Octreotide attenuates intestinal barrier damage by maintaining basal autophagy in Caco2 cells.

The intestinal mucosal barrier is of great importance for maintaining the stability of the internal environment, which is closely related to the occurrence and development of intestinal inflammation. Octreotide (OCT) has potential applicable clinical value for treating intestinal injury according to previous studies, but the underlying molecular mechanisms have remained elusive. This article is based on a cell model of inflammation induced by lipopolysaccharide (LPS), aiming to explore the effects of OCT in protecting intestinal mucosal barrier function. A Cell Counting Kit­8 assay was used to determine cell viability and evaluate the effectiveness of OCT. Gene silencing technology was used to reveal the mediated effect of somatostatin receptor 2 (SSTR2). The changes in intestinal permeability were detected through trans­epithelial electrical resistance and fluorescein isothiocyanate­dextran 4 experiments, and the alterations in tight junction proteins were detected using immunoblotting and reverse transcription fluorescence­quantitative PCR technology. Autophagosomes were observed by electron microscopy and the dynamic changes of the autophagy process were characterized by light chain (LC)3­II/LC3­I conversion and autophagic flow. The results indicated that SSTR2­dependent OCT can prevent the decrease in cell activity. After LPS treatment, the permeability of monolayer cells decreased and intercellular tight junctions were disrupted, resulting in a decrease in tight junction protein zona occludens 1 in cells. The level of autophagy­related protein LC3 was altered to varying degrees at different times. These abnormal changes gradually returned to normal levels after the combined application of LPS and SSTR2­dependent OCT, confirming the role of OCT in protecting intestinal barrier function. These experimental results suggest that OCT maintains basal autophagy and cell activity mediated by SSTR2 in intestinal epithelial cells, thereby preventing the intestinal barrier dysfunction in inflammation injury.

Induced acute hyperglycemia modifies the barrier function of the intestinal epithelium by tissue inflammation and tight junction disruption resulting in hydroelectrolytic secretion in an animal model.

Diabetic-metabolic syndrome (MetS-D) has a high prevalence worldwide, in which an association with the rupture of the intestinal epithelium barrier function (IEBF) has been pointed out, but the functional and morphological properties are still not well understood. This study aimed to evaluate the impact of acute hyperglycemia diabetes on intestinal tight junction proteins, metabolic failure, intestinal ion and water transports, and IEBF parameters. Diabetes was induced in male Rattus norvegicus (200-310 g) with 0.5 mL of streptozotocin (70 mg/kg). Glycemic and clinical parameters were evaluated every 7 days, and intestinal parameters were evaluated on the 14th day. The MetS-D animals showed a clinical pattern of hyperglycemia, with increases in the area of villi and crypts, lactulose:mannitol ratio, myeloperoxidase (MPO) activity, and intestinal tissue concentrations of malondialdehyde (MDA), but showed a reduction in reduced glutathione (GSH) when these parameters were compared to the control. The MetS-D group had increased secretion of Na+, K+, Cl-, and water compared to the control group in ileal tissue. Furthermore, we observed a reduction in mRNA transcript of claudin-2, claudin-15, and NHE3 and increases of SGLT-1 and ZO-1 in the MetS-D group. These results showed that MetS-D triggered intestinal tissue inflammation, oxidative stress, complex alterations in gene regulatory protein transcriptions of intestinal transporters and tight junctions, damaging the IEBF and causing hydroelectrolyte secretion.

Aged polystyrene microplastics exacerbate alopecia associated with tight junction injuries and apoptosis via oxidative stress pathway in skin.

