Caracterização de xiloglucanas nativas e modificadas e efeitos sobre vias de ativação em macrófagos peritoniais de camundongos

Abstract

Resumo: Xiloglucanas de reserva foram obtidas de cotiledones moidos e deslipidificados de Copaifera langsdorffii (copaiba) (XGC), Hymeneae courbaril (jatoba) (XGJ) e Tamarindus indica (tamarindo) (XGT) atraves de extracao aquosa a 25 ¢XC. As proporcoes molares de Glc: Xyl: Gal foram de 2,4: 1,5: 1,0, para XGC, 3,8: 2,6: 1,0 para XGJ e 3,6: 2,4: 1,0 para XGT. Analises por HPSEC-MALLS/RI indicaram que todas as fracoes apresentaram-se homogeneas com valores de Mw para XGC, XGJ e XGT de 9,6 x 105, 9,1 x 105 e 9,1 x 105 g/mol, respectivamente. Parte das xiloglucanas de reserva obtidas foram tratadas com a enzima ƒÒ-galactosidase para obtencao das fracoes desgalactosiladas, denominadas XGCd, XGJd, XGTd, as quais apresentaram reducao na quantidade de unidades de galactose, de aproximadamente 70%, 76% e 60%, respectivamente. Para obtencao de xiloglucana fucosilada, folhas de C. langsdorffii, apos a remocao de pigmentos, lipideos e lignina, foram submetidas as extracoes sequenciais, aquosa e alcalinas. A fracao obtida da primeira extracao alcalina com KOH 4M (FCHB4) foi a que apresentou maior proporcao de Glc, Xyl, Gal e Fuc, entretanto, tambem foi observada a presenca de unidades de manose, arabinose e acido uronico. O perfil de eluicao obtido por HPSEC-MALLS/RI indicou uma fracao heterogenea. FCHB4 foi submetida a purificacao por cromatografia de troca ionica (FCHB4DS) e precipitacao com reativo de Fehling (PFHB4), obtendo uma fracao com composicao monossacaridica e espectro de 13C caracteristico de xiloglucanas fucosiladas. Os efeitos das xiloglucanas nativas e modificadas foram avaliados em macrofagos peritoneais de camundongos, apos a determinacao da viabilidade celular, em diferentes concentracoes, nos tempos de incubacao de 2 e 48h. A producao de O2„h- por macrofagos tratados com XGT, XGCd, XGJd e XGTd nao foi estatisticamente significativa, entretanto, quando as celulas foram incubadas com os polissacarideos na presenca de PMA, reduziram significativamente a producao de O2„h-. O aumento da capacidade fagocitica somente foi observado no tratamento com XGT. Porem, a producao de NO„h foi estimulada em diferentes intensidades, por todos os polissacarideos de reserva testados. Diferentes repostas quanto a producao de citocinas IL-1£], IL-6 e TNF-£\ foram observadas para as xiloglucanas nativas e modificadas. Na maxima concentracao, XGC aumentou IL-1£] e TNF-£\ em 1740 e 220% respectivamente, enquanto que XGCd nao mostrou tal estimulo. XGJ estimulou a producao de todas as citocinas. Com menor intensidade, XGJd estimulou IL-1£] e IL-6 em 104 e 40% a 50 £gg/mL. XGTd foi a unica fracao que ativou intensamente a producao das tres citocinas atingindo 1160, 76 e 203% para a producao de IL-1£], IL-6 e TNF-£\, respectivamente. A fracao nativa estimulou significantemente a producao de IL-6 e mostrou leve estimulo para producao de IL-1£] na maxima concentracao testada. A partir dos resultados obtidos, pode-se observar que cada polissacarideo testado apresentou caracteristicas singulares na ativacao de macrofagos, com intesidades e respostas distintas. Portanto, as xiloglucanas de reserva, nativas e modificadas podem ser classificadas como modificadoras da resposta biologica.

Abstract: Storage xyloglucans were obtained from milled and defatted cotyledons from Copaifera langsdorffii (copaiba) (XGC), Hymeneae courbaril (jatoba) (XGJ) and Tamarindus indica (tamarindo) (XGT) by aqueous extraction at 25oC. The fractions contained Glc:Xyl:Gal in molar ratio of 2,4: 1,5: 1,0, for XGC, 3,8: 2,6: 1,0 for XGJ and 3,6: 2,4: 1,0 for XGT. HPSEC-MALLS/RI analyses showed that all the fractions were homogeneous and that their Mw values were 9,6 x 105, 9,1 x 105 e 9,1 x 105 g/mol, respectively. Storage xyloglucans fractions were treated with ƒÒ-galactosidase enzyme to obtain desgalactosilated fraction named XGCd, XGJd XGTd, which showed a reduction of galactoses units reaching 70%, 76% e 60%, respectively. To obtain fucosilated xyloglucans, C. langsdorffii leaves, after pigment, fat and lignin remotion, were submitted to several aqueous and alkaline extractions. The fraction obtained from first extraction with 4M KOH (FCHB4), showed the highest ratio of Glc, Xyl, Gal and Fuc, however, it was also observed the presence of mannose, arabinose and uronic acid. HPSEC-MALLS/RI analysis revealed that this fraction was heterogeneous. FCHB4 was submitted to purification procedures using ion-exchange chromatography (FCHB4DS) and precipitation by Fehling solution (PFHB4), leading to a fraction containing a characteristic monosaccharide composition and presenting a 13C spectrum characteristic of fucosilated xyloglucan. The effects of native and modified xyloglucans on functional properties of peritoneal cavity macrophages were evaluated after celular viability determination, in different concentration, at 2 and 48h of incubation. The effects of XGT, XGCd, XGJd and XGTd on O2„h- production in absence of PMA were not statistically significant, however, showed significant reduction of O2„h- production in the PMA presence. The enhancement of phagocytic activity was only observed by XGT. The NO„h production was stimulated in different intensities for all tested storage polysaccharides. Different responses in IL-1£], IL-6 and TNF-£\ cytokines production were observed for native and modified xyloglucans. The maximum concentration of XGC increased IL-1£] and TNF-£\ in 1740 and 220%, respectively, while XGCd did not show this effect. XGJ promote the production of all cytokines. With less intensity, XGJd stimulated IL-1£] and IL-6 reaching 104 and 40% at 50 £gg/mL. XGTd had been the only fraction that intensely activated the three cytokines production, leading to 1160, 76 and 203% of increasing for the production of IL-1£], IL-6 and TNF- £\, respectively. The native fraction significantly stimulated the production of IL-6 and showed a slight stimulus for the production of IL-1£] in maximum concentration. From the obtained results, it was observed that each polysaccharide presents unique characteristics in the macrophages activation, with differents intesidades and response. Therefore storage xyloglucan, native and modified may be classified as biological response modifiers.