Zona Occludin Research Articles

Similarities and differences between brain and skin GNAQ p.R183Q driven capillary malformations.

Capillary malformations (CM) are congenital vascular irregularities of capillary and venous blood vessels that appear in the skin, leptomeninges of the brain, and the choroid of the eye in the disorder known as Sturge Weber Syndrome (SWS). More common are non-syndromic CM found only in the skin, without brain or ocular involvement. A somatic activating mutation in GNAQ (p.R183Q) is found in ~ 90% of syndromic and non-syndromic CM specimens and is present in CD31pos endothelial cells isolated from brain and skin CM specimens. Endothelial expression of the GNAQ p.R183Q variant is sufficient to form CM-like vessels in mice. Given the distinct features and functions of blood vessels in the brain versus the skin, we examined the features of CM vessels in both tissues to gain insights into the pathogenesis of CM. Herein, we present morphologic characteristics of CM observed in specimens from brain and skin. The GNAQ p.R183Q variant allelic frequency in each specimen was determined by droplet digital PCR. Sections were stained for endothelial cells, tight junctions, mural cells, and macrophages to assess the endothelium as well as perivascular constituents. CM blood vessels in brain and skin were enlarged, exhibited fibrin leakage and reduced zona occludin-1 and claudin-5, and were surrounded by MRC1pos/LYVE1pos macrophages. In contrast, the CMs from brain and skin differ in endothelial sprouting activity and localization of mural cells. These characteristics might be helpful in the development of targeted and/or tissue specific therapies to prevent or reverse non-syndromic and syndromic CM.

Similarities and differences between brain and skin GNAQ p.R183Q driven capillary malformations.

Capillary malformations (CM) are congenital vascular irregularities of capillary and venous blood vessels that appear in the skin, leptomeninges of the brain, and the choroid of the eye in the disorder known as Sturge Weber Syndrome (SWS). More common are non-syndromic CM found only in the skin, without brain or ocular involvement. A somatic activating mutation in GNAQ (p.R183Q) is found in ~90% of syndromic and non-syndromic CM specimens and is present in CD31pos endothelial cells isolated from brain and skin CM specimens. Endothelial expression of the GNAQ p.R183Q variant is sufficient to form CM-like vessels in mice. Given the distinct features and functions of blood vessels in the brain versus the skin, we examined the features of CM vessels in both tissues to gain insights into the pathogenesis of CM. Herein, we present morphologic characteristics of CM observed in specimen from brain and skin. The GNAQ p.R183Q variant allelic frequency in each specimen was determined by droplet digital PCR. Sections were stained for endothelial cells, tight junctions, mural cells, and macrophages to assess the endothelium as well as perivascular constituents. CM blood vessels in brain and skin were enlarged, exhibited fibrin leakage and reduced zona occludin-1, and were surrounded by MRC1pos/LYVE1pos macrophages. In contrast, the CMs from brain and skin differ in endothelial sprouting activity and localization of mural cells. These characteristics might be helpful in the development of targeted and/or tissue specific therapies to prevent or reverse non-syndromic and syndromic CM.

Protective effect of synbiotic combination of Lactobacillus plantarum SC-5 and olive oil extract tyrosol in a murine model of ulcerative colitis

BackgroundUlcerative colitisis (UC) classified as a form of inflammatory bowel diseases (IBD) characterized by chronic, nonspecific, and recurrent symptoms with a poor prognosis. Common clinical manifestations of UC include diarrhea, fecal bleeding, and abdominal pain. Even though anti-inflammatory drugs can help alleviate symptoms of IBD, their long-term use is limited due to potential side effects. Therefore, alternative approaches for the treatment and prevention of inflammation in UC are crucial.MethodsThis study investigated the synergistic mechanism of Lactobacillus plantarum SC-5 (SC-5) and tyrosol (TY) combination (TS) in murine colitis, specifically exploring their regulatory activity on the dextran sulfate sodium (DSS)-induced inflammatory pathways (NF-κB and MAPK) and key molecular targets (tight junction protein). The effectiveness of 1 week of treatment with SC-5, TY, or TS was evaluated in a DSS-induced colitis mice model by assessing colitis morbidity and colonic mucosal injury (n = 9). To validate these findings, fecal microbiota transplantation (FMT) was performed by inoculating DSS-treated mice with the microbiota of TS-administered mice (n = 9).ResultsThe results demonstrated that all three treatments effectively reduced colitis morbidity and protected against DSS-induced UC. The combination treatment, TS, exhibited inhibitory effects on the DSS-induced activation of mitogen-activated protein kinase (MAPK) and negatively regulated NF-κB. Furthermore, TS maintained the integrity of the tight junction (TJ) structure by regulating the expression of zona-occludin-1 (ZO-1), Occludin, and Claudin-3 (p < 0.05). Analysis of the intestinal microbiota revealed significant differences, including a decrease in Proteus and an increase in Lactobacillus, Bifidobacterium, and Akkermansia, which supported the protective effect of TS (p < 0.05). An increase in the number of Aspergillus bacteria can cause inflammation in the intestines and lead to the formation of ulcers. Bifidobacterium and Lactobacillus can regulate the micro-ecological balance of the intestinal tract, replenish normal physiological bacteria and inhibit harmful intestinal bacteria, which can alleviate the symptoms of UC. The relative abundance of Akkermansia has been shown to be negatively associated with IBD. The FMT group exhibited alleviated colitis, excellent anti-inflammatory effects, improved colonic barrier integrity, and enrichment of bacteria such as Akkermansia (p < 0.05). These results further supported the gut microbiota-dependent mechanism of TS in ameliorating colonic inflammation.ConclusionIn conclusion, the TS demonstrated a remission of colitis and amelioration of colonic inflammation in a gut microbiota-dependent manner. The findings suggest that TS could be a potential natural medicine for the protection of UC health. The above results suggest that TS can be used as a potential therapeutic agent for the clinical regulation of UC.