Microplastics (MPs) are pervasive pollutants in the natural environment and contribute to increased levels of illness in both animals and humans. However, thespecific impacts of MPs on skin damage and alopeciaare not yet well understood. In this study, we have examined the effects of two types of polystyrene MPs (pristine and aged) on skin and hair follicle damage in mice. UV irradiation changed the chemical and physical properties of the aged MPs, including functional groups, surface roughness, and contact angles. In both in vivo and in vitro experiments, skin and cell injuries related to oxidative stress, apoptosis, tight junctions (TJs), alopecia, mitochondrial dysfunction, and other damages were observed. Mechanistically, MPs and aged MPs can induce TJs damage via the oxidative stress pathway and inhibition of antioxidant-related proteins, and this can lead to alopecia. The regulation of cell apoptosis was also observed, and this is involved in the ROS-mediated mitochondrial signaling pathway. Importantly, aged MPs showed exacerbated toxicity, which may be due to their elevated surface irregularities and altered chemical compositions. Collectively, this study suggests a potential therapeutic approach for alopecia and hair follicle damage caused by MPs pollution.

The Role of ZO-2 in Modulating JAM-A and γ-Actin Junctional Recruitment, Apical Membrane and Tight Junction Tension, and Cell Response to Substrate Stiffness and Topography.

This work analyzes the role of the tight junction (TJ) protein ZO-2 on mechanosensation. We found that the lack of ZO-2 reduced apical membrane rigidity measured with atomic force microscopy, inhibited the association of γ-actin and JAM-A to the cell border, and instead facilitated p114RhoGEF and afadin accumulation at the junction, leading to an enhanced mechanical tension at the TJ measured by FRET, with a ZO-1 tension probe, and increased tricellular TJ tension. Simultaneously, adherens junction tension measured with an E-cadherin probe was unaltered. The stability of JAM-A and ZO-2 binding was assessed by a collaborative in silico study. The absence of ZO-2 also impacted the cell response to the substrate, as monolayers plated in 20 kPa hydrogels developed holes not seen in parental cultures and displayed a retarded elongation and formation of cell aggregates. The absence of ZO-2 was sufficient to induce YAP and Snail nuclear accumulation in cells cultured over glass, but when ZO-2 KD cells were plated in nanostructured ridge arrays, they displayed an increased abundance of nuclear Snail and conspicuous internalization of claudin-4. These results indicate that the absence of ZO-2 also impairs the response of cells to substrate stiffness and exacerbates transformation triggered by substrate topography.

Micronutrients at Supplemental Levels, Tight Junctions and Epithelial Barrier Function: A Narrative Review.

Disease modifiers, whether from cancer, sepsis, systemic inflammation, or microbial pathogens, all appear to induce epithelial barrier leak, with induced changes of the Tight Junctional (TJ) complex being pivotal to the process. This leak-and the ensuant breakdown of compartmentation-plays a central role in disease morbidity on many levels. Accumulation of lung water in the luminal compartment of airways was a major driver of morbidity and mortality in COVID-19 and is an excellent example of the phenomenon. Increasing awareness of the ability of micronutrients to improve basal barrier function and reduce barrier compromise in pathophysiology may prove to be a low-cost, safe, and easily administered prophylactic and/or therapeutic option amenable to large populations. The growing appreciation of the clinical utility of supplemental doses of Vitamin D in COVID-19 is but one example. This narrative review is intended to propose a general theory on how and why micronutrients-at levels above normal dietary intake-successfully remodel TJs and improve barrier function. It discusses the key difference between dietary/Recommended Daily Allowance (RDA) levels of micronutrients versus supplemental levels, and why the latter are needed in disease situations. It advances a hypothesis for why signal transduction regulation of barrier function may require these higher supplemental doses to achieve the TJ remodeling and other barrier element changes that are clinically beneficial.

Ginseng Oligopeptides Improve the Intestinal Physiology and Promote the Antioxidant Capacity of the Gut-on-a-Chip Model.

