Ex-vivo evaluation of effect of beta-acid hops (Humulus lupulus) on intestinal gene expression in broilers

Abstract

The objective of this study is to evaluate the capacity hops beta-acids to modulate the expression of genes related with intestinal inflammatory response, under normal conditions and bacterial challenge, establishing the relationship between intestinal microbiota-host-additive. The research will be conducted with an ex vivo model, that permit a study more detailed, i.e., without interference of variables that generally influence in studies of molecular biology. Broilers will be fed corn soybean meal based diet to meet the nutritional requirements. The ileum will be collected (2 cm) and immersed for 30 minutes in a buffer containing antimicrobials (penicillin, neomycin and streptomycin) with the objective of eliminate the present microbiota (i.e. contaminants). Them, the tissue will be washed with PBS and immersed during 1 hour in a cell culture medium (DMEM) according with following treatments: T1: control, only DMEM; T2: DMEM with addition of 60 mg L-1 hops beta-acids; T3: DMEM with addition of 240 mg L-1 hops beta-acids; T4: DMEM with addition of 240 mg L-1 hops beta-acids plus Gram-negative bacteria (Salmonella Typhimurium at concentration of 105 ufc/mL.) and T5: DMEM with addition of 240 mg L-1 hops beta-acids plus Gram-positive bacteria (Enterococcus faecalis at concentration of 105 ufc/mL.). The tissue will be washed with PBS and stored in Quiazol solution at -80oC for subsequent RNA extraction, cDNA synthesis and analysis of expression of genes encoding the following cytokines: IL-1²; IL-6; INF-³; IL-4, IL-10 using real time PCR (qPCR). The data of qPCR will be submitted to analyses of variance (ANOVA), and when significant will be used the Tukey-Kramer Test (P<0,05) using the SAS 9.3 (2012). The methodology for this research has not been established in Brazil, justifying the interest in using the internship to conduct the study, with can applied in future projects. (AU)