Reduced dietary Ca, Cu, Zn, Mn, and Mg bioavailability but increased Fe bioavailability with polyethylene microplastic ingestion in a mouse model: Changes in intestinal permeability and gut metabolites

Microplastics emerge as a new environmental and human health crisis. Minimal research exists on effects of microplastic ingestion on the oral bioavailability of minerals (Fe, Ca, Cu, Zn, Mn, and Mg) in the gastrointestinal tract via impacting intestinal permeability, mineral transcellular transporters, and gut metabolites. Here, mice were exposed to polyethylene spheres of 30 and 200 μm (PE-30 and PE-200) in diet (2, 20, and 200 μg PE g−1) for 35 d to determine the microplastic effects on mineral oral bioavailability. Results showed that for mice fed diet amended with PE-30 and PE-200 at 2–200 μg g−1, Ca, Cu, Zn, Mn, and Mg concentrations in the small intestine tissue were 43.3–68.8 %, 28.6–52.4 %, 19.3–27.1 %, 12.9–29.9 %, and 10.2–22.4 % lower compared to control mice, suggesting hampered bioavailability of these minerals. In addition, Ca and Mg concentrations in mouse femur were 10.6 % and 11.0 % lower with PE-200 at 200 μg g−1. In contrast, Fe bioavailability was elevated, as suggested by significantly (p < 0.05) higher Fe concentration in the intestine tissue of mice exposed to PE-200 than control mice (157–180 vs. 115 ± 7.58 μg Fe g−1) and significantly (p < 0.05) higher Fe concentrations in liver and kidney with PE-30 and PE-200 at 200 μg g−1. Following PE-200 exposure at 200 μg g−1, genes coding for duodenal expression of tight junction proteins (e.g., claudin 4, occludin, zona occludins 1, and cingulin) were significantly up-regulated, possibility weakening intestinal permeability to Ca, Cu, Zn, Mn, and Mg ions. The elevated Fe bioavailability was possibly related to microplastic-induced greater abundances of small peptides in the intestinal tract, which inhibited Fe precipitation and elevated Fe solubility. Results showed that microplastic ingestion may cause Ca, Cu, Zn, Mn, and Mg deficiency but Fe overload via altering intestinal permeability and gut metabolites, posing a threat to human nutrition health.

