Protein Expression Levels Of Occludin Research Articles

The Novel-m0230-3p miRNA Modulates the CSF1/CSF1R/Ras Pathway to Regulate the Cell Tight Junctions and Blood-Testis Barrier in Yak.

The yak (Bos grunniens) is a valuable livestock animal endemic to the Qinghai-Tibet Plateau in China with low reproductive rates. Cryptorchidism is one of the primary causes of infertility in male yaks. Compared with normal testes, the tight junctions (TJs) of Sertoli cells (SCs) and the integrity of the blood-testis barrier (BTB) in cryptorchidism are both disrupted. MicroRNAs are hairpin-derived RNAs of about 19-25 nucleotides in length and are involved in a variety of biological processes. Numerous studies have shown the involvement of microRNAs in the reproductive physiology of yak. In this study, we executed RNA sequencing (RNA-seq) to describe the expression profiles of mRNAs and microRNAs in yaks with normal testes and cryptorchidism to identify differentially expressed genes. GO and KEGG analyses were used to identify the biological processes and signaling pathways which the target genes of the differentially expressed microRNAs primarily engaged. It was found that novel-m0230-3p is an important miRNA that significantly differentiates between cryptorchidism and normal testes, and it is down-regulated in cryptorchidism with p < 0.05. Novel-m0230-3p and its target gene CSF1 both significantly contribute to the regulation of cell adhesion and tight junctions. The binding sites of novel-m0230-3p with CSF1 were validated by a dual luciferase reporter system. Then, mimics and inhibitors of novel-m0230-3p were transfected in vitro into SCs, respectively. A further analysis using qRT-PCR, immunofluorescence (IF), and Western blotting confirmed that the expression of cell adhesion and tight-junction-related proteins Occludin and ZO-1 both showed changes. Specifically, both the mRNA and protein expression levels of Occludin and ZO-1 in SCs decreased after transfection with the novel-m0230-3p mimics, while they increased after transfection with the inhibitors, with p < 0.05. These were achieved via the CSF1/CSF1R/Ras signaling pathway. In summary, our findings indicate a negative miRNA-mRNA regulatory network involving the CSF1/CSF1R/Ras signaling pathway in yak SCs. These results provide new insights into the molecular mechanisms of CSF1 and suggest that novel-m0230-3p and its target protein CSF1 could be used as potential therapeutic targets for yak cryptorchidism.

Comparative analysis of co-culture and monoculture models in simulating diabetic neurovascular dysfunction: insights into diabetic retinopathy.

Interaction between retinal vascular endothelial cells and neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR). This study aims to compare an in vitro model over a monoculture model to simulate the neurovascular coupling under the hyperglycemic microenvironment of diabetes. Rat retinal vascular endothelial cells (RRMECs) and ganglion cells (RGCs) were seeded mono- or co-cultured in a normal (NG, 5.5 mM) and high (HG, 75 mM) glucose concentrations culture medium. Cell viability was detected by the cell counting kit-8 (CCK-8) assay. The ability of migration and lumen formation of RRMECs were determined by scratch wound, transwell migration, and lumen formation assays. The apoptosis index of cells was calculated and detected by propidium iodide (PI)/Hoechst staining. Quantitative and morphological analysis of RGCs was performed through the labeling of RGCs by brain-specific homeobox/POU domain protein 3A (BRN3A) and anti-beta-III tubulin (TUJ1). The gene and protein expression levels of occludin (OCLN) and zonula occludens-1 (ZO-1) were evaluated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The viability, migration, and lumen formation abilities of RRMECs in the HG group significantly increased (P<0.05) in both mono- and co-culture models. Migration and lumen formation abilities of RRMECs in the co-culture with HG were lower than that in the monoculture group (P<0.05). The viability of RGCs cells with HG significantly decreased in both mono- and co-culture models (Pmono<0.001, Pco<0.001), the apoptosis index of RGCs in the co-culture with HG was higher than that in the monoculture (P=0.010). The protein and gene expression of OCLN, and ZO-1 in RRMECs significantly decreased with HG culture medium in both culture models (P<0.05). In the HG group, the protein and gene expression level of the ZO-1 and OCLN of RRMECs significantly decreased in the co-culture model than that in the monoculture model (P<0.05). Compared with mono cell culture, the established co-culture in vitro system for diabetic neurovascular dysfunction can better stimulate the micro-environment of the retinal neurovascular unit.

