Occludin mRNA Research Articles

The effects of subacute ruminal acidosis on rumen epithelium barrier function in dairy goats

The effects of subacute ruminal acidosis (SARA) on the electrophysiological and rumen epithelium barrier functions and its mechanism using dairy goats as a ruminant model were investigated. Eight ruminal cannulated female Sannan dairy goats of mid-lactating period (body weight 43.58 ± 2.77 kg) were selected. The experiment lasted 60 d, and divided equally into 4 periods. During each phase, animals were fed with 4 diets in which non-fiber carbohydrates/neutral detergent fiber ratios (NFC/NDF) were either 1.40, 1.78, 2.31, or 3.23 respectively. Ruminal fluid samples were collected for volatile fatty acids analyzed. Ruminal pH was measured continuously for 72 h. Ruminal tissue samples from the ventral sac were analyzed by Ussing chamber, where electrophysiological properties were determined in parallel with the permeability of marker molecules of different sizes, fluorescein isothiocyanate (FITC) and horseradish peroxidase(HRP), from luminal to apical side. Additionally, ruminal epithelial junction (TJs) proteins gene expressions were evaluated using quantitative real-time PCR and Western blot. The ruminal acetate concentration and Acetate/Propionate ratio (A/P) decreased, and the concentration of propionate, butyrate and total volatile fatty acids (VFA) increased with the increase of dietary NFC/NDF ratio. Rumen epithelial short- circuit current (Isc) and tissue conductance (Gt) increased in SARA goats compared with control goats, and the mucosal to serosal flux of HRP and FITC increased as well, indicating a higher permeability for SARA goats. SARA increased mRNA and protein expressions of Claudin-4 compared with control group. The mRNA and protein expressions of Claudin-1, Claudin-7, Occludin, ZO-1 and Connxin-43 in SARA group were lower than those in control. The mRNA and protein expressions of desmoglein-1 in SARA group were significantly lower than in the control group. Hence, dairy goats suffering from SARA could result in damaged ruminal epithelial barrier functions, resulting in increase of ruminal epithelial permeability.

RhoA/ROCK-2 Pathway Inhibition and Tight Junction Protein Upregulation by Catalpol Suppresses Lipopolysaccaride-Induced Disruption of Blood-Brain Barrier Permeability.

Lipopolysaccaride (LPS) directly or indirectly injures brain microvascular endothelial cells (BMECs) and damages the intercellular tight junction that gives rise to altered blood-brain barrier (BBB) permeability. Catalpol plays a protective role in LPS-induced injury, but whether catalpol protects against LPS-caused damage of BBB permeability and the underlying mechanism remain to be delineated. Prophylactic protection with catalpol (5 mg/kg, i.v.) consecutively for three days reversed the LPS-induced damage of BBB by decreased Evans Blue (EB) leakage and restored tight junctions in C57 mice. Besides, catalpol co-administrated with LPS increased BMECs survival, decreased their endothelin-1, TNF-Α and IL-6 secretion, improved transmembrane electrical resistance in a time-dependent manner, and in addition increased the fluorescein sodium permeability coefficient of BMECs. Also, transmission electron microscopy showed catalpol protective effects on tight junctions. Fluorescence staining displayed that catalpol reversed the rearrangement of the cytoskeleton protein F-actin and upregulated the tight junction protein of claudin-5 and ZO-1, which have been further demonstrated by the mRNA and protein expression levels of ZO-1, ZO-2, ZO-3, claudin-5, and occludin. Moreover, catalpol concurrently downregulated the mRNA and protein levels of RhoA, and ROCK2, the critical proteins in the RhoA/ROCK2 signaling pathway. This study thus indicated that catalpol, via inhibition of the RhoA/ROCK2 signaling pathway, reverses the disaggregation of cytoskeleton actin in BMECs and prevents down-regulation of junctional proteins, such as claudin-5, occludin, and ZO-1, and decreases endothelin-1 and inflammatory cytokine secretion, eventually alleviating the increase in LPS-induced BBB permeability.

Increased macrophages and\xa0changed brain endothelial cell gene expression in the frontal cortex of people with schizophrenia displaying inflammation

