Claudin-4 Modulates Autophagy via SLC1A5/LAT1 as a Mechanism to Regulate Micronuclei.

Genome instability is a hallmark of cancer crucial for tumor heterogeneity and is often a result of defects in cell division and DNA damage repair. Tumors tolerate genomic instability, but the accumulation of genetic aberrations is regulated to avoid catastrophic chromosomal alterations and cell death. In ovarian cancer tumors, claudin-4 is frequently upregulated and closely associated with genome instability and worse patient outcomes. However, its biological association with regulating genomic instability is poorly understood. Here, we used CRISPR interference and a claudin mimic peptide to modulate the claudin-4 expression and its function in vitro and in vivo. We found that claudin-4 promotes a tolerance mechanism for genomic instability through micronuclei generation in tumor cells. Disruption of claudin-4 increased autophagy and was associated with the engulfment of cytoplasm-localized DNA. Mechanistically, we observed that claudin-4 establishes a biological axis with the amino acid transporters SLC1A5 and LAT1, which regulate autophagy upstream of mTOR. Furthermore, the claudin-4/SLC1A5/LAT1 axis was linked to the transport of amino acids across the plasma membrane as one of the potential cellular processes that significantly decreased survival in ovarian cancer patients. Together, our results show that the upregulation of claudin-4 contributes to increasing the threshold of tolerance for genomic instability in ovarian tumor cells by limiting its accumulation through autophagy. SIGNIFICANCE: Autophagy regulation via claudin-4/SLC1A5/LAT1 has the potential to be a targetable mechanism to interfere with genomic instability in ovarian tumor cells.

Expression Patterns of Grainyhead-Like 2 and Ovo-Like 2 in Mouse Mammary Gland Development During Pregnancy, Lactation, and Weaning.

Grainyhead-like 2 (Grhl2) is a transcription factor that regulates cell adhesion genes in mammary ductal development and serves as a repressor of the epithelial-mesenchymal transition. Conversely, Ovo-like2 (Ovol2) is a target gene of Grhl2 but functions as a substitute in Grhl2-deficient mice, facilitating successful epithelial barrier formation and lumen expansion in kidney-collecting ductal epithelial cells. Our objective was to examine the expression patterns of Grhl2, Ovol2, and their associated genes during the intricate phases of mouse mammary gland development. The mRNA expression of Grhl2 and Ovol2 increased after pregnancy. We observed Grhl2 protein presence in the epithelial cell's region, coinciding with acini formation, and its signal significantly correlated with E-cadherin (Cdh1) expression. However, Ovol2 was present in the epithelial region without a correlation with Cdh1. Similarly, Zeb1, a mesenchymal transcription factor, showed Cdh1-independent expression. Subsequently, we explored the interaction between Rab25, a small G protein, and Grhl2/Ovol2. The expressions of Grhl2 and Ovol2 exhibited a strong correlation with Rab25 and claudin-4, a tight junction protein. These findings suggest that Grhl2 and Ovol2 may collaborate to regulate genes associated with cell adhesion and are crucial for maintaining epithelial integrity during the different phases of mammary gland development.

Structural and biophysical insights into targeting of claudin-4 by a synthetic antibody fragment.

Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties-these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.

Disruption of pulmonary microvascular endothelial barrier by dysregulated claudin-8 and claudin-4: uncovered mechanisms in porcine reproductive and respiratory syndrome virus infection.

The pulmonary endothelium is a dynamic and metabolically active monolayer of endothelial cells. Dysfunction of the pulmonary endothelial barrier plays a crucial role in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), frequently observed in the context of viral pneumonia. Dysregulation of tight junction proteins can lead to the disruption of the endothelial barrier and subsequent leakage. Here, the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) served as an ideal model for studying ALI and ARDS. The alveolar lavage fluid of pigs infected with HP-PRRSV, and the supernatant of HP-PRRSV infected pulmonary alveolar macrophages were respectively collected to treat the pulmonary microvascular endothelial cells (PMVECs) in Transwell culture system to explore the mechanism of pulmonary microvascular endothelial barrier leakage caused by viral infection. Cytokine screening, addition and blocking experiments revealed that proinflammatory cytokines IL-1ß and TNF-α, secreted by HP-PRRSV-infected macrophages, disrupt the pulmonary microvascular endothelial barrier by downregulating claudin-8 and upregulating claudin-4 synergistically. Additionally, three transcription factors interleukin enhancer binding factor 2 (ILF2), general transcription factor III C subunit 2 (GTF3C2), and thyroid hormone receptor-associated protein 3 (THRAP3), were identified to accumulate in the nucleus of PMVECs, regulating the transcription of claudin-8 and claudin-4. Meanwhile, the upregulation of ssc-miR-185 was found to suppress claudin-8 expression via post-transcriptional inhibition. This study not only reveals the molecular mechanisms by which HP-PRRSV infection causes endothelial barrier leakage in acute lung injury, but also provides novel insights into the function and regulation of tight junctions in vascular homeostasis.

