Influence of Nutritional Strategies on Performance, Gut Barrier Function and Microbiota Composition in Weaned Piglets.

1046 A Diet Rich in Arabinoxylan and Resistant Starch Increases Colonic Butyrate Concentration and Expression of Tight Junction Protein Occludin and Decreases Fecal Calprotectin in Subjects With Metabolic Syndrome

The present study aimed to investigate the individual and combined effects of food and biliopancreatic secretions on intestinal morphology, tight junction (TJ) protein expression and microbial composition in a rat model. A total of 30 male Sprague-Dawley rats were randomly allocated into three groups: Control, biliopancreatic diversion and jejunal bypass. Jejunal segments were surgically manipulated to selectively exclude biliopancreatic secretions, food or both. Corresponding control segments were also collected. Intestinal samples were analysed for villus height/crypt depth ratios and intraepithelial lymphocyte (IEL) counts using H&E staining, as well as TJ protein expression (occludin, claudin-1, zonula occludens-3) measured using immunohistochemistry. In addition, portal venous blood samples were obtained to assess serum lipopolysaccharide and citrulline levels using ELISA. Furthermore, quantitative bacterial cultures were performed on jejunal tissues. The villus height/crypt depth ratio was significantly reduced in segments deprived of both bile and food (P=0.007), and IEL counts were significantly elevated in food- and bile-deficient segments (P<0.001). Moreover, claudin-1 expression was significantly lower in food-deficient segments (surgically created intestinal segment that is bypassed from luminal nutrient flow)(BPS1 and JPS2) (P=0.035), whereas occludin expression was increased in the presence of food (P=0.016). In conclusion both food and biliopancreatic secretions are essential in maintaining intestinal epithelial morphology and TJ integrity. Food deprivation induced more pronounced changes in intestinal integrity than the absence of biliopancreatic secretions. The present findings highlight the synergistic role of luminal nutrition and digestive secretions in preserving gut barrier function.

Claudin-4 Levels Are Associated with Intact Alveolar Fluid Clearance in Human Lungs

IntroductionThis study investigates the effects of α-GML on intestinal epithelial tight junction (TJ) protein expression and its molecular mechanisms. Recognizing the critical role of TJ proteins in intestinal barrier function and the potential of α-GML to enhance this barrier, we employ the IPEC-J2 cell model. Our aim is to validate the regulatory impact ofα-GML on TJ protein expression and elucidate the underlying signaling pathways, thereby offering new strategies for intestinal health maintenance.MethodsUtilized Data-Independent Acquisition (DIA) analysis to identify optimal targets of α-GML in modulating Tight Junction (TJ) protein expression. Treated cells with specific inhibitors of PKC and MAPK to assess their role in TJ regulation by α-GML. Co-treated cells with the MAPK inhibitor SCH772984 and α-GML to study the effects on p-ATF-2 expression. Evaluated the effects of SCH772984 and ATF-2 overexpression on the protein expression levels of phosphorylated ATF-2, ZO-1, and OCLN.ResultsRevealed that α-GML’s modulation of TJ proteins might involve the PKC/MAPK signaling pathway, leading to ATF-2 phosphorylation. Both PKC and MAPK inhibitors reduced TJ protein expression (p < 0.05, p < 0.01 or p < 0.001), indicating their involvement in α-GML’s regulation. SCH772984 counteracted α-GML-induced upregulation of p-ATF-2 (p < 0.05), suggesting MAPK’s role in this process. Identified potential ATF-2 binding sites on ZO-1 and OCLN promoters. ATF-2 significantly enhanced ZO-1 promoter activity (p < 0.001). SCH772984 reduced phosphorylated ATF-2, ZO-1, and OCLN levels (p < 0.05 or p < 0.01), while ATF-2 overexpression rescued this decrease (p < 0.05 or p < 0.01), confirming ATF-2’s role in TJ protein upregulation via the MAPK pathway.DiscussionOur study indicated that α-GML enhanced the expression of TJ proteins through the PKC/MAPK/ATF-2 pathway, thereby enhancing the barrier function of intestinal epithelial cells.