Estrogen deficiency aggravates fluoride-induced small intestinal mucosa damage and junctional complexes proteins expression disorder in rats

To investigate the effect of estrogen deficiency on the small intestinal mucosal barrier induced by fluoride (F), F exposure models of ovariectomy (OVX) rats (surgically removed ovaries) and non-OVX rats (normal condition) were established by adding sodium fluoride (NaF) (0, 25, 50, and 100 mg/L, calculated by F ion) in drinking water for 90 days. The intestinal mucosal histomorphology, mucosal barrier function, and protein expression levels of tight junctions (TJs), adhesion junctions (AJs), and desmosomes were evaluated in the duodenum, jejunum, and ileum. Hematoxylin-eosin (HE) staining and 5-Bromo-2-deoxyUridine (BrdU) measurement showed that excessive F-induced damage to intestinal epithelial cells and inhibited the proliferation of intestinal epithelial cells, eventually decreasing the number of goblet cells and decreasing glycoprotein secretion, as indicated by Alcian blue and periodic acid-Schiff (AB-PAS) and periodic acid-Schiff (PAS) staining. Further immunofluorescence analysis demonstrated that excessive F decreased the protein expression levels of occludin, zonula occludens-1 (ZO-1), E-cadherin, and desmoplakin (P < 0.05, P < 0.01) and enhanced the expression of claudin-2 (P < 0.01), suggesting that cell-to-cell junctions were disrupted. Collectively, F exposure impaired the small intestinal mucosal barrier by inducing damage to intestinal epithelial cells and inhibiting intestinal epithelial cell proliferation. Disorders in the junctional complex protein expression blocked the synergy between intercellular communication and aggravated mucosal injury. In particular, estrogen deficiency exacerbated F-induced enterotoxicity, which provides new explanations for the development and severity of intestinal disease in postmenopausal women with high-F areas.

Pathological mechanism of intestinal mucosal barrier injury of large intestine dampness-heat syndrome rats and the protective effect of Yujin powder

Large intestine dampness-heat syndrome (LIDHS) is frequently-occurring in the inflammatory intestinal disease of animals and human. Yujin powder (YJP) is a classical prescription for treating LIDHS. To explore the pathological mechanism of intestinal mucosal barrier injury of LIDHS and the protection of YJP, the LIDHS rat model was established through imitating the inducing conditions of LIDHS and treated with YJP. The integrity of ileal and colonic mucosa was detected through histopathological examination. The serum DAO, D-LA and ET levels were detected by ELISA. The mRNA and protein expression levels of Occludin, ZO-1 and MUC2 in ileum and colon were detected using RT-PCR and immunohistochemistry methods, respectively. The results showed that the ileal and colonic epithelium of LIDHS rats were destroyed; the serum DAO, D-LA and ET levels were significantly increased; the mRNA and protein expression levels of Occludin, ZO-1 and MUC2 in ileum and colon were all abnormally expressed. After treatment with YJP, the mucosal integrity was restored; the levels of serum DAO, D-LA and ET, mRNA and protein levels of Occludin and ZO-1 in ileum and colon and MUC2 in ileum were back-regulated; however, MUC2 level in colon was further increased. The results demonstrated that the intestinal mucosal barrier was damaged in LIDHS rats and Occludin, ZO-1 and MUC2 were abnormally expressed, and YJP could repair the intestinal mucosal barrier through up-regulating the expression of Occludin and ZO-1 in ileum and colon as well as MUC2 in colon and down-regulating MUC2 in ileum.

Effects of Ganoderic Acid A on Gastrointestinal Motility and Brain-Gut Peptide in Rats with Functional Dyspepsia.

Objective The therapeutic effect of drugs for functional dyspepsia (FD) is still limited. Ganoderic acid A (GAA) has anti-inflammatory and cellular protective activities. The aim of this study is to explore the therapeutic effect of GAA on FD. Methods The FD rat model was established via tail damping and forced exercise fatigue. The gastric emptying rate and intestinal propulsion rate of the rats in each group were then detected, and the pathological damage of gastric antrum and duodenum tissues was observed by hematoxylin-eosin (HE) staining. An enzyme-linked immunosorbent assay (ELISA) was conducted to determine the levels of motilin (MTL), vasoactive intestinal peptide (VIP), leptin, gastrin (GAS), calcitonin gene-related peptide (CGRP), and somatostatin (SS) in plasma, and Western blot was used to detect the protein expression levels of occludin, zonula occluden-1 (ZO-1), and junctional adhesion molecule-1 (JAM-1) in the duodenal tissue. Results Treatment with GAA significantly raised the gastric emptying rate and intestinal propulsion rate of FD rats and histologically alleviated the gastric and duodenal damage. Meanwhile, GAA positively regulated the secretion of brain-gut proteins, such as upregulation of MTL, GAS, and SS and downregulation of VIP, leptin, and CGRP. In addition, GAA treatment increased the protein expression levels of occludin, ZO-1, and JAM-1 in the duodenal tissue of the FD rats. Conclusion GAA may exhibit protective effects on FD by regulating the secretion of brain-gut peptide, protecting the intestinal barrier and improving gastrointestinal motility.

