Zonula Occludens-1 Protein Levels Research Articles

Human Milk Exosomes Induce ZO-1 Expression via Inhibition of REDD1 Expression in Human Intestinal Epithelial Cells.

Human milk exosomes (HMEs) enhance intestinal barrier function and contribute to an improvement in inflammation and mucosal injury, such as necrotizing enteritis (NEC), in infants. Here, we aimed to elucidate the intracellular factors involved in HME-induced expression of zonula occludens-1 (ZO-1), a tight junction protein, in Caco-2 human intestinal epithelial cells. HME treatment for 72 h significantly increased transepithelial electrical resistance in these cells. The mean ZO-1 protein levels in cells treated with HME for 72 h were significantly higher than those in the control cells. The mRNA and protein levels of regulated in development and DNA damage response 1 (REDD1) in HME-treated cells were significantly lower than those in the control cells. Although HME treatment did not increase the mechanistic target of rapamycin (mTOR) level in Caco-2 cells, it significantly increased the phosphorylated mTOR (p-mTOR) level and p-mTOR/mTOR ratio. The ZO-1 protein levels in cells treated with an inducer of REDD1, cobalt chloride (CoCl2) alone were significantly lower than those in the control cells. However, ZO-1 protein levels in cells co-treated with HME and CoCl2 were significantly higher than those in cells treated with CoCl2 alone. Additionally, REDD1 protein levels in cells treated with CoCl2 alone were significantly higher than those in the control cells. However, REDD1 protein levels in cells co-treated with HME and CoCl2 were significantly lower than those in cells treated with CoCl2 alone. This HME-mediated effect may contribute to the development of barrier function in the infant intestine and protect infants from diseases.

Effects of butyphthalide on microglia polarization after intracerebral hemorrhage and the underlying mechanisms.

Because intracerebral hemorrhage (ICH) has high morbidity, disability and mortality, it is significant to find new and effective treatments for ICH. This study aims to explore the effect of butyphthalide (NBP) on neuroinflammation secondary to ICH and microglia polarization. A total of 48 healthy male SD rats were randomly divided into 6 groups: a sham 24 h group, a sham 72 h group, an ICH 24 h group, an ICH 72 h group, an ICH+NBP 24 h group, and an ICH+NBP 72 h group (8 rats per group). After operation, the neurological deficiencies were assessed based on improved Garcia scores and corner test. The expressions of Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), aquaporin-4 (AQP4), zonula occludens-1 (ZO-1), occludin, CD68, CD86, and CD206 were observed by Western blotting. Inflammatory cytokines were detected by ELISA. The immunofluorescence was to detect the polarization of microglia. 1) Compared with the sham groups, the expression of TLR4 (24 h: P<0.05; 72 h: P<0.01), NF-κB (both P<0.01) and Nrf2 (both P<0.01) in the perihematoma of the ICH group was increased, leading to microglia activation (P<0.01). The expressions of IL-6 (24 h: P<0.05; 72 h: P<0.01) and TNF-α (both P<0.01), the pro-inflammatory cytokines were up-regulated, and the expression of anti-inflammatory cytokine IL-4 was down-regulated (both P<0.01). Besides, the expression of AQP4 was enhanced (both P<0.01). The protein level of tightly connected proteins (including ZO-1, occludin) was decreased (all P<0.01). The neurological function of the rats in the ICH group was impaired in the 2 time points (both P<0.01). 2) Compared with the sham group at 24 h and 72 h after the intervention of NBP, the expressions of TLR4 (both P<0.05) and NF-κB (both P<0.01) were significantly declined, and the expression of Nrf2 was further enhanced (both P<0.05) in the perihematoma of the ICH+NBP group. Furthermore, the expression of M1 microglia marker was inhibited (P<0.05), and the polarization of microglia to the M2 phenotype was promoted (P<0.01). 3) In terms of inflammation after ICH, the IL-4 expression in the ICH+NBP group was increased compared with the ICH group (24 h: P<0.05; 72 h: P<0.01); the expression of IL-6 was decreased significantly in the ICH+NBP 72 h group (P<0.01); the level of AQP4 was declined significantly in the ICH+NBP 24 h group (P<0.05), there was a downward trend in the 72-hour intervention group but without significant statistical difference. 4) Compared with the ICH group, the ZO-1 protein levels were increased (24 h: P<0.05; 72 h: P<0.01), and the symptoms of nerve defect were improved eventually (both P<0.05) in the ICH+NBP groups. After ICH, the TLR4/NF-κB pathway is activated. The M1 microglia is up-regulated along with the release of detrimental cytokines, while the anti-inflammatory cytokines are down-regulated. The expression of AQP4 is increased, the tight junction proteins from the blood-brain barrier (BBB) is damaged, and the neurological function of rats is impaired. On the contrary, NBP may regulate microglia polarization to M2 phenotype and play a role in the neuroprotective effect mediated via inhibiting TLR4/NF-κB and enhancing Nrf2 pathways, which relieves the neuroinflammation, inhibits the expression of AQP4, repairs BBB, and improves neurological functional defects.

