Hypoxia impairs urothelial barrier function by inhibiting the expression of tight junction proteins in SV-HUC-1 cells.

Hypoxia plays an important role in the pathological process of bladder outlet obstruction. Previous research has mostly focused on the dysfunction of bladder smooth muscle cells, which are directly related to bladder contraction. This study delves into the barrier function changes of the urothelial cells under exposure to hypoxia. Results indicated that after a 5-day culture, SV-HUC-1 formed a monolayer and/or bilayer of cell sheets, with tight junction formation, but no asymmetrical unit membrane was observed. qPCR and western blotting revealed the expression of TJ-associated proteins (occludin, claudin1 and ZO-1) was significantly decreased in the hypoxia group in a time-dependent manner. No expression changes were observed in uroplakins. When compared to normoxic groups, immunofluorescent staining revealed a reduction in the expression of TJ-associated proteins in the hypoxia group. Transepithelial electrical resistance (TEER) revealed a statistically significant decrease in resistance in the hypoxia group. Fluorescein isothiocyanate-conjugated dextran assay was inversely proportional to the results of TEER. Taken together, hypoxia down-regulates the expression of TJ-associated proteins and breaks tight junctions, thus impairing the barrier function in human urothelial cells.

The impact of microplastics polystyrene on the microscopic structure of mouse intestine, tight junction genes and gut microbiota.

Microplastics, which are tiny plastic particles less than 5 mm in diameter, are widely present in the environment, have become a serious threat to aquatic life and human health, potentially causing ecosystem disorders and health problems. The present study aimed to investigate the effects of microplastics, specifically microplastics-polystyrene (MPs-PS), on the structural integrity, gene expression related to tight junctions, and gut microbiota in mice. A total of 24 Kunming mice aged 30 days were randomly assigned into four groups: control male (CM), control female (CF), PS-exposed male (PSM), and PS-exposed female (PSF)(n = 6). There were significant differences in villus height, width, intestinal surface area, and villus height to crypt depth ratio (V/C) between the PS group and the control group(C) (p <0.05). Gene expression analysis demonstrated the downregulation of Claudin-1, Claudin-2, Claudin-15, and Occludin, in both duodenum and jejunum of the PS group (p < 0.05). Analysis of microbial species using 16S rRNA sequencing indicated decreased diversity in the PSF group, as well as reduced diversity in the PSM group at various taxonomic levels. Beta diversity analysis showed a significant difference in gut microbiota distribution between the PS-exposed and C groups (R2 = 0.113, p<0.01), with this difference being more pronounced among females exposed to MPs-PS. KEGG analysis revealed enrichment of differential microbiota mainly involved in seven signaling pathways, such as nucleotide metabolism(p<0.05). The relative abundance ratio of transcriptional pathways was significantly increased for the PSF group (p<0.01), while excretory system pathways were for PSM group(p<0.05). Overall findings suggest that MPs-PS exhibit a notable sex-dependent impact on mouse gut microbiota, with a stronger effect observed among females; reduced expression of tight junction genes may be associated with dysbiosis, particularly elevated levels of Prevotellaceae.

Mfsd2a regulates the blood-labyrinth-barrier formation and function through tight junctions and transcytosis.

The Blood-Labyrinth Barrier (BLB) is pivotal for the maintenance of lymphatic homeostasis within the inner ear, yet the intricacies of its development and function are inadequately understood. The present investigation delves into the contribution of the Mfsd2a molecule, integral to the structural and functional integrity of the Blood-Brain Barrier (BBB), to the ontogeny and sustenance of the BLB. Our empirical findings delineate that the maturation of the BLB in murine models is not realized until approximately two weeks post-birth, with preceding stages characterized by notable permeability. Transcriptomic analysis elucidates a marked augmentation in Mfsd2a expression within the lateral wall of the cochlea in specimens exhibiting an intact BLB. Moreover, both in vitro and in vivo assays substantiate that a diminution in Mfsd2a expression detrimentally impacts BLB permeability and structural integrity, principally via the attenuation of tight junction protein expression and the enhancement of endothelial cell transcytosis. These insights underscore the indispensable role of Mfsd2a in ensuring BLB integrity and propose it as a viable target for therapeutic interventions aimed at the amelioration of hearing loss.