Jiawei Baitouweng Decoction affects intestinal mucosal tight junction proteins in rats with ulcerative colitis through p38 MAPK-MLCK signaling pathway

The aim of this paper was to explore the effect and mechanism of Jiawei Baitouweng Decoction(JWBTW) against ulcerative colitis(UC) from the perspective of intestinal mucosal tight junction proteins. From 60 SPF-grade male SD rats, 10 were randomly selected as the blank control, and the remaining 50 were treated with 3% dextran sodium sulfate(DSS) solution to induce UC and then randomized into the model group, mesalazine group, and low-, medium-, and high-dose JWBTW( L-JWBTW, M-JWBTW and H-JWBTW) groups, with 10 rats in each group. After successive medication for 14 days, the rat general conditions like body weight and stool were observed and the disease activity index(DAI) was calculated. The pathological changes in colon tissue was observed under a microscope for injury severity scoring and histopathological scoring. The serum endotoxin content was determined by limulus assay, followed by the measurement of protein expression levels of ZO-1, occludin, claudin-1, p38 MAPK, MLCK, MLC2 and p-MLC in colon tissue by Western blot. The results showed that compared with the blank group, the model group exhibited significantly reduced body weight, elevated DAI, injury severity and histopathological scores and serum endotoxin content, up-regulated protein expression levels of p38 MAPK, MLCK, MLC2 and p-MLC, and down-regulated ZO-1, occludin and claudin-1. Compared with the model group,mesalazine and JWBTW at each dose obviously increased the body weight, lowered the DAI, injury severity and histopathological scores and serum endotoxin content, down-regulated the protein expression levels of p38 MAPK, MLCK, MLC2 and p-MLC, and up-regulated the ZO-1, occludin and claudin-1, with the most obvious changes noticed in the H-JWBTW group. All these have indicated that JWBTW exerts the therapeutic effect against UC by inhibiting the activation of p38 MAPK/MLCK pathway, reversing the protein expression levels of occludin, claudin-1 and ZO-1, decreasing the serum endotoxin content, promoting the repair of intestinal mucosal mechanical barrier, maintaining the integrity of tight junctions, and reducing the permeability of intestinal mucosa.

Protective Effect of Conditioned Media of Human Fetal Dermal Mesenchymal Stem Cells Can Inhibit Burn-induced Microvascular Hyperpermeability.

Burns often cause loss of skin barrier protection, fluid exudation, and local tissue edema, which hinder functional recovery. Effectively improving the quality of deep burn wound healing, shortening the wound healing time, and reducing tissue fluid leakage are urgent problems in the medical field. Human mesenchymal stem cells (MSCs) can effectively stabilize vascular endothelial injury. Fetal dermal MSCs (FDMSCs) are a newly discovered source of MSCs derived from the skin of accidentally aborted fetuses. However, the effect of FDMSCs on vascular permeability remains poorly understood. In this study, conditioned media from FDMSCs (F-CM) extracted from fetal skin tissue was prepared. The effect of F-CM on vascular permeability was evaluated using the internal circulation method FITC-dextran in vivo, and several in vitro assays, including cell viability assay, transwell permeability test, immunofluorescence, and western blotting. Altogether, our results demonstrate that F-CM could inhibit burn-induced microvascular hyperpermeability by increasing the protein expression levels of occludin and VE-cadherin, while restoring the expression of endothelial F-actin, and providing the foundation of a novel therapy for the treatment of burns with F-CM.

Roles of Crosstalk between Astrocytes and Microglia in Triggering Neuroinflammation and Brain Edema Formation in 1,2-Dichloroethane-Intoxicated Mice.

