Expression Of Zona Occludens-1 Research Articles

Prevotella melaninogenica disrupted oral epithelial barrier function via myosin light chain kinase.

Our previous studies have found that the composition ratio of Prevotella melaninogenica (Pm) on buccal mucosa surface of oral lichen planus (OLP) patients increased significantly compared with control. Furthermore, Pm could invade the epithelium of OLP patients. This study aimed to further explore the impact of Pm on oral keratinocytes. The Pm-human oral keratinocyte (HOK) co-culture model was established to detect monolayer permeability, zona occludens-1 (ZO-1) expression, and intracellular survival of Pm. We performed RNA-seq followed by identification of differentially expressed genes (DEGs) and Gene Ontology (GO) analysis, with a particular focus on myosin light chain kinase (MLCK). An MLCK inhibitor ML-7 was utilized in Pm-HOK co-culture model to assess its effects on monolayer permeability and ZO-1 expression. HOK monolayer permeability was increased, and ZO-1 expression was decreased after co-culture (p < 0.05). Pm could survive in HOK cells. RNA-seq revealed MLCK was an upregulated common DEG. The expression of MLCK in the Pm-HOK co-culture model was upregulated. Inhibition of MLCK rescued the increased epithelial permeability, and ZO-1 expression was upregulated (p < 0.05). MLCK may be involved in disrupting epithelial barrier function by Pm.

Optimal timing for drug delivery into the hippocampus by focused ultrasound: A comparison of hydrophilic and lipophilic compounds

AimsPrevious studies have reported that focused ultrasound (FUS) helps modulate the blood-brain barrier (BBB). These studies have generally used the paracellular pathway owing to tight junction proteins (TJPs) regulation. However, BBB transport pathways also include diffusion and transcytosis. Few studies have examined transcellular transport across endothelial cells. We supposed that increased BBB permeability caused by FUS may affect transcytosis. We investigated drug delivery through transcytosis and paracellular transport to the brain after BBB modulation using FUS. Main methodsFUS and microbubbles were applied to the hippocampus of rats, and were euthanized at 1, 4, 24, and 48 h after sonication. To investigate paracellular transport, we analyzed TJPs, including zona occludens-1 (ZO-1) and occludin. We also investigated caveola-mediated transcytosis by analyzing caveola formation and major facilitator superfamily domain-containing 2a (Mfsd2a) levels, which inhibit caveola vesicle formation. Key findingsOne hour after FUS, ZO-1 and occludin expression was the lowest and gradually increased over time, returning to baseline 24 h after FUS treatment. Compared with that of TJPs, caveola formation started to increase 1 h after FUS treatment and peaked at 4 h after FUS treatment before returning to baseline by 48 h after FUS treatment. Decreased Mfsd2a levels were observed at 1 h and 4 h after FUS treatment, indicating increased caveola formation. SignificanceFUS induces BBB permeability changes and regulates both paracellular transport and caveola-mediated transcytosis. However, a time difference was observed between these two mechanisms. Hence, when delivering drugs into the brain after FUS, the optimal drug administration timing should be determined by the mechanism by which each drug passes through the BBB.

Intestinal Epithelial Cell-specific Knockout of METTL3 Aggravates Intestinal Inflammation in CLP Mice by Weakening the Intestinal Barrier.

Many studies have demonstrated that the expression of methyltransferase- like 3 (METTL3) is altered in various inflammatory diseases. Its specific mechanistic role in the intestinal inflammatory response during sepsis remains limited and requires further investigation. Explore the potential mechanism of METTL3 in the intestinal inflammatory response during sepsis. Immunohistochemical analysis was utilized to detect the expression of METTL3 in the necrotic intestine of patients with intestinal necrosis and the small intestine of cecal ligation and puncture (CLP) mice. Mice were subjected to the CLP and Sham surgeries, intestine tissue was harvested and performed HE staining, and ELISA to examine intestinal inflammatory responses, while TUNEL staining was applied to detect intestinal cell apoptosis. Additionally, ELISA was used to detect diamine oxidase (DAO) and intestinal fatty acid binding protein (I-FABP) levels in intestinal tissue. Immunohistochemistry and RT-qPCR were also employed to examine the mRNA and protein expression levels of Zona Occludens 1 (ZO-1) and Claudin-1. Finally, transcriptomic sequencing was performed on the small intestine tissues of METTL3 Knock-out (KO) and Wild-type (WT) mice in response to sepsis. METTL3 exhibited lower expression level in the necrotic intestine of patients and the small intestine of CLP mice. Loss of METTL3 in CLP mice triggered significantly higher expression of TNF-α and IL-18, down-regulated expression of ZO-1 and claudin-1, and decreased expression of DAO and I-FABP in the intestinal tissue. KEGG enrichment analysis showed that the differential genes were significantly enriched in immune-related pathways. This study reveals a novel mechanism responsible for exacerbated intestinal inflammation orchestrated by METTL3. Particularly, METTL3 null mice displayed decreased ZO- 1 and Claudin-1 expression, which largely hampered intestinal epithelial barrier function, resulting in bacterial and toxin translocation and intestinal immune activation and inflammation against sepsis.