Abstract 164: Development of a living biobank of breast cancer organoids

Abstract Introduction: Breast cancer is one of the most frequently diagnosed cancers among women, with a high mortality rate. More than 20 different subtypes of breast cancer are identified. Advancement in patient-derived organoid technology makes it possible to preserve cellular, structural, and tissue microenvironment, which mimics the tissue in vivo. The present study aims to develop a cryopreserved (living) tumor biobank from patient-derived tumor breast tissues. Eventually, biobank organoids will be used to test treatment options outside of patients, in a dish, and provide information on what treatments the patient responds to. Method: Patient-derived organoids (PDOs) were bio-fabricated using an unsorted tumor and normal cell suspension in a collagen-based hydrogel. Immune-enhanced tumor organoids (iTOs) were also bio-fabricated using patient-matched PMBCs and tumor cells. Breast cancer living biobanks were developed by freezing organoids in cryopreservation solutions and reestablishing them in growth media compatible with breast organoids. Histological staining for hormone receptors and Her2 receptor markers was performed. Organoids were cultured for seven days, followed by treatment with chemotherapy (Paclitaxel, Doxorubicin, and Sulphonamide) and immunotherapy (Nivolumab, Pembrolizumab, and Atezolizumab), and assessed for cell viability. Results: H&amp;E staining showed the characteristics of the breast tissue with well-defined acini. In tumor organoids, acini were somewhat perturbed compared to normal breast organoids. Immunofluorescence staining showed the expression of breast biomarkers, including EGF receptor 2 (HER2), Progesterone receptor (PR), and Estrogen receptor (ER). Zona occludin 2 and keratin 19 expression in luminal cells and expression of Keratin 14 and P63 in basal cells, suggesting correct polarization in the organoids. Immunofluorescence staining of iTOs, with T cell markers including CD3, CD4, and CD8, indicated that immune cells remained viable in the iTOs. Drug responses to Doxorubicin, Paclitaxel, Sulphonamide and a combination of Doxorubicin-Paclitaxel showed significant inhibition of cell growth in normal and tumor organoids (p&amp;lt; 0.04). Treatment of breast iTOs showed moderate responses to nivolumab (n=11) and pembrolizumab (n=5), with a 63%-100% efficacy (P &amp;lt; 0.05) respectively. No responses were observed for Atezolizumab (n=5). Drug responses of biobank organoids showed comparable drug sensitivities to their fresh, non-frozen counterparts, suggesting that biobank organoids retain physiological relevant information and can be used for testing new drug therapies. Conclusion: Breast cancer organoids recapitulate the histological features of breast tissue in culture and respond to chemotherapy and immunotherapy. In the future, patient-derived breast tumor organoids can be cryopreserved and serve as living biobanks to provide a platform for personalized medicine. Citation Format: Nadeem Wajih, Richard Erali, Edward A. Levine, Konstantinos Votanopoulos, Shay Soker. Development of a living biobank of breast cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 164.

Abstract P3-09-02: Pateint Derived Breast Cancer Organoids as a Model for Testing Personalized Therapies

Abstract Introduction: Breast cancer is one of the most frequently diagnosed cancer in women with a high mortality rate. More than 20 different subtypes of breast cancer are identified. Advancement in patient-derived organoid technology makes it possible to preserve cellular, structural, and tissue microenvironment which mimics the tissue in vivo. Method: We developed patient derived breast organoids both from normal and cancer biopsies recapitulate the structure of breast tissue. Breast biopsies were enzymatically digested to yield a single cell suspension. About 1 × 105 cells were encapsulated in a specialized hydrogel mixture. Immune enhanced breast tumor organoids (iTOs) included 2 × 105 patient matched PMBCs. Results: Histological analyses of the breast organoids shows the characteristics of the breast tissue with well-defined acini in H&amp;E. In tumor organoids acini were somewhat perturbed compared to normal breast organoids. Immuno-fluorescence staining shows the expression of breast biomarkers including EGF receptor 2 (HER2), Progesterone receptor (PR), Estrogen receptor (ER). Zona occludin 1 and 2 and keratin 19 expression in luminal cells and expression of Keratin 14 and P63 in basal cells suggested correct polarization in the organoids. Immunofluorescence staining of (iTOs), with T cell markers including CD3, CD4, and CD8 indicated that immune cells remained viable in the iTOs. Drug responses to Doxorubicin, Paclitaxel and a combination of Doxorubicin-Paclitaxel showed significant inhibition of cell growth in normal and tumor organoids (p&amp;lt; 0.04, n=11) except for Paclitaxel failed to inhibit tumor cell growth. However, immunotherapy with nivolumab (anti PD-1) showed no significant effect on cell growth. Conclusions: Pateint Derived Breast cancer organoids recapitulate the histological features of breast tissue in culture and response to chemotherapies. In the future, patient-derived tumor organoids can provide a platform for personalized medicine. Citation Format: Nadeem Wajih, Richard Erali, Steven Forsythe, Shay Soker, Konstantinos Votanopoulos. Pateint Derived Breast Cancer Organoids as a Model for Testing Personalized Therapies [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-09-02.

Transplantation of Human Placenta Derived Mitochondria Promotes Cell Communication in Endometrium in a Murine Model of Disturbed Endometrium.