We have previously reported that the activation of astrocytes and microglia may lead to the overproduction of proinflammatory mediators, which could induce neuroinflammation and cause brain edema in 1,2-dichloroethane (1,2-DCE)-intoxicated mice. In this research, we further hypothesized that astrocyte–microglia crosstalk might trigger neuroinflammation and contribute to brain edema in 1,2-DCE-intoxicated mice. The present research revealed, for the first time, that subacute intoxication with 1,2-DCE might provoke the proinflammatory polarization of microglia, and pretreatment with minocycline, a specific inhibitor of microglial activation, may attenuate the enhanced protein levels of ionized calcium-binding adapter molecule1 (Iba-1), cluster of differentiation 11b (CD11b), glial fibrillary acidic protein (GFAP), soluble calcium-binding protein 100B (S100B), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase-9 (MMP-9), Toll-like receptor 4 (TLR4), MyD88, and p-p65, and ameliorate the suppressed protein expression levels of occludin and claudin 5; we also observed changes in water content and made pathological observations on edema in the brains of 1,2-DCE-intoxicated mice. Moreover, pretreatment with fluorocitrate, an inhibitor of reactive astrocytes, could also reverse the alteration in protein expression levels of GFAP, S100B, Iba-1, CD11b, TNF-α, IL-6, iNOS, VCAM-1, ICAM-1, MMP-9, occludin, and claudin 5 in the brain of 1,2-DCE intoxicated mice. Furthermore, pretreatment with melatonin, a well-known anti-inflammatory drug, could also attenuate the above-mentioned changes in the brains of 1,2-DCE-intoxicated mice. Altogether, the findings from this research indicated that microglial activation might play an important role in triggering neuroinflammation, and hence may contribute to brain edema formation; additionally, the findings suggested that molecular crosstalk between reactive astrocytes and activated microglia may amplify the neuroinflammatory reaction, which could induce secondary brain injury in 1,2-DCE-intoxicated mice.

Effects of Qing Hua Chang Yin on lipopolysaccharide‑induced intestinal epithelial tight junction injury in Caco‑2 cells

Disruption of the intestinal mucosal barrier integrity is a pathogenic process in inflammatory bowel disease (IBD) development, and is therefore considered a drug discovery target for IBD. The well-known traditional Chinese formulation Qing Hua Chang Yin (QHCY) has been suggested as a potential therapeutic agent for the treatment of ulcerative colitis. However, the possible underlying molecular mechanisms regarding its therapeutic effect remain unclear. Consequently, the present study investigated the effects of QHCY on lipopolysaccharide (LPS)-induced loss of intestinal epithelial barrier integrity in vitro using the Caco-2 cell model of intestinal epithelium. QHCY reversed the LPS-induced decrease in transepithelial electrical resistance and significantly alleviated the increased fluorescently-labeled dextran 4 flux caused by LPS. Moreover, QHCY upregulated the mRNA and protein expression levels of occludin, zona occludens-1 and claudin-1 in LPS-exposed Caco-2 cells. In conclusion, QHCY was able to protect intestinal epithelial barrier integrity following an inflammatory insult; the protective effects of QHCY may be mediated by modulation of the expression of tight junction proteins.

Role of TLR4/NF-κB pathway in the damage of acute hypobaric hypoxia to small intestinal mucosa in rats.

The purpose of this study was to investigate the effect of acute hypobaric hypoxia (HH) exposure on small intestinal mucosa in rats and its underling mechanism. The pathological changes of rat small intestine mucosa were detected by HE staining. The expressions of TLR4 and NF-κB in the small intestine were detected by immunohistochemistry (IHC). The levels of Zonulin, TNF-α, IL-1β, and IL-6 in the serum were detected by ELISA. The proportion of natural killer (NK) cells and dendritic cell population in the small intestine was analyzed by flow cytometry. The mRNA and protein levels of TLR4, NF-κB, occludin, HIF-1α, and iNOS were detected by RT-qPCR and Western blot, respectively. Compared with the Control group, the HH groups had different degrees of injury in intestinal mucosa. Meanwhile, in the HH groups, it was also found the increased levels of Zonulin, TNF-α, IL-1β and IL-6 in the serum, the increased CD4+/CD8+T cells ratio and small intestine NK cells population, the increased mRNA and protein expression levels of small intestine TLR4, NF-κB, HIF-1α and iNOS, and the decreased mRNA and protein expression levels of occludin. Acute HH may damage the intestinal mucosa of rats by inducing TLR4/NF-κB pathway overexpression.