Ultrastructural examination of testicular tissues and evaluation of ZO-1 protein levels obtained from azoospermic cases

Spermatogenesis is a process that involves cellular and biochemical processes. The purpose of this study is to understand the role of tight junctions in spermatogenesis by evaluating Zonula occludens-1 (ZO-1) protein levels of cells from the testicular sperm extraction (TESE) biopsies in patients with azoospermia. The study included 40 infertile men with azoospermia. They were divided into two groups, as spermatozoa positive (n=20) and spermatozoa negative (n=20). Testis tissues were examined morphologically, immunohistochemically and ultrastructurally by using transmission electron microscopy (TEM). The structure of seminiferous tubules was deteriorated, degenerated and hyalinized in spermatozoa negative group. ZO-1 expression level was normal in blood-testis barrier of spermatozoa positive group in comparing to lateral surfaces of Sertoli cells spermatozoa negative group. The ultrastructural results were also supported with light microscopy findings. These findings suggest that decrease of the ZO-1 protein expression level in Sertoli cells might be one of the factors involved in the impairment of spermatogenesis in spermatozoa negative group. Therefore, we suggest that tight junctions may have crucial roles in spermatogenesis.

Alteration of fecal tryptophan metabolism correlates with shifted microbiota and may be involved in pathogenesis of colorectal cancer.

BACKGROUNDGut tryptophan (Trp) metabolites are produced by microbiota and/or host metabolism. Some of them have been proven to promote or inhibit colorectal cancer (CRC) in vitro and animal models. We hypothesized that there is an alteration of gut Trp metabolism mediated by microbiota and that it might be involved in the pathogenesis of cancer in patients with CRC.AIMTo investigate the features of Trp metabolism in CRC and the correlation between fecal Trp metabolites and gut microbiota.METHODSSeventy-nine patients with colorectal neoplastic lesions (33 with colon adenoma and 46 with sporadic CRC) and 38 healthy controls (HCs) meeting the inclusion and exclusion criteria were included in the study. Their demographic and clinical features were collected. Fecal Trp, kynurenine (KYN), and indoles (metabolites of Trp metabolized by gut microbiota) were examined by ultraperformance liquid chromatography coupled to tandem mass spectrometry. Gut barrier marker and indoleamine 2,3-dioxygenase 1 (IDO1) mRNA were analyzed by quantitative real-time polymerase chain reaction. Zonula occludens-1 (ZO-1) protein expression was analyzed by immunohistochemistry. The gut microbiota was detected by 16S ribosomal RNA gene sequencing. Correlations between fecal metabolites and other parameters were examined in all patients.RESULTSThe absolute concentration of KYN [1.51 (0.70, 3.46) nmol/g vs 0.81 (0.64, 1.57) nmol/g, P = 0.036] and the ratio of KYN to Trp [7.39 (4.12, 11.72) × 10-3 vs 5.23 (1.86, 7.99) × 10-3, P = 0.032] were increased in the feces of patients with CRC compared to HCs, while the indoles to Trp ratio was decreased [1.34 (0.70, 2.63) vs 2.46 (1.25, 4.10), P = 0.029]. The relative ZO-1 mRNA levels in patients with CRC (0.27 ± 0.24) were significantly lower than those in HCs (1.00 ± 0.31) (P < 0.001), and the relative IDO1 mRNA levels in patients with CRC [1.65 (0.47-2.46)] were increased (P = 0.035). IDO1 mRNA levels were positively associated with the KYN/Trp ratio (r = 0.327, P = 0.003). ZO-1 mRNA and protein levels were positively correlated with the indoles/Trp ratio (P = 0.035 and P = 0.009, respectively). In addition, the genera Asaccharobacter (Actinobacteria) and Parabacteroides (Bacteroidetes), and members of the phylum Firmicutes (Clostridium XlVb, Fusicatenibacter, Anaerofilum, and Anaerostipes) decreased in CRC and exhibited a positive correlation with indoles in all subjects.CONCLUSIONAlteration of fecal Trp metabolism mediated by microbiota is associated with intestinal barrier function and tissue Trp metabolism, and may be involved in the pathogenesis of CRC.