Blood-brain barrier dysfunction and decreased transcription of tight junction proteins in epileptic dogs.

BACKGROUND: Epilepsy in dogs and humans is associated with blood-brain barrier (BBB) dysfunction (BBBD), which may involve dysfunction of tight junction (TJ) proteins, matrix metalloproteases, and astrocytes. Imaging techniques to assess BBB integrity, to identify potential treatment strategies, have not yet been evaluated in veterinary medicine. HYPOTHESIS: Some dogs with idiopathic epilepsy (IE) will exhibit BBBD. Identifying BBBD may improve antiepileptic treatment in the future. ANIMALS: Twenty-seven dogs with IE and 10 healthy controls. METHODS: Retrospective, prospective cohort study. Blood-brain barrier permeability (BBBP) scores were calculated for the whole brain and piriform lobe of all dogs by using dynamic contrast enhancement (DCE) magnetic resonance imaging (MRI) and subtraction enhancement analysis (SEA). Matrix metalloproteinase-9 (MMP9) activity in serum and cerebrospinal fluid (CSF) was measured and its expression in the piriform lobe was examined using immunofluorescent staining. Gene expression of TJ proteins and astrocytic transporters was analyzed in the piriform lobe. RESULTS: The DCE-MRI analysis of the piriform lobe identified higher BBBP score in the IE group when compared with controls (34.5% vs 26.5%; P = .02). Activity and expression of MMP9 were increased in the serum, CSF, and piriform lobe of IE dogs as compared with controls. Gene expression of Kir4.1 and claudin-5 in the piriform lobe of IE dogs was significantly lower than in control dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Our findings demonstrate BBBD in dogs with IE and were supported by increased MMP9 activity and downregulation of astrocytic potassium channels and some TJ proteins. Blood brain barrier dysfunction may be a novel antiepileptic therapy target.

Intestinal health of squab pigeons responded to parental dietary protein levels during breeding period.

The objective of this study was to determine the effects of dietary crude protein (CP) levels on intestinal antioxidant status, tight junction proteins expression, and amino acids transporters levels in squabs. A total of 180 pairs of White King parent pigeons approximately 10 mo old were randomly assigned to 5 groups with 6 replications of 6 pairs of parental pigeons each, and were fed with 14, 15, 16, 17, and 18% CP diets for 46 d, respectively. Dietary increasing CP levels increased final body weight (linear and quadratic, P < 0.05), serum urea nitrogen (linear, P<0.05) and triglyceride levels (quadratic, P < 0.05), and reduced kidney relative weight (quadratic, P < 0.05) in squabs. Final body weight of squabs in the 18% CP diet group was higher than that of the 14, 15, and 16% CP diet groups (P < 0.05) but was similar to that of the 17% CP diet group (P > 0.05). Increasing dietary CP levels reduced intestinal malondialdehyde contents (linear and quadratic, P < 0.05) and jejunal total superoxide dismutase (T-SOD) activity (linear, P < 0.05), and enhanced (linear and quadratic, P<0.05) ileal catalase and T-SOD activities in squabs, and these effects were more prominent in the 17% CP diet group. Graded CP levels up-regulated the mRNA expression of intestinal zonula occludens 1 (linear, P < 0.05), solute carrier family 7 members 9 (linear, P < 0.05) and claudin 1 (CLDN1, linear and quadratic, P < 0.05), ileal CLDN3 and solute carrier family 6 members 14 (linear, P < 0.05) but lowered jejunal solute carrier family 6 member 14 (quadratic, P<0.05) mRNA expression in squabs. The effects of dietary CP levels on intestinal tight junction proteins expression were more apparent when its supplemental levels were 18%. These results suggested that increasing parental dietary CP levels ranged from 14 to 18% during breeding period improved growth and intestinal function of squabs, with its recommended level being 17%.

Autolyzed yeast and sodium butyrate supplemented alone to diets promoted improvements in performance, intestinal health and nutrient transporter in weaned piglets.