Effects of emodin on intestinal mucosal barrier by the upregulation of miR-218a-5p expression in rats with acute necrotizing pancreatitis

Emodin is an effective component in rhubarb to cure intestinal dysfunction, but the specific mechanism remains unknown. This study aimed to evaluate the protective effects of emodin on intestinal dysfunction caused by acute severe pancreatitis and reveal the functional mechanism of emodin in the treatment of this condition. An acute severe pancreatitis model was prepared using taurocholate. In the treatment group, 50 mg/kg emodin was injected intravenously 2 h before the induction of acute severe pancreatitis at an interval of 8 h. After 24 h, the gene expression and protein levels of miR-218a-5p, RhoA, ROCK1, Akt, Notch1, Bax, Bcl-2, Fas, FasL, caspase-3, and caspase-9 were determined through reverse transcription polymerase chain reaction and Western blot analysis. The protein levels of occludin, zonula occludens-1 (ZO-1), and E-cadherin in the intestinal tract were also determined through Western blot analysis. The effects of miR-218a-5p on the apoptosis of rat intestinal epithelial cell-18 were observed through flow cytometry. The effects of emodin on intestinal cell apoptosis induced by acute severe pancreatitis were observed via TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling). Pathological changes in the pancreas and intestine of rats in each group were observed through hematoxylin and eosin staining. After 24 h of acute severe pancreatitis induced by taurocholate, emodin reduced the expression of miR-218a-5p in the intestinal tract; increased the expression of Notch1 and Bcl-2; decreased the expression levels of RhoA, ROCK1, Akt, Bax, Fas, FasL, caspase-3, and caspase-9; inhibited the intestinal cell apoptosis caused by acute severe pancreatitis; increased the protein expression levels of occludin, zonula occludens-1 (ZO-1), and E-cadherin in the intestinal tract; and alleviated intestinal dysfunction caused by acute severe pancreatitis. Emodin could regulate Notch1 and RhoA/ROCK pathways by regulating the miR-218a-5p expression in the intestine. It could also inhibit intestinal cell apoptosis induced by acute severe pancreatitis and improve the intestinal dysfunction caused by severe acute pancreatitis.

Effects of cigarette smoke on barrier function and tight junction proteins in the bronchial epithelium: protective role of cathelicidin LL-37

BackgroundAirway epithelial barrier function is maintained by the formation of tight junctions (TJs) and adherens junctions (AJs). Inhalation of cigarette smoke causes airway epithelial barrier dysfunction and may contribute to the pathogenesis of chronic lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). We assessed the effects of cigarette smoke on barrier function and expression of multiple TJ and AJ proteins in the bronchial epithelium. We also examined whether treatment with glucocorticosteroids (GCSs), long-acting β2-agonists (LABAs), and human cathelicidin LL-37 can protect against cigarette smoke extract (CSE)-induced barrier dysfunction.MethodsCalu-3 cells cultured at the air-liquid interface were pretreated with or without GCSs, LABAs, GCSs plus LABAs, or LL-37, and subsequently exposed to CSE. Barrier function was assessed by transepithelial electronic resistance (TEER) measurements. Gene and protein expression levels of TJ and AJ proteins were analyzed by quantitative PCR and western blotting, respectively. Immunofluorescence staining of TJ and AJ proteins was performed.ResultsCSE decreased TEER and increased permeability in a concentration-dependent manner. CSE suppressed gene expression of claudin-1, claudin-3, claudin-4, claudin-7, claudin-15, occludin, E-cadherin, junctional adhesion molecule-A (JAM-A) and zonula occludens-1 (ZO-1) within 12 h post-CSE exposure, while suppressed protein expression levels of occludin at 12 h. CSE-treated cells exhibited discontinuous or attenuated immunostaining for claudin-1, claudin-3, claudin-4, occludin, ZO-1, and E-cadherin compared with untreated cells. GCS treatment partially restored CSE-induced TEER reduction, while LABA treatment had no effect. GCS and LABA combination treatment had no additive effect on CSE-induced TEER reduction and gene suppression of TJ and AJ proteins. Human cathelicidin LL-37 counteracted CSE-induced TEER reduction and prevented disruption of occludin and ZO-1. LL-37 also attenuated CSE-induced decreases in gene and protein expression levels of occludin.ConclusionsCSE caused airway epithelial barrier dysfunction and simultaneously downregulated multiple TJ and AJ proteins. GCS and LABA combination treatment had no additive effect on CSE-induced TEER reduction. LL-37 counteracted CSE-induced TEER reduction and prevented disruption of occludin and ZO-1. Use of LL-37 to counteract airway epithelial barrier dysfunction may have significant benefits for respiratory diseases such as asthma and COPD.