Effect of electroacupuncture on intestinal calcium absorption and expression of transmembrane calcium transport-related receptors in ovariectomized rats

To observe the effect of electroacupuncture (EA) on bone mineral density (BMD) and expression of calcium transport-related receptors in ovariectomized rats, so as to explore its mechanisms underlying improvement of osteoporosis. Thirty-two three-month female SD rats were randomly divided into control, sham operation, model and EA groups (n=8 in each group). The ovariectomy model was established by resection of bilateral ovaries. EA (2 Hz/15 Hz, 2 mA) was applied to "Guanyuan"(CV4)+"Sanyinjiao"(SP6) or "Shenshu"(BL23)+"Housanli"(ST36) alternatively for 20 min, once daily (except the weekends) for 12 weeks. BMD of the tibia and femur were detected by using a bone densitometer. The expression levels of transient receptor potential vanillic receptor 5 (TRPV5), transient receptor potential vanillic receptor 6 (TRPV6), sodium calcium exchanger 1 (NCX1), membrane calcium adenosine triphosphate hydrolase (PMCA1b), zonula occludens 1 (ZO-1), occludin and claudin in intestinal mucosa were detected by quantitative real-time PCR and Western blot, respectively. Following modeling, BMD of both tibia and femur, and expression levels of TRPV5, PMCA1b, ZO-1, occludin and claudin mRNAs were significantly decreased in the model group compared with the control and sham operation groups (P<0.05, P<0.01)，while the mRNAs levels of TRPV6 and NCX1 in the model group were increased (P<0.01). After the treatment, BMD of both tibia and femur, and expression levels of TRPV5, PMCA1b, ZO-1 and occludin mRNAs were significantly increased(P<0.01，P<0.05)，and TRPV6 and NCX1 were significantly decreased(P<0.01，P<0.05) in the EA group than those in the model group. Compared with the control and sham operation groups, protein expression levels of TRPV5, PMCA1b, ZO-1, occludin and claudin in the model group were significantly decreased (P<0.05), while the protein levels of TRPV6 and NCX1 in the model group were increased (P<0.05). After EA intervention, modeling-induced decrease of TRPV5, PMCA1b, ZO-1, occludin and claudin protein levels and increase of TRPV6 and NCX1 protein levels were all completely reversed in the EA group relevant to the model group (P<0.05). EA intervention can improve bone mineral density and up-regulate the mRNA and protein expression of TRPV5, PMCA1b, ZO-1 and occludin, which may be one of the mechanisms of acupuncture in the treatment of osteoporosis.