Elevated pro-inflammatory cytokines exist in both blood and brain of people with schizophrenia but how this affects molecular indices of the blood–brain barrier (BBB) is unclear. Eight mRNAs relating to BBB function, a microglia and three immune cell markers were measured by qPCR in the prefrontal cortex from 37 people with schizophrenia/schizoaffective disorder and 37 matched controls. This cohort was previously grouped into “high inflammation” and “low inflammation” subgroups based on cortical inflammatory-related transcripts. Soluble intercellular adhesion molecule-1 (sICAM1) was measured in the plasma of 78 patients with schizophrenia/schizoaffective disorder and 73 healthy controls. We found that sICAM1 was significantly elevated in schizophrenia. An efflux transporter, ABCG2, was lower, while mRNAs encoding VE-cadherin and ICAM1 were higher in schizophrenia brain. The “high inflammation” schizophrenia subgroup had lower ABCG2 and higher ICAM1, VE-cadherin, occludin and interferon-induced transmembrane protein mRNAs compared to both “low inflammation” schizophrenia and “low inflammation” control subgroups. ICAM1 immunohistochemistry showed enrichment in brain endothelium regardless of diagnosis and was localised to astrocytes in some brains. Microglia mRNA was not altered in schizophrenia nor did it correlate with ICAM1 expression. Immune cell mRNAs were elevated in “high inflammation” schizophrenia compared to both “low inflammation” schizophrenia and controls. CD163+ perivascular macrophages were identified by immunohistochemistry in brain parenchyma in over 40% of “high inflammation” schizophrenia brains. People with high levels of cytokine expression and schizophrenia display changes consistent with greater immune cell transmigration into brain via increased ICAM1, which could contribute to other neuropathological changes found in this subgroup of people.

Abstract 025: Cellular Mechanisms of Blood-Brain Barrier Disruption in Ang II-Induced Hypertension

The blood-brain barrier (BBB) is critically important for brain health by regulating molecular exchanges between blood and brain. Hypertension (HTN) induces breakdown of the BBB which may contribute to its deleterious effect on the brain, but the cellular bases of the BBB opening remain to be established. We used a model of angiotensin II (AngII) HTN (600ng/kg/min x 2 weeks; n>5/group) to investigate the mechanisms of the BBB opening. BBB permeability was assessed spectrophotometrically in C57BL/6J mice using 3000 MW FITC-dextran as a tracer. HTN increased BBB permeability in Ang II HTN (29.8 ± 0.9 vs 18.1 ± 0.5 ng/g in control, p<0.01), an effect partially ameliorated by the angiotensin type 1 receptor (AT1R) antagonist losartan in the drinking water (22.9 ± 0.6 ng/g) but not by hydralazine + hydrochlorothiazide (27.4 ± 0.7 ng/g), suggesting involvement of AT1R and not elevated blood pressure. Next, we sought to identify the mechanisms of the breakdown of the BBB in HTN. First, we used electron microscopy to examine the ultrastructure of endothelial tight junctions (TJ) and assess vesicular transport, key components of the BBB. HTN reduced the length (-25%) and complexity (-11%) of TJ, and increased the number of endothelial vesicles (2.22 vs 1.42 vesicles/endothelial area, p<0.05). The TJ remodeling was associated with a reduction in occludin (-17%, p<0.01) and claudin-5 (-30%, p<0.05) mRNA in microvascular preparations. Additionally, the expression of Mfsd2a, a lipid transporter that also suppresses vesicular transcytosis, was markedly attenuated (-43%, p<0.01). Taken together, these data suggest a strong effect of Ang II on cerebral endothelial cells to induce BBB opening. Since perivascular macrophages (PVM) mediate cerebral endothelial dysfunction in HTN (J Clin Invest 2016;126:4674), we tested their involvement in the BBB opening. PVM depletion with icv clodronate or deletion of AT1 receptors in PVM partially attenuated the BBB opening in Ang II HTN (p<0.05). Thus, we conclude that AT1R in cerebral endothelial cells and PVM mediate the BBB opening by targeting both paracellular and vesicular transport. Such increase in BBB permeability to circulating agents may contribute to the cerebrovascular and cognitive dysfunction associated with HTN.

Glutamine alleviates heat stress-induced impairment of intestinal morphology, intestinal inflammatory response, and barrier integrity in broilers

The aim of this study was to investigate the protective effect of glutamine (Gln) on the intestinal morphology, intestinal inflammatory response, and barrier integrity in broilers exposed to high ambient temperature. Three-hundred-sixty 21-d-old Arbor Acres broilers (half male and half female) were randomly allocated to 4 treatment groups in a completely randomized design, each of which included 6 replicates with 15 birds per replicate, for 21 d. The 4 treatment groups were as follows: the control group, in which birds were kept in a thermoneutral room at 22 ± 1°C (no stress, NS; fed a basal diet); the heat stress group (36 ± 1°C for 10 h/d from 08:00 to 18:00 h and 22 ± 1°C for the remaining time, heat stress (HT); fed a basal diet); and heat stress + Gln group (0.5 and 1.0% Gln, respectively). Compared to the NS group, broilers in the HT group had lower villus height (P < 0.05), higher crypt depth (P < 0.05), higher D-lactic acid and diamine oxidase (DAO) activity (P < 0.05), higher soluble intercellular adhesion molecule-1 (sICAM-1) concentration (P < 0.05), higher tumor necrosis factor (TNF)-α/interleukin (IL)-10 (P < 0.05), and lower tight junction protein expression levels (P < 0.05). Compared with birds in the HT, birds in the HT + Gln group exhibited increased villus height (P < 0.05), decreased D-lactate and DAO activity (P < 0.05), decreased sICAM-1 concentration (P < 0.05), and mediate the secretion of cytokines (P < 0.05), as well as increased zonula occludens-1 (ZO-1), claudin-1, and occludin mRNA expression levels (P < 0.05). In conclusion, these results indicate that supplementation with Gln was effective in partially ameliorating the adverse effects of heat stress on intestinal barrier function in broilers by promoting epithelial cell proliferation and renewal, modifying the function of the intestinal mucosa barrier, and regulating the secretion of cytokines.