During ageing, the permeability of the intestinal barrier increases, the integrity of the intestinal barrier decreases, and the physiology of intestinal cells changes. Furthermore, intestinal inflammation and excessive oxidative stress are both likely to cause systemic diseases. Ginseng oligopeptides have a positive significant effect in terms of improving human health and delaying ageing, but their role in the ageing of the intestine has not been studied much. In our experiment, we constructed a gut-on-a-chip model and induced senescence of the chip with H2O2 so as to explore the effects of ginseng oligopeptides on the senescent intestine. The experimental results showed that ginseng oligopeptides had no obvious effects on the integrity of the intestine, including the TEER value and the expression of tight junction proteins. However, ginseng oligopeptides might have other positive effects, such as inhibiting excessive cell proliferation, promoting mucin secretion, and increasing the antioxidant capacity of the intestine, to improve intestinal health.

ITF2357 regulates NF-κB signaling pathway to protect barrier integrity in retinal pigment epithelial cells.

The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 µM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1ß, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.

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.

Tight junction membrane proteins regulate the mechanical resistance of the apical junctional complex.

Epithelia must be able to resist mechanical force to preserve tissue integrity. While intercellular junctions are known to be important for the mechanical resistance of epithelia, the roles of tight junctions (TJs) remain to be established. We previously demonstrated that epithelial cells devoid of the TJ membrane proteins claudins and JAM-A completely lack TJs and exhibit focal breakages of their apical junctions. Here, we demonstrate that apical junctions fracture when claudin/JAM-A-deficient cells undergo spontaneous cell stretching. The junction fracture was accompanied by actin disorganization, and actin polymerization was required for apical junction integrity in the claudin/JAM-A-deficient cells. Further deletion of CAR resulted in the disruption of ZO-1 molecule ordering at cell junctions, accompanied by severe defects in apical junction integrity. These results demonstrate that TJ membrane proteins regulate the mechanical resistance of the apical junctional complex in epithelial cells.

Molecular Dynamics Simulations of Claudin-10a and -10b Ion Channels: With Similar Architecture, Different Pore Linings Determine the Opposite Charge Selectivity.

Claudin polymers constitute the tight junction (TJ) backbone that forms paracellular barriers, at least for bigger solutes. While some claudins also seal the barrier for small electrolytes, others form ion channels. For cation-selective claudin-15 and claudin-10b, structural models of channels embedded in homo-polymeric strands have been suggested. Here, we generated a model for the prototypic anion-selective claudin-10a channel. Based on previously established claudin-10b models, dodecamer homology models of claudin-10a embedded in two membranes were analyzed by molecular dynamics simulations. The results indicate that both claudin-10 isoforms share the same strand and channel architecture: Sidewise unsealed tetrameric pore scaffolds are interlocked with adjacent pores via the ß1ß2 loop of extracellular segment 1. This leads to TJ-like strands with claudin subunits arranged in four joined rows in two opposing membranes. Several but not all cis- and trans-interaction modes are indicated to be conserved among claudin-10a, -10b, and -15. However, pore-lining residues that differ between claudin-10a and -10b (i.e., R33/I35, A34/D36, K69/A71, N54/D56, H60/N62, R62/K64) result in opposite charge selectivity of channels. This was supported by electric field simulations for both claudins and is consistent with previous electrophysiological studies. In summary, for the first time, a structural and mechanistic model of complete and prototypic paracellular anion channels is provided. This improves understanding of epithelial paracellular transport.

Computational Models of Claudin Assembly in Tight Junctions and Strand Properties.

Claudins are one of the major components of tight junctions (TJs) that polymerize within the cell membrane and form interactions between cells. Some claudins seal the paracellular space, limiting paracellular flux, while others form selectively permeable ion channels that control the paracellular permeability of small ions. Claudin strands are known to be dynamic and reshape within TJs to accommodate large-scale movements and rearrangements of epithelial tissues. Here, we summarize the recent computational and modeling studies on claudin assembly into tetrameric ion channels and their polymerization into µm long strands within the membrane. Computational studies ranging from all-atom molecular dynamics, coarse-grained simulations, and hybrid-resolution simulations elucidate the molecular nature of claudin assembly and function and provide a framework that describes the lateral flexibility of claudin strands.