Brain-derived neurotrophic factor (BDNF) may play a vital role in the homeostatic regulation of intestinal barrier integrity. We aimed to investigate the physiological role of BDNF in maintaining the intestinal epithelial barrier using postinflammatory irritable bowel syndrome (PI-IBS) mice and explore the underlying molecular mechanisms using intestinal epithelial cells in vitro. Postinflammatory-IBS mice were induced by intrarectal administration of trinitrobenzene sulfonic acid and allowed to recover for 28days. Frequency of defecation, fecal water content, colonic epithelial integrity and expressions of BDNF and tight junction (TJ) proteins (occludin, ZO-1, claudin-1, claudin-2) of the PI-IBS mice were investigated. Based on the results of animal studies, we further performed RT-PCR and Western blots to assess how BDNF stimulation and BDNF knockdown impacted TJ proteins in the ht-29 intestinal epithelial cells. Water content of stools was significantly increased in the PI-IBS mice compared with controls. Colonic mucosa from the PI-IBS mice displayed epithelial barrier defects and exhibited increased protein expressions of BDNF and claudin-2 and decreased protein expressions of occludin, ZO-1 and claudin-1. Furthermore, a siRNA against BDNF in the ht-29 cells could effectively suppress BDNF gene and protein expressions, and subsequently reduce TJ gene and protein levels. When the ht-29 cells were incubated with different doses of exogenous BDNF, significant increases of occludin, ZO-1 and claudin-1 and decreases of claudin-2 protein were observed. BDNF may play a role in regulating intestinal epithelial barrier via affecting the expression of TJ proteins.

Gastrodia elata and parishin ameliorate aging induced ‘leaky gut’ in mice: Correlation with gut microbiota

Probiotic Bacteria Induce Maturation of Intestinal Claudin 3 Expression and Barrier Function

Objective To investigate the distribution and expression of tight junction proteins (including elaudin 1, occludin,ZO-1 and JAM-1) in mucosa of rats with reflux esophagitis (RE), and its underline mechanism in pathogenesis of RE. Methods Two hundred and twenty 8-week-old male Wistar rats were divided into sham operation control group (n=10), acid reflux group (n=70), alkaline reflux group (n=70) and mixed reflux group (n=70). The rats were sacrificed at day 3, 6, 9 and 14 after operation. The successful rate of modeling was assessed by evidence of inflammation in middle and low esophagus. Transmission electron microscopy (TEM) was used to observe the morphological changes of tight junction in esophageal epithelium. The mRNA and protein expressions of tight junction proteins were detected by Western blotting and RT-PCR, respectively. And interleukin (IL)-6 expression was measured by immunohistochemistry. Results At day 14 after the procedure, RE model was established in all executed rats. Successful rate of 100% was achieved. The microscopic observation showed that mucosa was damaged and thickened as the disease progressed. With TEM observation, widened intercellular space was noticed with fewer desmosomes. Elevated expressions of IL-6 and tight junction proteins were found in three model groups compared with control group. Whereas the expression of tight junction proteins in individual cells was gradually decreased with continuing hyperplasia in the basal layer. The mRNA and protein expressions of IL-6 and tight junction proteins were increased gradually as disease progressed. Conclusions The highly expression of tight junction proteins, which involves in the mechanism of RE by playing the role of positive regulation and synergism, may be early molecular event in development of RE. And IL-6 is an inflammatory factor in this process. Key words: Esophagitis; Tight junctions; Interleukin-6