Metabolism of Caprine Milk Carbohydrates by Probiotic Bacteria and Caco-2:HT29⁻MTX Epithelial Co-Cultures and Their Impact on Intestinal Barrier Integrity.

The development and maturation of the neonatal intestine is generally influenced by diet and commensal bacteria, the composition of which, in turn, can be influenced by the diet. Colonisation of the neonatal intestine by probiotic Lactobacillus strains can strengthen, preserve, and improve barrier integrity, and adherence of probiotics to the intestinal epithelium can be influenced by the available carbon sources. The goal of the present study was to examine the role of probiotic lactobacilli strains alone or together with a carbohydrate fraction (CF) from caprine milk on barrier integrity of a co-culture model of the small intestinal epithelium. Barrier integrity (as measured by trans epithelial electrical resistance (TEER)), was enhanced by three bacteria/CF combinations (Lactobacillus rhamnosus HN001, L. plantarum 299v, and L. casei Shirota) to a greater extent than CF or bacteria alone. Levels of occludin mRNA were increased for all treatments compared to untreated co-cultures, and L. plantarum 299v in combination with CF had increased mRNA levels of MUC4, MUC2 and MUC5AC mucins and MUC4 protein abundance. These results indicate that three out of the four probiotic bacteria tested, in combination with CF, were able to elicit a greater increase in barrier integrity of a co-culture model of the small intestinal epithelium compared to that for either component alone. This study provides additional insight into the individual or combined roles of microbe–diet interactions in the small intestine and their beneficial contribution to the intestinal barrier.

Sodium Butyrate Improves High-Concentrate-Diet-Induced Impairment of Ruminal Epithelium Barrier Function in Goats.

We investigated the effect of sodium butyrate feeding on the disruption of ruminal epithelium barrier function in goats fed a high-concentrate diet. A total of 18 male Boer goats (live weight of 31.75 ± 1.35 kg, aged 1 year) were randomly assigned to three groups, which were fed a low-concentrate diet (LC), a high-concentrate diet (HC), or a high-concentrate diet with 1% sodium butyrate by weight (SH) for 9 weeks. We found that the pH of rumen fluid in the SH and LC groups was higher than that in the HC group. The activity of protein kinase C (PKC) kinase in the rumen epithelium was higher in the HC group than that in the LC and SH groups. The mRNA expression and phosphorylated protein levels of mitogen-activated protein kinases (MAPKs) in the rumen epithelium were lower in the SH and LC groups than those in the HC group. The DNA methylation rate of occludin was higher in the HC group than that in the SH and LC groups. The mRNA and protein expression of claudin-1, claudin-4, occludin, and zona occludin-1 was greater in the SH and LC groups than that in the HC group. In addition, sodium butyrate mitigated damage to the rumen epithelium caused by the HC diet. Together, our results suggest that the supply of sodium butyrate reverses the damage of rumen epithelium tight junction by inhibiting PKC and MAPK signaling pathways and is protective to the rumen epithelium during subacute rumen acidosis.

Bletilla striata polysaccharide has a protective effect on intestinal epithelial barrier disruption in TAA-induced cirrhotic rats.