Inhibiting ferroptosis in brain microvascular endothelial cells: A potential strategy to mitigate polystyrene nanoplastics‒induced blood‒brain barrier dysfunction

Polystyrene nanoplastics (PS-NPs), a group of ubiquitous pollutants, may injure the central nervous system through the blood‒brain barrier (BBB). However, whether exposure to PS-NPs contributes to BBB disruption and the underlying mechanisms are still unclear. In vivo, we found that PS-NPs (25 mg/kg BW) could significantly increase BBB permeability in mice and downregulate the distribution of the tight junction-associated protein zona occludens 1 (ZO-1) in brain microvascular endothelial cells (BMECs). Using an in vitro BBB model, exposure to PS-NPs significantly reduced the transendothelial electrical resistance and altered ZO-1 expression and distribution in a dose-dependent manner. RNA-seq analysis and functional investigations were used to investigate the molecular pathways involved in the response to PS-NPs. The results revealed that the ferroptosis and glutathione metabolism signaling pathways were related to the disruption of the BBB model caused by the PS-NPs. PS-NPs treatment promoted ferroptosis in bEnd.3 cells by inducing disordered glutathione metabolism in addition to Fe2+ and lipid peroxide accumulation, while suppressing ferroptosis with ferrostatin-1 (Fer-1) suppressed ferroptosis-related changes in bEnd.3 cells subjected to PS-NPs. Importantly, Fer-1 alleviated the decrease in ZO-1 expression in bEnd.3 cells and the exacerbation of BBB damage induced by PS-NPs. Collectively, our findings suggest that inhibiting ferroptosis in BMECs may serve as a potential therapeutic target against BBB disruption induced by PS-NPs exposure.

Sodium butyrate alleviates heat stress-induced damage to morphology, anti-oxidant status, inflammatory response and barrier integrity in jejunum of broilers

To evaluate the protective effects of sodium butyrate (SB) on intestinal morphology, anti-oxidant status, inflammatory response and barrier integrity in male broilers under heat stress. A total of 180 21-d-old male Arbour Acres broilers with similar body weight were used for this 21-d experiment. Broilers were randomly allocated to three groups: CON group, basal diet and raised under 24 °C; HS group, basal diet and raised under heat stress condition (34 °C from 10:00 to 18:00 and 24 °C for the rest time); HS-SB group, basal diet with 1200 mg/kg SB and raised under heat stress condition. Compared to the HS group, SB supplementation increased average daily gain (ADG), villus height: crypt depth ratio (VH: CD), VH, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activity, and interleukin-10 (IL-10) level, decreased feed conversion ratio (FCR), CD, reactive oxygen species (ROS), IL-1β and tumour necrosis factor-alpha (TNF-α) level, up-regulated the mRNA expression of nuclear factor erythroid 2-related factor 2, haem oxygenase 1, GSH 1 (GPX1), SOD and CAT, down-regulated the mRNA expression of toll-like receptor 4 (TLR4), nuclear transcription factors, IL-1β, and TNF-α in jejunum of heat-stressed broilers. Furthermore, SB supplementation up-regulated the mRNA expression of occluding, claudin-1 and zona occludens-1 (ZO-1) in jejunum of heat-stressed broilers, accompanied with decreasing serum endotoxin and D-lactic acid concentration, and diamine oxidase (DAO) activity. There results suggested that SB supplementation could alleviate heat stress-induced jejunal tight junction structure dysfunction by up-regulated the expression of occluding, claudin-1 and ZO-1, presumably through alleviating oxidative stress and inflammatory response. HIGHLIGHTS Sodium butyrate alleviated heat stress-induced jejunal morphology damage. Sodium butyrate alleviated heat stress-induced jejunal oxidative damage. Sodium butyrate alleviated heat stress-induced jejunal inflammatory response. Sodium butyrate alleviated heat stress-induced tight junction structure dysfunction.