Herein, we investigated the regenerative potential of functional mitochondria to restore endometrial injury. The endometrium was disturbed with an intrauterine injection of 95% ethanol. Regeneration of the disturbed endometrium was achieved by transplantation of human placenta derived mitochondria followed by thrombin activated platelet rich plasma (hMTx). The transplantation method provided a biomimetic gel layer that stabilized and supported the functionality of the transplanted mitochondria to flourish regeneration of the disturbed endometrium. The presence of engrafted Rhodamine B labelled mitochondria was quantified at 12, 24, 48, and 72h after transplantation. Detection of human-specific mitochondria mRNA in recipient rat uterus showed significant up-regulation of MT ATP-8, MT COX-1, MT COX -3, MT COX -2, MT ATP-6 (p = 0.009) in the hMTx treated group compared to the disturbed endometrium group. The hMTx group demonstrated showed regeneration through increased expressions of α-SMA, CK-18, CK-19, Connexin-40, E Cadherin, Claudin-1, Zona Occludin as compared with disturbed endometrium group. Experimental hMTx endometrial cells had significantly higher values of activities of NADH, NADPH, Cytochrome B5, Cytochrome P450, Complex I, Complex II, Complex III, Complex IV compared with disturbed endometrium indicating the regeneration of damaged endometrial cells at 72h. Intrauterine hMTx was accounted to improve endometrial junction protein thus regeneration in the disturbed endometrium. Our Data provide the first evidence that hMTx promotes endometrial regeneration in the disturbed endometrium, paving the way for the development of a novel approach to human endometrial regeneration.

Extracellular Alterations in pH and K+ Modify the Murine Brain Endothelial Cell Total and Phospho-Proteome.

Pathologies of the blood–brain barrier (BBB) have been linked to a multitude of central nervous system (CNS) disorders whose pathology is poorly understood. Cortical spreading depression (CSD) has long been postulated to be involved in the underlying mechanisms of these disease states, yet a complete understanding remains elusive. This study seeks to utilize an in vitro model of the blood–brain barrier (BBB) with brain endothelial cell (b.End3) murine endothelioma cells to investigate the role of CSD in BBB pathology by characterizing effects of the release of major pronociceptive substances into the extracellular space of the CNS. The application of trans-endothelial electrical resistance (TEER) screening, transcellular uptake, and immunoreactive methods were used in concert with global proteome and phospho-proteomic approaches to assess the effect of modeled CSD events on the modeled BBB in vitro. The findings demonstrate relocalization and functional alteration to proteins associated with the actin cytoskeleton and endothelial tight junctions. Additionally, unique pathologic mechanisms induced by individual substances released during CSD were found to have unique phosphorylation signatures in phospho-proteome analysis, identifying Zona Occludins 1 (ZO-1) as a possible pathologic “checkpoint” of the BBB. By utilizing these phosphorylation signatures, possible novel diagnostic methods may be developed for CSD and warrants further investigation.

Variations in the Composition of Human Milk Oligosaccharides Correlates with Effects on Both the Intestinal Epithelial Barrier and Host Inflammation: A Pilot Study.

Background: Human milk oligosaccharides are complex, non-digestible carbohydrates that directly interact with intestinal epithelial cells to alter barrier function and host inflammation. Oligosaccharide composition varies widely between individual mothers, but it is unclear if this inter-individual variation has any impact on intestinal epithelial barrier function and gut inflammation. Methods: Human milk oligosaccharides were extracted from the mature human milk of four individual donors. Using an in vitro model of intestinal injury, the effects of the oligosaccharides on the intestinal epithelial barrier and select innate and adaptive immune functions were assessed. Results: Individual oligosaccharide compositions shared comparable effects on increasing transepithelial electrical resistance and reducing the macromolecular permeability of polarized (Caco-2Bbe1) monolayers but exerted distinct effects on the localization of the intercellular tight junction protein zona occludins-1 in response to injury induced by a human enteric bacterial pathogen Escherichia coli, serotype O157:H7. Immunoblots showed the differential effects of oligosaccharide compositions in reducing host chemokine interleukin 8 expression and inhibiting of p38 MAP kinase activation. Conclusions: These results provide evidence of both shared and distinct effects on the host intestinal epithelial function that are attributable to inter-individual differences in the composition of human milk oligosaccharides.

Short-Chain Naphthoquinone Protects Against Both Acute and Spontaneous Chronic Murine Colitis by Alleviating Inflammatory Responses

Ulcerative colitis (UC) is characterised by chronic, relapsing, idiopathic, and multifactorial colon inflammation. Recent evidence suggests that mitochondrial dysfunction plays a critical role in the onset and recurrence of this disease. Previous reports highlighted the potential of short-chain quinones (SCQs) for the treatment of mitochondrial dysfunction due to their reversible redox characteristics. We hypothesised that a recently described potent mitoprotective SCQ (UTA77) could ameliorate UC symptoms and pathology. In a dextran sodium sulphate- (DSS-) induced acute colitis model in C57BL/6J mice, UTA77 substantially improved DSS-induced body weight loss, disease activity index (DAI), colon length, and histopathology. UTA77 administration also significantly increased the expression of tight junction (TJ) proteins occludin and zona-occludin 1 (ZO-1), which preserved intestinal barrier integrity. Similar responses were observed in the spontaneous Winnie model of chronic colitis, where UTA77 significantly improved DAI, colon length, and histopathology. Furthermore, UTA77 potently suppressed elevated levels of proinflammatory cytokines and chemokines in colonic explants of both DSS-treated and Winnie mice. These results strongly suggest that UTA77 or its derivatives could be a promising novel therapeutic approach for the treatment of human UC.