It has been reported that intestinal epithelial barrier dysfunction serves an important role in the development of liver cirrhosis. However, at present there is no satisfactory treatment for intestinal mucosal lesions and ulcers associated with cirrhosis. The aim of the present study was to investigate the effect of Bletilla striata polysaccharide (BSP) on intestinal epithelial barrier disruption in rats with thioacetamide (TAA)-induced liver cirrhosis. Rats were randomly divided into 5 groups (n=10): BSP low dosage (15 mg/kg), BSP middle dosage (30 mg/kg), BSP high dosage (60 mg/kg), experiment and control groups. All groups except control group were administered with TAA (200 mg/kg/day) to induce liver cirrhosis. Following modeling, rats in the low, middle and high-dose BSP groups received BSP. ELISA kits were used to measure the endotoxin, alanine transaminase (ALT) and aspartate transaminase (AST) levels in the portal vein, while interleukin (IL)-6 and tumor necrosis factor (TNF)-α expression in the ileal tissue was measured. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to detect the expression of zonula occludens (ZO)-1 and occludin mRNA and protein, respectively. Intestinal epithelial tissue pathology was detected using hematoxylin-eosin (HE) staining. Immunohistochemistry was performed to assess the expression of ZO-1 and occludin in intestinal epithelial tissues. Following treatment with BSP, ALT, AST and endotoxin levels in the portal vein, as well as IL-6 and TNF-α expression in ileal tissues, were significantly decreased compared with model group (P<0.05 or 0.01). Furthermore, BSP treatment upregulated the expression of ZO-1 and occludin mRNA and protein compared with the model group (P<0.05 or 0.01) and cytoplasmic staining for these proteins was increased. The degree of intestinal epithelial tissue pathological damage was significantly reduced in the BSP groups. In conclusion, BSP is able to reduce endotoxin levels, inhibit the inflammatory cytokines IL-6 and TNF-α and elevate expression of ZO-1 and occludin at tight junctions. Together, these results suggest a novel protective agent for the restoration of intestinal epithelial barrier disruption.

Abstract 4476: Role of MAP-Kinase suppressor in IBD

Abstract BACKGROUND &amp; AIMS: Endogenous suppressor of Mitogen Activated Kinase Pathway (MAPK) are considered cancer suppressors. In this regard , Sprouty (SPRY) proteins that are endogenous suppressor of receptor tyrosine kinase signaling are reported to be tumor suppressors in various cancers, whereas we discovered that SPRY2 augments Epithelial Mesenchymal Transition (EMT) in colorectal cancer (CRC) [Oncogene, 2016 Jun 16; 35(24): 3151-62]. Significance of SPRY proteins in Inflammatory Bowel Disease (IBD) is not clearly understood. MATERIALS &amp; METHODS: Biopsies from Crohn's Colitis patients were assessed for SPRY, Beta-catenin, Occludin and ZO1 expression. Tight junction profiler was employed to assess expression of integrin proteins in CaCO2 colon cancer cell line. SPRY was suppressed in colon cancer cells by siRNA and CRISPR/Cas9 technology and changes were assessed by immunoblotting, real time PCR, phase-contrast and confocal microscopy. Spry1 and Spry2 floxed mice were utilized to dissect SPRY functions in mesenchymal cells. RESULTS: SPRY, E-cadherin, Beta-catenin and Occludin mRNA transcripts were significantly different in IBD patients (p&amp;lt;0.05). No significant changes in ZO1 mRNA transcripts were noted. Suppression of SPRY significantly decreased expression of several integrins in CaCO2 cells (p&amp;lt;0.05) . Treatment of CaCO2 cells with Dextran Sodium Sulfate (DSS) significantly altered SPRY, Beta-catenin, Occludin and ZO1 expressions that was reversed by SPRY suppression. Spry1-/- and Spry2-/- double mutant murine embryonic fibroblasts (MEFs) demonstrated restoration of several epithelial markers which protected cells from DSS-induced changes. CONCLUSIONS: SPRY expression in colonic epithelium regulates IBD. SPRY has prognostic value in IBD. Citation Format: Sharad Khare, Qiong Zhang. Role of MAP-Kinase suppressor in IBD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4476.

GABAA Receptor Subtypes Regulate Stress-Induced Colon Inflammation in Mice

Psychological stress, in early life or adulthood, is a significant risk factor for inflammatory disorders, including inflammatory bowel diseases. However, little is known about the mechanisms by which emotional factors affect the immune system. γ-Aminobutyric acid type A receptors (GABAARs) regulate stress and inflammation, but it is not clear whether specific subtypes of GABAARs mediate stress-induced gastrointestinal inflammation. We investigated the roles of different GABAAR subtypes in mouse colon inflammation induced by 2 different forms of psychological stress. C57BL/6J mice were exposed to early-life stress, and adult mice were exposed to acute-restraint stress; control mice were not exposed to either form of stress. We collected colon tissues and measured contractility using isometric tension recordings; colon inflammation, based on levels of cluster of differentiation 163 and tumor necrosis factor messenger RNA (mRNA) and protein and myeloperoxidase activity; and permeability, based on levels of tight junction protein 1 and occludin mRNA and protein. Mice were given fluorescently labeled dextran orally and systemic absorption was measured. We also performed studies of mice with disruption of the GABAAR subunit α3 gene (Gabra3-/- mice). Mice exposed to early-life stress had significantly altered GABAAR-mediated colonic contractility and impaired barrier function, and their colon tissue had increased levels of Gabra3 mRNA compared with control mice. Restraint stress led to colon inflammation in C57/BL6J mice but not Gabra3-/- mice. Colonic inflammation was induced invitro by an α3-GABAAR agonist, showing a proinflammatory role for this receptor subtype. In contrast, α1/4/5-GABAAR ligands decreased the expression of colonic inflammatory markers. We found stress to increase expression of Gabra3 and induce inflammation in mouse colon, together with impaired barrier function. The invitro pharmacologic activation of α3-GABAARs recapitulated colonic inflammation, whereas α1/4/5-GABAAR ligands were anti-inflammatory. These proteins might serve as therapeutic targets for treatment of colon inflammation or inflammatory bowel diseases.