YES-associated protein-regulated Smad7 worsen epithelial barrier injury of chronic sinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) was potentially due to the epithelial barrier injury. YES-associated protein (YAP) is a multifunctional transcriptional factor and plays versatile roles in the regulation and maintenance of epithelial barrier in different organs and tissues. The purpose of this study is to elucidate possible effect and mechanism of YAP on the epithelial barrier of CRSwNP. Patients were divided into CRSwNP group (n = 12) and control group (n = 9). The location of YAP, PDZ-binding transcriptional co-activator (TAZ), and Smad7 were estimated by immunohistochemistry and immunofluorescence. Meanwhile, the expression of YAP, TAZ, Zona occludens-1 (ZO-1), E-cadherin, and transforming growth factor-beta1 (TGF-β1) were detected by Western blot. After primary human nasal epithelial cells were treated with YAP inhibitor, the expression level of YAP, TAZ, ZO-1, E-cadherin, TGF-β1, and Smad7 were measured by Western blot. Compared with the control group, the protein levels of YAP, TAZ, and Smad7 were significantly upregulated, while TGF-β1, ZO-1, and E-cadherin were downregulated in CRSwNP. YAP and Smad7 demonstrated lower levels, while the expression of ZO-1, E-cadherin, and TGF-β1 rose slightly after YAP inhibitor treatment in primary nasal epithelial cells. Higher level of YAP may lead to CRSwNP epithelial barrier injury via the TGF-β1 signaling pathway, and the inhibition of YAP can partially reverse epithelial barrier function.

Endogenous FGF21 attenuates blood-brain barrier disruption in penumbra after delayed recanalization in MCAO rats through FGFR1/PI3K/Akt pathway.

Restoration of blood circulation within "time window" is the principal treating goal for treating acute ischemic stroke. Previous studies revealed that delayed recanalization might cause serious ischemia/reperfusion injury. However, plenty of evidences showed delayed recanalization improved neurological outcomes in acute ischemic stroke. This study aims to explore the role of delayed recanalization on blood-brain barrier (BBB) in the penumbra (surrounding ischemic core) and neurological outcomes after middle cerebral artery occlusion (MCAO). Recanalization was performed on the 3rd day after MCAO. BBB disruption was tested by Western blotting, Evans blue dye, and immunofluorescence staining. Infarct volume and neurological outcomes were evaluated on the 7th day after MCAO. The expression of fibroblast growth factor 21 (FGF21), fibroblast growth factor receptor 1 (FGFR1), phosphatidylinositol-3-kinase (PI3K), and serine/threonine kinase (Akt) in the penumbra were observed by immunofluorescence staining and/or Western blotting. The extraversion of Evans blue, IgG, and albumin increased surrounding ischemic core after MCAO, but significantly decreased after recanalization. The expression of Claudin-5, Occludin, and zona occludens 1 (ZO-1) decreased surrounding ischemic core after MCAO, but significantly increased after recanalization. Infarct volume reduced and neurological outcomes improved following recanalization (on the 7th day after MCAO). The expressions of Claudin-5, Occludin, and ZO-1 decreased surrounding ischemic core following MCAO, which were up-regulated corresponding to the increases of FGF21, p-FGFR1, PI3K, and p-Akt after recanalization. Intra-cerebroventricular injection of FGFR1 inhibitor SU5402 down-regulated the expression of PI3K, p-Akt, Occludin, Claudin-5, and ZO-1 in the penumbra, which weakened the beneficial effects of recanalization on neurological outcomes after MCAO. Delayed recanalization on the 3rd day after MCAO increases endogenous FGF21 in the penumbra and activates FGFR1/PI3K/Akt pathway, which attenuates BBB disruption in the penumbra and improves neurobehavior in MCAO rats.

Cellular Uptake and Transport Mechanism of 6-Mercaptopurine Nanomedicines for Enhanced Oral Bioavailability.

Nanomedicines have significant advantages in enhancing the oral bioavailability of drugs, but a deeper understanding of the underlying mechanisms remains to be interpreted. Hence, the present study aims to explain the uptake and trafficking mechanism for 6-MP nanomedicines we previously constructed. 6-MP loaded poly(lactide-co-glycolide) (PLGA) nanomedicines (6-MPNs) were prepared by the multiple emulsion method. The transcytosis mechanism of 6-MPNs was investigated in Caco-2 cells, Caco-2 monolayers, follicle associated epithelium (FAE) monolayers and rats, including transmembrane pathway, intracellular trafficking, paracellular transport and the involvement of transporter. Pharmacokinetics in rats showed that the area under the curve (AUC) of 6-MP in the 6-MPNs group (147.3 ± 42.89 μg/L·h) was significantly higher than that in the 6-MP suspensions (6-MPCs) group (70.31 ± 18.24 μg/L·h). The uptake of 6-MPNs in Caco-2 cells was time-, concentration- and energy-dependent. The endocytosis of intact 6-MPNs was mediated mainly through caveolae/lipid raft, caveolin and micropinocytosis. The intracellular trafficking of 6-MPNs was affected by endoplasmic reticulum (ER)-Golgi complexes, late endosome-lysosome and microtubules. The multidrug resistance associated protein 4 (MRP4) transporter-mediated transport of free 6-MP played a vital role on the transmembrane of 6-MPNs. The trafficking of 6-MPNs from the apical (AP) side to the basolateral (BL) side in Caco-2 monolayers was obviously improved. Besides, 6-MPNs affected the distribution and expression of zona occludens-1 (ZO-1). The transport of 6-MPNs in FAE monolayers was concentration- and energy-dependent, while reaching saturation over time. 6-MPNs improved the absorption of the intestinal Peyer's patches (PPs) in rats. 6-MPNs improve the oral bioavailability through multiple pathways, including active transport, paracellular transport, lymphatic delivery and MRP4 transporter. The findings of current study may shed light on the cellular uptake and transcellular trafficking mechanism of oral nanomedicines.

