Epithelial Cell Integrity Research Articles

Nutraceutical Impact of Pumpkin Seed Oil on Expression Levels of EZH-2 and KRT-14 Genes against DSS-induced Inflammatory Bowel Disease in the Rat Model.

Inflammatory bowel disease is a collection of intestinal disorders that cause inflammation in the digestive tract. Prolonged inflammation in the gastrointestinal tract is a major risk factor for colorectal cancer. The objective of this study was to fucus on gene expression levels of (KRT-14; associated with epithelial cell integrity) and enhancer of zeste homolog-1 (EZH-2; involved in cellular proliferation) in a IBD rat model in order to rule out impact of nutraceuticals (pumpkin seed oil; PSO) as a complementary approach to conventional treatments of IBD. In the current study, IBD was induced using dextran sodium sulfate (DSS). Following acclimatization, rats were separated into three groups: the negative control, the positive control, and the treatment group. The DSS (1 ml/kg bw) was given to the positive control and treatment groups. Negative control was given only a normal diet. Pumpkin seed oil (PSO) was given orally as a treatment (0.5 ml/kg bw). Blood and colon tissue were obtained on the 5th, 10th, 14th, and 18th days. Physical parameters, hematology, biochemical assays, gene expression, and histopathology were carried out. After statistical analyses, macroscopic parameters showed significant differences. Biochemical analyses revealed a significant (P ≤ 0.05) decrease in serum potassium concentrations, total cholesterol, triglycerides, total proteins, total oxidants status, and C-reactive proteins in PSO treated group as compared with positive control. Gene expression levels of KRT-14 and EZH2 were significantly (P ≤ 0.05) upregulated in PSO treated group as compared to positive control group. Histopathology revealed that pumpkin seed oil preserved the structural integrity of colon.

SLAMF8 Disrupts Epithelial Barrier in Chronic Rhinosinusitis with Nasal Polyps via M1 Macrophage Polarization.

Recent studies show that M1 macrophages accumulate predominantly in non-eosinophilic chronic rhinosinusitis with nasal polyps (neCRSwNP). However, the precise mechanisms regulating M1 macrophages and their impact on the epithelial barrier remain unclear. We aim to investigate the expression and regulatory role of SLAMF8, a molecule exclusively expressed in myeloid cells, in M1 macrophage polarization and its potential contribution to neCRSwNP development. We evaluated SLAMF8 expression and its correlation with clinical variables using real-time quantitative polymerase chain reaction and Western blot in sinonasal mucosa samples from CRSwNP and control subjects. Immunofluorescence staining confirmed the co-expression of SLAMF8 with macrophages. After SLAMF8 knockdown, we explored the influence on macrophage M1 polarization and the effect on epithelial-mesenchymal transition (EMT) process and tight junction integrity in epithelial cells through an indirect co-culture system of M1 macrophages with human nasal epithelial cells. SLAMF8 was highly expressed on M1 macrophages in polyp tissues, notably in neCRSwNP, and correlated with disease severity indices only in neCRSwNP. SLAMF8 knockdown in THP-1 cells reduced M1 macrophage markers (CD86, iNOS, and NLRP3) and decreased secretion of inflammatory cytokines (IL-1β, IL-6, and TNF-α). Co-culture with M1 macrophage supernatant after SLAMF8 knockdown enhanced epithelial viability, reduced EMT and apoptosis, and upregulated tight junction markers Occludin and Claudin-4 in nasal epithelial cells. SLAMF8 elevation correlates with the EMT, epithelial tight junction and disease severity in neCRSwNP. The SLAMF8 upregulation promotes M1 macrophage polarization, by which facilitates EMT and impairs nasal epithelial barrier function. SLAMF8 may represent a novel therapeutic target for neCRSwNP.

Poliprotect®, a Medical Device Made of Substances, Potently Protects the Human Esophageal Mucosa Challenged by Multiple Agents: Evidence from In Vitro and Ex Vivo Electrophysiological Models.

