Dietary supplementation of microencapsulated botanicals and organic acids enhances the expression and function of intestine epithelial digestive enzymes and nutrient transporters in broiler chickens.

Organic acids and botanicals have shown protective effects on gut barrier and against inflammation in broilers. However, their effects on intestinal digestive enzymes and nutrients transporters expression and functions have not been fully studied. The objective of this study was to understand how a microencapsulated blend of botanicals and organic acids affected intestinal enzyme activities and nutrient transporters expression and functions in broilers. A total of 288 birds were assigned to a commercial control diet or diet supplemented with 500 g/MT (metric ton) of the microencapsulated additive. Growth performance was recorded weekly. At d 21 and d 42, jejunum and ileum were isolated for enzyme (maltase, sucrase, and aminopeptidase) and transporter (SGLT1, GLUT2, GLUT1, EAAT3, B0AT1, and PepT1) analyses. Jejunum specific nutrients (glucose, alanine, and glutamate) transport activities were evaluated by Ussing chamber. Protein expression of nutrient transporters in small intestine were measured in mucosa and brush-border membrane (BBM) samples by western blot. Intestinal gene expression of the transporters was determined by RT-PCR. Statistical analysis was performed using Student's t-test comparing the supplemented diet to the control. The feed efficiency was significantly improved through the study period in the supplemented group (P ≤ 0.05). Significant changes of intestinal histology were shown in both jejunum (P ≤ 0.10) and ileum (P ≤ 0.05) after 21 d of treatment. At d21, jejunal maltase activity was upregulated (P ≤ 0.10). The Ussing chamber transport of glucose and alanine was increased, which was in line with increased gene expression (GLUT2, GLUT1, EAAT3, and B0AT1) (P ≤ 0.10 and P ≤ 0.05, respectively) and BBMV protein levels (B0AT1, P < 0.10). At d21, ileal sucrase and maltase activities were upregulated (P ≤ 0.05). Increased expressions of GLUT1, EAAT3, and B0AT1 were observed in both mRNA and protein levels (P ≤ 0.05). Similar pattern of changes was also shown at d42 of age. Our results suggest that feeding microencapsulated additives improves intestinal nutrient digestion and transporter expression and function in broilers, thereby enhancing feed efficiency.

A blend of selected botanicals maintains intestinal epithelial integrity and reduces susceptibility to Escherichia coli F4 infection by modulating acute and chronic inflammation in vitro.

In the pig production cycle, the most delicate phase is weaning, a sudden and early change that requires a quick adaptation, at the cost of developing inflammation and oxidation, especially at the intestinal level. In this period, pathogens like enterotoxigenic Escherichia coli (ETEC) contribute to the establishment of diarrhea, with long-lasting detrimental effects. Botanicals and their single bioactive components represent sustainable well-recognized tools in animal nutrition thanks to their wide-ranging beneficial functions. The aim of this study was to investigate the in vitro mechanism of action of a blend of botanicals (BOT), composed of thymol, grapeseed extract, and capsicum oleoresin, in supporting intestinal cell health during inflammatory challenges and ETEC infections. To reach this, we performed inflammatory and ETEC challenges on Caco-2 cells treated with BOT, measuring epithelial integrity, cellular oxidative stress, bacterial translocation and adhesion, gene expression levels, and examining tight junction distribution. BOT protected enterocytes against acute inflammation: while the challenge reduced epithelial tightness by 40%, BOT significantly limited its drop to 30%, also allowing faster recovery rates. In the case of chronic inflammation, BOT systematically improved by an average of 25% the integrity of challenged cells (p < 0.05). Moreover, when cells were infected with ETEC, BOT maintained epithelial integrity at the same level as an effective antibiotic and significantly reduced bacterial translocation by 1 log average. The mode of action of BOT was strictly related to the modulation of the inflammatory response, protecting tight junctions' expression and structure. In addition, BOT influenced ETEC adhesion to intestinal cells (-4%, p < 0.05), also thanks to the reduction of enterocytes' susceptibility to pathogens. Finally, BOT effectively scavenged reactive oxygen species generated by inflammatory and H2O2 challenges, thus alleviating oxidative stress by 40% compared to challenge (p < 0.05). These results support the employment of BOT in piglets at weaning to help manage bacterial infections and relieve transient or prolonged stressful states thanks to the modulation of host-pathogen interaction and the fine-tuning activity on the inflammatory tone.