This study investigated the effects of supplemental nucleotides, autolyzed yeast (Saccharomyces cerevisiae), and sodium butyrate in diets for nursery pigs on growth performance, diarrhea incidence, blood profile, intestinal morphology, mRNA expression of nutrient transporters, inflammatory markers, antioxidant profile, and tight junction proteins in the small intestine. One hundred eighty 21-day-old pigs (5.17 ± 0.57 kg) were assigned in a randomized block design to 1 of 4 dietary treatments: (1) CON: control, basal diet, (2) NUC: CON + nucleotides, (3) YSC: CON + lysed yeast S. cerevisiae, (4) ASB: CON + acidifier sodium butyrate. Pigs were fed for 24 days, phase 1 (21-32 days) and 2 (32-45 days). During phase 1, YSC and ASB improved average daily gain (ADG) and feed conversion (FC) compared with CON. At the overall period, ASB improved ADG and YSC improved FC compared with CON. The NUC diet did not affect growth performance. The ASB increased ileal villus height compared to CON. The YSC and ASB reduced the number of Peyer's patches in the ileum compared with CON. The YSC increased mRNA expression of nutrient transporters (SMCT2, MCT1, and PepT1), tight junction proteins (OCL and ZO-1), antioxidants (GPX), and IL1-ß in the jejunum compared with CON. The ASB increased mRNA expression of nutrient transporters (SGLT1 and MCT1), tight junction proteins (OCL and ZO-1), and antioxidants (GPX and SOD) compared with CON. In conclusion, autolyzed yeast and sodium butyrate promoted growth performance by improving the integrity of the intestinal barrier, the mRNA expression of nutrient transporters, and antioxidant enzymes in the jejunum of nursery pigs whereas supplementation of nucleotides did not show such effects.

Expression of genes related to ileal barrier function in heritage and modern broiler chickens.

1. An experiment was conducted to determine differences in the expression of genes encoding intestinal barrier proteins between fast, medium and slow-growing chickens. Chicken breeds Athens Canadian Random Bred (ACRB), Longenecker's Heritage (LHR), RedBro, Hubbard H1 (HH1), Cobb500 and Ross708 were raised from hatch for 35 d.2. Ileal samples were collected at embryonic day E19 (-2 days post-hatch), hatch and d 7, 14, 21, 28 and 35 post-hatch to assess the expression of genes encoding tight junction proteins (claudins, CLDN; occludin, OCLN; zonula occludens, ZO; and junctional adhesion molecules, JAM), mucin (Muc2), immunoglobulin A (IgA), polymeric immunoglobulin receptor (pIgR) and fatty acid binding protein (FABP2).3. Expression of CLDN-1 was increased (p < 0.0001) in LHR compared to Cobb500 while CLDN-5 was increased (p < 0.0001) in ACRB, HH1, RedBro and Ross708 compared to LHR as well as in ACRB compared to Cob500. Occludin was upregulated (p = 0.01) in ACRB and LHR compared to Ross708 at d 14 post-hatch. Expression of ZO-1 was upregulated (p = 0.001) in LHR compared to Ross708, HH1 and Cobb500. Tight junction genes, except CLDN-4, JAM-2 and JAM-3 were downregulated (p < 0.0001) at hatch and d 7 post-hatch. Expression of Muc2 was increased (p < 0.0001) in LHR compared to RedBro and from -2 d to d 7 post-hatch.4. Immunoglobulin A was increased (p = 0.001) in LHR compared to Ross708 and HH1 at -2 d post-hatch and in LHR compared to ACRB, Cobb500 and Ross708 at hatch. In addition, IgA expression was increased in all breeds at d 14 post-hatch while pIgR was upregulated (p = 0.02) in Cobb500 and Ross708 compared to ACRB, HH1, LHR and RedBro at hatch.5. The gene expression patterns suggest that selection for growth may have not induced changes in junctional complexes and immune defence genes. However, the results confirmed that the expression of these genes is age dependent.

A Novel Lactobacillus brevis Fermented with a Vegetable Substrate (AL0035) Counteracts TNBS-Induced Colitis by Modulating the Gut Microbiota Composition and Intestinal Barrier.