Evaluation of long-term rt-PA effects on bEnd.3 endothelial cells under ischemic conditions; changes in ZO-1 expression and glycosylation of the bradykinin B2 receptor

Recombinant tissue plasminogen activator (rt-PA) has proven effective in the treatment of acute ischemic stroke, despite the increased risk of hemorrhagic transformation (HT), its major associated complication. Although it is known that HT is related to blood brain barrier (BBB) disruption, the underlying mechanisms are not well established. We assessed time-dependent effects of rt-PA on the bEnd.3 murine brain endothelial cell line subjected either to normoxia or to 2.5 h of oxygen and glucose deprivation (OGD), evaluating a longer period than has previously been done, beyond 6 h post-reoxygenation. Parameters of cell viability, metabolic activity, ionic and transcellular permeability, as well as levels of claudin-5, zonula occludens-1 (ZO-1) and bradykinin B2 receptor (B2R) protein expression were analyzed at 24, 48 and 72 h post-reoxygenation with or without the administration of rt-PA. rt-PA treatment increased both the ionic and transcellular permeability until 72 h and did not modify cell viability or metabolic activity or the expression of claudin-5, ZO-1 and B2R under normoxia at any analyzed time. Under OGD conditions, rt-PA exacerbated OGD effects on metabolic activity from 48 to 72 h, increased transcellular permeability from 24 to 72 h, significantly decreased ZO-1 protein levels at the plasma membrane and increased B2R glycosylation at 72 h post-reoxygenation. Our findings suggest that a long-term analysis is necessary to elucidate time-dependent molecular mechanisms associated to BBB breakdown due to rt-PA administration under ischemia. Thus, protective BBB therapies after ischemic stroke and rt-PA treatment should be explored at least until 72 h after OGD and rt-PA administration.

Ghrelin ameliorates blood-brain barrier disruption during systemic hypoxia.

What is the central question of this study? Is an anti-oedematous effect of ghrelin associated with increased expression of tight junction proteins in the hypoxic brain? What is the main finding and its importance? We showed that injection of ghrelin during acute and chronic systemic hypoxia is associated with increased expression of tight junction proteins and protection of the blood-brain barrier. Ghrelin appears to be a new therapeutic strategy for protection of the blood-brain barrier from disruption and prevention of brain oedema in hypoxic conditions. The blood-brain barrier, which serves to protect the homeostasis of the CNS, is formed by tight junction proteins. Several studies have indicated that systemic hypoxia leads to cerebral oedema through disruption of tight junction proteins, such as occludin and zonula occludens-1 (ZO-1). According to our previous studies, ghrelin attenuates cerebral oedema in the hypoxic brain. However, the mechanism is not completely understood. The present study was designed to determine the effect of ghrelin on occludin and ZO-1 in the hypoxic brain. Adult male Wistar rats were divided into acute and chronic control, acute or chronic hypoxia, and ghrelin-treated acute or chronic hypoxia groups. Hypoxic groups were kept in a hypoxic chamber (10-11% O2 ) for 2 (acute) or 10 days (chronic). Effects of ghrelin on occludin and ZO-1 protein levels were assessed using Western blotting. Western blot analysis revealed that the protein expression of ZO-1 and occludin decreased significantly in acute and chronic hypoxia. Ghrelin significantly increased ZO-1 protein expression in both acute and chronic hypoxia (P<0.05). Ghrelin also increased occludin protein expression in chronic hypoxia (P<0.05) but did not effectively change it in acute hypoxia. Our data showed that ghrelin injection maintains occludin and ZO-1 tight junction proteins, which may improve the integrity of the blood-brain barrier in hypoxic conditions.

TGFβ1-Induced LncRNA UCA1 Upregulation Promotes Gastric Cancer Invasion and Migration.