The integrity of esophageal epithelial cells in patients with gastroesophageal reflux disease (GERD) or GERD-like symptoms is the first mechanism of protection to decrease the sensitivity to gastric reflux and heartburn symptoms. We investigated the protective effects of Poliprotect® (PPRO), a CE-marked medical device, on esophageal epithelial integrity using in vitro and ex vivo models. In vitro, the protective effects of PPRO were tested on Caco-2 cells. PPRO demonstrated safety and protection against oxidative damage induced by hydrogen peroxide. It also preserved epithelial integrity by maintaining transepithelial electrical resistance (TEER) against damage from calcium removal or bile acid exposure (taurodeoxycholic acid, TDCA). Ex vivo, esophageal biopsies from patients subjected to endoscopy were mounted in Ussing chambers and exposed to damaging agents (HCl or HCl + TDCA). Untreated biopsies (control) showed significant loss of epithelial resistance (up to -33%). In contrast, low concentrations of PPRO (50-100 µg/mL) provided strong protection against these damages (p < 0.001), even after 60 min of washing. Histological analysis confirmed the barrier-enhancing effect of PPRO. Overall, PPRO effectively protected the esophageal epithelium from damage in both models, suggesting its potential role in alleviating GERD or GERD-like symptoms by strengthening mucosal barriers and reducing epithelial sensitivity to reflux.

Glycosylation of oyster peptides by COS ameliorates zinc deficiency-induced syndromes: intestinal inflammation and imbalance of the gut microbiota in vivo.

Zinc is essential for maintaining the integrity and repair of small intestinal epithelial cells while zinc deficiency could induce the inflammatory infiltration and imbalance of intestinal flora in the intestine. In this study, glycosylation between oyster protein hydrolysate (OPH) and chitosan oligosaccharide (COS) was conducted and used as the carrier of zinc ions (OCZn). The results of zeta potential and particle size distribution showed that the OPH-COS successfully bound to zinc ions to form OCZn with a surface zinc content of 0.56% (scanning electron microscopy). In addition, OCZn was found to exhibit good intestinal digestion by in vitro simulated digestion microscopy, while TSQ fluorescence staining revealed the presence of free zinc ions released from OCZn in the intestinal cells. In the zinc deficiency-induced mouse model, a moderate dose of OCZn (zinc: 6.96 mg kg-1) showed significant restorative effects on colonic inflammation (IL-1β: 28.20 pg per mg·protein, IL-6: 27.73 pg per mg·protein), protein expressions of HO-1 and ZO-1, oxidative stress (the liver and kidneys), and imbalance of the gut microbiota, increasing microbial diversity and abundance (ratio of Firmicutes/Bacteroides). Zinc deficiency triggered the abundance of Proteobacteria (risk of diseases), while the dominant bacteria were mainly restored to Bacteroides, Parabacteroides, Alistipes, Alloprevotella, and Muribaculaceae following the administration of OCZn. This study provided a theoretical basis for improving the inflammatory infiltration of the colon and the imbalance of intestinal flora caused by zinc deficiency.

MiR-195-5p Inhibits Colon Cancer Progression via KRT23 Regulation.

KRT23 was recently discovered as an epithelial-specific intermediate filament protein in the type I keratin family. Many studies have underlined keratin's involvement in several biological processes as well as in the pathogenesis of different diseases. Specifically, KRT23 was reported to affect the structural integrity of epithelial cells and to trigger cellular signaling leading to the onset of cancer. The aim of this study is to characterize a novel mechanism based on miR-195-5p/KRT23 in colorectal cancer. KRT23 mRNA and protein expression were characterized in FFPE sections from patients with CRC. The effects of miR-195-5p on KRT23 expression at the mRNA and protein levels were assessed by transient transfection experiments with mimic and inhibitor molecules. Cell attachment/detachment, migration, invasion, clone formation, and apoptosis were evaluated in human CRC cell lines after miR-195-5p mimic transfection. We identified KRT23 as a putative target of miR-195-5p, a microRNA that we previously demonstrated to be reduced in CRC. We have proved the KRT23 expression deregulation in the tumoral section compared to adjacent normal mucosa in patients with CRC, according to the data derived from the public repository. We proved that the gain of miR-195-5p decreased the KRT23 expression. Conversely, we demonstrated that the inhibition of miR-195-5p led to an increase in KRT23 expression levels. We have demonstrated the in vitro effectiveness of miR-195-5p on CRC progression and that the in vivo intraperitoneal delivery of miR-195-5p mimic lowered colonic KRT23 mRNA and protein expression. These findings highlight a new regulatory mechanism by miR-195-5p in CRC affecting the keratin intermediate filaments and underline the miR-195-5p potential clinical properties.

Hyperosmotic Stress Induces the Expression of Organic Osmolyte Transporters in Porcine Intestinal Cells and Betaine Exerts a Protective Effect on the Barrier Function.