Traumatic brain injury (TBI) can lead to several physiologic complications including gastrointestinal dysfunction. Specifically, TBI can induce an increase in intestinal permeability, which may lead to bacterial translocation, sepsis, and eventually multi-system organ failure. However, the exact mechanism of increased intestinal permeability following TBI is unknown. We hypothesized that expression of tight junction protein ZO-1 and occludin, responsible for intestinal architectural and functional integrity, will decrease following TBI and increase intestinal permeability. BALB/c mice underwent a weight drop TBI model following anesthesia. Brain injury was confirmed by a neurologic assessment and gross brain pathology. Six hours following injury, FITC-dextran (25 mg 4.4 kDa FITC-dextran) was injected into the intact lumen of the isolated ileum. Intestinal permeability was measured in plasma 30 min following injection, by using spectrophotometry to determine plasma FITC-dextran concentrations. Whole ileum extracts were used to measure expression of tight junction proteins ZO-1 and occludin by Western blot. TBI caused a significant increase in intestinal permeability (110.0 microg/mL +/-22.2) compared to sham animals (29.4 microg/mL +/- 9.7) 6 h after injury (p = 0.016). Expression of ZO-1 was decreased by 49% relative to sham animals (p < 0.02), whereas expression of occludin was decreased by 73% relative to sham animals (p < 0.001). An increase in intestinal permeability corresponds with decreased expression of tight junction proteins ZO-1 and occludin following TBI. Expression of intestinal tight junction proteins may be an important factor in gastrointestinal dysfunction following brain injury.

The aim of this experiment was to investigate the effects of Ageratina adenophora on the expression of epithelium tight junction proteins and inflammatory factors in the rumen of goats. Twelve goats were randomly divided into three groups. The first group was the blank control group (n = 3, C) which was fed normal diet. The second group was fistulas control group (n = 3, RFC), which was fitted with rumen fistulas, and fed normal diet. The third group was the A. adenophora test group (n = 6, AA), which was fitted with rumen fistulas and fed a mixture of 60% of normal diet and 40% of A. adenophora grass powder. The feeding experiment lasted for 90d, after which all goats were sacrificed and samples were collected from the rumen dorsal sac and ventral sac. The relative expression of mRNA of inflammatory factors in the rumen epithelium (tumor necrosis factor alpha [TNF-α], interferon gamma [IFN-γ], interleukin 1 beta [IL-1β], IL-2, IL-4, IL-6, and IL-10) and tight junction protein genes (occludin, claudin-1, and ZO-1) was measured by quantitative real-time fluorescence PCR. Expression of tight junction proteins in the rumen epithelium was measured by Western blot. A correlation was established between the expression of inflammatory factors and tight junction protein genes using Graph Pad Prism. The results showed that A. adenophora caused a significant increase in the mRNA expression levels of TNF-α, IFN-γ, IL-1β, IL-2, IL-6, and IL-10 in the rumen epithelial (P < 0.05 or P < 0.01). The expression of tight junction proteins at both gene and protein levels was significantly decreased (P < 0.05 or P < 0.01). Furthermore, the correlation analysis revealed that the changes in tight junction protein expression in the test group were closely related to the upregulation of the expression of inflammatory factors TNF-α and IFN-γ in rumen epithelial cells. In conclusion, the expression of inflammatory factors was increased and the expression of tight junction proteins was decreased in goats after feeding on A. adenophora, which caused some damage to the rumen epithelium.

The Pivotal Mediating Role of Adenosine Monophosphate-Activated Protein Kinase (AMPK) in Liver Tight Junctions and Liver Regeneration of a Partial-Hepatectomy Mouse Model