According to recent studies, long noncoding RNA urothelial carcinoma associated 1 (UCA1) is involved in the development and progression of many malignant tumors, including gastric cancer (GC). We validated the detailed role of UCA1 in human GC cell lines and GC tissues so as to determine its exact function and the underlying mechanism of GC invasion and migration. In our research, lncRNA-UCA1 was specifically upregulated in GC tissues and cell lines, and augmented GC cell proliferation, and invasive and migratory capabilities. High UCA1 expression in GC was related with poorer prognosis (poorer invasion depth, lymph node metastasis, advanced TNM [T is for the original (primary) tumor, N for nearby (regional) lymph nodes that are involved, and M for distant metastasis] stage, and shorter overall survival). Epithelial mesenchymal transition (EMT), associated with malignancy of cancers, was reported to be responsible for invasion and migration of cancer cells. Transforming growth factor β1 (TGFβ1)-induced EMT was well evaluated. UCA1 silence reduced the protein levels of EMT-related factors, vimentin and snail, while promoted E-cadherin and zonula occludens-1 protein levels in GC cells; the effect of UCA1 could be partly restored by TGFβ1 treatment. Taken together, UCA1 might regulate the tumor proliferation, invasion, and metastasis under TGFβ1 induction. Taken together, UCA1 might present a potential oncogenic factor by promoting GC cell proliferation, invasion, and migration. UCA1 could serve as a novel biomarker for prognosis and a novel therapeutic target of GC treatment.

Baicalin Protects against TNF-α-Induced Injury by Down-Regulating miR-191a That Targets the Tight Junction Protein ZO-1 in IEC-6 Cells.

Tumor necrosis factor-alpha (TNF-α) plays an important role in the developing process of inflammatory bowel disease. Tight junction protein zonula occludens-1 (ZO-1), one of epithelial junctional proteins, maintains the permeability of intestinal barrier. The objective of this study was to investigate the mechanism of the protective effect of baicalin on TNF-α-induced injury and ZO-1 expression in intestinal epithelial cells (IECs). We found that baicalin pretreatment significantly improved cell viability and cell migration following TNF-α stimulation. miR-191a inhibitor increased the protective effect of baicalin on cell motility injured by TNF-α. In addition, miR-191a down-regulated the mRNA and protein level of its target gene ZO-1. TNF-α stimulation increased miR-191a expression, leading to the decline of ZO-1 mRNA and protein. Moreover, pretreatment with baicalin reversed TNF-α induced decrease of ZO-1 and increase of miR-191a, miR-191a inhibitor significantly enhanced ZO-1 protein expression restored by baicalin. These results indicate that baicalin exerts a protective effect on IEC-6 (rat small intestinal epithelial cells) cells against TNF-α-induced injury, which is at least partly via inhibiting the expression of miR-191a, thus increasing ZO-1 mRNA and protein levels.

Effects of interleukin-13 and montelukast on the expression of zonula occludens-1 in human podocytes.

PurposeThe aim of this study was to investigate whether pathologic changes in zonula occludens-1 (ZO-1) are induced by interleukin-13 (IL-13) in the experimental minimal-change nephrotic syndrome (MCNS) model and to determine whether montelukast, a leukotriene receptor antagonist, has an effect on ZO-1 restoration in cultured human podocytes.Materials and MethodsHuman podocytes cultured on bovine serum albumin-coated plates were treated with different doses of IL-13 and montelukast and then examined for distribution using confocal microscopy and for ZO-1 protein levels using Western blotting.ResultsZO-1 was internalized and shown to accumulate in the cytoplasm of human podocytes in an IL-13 dose-dependent manner. High doses (50 and 100 ng/mL) of IL-13 decreased the levels of ZO-1 protein at 12 and 24 h (both p<0.01; n=3), which were significantly reversed by a high dose (0.5 µM) montelukast treatment (p<0.01; n=3).ConclusionOur results suggest that IL-13 alters the expression of ZO-1, and such alterations in the content and distribution of ZO-1 may be relevant in the pathogenesis of proteinuria in the MCNS model.