Crohn's and ulcerative colitis are common conditions associated with inflammatory bowel disease as well as intestinal flora and epithelial barrier dysfunction. A novel fermented Lactobacillus brevis (AL0035) herein assayed in a trinitro benzene sulfonic acid (TNBS)-induced colitis mice model after oral administration significantly counteracted the body weight loss and improves the disease activity index and histological injury scores. AL0035 significantly decreased the mRNA and protein expression of different pro-inflammatory cytokines (TNFalpha, IL-1beta, IL-6, IL-12, IFN-gamma) and enhanced the expression of IL-10. In addition, the probiotic promoted the expression of tight junction proteins, such as ZO-1, keeping the intestinal mucosal barrier function to attenuate colitis symptoms in mice. Markers of inflammation cascade such as myeloperoxidase (MPO) and PPAR-gamma measured in the colon were also modified by AL0035 treatment. AL0035 was also able to reduce different lymphocyte markers' infiltration in the colon (GATA-3, T-Bet, NK1.1) and monocyte chemoattractant protein-1 (MCP-1/CCL2), a key chemokine involved in the migration and infiltration of monocytes/macrophages in the immunological surveillance of tissues and inflammation. In colonic microbiota profile analysis through 16S rRNA sequencing, AL0035 increased the microbial diversity depleted by TNBS administration and the relative abundance of the Lactobacillaceae and Lachnospiraceae families, whereas it decreased the abundance of Proteobacteria. Altogether, these data indicated that AL0035 could lower the severity of colitis induced by TNBS by regulating inflammatory cytokines, increasing the expression of tight junction proteins and modulating intestinal microbiota, thus preventing tissue damage induced by colitis.

Fentanyl dysregulates neuroinflammation and disrupts blood-brain barrier integrity in HIV-1 Tat transgenic mice.

Opioid overdose deaths have dramatically increased by 781% from 1999 to 2021. In the setting of HIV, opioid drug abuse exacerbates neurotoxic effects of HIV in the brain, as opioids enhance viral replication, promote neuronal dysfunction and injury, and dysregulate an already compromised inflammatory response. Despite the rise in fentanyl abuse and the close association between opioid abuse and HIV infection, the interactive comorbidity between fentanyl abuse and HIV has yet to be examined in vivo. The HIV-1 Tat-transgenic mouse model was used to understand the interactive effects between fentanyl and HIV. Tat is an essential protein produced during HIV that drives the transcription of new virions and exerts neurotoxic effects within the brain. The Tat-transgenic mouse model uses a glial fibrillary acidic protein (GFAP)-driven tetracycline promoter which limits Tat production to the brain and this model is well used for examining mechanisms related to neuroHIV. After 7 days of fentanyl exposure, brains were harvested. Tight junction proteins, the vascular cell adhesion molecule, and platelet-derived growth factor receptor-ß were measured to examine the integrity of the blood brain barrier. The immune response was assessed using a mouse-specific multiplex chemokine assay. For the first time in vivo, we demonstrate that fentanyl by itself can severely disrupt the blood-brain barrier and dysregulate the immune response. In addition, we reveal associations between inflammatory markers and tight junction proteins at the blood-brain barrier.

Functional Analysis of Membrane-Associated Scaffolding Tight Junction (TJ) Proteins in Tumorigenic Characteristics of B16-F10 Mouse Melanoma Cells.

Tight junction (TJ) proteins (Tjps), Tjp1 and Tjp2, are tight junction-associated scaffold proteins that bind to the transmembrane proteins of tight junctions and the underlying cytoskeleton. In this study, we first analyzed the tumorigenic characteristics of B16-F10 melanoma cells, including cell proliferation, migration, invasion, metastatic potential, and the expression patterns of related proteins, after the CRISPR-Cas9-mediated knockout (KO) of Tjp genes. The proliferation of Tjp1 and Tjp2 KO cells significantly increased in vitro. Other tumorigenic characteristics, including migration and invasion, were significantly enhanced in Tjp1 and Tjp2 KO cells. Zonula occludens (ZO)-associated protein Claudin-1 (CLDN-1), which is a major component of tight junctions and functions in controlling cell-to-cell adhesion, was decreased in Tjp KO cells. Additionally, Tjp KO significantly stimulated tumor growth and metastasis in an in vivo mouse model. We performed a transcriptome analysis using next-generation sequencing (NGS) to elucidate the key genes involved in the mechanisms of action of Tjp1 and Tjp2. Among the various genes affected by Tjp KO-, cell cycle-, cell migration-, angiogenesis-, and cell-cell adhesion-related genes were significantly altered. In particular, we found that the Ninjurin-1 (Ninj1) and Catenin alpha-1 (Ctnna1) genes, which are known to play fundamental roles in Tjps, were significantly downregulated in Tjp KO cells. In summary, tumorigenic characteristics, including cell proliferation, migration, invasion, tumor growth, and metastatic potential, were significantly increased in Tjp1 and Tjp2 KO cells, and the knockout of Tjp genes significantly affected the expression of related proteins.