Effects of microencapsulated blend of organic acids and botanicals on growth performance, intestinal barrier function, inflammatory cytokines, and endocannabinoid system gene expression in broiler chickens.

With restricted usage of growth-promoting antibiotics, identifying alternative feed additives that both improve intestinal barrier function and reduce inflammation is the center to improve chickens' health. This study examined the effects of a microencapsulated feed additive containing citric acid, sorbic acids, thymol, and vanillin on intestinal barrier function and inflammation status. A total of 240 birds were assigned to either a commercial control diet or control diet supplemented with 500 g/MT of the microencapsulated additive product. Birds were raised by feeding a 2-phase diet (starter, d 1 to d 21; and grower, d 15 to d 42). Growth performance was recorded weekly. At d 21 and d 42, total gastrointestinal tract permeability was evaluated by FITC-dextran (FD4) oral gavage. Jejunum-specific barrier functions were evaluated by Ussing chamber. Intestinal gene expression of selected epithelial cell markers, tight junction (TJ) proteins, inflammatory cytokines, and endocannabinoid system (ECS) markers were determined by RT-PCR. Statistical analysis was performed using Student t test. Results showed significant improvement of feed efficiency in the birds supplemented with the blend of organic acids and botanicals. At d 21, both oral and jejunal FD4 permeability were lower in the supplemented group. Jejunal transepithelial resistance was higher in the supplemented birds. At d 21, expression of TJs mRNA (CLDN1 and ZO2) was both upregulated in the jejunum and ileum of supplemented birds, while CLDN2 was downregulated in cecum. Proliferating cell marker SOX9 was higher expressed in jejunum and ceca. Goblet cell marker (MUC2) was upregulated, while Paneth cell marker (LYZ) was downregulated in the ileum. Proinflammatory cytokine expressions of IL1B, TNFA, and IFNG were downregulated in jejunum, while anti-inflammatory IL10 expression was higher in jejunum, ileum, cecum, and cecal tonsil. The ECS markers expressions were upregulated in most intestinal regions. Together, these results demonstrated that the blend of organic acids and botanical supplementation reduced inflammation, improved the TJs expression and intestinal barrier function, and thus improved chicken feed efficiency. The activated ECS may play a role in reducing intestinal tissue inflammation.

Phenol-Rich Botanicals Modulate Oxidative Stress and Epithelial Integrity in Intestinal Epithelial Cells.

Botanicals are mainly known for their role as antimicrobials and anti-inflammatories. Thus, the dual purpose of the study was to verify the antioxidant potential of the tested botanicals and to evaluate their possible modulation of intestinal barrier integrity. As the effects of various phenol-rich extracts were screened, the human Caco-2 cell line was determined to be most suitable for use as the in vitro model for the intestinal epithelium. The tested botanicals, all approved as feed additives, are ginger essential oil, tea tree oil, grape seed extract, green tea extract, olive extract, chestnut extract, pomegranate extract, thyme essential oil, and capsicum oleoresin. The cells were treated with incremental doses of each botanical, followed by measurements of transepithelial electrical resistance (TEER), gene expression of tight junctions (TJs), and reactive oxygen species (ROS). The results showed how different phenol-rich botanicals could modulate barrier functions and oxidative stress in different ways. Interestingly, all the botanicals tested exerted an antioxidant potential by dropping the cytoplasmatic ROS, while the beneficial effect was exerted at different concentrations for each botanical. Our data support the role of plant extracts and essential oils in controlling gut barrier function and in reducing the negative effects of oxidative stress in intestinal epithelial cells, thereby supporting gut barrier functionality.

Cannabinoid and Cannabinoid-Related Receptors in the Myenteric Plexus of the Porcine Ileum.

