Intestinal Epithelial Tight Junction Proteins Research Articles

Age-Associated Remodeling of the Intestinal Epithelial Barrier

Disorders of the gastrointestinal tract are common in the elderly people; however, the precise trait(s) of aging that contribute to the vulnerability of the gastrointestinal tract are poorly understood. Recent evidence suggests that patients with gastrointestinal disorders have increased intestinal permeability. Here, we address the hypothesis that disruption of the intestinal barrier is associated with aging. Our results demonstrated that permeability was significantly higher in colonic biopsies collected from old baboons compared with young baboons. Additionally, colonic tissue from the older animals had decreased zonula occluden-1, occludin, and junctional adhesion molecule-A tight junction protein expression and increased claudin-2 expression. Upregulation of miR-29a and inflammatory cytokines IFN-γ, IL-6, and IL-1β was also found in colonic biopsies from old baboons relative to young baboons. These results show for the first time that a pivotal contributing factor to geriatric vulnerability to gastrointestinal dysfunction may be increased colonic permeability via age-associated remodeling of intestinal epithelial tight junction proteins.

Probiotics Can Alleviate Cardiopulmonary Bypass-Induced Intestinal Mucosa Damage in Rats

Cardiopulmonary bypass (CPB) is commonly applied to support circulation during heart surgery but frequently causes adverse effects. The purpose of this study was to examine the potential of probiotics to improve small intestinal mucosa barrier function after CPB. Twenty-four adult male SD rats were randomly divided into sham-operated (S), CPB-operated (CPB), and probiotic-fed (Y) groups. Diamine oxidase (DAO) activity and concentrations of D-lactic acid, endotoxin, TNFα, and IL-6 were measured in portal vein blood. IgA concentrations were determined in plasma and the small intestine. Vena cava blood and tissue samples were used to monitor bacterial growth. Intestinal epithelial ultrastructure was analyzed by transmission electron microscopy (TEM). Occludin and ZO-1 expression levels in the intestinal epithelium were detected by western blotting and immunohistochemistry, respectively. D-lactic acid, endotoxin, TNFα and IL-6 levels, DAO activity, and bacterial translocation rate were increased (P < 0.05) in CPB and Y compared to the S group. The above indices were relatively lower (P < 0.05) in Y than in CPB. Plasma and small intestinal IgA levels were significantly lower (P < 0.05) in CPB, while in Y they were significantly increased (P < 0.05) but lower than in S (P < 0.05). These results were confirmed by TEM. Consistently, occludin and ZO-1 expression levels were significantly higher in Y than in CPB (P < 0.05) but still lower compared to S (P < 0.05). Pre-administration of probiotics can improve, to some extent, intestinal barrier function after CPB in rats, and this effect is likely related to inhibition of the CPB-induced inflammatory response, improvement in local intestinal immune function, and increased expression of intestinal epithelial tight junction proteins.