Background/objectives: The porcine intestinal epithelium plays a fundamental role as a defence interface against pathogens. Its alteration can cause severe inflammatory conditions and diseases. Hyperosmotic stress under physiological conditions and upon pathogen challenge can cause malabsorption. Different cell types counteract the osmolarity increase by accumulating organic osmolytes such as betaine, taurine, and myo-inositol through specific transporters. Betaine is known for protecting cells from hyperosmotic stress and has positive effects when fed to pigs. The aim of this study is to demonstrate the modulation of osmolyte transporters gene expression in IPEC-J2 during osmolarity changes and assess the effects of betaine. Methods: IPEC-J2 were seeded in transwells, where differentiate as a polarized monolayer. Epithelial cell integrity (TEER), oxidative stress (NO) and gene expression of osmolyte transporters, tight junction proteins (TJp) and pro-inflammatory cytokines were evaluated. Results: Cells treated with NaCl hyperosmolar medium (500 mOsm/L) showed a TEER decrease at 3 h and detachment within 24 h, associated with an osmolyte transporters reduction. IPEC-J2 treated with mannitol hyperosmolar medium (500 mOsm/L) upregulated taurine (TauT), myo-inositol (SMIT) and betaine (BGT1) transporters expression. A decrease in TJp expression was associated with a TEER decrease and an increase in TNFα, IL6, and IL8. Betaine could attenuate the hyperosmolarity-induced reduction in TEER and TJp expression, the NO increase and cytokines upregulation. Conclusions: This study demonstrates the expression of osmolyte transporters in IPEC-J2, which was upregulated upon hyperosmotic treatment. Betaine counteracts changes in intracellular osmolarity by contributing to maintaining the epithelial barrier function and reducing the inflammatory condition. Compatible osmolytes may provide beneficial effects in therapies for diseases characterized by inflammation and TJp-related dysfunctions.

A multispecies bacterial-based direct-fed microbial alleviates Salmonella invasion and supports in vitro epithelial integrity.

Managing bacterial infections is of great importance in livestock production, particularly those caused by Salmonella enterica serovars Typhimurium or Dublin, which can impact both animal health and performance, as well as human food safety. Direct-fed microbials (DFM) can support gastrointestinal function and alleviate the potential negative effects of bacterial infections. In the present study, the capacity of a multispecies bacterial-based DFM containing Ligilactobacillus (formerly Lactobacillus) animalis 506, Propionibacterium freudenreichii 507, Bacillus licheniformis 809, and B. subtilis 597 to reduce S. Typhimurium ATCC14028 invasion was investigated using a co-incubation model with the HT29-MTX-E12 cell line (experiment 1). Next, a possible antagonistic effect of the DFM against S. Dublin ATCC 41286 was evaluated using an in vitro agar well diffusion method following a co-incubation of 48 h (experiment 2). At last, a series of experiments were performed to evaluate how different doses (6.25 × 106, 2.50 × 107, or 1.00 × 108 CFU/well) of the DFM would support the integrity of intestinal epithelial cells challenged or not with S. Typhimurium ATCC14028 or hydrogen peroxide under a transepithelial electrical resistance (TEER) assay with Caco-2 cells (experiments 3 and 4). In experiment 1, BDP significantly (P < 0.001) reduced by 90.8% the invasion of S. Typhimurium into HT29-MTX-E12 cells, whereas viability of the potentially harmful bacteria was reduced by 21.0% (P < 0.0001). In experiment 2, the antagonistic properties of BDP towards S. Dublin were confirmed by the detection of a clear inhibition zone (size = 8.6 mm). Lastly, without challenge, the lowest dose of the DFM (6.25 × 106 CFU) provided the greatest support to the cells (treatment × hour; P < 0.0001). However, when the cells were challenged with S. Typhimurium, all doses alleviated the loss of integrity caused by the pathogen (treatment × hour; P < 0.0001). In cells challenged with hydrogen peroxide, the greater dose (1.00 × 108 CFU) supported the cells for a longer period of time (treatment × hour; P < 0.0001). These in vitro findings set the stage for exploring the potential benefits of using a novel DFM as a promising tool and strategy to mitigate S. enterica infections in ruminants and improve animal health, food safety, and public health. Further, in vivo confirmation needs to be developed to validate these preliminary in vitro results.

