Epithelial Permeability Research Articles

Enhanced Transepithelial Riboflavin Delivery Across the Cornea Using Magnetic Nanocarriers.

Purpose: Keratoconus is a progressive corneal ectasia characterized by irregular astigmatism, leading to corneal scarring and decreased vision. Corneal cross-linking (CXL) is the standard treatment to halt disease progression, but its effectiveness in transepithelial (epithelium-on, epi-on) approaches is limited by the low permeability of the corneal epithelium to riboflavin (Rb). This study aimed to enhance transepithelial Rb penetration in ex vivo bovine corneas using Rb-modified tannic acid-coated superparamagnetic iron oxide nanoparticles (Rb-TA-SPIONs) under an external magnetic field. Methods: SPIONs were synthesized via co-precipitation, modified with TA and Rb, and characterized by physicochemical techniques. The average size of the Rb-TA-SPIONs was 46 ± 5.3 nm, with a saturation magnetization of 55.9 emu/g. Ex vivo experiments involved the application of 0.1% Rb to bovine corneas, and penetration was evaluated under epi-on conditions with iontophoresis (1-5 mA, 5 min). In addition, a 0.1% Rb-containing nanocarrier solution was tested under magnetic fields of 1-300 Gauss. Results: Results showed increased Rb penetration with rising electric current density and Rb-TA-SPION penetration with stronger magnetic fields, compared with epi-on control groups. Specifically, Rb penetration increased from 0.036% (P ≤ 0.01) at 1 mA to 0.059% (P ≤ 0.001) at 5 mA in the iontophoresis group and from 0.035% (P ≤ 0.001) at 1 G to 0.054% (P ≤ 0.001) at 300 G in the magnetic group. Conclusion: These findings indicate that magnetic nanoparticle-assisted Rb delivery, guided by an external magnetic field, could improve potential CXL efficacy by enhancing Rb penetration and corneal permeability.

Modulating the elasticity of milk exosome-based hybrid vesicles to optimize transepithelial transport and enhance oral peptide delivery.

To address challenges such as limited loading capacity, restricted targeting precision, and low yield of natural exosomes as drug carriers, the fusion of liposomes and exosomes to create hybrid vesicles has emerged as a viable solution approach. While current research mainly focuses on designing functionalized liposomes, less attention is given to how liposome membrane materials affect the elasticity of these hybrids and their delivery efficiency. This study utilized milk exosomes (mExos) as model exosomes, and generated hybrid vesicles with varying elasticity through the fusion of phospholipids with differing chain lengths, examining the disparities among various hybrid vesicles in their ability to overcoming the gastrointestinal barriers. It was observed that while hard hybrid vesicles exhibited reduced mucus penetration compared to soft hybrid vesicles, they demonstrated a notably higher efficacy in traversing the epithelial cell barrier. The enhanced transepithelial cell capability of hard vesicles can be attributed to their reduced tendency to aggregate in the lysosome through the down-regulated clathrin-mediated endocytosis pathway, as well as by the strengthening of the endoplasmic reticulum-Golgi exocytosis pathway due to their rigid characteristics. In comparison to soft hybrid vesicles, semaglutide (SET) loaded hard hybrid vesicles demonstrated improved in vivo epithelial permeability, enhanced oral bioavailability, and better therapeutic effectiveness. This study could provide valuable insights for determining the optimal elasticity of exosome-liposome hybrid vesicles in the development of oral nanocarriers.

Disrupting of IGF2BP3-stabilized CLDN11 mRNA by TNF-α increases intestinal permeability in obesity-related severe acute pancreatitis

BackgroundObesity is a significant risk factor for severe acute pancreatitis (SAP) and is typically associated with increased intestinal permeability. Understanding the role of specific molecules can help reduce the risk of developing SAP. Claudin 11 (CLDN11), a member of the Claudin family, regulates the permeability of various internal barriers. However, the role and mechanism of CLDN11 in the intestinal permeability of obesity-related SAP remain unclear.MethodsWe evaluated intestinal permeability and the expression of CLDN11 in experimental obesity-related SAP. A recombinant adeno-associated virus carrying CLDN11 was used to treat experimental obesity-related SAP. The interaction between CLDN11 mRNA and insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) protein was predicted through bioinformatics analysis and validated by RNA immunoprecipitation and RNA pull-down assay. Additionally, tumor necrosis factor-α (TNF-α) treatment in Caco-2 cells was conducted, and the IGF2BP3/CLDN11 axis was detected. Moreover, we conducted anti-TNFα therapy and evaluated intestinal permeability and pancreatic inflammation in experimental obesity-related SAP.ResultsDownregulation of CLDN11 was observed in the intestinal epithelial cells of experimental obesity-related SAP. When the expression of CLDN11 in intestinal epithelial cells of experimental obesity-related SAP was increased exogenously, intestinal epithelial permeability and pancreatic inflammation were relieved. Overexpression of CLDN11 reduced the paracellular permeability of Caco-2 monolayer cells, while knockdown of CLDN11 increased it. IGF2BP3 bound to and regulated the stability of CLDN11 mRNA. TNF-α treatment downregulated IGF2BP3 and CLDN11 in vitro. Anti-TNFα therapy reduced intestinal permeability, alleviated pancreatitis, and improved the expression of IGF2BP3 and CLDN11 in intestinal epithelial cells in experimental obesity-related SAP.ConclusionCLDN11 regulates intestinal permeability in obesity-related SAP. Mechanistically, an increase in TNF-α impaired the stability of IGF2BP3-dependent CLDN11 mRNA in obesity-related SAP.