Single-cell transcriptomics reveal metastatic CLDN4+ cancer cells underlying the recurrence of malignant pleural effusion in patients with advanced non-small-cell lung cancer.

BACKGROUND: Recurrent malignant pleural effusion (MPE) resulting from non-small-cell lung cancer (NSCLC) is easily refractory to conventional therapeutics and lacks predictive markers. The cellular or genetic signatures of recurrent MPE still remain largely uncertain. METHODS: 16 NSCLC patients with pleural effusions were recruited, followed by corresponding treatments based on primary tumours. Non-recurrent or recurrent MPE was determined after 3-6 weeks of treatments. The status of MPE was verified by computer tomography (CT) and cytopathology, and the baseline pleural fluids were collected for single-cell RNA sequencing (scRNA-seq). Samples were then integrated and profiled. Cellular communications and trajectories were inferred by bioinformatic algorithms. Comparative analysis was conducted and the results were further validated by quantitative polymerase chain reaction (qPCR) in a larger MPE cohort from the authors' centre (n = 64). RESULTS: The scRNA-seq revealed that 33 590 cells were annotated as 7 major cell types and further characterized into 14 cell clusters precisely. The cell cluster C1, classified as Epithelial Cell Adhesion Molecule (EpCAM)+ metastatic cancer cell and correlated with activation of tight junction and adherence junction, was significantly enriched in the recurrent MPE group, in which Claudin-4 (CLDN4) was identified. The subset cell cluster C3 of C1, which was enriched in recurrent MPE and demonstrated a phenotype of ameboidal-type cell migration, also showed a markedly higher expression of CLDN4. Meanwhile, the expression of CLDN4 was positively correlated with E74 Like ETS Transcription Factor 3 (ELF3), EpCAM and Tumour Associated Calcium Signal Transducer 2 (TACSTD2), independent of driver-gene status. CLDN4 was also found to be associated with the expression of Hypoxia Inducible Factor 1 Subunit Alpha (HIF1A) and Vascular Endothelial Growth Factor A (VEGFA), and the cell cluster C1 was the major mediator in cellular communication of VEGFA signalling. In the extensive MPE cohort, a notably increased expression of CLDN4 in cells from pleural effusion among patients diagnosed with recurrent MPE was observed, compared with the non-recurrent group, which was also associated with a trend towards worse overall survival (OS). CONCLUSIONS: CLDN4 could be considered as a predictive marker of recurrent MPE among patients with advanced NSCLC. Further validation for its clinical value in cohorts with larger sample size and in-depth mechanism studies on its biological function are warranted. TRIAL REGISTRATION: Not applicable.

Transcriptional Levels of Intercellular Junction Proteins in an Alveolar Epithelial Cell Line Exposed to Irradiation or Bleomycin.

We performed a comparative study of the effects of X-ray irradiation and bleomycin on the mRNA levels of E-cadherin and tight junction proteins (claudin-3, claudin-4, claudin-18, ZO-2, and occludin) in an alveolar epithelial cell line L2. Irradiation decreased claudin-4 levels and increased occludin levels, while the levels of other mRNAs remained unchanged. Bleomycin increased the expression levels of all proteins examined except claudin-3. Irradiation and bleomycin have different effects on the expression level of intercellular junction proteins, indicating different reactions triggered in alveolar epithelial cells and a great prospects of further comparative studies.

Development of tight junction-strengthening compounds using a high-throughput screening system to evaluate cell surface-localized claudin-1 in keratinocytes.