Vitamin A supplementation improves the intestinal mucosal barrier and facilitates the expression of tight junction proteins in rats with diarrhea.

The aim of this study is to investigate the specific effects of vitamin A (VA) on diarrhea in rats and its potential targets to protect the intestinal mucosa. Specific pathogen-free Sprague Dawley rats were fed a VA deficient (VAD) or VA normal (VAN) diet for 4 wk. Then, half of the VAN rats were treated with a VAN diet and the other half with a lactose VAN diet. VAD rats were randomly assigned to one of four groups and fed a VAD diet, lactose VAD diet, VAN diet with VA supplementation (VAS) via daily intragastric administration, or a lactose VAN diet with daily VAS. Rat weight and degree of diarrhea were evaluated daily. After 15 d, the serum retinol level was measured by high-performance liquid chromatography, and the serum diamine oxidase (DAO) and zonulin concentrations were analyzed by enzyme-linked immunosorbent assays. The small intestine mucosal pathology was observed by hematoxylin and eosin staining. Western blotting was performed to detect the protein expression levels of occludin and claudin-1 in the intestinal mucosa, and the zonula-occludens 1 expression was assessed using immunohistochemistry. VAD limited weight gain in rats and increased the degree of diarrhea. The serum retinol levels and the level of tight junction (TJ) proteins claudin-1 and occludin and grip strength were affected by the interaction between lactose-induced diarrhea and the VA diet. Diarrhea, independent of VAD, significantly decreased rat weight, increased serum DAO levels, damaged small intestine villi, and impaired zonula-occludens 1 protein expression. VAD significantly increased the concentration of zonulin independently of diarrhea, but VAS increased the serum retinol level, reduced the severity of diarrhea, increased the expression levels of the TJ proteins, facilitated the restoration of the small intestine villi that were damaged by the diarrhea, and decreased the concentrations of serum DAO and zonulin. VAD may aggravate the degree of diarrhea and intestinal mucosal damage during the duration of diarrhea, and VAS helps relieve diarrhea and improves intestinal damage likely by regulating the expression of TJ proteins. Therefore, VA plays a pivotal role in the protection of the intestinal mucosa during instances of diarrhea.

Astilbin from Engelhardtia chrysolepis enhances intestinal barrier functions in Caco-2 cell monolayers

Astilbin, which is one of polyphenolic compounds isolated from the leaves of Engelhardtia chrysolepis HANCE (Chinese name, huang-qui), is available as the effective component in food and cosmetics because of its anti-oxidant and anti-inflammatory effects. The tight junction (TJ) proteins, which protect the body from foreign substances, are related to adhesion between a cell and a cell. Previously, the enhancement of TJ's functions induced by aglycones of flavonoids has been demonstrated, but the effects of the glycosides such as astilbin have not been observed yet. In this study, we investigated the effects of astilbin on the TJ's functions, and human colon carcinoma Caco-2 cell monolayers were used to evaluate the effects of astilbin on transepithelial electrical resistance (TER) value and the mRNA and proteins expressions of TJ-related molecules. Astilbin increased the TER value, mRNA expression levels of claudin-1 and ZO-2, and protein expression levels of occludin and ZO-2 in Caco-2 cells. Astilbin also increased the TER value in Caco-2 cells co-stimulated with TNF-α plus IFN-γ, and moreover upregulated the protein expression of TJ-related molecules in Caco-2 cells co-treated with TNF-α plus IFN-γ. These results suggest that astilbin can enhance the expressions of TJ-related molecules, leading to upregulation of the barrier functions in the intestinal cells.

Propofol protects against blood-spinal cord barrier disruption induced by ischemia/reperfusion injury.

