Search Results (207)

Background: Endothelial cells (EC), crucial components of the vascular system, are adaptable cells that maintain homeostasis and respond to pathological events through structural and functional plasticity. Hepatocyte growth factor (HGF) is a multifunctional cytokine that has been demonstrated to have protective and disruptive influence on the blood barrier function. In endothelial biology, its role is also poorly characterized. The present study explores the impact of supraphysiological concentrations of HGF on mouse brain endothelial cells (MBECs), scrutinizing how it alters their integrity and morphology. Methods: Two groups of MBECs—control (CTR) and experimental (EXP)—were analyzed at two time points: early passage (p5) and late passage (p41). The EXP-groups (p5 and p41) were treated with HGF at a concentration of 4 µL/mL. Cellular morphology was assessed with brightfield microscopy; protein expression and localization of the tight junction marker (ZO-1) and the endothelial marker (Factor VII related antigen/von Willebrand factor, vWf) were analyzed using Western blotting, immunocytochemistry, and confocal microscopy. Intercellular barrier function was estimated via Transendothelial Electric Resistance (TEER) and Transendothelial Dextran Permeability (TEDP) assays. Results: Microscopical analysis demonstrated a change in the morphology of the MBECs from a longitudinal, spindle-like shape to a rounded, more spheroid, cobblestone-like morphology under high-dose HGF treatment. Western blotting revealed a progressive decrease of ZO-1 expression in the EXP-groups. The expression of vWf did not show significant differences. Qualitative immunocytochemical staining: vWf showed consistent expression across all groups. ZO-1 displayed a punctate, well-defined membrane and cytoplasmic localization pattern in the CTR-groups at p5 and p41. In contrast, the p5 EXP-group demonstrated a shift to a more diffuse cytoplasmic pattern. At p41, the EXP-group displayed a markedly reduced ZO-1 signal with no clear-cut membrane localization. Confocal analysis: ZO-1: punctate membrane-associated localization in CTR-groups at p5 and 41. The EXP-groups at p5 and p41 confirmed the diffuse cytoplasmic ZO-1 distribution. Phalloidin: well-organized actin cytoskeleton in CTR-groups, but rearrangement and stress fiber disorganization in the EXP-groups, especially at p41. The merged images confirmed reduced co-localization of ZO-1 with actin structures. Barrier function: TEER values dropped significantly in HGF-treated cells. TEDP to small and medium molecular weight dextran increased markedly under HGF treatment. Conclusions: Our data demonstrate that supraphysiological doses of HGF in an in vitro MBEC-barrier-like model disrupt TJ organization, leading to morphological changes and functional weakening of the MBEC-barrier-like structure, as shown by uncoupling between ZO-1/F-actin cytoskeleton, reduced TEER, and increased size-selective paracellular permeability (TEDP). Full article

Intercellular junctions are involved in the regulation of epithelial function and remodeling in the female reproductive system; however, their importance in the avian oviduct is poorly known. The aim of this study was: first, to provide information on the expression and localization of key tight (occludin, claudin 1, 4, 5, junctional adhesion molecule [JAM] 2, 3) and adherens (E-cadherin, β-catenin) junction proteins in the hen oviduct, and second, to compare expression and localization of these molecules between laying and subjected to fasting-induced pause in laying hens. Tissue samples from all oviductal segments, i.e., infundibulum, magnum, isthmus, shell gland, and vagina were collected on the sixth day of the experiment from the control hens and hens that had been fasted for five consecutive days. Specific oviductal part-dependent expression patterns of examined genes (by quantitative real-time polymerase chain reaction [qRT-PCR]) and/or proteins (by Western blotting) were found, with the highest mRNA transcript and protein abundances in the infundibulum, shell gland, and vagina, and the lowest in the magnum. Fasting-induced partial regression of the oviduct was accompanied by alterations in mRNA transcript and protein abundances of examined molecules. Reduced staining intensity of immunoreaction (analyzed by immunofluorescence) for occludin, E-cadherin, and β-catenin proteins was observed in the oviduct of non-laying hens. Our results indicate the potential involvement of these proteins in controlling intercellular communication, cell signaling, paracellular permeability, and mucosal barrier functionality, which impact the functioning of the hen oviduct. Furthermore, our observations provide novel insights into the molecular composition of tight and adherens junctions and its contribution to the remodeling of the oviduct during its regression induced by fasting. Full article