TLR4/PKCα/occludin signaling pathway may be related to blood‑brain barrier damage.

Abnormal blood-brain barrier (BBB) is a common pathological feature in brain damage. In the present study, a brain microvascular endothelial cell (BMEC) model was established to determine the role of the toll‑like receptor 4 (TLR4)/protein kinase Cα (PKCα)/occludin signaling pathway in BBB dysfunction. Three small interfering (si)RNAs directed against PKCα were designed to investigate the molecular mechanisms of PKCα underlying BBB damage. BMECs were divided into 4 groups: Control group, TAK‑242 (a TLR4 inhibitor) group, PKCα‑siRNA group and TAK‑242+PKCα‑siRNA group. The results indicated that siRNA‑3 was the most effective at silencing PKCα gene expression. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis indicated no significant difference of TLR4 mRNA expression levels between three different treated groups and the Control group. However, PKCα mRNA expression in the PKCα‑siRNA and TAK‑242+PKCα‑siRNA groups were significantly decreased compared with that in Control and TAK‑242 groups. In addition, occludin mRNA expression in PKCα‑siRNA and TAK‑242+PKCα‑siRNA groups were significantly higher compared with the Control group. Meanwhile, occluding expressions in three treated groups were also significantly higher compared with the Control group. Furthermore, TAK‑242 treatment, PKCα‑siRNA treatment, and TAK‑242+PKCα‑siRNA treatment could promote occludin junctional labeling compared with the Control group. The permeability of PKCα‑siRNA and TAK‑242+PKCα‑siRNA groups was significantly promoted compared with the control group. The TLR4/PKCα/occludin signaling pathway was closely related to BBB damage. The present study will lead to an improved molecular understanding of BBB damage in the future.

Deregulation of small intestine permeability by miRNA-21 in liver disease

The gut-liver axis influences a wide variety of liver diseases. High fat, choline-deficient (HFCD) diet and common bile duct ligation (BDL) are experimental models of liver disease associated with early increased small intestinal permeability (SIP), disturbed bile acid homeostasis and gut microbiota (GM) dysbiosis. Here we investigate the impact of whole body genetic deletion or therapeutic modulation of miR-21 in SIP as well as in the GM, which may influence miR-21 effects in the liver. We have previously demonstrated that miR -21 contributes to BDL-induced liver injury and fibrosis. Here we reveal that occludin and claudin-1 mRNA levels were decreased in small intestine of WT mice after BDL, suggesting increased SIP. Moreover, zonula occludens (ZO-1) and occludin mRNA levels were also decreased in HFCD-fed mice. Strikingly, both genetic deletion of miR-21 and Anti-miR-21 treatment reverted mRNA levels of tight junction proteins to control. Moreover, the GM of WT mice became dysbiotic, with decreased Bacteroidetes and increased Proteobacteria and Firmicutes. In miR-21 KO mice, the GM was not affected by BDL surgery. Our results suggest miR-21 involvement in liver injury by impacting on liver and intestinal targets.

Effects of Lactobacillus acidophilus on the growth performance and intestinal health of broilers challenged with Clostridium perfringens

