Objective: To study the mechanisms of cold exposure mediated ileum mechanical barrier injury in mice. Methods: Twenty mice were randomly divided into the control and cold exposure groups. Both the control and cold exposure groups were placed in the climate room with (24±2)℃ and 40% humidity. The mice in the cold exposure group were moved to the climate room at (4±2)℃ every day for 3 hours for three consecutive weeks. Three weeks later, the ileum tissues of mice were collected. Changes in ileum tissue structure were observed by hematoxylin-eosin staining and Masson staining. The related protein expression levels of the tight junction, inflammatory cytokines, and the NF-κB pathway were detected by Western blot. Results: Compared with the control group, the circular muscle layer of the ileum in cold exposed mice became thin, a large number of inflammatory cells infiltrated, the length of villi became short, the depth of recess was increased, and tissue fibrosis appeared. The expression levels of ideal tight junction-associated proteins in cold exposed mice were decreased significantly (P＜0.05), while the protein expression levels of IL-1β, IL-6 and phosphorescent p65 were increased significantly (P＜0.05). Conclusion: Cold exposure can damage the tight junction of the mouse ileum, destroy the integrity of the mechanical barrier and activate the NF-κB signaling pathway to promote the occurrence of the inflammatory response.
Animals ; Cytokines/metabolism* ; Ileum/metabolism* ; Intestinal Mucosa ; Mice ; NF-kappa B/metabolism* ; Tight Junctions/metabolism*

OBJECTIVETo investigate the expression of occludin, ZO-1, MMP-2, and MMP-9 in cerebral microvasculature following Pulse Electromagnetic Field (PEMF) induced BBB permeability change.

METHODSSprague-Dawley rats were randomized into PEMF and sham exposed groups (n = 8). After exposure to PEMF at 0.5, 1, 3, 6, and 12 h, BBB permeability was measured by Evans-Blue extravasation. The expression of occludin, ZO-1, MMP-2, and MMP-9 were detected by real-time quantitative reverse transcriptase PCR and western blotting. MMP-2 and MMP-9 activity were detected by EnzChek gelatinase assay.

RESULTSCompared with the sham group, PEMF exposure led to increased permeability of the BBB to EB, which was prolonged after exposure. BBB permeability became progressively more severe, and recovered at 6 h. The gene and protein expression of occludin and ZO-1 were significantly decreased, while MMP-2 and MMP-9 expression were significantly increased after exposure to PEMF. All levels of expression recovered 12 h following PEMF.

CONCLUSIONChanges to BBB permeability were related to the alteration expression of tight junction proteins and matrix metalloproteinase after exposure to PEMF.

Animals ; Blood-Brain Barrier ; Electromagnetic Fields ; Male ; Matrix Metalloproteinases ; metabolism ; Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tight Junctions ; metabolism

BACKGROUND: The information on nasal transport and the metabolism of peptides have been obtained from pharmacokinetic investigations in experimental animals. However, there are no transport and metabolic studies of human nasal epithelial cells. In this study, the permeability characteristics and the metabolic properties of in vitro human nasal cell monolayers were investigated. Material and METHODS: Normal human inferior nasal conchal tissue samples were obtained from patients undergoing endoscopic nasal cavitary surgery. The specimens were cultured in a transwell using an air-liquid interface (ALI) culture, and the transepithelial electrical resistance (TEER) value of the blank filter and confluent cell monolayers were measured. To determine the % leakage of mannitol, 4µmol 14C-labelled mannitol was added and the % leakage was measured every 10 minute for 1 hour. RESULT: Human nasal epithelial cells in the primary culture grew to a confluent monolayer within 7 days and expressed microvilli. The tight junction between the cells was confirmed by transmission electron microscopy. The TEER value of the blank filter, fifth day and seventh day reached 108.5 ohm.cm2, 141 ohm.cm2 and 177.5 ohm.cm2, respectively. Transcellular % leakage of the 14C-mannitol at 10, 20, 30, 40, 50 and 60 minutes was 35.67±5.43, 34.42±5.60, 32.75±5.71, 31.76±4.22, 30.96±3.49 and 29.60±3.68 %, respectively. CONCLUSION: The human nasal epithelial monolayer using ALI using techniques is suitable for a transcellular permeability study. The data suggests that human nasal epithelial cells in as ALI culture technique shows some promise for a nasal transport and metabolism study.
Animals ; Culture Techniques* ; Electric Impedance ; Epithelial Cells* ; Humans* ; Mannitol ; Metabolism ; Microscopy, Electron, Transmission ; Microvilli ; Peptides ; Permeability ; Tight Junctions

OBJECTIVE: To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD). METHODS: Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins. RESULTS: In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa. CONCLUSION Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.
Humans ; Mice ; Animals ; Caco-2 Cells ; beta Catenin/metabolism* ; Culture Media, Conditioned/pharmacology* ; Tight Junctions/metabolism* ; Intestinal Mucosa ; Inflammatory Bowel Diseases ; Tight Junction Proteins/metabolism* ; Inflammation/metabolism* ; Fibroblasts/metabolism* ; Mice, Inbred C57BL ; Glycoproteins/metabolism* ; Wnt Proteins/pharmacology* ; Frizzled Receptors/metabolism*