Gut barrier dysfunction and increased intestinal permeability are closely linked to the pathogenesis of type 1 diabetes and its complications. Streptozotocin (STZ)-induced diabetic mice, which mimic β-cell destruction and insulin deficiency, provide a widely used model for studying type 1 diabetes-associated intestinal barrier impairment. However, the cellular pathways mediating this dysfunction, particularly the role of goblet cells, remain incompletely elucidated. This study aimed to investigate the association between the gut barrier function and diabetes. Using real-time intravital multiphoton microscopy, we investigated intestinal barrier integrity in STZ-induced type 1 diabetic mice. Three groups were analysed: the control, STZ-diabetic, and STZ-diabetic mice treated with fructooligosaccharide (FOS) for 1 week. Intestinal permeability was assessed by measuring fluorescein isothiocyanate (FITC)-dextran concentrations in the portal vein and visualising translocation into villi. Epithelial morphology was examined, focusing on goblet cell density and leakage pathways. STZ-diabetic mice demonstrated a significant increase in intestinal permeability, evidenced by elevated FITC-dextran levels in the portal vein and villi. Multiphoton imaging revealed a notable rise in the goblet cell-to-enterocyte ratio in diabetic mice, while the gap density remained unchanged. The predominant route of macromolecular leakage in STZ-diabetic mice was via goblet cells rather than by paracellular gaps. One-week FOS supplementation significantly reduced goblet cell density and partially restored barrier function without altering the distribution of leakage pathways. These findings highlight goblet cell-mediated transcellular leakage as a major mechanism of gut barrier dysfunction in type 1 diabetic mice. Short-term FOS treatment partially reverses these alterations. Targeting goblet cell function may offer a promising therapeutic strategy to restore gut barrier integrity in diabetes. Full article

Botulinum toxin type A (BTXA) is widely used for the treatment of sialorrhea; however, its mechanism remains unclear. Tight junctions (TJs) are limiting factors for salivary secretion through the paracellular pathway in the salivary gland, among which claudin-1 (Cldn1) is a TJ protein that mainly plays a barrier role. This study observed that Cldn1 was upregulated in BTXA-treated rats’ submandibular glands and SMG-C6 cells. Knockdown of Cldn1 reversed the BTXA-induced reduction in paracellular permeability. The transcription factor specificity protein-1 (Sp1), which binds to the Cldn1 promoter, was also upregulated by BTXA, and its expression was linked to the ERK1/2 pathway. Inhibition of ERK1/2 by U0126 reversed the BTXA-induced upregulation of Sp1 and Cldn1, as well as the reduction in paracellular permeability. MiR-124-3p, which directly targets Sp1, was downregulated by BTXA, but its overexpression counteracted Sp1 and Cldn1 upregulation. Although miR-124-3p did not affect ERK1/2 phosphorylation, ERK1/2 inhibition reversed the BTXA-induced decrease in miR-124-3p expression. These findings reveal a regulatory pathway through which BTXA reduces paracellular permeability in SMG-C6 cells via the ERK1/2/miR-124-3p/Sp1/Cldn1 axis. Full article

Claudin 5 (Cldn5) is a critical tight junction protein essential for maintaining paracellular barrier integrity across endothelial and epithelial cells in barrier-forming tissues, including the blood–brain barrier and blood–retinal barrier. Cldn5 plays a central role in regulating vascular permeability, immune responses, and tissue homeostasis. The complex distribution and organ-specific regulation of Cldn5 underscore its potential as a promising therapeutic target. This review comprehensively analyzes the role of Cldn5 in endothelial and epithelial barrier function, its regulation of vascular permeability, and the discrepancies in the literature regarding its expression, regulation, and function in both physiological and pathological conditions across multiple organ systems, including the retina, brain, lung, heart, gut, kidney, liver, skin, and peripheral nerves, while emphasizing its tissue-specific expression patterns. We discuss how both reduced and excessive expressions of Cldn5 can disrupt barrier integrity and contribute to the pathogenesis of ischemic retinopathies, neuroinflammation, cardiovascular injury, and other forms of barrier dysfunction. Furthermore, we explore the dual role of Cldn5 as both a biomarker and a therapeutic target, highlighting emerging strategies such as RNA silencing, pharmacological stabilizers, and transcriptional modulators in controlling barrier leakage in disease conditions. Full article