Spatio-temporally restricted blood–brain barrier disruption after intra-amygdala kainic acid-induced status epilepticus in mice

Mesial temporal lobe epilepsy (MTLE) is the most common, intractable seizure disorder in adults. Blood-brain barrier (BBB) disruption, including interruption of endothelial tight cell junctions and serum protein and immunoglobulin G (IgG) extravasation into brain parenchyma, has been reported in experimental and human MTLE and implicated in disease pathogenesis. Triggering status epilepticus in mice by intra-amygdala microinjection of kainic acid produces damage mainly within the CA3 subfield of the ipsilateral hippocampus, and recurrent spontaneous seizures emerge during the following week. To investigate whether BBB impairment is a feature of this model, we characterized endothelial tight cell junction proteins and IgG and albumin in the hippocampus up to three weeks after status epilepticus. Hippocampal microvessels displayed a reduction in continuous staining for zonula occludens 1 (ZO-1), the main tight junction protein, after status epilepticus and in epileptic mice, although western blotting found ZO-1 protein levels in the hippocampal subfields were not different from controls at any time. Increased IgG and albumin were detected in damaged and non-damaged ipsilateral hippocampal subfields, mainly 4-24h after status epilepticus, although increased serum protein extravasation was also found in the CA3 subfield in epileptic mice. Thus, BBB opening or damage occurs mainly in the period shortly after status epilepticus but may also persist within the CA3 subfield as a feature of the pathophysiology of chronic epilepsy in this model.

Breakdown of the Gut Barrier in Patients with Multiple Organ Dysfunction Syndrome is Attenuated by Continuous Blood Purification: Effects on Tight Junction Structural Proteins

Breakdown of the gut barrier increases intestinal permeability and allows movement of intraluminal contents across the mucosa, which can lead to distant organ injury and multiple organ dysfunction syndrome (MODS). Intestinal permeability is associated with alterations in cellular tight junctions involving the structural proteins occludin and zonula occludens-1 (ZO-1). The aim of this study was to investigate the effect of continuous blood purification (CBP) on gut barrier function in patients with MODS. Serum diamine oxidase (DAO) and endotoxin, epithelial monolayer permeability, and transepithelial electrical resistance (TER) were used as markers for the assessment of gut barrier function in 22 patients with MODS who underwent continuous venovenous hemofiltration (CWH) for 24 hours. Blood samples were taken from patients at 0, 6, 12, and 24 hours during CWH therapy. Serum DAO and endotoxin were determined by spectrophotography. Permeability and TER were assessed using Caco-2 cell monolayers. Occludin and ZO-1 protein levels were analyzed by immunoblotting and immunofluorescence staining. And inducible nitric oxide synthase (iNOS) mRNA levels and nitric oxide (NO) production were determined by real-time PCR and spectrophotography, respectively. Gut barrier dysfunction was evident in patients with MODS compared with normal controls. Serum DAO, endotoxin levels, and epithelial permeability were elevated, while TER was decreased in patients with MODS, and this change was more pronounced in nonsurvivors. Breakdown and reorganization of occludin and ZO-1 away from tight junctions was found in all MODS patients. After CBP treatment, APACHE II and MODS scores improved significantly. Serum DAO and endotoxin levels and epithelial permeability also diminished, while TER increased in all patients; CBP significantly attenuated breakdown and reorganization of tight junction proteins, and also attenuated the inflammation-induced increase in iNOS mRNA expression and NO production. Breakdown of gut barrier function is present in patients with MODS and may be correlated with poor outcomes in the disease. CBP can not only improve the general conditions, as measured by the APACHE II score, but also improve gut barrier dysfunction by attenuating the breakdown and reorganization of occludin and ZO-1. This beneficial effect of CBP on gut barrier dysfunction is associated with down-regulation of iNOS.