P0204 Impact of crypt niche factors and oxygen gradient on intestinal epithelial differentiation and inflammatory response

Abstract Background The gastrointestinal mucosa, with its rich vascularization juxtaposed to the anaerobic lumen, creates a steep physiological oxygen gradient along the intestinal crypt. This gradient, together with soluble stromal factors, is critical for maintaining barrier homeostasis. In inflammatory bowel disease (IBD), active inflammation disturbs stem cell niche environment and oxygen perfusion 1. However, the impact of niche factors and oxygen gradients on epithelial behaviour in health and IBD remains poorly understood. Here, we aim to elucidate how crypt niche factors and oxygen tension influence epithelial differentiation, barrier permeability and response to pro-inflammatory stimuli. Methods We used air-liquid interface (ALI) cultures derived from human intestinal organoids as a physiologically relevant crypt model 2. Differentiation was monitored across four culture conditions with progressively reduced stem cell niche factors (including Wnt3A and Rspondin), and upon exposure to IBD-associated cytokines IFN-ɣ, IL22 and IL17A. Submerged cultures were used to model low oxygen tension during differentiation. Epithelial differentiation kinetics and response to inflammatory stimuli were assessed using transcriptional analysis, trans-epithelial resistance measurement and morphological evaluation. Results Transition of the epithelial cultures to ALI conditions resulted in transient over-expression of stem/transit-amplifying (TA) markers (LGR5, MKI67), with expression levels correlating to stem cell niche factor availability (Fig. 1A). Culture conditions significantly influenced the differentiation kinetics, with progressive up-regulation of differentiation markers (AQP8, ZG16, CHGA) under decreasing stem cell niche factor availability (Fig. 1B). These factors also affected epithelial permeability (Fig. 1C) and morphology (Fig. 1D). In submerged cultures, differentiation and polarization were entirely inhibited, while stem/TA marker expression showed only partial perturbations (Fig. 1E). Oxygen tension variations further modulated epithelial responses to IFN-ɣ, IL22 and IL17A. Notably, submerged cultures exhibited enhanced expression of IFN-ɣ- and IL22-specific targets (CXCL10 and OLFM4, respectively) (Fig. 1F), despite the limited effect of cytokine stimulation on barrier integrity (Fig. 1G). Conclusion In conclusion, our findings indicate that crypt niche factors and oxygen gradients distinctly regulate epithelial behaviour, with the differentiated compartment being particularly sensitive to oxygen availability. These results suggest that diminished oxygen tension, as commonly observed in active IBD, may exacerbate epithelial barrier dysfunction and amplify the epithelial responses to pro-inflammatory stimuli.

P0221 The role of caffeine and its metabolites in the modulation of intestinal inflammation and epithelial barrier integrity