Protective Effect of Purinergic P2X7 Receptor Inhibition on Acrolein-Induced Urothelial Cell Damage.

Patients undergoing chemotherapy with cyclophosphamide experience cystitis due to excretion of a toxic metabolite, acrolein. Cystitis, an inflammation of the bladder, is associated with damage to the integrity of the urothelial barrier. The purinergic P2X7 receptor (P2X7R) is increasingly recognized for its role in inflammation and cell death. P2X7R is expressed abundantly on the bladder urothelium. The aim of this study was to investigate the role of P2X7R in acrolein-induced inflammatory damage in primary cultured porcine bladder urothelial cells. Confluent urothelial cells in culture were treated with acrolein to induce damage; also, with the P2X7R selective antagonist, A804598. Cell viability assay, immunocytochemistry, and trans-epithelial electrical resistance (TEER) studies were carried out to investigate the effect of treatments on urothelial cell function. Acrolein induced a significant reduction in urothelial cell viability, which was protected by the presence of A804598 (10 µM). The urothelial barrier function, indicated by TEER values, was also significantly reduced by acrolein, whereas pre-incubation with P2X7R antagonist significantly protected the urothelial cell barrier from acrolein-induced TEER reduction. The structure of urothelial cell tight junctions was similarly impacted by acrolein treatment, showing the fragmentation of zona occludens-1 (ZO-1) immunoreactivity. Pre-treatment of cells with A804598 countered against the actions of acrolein and maintained ZO-1 expression level and cell structure. The damaging effect of acrolein on urothelial cells integrity could be impaired by inhibition of P2X7R, therefore P2X7R blockade may be a possible therapy in patients with bladder cystitis caused by cyclophosphamide treatment.

Enterococcus faecium HDRsEf1 inhibits Lipopolysaccharide-induced downregulation of zona occludens -1 expression via toll-like receptor 2/4-mediated c-Jun N-terminal kinase/activator protein-1 signalling pathways.

Zona occludens-1 (ZO-1) is a key regulatory tight junction protein that plays an important role in maintaining gastrointestinal health. In this study, we investigated the protective effect and regulation mechanism of the probiotic Enterococcus faecium HDRsEf1 on tight junction protein ZO-1 at the cellular and molecular levels. We established lipopolysaccharide (LPS)-induced intestinal epithelial cell injury model and detected the protective effect of HDRsEf1 on ZO-1 in IPEC-J2 cells by real-time polymerase chain reaction and Western blot. The results showed that HDRsEf1 inhibited the downregulation of ZO-1 expression induced by LPS. HDRsEf1 stabilized the destruction of the ZO-1 structure caused by LPS in an immunofluorescence assay. Through gene overexpression and siRNA interference tests, we found that transcription factor activator protein 1 (AP-1) inhibited the level of ZO-1 expression. Silencing experiment further supported that the protective effect of HDRSEF1 might be mediated by suppression of LPS-provoked activation of apoptosis signal-regulating kinase 1 (ASK1)/mitogen-activated protein kinase kinase 7 (MKK7)/c-Jun N-terminal kinase (JNK) signalling pathways. In addition, HDRsEf1 could stabilize ZO-1 expression by increasing toll-like receptor 2 (TLR2) expression and competing with LPS for the TLR4 binding site. More interestingly, we also found that HDRsEf1 could stabilize ZO-1 expression through inhibiting the production of tumour necrosis factor-α (TNF-α) induced by LPS. HDRsEf1 could protect the IPEC-J2 cell against LPS induced downregulation of ZO-1 expression by inhibiting the activation of TLR2/4-mediated JNK-AP-1 and signalling cascade and the production of TNF-α. This study can provide a theoretical basis for probiotics to regulate the expression of intestinal tight junction proteins, and supply technical support for probiotics to prevent and treat animal intestinal infectious diseases.