Astaxanthin mediated regulation of VEGF through HIF1α and XBP1 signaling pathway: An insight from ARPE-19 cell and streptozotocin mediated diabetic rat model

Breakdown of outer blood-retina barrier (BRB) has been associated with the pathogenesis of diabetic retinopathy (DR) and diabetic macular edema (DME). Vascular endothelial growth factor (VEGF) might play a detrimental role in the pathogenesis of DME, a major clinical manifestation of DR. In the present study, we investigated the inhibitory mechanism of astaxanthin on VEGF and its upstream signaling pathways under in vitro and in vivo conditions. Astaxanthin has been observed to downregulate VEGF expression under hyperglycemic (HG) and CoCl2 induced hypoxic conditions in ARPE-19 cells. There were compelling pieces of evidence for the involvement of transcription factors like HIF1α and XBP1 in the upregulation of VEGF under HG and hypoxic conditions. Thus, we investigated the role of astaxanthin in the expression and nuclear translocation of HIF1α and XBP1. The activation and translocation of HIF1α and XBP1 induced by HG or CoCl2 conditions were hindered by astaxanthin. Additionally, treatment with HIF1α siRNA and IRE1 inhibitor STF-083010 also inhibited the expression of VEGF induced by HG and CoCl2 conditions. These results indicated that the anti-VEGF property of astaxanthin might be associated with the downregulation of HIF1α and XBP1. Furthermore, astaxanthin mitigated the enhanced migration of retinal pigment epithelial (RPE) cells under DR conditions. As well, astaxanthin protected disorganization of zona occludin-1 (ZO-1) tight junction protein in RPE and reduced HG or hypoxic induced permeability of RPE cells. In streptozotocin-induced diabetic rat model, astaxanthin reduced the expression of HIF1α, XBP1, and VEGF as well as protected the abnormalities in the retinal layers induced by diabetes condition. Thus, astaxanthin may be used as a potential nutraceutical to prevent or treat retinal dysfunction in diabetic patients.

Quercetin: a savior of alveolar barrier integrity under hypoxic microenvironment

ABSTRACT High altitude pulmonary edema (HAPE) is generally characterized by the loss of alveolar epithelial barrier integrity. The current study was undertaken to assess the noninvasive approaches of HAPE diagnosis and to evaluate the prophylactic potential of quercetin in preventing alveolar junction impairments. Male SD rats fed with quercetin 1 h prior to hypoxia (7,620 m, for 6 h) were selected. PET/CT imaging was performed to visualize the lung uptake of 18F-FDG in animals under hypoxia. Further, oxidant status, catalase activity, hematological & blood gas parameters were evaluated. Moreover, tight junction (TJ) proteins (ZO-1, JAM-C, Claudin-4, and occludin) expression analysis was accomplished using immune-blotting. The structural differences in lung epithelia were noted by TEM imaging. Quercetin prophylaxis has significantly reduced the FDG uptake in rat lungs under hypoxia. It has also dramatically alleviated the protein oxidation followed by an elevation in catalase activity in the lungs under hypoxia. The TJ protein expression in the lungs has also been restored to normal upon quercetin pre-treatment. Concomitantly, the quercetin preconditioning has elicited the stable blood gas and hematological parameters under hypoxia. The observations from TEM imaging have also implicated the normal lung epithelial structures in the quercetin pretreated animals under hypoxia. Quercetin prophylaxis has significantly restored alveolar epithelium integrity by abating oxidative stress in the lungs under hypoxia. Abbreviations: CT- Computed Tomography 18F-FDG- Fluorodeoxyglucose (18FHAPE- High Altitude Pulmonary EdemaHb- HemoglobinHCT- HematocritHCO3 – BicarbonateJAM- Junctional Adhesion MoleculeKBq- Killo BecquerelPaO2 - Partial pressure of arterial oxygenPaCO2 - Partial pressure of arterial carbon di-oxidePET- Positron Emission TomographyRBC- Red Blood CorpusclesSD- Sprague DawleyTJ- Tight JunctionsTEM- Transmission Electron MicroscopyWBC- White Blood CorpusclesZO- Zona Occludin.

