractions, U50 and U70, in 100 µg/mL reduced IL-6 in 26% and TNF-a in 18% and 26%, respectively. The fraction U70 markedly reduced the NF-kB translocation in 100 µg/mL concentration when compared with the control. The lipid droplets biogenesis in macrophages RAW 264.7 and peritoneal stimulated and incubated with U70 were reduced when compare with stimulated standard. The U70 fraction reduced the lipid peroxidation between 33 and 55% in the concentrations between 12,5 and 200 µg/mL. The results suggest that U70 fraction have potencial anti-inflammatory activity in vitro, being necessary the investigation of action mechanisms, thus the application in animals and cellular models for inflammatory cronic diseases.