Impact of maternal live yeast supplementation to sows on intestinal inflammatory cytokine expression and tight junction proteins in suckling and weanling piglets.

Recent studies have highlighted the importance of maternal nutrition during gestation and lactation in modulating the gastrointestinal development and health of offspring. Therefore, the objective of this study was to determine the effects of live yeast (LY) supplementation to sows during late gestation and throughout lactation on markers of gut health of piglets prior to weaning and immediately postweaning. On day 77 of gestation, forty sows were allotted based on parity and expected farrowing dates to two dietary treatments: without (CON) or with (LY) supplementation at 0.05% and 0.1% of diet during gestation and lactation, respectively. On postnatal days (PND) 0, 10, 18, and postweaning days (PWD) 7 and 14, one piglet from each of 10 sows per treatment were selected for intestinal tissue collection (n = 10). Real-time PCR and western blotting analyses were used to determine the mucosal expression of immune and antioxidant-regulatory genes and tight junction markers of gut health in the duodenum, jejunum, and ileum. Inflammatory and tight junction markers on PND 0 were not affected by maternal dietary treatment. On PND 18, maternal LY supplementation increased (P < 0.05) mRNA expression of interleukin (IL)-6 and tended (P = 0.08) to increase expression of IL-10 in the ileal muocsa. Maternal LY supplementation also increased (P < 0.05) expression of IL-1ß in the ileal mucosa on PWD 14. Likewise, expression of superoxide dismutase (SOD) 1 was increased (P < 0.05) by LY on PND 10, 18, and PWD 14, with a tendency (P = 0.09) for a greater mRNA abundance of catalase on PND 14 in the ileal mucosa. Compared to CON piglets, LY piglets had a higher (P < 0.05) protein abundance of E-cadherin in the jejunal mucosa on PND 0, PWD 7, and PWD 14. Levels of occludin and claudin-4 were also higher (P < 0.05) in the jejunum of LY piglets on PWD 14. No differences were found in jejunal histomorphological measurements between treatments. In conclusion, this study shows that maternal LY supplementation affects key markers of gut health and development in the offspring that may impact the future growth potential and health of newborn piglets.

Increasing evidence supports the benefits of improving sow nutrition during gestation and lactation to promote gastrointestinal development and overall health of piglets. The objective of this research was to investigate the effects of maternal live yeast (LY) supplementation to sows during late gestation and lactation periods on the intestinal health of suckling and weaned piglets. Sows were fed LY during gestation and lactation and piglets were killed for sampling at different time points to track the temporal effect of maternal LY supplementation on changes in markers of intestinal health and development on postnatal days 0, 10, and 18, and postweaning days 7 and 14. Results showed that maternal LY supplementation affected several markers of health and development in the offspring, especially the expression of tight junction proteins, inflammatory cytokines, and antioxidant enzymes. These results indicate that nutritional intervention during gestation and lactation could serve as an effective strategy for raising piglets with better health and growth performance.

A critical review on intestinal mucosal barrier protection effects of dietary polysaccharides.