Inclusion formation in neonatal enterocytes lacking Myosin Vb is associated with apical accumulation of tight junction proteins

BackgroundMicrovillus inclusion disease (MVID) is caused by inactivating mutations in the molecular motor Myosin Vb (Myo5B). MVID is characterized by the presence of intracellular inclusions lined by microvilli in enterocytes. In germline Myosin Vb knockout mice (Myo5b KO) inclusions form from invagination of the apical brush border via apical bulk endocytosis. Crumbs 3 regulates apical membrane formation, intestinal epithelial polarity and tight junction assembly. As a result, we speculate that Crumbs 3 and junctional proteins are involved in inclusion formation in our Myo5b KO mice. While some aspects of inclusion formation and altered enterocyte polarity have been previously characterized in Myo5b KO mice, the localization and role of tight junction proteins during inclusion formation is currently unknown. Thus, we sought to elucidate the role of junctional proteins during the formation and excision of inclusions from the apical membrane in Myo5b KO mice.Methods &amp; ResultsIntestinal tissue collected from neonatal control and Myo5b KO littermates were analyzed by immunofluorescence to determine the localization of apical proteins and tight junction proteins. In the neonatal intestine the tight junction protein, Claudin 2, provides a pathway for the paracellular transport of sodium, potassium and water. Immunostaining for Claudin 2, which is expressed in neonatal villus enterocytes, showed normal localization at the apical junctional complex of neighboring intestinal epithelial cells in control mice. In Myo5b KO mice, Claudin 2 was present intracellularly as puncta and was associated with inclusions, identified by gamma‐actin staining, that were forming at the apical membrane. Occludin immunofluorescence revealed a similar pattern in Myo5b KO mice in which occludin appeared to be concentrated immediately above the site of inclusion formation. In control mice, zona occludin‐1 (ZO‐1) was observed at the apical junctional complex between intestinal cells as expected. In contrast, ZO‐1 appeared concentrated over inclusions that were still associated with the brush border in Myo5b KO mice. Finally, immunostaining for Crumbs 3 showed Crumbs 3 present on the apical brush border in enterocytes in control mice, while Myo5b KO mice exhibited Crumb 3‐positive inclusions below the apical membrane.ConclusionsCollectively, these data demonstrate that inclusion formation in neonatal Myo5b KO enterocytes is associated with collection of junctional proteins. The data suggest two possible mechanisms: either inclusions are forming at the apical junction of intestinal epithelial cells or alternatively inclusion formation recruits junctional components.(A) Claudin 2 (red) and Occludin (green) are restricted to the apical junctional complex and gamma‐actin (white) labels the apical membrane in control enterocytes. In Myo5b KO enterocytes gamma‐actin rich inclusions forming from the brush border had an accumulation of Claudin 2 and Occludin. (B) Myo5b KO enterocytes showed ZO‐1 enrichment (red) immediately above forming inclusions. (C &amp; D) In Myo5b KO mice Crumbs 3 (red) was closely associated with forming and internalized inclusions.Figure 1

Protective Effect of Naringin on In Vitro Gut-Vascular Barrier Disruption of Intestinal Microvascular Endothelial Cells Induced by TNF-α.

Naringin is a polymethoxylated flavonoid commonly found in citrus species and has therapeutic potential in intestinal disorders. However, the effect and mechanism of naringin on gut-vascular barrier disruption has not yet been reported. This study aimed to investigate the distinguishing and selectively protective effects of naringin on tumor necrosis factor (TNF)-α-induced gut-vascular barrier disruption and elucidate the potential mechanism. In the present study, an in vitro gut-vascular barrier model composed of rat intestinal microvascular endothelial cells (RIMVECs) was studied. Evans blue-albumin efflux assay showed that naringin (50 μM) evidently protected the integrity of RIMVEC monolayer barriers against TNF-α-induced disruption. Naringin maintained the expression and distribution of tight junction proteins including zona occludin-1, occludin, claudin-1, and claudin-2. Additionally, naringin protected RIMVECs from TNF-α-induced apoptosis and cell migration suppression (41.1 ± 2.2 vs 51.1 ± 3.5%; 61.0 ± 5.1 vs 72.2 ± 6.2%). Our results indicate that naringin effectively ameliorates gut-vascular barrier disruption.

Konjac glucomannan polysaccharide and inulin oligosaccharide enhance the colonic mucosal barrier function and modulate gut-associated lymphoid tissue immunity in C57BL/6J mice.

Both konjac glucomannan (KGM) and inulin oligosaccharide have been shown to improve bowel function, but their effects on the mucosal barrier function and immunity are not fully understood. The aim of the present study was to determine the effects of a low-level supplementation of dietary fibres on the colonic mucosal barrier function, antioxidant enzyme defence and immunity. C57BL/6J mice (6 weeks of age, eight per group) were randomly assigned to consume one of the following diets: control or control diet supplemented with 2 % (w/w) of KGM, inulin oligosaccharide (degree polymerisation = 8) or KGM+inulin (1 %, w/w each (K+I)). Fresh faeces were collected on days 19-21. Mice were killed on day 22 after fasting. Segments of colon tissues were processed for histological procedure and stained for acidic mucins and tight junction protein marker zona occludin-1 (ZO-1). The remaining tissues were processed to determine the gene expression of mucin 2, tight junction proteins, antioxidant enzymes and cytokines. The plasma cytokines were measured. Results indicated that KGM, inulin and K+I significantly increased the mucosal layer thickness, mucin density (granule number/crypt) and gene expression of Muc2 as compared with the control. All fibre treatments increased the gene expressions of ZO-1, occludin, glutathione peroxidase, glutathione S-transferase π, catalase and IL-10. In addition, all fibre treatments increased faecal butyrate and probiotics, and plasma IL-10 concentrations. In conclusion, supplementation of low-level, 2 % (w/w), of K+I was sufficient to enhance the mucosal barrier function and anti-inflammatory status.