Tight junctions (TJs) are important factors constituting the physical barriers of the skin, and their suppression has been described in various conditions, such as aged skin and atopic dermatitis lesions. However, the methods for improving skin TJ function remain insufficient. Therefore, to obtain compounds that can improve TJ function, we developed a novel high-throughput screening system termed live-cell immunostaining to evaluate cell surface-localized claudin-1 (CLDN1) with high selectivity using normal human epidermal keratinocytes (NHEKs). Heparinoid and phospho-pyridoxal (p-Pyr), a metabolite of pyridoxine, were identified as hit compounds. In addition, heparinoid was strongly suggested to increase CLDN1 expression by inhibiting epidermal growth factor receptor signaling. By contrast, p-Pyr did not enhance CLDN1 expression, but it accelerated the translocation of CLDN1 to the cell surface. Finally, we confirmed that heparinoid and p-Pyr improved barrier function in NHEKs in a transepithelial electrical resistance assay. In conclusion, heparinoid and p-Pyr could potentially ameliorate skin conditions by improving TJ function.

Canonical and Non-Canonical Localization of Tight Junction Proteins during Early Murine Cranial Development.

This study investigates the intricate composition and spatial distribution of tight junction complex proteins during early mouse neurulation. The analyses focused on the cranial neural tube, which gives rise to all head structures. Neurulation brings about significant changes in the neuronal and non-neuronal ectoderm at a cellular and tissue level. During this process, precise coordination of both epithelial integrity and epithelial dynamics is essential for accurate tissue morphogenesis. Tight junctions are pivotal for epithelial integrity, yet their complex composition in this context remains poorly understood. Our examination of various tight junction proteins in the forebrain region of mouse embryos revealed distinct patterns in the neuronal and non-neuronal ectoderm, as well as mesoderm-derived mesenchymal cells. While claudin-4 exhibited exclusive expression in the non-neuronal ectoderm, we demonstrated a neuronal ectoderm specific localization for claudin-12 in the developing cranial neural tube. Claudin-5 was uniquely present in mesenchymal cells. Regarding the subcellular localization, canonical tight junction localization in the apical junctions was predominant for most tight junction complex proteins. ZO-1 (zona occludens protein-1), claudin-1, claudin-4, claudin-12, and occludin were detected at the apical junction. However, claudin-1 and occludin also appeared in basolateral domains. Intriguingly, claudin-3 displayed a non-canonical localization, overlapping with a nuclear lamina marker. These findings highlight the diverse tissue and subcellular distribution of tight junction proteins and emphasize the need for their precise regulation during the dynamic processes of forebrain development. The study can thereby contribute to a better understanding of the role of tight junction complex proteins in forebrain development.

Autoinducer-2 promotes the colonization of Lactobacillus rhamnosus GG to improve the intestinal barrier function in a neonatal mouse model of antibiotic-induced intestinal dysbiosis.

BACKGROUND: Human health is seriously threatened by antibiotic-induced intestinal disorders. Herein, we aimed to determine the effects of Autoinducer-2 (AI-2) combined with Lactobacillus rhamnosus GG (LGG) on the intestinal barrier function of antibiotic-induced intestinal dysbiosis neonatal mice. METHODS: An antibiotic-induced intestinal dysbiosis neonatal mouse model was created using antibiotic cocktails, and the model mice were randomized into the control, AI-2, LGG, and LGG + AI-2 groups. Intestinal short-chain fatty acids and AI-2 concentrations were detected by mass spectrometry and chemiluminescence, respectively. The community composition of the gut microbiota was analyzed using 16S rDNA sequencing, and biofilm thickness and bacterial adhesion in the colon were assessed using scanning electron microscopy. Transcriptome RNA sequencing of intestinal tissues was performed, and the mRNA and protein levels of HCAR2 (hydroxycarboxylic acid receptor 2), claudin3, and claudin4 in intestinal tissues were determined using quantitative real-time reverse transcription PCR and western blotting. The levels of inflammatory factors in intestinal tissues were evaluated using enzyme-linked immunosorbent assays (ELISAs). D-ribose, an inhibitor of AI-2, was used to treat Caco-2 cells in vitro. RESULTS: Compared with the control, AI-2, and LGG groups, the LGG + AI-2 group showed increased levels of intestinal AI-2 and proportions of Firmicutes and Lacticaseibacillus, but a reduced fraction of Proteobacteria. Specifically, the LGG + AI-2 group had considerably more biofilms and LGG on the colon surface than those of other three groups. Meanwhile, the combination of AI-2 and LGG markedly increased the concentration of butyric acid and promoted Hcar2, claudin3 and claudin4 expression levels compared with supplementation with LGG or AI-2 alone. The ELISAs revealed a significantly higher tumor necrosis factor alpha (TNF-α) level in the control group than in the LGG and LGG + AI-2 groups, whereas the interleukin 10 (IL-10) level was significantly higher in the LGG + AI-2 group than in the other three groups. In vitro, D-ribose treatment dramatically suppressed the increased levels of Hcar2, claudin3, and claudin4 in Caco-2 cells induced by AI-2 + LGG. CONCLUSIONS: AI-2 promotes the colonization of LGG and biofilm formation to improve intestinal barrier function in an antibiotic-induced intestinal dysbiosis neonatal mouse model.