BackgroundClostridium perfringens is the main etiological agent of necrotic enteritis. Lactobacilli show beneficial effects on intestinal health in infectious disease, but the protective functions of lactobacilli in C. perfringens-infected chickens are scarcely described. This study examined the effects of Lactobacillus acidophilus (L. acidophilus) on the growth performance and intestinal health of broiler chickens challenged with Clostridium perfringens (C. perfringens) over a 28-day period. Using a 2 × 2 factorial arrangement of treatments, a total of 308 1-day-old male Arbor Acres broiler chicks were included to investigate the effects of Lactobacillus acidophilus (L. acidophilus) on the growth performance and intestinal health of broiler chickens challenged with Clostridium perfringens (C. perfringens) during a 28-day trial.ResultsDuring infection (d 14–21), C. perfringens challenge decreased the average daily gain (P < 0.05), and increased feed conversion ratio and the mortality rate (P < 0.05). However, dietary supplementation with L. acidophilus increased the body weight of C. perfringens-infected broilers on d 21 (P < 0.05), and tended to decrease the mortality (P = 0.061). C. perfringens challenge decreased the villus height (P < 0.05), the ratio of villus height to crypt depth (P < 0.05) and OCLN (occludin) mRNA expression (P < 0.05), and increased the pro-inflammatory cytokine expression in the spleen and jejunum, the intestinal populations of C. perfringens and Escherichia (P < 0.05), and the serum content of endotoxin (P < 0.05), regardless of L. acidophilus supplementation. In contrast, dietary L. acidophilus reducedthe intestinal lesion score of challenged broilers (P < 0.05), the mRNA expression of pro-inflammatory cytokines, ileal populations of Escherichia and serum endotoxin content (P < 0.05), but increased the intestinal Lactobacillus populations (P < 0.05), irrespective of C. perfringens challenge.ConclusionDietary addition of L. acidophilus could improve the intestinal health and reduce the mortality of broilers suffering from necrotic enteritis.

Effect of interleukin-1β on occludin mRNA expression in the duodenal and colonic mucosa of dogs with inflammatory bowel disease.

BackgroundMucosal imbalance of interleukin (IL)‐1β and IL‐1 receptor antagonist (Ra) has been reported in the duodenal mucosa of dogs with inflammatory bowel disease (IBD). However, the imbalance in the colonic mucosa and its role in duodenitis and colitis in IBD of dogs remain unclear.ObjectivesTo measure the expression of IL‐1β and IL‐1Ra proteins in the colonic mucosa of dogs with IBD, and to determine the effect of IL‐1β on expression of occludin (ocln) mRNA, a tight junction component, in the duodenal and colonic mucosa of dogs with IBD.AnimalsTwelve dogs with IBD and 6 healthy dogs.MethodsIL‐1β and IL‐1 Ra proteins in the colonic mucosa were quantified by ELISA in 7 of the 12 dogs with IBD. Expression of ocln mRNA in the duodenal and colonic mucosa was examined in the 12 dogs by real‐time PCR.ResultsThe ratio of IL‐1β to IL‐1Ra in the colonic mucosa was significantly higher in dogs with IBD than in healthy dogs. The ex vivo experiment determined that IL‐1β suppressed expression of ocln mRNA in the colonic mucosa, but not in the duodenal mucosa, of healthy dogs. Expression of ocln mRNA in the colonic mucosa, but not in the duodenal mucosa, was significantly lower in dogs with IBD than in healthy dogs.Conclusions and Clinical ImportanceA relative increase in IL‐1β may attenuate ocln expression, leading to intestinal barrier dysfunction and promotion of intestinal inflammation in the colonic mucosa, but not in the duodenal mucosa, of dogs with IBD.

Glucagon-Like Peptide-2 Improve Intestinal Mucosal Barrier Function in Aged Rats.

Glucagon-like peptide-2 (GLP-2) plays a major role in repairing impaired intestinal mucosa, but its mechanism in the improvement of intestinal barrier function during the aging process remains unclear. In this study, 26-month-old male Sprague-Dawley rats were randomized to control group and GLP-2 group treated with a dose of 250 μg•kg-1•d-1 by intraperitoneal injection. After 14 days of treatment, intestinal mucosal morphometric changes were observed by light microscopy and transmission electron microscopy (TEM). Small intestinal permeability was evaluated by fluorescein isothiocyanate (FITC)-labeled dextran. The mRNA and protein expression of Zonula Occludens-1 (ZO-1), occludin, claudin-1 and the GLP-2 receptor (GLP-2R) were detected by Real-time PCR and Western blot. Our results showed that GLP-2 administration significantly improved the age-related atrophy of intestinal mucosa and villi and increased small intestinal permeability. The mRNA and protein expression of ZO-1and occludin in ileum were up regulated in the GLP-2-treated old rats. In addition, the serum GLP-2 levels were negatively correlated with small intestinal permeability measured by FITC-dextran levels (r=-0.610, P<0.01). Taking all these data together, it is concluded that GLP-2 improved small intestinal epithelial barrier function in aged rats mainly by facilitating intestinal mucosa growth, alleviating the increased small intestinal permeability and increasing ZO-1 and occludin expression. Our observations provide evidence for the clinical significance of GLP-2 in preventing the intestinal epithelial barrier dysfunction during aging.

Quorum-sensing molecule dihydroxy-2,3-pentanedione and its analogs as regulators of epithelial integrity.