Studies have shown that dietary polysaccharides, which are widely present in natural foods, have an important impact on the intestinal mucosal barrier. Dietary polysaccharides can maintain the intestinal barrier function through multiple mechanisms. The intestinal barrier is composed of mechanical, chemical, immune, and biological barriers, and dietary polysaccharides, as a bioactive component, can promote and regulate these four barriers. Dietary polysaccharides can enhance the expression of tight junction proteins and mucins such as occludin-1 and zonula occludens-1 (ZO-1) between intestinal epithelial cells, inhibit inflammatory response and oxidative stress, increase the growth of beneficial bacteria, produce beneficial metabolites such as short chain fatty acids (SCFAs), and promote the proliferation and metabolism of immune cells. Given the critical role of the intestinal mucosal system in health and disease, the protective effects of dietary polysaccharides may be potentially valuable for the prevention and treatment of gut-related diseases. Therefore, it is of great significance to further study the mechanism and application prospects of the intestinal mucosal barrier derived from plant, animal, fungal and bacterial sources.

Regulation of tight junction proteins and cell death by peroxisome proliferator-activated receptor γ agonist in brainstem of hypertensive rats.

The decrease in tight junction proteins and their adapter proteins in the hypertensive brain is remarkable. Here, we aimed to investigate tight junction proteins and peroxisome proliferator-activated receptor (PPARγ) activation as well as inflammation factors and cell death proteins in the brainstem of hypertension models, namely spontaneously hypertensive rats (SHR) and borderline hypertensive rats (BHR). At first, SHR and BHR groups were treated with PPARγ agonist, pioglitazone. Then, occludin, claudin-1, claudin-2, claudin-12, ZO-1, and NF-κB p65 gene expression levels; pIKKß, NF-κB p65, TNF, IL-1ß, caspase-3, caspase-9 levels, and PARP-1 cleavage were evaluated. Significantly lower pIKKß, NF-κB p65, TNF, and IL-1ß levels were measured in pioglitazone-treated SHR. Results from this study confirm higher occludin (1.35-fold), claudin-2 (7.45-fold), claudin-12 (1.12-fold), and NF-κB p65 subunit (4.76-fold) expressions in the BHR group when compared to the SHR group. Pioglitazone was found effective in terms of regulating gene expression in SHR. Pioglitazone significantly increased occludin (8.17-fold), claudin-2 (2.41-fold), and claudin-12 (1.85-fold) mRNA levels, which were accompanied by decreased cleaved caspase-3, caspase-9 levels, PARP-1 activation, and proinflammatory factor levels in SHR (p ˂ 0.05). Our work has led us to conclude that alterations in tight junction proteins, particularly occludin, and cell death parameters in the brainstem following PPARγ activation may contribute to neuroprotection in essential hypertension.

Ageratina adenophora damages the rumen epithelium via inducing the expression of inflammatory factors in goats.

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 90 d, 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-1ß], 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-1ß, 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 article aims to investigate the toxic effects of Ageratina adenophora, an invasive plant on the integrity of the rumen epithelium by measuring the changes in the expression of inflammatory factors and tight junction proteins after the consumption of A. adenophora in goats. The results showed that A. adenophora causes damage to the rumen epithelium by increasing the expression of pro-inflammatory markers like TNF-α and IFN-γ and reducing the expression of tight junction proteins such as occludin and claudin-1 in goats.

D-Mannose promotes recovery from experimental colitis by inducing AMPK phosphorylation to stimulate epithelial repair.

Delayed mucosal healing and impaired intestinal epithelial barrier function have been implicated in the pathogenesis of ulcerative colitis (UC). Accordingly, restoration of epithelial barrier function as a means to reshape mucosal homeostasis represents an important strategy for use in the treatment of UC. In this study, we examined the role and mechanisms of D-mannose in the recovery of colitis as assessed in both animal and cell models. We found that D-mannose ameliorated inflammation, promoted mucosal healing in the colon and therefore was able to induce the recovery of UC. Furthermore, D-mannose increased the expression of tight junction (TJ) proteins and reduced the intestinal permeability during the recovery of colitis. Moreover, D-mannose inhibited M1 macrophage polarization and promoted M2 macrophage polarization via inducing AMPK phosphorylation while reducing mTOR phosphorylation in both models. In addition, increased TJ protein expression and decreased paracellular permeability were observed in NCM460 cells when incubated with the supernatants of D-mannose-treated RAW264.7 cells, suggesting that M1/M2 polarization induced by D-mannose modulates the expression of TJ proteins. Further study showed that D-mannose significantly upregulated the expression of TJ proteins in DSS-treated NCM460 cells by inducing AMPK phosphorylation, indicating a direct protective effect on epithelial cells. Finally, the protective effects of D-mannose were significantly abrogated by the presence of compound C, an AMPK inhibitor. Taken together, our data indicate that D-mannose can alleviate inflammation and foster epithelial restitution in UC recovery by inducing the TJ protein expression, which are achieved by inducing AMPK phosphorylation in the epithelium and/or macrophages.