Fine particulate matter (PM2.5) is an environmental factor that triggers gastrointestinal diseases. However, the effects of PM2.5 on intestinal function are not fully understood. This study established an environmental exposure cell model to explore PM2.5-induced intestinal permeability alteration and its mechanisms. Intestinal barrier permeability was evaluated via trans-epithelial electrical resistance (TEER) measurement and FITC–dextran paracellular penetration analysis, followed by detection of intercellular junction protein β-catenin and its coding gene CTNNB1. Expression of inflammatory cytokines (TNF-α, IL-6) and phosphorylation of PI3K and AKT were assessed using quantitative real-time polymerase chain reaction and Western blot, respectively. Reactive oxygen species (ROS) and malondialdehyde were measured using commercial kits to observe cellular oxidative stress. The results showed that PM2.5 impaired the intestinal barrier, as indicated by reduced TEER, increased FITC–dextran penetration, down-regulated expression of β-catenin and CTNNB1. Additionally, compared with the control, inflammatory cytokines and oxidative stress markers were significantly elevated after PM2.5 exposure. The ratio of p-PI3K/PI3K and p-AKT/AKT was also up-regulated in PM2.5-exposed Caco-2 cells. Pretreatment with PI3K inhibitor LY294002 and ROS scavenger NAC modulated β-catenin expression, reduced inflammation/ROS, and alleviated the hyperpermeability of Caco-2 cells. Thus, our results reveal that PM2.5 induces PI3K/AKT-mediated inflammation and ROS generation in Caco-2 cells, leading to intestinal barrier impairment. Full article

A comprehensive UPLC-MS/MS method was developed and validated for the simultaneous separation and quantification of atenolol, propranolol, quinidine, and verapamil, using established intestinal permeability standards in the Caco-2 cell monolayer model. This in vitro model is widely accepted for predicting intestinal drug permeability and is formally recognized by global regulatory agencies, including the FDA, EMA, and WHO, as a surrogate for assessing drug permeability in biowaiver applications under the Biopharmaceutics Classification System (BCS) framework. Despite its regulatory importance, standardized methods for the simultaneous quantification of key permeability markers remain scarce. The selected compounds represent distinct transport pathways: paracellular (atenolol), passive transcellular (propranolol, verapamil), and P-glycoprotein-mediated efflux (quinidine). Method validation followed FDA guidelines and demonstrated high selectivity, linearity (r² > 0.998), precision, and accuracy. Solid-phase extraction enhanced recovery and reduced matrix effects. Application to Caco-2 permeability assays confirmed expected transport profiles, including P-gp inhibition effects with verapamil. By integrating multiple analytes in a single workflow, the method improves analytical throughput, supports mechanistic interpretation, and ensures consistency across assays. This advanced separation strategy, combined with sensitive mass spectrometric detection, supports regulatory and BCS-based classification studies, contributing to the standardization of permeability assessments in drug development. Full article

Background/Objectives: The oral cavity represents a convenient route of administration for drugs that exhibit significant hepatic first-pass extraction. In this study, the mucosal permeation properties of selected active pharmaceutical ingredients (APIs) incorporated into oral cavity drug products that are approved by the U.S. Food and Drug Administration were quantified using the human-derived sublingual HO-1-u-1 and buccal EpiOral™ in vitro tissue models. Methods: Epithelial barrier properties were monitored using propranolol and Lucifer Yellow as prototypic transcellular and paracellular markers. APIs were dissolved in artificial saliva, pH 6.7, and transepithelial flux from the apical to the basolateral compartment was quantified using HPLC. Results: Apparent permeability coefficients (Papp) calculated for these APIs in the sublingual HO-1-u-1 tissue model varied from Papp = 2.72 ± 0.06 × 10⁻⁵ cm/s for asenapine to Papp = 6.21 ± 2.60 × 10⁻⁵ cm/s for naloxone. In contrast, the buccal EpiOral™ tissue model demonstrated greater discrimination power in terms of permeation properties for the same APIs, with values ranging from Papp = 3.31 ± 0.83 × 10⁻⁷ cm/s for acyclovir to Papp = 2.56 ± 0.68 × 10⁻⁵ cm/s for sufentanil. The tissue-associated dose fraction recovered at the end of the transport experiment was significantly increased in the buccal EpiOral™ tissue model, reaching up to 8.5% for sufentanil. Conclusions: Experimental permeation data collected for selected APIs in FDA-approved oral cavity products will serve as a training set to aid the development of predictive computational models for improving algorithms that describe drug absorption from the oral cavity. Following a robust in vitro–in vivo correlation analysis, it is expected that such innovative in silico modeling strategies will the accelerate development of generic oral cavity products by facilitating the utility of model-integrated evidence to support decision making in generic drug development and regulatory approval. Full article