Abstract Background Methylxanthine caffeine and its metabolites are widely consumed bioactive compounds that are known to influence various physiological processes 1. However, their impact on gut health, particularly in modulating inflammation and epithelial permeability, remains underexplored. In this study we aimed to investigate the effects of caffeine and its primary metabolites paraxanthine, theobromine and theophylline on inflammatory responses and barrier integrity in gut epithelial models, providing insights into their potential role in gastrointestinal health in the context of Inflammatory bowel disease (IBD). Methods Primary screening of different concentrations of caffeine and its metabolites paraxanthine, theobromine and theophylline was performed in Caco-2 cells. Selected metabolites were further tested on inflamed 3D intestinal epithelial organoids derived from ulcerative colitis patient (n = 1) and non-IBD individual (n = 1). Inflammation was induced for 24 h using TNF-α and IFN-γ cytokine mix. The effects on inflammation status and epithelial barrier integrity were tested through targeted gene expression analysis using RT-PCR, while the effects on generation of reactive oxygen species were investigated by Flow cytometry and DHE assay. Results Primary screening revealed that both caffeine and its metabolite paraxanthine affected the expression of genes encoding inflammatory cytokines (TNF-α, CXCL9; p < 0.05) and tight junction proteins (OCLN, ZO-1, CLDN1; p < 0.05) and were selected for further analysis in 3D intestinal organoids. Neither caffeine nor paraxanthine reduced the expression of inflammatory cytokine-encoding genes in inflamed intestinal organoids. However, both metabolites significantly restored the expression of tight junction protein-coding genes OCLN, ZO-1 and CLDN1 (p < 0.05). A tendency of reduction in reactive oxygen species production (by 11 %) was also observed after exposure with paraxanthine. Conclusion Methylxanthine caffeine and its primary metabolite paraxanthine might have a positive effect on gut epithelial barrier function through restoration of the expression of tight junction protein-coding genes. Study was funded by the European Union and the State Secretariat for Education, Research and Innovation (SERI) (project: miGut-Health, grant no. 101095470). References Reddy V. S. et al. (2024) Pharmacology of caffeine and its effects on the human body. European Journal of Medicinal Chemistry Reports, 10 (100138). https://doi.org/10.1016/j.ejmcr.2024.100138.

P0098 Effect of the dietary emulsifier κ-carrageenan on the inflammatory state and permeability of intestinal epithelial cells from patients with Crohn’s disease

Abstract Background The consumption of ultra-processed foods has increased worldwide and is associated with the rise in inflammatory bowel disease (IBD)1. However, any causative factors and their underlying mechanisms are yet to be identified. Interestingly, our group found a higher consumption of the dietary emulsifier carrageenan (CGN) in Belgian IBD patients, compared to healthy controls2. This study aimed to elucidate whether different types of CGN can alter the permeability and inflammatory state of the intestinal epithelium in patients with Crohn’s disease (CD). Methods Confluent Caco-2/HT29-MTX cocultures (n=4) were exposed to either κ-, ι-, or λ-CGN (100 µg/ml) for 24 hours. Confluent epithelial monolayers derived from colonic organoids3 of 5 CD patients were exposed to κ-CGN (100 µg/ml), the most abundant subtype in foods, for 48 hours. Two independent experiments were performed per patient (n = 10). In both models, inflammation was established by adding an inflammatory mix (100 ng/mL TNF-α, 20 ng/mL IL-1β, and 1 µg/mL flagellin)4 to the basolateral side, 24 hours prior to CGN exposure. Changes in permeability were measured by transepithelial electrical resistance (TEER) at predetermined timepoints (0, 12, 24, 48 hours). In organoid-derived monolayers, cytokines were quantified in the apical and basolateral supernatant using the V-PLEX Proinflammatory Panel 1 kit (Meso Scale Diagnostics) and gene expression was analysed with RT-qPCR (Taqman probes, run on Viia7) (n = 8; 2 samples excluded due to low RNA concentrations). Results In organoid-derived monolayers, κ-CGN increased the expression of TNF, IL8 and IL1B, both in inflamed and non-inflamed monolayers (n=8; Repeated measures (RM) one-way ANOVA), compared to the control (Fig. 1A). Release of the cytokines IL-6, IL-13, IL-4, IL-2, and IL-10 in the apical supernatant was increased after 48 hours of κ-CGN stimulation (n=8; RM one-way ANOVA) (Fig. 1B). κ-CGN exposure did not affect TEER (n=10; RM one-way ANOVA) (Fig. 2B+C), but induced an upregulation of tight junction markers ZO1 and OCLN, and resulted in a downregulation of MUC5AC and upregulation of MUC5B (n=8; RM one-way ANOVA or Friedman test) (Fig. 2D). Additionally, none of the CGN subtypes altered permeability of non-inflamed or inflamed Caco-2/HT29-MTX cocultures (n=4; Kruskal-Wallis test), compared to the unexposed control (Fig. 2A). Conclusion Dietary κ-CGN caused upregulation of inflammatory markers and affected cytokine release of intestinal epithelial cells from patients with CD, while permeability remained unaltered. When inflammation was already present, this pro-inflammatory effect was more pronounced, suggesting a role for dietary CGN in perpetuating inflammation during active CD.

Human cathelicidin LL-37 rapidly disrupted colonic epithelial integrity.