Propofol has been shown to exert neuroprotective effects on the injured spinal cord. However, the effect of propofol on the blood-spinal cord barrier (BSCB) after ischemia/reperfusion injury (IRI) is poorly understood. Therefore, we investigated whether propofol could maintain the integrity of the BSCB. Spinal cord IRI (SCIRI) was induced in rabbits by infrarenal aortic occlusion for 30 minutes. Propofol, 30 mg/kg, was intravenously infused 10 minutes before aortic clamping as well as at the onset of reperfusion. Then, 48 hours later, we performed histological and mRNA/protein analyses of the spinal cord. Propofol decreased histological damage to the spinal cord, attenuated the reduction in BSCB permeability, downregulated the mRNA and protein expression levels of matrix metalloprotease-9 (MMP-9) and nuclear factor-κB (NF-κB), and upregulated the protein expression levels of occludin and claudin-5. Our findings suggest that propofol helps maintain BSCB integrity after SCIRI by reducing MMP-9 expression, by inhibiting the NF-κB signaling pathway, and by maintaining expression of tight junction proteins.

Vitamin D/VDR signaling attenuates lipopolysaccharide‑induced acute lung injury by maintaining the integrity of the pulmonary epithelial barrier.

Vitamin D and its receptor have a protective effect on epithelial barriers in various tissues. Low levels of vitamin D are associated with numerous pulmonary diseases, including acute lung injury (ALI) and acute respiratory distress syndrome. The present study investigated whether the vitamin D/vitamin D receptor (VDR) pathway may ameliorate lipopolysaccharide (LPS)-induced ALI through maintaining the integrity of the alveolar epithelial barrier. This was investigated by exposing wild-type (WT) and VDR knockout C57BL/6J mice to LPS, then comparing the healthy and LPS-treated mice lungs and bronchoalveolar lavage fluid (BALF). More specifically, lung histology, mRNA levels of proinflammatory cytokines and chemokines, and protein expression levels of tight junction proteins were determined. In addition, a vitamin D analog (paricalcitol) was administered to WT mice in order to investigate the effect of vitamin D on the alveolar epithelial barrier following exposure to LPS. VDR knockout mice exhibited severe lung injuries (P<0.001), increased alveolar permeability [demonstrated by a higher wet-dry ratio of lung weight (P<0.05), greater expression levels of BALF protein (P<0.001) and fluorescein isothiocyanate-conjugated 4 kDa dextran (P<0.001) leakage into the alveolar space], elevated proinflammatory cytokine and chemokine mRNA levels, as demonstrated by reverse transcription-quantitative polymerase chain reaction (P<0.05), and decreased protein and mRNA expression levels of occludin (P<0.01) and zonula occludens-1 (ZO-1; P<0.01) compared with WT mice. Paricalcitol treatment partially inhibited these pathological changes in WT mice by maintaining the mRNA and protein expression levels of occludin (P<0.01) and ZO-1 (P<0.05). A lack of VDRs in the pulmonary epithelial barrier appeared to compromise its defense, leading to more severe LPS-induced lung injury. Furthermore, vitamin D treatment alleviated LPS-induced lung injury and preserved alveolar barrier function. Therefore vitamin D treatment may present as a potential therapeutic strategy in ALI and acute respiratory distress syndrome.

Uterine expression of tight junctions in the Canine uterus

Tight junctions (TJs) form continuous intercellular contacts in intercellular junctions. TJs involve integral proteins such as occludin (OCLN) and claudins (CLDNs) as well as peripheral proteins such as zona occludens-1 (ZO-1) and junctional adhesion molecules (JAMs). TJs control paracellular transportation across cell-to-cell junctions. Although TJs have been studied for several decades, comparison of the transcriptional-translational levels of these molecules in canine organs has not yet been performed. In this study, we examined uterine expression of CLDNs, OCLN, junction adhesion molecule-A, and ZO-1 in canine. Expression levels of canine uterine TJ proteins, including CLDN1, 2, 4, 5, JAM-A, ZO-1, and OCLN, were measured using reverse transcription PCR, real-time PCR, and Western blotting, whereas TJs distribution was determined by immunohistochemistry. The mRNA and protein expression levels of OCLN, CLDN-1, 4, JAM-1, and ZO-1 were identified in the uterus. Immunohistochemistry demonstrated that TJs were localized to the endometrium and/or myometrium of the uterus. Our results show that canine TJ proteins, including CLDNs, OCLN, JAM-A, and ZO-1, were expressed in the canine uterus. Taken together, these proteins may perform unique physiological roles in the uterus. Therefore, these findings may serve as a basis for further studies on TJ proteins and their roles in the physiological or pathological condition of the canine uterus.