Background: PTEN is a tumor suppressor that controls many pathophysiological pathways, including cell proliferation, differentiation, apoptosis and invasiveness. Although PTEN down-modulation is a critical event in neoplastic progression, it becomes apparent that transient and local inhibition of PTEN activity might be beneficial for the healing process. Methods: In the present study, we investigated the impact of PTEN invalidation in mouse intestinal epithelium under a physiological condition and after dextran sulfate sodium (DSS) treatment to induce experimental colitis. PTEN conditional knockout was induced in intestinal epithelial cells after crossing villin-Cre and PTEN^flox/flox mice. Results: PTEN invalidation alleviates experimental colitis induced by DSS, as evidenced by decreased weight loss during the acute phase, the lower expression of inflammation markers, including the proinflammatory cytokines IFN-γ, CXCL1 and CXCL2, reduced mucosal lesions, and faster recovery after resolution of inflammation. This protective effect might result in part from the sustained proliferation of colonic epithelium, leading to hyperplasia and increased colonic crypt depth under physiological conditions, which was further exacerbated in the vicinity of mucosal injury induced by DSS treatment. Furthermore, PTEN knockout decreased paracellular permeability, thereby enhancing the intestinal barrier function. This process was associated with the reinforcement of claudin-3 immunostaining, especially on the surface epithelium of villin-Cre PTEN^flox/flox mice. Conclusions: PTEN inactivation exerts a protective effect on the onset of colitis, and the transient and local down-modulation of PTEN might constitute an approach to drive recovery following acute intestinal inflammation. Full article

Objectives: Ex vivo nasal mucosa models provide physiologically relevant platforms for evaluating nasal drug permeability; however, their application is often limited by high experimental variability and the absence of standardized methodologies. This study aimed to improve experimental design by addressing two major limitations: the confounding effects of mucosal thickness and the questionable reliability of fluorescein sodium (Flu-Na) as an integrity marker for chemically induced mucosal injury. Methods: Permeability experiments were conducted using porcine nasal tissues mounted in Franz diffusion cells, with melatonin and Flu-Na as model compounds. Tissues of varying thickness were collected from both intra- and inter-individual sources, and a numerical simulation-based method was employed to normalize apparent permeability coefficients (Papp) to a standardized mucosal thickness of 0.80 mm. The effects of thickness normalization and chemically induced damage were systematically evaluated. Results: Thickness normalization substantially reduced variability in melatonin Papp, particularly within same-animal comparisons, thereby improving statistical power and data reliability. In contrast, Flu-Na exhibited inconsistent correlations across different pigs and failed to reflect the expected increase in permeability following isopropyl alcohol (IPA)-induced epithelial damage. These results suggest that the relationship between epithelial injury and paracellular transport may be non-linear and not universally applicable under ex vivo conditions, limiting the suitability of Flu-Na as a standalone marker of mucosal integrity. Conclusions: The findings highlight the importance of integrating mucosal thickness correction into standardized experimental protocols and call for a critical reassessment of Flu-Na in nasal drug delivery research. Full article