Immunohistochemical Expression of Claudin-1 and Claudin-4 in Urothelial Carcinoma of the Urinary Bladder.

BACKGROUND: In Egypt, bladder cancer occupies the second rankamong reported cancers in men. Claudins are tight junctions that have a critical role in tumor pathogenesis, invasion, progression, and metastasis and currentlyare a focus of interest for targeting therapies. OBJECTIVES: We aimed to evaluatethe immunohistochemical expression of Claudin-1 and Claudin-4 in urinary bladder urothelial carcinoma and investigate the relationshipbetweenthe expressed Claudins with differentclinicopathological parameters. METHODS: Claudin-1 and Claudin-4 immunohistochemical expression was studied in 62 cases of urinary bladder urothelial carcinomas. The cases were classified into two categories; low and high Claudin-1 and Claudin-4 expression. RESULTS: High Claudin-1 expression was detected in67.7% of the studied urothelial carcinomas while 32.3% showed low expression. Claudin-1 expression was reduced significantly with high tumor grade, non-papillary tumors, muscle invasion, schistosomal infestation, and perineural invasion (p-value < 0.05). Claudin-4 high expression was detected in 82.3% of our cases while low expression was detected in 17.7%. Claudin-4 reduced expression was significantly associated with non-papillary tumors, muscle invasion, advanced T stages, and associated lympho-vascular emboli (P-value < 0.05). CONCLUSION: According to the results ofthe present study, the reduced expressions of Claudin-1 and Claudin-4 provide clues concerning the progression of urothelial carcinoma. Consequently, it is thought that Claudin-1 and Claudin-4 could help to differentiatelow-grade from high-grade and muscle-invasive from non-muscle-invasive urothelial carcinomas. In addition, it can be introduced as a possible therapeutic target.

Quercetin Improves Barrier Properties in Porcine Small Intestine but Not in Peyer's Patches.

Peyer's patches (PPs) are part of the gut-associated lymphatic tissue (GALT) and represent the first line of the intestinal immunological defense. They consist of follicles with lymphocytes and an overlying subepithelial dome with dendritic cells and macrophages, and they are covered by the follicle-associated epithelium (FAE). A sealed paracellular pathway in the FAE is crucial for the controlled uptake of luminal antigens. Quercetin is the most abundant plant flavonoid and has a barrier-strengthening effect on tight junctions (TJs), a protein complex that regulates the paracellular pathway. In this study, we aimed to analyze the effect of quercetin on porcine PPs and the surrounding villus epithelium (VE). We incubated both tissue types for 4 h in Ussing chambers, recorded the transepithelial electrical resistance (TEER), and measured the unidirectional tracer flux of [3H]-mannitol. Subsequently, we analyzed the expression, protein amount, and localization of three TJ proteins, claudin 1, claudin 2, and claudin 4. In the PPs, we could not detect an effect of quercetin after 4 h, neither on TEER nor on the [3H]-mannitol flux. In the VE, quercetin led to a higher TEER value, while the [3H]-mannitol flux was unchanged. The pore-forming claudin 2 was decreased while the barrier-forming claudin 4 was increased and the expression was upregulated. Claudin 1 was unchanged and all claudins could be located in the paracellular membrane by immunofluorescence microscopy. Our study shows the barrier-strengthening effect of quercetin in porcine VE by claudin 4 upregulation and a claudin 2 decrease. Moreover, it underlines the different barrier properties of PPs compared to the VE.