The intestinal barrier, held together by epithelial cells and intercellular tight junction (TJ) proteins, prevents the penetration of microbial pathogens. Concurrently, intestinal epithelial cells secrete antimicrobial peptides, including cathelicidin. Cathelicidin has direct antibacterial and immunomodulatory functions, although its role in intestinal integrity remains elusive. In this study, we demonstrate that direct stimulation of human colonic epithelial (T84) cells with human cathelicidin, LL-37, resulted in a rapid and transient increase in epithelial cell permeability. This increased permeability was associated with the TJ proteins occludin and claudin-2 degradation, mediated by these specific proteins' endocytosis and lysosomal degradation. While murine cathelicidin (CRAMP) failed to modify T84 cell permeability, LL-37 degraded TJ proteins in murine rectal epithelial (CMT-93) cells. The stimulus of (CMT-93) cells with LL-37 aggravated the cell permeability and furthered TJ degradation provoked by the intestinal pathogen, attaching/effacing (A/E) Citrobacter rodentium (C. rodentium). The number of C. rodentium that colonized CMT-93 cells was not severely impacted by the presence of LL-37. While a temporary disruption of tight junctions by LL-37 may lead to a 'leaky gut,' this study demonstrates that LL-37 increases epithelial cell permeability by degrading TJ proteins occludin and claudin-2 through endocytosis and lysosomal degradation. These immunomodulatory actions occurring at concentrations lower than those microbicidal uncover a new guise for cathelicidin modulating the epithelial barrier against A/E pathogens. Recognizing native cathelicidin's functions in a specified disease setting (e.g., colitis) will help establish it as an anti-infectious immunomodulator.

α-Bisabolol alleviates diesel exhaust particle-induced lung injury and mitochondrial dysfunction by regulating inflammatory, oxidative stress, and apoptotic biomarkers through the c-Jun N-terminal kinase signaling pathway.

Exposure to particulate matter ≤2.5μm in diameter (PM2.5) is associated with adverse respiratory outcomes, including alterations to lung morphology and function. These associations were reported even at concentrations lower than the current annual limit of PM2.5. Inhalation of PM2.5, of which diesel exhaust particles (DEPs) is a major component, induces lung inflammation and oxidative stress. α-Bisabolol (BIS) is a bioactive dietary phytochemical with various pharmacological properties, including anti-inflammatory and antioxidant actions. Here, we evaluated the possible protective effects of BIS on DEP-induced lung injury. Mice were exposed to DEPs (20 µg/mouse) or saline (control) by intratracheal instillation. BIS was administered orally at two doses (25 and 50mg/kg) approximately 1h before DEP exposure. Twenty-fourhours after DEP administration, multiple respiratory endpoints were evaluated. BIS administration was observed to prevent DEP-induced airway hyperreactivity to methacholine; influx of macrophages, neutrophils, and lymphocytes in the bronchoalveolar lavage fluid; and increases in epithelial and endothelial permeabilities. DEP exposure caused increases in the levels of myeloperoxidase, proinflammatory cytokines, and oxidative stress markers in lung tissue homogenates, and all these effects were abated by BIS treatment. The activities of mitochondrial complexes I, II, III, and IV were markedly increased in the lungs of mice exposed to DEPs, and these effects were significantly reduced in the BIS-treated group. Intratracheal instillation of DEPs induced DNA damage and increase in the apoptotic marker cleaved caspase-3. The latter effects were prevented in mice treated with BIS and exposed to DEPs. Moreover, BIS mitigated DEP-induced increase in the expression of phospho-c-Jun N-terminal kinase (JNK) in a dose-dependent manner. BIS markedly alleviated DEP-induced lung injury by regulating the inflammatory, oxidative stress, and apoptotic biomarkers through the JNK signaling pathway. Following additional studies, BIS may be considered as a plausible protective agent against inhaled-particle-induced pulmonary adverse effects.

Clinically relevant cell culture model of inflammatory bowel diseases for identification of new therapeutic approaches.

Inflammatory Bowel Diseases (IDB) are chronic disorders characterized by gut inflammation, mucosal damage, increased epithelial permeability and altered mucus layer. No accurate in vitro model exists to simulate these characteristics. In this context, drug development for IBD or intestinal inflammation requires in vivo evaluations to verify treatments efficacy. A new model with altered mucus layer composition; altered epithelial permeability and pro-inflammatory crosstalk between immune and epithelial cells will be developed to enhance in vitro models for studying IBD treatments. The effects of dextran sulfate sodium and/or lipopolysaccharides on intestinal permeability, cytokines synthesis (IL-6, IL-8, TNF-α and IL-1β), mucins (MUC2, MUC5AC) and tight junction proteins expression (Claudin-1, ZO-1 and Occludin) were investigated in a tri-coculture model combining differentiated Caco-2/HT29-MTX cells and THP-1 cells. Two anti-inflammatory agents were evaluated to assess the model's therapeutic strategy applicability (corticoids and pro-resolving factors). Two in vitro models have been developed. The first model, characterized by increased permeability of the epithelial layer and subsequent secretion of inflammatory cytokines, can reproduce the different phases of inflammation, and enables the evaluation of preventive treatments. The second model simulates the acute phase of inflammation and allows for the assessment of curative treatments. Both models demonstrated reversibility when treated with betamethasone and pro-resolving factors. These in vitro models are valuable for selecting therapeutic agents prior to their application in in vivo models. They enable the assessment of agents' anti-inflammatory effects and their ability to permeate the inflamed epithelial layer and interact with immune cells.