Background: In inflammatory bowel diseases (IBD) intestinal permeability is increased. The effect of VSL#3, a mixture of 8 probiotic bacteria, on the epithelial barrier was studied in a murine model of colitis. Material and Methods: Three groups, each with 14 balb/c mice, were studied: healthy controls, acute colitis + placebo, and acute colitis + 15 mg VSL#3 daily (induction of colitis with Dextrane-Sodium-Sulphate (DSS) 3.5 % in drinking water for 7 days; placebo/VSL#3 via gastric tube once daily). The inflammation was assessed by a daily disease activity index (DAI) and a histological inflammation score. Colonic permeability to Evans Blue in vivo was measured (extinction/gram colonic tissue). Epithelial apoptotic ratio was assessed by immunofluorescence for cleaved caspase 3. The expression of epithelial tight junction and adherens junction proteins was studied by immunofluorescence and Western blot. Results: VSL#3 reduced the DAI on days 5–7 vs. placebo (day 7: healthy controls 0 ± 0, DSS + placebo 8.4 ± 0.4, DSS + VSL#3 5.4 ± 0.9; p = 0.012), and histological inflammation scores (healthy controls 0.9 ± 0.3, DSS + placebo 14.6 ± 2.5, DSS + VSL#3 8.4 ± 1.8; p = 0.011). In DSS colitis + placebo, colonic permeability was increased compared to controls (5.7 ± 1.7 vs. 0.4 ± 0.1; p < 0.001), whereas this increase was prevented by VSL#3 (0.3 ± 0.1; p = 0.003 vs. DSS + placebo, N.S. vs. healthy controls). VSL#3 prevented the DSS induced increase of epithelial apoptotic ratio (healthy controls 1.58 ± 0.01/1000 cells, DSS + placebo 13.35 ± 1.29/1000 cells, DSS + VSL#3 1.67 ± 0.10/1000 cells; p = 0.012 vs. DSS + placebo, N.S. vs. controls). In DSS colitis + placebo, immunofluorescence revealed a reduced apical expression of tight junction proteins occludin, ZO-1, claudin-1, and -5, whereas VSL#3 therapy preserved the physiological expression pattern. No changes were observed for claudin-3, and for adherens junction proteins β-catenin and E-cadherin in the three groups. Western blots showed a quantitatively reduced expression of occludin, claudin-1, -2 and -4 for DSS + placebo. VSL#3 prevented this decrease in expression. Conclusion: The probiotic mixture VSL#3 preserves the epithelial barrier by preventing the redistribution and the decrease in expression of tight junction proteins, and by reducing the epithelial apoptotic ratio. This may lead to the anti-inflammatory effect observed in this IBD model.

GLP-2 enhances barrier formation and attenuates TNFα-induced changes in a Caco-2 cell model of the intestinal barrier

Background High prevalence of vitamin D deficiency has been found among Crohn’s disease (CD) patients. Vitamin D probably participates in the pathogenesis of CD, but this idea remains controversial. This study was to investigate the levels of vitamin D in CD patients and analyze the relationship between vitamin D and intestinal inflammation. Methods Vitamin D levels were measured by chemiluminescence immunoassay in 198 CD patients (96 in active, 102 in remission) and 100 healthy controls. The correlation between vitamin D levels and clinical parameters was analysed. The expression of intestinal tight junction (TJ) proteins in CD patients was measured by immunofluorescence staining. Treg and Th17 percentages in the peripheral blood were determined by flow cytometry. Results CD patients exhibited significantly lower 25(OH)D levels than healthy controls, especially in active CD patients. Serum 25(OH)D levels in CD patients were negatively correlated with the CD activity index (CDAI), the simple endoscopic score for CD (SES-CD), and inflammatory markers, including erythrocyte sedimentation rate (ESR), platelet (PLT) count and faecal calprotectin (FC) levels. Moreover, in patients with vitamin D deficiency, the expression of TJ proteins (Occludin, claudin-1, ZO-1 and JAM-1) in the intestinal mucosa was reduced, and Treg cells in the peripheral blood were decreased, while Th17 cells were increased compared to those with vitamin D sufficiency and controls. Conclusions Vitamin D deficiency in CD patients is common. Vitamin D is associated with disease activity and intestinal inflammation, which may affect the Treg/Th17 balance and the expression of gut TJ proteins.

A high-grain diet alters the omasal epithelial structure and expression of tight junction proteins in a goat model