Effect of branched-chain amino acids on the inflammatory response modulation induced by LPS in Caco-2 intestinal cells

Abstract

Background: Inflammation is a present factor in the pathophysiology of acute and chronic diseases, and the gut, a susceptible organ to dysfunction and dysbiosis, that is in constant exposure to microorganisms it may be associated with this mechanism. In contrast, the branched-chain amino acids (BCAA) may have anti-inflammatory and antioxidant effects on intestinal cell lines. Objective: Investigate the effects of BCAA in vitro on the inflammatory response modulation induced by lipopolysaccharides (LPS) in the Caco-2 intestinal cells model. Methods: The Caco-2 cells will be cultured and stored in bottles and expression plates (n=6) until they reach 80% confluence. The cultured cells will be divided into six groups: two control groups - control with Dulbecco's Modified Eagle's Medium media without BCAA (DMEM-0) and with BCAA (DMEM-P) -, leucine group (LEU), isoleucine group (ISO), valine group (VAL) and BCCA association (LIV). A pre-treatment (PT) protocol will be adopted with BCAA and LPS. The inflammatory cytokines dosage (IL-1beta, IL-6, IL-8, IL-10 e TNF-alpha) will be carried out through the ELISA method. The protein expression analysis (mTOR, AKT, AMPK, NF-kB, TAK-1, IKK-beta, JNK e IkB-alpha) will be done by Western Blotting. Components of the antioxidant system related to glutathione (GSH, GSSH, GPx) will be evaluated using a commercial kit. For statistical analysis, the one-way ANOVA will be performed, followed by Tukey's post hoc test. The results will be presented as mean ± standard deviation and p <0.05 will be considered. Expected results: It is expected to understand the potential mechanisms of BCAA action - in isolation and associated with each other - concerning the inflammatory response modulation induced by LPS. It is believed that these amino acids will modulate proteins of the inflammatory pathway through the Caco-2 cells experimental model. (AU)