An environment-responsive platform based on acid-resistant metal organic framework for efficient oral insulin delivery

Oral insulin delivery is considered a revolutionary alternative to daily subcutaneous injections in terms of compliance and convenience. However, significant challenges remain in terms of inactivation in gastrointestinal environment and limited permeation across the intestinal epithelium. Herein, we used acid-resistant metal-organic framework (PCN-222) to load insulin and modified the exterior with sodium dodecyl sulfate (SDS) to achieve efficient oral insulin delivery. The PCN-222 nanocarrier with ordered mesoporous cage structure and suitable pore size achieved a high insulin loading of 75 %. The SDS on the surface of nanocarrier reduces its hydrophilicity while reversibly altering cell morphology and increasing epithelial cell permeability, thereby promoting intestinal epithelial absorption. The constructed particle (I@P@S) was encapsulated in sodium alginate (SA) microspheres to protect it from gastric acid degradation and releases it upon entry into the intestinal tract. Through an uptake pathway dominated by clathrin-mediated endocytosis, the released I@P@S realized efficient intestinal permeability and controlled insulin release under physiological conditions due to the phosphate sensitivity of PCN-222, leading to an in vivo bioavailability of 12.9 %. This work provides a valuable reference for the design of oral insulin delivery systems.

Oral multistage nanomedicine for synergistic chemo/chemodynamic/near-infrared-II photothermal cancer therapy.

The oral administration of drugs for cancer therapy can maintain optimal blood concentrations, is biologically safe and simple, and is preferred by many patients. However, the complex lumen environment, mucus layer, and intestinal epithelial cells are biological barriers that hinder the absorption of orally administered drugs. In this study, sea urchin-like manganese-doped copper selenide nanoparticles (Mn-Cu2-xSe NPs) were designed using an anion exchange method and coated with calcium alginate and chitosan (AC) to form Mn-Cu2-xSe@AC capsules. The pH-responsive swelling behavior of the AC protective layer aided doxorubicin (DOX)-loaded Mn-Cu2-xSe NPs in overcoming multiple biological barriers, maintained their stability in gastric acid, and facilitated the release of the NPs in the small intestine. The intestinal epithelial cell permeability of DOX/Mn-Cu2-xSe NPs was confirmed using a monolayer absorption model involving Caco-2 human epithelial cells. The released DOX/Mn-Cu2-xSe NPs smoothly passed through the mucus layer, and were absorbed by intestinal epithelial cells. In mice, the NPs circulated in the blood and passively targeted the tumors through blood circulation by enhancing the permeability and retention effect to achieve significant tumor suppression and reduce damage to normal tissues. In addition, the unique sea urchin-like morphology of Mn-Cu2-xSe NPs enhanced the absorption in the near-infrared-II (NIR-II) window for photothermal therapy, realized the near-infrared-stimulated response release of DOX for increased chemotherapy, and promoted the Fenton-like effect because of the doping of manganese ions for chemodynamic therapy. These effects could permit the development of various synergistic cancer treatments. The use of DOX/Mn-Cu2-xSe@AC capsules as a multistage oral drug delivery system may overcome the sequential absorption barriers that currently hinder chemotherapy, chemodynamic, and photothermal therapies.

Targeting the Gut-Heart Axis Improves Cardiac Remodeling in a Clinical Scale Model of Cardiometabolic Syndrome.

Poor diet, gut dysbiosis, and systemic inflammation constitute a gut-heart axis (GHA) that may affect heart failure with preserved ejection fraction. Clinical scale models to interrogate this axis are limited. Here, we show the full extent of the GHA-gut barrier function loss, systemic and microvascular inflammation, and its colocalization with apoptosis (left atrium) and hypertrophy (left ventricle). Gut barrier function primacy in regulating the GHA is supported by a synbiotic intervention that shuts down gut epithelial permeability, markedly decreasing systemic inflammation and, remarkably, cardiac structural changes in left heart chambers. These data support a new paradigm for targeting heart failure with preserved ejection fraction via the GHA.

Bioenergetic adaptations of small intestinal epithelial cells reduce cell differentiation enhancing intestinal permeability in obese mice.