Acrylamide, a harmful substance generated during the normal thermal treatment of foods, has been shown to adversely affect human health, particularly the vital intestinal barrier function. Meanwhile, natural polysaccharides are recognized to exert an important biofunction in the intestine by protecting barrier integrity. In this study, the non-starch, water-soluble, and nondigestive yam polysaccharide (YP) was extracted from fresh Chinese yam, while two selenylated derivatives with different extents of selenylation were prepared via the HNO₃-Na₂SeO₃ reaction system, and designated as YPSe-I and YPSe-II, respectively. Their protective activities and the associated molecular mechanisms of these substances against acrylamide-induced damage in rat intestinal epithelial (IEC-6) cells were thereby investigated. The experimental results demonstrated that the selenium contents of YPSe-I and YPSe-II were 0.80 and 1.48 g/kg, respectively, whereas that of the original YP was merely 0.04 g/kg. In IEC-6 cells, in comparison with YP, both YPSe-I and YPSe-II showed higher efficacy than YP in alleviating acrylamide-induced cell toxicity through promoting cell viability, suppressing the release of lactate dehydrogenase, and decreasing the generation of intracellular reactive oxygen species. Both YPSe-I and YPSe-II could also manifest higher effectiveness than YP in maintaining cell barrier integrity against the acrylamide-induced barrier disruption. The mentioned barrier protection was achieved by increasing transepithelial electrical resistance, reducing paracellular permeability, facilitating the distribution and expression of F-actin between the cells, and up-regulating the production of three tight junctions, namely ZO-1, occludin, and claudin-1. Additionally, acrylamide was observed to trigger the activation of the MAPK signaling pathway, thereby leading to cell barrier dysfunction. In contrast, YPSe-I and particularly YPSe-II were capable of down-regulating two MAPK-related proteins, namely p-p38 and p-JNK, and thereby inhibiting the acrylamide-induced activation of the MAPK signaling pathway. Moreover, YPSe-II in the cells was consistently shown to provide greater barrier protection than YPSe-I. In conclusion, chemical selenylation of YP could cause higher activity in mitigating acrylamide-induced cytotoxicity and intestinal barrier dysfunction, while the efficacy of activity enhancement was positively affected by the selenylation extent. Full article

Background/Objectives: The low permeability of the blood-brain barrier (BBB) represents a significant challenge to effective systemic chemotherapy for primary and metastatic brain cancers. Kinin receptors play a crucial role in modulating BBB permeability, and their agonist analogs have been explored in preclinical animal models to enhance drug delivery to the brain. In this study, we investigated whether des-Arg⁹-bradykinin (DBK), a physiological agonist of kinin B₁ receptor (B1R), acts as a brain drug delivery adjuvant by promoting the transient opening of the BBB. Methods: Human brain microvascular endothelial cells (HBMECs) were treated with DBK in the culture medium and in conditioned media from glioblastoma cell lines, namely T98G (CMT98G) and U87MG (CMU87). Immunofluorescence, RT-qPCR, in-cell Western assay, and proximity ligation assay (PLA) were performed to analyze BBB components, kinin receptors and TLR4, a receptor associated with the kinin pathway and inflammation. The effect of DBK on enhancing paracellular molecule transport was evaluated using Evans blue dye (EB) quantification in a cell culture insert assay and in an in vivo model, where mice with and without brain tumors were treated with DBK. To assess the functional impact of the transient BBB opening induced by DBK, the chemotherapeutic drug doxorubicin (DOX) was administered. Results: Treatment with DBK facilitates the presence of EB in the brain parenchyma by transiently disrupting the BBB, as further evidenced by the increased paracellular passage of the dye in an in vitro assay. B1R activation by DBK induces transient BBB opening lasting less than 48 h, enhancing the bioavailability of the DOX within the brain parenchyma and glioma tumor mass. The interaction between B1R and TLR4 is disrupted by the secreted factors released by glioblastoma cells, as conditioned media from T98G and U87 reduce TLR4 staining in endothelial cells without affecting B1R expression. Conclusions: These results further support the potential of B1R activation as a strategy to enhance targeted drug delivery to the brain. Full article