HDAC3 promotes Sertoli cell maturation and maintains the blood-testis barrier dynamics.

Germ cell development depends on the capacity of somatic Sertoli cells to undergo differentiation into a mature state and establish a germ cell-specific blood-testis barrier (BTB). The BTB structure confers an immunological barrier for meiotic and postmeiotic germ cells, and its dynamic permeability facilitates a transient movement of preleptotene spermatocytes through BTB to enter meiosis. However, the regulatory factors involved in Sertoli cell maturation and how BTB dynamics coordinate germ cell development remain unclear. Here, we found a histone deacetylase HDAC3 abundantly expresses in Sertoli cells and localizes in both cytoplasm and nucleus. Sertoli cell-specific Hdac3 knockout in mice causes infertility with compromised integrity of blood-testis barrier, leading to germ cells unable to traverse through BTB and an accumulation of preleptotene spermatocytes in juvenile testis. Mechanistically, nuclear HDAC3 regulates the expression program of Sertoli cell maturation genes, and cytoplasmic HDAC3 forms a complex with the gap junction protein Connexin 43 to modulate the BTB integrity and dynamics through regulating the distribution of tight junction proteins. Our findings identify HDAC3 as a critical regulator in promoting Sertoli cell maturation and maintaining the homeostasis of the blood-testis barrier.

Sodium butyrate ameliorated diabetic nephropathy-associated tubulointerstitial inflammation by modulating the tight junctions of renal tubular epithelial cells.

As one of the most significant pathological changes of diabetic nephropathy (DN), tubulointerstitial fibrosis (TIF) had a close relationship with tubulointerstitial inflammation (TI), and the occurrence of TI could have resulted from the disrupted tight junctions (TJs) of renal tubular epithelial cells (RTECs). Studies have demonstrated that sodium butyrate (NaB), a typical short chain fatty acid (SCFA), played an important regulatory role in intestinal TJs and inflammation. In this study, our in vivo and in vitro results showed that accompanied by TI, renal tubular TJs were gradually disrupted in the process of DN-related TIF. In HG and LPS co-cultured HK-2 cells and db/db mice, NaB treatment regained the TJs of RTECs via the sphingosine 1-phosphate receptor-1 (S1PR1)/AMPK signaling pathway, relieving inflammation. Small interfering RNA of S1PR1, S1PR1 antagonist W146 and agonist SEW2871, and AMPK agonist AICAR were all used to further confirm the essential role of the S1PR1/AMPK signaling pathway in NaB's TJ protection in RTECs in vitro. Finally, NaB administration not only improved the renal function and TIF, but also relieved the TI of db/db mice. These findings suggested that the use of NaB might be a potential adjuvant treatment strategy for DN-associated TIF, and this protective effect was linked to the TJ modulation of RTECs via the S1PR1/AMPK signaling pathway, leading to the improvement of TI.

Removal of Expression of Concern: Sodium butyrate ameliorated diabetic nephropathy-associated tubulointerstitial inflammation by modulating tight junction of renal tubular epithelial cells.

Removal of Expression of Concern for 'Sodium butyrate ameliorated diabetic nephropathy-associated tubulointerstitial inflammation by modulating tight junction of renal tubular epithelial cells' by Tingting Yang et al., Food Funct., 2022, Accepted Manuscript, https://doi.org/10.1039/D2FO00940D.

Claudin-2 upregulation enhances intestinal permeability, immune activation, dysbiosis, and mortality in sepsis.