Obesity and overweight are associated with low-grade inflammation induced by adipose tissue expansion and perpetuated by altered intestinal homeostasis, including increased epithelial permeability. Intestinal epithelium functions are supported by intestinal epithelial cells (IEC) mitochondria function. Here, we report that diet-induced obesity (DIO) in mice induces lipid metabolism adaptations favoring lipid storage in IEC together with reduced number, altered dynamics and diminished oxidative phosphorylation activity of IEC mitochondria. Using the jejunal epithelial cell line IPEC-J2, we showed that IEC lipid metabolism and oxidative stress machinery adaptations preceded mitochondrial bioenergetic ones. Moreover, we unraveled the intricate link between IEC energetic status and proliferation / differentiation balance since enhancing mitochondrial function with the AMPK activator AICAR in jejunal organoids reduced proliferation and initiated IEC differentiation and conversely. We confirmed that the reduced IEC mitochondrial function observed in DIO mice was associated with increased proliferation and reduced differentiation, promoting expression of the permissive Cldn2 in the jejunal epithelium of DIO mice. Our study provides new insights into metabolic adaptations of IEC in obesity by revealing that excess lipid intake diminishes mitochondrial number in IEC, reducing IEC differentiation that contribute to increased epithelial permeability.

The Mucosal Protection in the Treatment of Erosive Reflux Esophagitis: Mechanisms for Restoring Epithelial Permeability. A Randomized Clinical Trial.

Gastroesophageal reflux disease (GERD) is widespread in the population and is characterized by the risk of developing Barrett's esophagus and associated adenocarcinoma. Key factors in the progression of the disease are not only the frequency and duration of reflux episodes, but also the resistance of the esophageal mucosa to aggressive reflux molecules. Assessment of the state of tight junction proteins, the rate of their recovery under the influence of various treatment regimens is an urgent task for choosing optimal approaches to curing patients with GERD. The purpose of the study was to evaluate the efficacy and safety of the combination therapy with a proton pump inhibitor (PPI) and a topical mucosal protective agent (MPA) to relieve GERD symptoms and achieve a complete remission of reflux esophagitis faster. 60 patients (38 men and 22 women with an average age 41.5 years) with GERD duration of 5 years, took part in an open randomized prospective clinical study. Participants were randomized in a 1:1 ratio to be divided into the main group taking MPA and PPI and a comparison group taking a PPI only. The duration of therapy was 4 weeks. Before the treatment onset and after the end of treatment, the occurrence and severity of GERD symptoms were assessed, an endoscopic examination was performed and biopsy specimens were taken from the edge of the erosive area and the area of unchanged esophageal mucosa. The severity of histological signs of esophagitis, the expression of tight junction proteins and the proliferation marker Ki-67 were assessed. A more effective relief of GERD symptoms was documented when using combination therapy PPIs and MPAs. A more pronounced reduction in macroscopic changes was found in patients with erosive esophagitis taking PPIs and MPAs. The use of MPAs in addition to PPIs made it possible to achieve the primary and secondary endpoints more often, which suggested a high efficiency of the drug in combination with PPIs in the treatment of erosive esophagitis. An individualized approach based on the combination therapy of PPIs and MPAs can improve the effectiveness of treatment in achieving clinical, endoscopic and, more importantly, histological remission in a patient with erosive GERD.

Co-exposure to polyethylene microplastics and house dust mites aggravates airway epithelial barrier dysfunction and airway inflammation via CXCL1 signaling pathway in a mouse model.

Environmental pollutants have been found to contribute to the development and acute exacerbation of asthma. Microplastics (MPs) have received widespread attention as an emerging global pollutant. Airborne MPs can cause various adverse health effects. Due to their hydrophobicity, MPs can act as a carrier for other pollutants, pathogens, and allergens. This carrier effect of MPs may adsorb allergens and thus make the body exposed to MPs and a large number of allergens simultaneously. We hypothesized that co-exposure to inhaled MPs and aeroallergens may promote the development of airway inflammation of asthma by disrupting the airway epithelial barrier. The effects of co-exposure to Polyethylene microplastics (PE-MPs) and allergens on allergic airway inflammation and airway epithelial barrier were examined in a mouse model of asthma. The mice were divided into four groups: (i) Control group, treated only with PBS; (ii) MP group, exposed to PE-MPs and PBS; (iii) HDM group, mice were sensitized and challenged with HDM, and intranasally treated with PBS; (iv) HDM+MP group, mice were sensitized and challenged with HDM, and intranasally treated with PE-MPs. Histology and ELISA assays were used to evaluate the severity of airway inflammation. FITC-dextran permeability assay, immunofluorescence assay, and RT-PCR were used to evaluate the airway epithelial barrier function and the expression of relevant molecules. Transcriptomics analysis with lung tissue sequencing was conducted to identify possible pathways responsible for the effects of PE-MPs. Co-exposure of mice to PE-MPs and HDM induced a higher degree of inflammatory cell infiltration, bronchial goblet cell hyperplasia, collagen deposition, allergen sensitization, and Th2 immune bias than exposure to HDM alone. Co-exposure to PE-MPs and HDM aggravated oxidative stress injury in the lung and the production of cytokine IL-33 in the BALF. In addition, co-exposure of mice to PE-MPs and HDM resulted in a more pronounced decrease in the expression of relevant molecules of the airway epithelial barrier and more significant increase in the permeability of airway epithelia. Lung tissue transcriptomics analysis revealed that PE-MPs exposure was associated with CXCL1 signaling and neutrophil activation. Co-exposure to MPs and HDM may promote airway inflammation and airway epithelial barrier disruption and induce immune responses characterized by CXCL1 signaling and neutrophilic inflammation.

