Peptídeos e proteínas anti-inflamatórios e funcionalidade da barreira intestinal. I: efeito do inibidor de tripsina isolado de tamarindo na permeabilidade intestinal

Abstract

A healthy intestinal barrier protects the individual against antigen translocation, ensuring a controlled inflammatory environment. The first two chapters of this thesis refer to a systematic review (SR), which aimed to understand the mechanisms of action of antiinflammatory molecules that reduce TNF-α and their effects on the intestinal barrier in animal models. The protocol of the first chapter was registered in PROSPERO and guided the elaboration of the SR, presented in the second chapter. The articles were selected according to the PICO strategy (Population, Intervention, Comparison/Control and Outcomes), from PubMed, Scopus, Web of Science, EMBASE and ScienceDirect databases. Twenty-five articles were included and the risk of bias assessment was performed using the SYRCLE tool. The results show that the anti-inflammatory molecules that acted by reducing TNF-α acted mainly on the TNF-TNFR1/TNFR2 and TLR4/MD2 complex signaling pathways, and consequently on the NF-κB pathway, improving the signs of inflammatory diseases and the macroscopic, histological and intestinal permeability aspects. In the third chapter, the proposal and methodological validation of an inflammation model with TNF-α (50 ng/mL/48h, on the basolateral side) in co-culture of Caco-2:HT29-MTX intestinal cells is presented. In the fourth chapter, the trypsin inhibitor isolated from tamarind seeds (TTI) was evaluated in vitro regarding its interaction with bacterial lipopolysaccharide (LPS) and its action against human neutrophil elastase (HNE). In addition, the effects on the integrity and functionality of the intestinal barrier in cell culture and in an experimental obesity model were evaluated, including hematological, biochemical and inflammatory parameters. The study in cells was carried out in the inflammation model validated in the third chapter and evaluated cell viability and the production of reactive oxygen species by contacting the monolayers with the TTI. In addition, transepithelial electrical resistance and permeability in monolayers were evaluated after induction of inflammation and treatment with ITT (1.0 mg/mL, after or during the inflammatory stimulus). For the animal study, obese Wistar rats (n=15) were divided into three groups: no treatment group (n=5), group treated with a nutritionally adequate diet (n=5) and group treated with TTI (25 mg/ kg/day, n=5) for ten days. TTI did not interact with LPS, but showed inhibition against HNE (93%). In the cell study, TTI did not alter cell viability and did not present pro- or antioxidant properties, as well as it did not prevent damage or restore the monolayers integrity. In the in vivo experiment, TTI-treated rats had significantly lower plasma concentrations of inflammatory cytokines, and of other obesity-related parameters, such as total leukocyte count, fasting blood glucose, and LDL-c, compared to animals treated with a nutritionally adequate diet. They also showed better histopathological and histomorphometric results in the small intestine, although there were no significant differences between the groups regarding most semiquantitative parameters, intestinal permeability and concentration of inflammatory cytokines in the intestine. Thus, it is concluded that TTI, at the concentrations tested, was safe for cell cultures and reduced systemic inflammation in Wistar rats, which possibly reflected in the improvement of the morphology of the intestinal epithelium in the treated animals. In view of the results, it is concluded that the action of the TTI on the intestinal epithelium is probably related to inhibitory activity against HNE.