Avaliação da resposta inflamatória local após isquemia e reperfusão intestinal em ratos: efeito do tratamento com estradiol.

Abstract

RICARDO-DA-SILVA, F.Y. Local inflammatory response following intestinal ischemia and reperfusion in male rats: effect of estradiol treatment effect. 2015. 130 p. Masters thesis (Pharmacology) – Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo, 2015. Intestinal ischemia/reperfusion (IR) causes local and remote injuries. Experimental evidence indicates that female rats are more resistant to IR inflammatory effects and this could likely be due the protective effects of estradiol. However, the role of estradiol on the control of inflammation induced by by intestinal IR in male, is still not completely elucidated. In order to investigate the inflammatory response in the intestine and its systemic repercussions, groups of male rats (Wistar, 60 days, 160 g) were submitted to intestinal IR. Under anesthesia, intestinal ischemia was induced by obstruction of superior mesenteric artery (SMA) during 45 min. After 2 h of intestinal reperfusion the animals were submitted to euthanasia and the experiments were conducted. Groups of animals received estradiol treatment (280 μg/kg) 30 min after the induction of intestinal ischemia (E30) (therapeutic treatment) or (preventive treatment) 24 h before the induction of intestinal ischemia (E24). As controls rats were falsely submitted to instestinal IR (Sham) and as reference we used non manipulated rats (Naive). Vascular and mucosal permeability from intestine were quantified by Evans blue dye and dextran-FITC extravasation, respectively. The recruitment/activation of neutrophils was determined by myeloperoxidase activity, the intestinal motility and ileum ex vivo reactivity by activated coal transit in the intestine and by dose response curve to methacholine, respectively) were also evaluated. In a parallel set of experiments, the intestine of rats submitted to intestinal IR or sham was isolated in a plastic bag and the volume and content of the fluid accumulated inside during intestinal IR period were analyzed. The levels of IL-10, TNF-α, leukocytes and Evans blue dye extravaseted were quantified. In order to investigate the systemic repercussions of intestinal IR, we determined the number of blood leukocytes, bone marrow cells, tissue injury markers (alkaline phosphatase and lactate dehydrogenase (LDH)), cytokines (IL-1β, IL-6, IL12p70, IL-17, IL-10,IFN-γ), factors (VEGF, TNF-α e GM-CSF) and chemokines (MIP-1α, MCP-1, GRO/KC e IP-10) in serum of those animals. In vitro studies about neutrophils migration were also conducted. Our data showed that intestinal IR increased intestinal vascular and mucosal permeability, recruitment/activation of neutrophils, intestinal motility and ex vivo reactivity to methacoline. There was a reduction of exsudate intestinal fluid volume that was accompanied by an increase of TNF-α and IL-10 levels and leukocytes numbers. Systemically, intestinal IR increased the white blood cell counts and reduced the bone marrow cell counts. Serum alkaline phosphatase, LDH and all quantified cytokines and chemokines were found increased after intestinal I/R. Regarding the in vitro migration assay we observed that the neutrophil migration after intestinal IR was increased. Estradiol treatment, showed that both (E30 and E24) reduced TNF-α and leukocytes in the intestinal fluid, neutrophils recruitment/activation, LDH and IL-1β, IL-10, VEGF, MIP-1α, MCP-1, GRO/KC e IP-10. Preventive treatment (E24) reduced the intestinal vascular permeability but increased intestinal motility and bone marrow cell counts. On the other hand, therapeutic treatment (E30) reduced the intestinal mucosal permeability, white blood cell counts and serum concentrations of alkaline phosphatase, IL-6, TNF-α and GM-CSF. Overall, our results suggest that the systemic and local repercussions of intestinal IR were attenuated by estradiol administration in the context of therapeutic and preventive treatment of the rats. Since that intestinal inflammation could lead to important systemic consequences, our data allow us to suggest that estradiol could be an interesting pharmacological alternative in the control and understanding of the mechanisms triggered by intestinal ischemic events followed by reperfusion.