Butyric acid alleviates LPS-induced intestinal mucosal barrier damage by inhibiting the RhoA/ROCK2/MLCK signaling pathway in Caco2 cells.

Butyric acid (BA) can potentially enhance the function of the intestinal barrier. However, the mechanisms by which BA protects the intestinal mucosal barrier remain to be elucidated. Given that the Ras homolog gene family, member A (RhoA)/Rho-associated kinase 2 (ROCK2)/Myosin light chain kinase (MLCK) signaling pathway is crucial for maintaining the permeability of the intestinal epithelium, we further investigated whether BA exerts a protective effect on epithelial barrier function by inhibiting this pathway in LPS-induced Caco2 cells. First, we aimed to identify the optimal treatment time and concentration for BA and Lipopolysaccharide (LPS) through a CCK-8 assay. We subsequently measured Trans-epithelial electrical resistance (TEER), FITC-Dextran 4 kDa (FD-4) flux, and the mRNA expression of ZO-1, Occludin, RhoA, ROCK2, and MLCK, along their protein expression levels, and average fluorescence intensity following immunofluorescence staining. We then applied the ROCK2 inhibitor Y-27632 and reevaluated the TEER, FD-4 flux, and mRNA, and protein expression of ZO-1, Occludin, RhoA, ROCK2, and MLCK, as well as their distribution in Caco2 cells. The optimal treatment conditions were determined to be 0.2 mmol/L BA and 5 μg/mL LPS for 24 hours. Compared with LPS treatment alone, BA significantly mitigated the reduction in the TEER, decreased FD-4 flux permeability, increased the mRNA expression of ZO-1 and Occludin, and normalized the distribution of ZO-1 and Occludin in Caco2 cells. Furthermore, BA inhibited the expression of RhoA, ROCK2, and MLCK, and normalized their localization within Caco2 cells. Following treatment with Y-27632, the epithelial barrier function, along with the mRNA and protein expression and distribution of ZO-1 and Occludin were further normalized upon inhibition of the pathway. These findings contribute to a deeper understanding of the potential mechanisms through which BA attenuates LPS-induced impairment of the intestinal epithelial barrier.

Dietary Carrageenan Amplifies the Inflammatory Profile, but not Permeability, of Intestinal Epithelial Cells from Patients With Crohn’s Disease

Abstract Background The consumption of ultra-processed foods has increased significantly worldwide and is associated with the rise in inflammatory bowel diseases. However, any causative factors and their underlying mechanisms are yet to be identified. This study aimed to further elucidate whether different types of the dietary emulsifier carrageenan (CGN) can alter the permeability and inflammatory state of the intestinal epithelium. Methods Caco-2/HT29-MTX cocultures (n = 4) were exposed to either κ-, ι-, or λ-CGN (100 µg mL–1) for 24 hours. Organoid-derived monolayers from patients with Crohn’s Disease (CD) were exposed to κ-CGN (100 µg mL–1) for 48 hours (n = 10). In both models, an inflamed condition was established by adding a mix of inflammatory stimuli. Changes in permeability were measured by transepithelial electrical resistance (TEER). In the organoid-derived monolayers, cytokines were quantified in the apical and basolateral supernatant and gene expression was analyzed with RT-qPCR. Results None of the CGN subtypes altered permeability of non-inflamed or inflamed Caco-2/HT29-MTX cocultures. In organoid-derived monolayers, κ-CGN did not affect TEER, but induced alterations in the gene expression of tight junctions and mucus proteins. Expression of TNF, IL8, and IL1B increased upon κ-CGN stimulation, both in inflamed and non-inflamed monolayers. Cytokine release in the supernatant was increased by κ-CGN for IL-6, IL-13, IL-4, IL-2, and IL-10. Conclusions Dietary CGN caused upregulation of inflammatory markers and affected cytokine release of intestinal epithelial cells from CD patients, while permeability remained unaltered. When inflammation was already present, this pro-inflammatory effect was more pronounced, suggesting a role for dietary CGN during active CD.