Telmisartan Increases the Permeability of Endothelial Cells through Zonula Occludens-1

Angiotensin receptor-1 blockers (ARBs) have been used for the treatment of atherosclerosis. Tight junctions are very important in the regulation of cellular permeability which may have a profound role in atherosclerosis. The relationship between them is unresolved. The expression and distribution of zonula occludens-1 (ZO-1), the permeability of cultured endothelial cells, and the activity of phosphatidylinositol 3-kinase (PI3K) were all detected with various methods after cultured endothelial cells were exposed to valsartan and the special ARB telmisartan or combined with PI3K inhibitor wortmannin or the peroxisome proliferator-activated receptor gamma antagonist GW9662. We found that telmisartan but not valsartan downregulated ZO-1 mRNA and protein levels, disrupted the distribution of ZO-1 in cultured endothelial cells, and increased the permeability of endothelial cells in a dose-dependent manner. When the PI3K inhibitor wortmannin was used, the effect induced by telmisartan was at least partly reversed. The role of the PI3K signaling pathway was further confirmed in the PI3K activity assay. GW9662 significantly blocked telmisartan-induced ZO-1 changes. Our results suggest that telmisartan disrupts the continuous pericellular distribution of ZO-1, downregulates the expression of ZO-1 in endothelial cells, and increases the permeability of endothelial cells at least partly through PI3K and the peroxisome proliferator-activated receptor gamma-dependent pathway.

Phosphodiesterase inhibition attenuates alterations to the tight junction proteins occludin and ZO-1 in immunostimulated Caco-2 intestinal monolayers

Aims Under normal conditions, the intestinal mucosa acts as a local barrier to prevent the influx of luminal contents. The intestinal epithelial tight junction is comprised of several membrane associated proteins, including zonula occludens-1 (ZO-1) and occludin. Disruption of this barrier can lead to the production of pro-inflammatory mediators and ultimately multiple organ failure. We have previously shown that Pentoxifylline (PTX) decreases histologic gut injury and pro-inflammatory mediator synthesis. We hypothesize that PTX prevents the breakdown of ZO-1 and occludin in an in vitro model of immunostimulated intestinal cell monolayers. Main methods Caco-2 human enterocytes were grown as confluent monolayers and incubated under control conditions, or with PTX (2 mM), Cytomix (TNF-α, IFN-γ, IL-1), or Cytomix + PTX for 24 h. Occludin and ZO-1 protein levels were analyzed by Western blot. Confocal microscopy was used to assess the cytoplasmic localization of ZO-1 and occludin. Key findings Cytomix stimulation of Caco-2 cells resulted in a 50% decrease in both occludin and ZO-1 protein. Treatment with Cytomix + PTX restored both occludin and ZO-1 protein to control levels. Confocal microscopy images show that Cytomix caused an irregular, undulating appearance of ZO-1 and occludin at the cell junctions. Treatment with PTX prevented the Cytomix-induced changes in ZO-1 and occludin localization. Significance Treatment with PTX decreases the pro-inflammatory cytokine induced changes in the intestinal tight junction proteins occludin and ZO-1. Pentoxifylline may be a useful adjunct in the treatment of sepsis and shock by attenuating intestinal barrier breakdown.

Effect of Alcohol on miR‐212 Expression in Intestinal Epithelial Cells and Its Potential Role in Alcoholic Liver Disease