A-239 Cxcl10 induced gut dysbiosis exacerbates acute lung injury and secondary infection in influenza by inhibiting il22

Abstract Background Secondary infections are the most cause of severe pulmonary injury and death in influenza. CXCL10, known as interferon γ-induced protein 10(IP-10), is highly upregulated in response to IFN-γstimulation during influenza. Previous studies show that CXCL10 contributes to bacteria co-infections and worse finales in influenza. However, the underlying mechanisms by which CXCL10 facilitate secondary infections remains unclear. Emerging evidences show that influenza virus infections disturb the composition and function of gut microbiota, which in turn influences the infection process. Herein, we aimed to explore whether CXCL10 could affect the progress of influenza infection and secondary infection in a gut microbiota dependent manner. Methods CXCL10 knockout mice were used to determine the role of CXCL10 in staphylococcus aureus co-infection after influenza A virus (IAV) infection. Lung tissues from CXCL10 knockout mice were collected for Transcriptomics sequencing. Feces from mice and clinicals including IAV patients and healthy individuals were collected for Metagenomic sequencing. Paired serum samples were collected for Metabolomic sequencing. QRT-PCR was performed to determine the expression of inflammatory factors in lung tissues from CXCL10 knockout mice. Elisa assays were used to measure the serum expression levels of CXCL10 and IL22. Flow cytometry was applied to detect the proportion of IL22+ cells. Fecal microbiota transplantation (FMT) was applied to confirm that CXCL10 plays an adverse role in acute lung injury and staphylococcus aureus co-infection after IAV infection by disrupting intestinal microbiome homeostasis. Results CXCL10 aggravated acute lung injury and promoted staphylococcus aureus coinfection after IAV infection. Transcriptomics sequencing results suggests improved cell membrane integrity and regeneration of epithelial cells after IAV infection in CXCL10 knockout mice with an upregulation of integrity proteins. CXCL10 knockout significantly increased the expression levels of IL22 without influencing virus duplication. rh-IL22 protects lung epithelial cell from staphylococcus aureus co-infection in influenza and increased the expression of tight junction proteins. The gut microbiome shows different structures between wild type mice and knockout mice, as well as clinical samples according to serum CXCL10 expression levels. We found some genera were markedly increased in knockout mice and patients with low serum CXCL10 expression levels including Lactobacillus, Lachnospiraceae, Oscillibacter, Odoribacter, Eubacterium. FMT from patients with low serum CXCL10 levels and healthy individuals ameliorating lung pathological damage. Conclusions This study interpreted that CXCL10 exacerbates acute lung injury and secondary infection in influenza by inducing gut dysbiosis, suggesting that balancing the gut flora could be assistant treatments to protect from acute lung injury and secondary infections in influenza. And IL22 may be a potential mediating factor between gut-lung axis to improve epithelial integrity.

Eosinophilic gastrointestinal disorders and the role for the epithelium in pathogenesis and treatment.

This review aims to provide an overview of the current understanding of eosinophilic gastrointestinal disorders (EGIDs) and the role of the epithelium in influencing disease pathogenesis to inform and devise future therapeutic strategies. Changes in epithelial cell structure, functions, and integrity are observed in EGIDs. In eosinophilic esophagitis (EoE), the esophageal epithelium has been shown to play key roles in perpetuating the inflammatory response in EoE through the expression of pro-inflammatory cytokines and immunological cell-surface proteins. Similar mechanisms appear to exist in the other EGIDs, including eosinophilic gastritis (EoG), eosinophilic enteritis (EoN), and eosinophilic colitis (EoC). Because of the increasing rarity of each non-EoE EGID, research focusing on how the epithelium is modulating disease in each lower gastrointestinal compartment is still in its rudimentary stages. While there has been significant progress in understanding the role of the epithelium in EoE, further research is needed to obtain a better understanding of the mechanisms mediating epithelial-immune crosstalk in non-EoE EGIDs. Using EoE-epithelial cell research to inform future EGID investigations could lead to the development of new therapeutic interventions, such as targeted therapies to restore epithelial barrier function and reduce inflammation, to improve rare disease-patient quality of life.

Effect of β-Alanine Metabolite on Gut Integrity and Immunity in Commercial Broiler Chickens Infected with Eimeria maxima.