Intestinal epithelial expression of the tight junction protein claudin-2, which forms paracellular cation and water channels, is precisely regulated during development and in disease. Here, we show that small intestinal epithelial claudin-2 expression is selectively upregulated in septic patients. Similar changes occurred in septic mice, where claudin-2 upregulation coincided with increased flux across the paracellular pore pathway. In order to define the significance of these changes, sepsis was induced in claudin-2 knockout (KO) and wild-type (WT) mice. Sepsis-induced increases in pore pathway permeability were prevented by claudin-2 KO. Moreover, claudin-2 deletion reduced interleukin-17 production and T cell activation and limited intestinal damage. These effects were associated with reduced numbers of neutrophils, macrophages, dendritic cells, and bacteria within the peritoneal fluid of septic claudin-2 KO mice. Most strikingly, claudin-2 deletion dramatically enhanced survival in sepsis. Finally, the microbial changes induced by sepsis were less pathogenic in claudin-2 KO mice as survival of healthy WT mice injected with cecal slurry collected from WT mice 24 h after sepsis was far worse than that of healthy WT mice injected with cecal slurry collected from claudin-2 KO mice 24 h after sepsis. Claudin-2 upregulation and increased pore pathway permeability are, therefore, key intermediates that contribute to development of dysbiosis, intestinal damage, inflammation, ineffective pathogen control, and increased mortality in sepsis. The striking impact of claudin-2 deletion on progression of the lethal cascade activated during sepsis suggests that claudin-2 may be an attractive therapeutic target in septic patients.

Cellular junction dynamics and Alzheimer's disease: a comprehensive review.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder characterized by progressive neuronal damage and cognitive decline. Recent studies have shed light on the involvement of not only the blood-brain barrier (BBB) dysfunction but also significant alterations in cellular junctions in AD pathogenesis. In this review article, we explore the role of the BBB and cellular junctions in AD pathology, with a specific focus on the hippocampus. The BBB acts as a crucial protective barrier between the bloodstream and the brain, maintaining brain homeostasis and regulating molecular transport. Preservation of BBB integrity relies on various junctions, including gap junctions formed by connexins, tight junctions composed of proteins such as claudins, occludin, and ZO-1, as well as adherence junctions involving molecules like vascular endothelial (VE) cadherin, Nectins, and Nectin-like molecules (Necls). Abnormalities in these junctions and junctional components contribute to impaired neuronal signaling and increased cerebrovascular permeability, which are closely associated with AD advancement. By elucidating the underlying molecular mechanisms governing BBB and cellular junction dysfunctions within the context of AD, this review offers valuable insights into the pathogenesis of AD and identifies potential therapeutic targets for intervention.

Modulation of hepatic cellular tight junctions via coculture with cholangiocytes enables non-destructive bile recovery.

Estimation of the biliary clearance of drugs and their metabolites in humans is crucial for characterizing hepatobiliary disposition and potential drug-drug interactions. Sandwich-cultured hepatocytes, while useful for in vitro bile analysis, require cell destruction for bile recovery, limiting long-term or repeated dose drug effect evaluations. To overcome this limitation, we investigated the feasibility of coculturing a human hepatic carcinoma cell line (HepG2-NIAS cells) and a human cholangiocarcinoma cell line (TFK-1 cells) using the collagen vitrigel membrane in a variety of coculture configurations. The coculture configuration with physiological bile flow increased the permeability of fluorescein-labeled bile acids (CLF) across the HepG2-NIAS cell layer by approximately 1.2-fold compared to the HepG2-NIAS monoculture. This enhancement was caused by paracellular leakage due to the loosened tight junctions of HepG2-NIAS, confirmed by the use of an inhibitor for bile acid transporters, the increase of permeability of dextran, and the decrease of the transepithelial electrical resistance (TEER) value. Based on the results of loosening hepatic tight junctions via coculture with TFK-1 in the CLF permeability assay, we next attempted to collect the CLF accumulated in the bile canaliculi of HepG2-NIAS. The recovery of the CLF accumulated in the bile canaliculi was increased 1.4 times without disrupting hepatic tight junctions by the coculture of HepG2-NIAS cells and TFK-1 cells compared to the monoculture of HepG2-NIAS cells. This non-destructive bile recovery has the potential as a tool for estimating the biliary metabolite and provides valuable insights to improve in vitro bile analysis.

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.

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.