Elafibranor Inhibits Chronic Kidney Disease Progression in NASH Mice.

Identification of new pharmacological approaches to inhibit the excessive fat intake-induced steatohepatitis and chronic kidney disease (CKD) is important. High-fat diet (HFD)-induced steatohepatitis and CKD share common pathogenesis involving peroxisome proliferator-activated receptor (PPAR)-α and -δ. Elafibranor, a dual PPARα/δ agonist, can ameliorate the HFD-induced steatohepatitis. Nonetheless, the effects of HFD-induced CKD had not yet explored. This study investigated the effects of elafibranor (elaf) on the progression of HFD-induced CKD in mice. In vivo and in vitro renal effects were evaluated in HFD-elaf mice receiving 12 weeks of elafibranor (from 13th to 24th week of HFD feeding) treatment. In elafibranor-treated HFD mice, increased insulin sensitivity, reduced obesity and body fat mass, decreased severity of steatohepatitis, increased renal expression of PPARα, PPARδ, SIRT1, and autophagy (Beclin-1 and LC3-II) as well as glomerular/renal tubular barrier markers [synaptopodin (podocyte marker), zona occludin-1, and cubulin], reduced renal oxidative stress and caspase-3, and less urinary 8-isoprostanes excretion were observed. Aforementioned benefits of elafibranor were associated with low renal tubular injury and tubulointerstitial fibrosis scores, less albuminuria, low urinary albumin-to-creatinine ratio, and preserved glomerular filtration rate. Acute incubation of podocytes and HK-2 cells with elafibranor or recombinant SIRT1 reversed the HFD-sera-induced oxidative stress, autophagy dysfunction, cell apoptosis, barrier marker loss, albumin endocytosis, and reuptake reduction. Besides hepatoprotective and metabolic beneficial effects, current study showed that elafibranor inhibited the progression of HFD-induced CKD through activation of renal PPARα, PPARδ, SIRT1, autophagy, reduction of oxidative stress, and apoptosis in mice with steatohepatitis.

Rumen-derived lipopolysaccharide induced ruminal epithelium barrier damage in goats fed a high-concentrate diet

This study aimed to investigate the mechanism of lipopolysaccharide (LPS) released in the rumen on epithelium barrier function of goats fed a HC diet. Twelve Boer goats were randomly divided into two groups: low-concentrate(LC) diet and high-concentrate(HC) diet treatment. We found that the pH of rumen fluid in the HC group was lower than in the LC group (P < 0.05). The mRNA and protein expression levels of p38 mitogen-activated protein kinase (MAPK), extracellular regulated protein kinases (ERK), and c-Jun N-terminal kinase (JNK) in the rumen epithelium were lower in the LC group than the HC group (P < 0.05). Gene expression and protein levels of the tight junction proteins claudin-1, claudin-4, occludin, and Zona occludin-1 were all greater in the LC group than the HC group (P < 0.05). Staining of claudin-1, occludin and ZO-1 was became irregular. In conclusion, high concentrate diet feeding can impair rumen epithelium function and decrease tight junction protein expression through MAPK signaling pathway.

Roux-en-Y gastric bypass decreases endotoxemia and inflammatory stress in association with improvements in gut permeability in obese diabetic rats.