(1) Background: In a metabolomics analysis conducted to investigate the mechanisms behind the growth-promoting effects of probiotics in broilers, β-alanine was found to be significantly elevated. This led to the hypothesis that β-alanine could also contribute to growth-promoting effects in infected broilers. (2) Methods: An in vitro culture system was developed to assess β-alanine's impact on proinflammatory cytokine response in chicken macrophage cells, gut integrity in chicken intestinal epithelial cells, and muscle differentiation in quail muscle cells and primary chicken embryonic muscle cells. In vivo animal feeding studies were then conducted to investigate the effects of dietary β-alanine on various disease parameters in Eimeria maxima-infected broiler chickens. (3) Results: In vitro, β-alanine treatment significantly decreased the gene expression of cytokines in chicken macrophage cells and increased occuldin expression in chicken intestinal epithelial cells. Dietary β-alanine increased the body weight of chickens following Eimeria maxima infection in the H-ALA group. Dietary β-alanine also suppressed cytokines and increased JAM-2 and occludin expression in the H-ALA group compared to the infected group without β-alanine supplementation. (4) Conclusions: These results strongly support the positive effects of dietary β-alanine on intestinal immune responses and gut barrier function in broiler chickens infected with Eimeria maxima.

Irisin preserves mitochondrial integrity and function in tubular epithelial cells after ischemia-reperfusion-induced acute kidney injury.

A myokine secreted by skeletal muscles during exercise called irisin mitigates ischemia-reperfusion (I/R) injury in epithelial cells of various organs by limiting damage to mitochondria. We test whether irisin may preserve the mitochondrial integrity and function in renal tubular epithelial cells and protect against ischemia-reperfusion-induced acute kidney injury (AKI). We correlated serum irisin levels with serum creatinine and BUN levels from both AKI patients and healthy individuals. In mice with irisin administration, various renal injury markers such as serum creatinine, BUN, kidney injury molecule-1 (Kim-1), and neutrophil gelatinase-associated lipocalin (NGAL), and renal histopathology were assessed after I/R. To identify the potential mechanisms of the protective of irisin's protective effect, we perfused proximal tubules under confocal microscopy and analyzed kidney tissues by qPCR, western blot, and immunohistochemistry. Serum irisin correlated inversely with serum creatinine and BUN levels were significantly lower in AKI patients than in healthy subjects. Administering irisin to mice after I/R decreased biomarker levels for AKI including serum creatinine, BUN, Kim-1, NAGL and lessened histological changes. In kidney tissues of mice, irisin upregulated the mitochondrial autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3), the mitochondrial autophagy pathway-related proteins PTEN-induced putative kinase 1 (PINK1) and Parkinson's disease 2 parkin (PARK2) and downregulated the reactive substrate protein sequestosome 1 (P62) and mitochondrial membrane proteins translocase of outer mitochondrial membrane 20 (TOM20) and translocase of inner mitochondrial membrane 23 (TIM23). Irisin protects against renal I/R injury, which may involve the preservation of mitochondrial integrity and function.

A Review on Analytical Methods for Determination of Azithromycin

Azithromycin treatment has been associated with a decrease in ventilation time and death in several viral infections. It possesses immune-modulating properties, including the capacity to inhibit cytokine production, preserve the integrity of epithelial cells, and prevent lung fibrosis. Primary hepatic metabolism is the process by which drugs are broken down into inactive metabolites that keep their biological effects. These prompted numerous studies and publications that used a variety of analytical techniques to find, evaluate, and investigate azithromycin and its metabolites. This review aims to provide an overview of the various analytical techniques—such as voltammetry, flow injection, hyphenated mass spectrometry, and chromatography—that have been published for the years 1990 to 2020 in order to determine azithromycin. While azithromycin was most commonly quantified using high-performance liquid chromatography, the study's results indicate that when compared to alternative techniques, liquid chromatography-mass spectrometry had the highest sensitivity, with a limit of detection of 0.0005 µg/mL.

The ARDS microenvironment enhances MSC-induced repair via VEGF in experimental acute lung inflammation

Clinical trials investigating the potential of mesenchymal stromal cells (MSCs) for the treatment of inflammatory diseases, such as acute respiratory distress syndrome (ARDS), have been disappointing; with less than 50% of patients responding to treatment. Licensed MSCs show enhanced therapeutic efficacy in response to cytokine-mediated activation signals. There are two distinct sub-phenotypes of ARDS: hypo- and hyper-inflammatory. We hypothesised that pre-licensing MSCs in a hyper-inflammatory ARDS environment would enhance their therapeutic efficacy in acute lung inflammation (ALI). Serum samples from ARDS patients were segregated into hypo- and hyper-inflammatory categories based on IL-6 levels. MSCs were licensed with pooled serum from hypo- or hyper-inflammatory ARDS patients or healthy serum controls. Our findings show that hyper-inflammatory ARDS pre-licensed MSC conditioned medium (MSC-CMHyper) led to a significant enrichment in tight junction expression and enhanced barrier integrity in lung epithelial cells in vitro and in vivo; in a VEGF-dependent manner. Importantly, while both MSC-CMHypo and MSC-CMHyper significantly reduced IL-6 and TNFα levels in the bronchoalveolar lavage fluid (BALF) of LPS-induced ALI mice, only MSC-CMHyper significantly reduced lung permeability and overall clinical outcomes including weight loss and clinical score. Thus the hypo- and hyper-inflammatory ARDS environment may differentially influence MSC cytoprotective and immunomodulatory functions.

