Modulação de NOX2 pela aminoguanidina e as implicações na função microbicida de neutrófilos e na produção de espécies reativas por células endoteliais (HUVEC)

Abstract

It has been showed that aminoguanidine (AG), a known inhibitor of AGE (Advanced Glycation End Products) increases the activity of NOX2 (phagocyte system NADPH Oxidase) peritoneal neutrophils of diabetic rats. Additional experiments demonstrated that the same effect occurs in non-diabetic rat neutrophils. To elucidate the mechanism and significance of NOX2 increase in neutrophils by AG, it was used an experimental model of diabetic and non-diabetic rats and an in vitro model in which human neutrophils isolated from peripheral blood or HUVEC endothelial cell lineage. For this, Wistar rats diabetic and non-diabetic were treated or not with AG by gavage (100 mg / kg / day) for 50 days. Neutrophils were recruited and isolated from the peritoneum 4h after injection of sodium caseinate and were used to evaluate superoxide generation, ROS and phagocytic and candicida activity of these cells. In vitro model, neutrophils isolated from peripheral blood or HUVEC were incubated or not with AG or Metformin (MET) for 18h in CO2 incubator. In vivo treatment with AG showed an increase in superoxide production compared with groups treated with water, in both groups diabetics and non-diabetics. The increased activity and expression of NOX2 (p47phox and p67phox) by AG increased phagocytic and candicida activity of Candida albicans, however this increase was less pronounced in the diabetic group. Additionally, the results demonstrate the effectiveness of treatment with AG on preventing glycation of proteins, reducing HbA1c and AGEs, in addition to reducing levels of urea and maintain the creatinine levels in serum of animals. The treatment with AG reduced the blood pressure and the production of nitrates and antioxidant enzymes activity were maintained in animals. It was found that HUVEC treated with AG or MET at baseline, or stimulated with LPS had lower production of ROS. Human neutrophils incubated with AG increase ROS and superoxide while treatment with MET remained the production of both. In addition, treatment with AG increased phagocytosis and candicidal activity of human neutrophils at times evaluated. In addition, treatment with AG increased phagocytosis and candicida activity of human neutrophils at times evaluated. The MET treatment maintained the expression of subunits assessed, and treatment with AG caused increased expression of gp91. The release of IL-8 by human neutrophils was decreased by treatment with MET, and AG increased the release of IFN-y The release of IL-8 was reduced in human neutrophils treated with MET, and AG increased IFN-y . In HUVEC the release of IL-8 and IFN-y were maintained with both treatments. It can be suggested that the AG used to inhibit the formation of AGEs increased activity and NOX2 expression in human and rat neutrophils and that directly contributed to the increased microbicidal activity by neutrophils. In addition, the human neutrophils the increased of IFN-y may have contributed to the increased expression of gp91phox. In addition, the influence of AG in cells in differentiation process to more mature state may be different of in vitro effects on cells already mature.