Potential Toxicity and Mechanisms of T-2 and HT-2 Individually or in Combination on the Intestinal Barrier Function of Porcine Small Intestinal Epithelial Cells.

Under natural conditions, T-2 toxin can be easily metabolized to HT-2 toxin by deacetylation, and T-2 and HT-2 are usually co-contaminated in grain and feed at a high detected rate. Our previous information indicated that T-2 toxin could injure the function of the intestinal barrier, but the combined toxicity and mechanism of T-2 and HT-2 on the intestinal cells of porcines are still unknown. Therefore, we aimed to explore T-2 and HT-2 individually and combined on cellular viability, cell membrane integrity, the expression of tight junction-related proteins, and the generation of inflammatory factors in porcine intestinal epithelial cells (IPEC-J2). The results showed that T-2 and HT-2, individually or in combination, could induce a decrease in cell viability, an increase in LDH release and IL-1, IL-6, and TNF-α generation, and a decrease in the anti-inflammatory factor IL-10. Based on the analysis of immunofluorescence staining, real-time PCR, and western blotting, the tight junction protein expressions of Claudin-1, Occludin, and ZO-1 were significantly decreased in the T-2 and HT-2 individual or combination treated groups compared with the control. Furthermore, all the parameter changes in the T-2 + HT-2 combination group were much more serious than those in the individual dose groups. These results suggest that T-2 and HT-2, individually and in combination, could induce an intestinal function injury related to an inflammatory response and damage to the intestinal barrier function in porcine intestinal epithelial cells. Additionally, T-2 and HT-2 in combination showed a synergistic toxic effect, which will provide a theoretical basis to assess the risk of T-2 + HT-2 co-contamination in porcine feed.

Disruption of blood-brain barrier: effects of HIV Tat on brain microvascular endothelial cells and tight junction proteins.

Although the widespread use of antiretroviral therapy (ART) has prolonged the life span of people living with HIV (PLWH), the incidence of HIV-associated neurocognitive disorders (HAND) in PLWH is also gradually increasing, seriously affecting the quality of life for PLWH. However, the pathogenesis of HAND has not been elucidated, which leaves HAND without effective treatment. HIV protein transactivator of transcription (Tat), as an important regulatory protein, is crucial in the pathogenesis of HAND, and its mechanism of HAND has received widespread attention. The blood-brain barrier (BBB) and its cellular component brain microvascular endothelial cells (BMVECs) play a necessary role in protecting the central nervous system (CNS), and their damage associated with Tat is a potential therapeutic target of HAND. In this review, we will study the Tat-mediated damage mechanism of the BBB and present multiple lines of evidence related to BMVEC damage caused by Tat.

Aflatoxin M1 decreases the expression of genes encoding tight junction proteins and influences the intestinal epithelial integrity.

Aflatoxin M1 (AFM1) is a mycotoxin that is commonly found as a milk contaminant, and its presence in milk has been linked to cytotoxicity. The present study aimed to evaluate the acute cytotoxic effects of AFM1 on intestinal Caco-2 cells. Initially, we checked the morphology and viability of Caco-2 cells after treatment with different concentrations of AFM1 (5 ng/L, 50 ng/L, 250 ng/L, 500 ng/L, 1000 ng/L, and 2000 ng/L) for different time intervals (6 h, 12 h, and 24 h). It was found that AFM1 did not show any effect on cell morphology, but 10% decrease in viability above 1000 ng/L after 12 h. Furthermore, DCFDA assay showed increased ROS production after 6 h treatments. qPCR analysis showed an increased expression of epithelial-specific cytoskeleton marker genes, Cytokeratin, Villin, Vimentin, and JAM-1, and a decreased expression of tight junction protein genes, Claudin-1, Occludin, and ZO-1. Similarly, we found an increased expression of Cyp1a1 transcript with an increasing AFM1 concentration and incubation time. This gene expression analysis showed AFM1 can cause disruption of tight junctions between intestinal cells, which was further confirmed by a transwell experiment. In conclusion, consumption of AFM1-contaminated milk does not show any effect on cells morphology and viability but decreases the expression of intestinal barrier transcripts that may lead to the disruption of intestinal barrier function and leaky gut.