Microglial type I interferon signaling mediates chronic stress-induced synapse loss and social behavior deficits.

Inflammation and synapse loss have been associated with deficits in social behavior and are involved in pathophysiology of many neuropsychiatric disorders. Synapse loss, characterized by reduction in dendritic spines can significantly disrupt synaptic connectivity and neural circuitry underlying social behavior. Chronic stress is known to induce loss of spines and dendrites in the prefrontal cortex (PFC), a brain region implicated in social behavior. However, the underlying mechanisms are not well understood. In the present study, we investigated the role of type I Interferon (IFN-I) signaling in chronic unpredictable stress (CUS)-induced synapse loss and behavior deficits in mice. We found increased expression of type I IFN receptor (IFNAR) in microglia following CUS. Conditional knockout of microglial IFNAR in adult mice rescued CUS-induced social behavior deficits and synapse loss. Bulk RNA sequencing data show that microglial IFNAR deletion attenuated CUS-mediated changes in the expression of genes such as Keratin 20 (Krt20), Claudin-5 (Cldn5) and Nuclear Receptor Subfamily 4 Group A Member 1 (Nr4a1) in the PFC. Cldn5 and Nr4a1 are known for their roles in synaptic plasticity. Krt20 is an intermediate filament protein responsible for the structural integrity of epithelial cells. The reduction in Krt20 following CUS presents a novel insight into the potential contribution of cytokeratin in stress-induced alterations in neuroplasticity. Overall, these results suggest that microglial IFNAR plays a critical role in regulating synaptic plasticity and social behavior deficits associated with chronic stress conditions.

Matrix-assisted laser desorption ionization mass spectrometry imaging reveals the spatial distribution of compounds that may exacerbate inflammation in garden ginseng and ginseng under forest

Ginseng, a highly esteemed herbal medicine, has been utilized over 5000 years, predominantly in Far Eastern countries. Ginseng is categorized into garden ginseng (GG) and ginseng under forest (FG). However, in contrast to FG, excessive intake of GG may lead to potential adverse effects due to disruption of epithelial cell integrity, and the specific population groups that may be at higher risk. In this work, untargeted metabolomics were used to determine the heterogeneity between GG and FG, the data indicates that the content of Ethyl caffeate, Homoorientin, Citric acid and Quinic acid in GG were higher than in FG. Mass spectrometry imaging showed that ethyl caffeate and Homoorientin were concentrated on the brownish yellow exocarp of the primary root. Our experiments demonstrated that excessive exposure to ethyl caffeate and Homoorientin exacerbated the inflammatory response of HUVECs and reduced the expression of cell junctions. This suggest that the compounds causing adverse effects from excessive intake of GG are mainly concentrated in the yellow exocarp of the primary root of GG. These results suggest that untargeted metabolomics coupled with MALDI-MSI can visualize the spatial distribution of endogenous differential molecules of the same herb in different growth environments or developmental stages.

CTNNAL1 promotes the structural integrity of bronchial epithelial cells through the RhoA/ROCK1 pathway.