Xuanfu Daizhe Tang alleviates reflux esophagitis in rats by inhibiting the STAT1/TREM-1 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Reflux esophagitis (RE) is a common chronic inflammatory disease of the esophageal mucosa with a high prevalence and recurrence rate, for which a satisfactory therapeutic strategy is still lacking. Chinese medicine has its characteristics and advantages in treating RE, and the clinical application of Xuanfu Daizhe Tang (XDT) in treating RE has achieved sound therapeutic effects. However, there needs to be more research on its mechanism of action. AIM OF THE STUDY: The present work aimed to investigate the mechanism of XDT action in RE through the Signal Transducer and Activator of Transcription 1 (STAT1)/Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) pathway. MATERIALS AND METHODS: The main active components of XDT were analyzed by ultra-performance liquid chromatography-mass spectrometer (UPLC-MS). The effect of XDT on RE was evaluated in a rat model of RE induced by "Cardioplasty + pyloric ligation + Roux-en-Y esophagojejunostomy". Each administration group was treated by gavage. The degree of damage to the esophageal mucosa was evaluated by visual observation, and the Potential of Hydrogen (PH) method and Hematoxylin-eosin staining (HE) staining were performed. Serum levels of Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6), Tumor Necrosis Factor alpha (TNF-α), and Inducible Nitric Oxide Synthase (iNOS) were measured by ELISA. Quantitative Real-time PCR (qPCR), Western Blot (WB), and Immunofluorescence (IF) methods were used to detect Claudin-4, Claudin-5, TREM-1, and p-STAT1 in esophageal tissues for studying the mechanism of action and signaling pathway of XDT. Immunohistochemistry (IHC) analysis was used to detect the expression of TREM-1 and CD68 in esophageal tissues. Flow Cytometry (FC) was used to detect the polarization of macrophages in the blood. After conducting preliminary experiments to verify our hypothesis, we performed molecular docking between the active component of XDT and STAT1 derived from rats and parallel experiments with STAT1 inhibitor. The selective increaser of STAT1 transcription (2-NP) group was used to validate the mechanism by which XDT acts. RESULTS: XDT alleviated esophageal injury and attenuated histopathological changes in RE rats. XDT also inhibited the inflammatory response and decreased serum IL-1ß, IL-6, TNF-α, and iNOS levels in RE rats. qPCR and WB results revealed that XDT inhibited the expression of Claudin-4, Claudin-5, TREM-1, and STAT1 in the esophageal mucosa of RE rats. IHC and FC results showed that XDT reduced TREM-1 levels in esophageal tissues and polarized macrophages toward M2. The molecular docking results showed that rat-derived STAT1 can strongly bind to Isochronogenic acid A in XDT. The parallel experimental results of STAT1 inhibitor showed that XDT has anti-inflammatory effects similar to STAT1 inhibitors. The 2-NP group confirmed that XDT exerts its therapeutic effect on reflux esophagitis through the STAT1/TREM-1 pathway, with STAT1 as the upstream protein. CONCLUSIONS: This study suggests that XDT may treat reflux esophagitis by modulating the STAT1/TREM-1 pathway.

Effect of sodium butyrate treatment at the basolateral membranes on the tight junction barrier function via a monocarboxylate transporter in goat mammary epithelial cells.

In lactating mammary glands, tight junctions (TJs) prevent blood from mixing with milk and maintain epithelial cell polarity, which is important for milk production. This study aimed to investigate the effect of sodium acetate and sodium butyrate (SB) stimulation direction on the TJ barrier function, which is measured with regard to transepithelial electrical resistance and fluorescein flux, in goat mammary epithelial cells. The expression and localization of the TJ proteins claudin-3 and claudin-4 were examined using Western blotting and immunofluorescence. SB treatment in the lower chamber of cell culture inserts adversely affected the TJ barrier function, whereas sodium acetate barely had any effect, regardless of stimulation direction. In addition, SB treatment in the lower chamber significantly upregulated claudin-3 and claudin-4, whereas TJ proteins showed intermittent localization. Moreover, SB induced endoplasmic reticulum (ER) stress. ARC155858, a monocarboxylate transporter-1 inhibitor, alleviated the adverse impact of SB on TJs and the associated ER stress. Interestingly, sodium ß-hydroxybutyrate, a butyrate metabolite, did not affect the TJ barrier function. Our findings indicate that sodium acetate and SB influence the TJ barrier function differently, and excessive cellular uptake of SB can disrupt TJs and induce ER stress.