Quorum-sensing molecules regulate the behavior of bacteria within biofilms and at the same time elicit an immune response in host tissues. Our aim was to investigate the regulatory role of dihydroxy-2,3-pentanedione (DPD), the precursor of universal autoinducer-2 (AI-2), and its analogs (ethyl-DPD, butyl-DPD and isobutyl-DPD) in the integrity of gingival epithelial cells. Human gingival keratinocytes were incubated with four concentrations (10μmol L-1 , 1μmol L-1 , 100nmol L-1 and 10nmol L-1 ) of DPD and its analogs for 24hours. The numbers of viable cells were determined using a proliferation kit, matrix metalloproteinase (MMP)-2 and -9 activities were determined by gelatin zymography, and expression of occludin protein and occludin mRNA were determined by western blotting and RT-qPCR, respectively. Increased cell proliferation was observed in gingival keratinocytes incubated with 100nmol L-1 of butyl-DPD. MMP-9 activity was elevated in cells incubated with 10μmol L-1 of ethyl-DPD. On the other hand, MMP-2 activity did not show any significant change when gingival keratinocytes were incubated with or without DPD or analogs. Western blot analyses demonstrated five forms (105, 61, 52.2, 44 and 37kDa) of occludin. Incubation with 1μmol L-1 and 100nmol L-1 of DPD and with 10nmol L-1 of ethyl-DPD increased dimeric (105kDa) forms of occludin, while incubation with 100nmol L-1 of isobutyl-DPD increased monomeric (61kDa) forms. DPD and ethyl-DPD decreased, and 100nmol L-1 of isobutyl-DPD and 10nmol L-1 of butyl-DPD increased, the monomeric (52.2kDa and 44kDa) forms of occludin, whereas ethyl-DPD decreased and isobutyl-DPD increased, the low-molecular-weight (37kDa) forms. According to RT-qPCR analysis, the exposure of gingival keratinocytes to 10μmol L-1 of isobutyl-DPD up-regulated expression of occludin. The results indicate that isobutyl-DPD has the potential to enhance the integrity of the epithelium by stimulating the formation of occluding, without affecting the proliferation or gelatinolytic enzyme activities of the exposed cells. The modulatory effect of an AI-2 analog on the epithelial cell response is shown for the first time.

Effects of dietary dandelion extract on intestinal morphology, antioxidant status, immune function and physical barrier function of juvenile golden pompano Trachinotus ovatus

Intestinal morphology, antioxidant status, immune function and tight junction proteins mRNA expression were examined in golden pompano (Trachinotus ovatus) that fed respectively six diets containing dandelion extracts (DE) at 0, 0.5, 1, 2, 4 and 10 g kg−1 after 8 weeks feeding. The study indicated that dietary DE significantly improved intestinal antioxidant abilities by increasing SOD, CAT, T-AOC activities and up-regulating intestinal cat, gpx mRNA levels, but by decreasing MDA content and down-regulating intestinal keap1 mRNA levels in golden pompano. Meanwhile, dietary DE improved intestinal morphology, suggesting that enhances intestinal digestion and absorption, by increasing muscle thickness, villus length, villus width and villus number in the foregut and hindgut; as well as villus number, villus width and muscle thickness in the midgut (P < .05). Dietary DE enhanced intestinal barrier function by increasing intestinal zo-1 and occludin mRNA levels, but by decreasing the mRNA levels of claudin-12 and claudin-15. Furthermore, dietary DE improved intestinal immunity via increasing goblet cells numbers and regulating expression of immune-related genes. In conclusion, dietary DE supplementation promoted intestine health by improving intestine morphology, immunity, antioxidant abilities and intestinal barrier in golden pompano.

Lactobacillus plantarum Enhanced IL-22 Production in Natural Killer (NK) Cells That Protect the Integrity of Intestinal Epithelial Cell Barrier Damaged by Enterotoxigenic Escherichia coli.

Interleukin (IL)-22-producing Natural Killer (NK) cells protect the gut epithelial cell barrier from pathogens. A strain of probiotics, Lactobacillus plantarum (L. plantarum, LP), was previously found by our laboratory to significantly improve the mucosal barrier integrity and function of the small intestine in pigs. However, it was unclear whether LP benefited the intestinal mucosal barrier via interactions with the intestinal NK cells. The present study, therefore, was focused on the therapeutic effect of NK cells that were stimulated by LP on attenuating enterotoxigenic Escherichia coli (ETEC)-induced the damage to the integrity of the epithelial cell barrier. The results showed that LP can efficiently increase protein levels of the natural cytotoxicity receptor (NCR) family, and the expression levels of IL-22 mRNA and protein in NK cells. Transfer of NK cells stimulated by LP conferred protection against ETEC K88-induced intestinal epithelial barrier damage in NCM460 cells. We found that NK cells stimulated by LP could partially offset the reduction in NCM460 cell monolayers transepithelial electrical resistance (TEER) caused by ETEC K88, and increase ZO-1 and occludin mRNA and protein expressions by ETEC K88-infected NCM460 cells. Furthermore, adding NK cells that were stimulated by LP to ETEC K88-infected NCM460cells, IL-22R1, p-Stat3, and p-Tyk2 expression by NCM460 cells was increased. Mechanistic experiment showed that NK cells stimulated by LP lost the function of maintaining TEER of NCM460 cells challenged with ETEC K88, when polyclonal anti-IL-22 antibody was used to block IL-22 production. Collectively, our results suggested that LP stimulation of NK could enhance IL-22 production, which might be able to provide defense against ETEC-induced damage to the integrity of intestinal epithelial barrier.