Adhesion molecules play critical roles in maintaining the structural integrity of the airway epithelium in airways under stress. Previously, we reported that catenin alpha-like 1 (CTNNAL1) is downregulated in an asthma animal model and upregulated at the edge of human bronchial epithelial cells (HBECs) after ozone stress. In this work, we explore the potential role of CTNNAL1 in the structural adhesion of HBECs and its possible mechanism. We construct a CTNNAL1 ‒/‒ mouse model with CTNNAL1-RNAi recombinant adeno-associated virus (AAV) in the lung and a CTNNAL1-silencing cell line stably transfected with CTNNAL1-siRNA recombinant plasmids. Hematoxylin and eosin (HE) staining reveals that CTNNAL1 ‒/‒ mice have denuded epithelial cells and structural damage to the airway. Silencing of CTNNAL1 in HBECs inhibits cell proliferation and weakens extracellular matrix adhesion and intercellular adhesion, possibly through the action of the cytoskeleton. We also find that the expressions of the structural adhesion-related molecules E-cadherin, integrin β1, and integrin β4 are significantly decreased in ozone-treated cells than in vector control cells. In addition, our results show that the expression levels of RhoA/ROCK1 are decreased after CTNNAL1 silencing. Treatment with Y27632, a ROCK inhibitor, abolished the expressions of adhesion molecules induced by ozone in CTNNAL1-overexpressing HBECs. Overall, the findings of the present study suggest that CTNNAL1 plays a critical role in maintaining the structural integrity of the airway epithelium under ozone challenge, and is associated with epithelial cytoskeleton dynamics and the expressions of adhesion-related molecules via the RhoA/ROCK1 pathway.

Protective effect of zinc gluconate on intestinal mucosal barrier injury in antibiotics and LPS-induced mice

ObjectiveThe aim of the study is to investigate the function and mechanism of Zinc Gluconate (ZG) on intestinal mucosal barrier damage in antibiotics and Lipopolysaccharide (LPS)-induced mice.MethodsWe established a composite mouse model by inducing intestinal mucosal barrier damage using antibiotics and LPS. The animals were divided into five groups: Control (normal and model) and experimental (low, medium, and high-dose ZG treatments). We evaluated the intestinal mucosal barrier using various methods, including monitoring body weight and fecal changes, assessing pathological damage and ultrastructure of the mouse ileum, analyzing expression levels of tight junction (TJ)-related proteins and genes, confirming the TLR4/NF-κB signaling pathway, and examining the structure of the intestinal flora.ResultsIn mice, the dual induction of antibiotics and LPS led to weight loss, fecal abnormalities, disruption of ileocecal mucosal structure, increased intestinal barrier permeability, and disorganization of the microbiota structure. ZG restored body weight, alleviated diarrheal symptoms and pathological damage, and maintained the structural integrity of intestinal epithelial cells (IECs). Additionally, ZG reduced intestinal mucosal permeability by upregulating TJ-associated proteins (ZO-1, Occludin, Claudin-1, and JAM-A) and downregulating MLCK, thereby repairing intestinal mucosal barrier damage induced by dual induction of antibiotics and LPS. Moreover, ZG suppressed the TLR4/NF-κB signaling pathway, demonstrating anti-inflammatory properties and preserving barrier integrity. Furthermore, ZG restored gut microbiota diversity and richness, evidenced by increased Shannon and Observed features indices, and decreased Simpson’s index. ZG also modulated the relative abundance of beneficial human gut bacteria (Bacteroidetes, Firmicutes, Verrucomicrobia, Parabacteroides, Lactobacillus, and Akkermansia) and harmful bacteria (Proteobacteria and Enterobacter), repairing the damage induced by dual administration of antibiotics and LPS.ConclusionZG attenuates the dual induction of antibiotics and LPS-induced intestinal barrier damage and also protects the intestinal barrier function in mice.

Collagen 17A1 in the Urothelium Regulates Epithelial Cell Integrity and Local Immunologic Responses in Obstructive Uropathy

Collagen 17A1 (COL17A1), an epidermal hemidesmosome component, is ectopically induced in the urothelium of mouse and human renal pelvis (RP) in parallel with urinary tract-associated lymphoid structure development. Here, COL17A1 was induced in obstructive uropathy-prone ureter of humans and cats. To ascertain its function, murine urinary organs with unilateral ureteral obstruction (UUO) were analyzed during 1 week after surgery. One day after UUO, COL17A1 expression increased in urothelial cells of RP and ureter, and was positively correlated with renal tubulointerstitial lesions. A portion of RP where the smooth muscle layer from the ureter was interrupted was sensitive to urothelium deciduation and COL17A1 induction, showing urine leaked from the RP lumen into the parenchyma. After urine stimulation, cultured immune cells expressed Cxcl2, also up-regulated in CD11b+ cells following COL17A1 stimulation. One day after UUO, CXCL2+ CD11b+ cells infiltrated the urothelium-disrupted area. However, these numbers were significantly lower in Col17a1-deficient mice. COL17A1+ urothelial cells partially co-expressed cytokeratin-14, a progenitor cell marker for urothelium, whereas Col17a1-deficient mice had lower numbers of cytokeratin-14+ cells. Gene Ontology analysis revealed that expression of epithelial- and immune-associated genes was up-regulated and down-regulated, respectively, in the ureter of Col17a1-deficient mice 4 days after UUO. Thus, COL17A1 maintains urothelium integrity by regulating urothelial cell adhesion, proliferation, and differentiation, and activates local immune responses during obstructive uropathy in mammals.