Claudin-10 Decrease in the Submandibular Gland Contributes to Xerostomia.

Tight junction proteins play a crucial role in paracellular transport in salivary gland epithelia. It is clear that severe xerostomia in patients with HELIX syndrome is caused by mutations in the claudin-10 gene. However, little is known about the expression pattern and role of claudin-10 in saliva secretion in physical and disease conditions. In the present study, we found that only claudin-10b transcript was expressed in human and mouse submandibular gland (SMG) tissues, and claudin-10 protein was dominantly distributed at the apicolateral membranes of acini in human, rat, and mouse SMGs. Overexpression of claudin-10 significantly reduced transepithelial electrical resistance and increased paracellular transport of dextran and Na+ in SMG-C6 cells. In C57BL/6 mice, pilocarpine stimulation promoted secretion and cation concentration in saliva in a dose-dependent increase. Assembly of claudin-10 to the most apicolateral portions in acini of SMGs was observed in the lower pilocarpine (1 mg/kg)-treated group, and this phenomenon was much obvious in the higher pilocarpine (10 mg/kg)-treated group. Furthermore, 7-, 14-, and 21-wk-old nonobese diabetic (NOD) and BALB/c mice were used to mimic the progression of hyposalivation in Sjögren syndrome. Intensity of claudin-10 protein was obviously lower in SMGs of 14- and 21-wk-old NOD mice compared with that of age-matched BALB/c mice. In the cultured mouse SMG tissues, interferon-γ (IFN-γ) downregulated claudin-10 expression. In claudin-10-overexpressed SMG-C6 cells, paracellular permeability was decreased. Furthermore, IFN-γ stimulation increased p-STAT1 level, whereas pretreatment with JAK/STAT1 antagonist significantly alleviated the IFN-γ-induced claudin-10 downregulation. These results indicate that claudin-10 functions as a pore-forming component in acinar epithelia of SMGs, assembly of claudin-10 is required for saliva secretion, and downregulation of claudin-10 induces hyposecretion. These findings may provide new clues to novel therapeutic targets on hyposalivation.

Effect of Infliximab on Radiation-Induced Submandibular Gland Dysfunction in Rats.

Inflammatory response is one of the essential parts of various pathogenic mechanisms of radiation-induced salivary dysfunction. The effect of decreasing the levels of inflammatory cytokines on alleviating submandibular gland injuries after irradiation is unclear. This study aimed to explore the effect of the antibody against tumor necrosis factor-alpha, infliximab, on radiation-induced submandibular gland dysfunction in rats. Male Wistar rats received a single 20 Gy dose to the right submandibular gland region or sham irradiated. Meanwhile, the irradiated group was divided into infliximab treatment groups or untreated groups. Animals were euthanized at 1, 6, and 12 weeks postirradiation, and the irradiated submandibular gland was dissected for subsequent detection. Submandibular gland exposure caused obvious pathological changes. The increased levels of inflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1ß, and interleukin-6, represent an aggravated inflammatory response. The results of the western blot, reverse transcription-quantitative polymerase chain reaction, and immunofluorescence staining showed upregulated levels of claudin-1, claudin-3, and aquaporin 5 and downregulated levels of claudin-4. Moreover, nuclear factor kappa-B phosphorylation levels were also up-regulated. In subsequent experiments, we found that infliximab alleviated inflammatory response, up-regulated tumor necrosis factor-alpha, interleukin-1ß, and interleukin-6 levels, and improved claudin-1, claudin-3, claudin-4, and aquaporin 5 expression. Our results indicate that infliximab might improve the para-cellular pathway and trans-cellular pathway destruction by reducing the inflammatory.

ß-Sitosterol Enhances Lung Epithelial Cell Permeability by Suppressing the NF-κB Signaling Pathway.