Alcohol-induced gut leakiness is a key factor in alcoholic liver disease (ALD); it allows endotoxin to enter the circulation and initiate liver damage. Zonula occludens 1 (ZO-1) protein is a major component of tight junctions that regulates intestinal permeability. microRNAs (miRNAs) are recently discovered regulatory molecules that inhibit expression of their target genes. (i) to investigate the effect of alcohol on miRNA-212 (miR-212) and on expression of its predicted target gene, ZO-1, (ii) to study the potential role of miR-212 in the pathophysiology of ALD in man. Using a TaqMan miRNA assay system, we measured miR-212 expression levels in colon biopsy samples from patients with ALD and in Caco-2 cells (a human intestinal epithelial cell line) treated with or without EtOH. We measured ZO-1 protein levels using western blots. ZO-1 mRNA was assayed using real-time PCR. Intestinal barrier integrity was measured using fluorescein sulfonic acid clearance and immunofluorescent staining for ZO-1. Ethanol increased miR-212 expression, decreased ZO-1 protein levels, disrupted tight junctions, and increased the permeability of monolayers of Caco-2 cells. An miR-212 over-expression is correlated with hyperpermeability of the monolayer barrier. miR-212 levels were higher in colon biopsy samples in patients with ALD than in healthy controls; ZO-1 protein levels were lower. These data suggest a novel mechanism for alcohol-induced gut leakiness, one in which EtOH induces miR-212 over-expression which causes gut leakiness by down-regulating ZO-1 translation. This mechanism is a potential therapeutic target for leaky gut in patients with or at risk for ALD.

Gap junction remodelling in human heart failure is associated with increased interaction of connexin43 with ZO-1

AimsRemodelling of gap junctions, involving reduction of total gap junction quantity and down-regulation of connexin43 (Cx43), contributes to the arrhythmic substrate in congestive heart failure. However, little is known of the underlying mechanisms. Recent studies from in vitro systems suggest that the connexin-interacting protein zonula occludens-1 (ZO-1) is a potential mediator of gap junction remodelling. We therefore examined the hypothesis that ZO-1 contributes to reduced expression of Cx43 gap junctions in congestive heart failure.Methods and resultsLeft ventricular myocardium from healthy control human hearts (n = 5) was compared with that of explanted hearts from transplant patients with end-stage congestive heart failure due to idiopathic dilated cardiomyopathy (DCM; n = 5) or ischaemic cardiomyopathy (ICM; n = 5). Immunoconfocal and immunoelectron microscopy showed that ZO-1 is specifically localized to the intercalated disc of cardiomyocytes in control and failing ventricles. ZO-1 protein levels were significantly increased in both DCM and ICM (P = 0.0025), showing a significant, negative correlation to Cx43 levels (P = 0.0029). There was, however, no significant alteration of ZO-1 mRNA (P = 0.537). Double immunolabelling demonstrated that a proportion of ZO-1 label is co-localized with Cx43, and that co-localization of Cx43 with ZO-1 is significantly increased in the failing ventricle (P = 0.003). Interaction between the two proteins was confirmed by co-immunoprecipitation. The proportion of Cx43 that co-immunoprecipitates with ZO-1 was significantly increased in the failing heart.ConclusionOur findings suggest that ZO-1, by interacting with Cx43, plays a role in the down-regulation and decreased size of Cx43 gap junctions in congestive heart failure.

Markers for OLN-93 oligodendroglia differentiation

Oligodendrocytes are target cells in the pathogenesis of multiple sclerosis (MS), a chronic demyelinating disease of the central nervous system (CNS). During the course of the disease, inflammatory mediators may damage oligodendrocytes and their myelin sheaths. Differentiation of oligodendrocyte progenitors is an important step in the process of remyelination. In the present study, OLN-93 differentiation was studied in co-culture with C6 astrocytes as a natural source of growth and differentiation factors as well as after exposure to insulin-like growth factor-I (IGF-I). Morphological evaluation showed an increased degree of differentiation of OLN-93 cells after IGF-I administration, but not after co-culture with astrocytes. During early differentiation, 2′, 3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and zonula occludens-1 (ZO-1) tight junction protein expression were significantly increased. However, neither astrocyte co-culture nor exposure to IGF-I further increased the expression of these markers. Although reverse transcriptase-polymerase chain reaction revealed myelin basic protein (MBP) mRNA expression not to be affected during differentiation, we did find increased MBP protein expression by Western blotting. ZO-1 protein and DM20 mRNA levels were increased during the course of differentiation and after IGF-I administration. The present findings suggest that ZO-1 may be used as a marker for OLN-93 oligodendroglia differentiation.