Estrogenic regulation of claudin 5 and tight junction protein 1 gene expression in zebrafish: A role on blood-brain barrier?

The blood-brain barrier (BBB) is a physical interface between the blood and the brain parenchyma, playing key roles in brain homeostasis. In mammals, the BBB is established thanks to tight junctions between cerebral endothelial cells, involving claudin, occludin, and zonula occludens proteins. Estrogens have been documented to modulate BBB permeability. Interestingly, in the brain of zebrafish, the estrogen-synthesizing activity is strong due to the high expression of Aromatase B protein, encoded by the cyp19a1b gene, in radial glial cells (neural stem cells). Given the roles of estrogens in BBB function, we investigated their impact on the expression of genes involved in BBB tight junctions. We treated zebrafish embryos and adult males with 17ß-estradiol and observed an increased cerebral expression of tight junction and claudin 5 genes in adult males only. In females, treatment with the nuclear estrogen receptor antagonist (ICI182,780 ) had no impact. Interestingly, telencephalic injuries performed in males decreased tight junction gene expression that was partially reversed with 17ß-estradiol. This was further confirmed by extravasation experiments of Evans blue showing that estrogenic treatment limits BBB leakage. We also highlighted the intimate links between endothelial cells and neural stem cells, suggesting that cholesterol and peripheral steroids could be taken up by endothelial cells and used as precursors for estrogen synthesis by neural stem cells. Together, our results show that zebrafish provides an alternative model to further investigate the role of steroids on the expression of genes involved in BBB integrity, both in constitutive and regenerative physiological conditions. The link we described between capillaries endothelial cells and steroidogenic neural cells encourages the use of this model in understanding the mechanisms by which peripheral steroids get into neural tissue and modulate neurogenic activity.

Activation of the 5-hydroxytryptamine 4 receptor ameliorates tight junction barrier dysfunction in the colon of type 1 diabetic mice.

Hyperglycemia drives dysfunction of the intestinal barrier. 5-Hydroxytryptaine 4 receptor (5-HT 4R) agonists have been considered therapeutics for constipation in clnic. However, the roles of 5-HT 4R activation in mucosa should be fully realized. Here, we investigate the effects of 5-HT 4R activation on diabetes-induced disruption of the tight junction (TJ) barrier in the colon. Not surprisingly, the TJ barrier in diabetic mice with or without 5-HT 4R is tremendously destroyed, as indicated by increased serum fluorescein isothiocyanate (FITC)-dextran and decreased transepithelial electrical resistance (TER). Simultaneously, decreased expressions of TJ proteins are shown in both wild-type (WT) and 5-HT 4R knockout (KO) mice with diabetes. Notably, chronic treatment with intraperitoneal injection of a 5-HT 4R agonist in WT mice with diabetes repairs the TJ barrier and promotes TJ protein expressions, including occludin, claudin-1 and ZO-1, in the colon, whereas a 5-HT 4R agonist does not improve TJ barrier function or TJ protein expressions in 5-HT 4R KO mice with diabetes. Furthermore, stimulation of 5-HT 4R inhibits diabetes-induced upregulation of myosin light chain kinase (MLCK), Rho-associated coiled coil protein kinase 1 (ROCK1), and phosphorylated myosin light chain (p-MLC), which are key molecules that regulate TJ integrity, in the colonic mucosa of WT mice. However, such action induced by a 5-HT 4R agonist is not observed in 5-HT 4R KO mice with diabetes. These findings indicate that 5-HT 4R activation may restore TJ integrity by inhibiting the expressions of MLCK, ROCK1 and p-MLC, improving epithelial barrier function in diabetes.