Natural Epithelial Barrier Integrity Enhancers-Citrus medica and Origanum dayi Extracts.

Buccal drug delivery offers a promising alternative for avoiding gastrointestinal degradation and first-pass metabolism. However, enhancing the buccal epithelial barrier's permeability remains challenging. This study explores the effects of ethanolic extracts from Citrus medica var. Balady (CM), Citrus medica var. Calabria (CMC), and Origanum dayi (ORD) on buccal epithelium permeability in vitro using a TR146 cell-based model. The cell viability assay revealed that the extracts were non-toxic at the concentration range tested (<0.5% w/v). Surprisingly, none of the tested extracts significantly enhanced the buccal permeability of 40 kDa Fluorescein Isothiocyanate Dextran (FD40). However, the CMC and ORD extracts significantly reduced the epithelial permeability of FD40, mirroring the effects of hyaluronic acid (HA), a known barrier integrity enhancer. The total phenolic content (TPC) analysis suggested a potential link between the phenolic concentration and epithelial barrier reinforcement. The rapid colorimetric response method was applied to assess the interaction of these extracts with biological membranes. The results indicated that HA interacts with cellular membranes via lipid bilayer penetration, whereas the extracts likely influence the barrier integrity through alternative mechanisms, such as ligand-receptor interactions or extracellular matrix modulation. These findings highlight the potential of CMC and ORD extracts as natural agents to enhance buccal epithelial integrity. In conclusion, incorporating these extracts into formulations, such as hydrogels, could offer a cost-effective and biocompatible alternative to HA for improving buccal cavity health.

Functional constipation in children and probiotics: A review

Functional constipation (FC) is a common gastrointestinal (GI) disorder in children. In 25 % of patients, symptoms persist into adulthood. This condition is not only a complex medical problem but is also associated with financial and economic distress. Initially, FC was considered an intestinal motility dysfunction without structural and metabolic disorders. Pathophysiological mechanisms of FC development include stress, nutritional characteristics, insufficient fluid intake, food allergy, and dysbiosis, which determines the need for complex and long-term therapy. However, only 37% of patients fully comply with the recommendations of the attending physician, which indicates low adherence to treatment. Recent studies prove that motor disorders are associated with complex mechanisms of interaction of the gut microbiota (GM) and low-grade inflammation. Probiotics are seen as a key strategy to eliminate intestinal dysfunction. The effectiveness of probiotics is based on their ability to normalize the GM composition, as well as to correct the pH of the colon, improving the GI motility and thereby reducing the FC symptoms. The researchers agree that GM is a complex biological system that plays a central role in a developing organism, being a fundamental part of bidirectional functional axes. The main role in regulating the functioning of GM is given to the interaction between microbial groups, which forms the metabolic potential. The probiotic Lacticaseibacillus paracasei DG (LPDG) has a unique ability to regulate the composition of microbial GI colonization through modulation of GM metabolic activity. Studies showed the ability of LPDG to maintain colonization resistance, thereby preventing the development of epithelial permeability syndrome and an immunotropic effect, supporting the use of this probiotic as a pathogenetically based method of treating FC.

Development and Bioavailability Assessment of an Estriol-Containing Vaginal Hydrogel.

Genitourinary syndrome of menopause (GSM) affects a significant percentage of postmenopausal women and manifests as vaginal dryness, irritation, and urinary discomfort, typically treated with vaginal estrogens. Hydrogels are preferred over creams due to their superior comfort and mucoadhesive properties. This study introduces a novel vaginal gel formulation containing hydroxyethyl cellulose (HEC) and estriol-hydroxypropyl-β-cyclodextrin complex (E3-HPBCD) for the treatment of GSM. The estriol (E3) release profile of the gel was evaluated using a Franz diffusion cell system, and its permeability was tested on reconstructed human vaginal epithelium. Biocompatibility was assessed using (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT), lactate dehydrogenase (LDH) assays, and real-time cell analysis (RTCA) on human skin keratinocyte (HaCaT) cells, which showed increased cell viability and no obvious cytotoxicity. The results indicated that efficient E3 release and satisfactory epithelial permeability with HPBCD provide the bioavailability of E3. These results suggest the potential of the gel as a biocompatible and effective alternative for the treatment of GSM. Further studies are required to assess the long-term safety and clinical efficacy.