Probiotic Bacteria Prevent Gut Permeability During Heat Stress Iryna Sorokulova, Ludmila Globa, Oleg Pustovyy, Vitaly Vodyanoy Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL INTRODUCTION Various types of stress have a major impact on gastrointestinal physiology, causing dysfunction and diseases. One of the mechanisms connecting stress and gastrointestinal diseases is stress-induced effects on mucosal barrier function. METHODS Ethics Statement - All animal procedures were approved by the Auburn University Institutional Animal Care and Use. The study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Bacillus probiotic strain - B. subtilis BSB3 with promising probiotic activity was used in this study. Animals - Adult male Sprague–Dawley rats (Harlan Laboratories, USA) weighing 250–300 g were used. The animals were housed two in a cage under specific pathogen free conditions and were acclimatized for 2 days at temperature (21 ± 1 o C), humidity (50 ± 5%) and lighting (12-h day/12-h night) with free access to water and standard food. Experimental design. Rats were treated by oral gavage with B. subtilis probiotic or PBS twice a day for two days. Half of the rats of each group were exposed to heat stress (45 o C, relative humidity 55% for 25 min) while the remaining rats were placed at identical conditions but at 25 o C (Figure 1). Histological analysis –Small intestine samples were removed from each rat, fixed in Bouin’s Fixative, embedded in paraffin, sectioned at 6 micrometers, slide mounted, hematoxylin and eosin (H&E) stained, and cover-slipped. Intestinal villi height and total mucosal thickness were measured for each sample using a high resolution microscope system. Western Blotting - Intestinal tissues were homogenized in a lysis buffer containing 1% Triton X-100, 100 mM NaCl, 10 mM HEPES pH 7.6, 2 mM EDTA with protease and phosphatase inhibitors. Samples were centrifuged at 15,000xg for 30 min and supernatants were collected. A protein assay (Bio-Rad) were conducted to determine the protein concentration for each sample. Equal amount of proteins (50 µg) were separated by SDS–PAGE (10%) and transferred to nitrocellulose membranes. Membranes were blocked for 1 h in 5% non- fat dry milk in TBS/ 0.1% Tween-20 and were incubated overnight at 4 o C with primary antibodies against actin, claudin, occludin, and ZO-1. Membranes were washed with TBS/0.1% Tween-20 three times and were incubated with secondary antibody for one hour, then washed with TBS/0.1% Tween-20 three times. Blots were developed using a Kodak XOMAT 1000A film processor (Rochester, NY). The procedure were repeated at least four times for each protein. Bands were standardized to the density of β-actin and were represented as a OBJECTIVE The main aim of this study was to analyze the effect of probiotic bacteria on composition of tight junction (TJ) proteins, which act as a primary barrier to the diffusion through the intracellular space. ratio of each protein to β-actin. Statistics - All results were presented as mean and standard deviation. Statistical calculations and graph plotting were carried out using Microcal™ Origin version 6.0 (Northhampton, MA) and 2010 Microsoft Excel RESULTS Histology. Morphometric study of intestine showed that heat stress significantly inhibited villi height and total mucosal thickness in rats (Figures 2,3). Oral administration of BSB3 strain before stress protected intestine from the harmful effect of heat. Proteins expression. Pre-treatment of rats with probiotic resulted in significant increase of claudin expression in heat-stressed animals (Figure 4). ZO-1 expression was also higher in stressed animals, pretreated with probiotic (Figure 5). We found that expression of claudin and zonula occludens (ZO-1) was reduced in heat-stressed animals, pretreated with PBS. In group of rats received probiotic before exposure to heat stress, the level of tight junction proteins was significantly higher. Exposure of rats to heat resulted in increase of occludin and HSP 27 (Figures 6,7). Probiotic pretreatment normalized expression of these proteins. CONCLUSION: We can speculate that regulation of TJ proteins in rats exposed to heat stress is one of the mechanisms of probiotic action on animal protection against stress-related adverse effects. ACKNOWLEDGEMENT: This work was supported by Auburn University Funds # Figure 2. Histological Images of intestinal mucosa Figure 4. Claudin expression in the intestine of rats Figure 5. ZO-1 expression in the intestine of rats Figure 6. Expression of occludin in the intestine of rats Figure 7. HSP 27 expression in the intestine of rats Figure 3. Villi height in the intestine of rats from the different groups