BACKGROUND: The dysregulation between pro-inflammatory and anti-inflammatory responses during sepsis is a crucial factor in driving sepsis progression. Acute lung injury (ALI) resulting from excessive production and accumulation of inflammatory mediators in the lungs contributes to impaired lung barrier function. The activation of the NF-κB signaling pathway during inflammation leads to the transcriptional activation of multiple inflammatory genes. Given the plausible impact of NF-κB signaling suppression in mitigating lung injury, substantive evidence demonstrates beta-sitosterol (BS)'s proficient ability to block NF-κB activation. Therefore, the aim of the present investigation was to delve into the impacts of BS in the context of sepsis-induced acute lung injury, employing both a mouse model and a model involving lung epithelial cells. METHODS: Sepsis-induced lung injury was simulated in mice through cecum ligation and puncture (CLP). To emulate injury in murine lung epithelial (MLE-12) cells, an experiment involving lipopolysaccharide (LPS) was administered. Evaluation of alterations in lung tissue permeability encompassed techniques such as lung wet/dry (W/D) mass ratio, Evans blue staining, and quantification of total protein concentration in bronchoalveolar lavage fluid (BALF). Lung tissue histopathological shifts were ascertained via hematoxylin and eosin (HE) staining. Additionally, the concentrations of inflammatory cytokines IL-6 and TNF-α were quantified in every lung tissue and cell group by implementing enzyme-linked immunosorbent assay (ELISA). Protein quantification for signal biomarkers was carried out using Western blotting and immunofluorescence methodologies. In tandem, the assessment of MLE-12 cell permeability was conducted by evaluating fluorescein isothiocyanate (FITC)-dextran extravasation. RESULTS: BS mitigated lung tissue pathologies, reduced inflammatory factors, and lowered tissue and cell permeability. BS inhibited NF-κB signaling and increased claudin-4 and claudin-5 expression, enhancing septic lung epithelial cell permeability. CONCLUSIONS: Through suppressing the NF-κB signaling cascade, BS effectively curtails the levels of inflammatory mediators. Simultaneously, it orchestrates the modulation of claudin-4 and claudin-5 expression, culminating in the augmentation of lung epithelial cell barrier competence, thus improving sepsis-induced lung injury.

Hypoxic bone marrow mesenchymal stem cell-derived exosomal lncRNA XIST attenuates lipopolysaccharide-induced acute lung injury via the miR-455-3p/Claudin-4 axis.

Mesenchymal stem cell-derived exosomes and long non-coding RNAs (lncRNAs) have been identified to play a role in acute lung injury (ALI). In this study, we investigated whether exosomal lncRNAs could regulate ALI and the underlying mechanisms. Bone marrow mesenchymal stem cells (BM-MSCs) were pretreated with hypoxia or normoxia, and exosomes were subsequently extracted from normoxic BM-MSCs (Nor-exos) and hypoxic BM-MSCs (Hypo-exos). A rat model of ALI was established via an airway perfusion of lipopolysaccharide (LPS). Exosomes were administered via the tail vein to evaluate the in vivo effect of exosomes in ALI. LPS-exposed RLE-6TN cells were incubated with exosomes to explore their in vitro effect in ALI. A luciferase reporter assay was used to evaluate the interaction between lncRNA XIST and miR-455-3p, as well as miR-455-3p and Claudin-4. We found that the exosomes attenuated LPS-induced ALI and Hypo-Exos exerted a greater therapeutic effect compared with Nor-exos both in vitro and in vivo. Moreover, an abundance of lncRNA XIST was observed in Hypo-exos compared with Nor-exos. Mechanistically, LncRNA XIST functioned as a miR-455-3p sponge and targeted Claudin-4 in ALI. Our results provide novel insight into the role of exosomal lncRNA XIST for the treatment of ALI. Thus, hypoxic pretreatment may represent an effective method for improving the therapeutic effects of exosomes.

Increase in Paracellular Leakage of Amino Acids Mediated by Aging-Induced Reduction of Claudin-4 Expression.