Haloperidol Attenuates Lung Endothelial Cell Permeability In Vitro and In Vivo.

We previously reported that claudin-5, a tight junctional protein, mediates lung vascular permeability in a murine model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Recently, it has been reported that haloperidol, an antipsychotic medication, dose-dependently increases expression of claudin-5 in vitro and in vivo, in brain endothelium. Notably, claudin-5 is highly expressed in both brain and lung tissues. However, the effects of haloperidol on EC barrier function are unknown. We hypothesized that haloperidol increases lung EC claudin-5 expression and attenuates agonist-induced lung EC barrier disruption. Human pulmonary artery ECs were pretreated with haloperidol at variable concentrations (0.1–10 μM) for 24 h. Cell lysates were subjected to Western blotting for claudin-5, in addition to occludin and zona occludens-1 (ZO-1), two other tight junctional proteins. To assess effects on barrier function, EC monolayers were pretreated for 24 h with haloperidol (10 µM) or vehicle prior to treatment with thrombin (1 U/mL), with measurements of transendothelial electrical resistance (TER) recorded as a real-time assessment of barrier integrity. In separate experiments, EC monolayers grown in Transwell inserts were pretreated with haloperidol (10 µM) prior to stimulation with thrombin (1 U/mL, 1 h) and measurement of FITC-dextran flux. Haloperidol significantly increased claudin-5, occludin, and ZO-1 expression levels. Measurements of TER and FITC-dextran Transwell flux confirmed a significant attenuation of thrombin-induced barrier disruption associated with haloperidol treatment. Finally, mice pretreated with haloperidol (4 mg/kg, IP) prior to the intratracheal administration of LPS (1.25 mg/kg, 16 h) had increased lung claudin-5 expression with decreased lung injury as assessed by bronchoalveolar lavage (BAL) fluid protein content, total cell counts, and inflammatory cytokines, in addition to lung histology. Our data confirm that haloperidol results in increased claudin-5 expression levels and demonstrates lung vascular-protective effects both in vitro and in vivo in a murine ALI model. These findings suggest that haloperidol may represent a novel therapy for the prevention or treatment of ALI and warrants further investigation in this context.

Endothelial barrier dysfunction induced by anthracene and its nitrated or oxygenated derivatives at environmentally relevant levels

Polycyclic aromatic hydrocarbons (PAHs) are epidemiologically associated with cardiovascular diseases characterized by early key events involving in the disruption of endothelial barrier function. Whether PAHs can induce adverse cardiovascular outcome by directly destabilizing endothelial barrier function remains elusive. Herein, we investigated the effect of anthracene (ANT), 9-nitroanthracene (9-NANT), and 9,10-anthraquinone (9,10-AQ) on vascular endothelial barrier functions in human umbilical vein endothelial cells (HUVECs). The integrity of endothelial barrier in HUVECs was disturbed with a 1.15–1.42 fold increase in fluorescein leakage, and 21.8%–58.3% downregulated transendothelial electrical resistance. ANT, 9-NANT and 9,10-AQ promoted paracellular gap formation as revealed by transmission electron microscope. The disrupted cell junctions after 24 h exposure to ANT, 9-NANT and 9,10-AQ at 0.01 μM were indicated by the downregulated mRNA expression of vascular endothelial cadherin (VE-cadherin), zona occludens-1 (ZO-1) and occludin by 33.2%–71.4%, 19.1%–21.0%, and 31.9% respectively, and the downregulated protein expression of ZO-1 and occludin, and by the internalization of VE-cadherin. We demonstrated that ANT and its derivatives at environmentally relevant concentrations induced endothelial barrier dysfunction via the disruption of cell junctions, providing essential in vitro evidence on the association with their adverse cardiovascular outcomes.

MiRNA-155-5p Reduces Corneal Epithelial Permeability by Remodeling Epithelial Tight Junctions during Corneal Wound Healing