The Squid Ink Polysaccharides Protect Tight Junctions and Adherens Junctions from Chemotherapeutic Injury in the Small Intestinal Epithelium of Mice

Gastrointestinal mucositis and infection by chemotherapy treatment are associated with alterations in the functioning of the intestinal barrier, due to the potential damage induced by anticancer drugs on the epithelial tight junctions and adheren junction. We aimed to study the protective effect of dietary polysaccharides on chemotherapy-induced injury in the epithelial cells. In the current study, using mice that were intraperitoneally injected with 50 mg/kg cyclophosphamide for 2 days, we reveal that polysaccharides from the ink of Ommastrephes bartrami (OBP) enhanced the mRNA and protein expression levels of Occludin, zonulae occluden (ZO)-1, and E-cadherin. Immunohistochemistry staining of ZO-1 and E-cadherin confirmed the increase in the mRNA and protein levels. OBP also remarkably enhanced the mRNA expression of other tight junction proteins, ZO-2, ZO-3, claudin-2, and cingulin. Our results may have important implications in host defense, especially the immunopotentiation function of OBP on the cyclophosphamide-induced epithelial cell injury, as well as intestinal disorders involving inflammation and infection.

Seasonal changes in gastric mucosal factors associated with peptic ulcer bleeding.

A close association has been established between climate and peptic ulcer bleeding (PUB). The incidence of PUB in cold climates is significantly higher than that in hot climates. In this study, gastric mucosal damage and its barrier function (through associated barrier factors) in extreme climate conditions were examined to investigate the pathogenesis of PUB in extreme cold climates. Gastric juice and biopsy specimens were collected from 176 patients with peptic ulcer. Conventional hematoxylin and eosin staining was used to exclude malignant ulcers. Helicobacter pylori infections were detected by modified Giemsa staining. pH values of the gastric juice samples were obtained on-site by precise pH dipstick readings. The protein expression levels of heat shock protein (HSP) 70, occludin, nitric oxide synthase (NOS), epidermal growth factor (EGF) and EGF receptor (EGFR) in the gastric mucosa were detected by immunohistochemistry. No significant differences were identified between the high and low bleeding risk groups in the rates of H. pylori infection and the pH values of the gastric juices in the extreme hot or cold climates. Furthermore, no statistically significant differences were identified in the protein expression levels of occludin, NOS, EGF and EGFR between the high and low bleeding risk groups. In the extreme cold climate, the expression of HSP70 and the mucus thickness of the gastric antrum in the high bleeding risk group were significantly lower than those in the low bleeding risk group. The protein expression levels of occludin, HSP70, NOS and EGFR in the extreme cold climate were significantly lower than those in the extreme hot climate, whereas the gastric acid secretion was significantly higher in the extreme cold climate than that in the extreme hot climate. In conclusion, low expression of HSP70 in the gastric mucosa and reduced gastric mucus thickness may play key roles in the mechanism of PUB in extreme cold climates. The significant decrease in barrier factors and increase in damage in extreme cold climates may be associated with the seasonal pattern of peptic ulcers.

Expressions of tight junction proteins Occludin and Claudin-1 are under the circadian control in the mouse large intestine: implications in intestinal permeability and susceptibility to colitis.

Background & AimsThe circadian clock drives daily rhythms in behavior and physiology. A recent study suggests that intestinal permeability is also under control of the circadian clock. However, the precise mechanisms remain largely unknown. Because intestinal permeability depends on tight junction (TJ) that regulates the epithelial paracellular pathway, this study investigated whether the circadian clock regulates the expression levels of TJ proteins in the intestine.MethodsThe expression levels of TJ proteins in the large intestinal epithelium and colonic permeability were analyzed every 4, 6, or 12 hours between wild-type mice and mice with a mutation of a key clock gene Period2 (Per2; mPer2m/m). In addition, the susceptibility to dextran sodium sulfate (DSS)-induced colitis was compared between wild-type mice and mPer2m/m mice.ResultsThe mRNA and protein expression levels of Occludin and Claudin-1 exhibited daily variations in the colonic epithelium in wild-type mice, whereas they were constitutively high in mPer2m/m mice. Colonic permeability in wild-type mice exhibited daily variations, which was inversely associated with the expression levels of Occludin and Claudin-1 proteins, whereas it was constitutively low in mPer2m/m mice. mPer2m/m mice were more resistant to the colonic injury induced by DSS than wild-type mice.ConclusionsOccludin and Claudin-1 expressions in the large intestine are under the circadian control, which is associated with temporal regulation of colonic permeability and also susceptibility to colitis.