Irritable Bowel Syndrome (IBS) is a disorder of gut- brain interaction characterized by recurrent abdominal pain associated with altered bowel habits. The therapeutic options for IBS patients include the use of probiotics. The aim of this study was to assess the effect of a multi-strain probiotic made up by Lactobacillus rhamnosus LR 32, Bifidobacterium lactis BL 04, and Bifidobacterium longum BB 536 (Serobioma, Bromatech s.r.l., Milano, Italy) on an in vitro model of the intestinal epithelial barrier in the presence of mucosal mediators that are released by IBS patients. IBS (n = 28; IBS with predominant diarrhea, IBS-D = 10; IBS with predominant constipation, IBS-C = 9; and IBS with mixed bowel habits, IBS-M = 9) patients, diagnosed according to the Rome IV criteria, and asymptomatic controls (ACs, n = 7) were enrolled. Mucosal mediators that were spontaneously released by colonic biopsies were collected (supernatants). Two doses of Serobioma were tested with/without IBS/AC mediators. RNA was extracted from Caco-2 cells to evaluate the tight junction (TJ) expression. Serobioma (10⁶ CFU/mL) significantly reinforced the Caco-2 monolayer compared to growth medium alone (p < 0.05). IBS supernatants significantly increased Caco-2 paracellular permeability compared to the AC supernatants. The co-incubation of Caco-2 cells with IBS supernatants and Serobioma (10⁶ CFU/mL) avoided the paracellular permeability alterations that were induced by IBS supernatants alone (p < 0.001), and, in particular, IBS-D and IBS-M ones. The co-incubation of Serobioma (10⁶ CFU/mL) and IBS-D supernatants significantly increased ZO-1 expression compared to Caco-2 cells incubated with supernatants alone (p < 0.05), as confirmed via qPCR analyses. Serobioma (10⁶ CFU/mL) counteracts the paracellular permeability changes that are induced by IBS supernatants, in particular IBS-D and IBS-M supernatants, likely modulating ZO-1 expression. Full article

Epithelial linings are crucial for the maintenance of physiological barriers. The intestinal epithelial barrier (IEB) consists of enterocytes through tight junctions and mucus-secreting cells and can undergo physiological modifications throughout life. To reproduce as closely as possible the IEB main features over time, in vitro co-cultures of Caco2/HT-29 70/30 formed by parental Caco2 and HT-29 cells sub-cultivated for more than 40 passages were set up. The measurements of the transepithelial electrical resistance (TEER) identified two populations: physiological TEER co-cultures (PC) with values > 50 Ωcm² formed by parental cells with fewer than 40 passages, and leaky TEER co-cultures (LC) with values < 50 Ωcm² formed by parental cells with more than 40 passages. In LC, paracellular permeability increased in parallel. By immunofluorescence and Western blot analysis, an increase in claudin 2 was observed in LC vs. PC, with no differences in occludin expression. MUC-2 immunoreactivity was stronger in PC than in LC. LC also showed an enhanced vulnerability to TNFα+IFN-γ. These results reproduce the main morpho-functional modifications reported in the human leaky/aged gut and support the usefulness of our in vitro cell model for studying the molecular processes underlying these modifications and testing drug/nutraceutical treatments to ameliorate leaky gut aging. Full article

The blood–brain barrier (BBB) comprises distinct cell types, including endothelial cells, pericytes, and astrocytes, and is essential for central nervous system (CNS) homeostasis by selectively regulating molecular transport and maintaining integrity. In particular, astrocytes are essential for BBB function, as they maintain BBB integrity through their end-feet, which form a physical and biochemical interface that enhances endothelial cell function and barrier selectivity. Moreover, they secrete growth factors like vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β), which regulate tight junction (TJ) proteins (e.g., claudins and occludins) crucial for limiting paracellular permeability. Molecular motors like kinesins, dynein, and myosins are essential for these astrocyte functions. By facilitating vesicular trafficking and protein transport, they are essential for various functions, including trafficking of junctional proteins to support BBB integrity, the proper mitochondria localization within astrocyte processes for efficient energy supply, the polarized distribution of aquaporin (AQP)-4 at astrocyte end-feet for regulating water homeostasis across the BBB, and the modulation of neuroinflammatory responses. Moreover, myosin motors modulate actomyosin dynamics to regulate astrocyte process outgrowth, adhesion, migration, and morphology, facilitating their functional roles. Thus, motor protein dysregulation in astrocytes can compromise BBB function and integrity, increasing the risk of neurodegeneration. This review explores the complex interplay between astrocytes and molecular motors in regulating BBB homeostasis, which represents an attractive but poorly explored area of research. Full article