ABSTRACT Purpose Corneal epithelial cells play a vital role in the function of the cornea by forming a physical barrier to protect the eye from invasion by external pathogenic agents. A recent study showed that miR-155 promotes cutaneous wound healing. However, its function in corneal epithelial wound healing is unknown. The present study examined whether miR-155-5p reduces corneal epithelial permeability by remodeling epithelial tight junctions during corneal wound healing. Materials and Methods Rat corneal wounds were produced by removing the central corneal epithelium with a blunt scalpel blade under a dissecting microscope. One eye of each rat was treated with topical miR-155-5p, and the other eye was treated with topical agomir negative control for 3 days before and after corneal epithelial wounding. Corneal epithelial permeability was assessed by the macromolecular osmosis method. Expression of zona occludens 1 (ZO-1), occludin, and myosin light chain kinase (MLCK) and phosphorylation of myosin light chain (MLC) were detected by Western blot. Human corneal epithelial (HCE) cells were cultured in the upper chamber of Transwell filters, and transepithelial electrical resistance (TER) was measured using a voltohmmeter. The distribution of ZO-1 and occludin in HCE cells treated with miR-155-5p was determined by immunofluorescence. Results miR-155-5p significantly promoted the repair of corneal epithelial injury and reduced the permeability of the corneal epithelium. It significantly decreased expression of MLCK and phosphorylation of MLC and increased expression of the tight junction proteins ZO-1 and occludin in corneal epithelial cells during corneal wound healing. miR-155-5p significantly increased TER, decreased MLCK expression and MLC phosphorylation, increased ZO-1 and occludin expression, and promoted anchoring of tight junction proteins in the cell membrane and remodeling in HEC cells. Conclusions Our results suggest that miR-155-5p reduced corneal permeability and accelerated the recovery of corneal epithelial wounds by decreasing the expression of MLCK and phosphorylation of MLC and by remodeling tight junctions.

The Interplay Between E-Cadherin, Connexin 43, and Zona Occludens 1 in Retinal Pigment Epithelial Cells.

Cell-cell contact in retinal pigment epithelium (RPE) involves adherent junctions, gap junctions, and tight junctions, which are primarily composed by E-cadherin, zona occludens 1 (ZO-1), and connexin 43, respectively. Here, we aimed to explore the relationship and interplay between these junction-associated proteins. E-cadherin, connexin 43, and ZO-1 expression in human primary RPE in the early phase after TGF-β1 stimulation was detected. The knockdown of E-cadherin, ZO-1, and connexin 43 was performed to characterize the regulatory network involving these three proteins. Dye transfer and FITC-dextran permeability assays were conducted to observe the epithelial functional alterations. Transmission electron microscopy (TEM) was used to observe the ultrastructure of the cell-cell junctions in mouse RPE. The immunofluorescence staining and coimmunoprecipitation were performed to observe the colocalization and the physical association of E-cadherin, ZO-1, and connexin 43. Among these three components, E-cadherin appeared to be the first protein that was downregulated after TGF-β1 treatment. The ultrastructures of adherent junctions, gap junctions, and tight junctions could be observed in mouse RPE by TEM. E-cadherin, ZO-1, and connexin 43 were colocalized and physically bound to each other. The knockdown of one of these three proteins led to downregulation of the other two proteins and compromised epithelial function. E-cadherin, ZO-1, and connexin 43 were physically associated with each other and were mutually regulated. To enhance the understanding of cell-cell contacts, a holistic view is needed. Our results provide new insights in RPE disorders such as proliferative vitreoretinopathy.

Protective effect of genes modified stem cells on the tight junction of damaged small intestinal epithelium

Objective To investigate the role of CXC subfamily chemokine receptor 3 (CXCR3) and heme oxygenase-1 (HO-1) gene-modified bone marrow mesenchymal stem cells (BMMSCs) in protecting tight junctions of transplanted small intestine epithelium. Methods BMMSCs were isolated by whole bone marrow adherence and identified by detecting Cluster of Differentiation 29 (CD29), Cluster of Differentiation 90 (CD90), Classical class Ⅰ major histocompatibility complex (RT1-A), Cluster of Differentiation 34 (CD34), Cluster of Differentiation 45 (CD45) and Classical class Ⅱ major histocompatibility complex (RT1-B) by Flow cytometry. BMMSCs modified by HO-1 gene, CXCR3 gene and CXCR3 adding HO-1 genes were obtained by adenovirus transfection. Experimental models of rat small bowel transplantation rejection were established by using Brown Norway rats as donors and Lewis rats as receptors. The pathology of intestine was analyzed by Hematoxylin-eosin staining and transmission electron microscopy. The expression of zona occludens-1 (ZO-1) in small intestine was detected by Western blotting. Results BMMSCs were long-shuttle type as well as positive for CD29 (99.6%), CD90 (99.8%) and RT1-A (99.8%) but negative for CD34, CD45 and RT1-B. Experimental models of acute rejection of small bowel transplantation were obtained successfully. Compared with the NS group (3.66±0.20), BMMSCs group (3.05±0.59), CXCR3/BMMSCs group (2.76±0.49) and HO-1/BMMSCs group (2.86±0.80), (CXCR3+ HO-1)/BMMSCs significantly alleviated intestinal damage (2.43±0.85, t=-5.996, P<0.05, 7 d). Meanwhile, (CXCR3+ HO-1)/BMMSCs could more significantly protected the tight junction structure and the level of ZO-1 protein in transplanted small intestine than other groups (t=-9.436, P<0.05). Conclusion Compared with single-gene-modified, BMMSCs modified by CXCR3 adding HO-1 could protect the structure and function of tight junctions of transplanted small intestine with better effects. Key words: Bone marrow mesenchymal; CXC subfamily chemokine receptor 3; Heme oxygenase-1; Small intestine transplantation; Zona occludens-1

Protective effect of Saccharomyces boulardii on intestinal mucosal barrier of dextran sodium sulfate-induced colitis in mice.

