1. Disruption of the Gut Barrier Integrity Prevented by a Yeast Fermentate Product
Henri Alexandre Giblot Ducray1, Ludmila Globa1, Oleg Pustovyy1, Stuart Reeves2, Larry Robinson2, Vitaly Vodyanoy1, Iryna Sorokulova1 1Department of Anatomy, Physiology and Pharmacology, 109 Greene Hall, Auburn University, Auburn, AL 36849, USA 2Embria Health Sciences, 2105 SE Creekview Drive, Ankeny, IA 50021, USA
Introduction
The integrity of the gut barrier plays an essential role in health and disease. It prevents the translocation of the luminal content, particularly toxic components of Gram-negative bacteria, lipopolysaccharides (LPS), into circulation. Increased intestinal permeability accompanies various pathological conditions, such as inflammatory bowel diseases, irritable bowel syndrome, celiac disease, food allergy, obesity and other metabolic diseases. High-fat diet, modification of gut microbiota, and mucus layer alterations can impact the integrity of the gut barrier. Heat stress was shown to disrupt the intestinal barrier function and change the expression of tight junction (TJ) proteins, responsible for keeping the integrity of intestinal epithelial cells (Ulluwishewa et al, 2011). The integrity of the gut barrier is a key for intestinal homeostasis and overall for the health status of the host. It was shown that microbiota and their metabolites can regulate the gut barrier function. Prebiotics have been proposed as a promising approach to normalize microbiota, and, as a result, to improve gut barrier integrity. The main aim of this study was to evaluate the protective effect of a yeast fermentate product EpiCor (EH) on the integrity of the intestinal barrier during heat stress.
Methods
Experimental Design. Male rats were treated by oral gavage with EH prebiotic or PBS once a day for 14 days. On the 15th day, half of the rats of each group were exposed to heat stress conditions (45o C, relative humidity 55% for 25 min), while the other half were placed in identical conditions but at room temperature.
Histological analysis. Small intestine samples were removed from each rat, fixed in Bouin’s Fixative, embedded in paraffin, sectioned at 6 micrometers, slide mounted, stained, and cover- slipped. Phloxine-tartrazine and alcian blue techniques were used to analyze Paneth and goblet cells respectively. The amount of Paneth cells was counted for each sample using a high resolution microscope system. Goblet cells were analyzed as previously described (Trevizan et al, 2016).
Serum concentration of lipopolysaccharides (LPS) was analyzed by the Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific, Rockford, IL) using the Limulus Amebocyte Lysate (LAL) assay.
Western Blots. Small intestine samples were removed from each rat, homogenized, underwent electrophoresis, transferred to a nitrocellulose membrane, incubated with primary and secondary antibodies. Membranes were imaged by LiCor Odyssey scanner, and blots were analyzed by Image Studio 2.0 analytical software (LiCor, Lincoln, NE). Bands were standardized to the density of beta-actin.
Figure 1
Results
Conclusion
Supplementation of rats with prebiotics was effective in preventing the adverse effects of heat stress:
Morphological changes in the small intestine such as the reduction of Paneth and goblet cells count
Increased release of lipopolysaccharides into the blood stream
Decreased expression of tight junction proteins (occludin, claudin, ZO-1, and JAM-A )
Our results demonstrated efficacy of EH treatment in protecting the integrity of the intestinal barrier in the heat stress model. We speculate that EH could be used for improvement of the gut barrier function in conditions, accompanied by this abnormality, such as metabolic and other disease states.
Acknowledgements
This work was supported by Auburn University Funds # and by Embria Health Sciences LLC.
Control PBS
Control EH
Stress PBS
Stress EH
Control PBS
Control EH
Stress PBS
Stress EH
Figure 2: Concentration of serum LPS. In heat stressed rats pre-treated with PBS, there was a significant increase of serum LPS in comparison with control animals. Administration of prebiotic EH before exposure of rats to heat stress conditions prevented elevated release of LPS into circulation.
Figure 8: Tight junction proteins expression. CE- control rats were treated with EH and placed at 25 oC; SE- rats were pre-treated with EH and exposed to 45 oC; CP – control rats were treated with PBS and placed at 25 oC; SP – rats were pre-treated with PBS and exposed to 45 oC. Heat stress causes a decrease in expression of TJ proteins in rats pre-treated with PBS. Pre-treatment of animals with EH before heat stress significantly increase the expression of these proteins.
Figure 3: Paneth cells count. The number of Paneth cells was significantly lower in rats pre-treated with PBS before exposure to heat stress. Prebiotic supplementation before heat stress, prevented loss of Paneth cells in intestine of rats. Arrows indicate Paneth cells in crypts.
Figure 4: Goblet cells count. The number of goblet cells was significantly decreased in the heat-stressed rats pre-treated with PBS. Goblet cell count in intestines of heat stressed rats pre-treated with prebiotic was similar to the control groups of rats.
References
Ulluwishewa D, et al. (2011). Regulation of Tight Junction Permeability by Intestinal Bacteria and Dietary Components. Journal of Nutrition, 141(5):
Trevizan AR, et al. (2016). Experimental Parasitology, 165:22-29.