BACKGROUND: Claudins (CLDNs), major components of tight junctions, control paracellular permeabilities of mineral ions and wastes. The absorption of nutrients including glucose and amino acids (AAs) is regulated by intestinal epithelial cells. However, the role of CLDNs is not fully understood. OBJECTIVES: The purpose of this study was to clarify the effect of AA deprivation on the expression of AA transporters and CLDNs, as well as the role of CLDNs in the regulation of paracellular AA fluxes. METHODS: The messenger RNA and protein expression of various CLDNs were examined by real-time quantitative polymerase chain reaction and Western blot analyses, respectively. The AA selectivity of CLDNs was estimated using liquid chromatography-tandem mass spectrometry (LC-MS) analysis. RESULTS: The expression levels of some AA transporters, CLDN4, and CLDN15 were increased by AA deprivation in normal mouse colon-derived MCE301 cells. The expression of AA transporters and CLDN15 in the mouse colon was positively correlated with aging but the expression of CLDN4 was not. The AA deprivation-induced elevation of CLDN4 expression was inhibited by MHY1485, a mammalian target of rapamycin (mTOR) activator. Furthermore, CLDN4 expression was increased by rapamycin, an mTOR inhibitor. mTOR may be involved in the transcriptional activation of CLDN4. The fluxes of AAs from the basal to apical compartments were decreased and increased by CLDN4 overexpression and silencing, respectively. LC-MS analysis showed that the fluxes of all AAs, especially Lys, His, and Arg, were enhanced by CLDN4 silencing. CONCLUSIONS: CLDN4 is suggested to form a paracellular barrier to AAs, especially alkaline AAs, which is attenuated with aging.

Long non-coding RNA LOC103222771 promotes infection of porcine reproductive and respiratory syndrome virus in Marc-145 cells by downregulating Claudin-4.

Porcine reproductive and respiratory syndrome (PRRS) is an important swine disease caused by infection of porcine reproductive and respiratory syndrome virus (PRRSV), which leads to huge loss in swine industry. How to effectively control PRRS is challenging. Long non-coding RNA (lncRNA) are key regulator of viral infections and anti-virus immunological responses, therefore, further understanding of lncRNAs will aid to identification of novel regulators of viral infections and better design of prevention and control strategies to viral infection related diseases and immune disorders. We demonstrated that PRRSV infection upregulated the expression of lncRNA LOC103222771 in Marc-145 cells and porcine alveolar macrophage cells (PAMs) and that LOC103222771 is mainly located in cytoplasm. Knockdown of LOC103222771 could inhibit the PRRSV infection in Marc-145 cells. RNA-seq analysis and subsequent validation revealed increased expression of Claudin-4 (CLDN4) in Marc-145 when LOC103222771 was specifically downregulated，suggesting that LOC103222771 might be an upstream regulator of CLDN4, an important component of tight junctions for establishment of the paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. We and others showed that Downregulation of CLDN4 could boost the infection of PRRSV. Collectively, LOC103222771/CLDN4 signal axis might be a novel mechanism of PRRSV pathogenesis, implying a potential therapeutic target against PRRSV infection.

Treatment with Ligilactobacillus murinus lowers blood pressure and intestinal permeability in spontaneously hypertensive rats.

One feature of hypertension is a microbial imbalance with increased intestinal permeability. In this study, we examined whether an alteration in the microbiota affects blood pressure and intestinal permeability in spontaneously hypertensive rats (SHRs). We performed a 16S metagenome analysis of feces from 10- to 15-week-old SHRs using a synthetic long-read sequencing approach, and found a candidate for the microbiome treatment, Ligilactobacillus murinus (L. murinus), that was robustly decreased. Oral administration of L. murinus to SHRs for 2 weeks significantly inhibited blood pressure elevation and improved endothelium-dependent vasodilation but did not attenuate enhanced vascular contraction in SHR mesenteric arteries. The proximal colon of SHRs exhibited increased intestinal permeability with decreased levels of the tight junction protein claudin 4, morphological changes such as decreased intestinal crypts and elevated TNF-α levels, which was reversed by treatment with L. murinus. Consistent with these intestinal phenotypes, plasma lipopolysaccharides levels were elevated in SHR but decreased following L. murinus administration. We concluded that oral administration of L. murinus to SHRs exerts protective effects on intestinal permeability via restoration of claudin 4 expression and reversal of morphologic disorder, which may improve low-grade endotoxemia and thus reduce development of hypertension via recovery of endothelial vasodilating functions.