An important piece of evidence has shown that molecules acting on cannabinoid receptors influence gastrointestinal motility and induce beneficial effects on gastrointestinal inflammation and visceral pain. The aim of this investigation was to immunohistochemically localize the distribution of canonical cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) and the cannabinoid-related receptors transient potential vanilloid receptor 1 (TRPV1), transient potential ankyrin receptor 1 (TRPA1), and serotonin receptor 5-HT1a (5-HT1aR) in the myenteric plexus (MP) of pig ileum. CB1R, TRPV1, TRPA1, and 5-HT1aR were expressed, with different intensities in the cytoplasm of MP neurons. For each receptor, the proportions of the immunoreactive neurons were evaluated using the anti-HuC/HuD antibody. These receptors were also localized on nerve fibers (CB1R, TRPA1), smooth muscle cells of tunica muscularis (CB1R, 5-HT1aR), and endothelial cells of blood vessels (TRPV1, TRPA1, 5-HT1aR). The nerve varicosities were also found to be immunoreactive for both TRPV1 and 5-HT1aR. No immunoreactivity was documented for CB2R. Cannabinoid and cannabinoid-related receptors herein investigated showed a wide distribution in the enteric neurons and nerve fibers of the pig MP. These results could provide an anatomical basis for additional research, supporting the therapeutic use of cannabinoid receptor agonists in relieving motility disorders in porcine enteropathies.

Nature-Identical Compounds and Organic Acids Ameliorate and Prevent the Damages Induced by an Inflammatory Challenge in Caco-2 Cell Culture.

Bioactive compounds, such as organic acids (OA) and nature-identical compounds (NIC), can exert a role in the protection of intestinal mucosa functionality due to their biological properties. The aim of this study was to understand the role of 2 OA (citric and sorbic acid) and 2 NIC (thymol and vanillin), alone or combined in a blend (OA + NIC), on intestinal barrier functionality, either during homeostatic condition or during an inflammatory challenge performed with pro-inflammatory cytokines and lipopolysaccharides (LPS). The study was performed on the human epithelial cell line Caco-2, a well-known model of the intestinal epithelial barrier. The results showed how OA and NIC alone can improve transepithelial electrical resistance (TEER) and mRNA levels of tight junction (TJ) components, but OA + NIC showed stronger efficacy compared to the single molecules. When an inflammatory challenge occurred, OA + NIC blend was able both to ameliorate, and prevent, damage caused by the pro-inflammatory stimulus, reducing or preventing the drop in TEER and improving the TJ mRNA expression. The data support the role of OA + NIC in modulating gut barrier functionality and reducing the negative effects of inflammation in intestinal epithelial cells, thereby supporting the gut barrier functionality.

Thymol modulates the endocannabinoid system and gut chemosensing of weaning pigs.

BACKGROUND: The recent identification of the endocannabinoid system in the gastrointestinal tract suggests a role in controlling intestinal inflammation. In addition, the gut chemosensing system has therapeutic applications in the treatment of gastrointestinal diseases and inflammation due to the presence of a large variety of receptors. The purposes of this study were to investigate the presence of markers of the endocannabinoid system and the chemosensing system in the pig gut and, second, to determine if thymol modulates these markers. One hundred sixty 28-day-old piglets were allocated into one of 5 treatment groups (n = 32 per treatment): T1 (control), T2 (25.5 mg thymol/kg feed), T3 (51 mg thymol/kg feed), T4 (153 mg thymol/kg feed), and T5 (510 mg thymol/kg feed). After 14 days of treatment, piglets were sacrificed (n = 8), and then duodenal and ileal mucosal scrapings were collected. Gene expression of cannabinoid receptors (CB1 and CB2), transient receptor potential vanilloid 1 (TRPV1), the olfactory receptor OR1G1, diacylglycerol lipases (DGL-α and DGL-ß), fatty acid amine hydrolase (FAAH), and cytokines was measured, and ELISAs of pro-inflammatory cytokines levels were performed. RESULTS: mRNAs encoding all markers tested were detected. In the duodenum and ileum, the CB1, CB2, TRPV1, and OR1G1 mRNAs were expressed at higher levels in the T4 and T5 groups compared to the control group. The level of the FAAH mRNA was increased in the ileum of the T4 group compared to the control. Regarding the immune response, the level of the tumor necrosis factor (TNF-α) mRNA was significantly increased in the duodenum of the T5 group, but this increase was not consistent with the protein level. CONCLUSIONS: These results indicate the presence of endocannabinoid system and gut chemosensing markers in the piglet gut mucosa. Moreover, thymol modulated the expression of the CB1, CB2, TRPV1, and OR1G1 mRNAs in the duodenum and ileum. It also modulated the mRNA levels of enzymes involved in the biosynthesis and degradation of endocannabinoid molecules. Based on these findings, the effects of thymol on promoting gut health are potentially mediated by the activation of these receptors.