Respiratory syncytial virus infection influences tight junction integrity.

Respiratory syncytial virus (RSV) is an important risk factor of asthma development and is responsible for severe respiratory tract infections. However, the influence of RSV infection on barrier function of bronchial epithelial cells in vitro and in vivo is still unclear. The aim of this study was to analyse the role of RSV in tight junction (TJ) regulation and to compare epithelial integrity between asthmatic and healthy individuals upon RSV infection. Healthy and asthmatic human bronchial epithelial cells (HBECs) were differentiated at air-liquid interface (ALI) and infected with RSV and ultraviolet (UV)-irradiated RSV. TJ expression and their integrity were analysed by quantitative polymerase chain reaction (qPCR), transepithelial resistance (TER) and paracellular flux. To determine the effect in vivo, BALB/c mice were infected intranasally with RSV or UV-irradiated RSV A2. Bronchoalveolar lavage and TJ integrity were analysed on days 1, 2, 4 and 6 post-infection by qPCR, bioplex and confocal microscopy. RSV increased barrier integrity in ALI cultures of HBEC from healthy subjects, but no effect was found in HBECs from asthmatics. This was not associated with an increase in TJ mRNA expression. In vivo, RSV induced lung inflammation in mice and down-regulated claudin-1 and occludin mRNA expression in whole lungs. Surprisingly, RSV infection was not observed in bronchial epithelial cells, but was found in the lung parenchyma. Decreased expression of occludin upon RSV infection was visible in mouse bronchial epithelial cells in confocal microscopy. However, there was no regulation of claudin-1 and claudin-7 at protein level.

Long-term exposure to ethanol downregulates tight junction proteins through the protein kinase Cα signaling pathway in human cerebral microvascular endothelial cells.

Brain microvascular endothelial cells (BMECs) are the primary component of the blood-brain barrier (BBB). Tight junction (TJ) proteins, including claudin, occludin and zonula occludens (ZO)-1, ZO-2 and ZO-3, maintain the structural integrity of BMECs. Ethanol activates the assembly and disassembly of TJs, which is a process that is regulated by protein kinase C (PKC). In addition, ethanol treatment leads to the loss of structural integrity, which damages the permeability of the BBB and subsequently affects central nervous system homeostasis, thus allowing additional substances to enter the brain. However, the mechanisms underlying ethanol-induced loss of BBB structure remain unknown. It has been hypothesized that long-term exposure to ethanol reduces the expression of claudin-5, occludin and ZO-1 via the PKC signaling pathway, thereby affecting BBB structural integrity. In the current study, the human cerebral microvascular endothelial cell line, HCMEC/D3, was treated with 50, 100, 200 and 400 mM ethanol for 24, 48 and 72 h. Cell viability was determined using an MTS assay. The expression of claudin-5, occludin and ZO-1 protein and mRNA was measured using western blot analysis and reverse transcription-quantitative polymerase chain reaction, respectively. Following the pretreatment of HCMEC/D3 cells with the PKCα-specific inhibitor, safingol (10 µmol/l), the expression of claudin-5, occludin, ZO-1 and phosphorylated (p)-PKCα was measured using western blot analysis, and PKCα localization was determined by immunofluorescence. With increasing concentrations of ethanol, the expression of claudin-5, occludin and ZO-1 protein decreased, while the expression of claudin-5, occludin and ZO-1 mRNA increased. Exposure to ethanol significantly increased the expression of p-PKCα, whereas no significant effect on the expression of PKCα was observed. Following 48 h treatment with 200 mM ethanol, the expression of claudin-5, occludin and ZO-1 protein was significantly decreased when compared with the control. By contrast, the expression of p-PKCα was increased, and increased translocation of PKCα from the cytoplasm to the nuclear membrane and nucleus was observed. In addition, the results demonstrated that safingol significantly reversed these effects of ethanol. In conclusion, long-term exposure to ethanol downregulates the expression of claudin-5, occludin and ZO-1 protein in HCMEC/D3 s, and this effect may be mediated via activation of PKCα.