Effects of rhein on intestinal epithelial tight junction in IgA nephropathy

To investigate the effects of rhein on intestinal epithelial tight junction proteins in rats with IgA nephropathy (IgAN). Twenty-eight female Sprague-Dawley rats were randomly divided into four groups (7 per group): Control, IgAN, Rhein-treated, and Rhein-prevented. Bovine serum albumin, lipopolysaccharide and CCl4 were used to establish the rat model of IgA nephropathy. The Rhein-treated group was given rhein from week 7 until the rats were sacrificed. The Rhein-prevented group was given rhein from week 1. Animals were sacrificed at the end of week 10. We observed the changes in the intestinal epithelial tight junctions using transmission electron microscopy, and expression of intestinal epithelial tight junction proteins zona occludens protein (ZO)-1 and occludin by immunofluorescence using laser confocal microscopy. Changes in mRNA and protein expression of ZO-1 and occludin were measured by reverse transcriptase polymerase chain reaction and Western blotting. The ratio of urinary lactulose/mannitol was measured by high performance liquid chromatography (HPLC) for assessing the intestinal permeability. In the control group, the tight junctions lied between epithelial cells on the top of the outer side of the cell membrane, and appeared in dense dotted crystal structures, the neighboring cells were binded tightly with no significant gap, and the tight junction protein ZO-1 and occludin were evenly distributed in the intestinal epithelial cells at the top of the junction. Compared with the control group, in the IgAN group, the structure of the tight junction became obscured and the dotted crystal structures had disappeared; the fluorescence of ZO-1 and occludin was uneven and weaker (5.37 ± 1.27 vs 10.03 ± 1.96, P < 0.01; 4.23 ± 0.85 vs 12.35 ± 4.17, P < 0.01); the mRNA expression of ZO-1 and occludin decreased (0.42 ± 0.19 vs 0.92 ± 0.24, P < 0.01; 0.40 ± 0.15 vs 0.97 ± 0.25, P < 0.01); protein expression of ZO-1 and occludin was decreased (0.85 ± 0.12 vs 1.98 ± 0.43, P < 0.01; 0.72 ± 0.15 vs 1.38 ± 0.31, P < 0.01); and the ratio of urinary lactulose/mannitol increased (3.55 ± 0.68 vs 2.72 ± 0.21, P < 0.01). In the Rhein-prevented and Rhein-treated groups, compared with the IgAN group, the intestinal epithelial tight junctions were repaired; fluorescence of ZO-1 and occludin was stronger (11.16 ± 3.52 and 8.81 ± 2.30 vs 5.37 ± 1.27, P < 0.01; 10.97 ± 3.40 and 9.46 ± 2.40 vs 4.23 ± 0.85, P < 0.01); mRNA of ZO-1 and occludin increased (0.81 ± 0.17 and 0.64 ± 0.16 vs 0.42 ± 0.19, P < 0.01; 0.82 ± 0.22 and 0.76 ± 0.31 vs 0.40 ± 0.15, P < 0.01); protein expression of ZO-1 and occludin was increased (2.07 ± 0.41 and 1.57 ± 0.23 vs 0.85 ± 0.12, P < 0.01; 1.34 ± 0.21 and 1.15 ± 0.17 vs 0.72 ± 0.15, P < 0.01); and the ratio of urinary lactulose/mannitol decreased (2.83 ± 0.43 and 2.87 ± 0.18 vs 3.55 ± 0.68, P < 0.01). Rhein can enhance the expression of ZO-1 and occludin, repair damaged tight junctions, and protect the intestinal barrier.

Effects of Soybean Agglutinin on Intestinal Barrier Permeability and Tight Junction Protein Expression in Weaned Piglets

This study was developed to provide further information on the intestinal barrier permeability and the tight junction protein expression in weaned piglets fed with different levels of soybean agglutinin (SBA). Twenty-five weaned crossbred barrows (Duroc × Landrace × Yorkshire) were selected and randomly allotted to five groups, each group with five replicates. The piglets in the control group were not fed with leguminous products. 0.05, 0.1, 0.15 and 0.2% SBA was added to the control diet to form four experimental diets, respectively. After the experimental period of 7 days (for each group), all the piglets were anesthetized with excess procaine and slaughtered. The d-lactic acid in plasma and the Ileal mucosa diamine oxidase (DAO) was analyzed to observe the change in the intestinal permeability. The tight junction proteins occludin and ZO-1 in the jejunum tissue distribution and relative expression were detected by immunohistochemistry and Western Blot. The results illustrated that a high dose of SBA (0.1–0.2%) could increase the intestinal permeability and reduce piglet intestinal epithelial tight junction protein occludin or ZO-1 expression, while low dose of SBA (0.05% of total diet) had no significant affects. The contents of DAO, d-lactic acid, occludin or ZO-1, had a linear relationship with the SBA levels (0–0.2%) in diets. The high dose SBA (0.1–0.2%) could increase the intestinal permeability and reduce piglet intestinal epithelial tight junction protein occludin or ZO-1 expression, while low dose of SBA (0.05% of total diet) had no affects.

Enterohemorrhagic E. coli alters murine intestinal epithelial tight junction protein expression and barrier function in a Shiga toxin independent manner

Shiga toxin (Stx) is implicated in the development of hemorrhagic colitis and hemolytic-uremic syndrome, but early symptoms of enterohemorrhagic Escherichia coli (EHEC) infection such as nonbloody diarrhea may be Stx independent. In this study, we defined the effects of EHEC, in the absence of Stx, on the intestinal epithelium using a murine model. EHEC colonization of intestines from two groups of antibiotic-free and streptomycin-treated C57Bl/6J mice were characterized and compared. EHEC colonized the cecum and colon more efficiently than the ileum in both groups; however, greater amounts of tissue-associated EHEC were detected in streptomycin-pretreated mice. Imaging of intestinal tissues of mice infected with bioluminescent EHEC further confirmed tight association of the bacteria with the cecum and colon. Greater numbers of EHEC were also cultured from stool samples obtained from streptomycin-pretreated mice, as compared with those that received no antibiotics. Transmission electron microscopy shows that EHEC infection leads to microvillous effacement of mouse colonocytes. Hematoxylin and eosin staining of the colonic tissues of infected mice revealed a slight increase in the number of lamina propria polymorphonuclear leukocytes. Transmucosal electrical resistance, a measure of epithelial barrier function, was reduced in the colonic tissues of infected animals. Increased mucosal permeability to 4- kDa FITC-dextran was also observed in the colonic tissues of infected mice. Immunofluorescence microscopy showed that EHEC infection resulted in redistribution of the tight junction (TJ) proteins occludin and claudin-3 and increased the expression of claudin-2, whereas ZO-1 localization remained unaltered. Quantitative real-time PCR showed that EHEC altered mRNA transcription of OCLN, CLDN2, and CLDN3. Most notably, claudin-2 expression was significantly increased and correlated with increased intestinal permeability. Our data indicate that C57Bl/6J mice serve as an in vivo model to study the physiological effects of EHEC infection on the intestinal epithelium and suggest that altered transcription of TJ proteins has a role in the increase in intestinal permeability.