Background:The effect and mechanism of Saccharomyces boulardii (Sb) in inflammatory bowel disease are unclear. The objective of the study was to evaluate the impact of Sb on intestinal mucosal barrier and intestinal flora in a colitis mouse model.Methods:Forty C57BL/6J male mice were randomly assigned to five groups: normal control group (A), pathologic control group (B), Sb treatment group (C), mesalazine treatment group (D), and Sb combined with mesalazine treatment group (E). Colitis was induced by the addition of 2.5% (wt/vol) dextran sodium sulfate (DSS) in the drinking water ad libitum for 7 days. The general condition, weight change, stool property, and bloody stool level of mice were observed to evaluate the disease activity index. The expression of zona occludens-1 (ZO-1) and occludin in intestinal tissue were measured by immunohistochemistry. The level of tumor necrosis factor-α (TNF-α) and interleukin (IL)-8 in plasma was measured by enzyme linked immunosorbent assay. Inter-cellular tight junctions were observed by transmission electron microscopy. The feces and intestinal contents were collected sterilely, and intestinal flora was analyzed by 16S rRNA sequencing.Results:Compared with group B, Sb reduced the disease activity index and histological score of group C (disease activity index: group B 2.708 ± 0.628, group C 1.542 ± 0.616, PBC = 0.005; histological score: group B 9.875 ± 3.271, group C 4.750 ± 1.832, PBC = 0.005) in DSS-induced colitis in mice. Sb exerted a protect effect on the expression of ZO-1 (group B 2.075 ± 1.176, group C 4.225 ± 1.316, PBC = 0.019) and occludin (group B 2.200 ± 0.968, group C 3.525 ± 1.047, PBC = 0.023). Compared with group B, Sb decreased the level of TNF-α and IL-8 of group C (TNF-α: group B 716.323 ± 44.691 ng/L, group C 521.740 ± 90.121 ng/L, PBC = 0.001; IL-8: group B 128.992 ± 11.475 pg/mL, group C 106.283 ± 15.906 pg/mL, PBC = 0.012). Treatment with Sb preserved the tight junctions and ameliorated microvilli and inter-cellular space. Treatment with Sb also showed its own characteristics: a higher percentage of Bacteroidetes and a lower percentage of Firmicutes, with significant differences or a significant trend. The proportion of the S24-7 family was increased significantly in the Sb treatment group.Conclusions:Sb shows an anti-inflammatory effect and has a protective effect on the intestinal mucosal mechanical barrier. Sb may up-regulate the abundance of family S24-7 specifically, and maybe a mechanism underlying its function.

The effect of neutrophil serine proteases on human nasal epithelial cell barrier function.

The neutrophil serine proteases neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3) are implicated in the regulation of inflammatory conditions. Pseudomonas aeruginosa elastase (PE), also a serine protease, has been found to behave similarly to NE and has been proposed to assist the pathogen in evading the host immune response. The effect of serine proteases on human nasal epithelial barrier function requires further investigation to better understand the pathophysiology of inflammatory conditions. Purified human neutrophil serine proteases and PE were applied to primary human nasal epithelial cells grown at air-liquid interface (HNEC-ALI) cultures from 6 patients. Barrier integrity and function was assessed via transepithelial electrical resistance (TER), permeability assays, immunofluorescence of Zona occludens-1 (ZO-1), and ciliary beat frequency (CBF) measurements. Cytotoxicity assays were employed to assess cell viability. Interleukin 6 (IL-6) and IL-8 enzyme-linked immunosorbent assay (ELISA) assessed cytokine release from HNEC-ALI. The application of serine proteases showed detrimental effects on HNEC-ALI barrier integrity. Reduction in TER occurred with NE, CG, and PE with increased paracellular permeability with NE, CG, PR3, and PE. Discontinuous tight junctions with reduction in ZO-1 expression were identified using immunofluorescence. Neutrophil serine proteases were not toxic cells to the HNECs and had no detrimental effects on the CBF. Serine proteases derived from neutrophils and from P. aeruginosa showed detrimental effects on the mucosal barrier integrity with increased permeability, allowing for potential bacterial invasion. This finding may further assist in understanding the pathophysiology present in chronic inflammatory airway diseases.