Postoperative modulation of the gut microbiome has been suggested to contribute to the metabolic benefits after metabolic surgery, but the mechanisms underlying these metabolic benefits remain unknown. Previously, we reported that Roux-en-Y gastric bypass (RYGB) surgery in Zucker diabetic fatty (ZDF) rats increased the abundance of Proteobacteria and Gammaproteobacteria. However, theoretically, these Gram-negative bacteria may elevate lipopolysaccharide (LPS) levels. Therefore, in this study we further investigated the potential mechanisms by which RYGB improves glucose homeostasis, endotoxemia, and inflammatory stress in ZDF rats. Rats were divided into three groups: (a) an RYGB group (RY); (b) a sham-operated group pair-fed with the RY group; and (c) a sham-operated group fed ad libitum. Changes in LPS, cytokine levels, intestinal permeability (evaluated using the fluorescein isothiocyanate-dextran method), and intestinal epithelial tight junction proteins zona occludins (ZO)-1, occludin, and claudin-1 were assessed 10 weeks postoperatively. Rats that underwent RYGB exhibited sustained weight loss and reduced glucose, as well as lower cytokine and LPS concentrations, than rats in the control groups. In the colonic epithelium, ZO1 and claudin-1 (Cldn1) mRNA levels were higher in the RY than control groups. Intestinal permeability declined in the RY group and was positively correlated with LPS levels and negatively correlated with ZO-1, occludin, and claudin-1 expression. The results demonstrate that RYGB can reduce the extent of endotoxemia and inflammation, which is associated with improved tight junction integrity and intestinal barrier strength. These effects may explain why a low level of inflammation is maintained after RYGB and the postoperative increase in Gram-negative bacteria.

Linoorbitides and enterolactone mitigate inflammation-induced oxidative stress and loss of intestinal epithelial barrier integrity

Barrier integrity dysfunction and oxidative stress are considered hallmarks of inflammatory bowel disease (IBD) pathogenesis. Their mitigation continues to be a drug discovery target in IBD. Natural products may aid treatment of chronic inflammatory diseases, but their use in IBD requires a better understanding of whether individual bioactives may positively modulate disease course. This study investigated the ability of flax linoorbitides (LOBs) and enterolactone (ENL), to mitigate inflammation-induced loss of intestinal epithelial barrier integrity and oxidative stress in vitro. TNF-α with INF-γ and lipopolysaccharide (LPS) induced an inflammatory response in HCT-8 monoculture and Caco-2/RAW-264.7 coculture, respectively. Trans-Epithelial Electrical Resistance (TEER) and Lucifer Yellow rejection for barrier permeability were assessed in differentiated monolayers in the presence and absence of LOBs and ENL. Additionally, RAW 264.7 cells were used to assess protective effects upon induction of oxidative stress. In HCT-8 model, 200 nM of LOB-J, LOB-A, and ENL mitigated the inflammation-induced reduction in TEER with relative TEER values of 108.6%, 63.2%, and 64.2%, respectively, at 24 h relative to time zero. Similarly, at 24 h Caco-2/RAW-264.7 coculture TEER values ranged from ~200% - 243.4% for LOB-A, LOB-J, LOB-ACEJ, and ENL relative to TEER values of untreated cells. ENL and LOBs reduced malondialdehyde (MDA) lipid peroxidation in RAW 264.7 cells upon induction with lipopolysaccharide (LPS). ENL, but not LOBs, caused an increase in zona occludins 1 (ZO-1) protein expression in HCT-8 cells exposed to an inflammatory stimulus to levels comparable to negative control. Our results demonstrate after an inflammatory insult that ENL and the tested LOBs protect intestinal barrier integrity and reduce oxidative stress damage. In conclusion, use of different flax bioactives in the treatment of IBD warrants further investigation.

A cell penetrating peptide from SOCS-1 prevents ocular damage in experimental autoimmune uveitis

We describe an immunosuppressive peptide corresponding to the kinase inhibitory region (KIR) of the intracellular checkpoint protein suppressor of cytokine signaling 1 (SOCS-1) that binds to the phospho-tyrosine containing regions of the tyrosine kinases JAK2 and TYK2 and the adaptor protein MAL, and thereby inhibits signaling downstream from these signaling mediators. The peptide, SOCS1-KIR, is thus capable of downregulating overactive JAK/STAT or NF-kB signaling in somatic cells, including those in many compartments of the eye. Attachment of poly-arginine to this peptide (R9-SOCS1-KIR) allows it to penetrate the plasma membrane in aqueous media. R9-SOCS1-KIR was tested in ARPE-19 cells and was found to attenuate mediators of inflammation by blocking the inflammatory effects of IFNγ, TNFα, or IL-17A. R9-SOCS1-KIR and also protected against TNFα or IL-17A mediated damage to the barrier properties of ARPE-19 cells, as evidenced by immunostaining with the tight junction protein, zona occludin 1 (ZO-1), and measurement of transepithelial electrical resistance (TEER). Experimental autoimmune uveitis (EAU) was generated in B10. RIII mice using a peptide of interphotoreceptor retinal binding protein (IRBP161−180) as immunogen. Topical administration of R9-SOCS1-KIR, 2 days before (prophylactic), or 7 days after immunization (therapeutic) protected ocular structure and function as seen by fundoscopy, optical coherence tomography (OCT), and electroretinography (ERG). The ability R9-SOCS1-KIR to suppress ocular inflammation and preserve barrier properties of retinal pigment epithelium makes it a potential candidate for treatment of autoimmune uveitis.