Evaluating protective effects of botanicals under inflammation and oxidative stress in chicken apical-out enteroids

Botanicals (BOTs) are well known for their anti-inflammatory and antioxidant activities. They have been widely used as feed additives to reduce inflammation and improve intestinal functions in agricultural animals. However, the effects of BOTs on chicken intestinal epithelial functions are not fully understood. The 3D apical-out chicken enteroids recapitulate the intestinal tissue, and allow convenient access to the luminal surface, thus serving as a suitable model for investigating gut functions. The aim of this study was to identify the roles of BOTs in protecting the intestinal epithelium in chicken enteroids under challenging conditions. Apical-out enteroids were isolated from the small intestines of 18 days-old chicken embryos. Lipopolysaccharide (LPS, 10 µg/mL) and menadione (400 µM) challenges were performed in the media with or without BOTs. Paracellular Fluorescein isothiocyanate–dextran 4kD (FD4) permeability, inflammatory cytokine gene expression, and reactive oxygen species (ROS) generation were analyzed post-BOTs and challenges treatments. Statistical analysis was performed using one-way ANOVA and post hoc multiple comparisons among treatments. The results showed that the LPS challenge for 24 h induced a 50% increase in FD4 permeability compared with non-treated control; thymol, thyme essential oil, and phenol-rich extract significantly (P < 0.02) reduced FD4 permeability by 25%, 41%, and 48% respectively, in comparison with LPS treatment. Moreover, the gene expression of inflammatory cytokines was upregulated, tight junction proteins and defensins were downregulated (P < 0.05) after 6 h of LPS treatment, while these BOTs treatments significantly restored the LPS-induced gene expression alterations (P < 0.05). Menadione oxidative challenge for 1 h significantly increased the ROS level compared with unchallenged control. Enteroids treated with thymol and thyme essential oils showed 30% reduced ROS levels, while the phenol-rich extract reduced them by 60%, in comparison with the challenged group (P < 0.0001). These data confirmed the role of BOTs in supporting the barrier function and reducing the disruptive effects of inflammation and oxidation in the chicken intestine.