Flavonoids from Citrus aurantium ameliorate TNBS-induced ulcerative colitis through protecting colonic mucus layer integrity

Intestinal barrier injury is recognized as the main reason for ulcerative colitis (UC) which is a chronic inflammatory disease and the effective therapeutic methods remain limited. In this study, we explored the protection effects of naringenin, nobiletin and hesperetin from Citrus aurantium on colonic mucus layer integrity. The UC mice model was induced by trinitrobenzenesulfonic acid (TNBS). Forty mg/kg/day of naringenin, nobiletin and hesperetin were administrated orally for 7 and 3 days before and after TNBS treatment. Then body weight, disease activity index (DAI) score and colon length were recorded. Colons were collected for immunofluorescence, RT-PCR and western blot assay. Lipopolysaccharide (LPS)-induced cell-co-culture system of Caco-2 and RAW264.7 cells were used to determine activities on protecting intestinal epithelial monolayers damage. Our results showed that Naringenin, nobiletin and hesperetin alleviated body weight loss and colon shortness, decreased DAI score, and up-regulated claudin-2, occludin and zona occludens-1 (ZO-1) expression significantly. Meanwhile, the flavonoids enhanced trans epithelial electric resistance, decreased the permeability and up-regulated occludin and ZO-1 expression in LPS-damaged epithelial monolayers system. Hence, Naringnien, nobiletin and hesperetin show the regulatory effect on UC by protecting colonic mucosa layer integrity. It is suggested the flavonoids may be new supplements for the treatment of UC.

Extracellular matrix electrospun membranes for mimicking natural renal filtration barriers

Kidney diseases are recognized as a major health problem, which affect 10% of the population. Because currently available therapies have many limitations, some tissue engineering strategies have been emerging as promising approaches in this field.In this work, porcine kidneys were decellularized to obtain decellularized kidney extracellular matrix (dKECM).11dKECM (decellularized kidney extracellular matrix) Our results demonstrate a successful protocol of decellularization characterized by the removal of nucleic acid material and preservation of collagen and glycosaminoglycans. Blends of polycaprolactone (PCL)22PCL (polycaprolactone) and dKECM were prepared by electrospinning and characterized. The biological performance of the membranes was tested with a human kidney cell line (HK-2) for 7 days. It was observed that cellular metabolic activity, proliferation and protein content increased with an increase in dKECM concentrations (30, 50 and 70%). Additionally, the expression of zona occludens-1 was revealed on dKECM-containing membranes but not on pure PCL membranes.To the best of our knowledge this is the first time that natural extracellular matrix is used to mimic the kidney basement membrane as an in vitro model. This could be a valuable tool for regenerative nephrology and may have an impact on the development of kidney advanced therapies in the future.

Asparaginyl endopeptidase induces endothelial permeability and tumor metastasis via downregulating zonula occludens protein ZO-1

Zona occludens-1 (ZO-1) is a key component of tight junctions that govern the function of the endothelial barrier against tumor metastasis. Factors secreted by tumor cells contribute to the maintenance of tumor vascular networks. How tumor cell-derived protein signals regulate ZO-1 expression is unclear. Here, we explored the effect of tumor cell-secreted asparaginyl endopeptidase (AEP) on the permeability of endothelial cells in the tumor microenvironment. First, we confirmed the existence of AEP in conditioned medium (CM) from AEP-overexpressing MDA-MB-231 and 4T1 cells. Treatment with CM from AEP-overexpressing tumor cells increased the permeability and tumor cell transversal of an endothelial monolayer. Furthermore, CM from AEP-overexpressing tumor cells suppressed endothelial ZO-1 expression, as well as ZO-1-associated nucleic acid binding protein ZONAB. In addition, the level of phosphorylated STAT3 was increased by treatment with AEP-containing CM. A mutation of RGD or blocking integrin αvβ3 with antibody recovered the ZO-1 downregulation induced by AEP. In vivo, a lung metastatic mouse model showed increased endothelial permeability in the AEP-overexpressing group compared with the control group. An orthotopic tumor transplantation model was established using AEP-overexpression and compared with mice receiving control 4T1 cells. Compared with controls, overexpression of AEP increased lung metastatic foci and area, as well as vascular instability in primary tumors or lung metastatic sites. Moreover, endothelial ZO-1 was decreased in the AEP-overexpressing group. Taken together, our data show that tumor cell-derived AEP increases the permeability of endothelial barriers. Interactions between RGD and endothelial integrin αvβ3 mediate this effect by downregulating ZO-1.