Lack of Pkd1 enhances intestinal integrity and motility prior to accelerated kidney cyst growth

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disorder, primarily caused by PKD1 germline mutations. Polycystin-1, PKD1’s encoded protein, is essential to maintain kidney epithelial cell integrity, but its role in the gut has yet to be determined. Considering macrophage infiltration precedes accelerated cyst growth in Pkd1 knockout ( Pkd1KO) mice, and the gut is the body’s largest reservoir of inflammatory cells, we hypothesized that loss of Pkd1 would weaken the intestinal epithelial cell barrier and mount an inflammatory response, potentially preceding kidney damage. Plasma, urine, kidney, and intestines were collected from 11-12 week old male and female conditional Pkd1KO (CAGG-creER; tamoxifen-induced at 5-6 weeks) and flox (control) mice. Plasma was used to measure intestinal permeability as well as monocyte chemoattractant protein-1 (MCP-1) and lipocalin-2 (LCN-2) concentration. Urine was utilized to assess kidney injury, while tissues were used for RT-PCR and histology. Pkd1KO mice had significantly elevated kidney-to-body weight ratio, cyst ratio, and expression of tubular injury markers kidney injury molecule 1 (Kim1) and Lcn2 compared to flox mice. Despite this cellular injury, kidney inflammatory gene expression and urine osmolality did not differ between genotypes. No overt morphological changes were observed in the ileum or colon, nor were there differences in expression of proinflammatory chemokines or cytokines in either tissue. Plasma abundance of MCP-1 and LCN-2 was similar between genotypes. Epithelial permeability in the small and large intestines was significantly decreased in Pkd1KO versus flox mice. Moreover, Pkd1KO mice had a significantly reduced whole gut transit time compared to flox mice. This corresponded with increases in colonic expression of epithelial integrity marker occludin and water channels aquaporin 4 and aquaporin 8 in Pkd1KO mice. Together, these data insinuate that loss of Pkd1 in the intestines leads to a tighter intestinal barrier accompanied by increased water channel expression and gut motility. These findings appear unrelated to inflammatory involvement in early PKD. These studies were supported by NIH T32 DK007545 to RS, R01 DK132028 to TS, and R03 DK119717 to TS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Blends of Organic Acids Are Weaponizing the Host iNOS and Nitric Oxide to Reduce Infection of Piscirickettsia salmonis in vitro.

For the last 30 years, Piscirickettsia salmonis has caused major economic losses to the aquaculture industry as the aetiological agent for the piscirickettsiosis disease. Replacing the current interventions, based on antibiotics, with natural alternatives (e.g., organic acids) represents a priority. With this study, we aimed to better understand their biological mechanism of action in an in vitro model of infection with salmon epithelial cells (CHSE-214). Our first observation revealed that at the sub-inhibitory concentration of 0.5%, the organic acid blend (Aq) protected epithelial cell integrity and significantly reduced P. salmonis invasion. The MIC was established at 1% Aq and the MBC at 2% against P. salmonis. The sub-inhibitory concentration significantly increased the expression of the antimicrobial peptides Cath2 and Hepcidin1, and stimulated the activity of the innate immune effector iNOS. The increase in iNOS activity also led to higher levels of nitric oxide (NO) being released in the extracellular space. The exposure of P. salmonis to the endogenous NO caused an increase in bacterial lipid peroxidation levels, a damaging effect which can ultimately reduce the pathogen's ability to attach or multiply intracellularly. We also demonstrate that the increased NO release by the host CHSE-214 cells is a consequence of direct exposure to Aq and is not dependent on P. salmonis infection. Additionally, the presence of Aq during P. salmonis infection of CHSE-214 cells significantly mitigated the expression of the pro-inflammatory cytokines IL-1β, IL-8, IL-12, and IFNγ. Taken together, these results indicate that, unlike antibiotics, natural antimicrobials can weaponize the iNOS pathway and secreted nitric oxide to reduce infection and inflammation in a Piscirickettsia salmonis in vitro model of infection.