Claudin-1 and claudin-2 expression is elevated in inflammatory bowel disease and may contribute to early neoplastic transformation

Patients with inflammatory bowel disease (IBD) are at increased risk of developing colorectal adenocarcinoma. The factors that result in IBD-associated carcinogenesis are not understood. We hypothesized that altered expression of intestinal epithelial tight junction proteins might contribute to neoplastic progression. Semiquantitative immunohistochemical staining of human biopsies was used to assess expression of the tight junction proteins claudin-1, claudin-2, claudin-4, and occludin in IBD, IBD-associated dysplasia, acute, self-limited colitis (ASLC), and sporadic adenomas. Claudin-1 and claudin-2 expression was elevated in active IBD, adenomas, and IBD-associated dysplasia, but not ASLC. In contrast, claudin-4 expression was elevated in both active IBD and ASLC. Occludin expression was similar to control in all cases. Importantly, in IBD, claudin-1 and claudin-2 expression correlated positively with inflammatory activity. To investigate mechanisms underlying altered claudin expression, β-catenin activation was assessed as nuclear localization. Like claudin-1 and claudin-2, β-catenin was markedly activated in IBD, sporadic dysplasia, and IBD-associated dysplasia, but was only slightly activated in ASLC. Taken together, these data suggest that β-catenin transcriptional activity is elevated in chronic injury and that this may contribute to increased claudin-1 and claudin-2 expression. We speculate that increased claudin-1 and claudin-2 expression may be involved at early stages of transformation in IBD-associated neoplasia.

Secreted bioactive factors fromBifidobacterium infantisenhance epithelial cell barrier function

Live probiotic bacteria are effective in reducing gut permeability and inflammation. We have previously shown that probiotics release peptide bioactive factors that modulate epithelial resistance in vitro. The objectives of this study were to determine the impact of factors released from Bifidobacteria infantis on intestinal epithelial cell permeability and tight junction proteins and to assess whether these factors retain their bioactivity when administered to IL-10-deficient mice. B. infantis conditioned medium (BiCM) was applied to T84 human epithelial cells in the presence and absence of TNF-alpha and IFN-gamma. Transepithelial resistance (TER), tight junction proteins [claudins 1, 2, 3, and 4, zonula occludens (ZO)-1, and occludin] and MAP kinase activity (p38 and ERK) were examined. Acute effects of BiCM on intestinal permeability were assessed in colons from IL-10-deficient mice in Ussing chambers. A separate group of IL-1-deficient mice was treated with BiCM for 4 wk and then assessed for intestinal histological injury, cytokine levels, epithelial permeability, and immune response to bacterial antigens. In T84 cells, BiCM increased TER, decreased claudin-2, and increased ZO-1 and occludin expression. This was associated with enhanced levels of phospho-ERK and decreased levels of phospho-p38. BiCM prevented TNF-alpha- and IFN-gamma-induced drops in TER and rearrangement of tight junction proteins. Inhibition of ERK prevented the BiCM-induced increase in TER and attenuated the protection from TNF-alpha and IFN-gamma. Oral BiCM administration acutely reduced colonic permeability in mice whereas long-term BiCM treatment in IL-10-deficient mice attenuated inflammation, normalized colonic permeability, and decreased colonic and splenic IFN-gamma secretion. In conclusion, peptide bioactive factors from B. infantis retain their biological activity in vivo and are effective in normalizing gut permeability and improving disease in an animal model of colitis. The effects of BiCM are mediated in part by changes in MAP kinases and tight junction proteins.

Down-regulated expression of intestinal epithelial tight junction protein occludin in mice with fulminant hepatic failure

Down-regulated expression of intestinal epithelial tight junction protein occludin in mice with fulminant hepatic failure