Severe burn injury is often accompanied by intestinal epithelial tight junction barrier dysfunction, which is believed to be closely associated with postburn shock, inflammation, hypermetabolism, infection, organ dysfunction etc. Recent studies have documented the critical role of tight junction-associated protein regulation in intestinal epithelial barrier dysfunction induced by severe burn injury. Myosin light chain (MLC) phosphorylation regulated by both myosin light chain kinase, which can phosphorylate MLC directly, and Rho-associated kinase, which can inhibit MLC phosphatase and then induce MLC phosphorylation indirectly, play a critical role in intestinal epithelial tight junction barrier dysfunction which occurs in severe burn injury. Recent advances have provided new insights into the mechanisms and the therapeutic strategies of intestinal epithelial tight junction barrier dysfunction following severe burn injury.
Burns ; metabolism ; physiopathology ; Humans ; Intestinal Mucosa ; metabolism ; physiopathology ; Myosin-Light-Chain Kinase ; metabolism ; Permeability ; Phosphorylation ; Tight Junctions ; metabolism ; physiology ; rho-Associated Kinases ; metabolism

Objective: To explore the role of TGF-β1 in the proliferation and apoptosis of Sertoli cells and its effect on the expressions of tight junction-related proteins and genes in rats. METHODS: Rat Sertoli cells were isolated in vitro, primarily cultured, and divided into groups A (blank control), B (TGF-β1 receptor blocker), C (TGF-β1), and D (TGF-β1 + receptor blocker). The proliferation and apoptosis of the cells were detected by CCK-8 and flow cytometry, respectively. After establishment of the dual-chamber model for the primary culture of Sertoli cells, the trans-epithelia electrical resistance (TER) value was measured and the relative expressions of Occludin, ZO-1 and Claudin Ⅱ determined by RT-PCR and Western blot. RESULTS: The OD value of the proliferation of the Sertoli cells was markedly higher in group C than in groups A and D (0.79 ± 0.04 vs 0.66 ± 0.05 and 0.68 ± 0.02, P<0.05), with statistically significant differences among the four groups (F = 5.05, P <0.05). However, no remarkable difference with found among the four groups in the apoptosis rate of the cells (F = 1.13, P >0.05). The TER value was dramatically decreased in group C as compared with groups A and D (［176.37 ± 16.61］ vs ［281.42 ± 9.83］ and ［254.37 ± 13.55］ /cm2, P<0.01), with statistically significant differences among the four groups (F = 38.99, P<0.01). There were no remarkable differences among the four groups in the mRNA expressions of ZO-1 and Claudin Ⅱ (F = 0.49 and 0.93, P>0.05) or their protein expressions (F = 0.28 and 1.31, P>0.05). Both the mRNA and protein expressions of Occludin were markedly lower in group C than in A and D (P<0.01 and P<0.05), with statistically significant differences among the four groups (F = 6.86 and 6.87, P<0.01). CONCLUSIONS TGF-β1 can promote the proliferation of Sertoli cells in rats and act on the tight junction of the cells by regulating the expression of Occludin.
Animals ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Claudin-2 ; metabolism ; Male ; Occludin ; metabolism ; RNA, Messenger ; Rats ; Sertoli Cells ; cytology ; physiology ; Tight Junction Proteins ; metabolism ; Tight Junctions ; genetics ; metabolism ; Transforming Growth Factor beta1 ; physiology ; Zonula Occludens-1 Protein ; metabolism

OBJECTIVETo investigate the expressions of cadherin molecules CDH18 and PCDH17 in normal and azoospermic human testes and their significance.

METHODSWe studied the routine pathological slices of normal and non-obstructive azoospermic human testis tissues for changes in the tight junction of Sertoli-germ cells, and identified the differential gene expression profiles of the normal and azoospermic testis tissues using cDNA microarrays containing multiple cadherin molecules. The results were confirmed by Western blot.

RESULTSAbnormal tight junction of the Sertoli-germ cells was observed in 37.5% of the azoospermic testis samples, and obvious changes were seen in the expressions of some cadherin molecules, with down-regulation of CDH18 and PCDH17.

CONCLUSIONCadherin molecules such as CDH18 and PCDH17 may play a certain role in the development and progression of azoospermia, which might be related with the abnormal tight junction of the Sertoli-germ cells.

Adult ; Azoospermia ; metabolism ; Cadherins ; metabolism ; Cells, Cultured ; Down-Regulation ; Gene Expression Profiling ; Humans ; Male ; Oligonucleotide Array Sequence Analysis ; Sertoli Cells ; metabolism ; Testis ; cytology ; metabolism ; Tight Junctions ; Young Adult

OBJECTIVETo analyze the distribution and significance of occludin mRNA expression in human gastric cancer, as well as its relationship with gastric cancer pathology and multidrug resistance (MDR) in vivo.

METHODSIn situ hybridization (ISH) technique was used to evaluate the expression of occludin mRNA in 42 gastric carcinoma specimens obtained by surgery and 23 relatively normal gastric mucosa obtained by gastric endoscopy. All specimens had been stored in cryostatic section.

RESULTSOccludin mRNA was found positive in the cytoplasm of gastric glandulous epithelia as blue particles with intensive stain in 14 of 42 gastric carcinomas (33.3%), 23 of 42 paracancerous gastric tissues (54.8%), 14 of 23 relatively normal gastric tissues (60.9%), 9 of 16 well differentiated carcinomas (56.3%), 4 of 14 moderately differentiated carcinomas (28.6%), 1 of 10 poorly differentiated carcinomas (10.0%) and none of 2 mucosal carcinomas. There were significant differences in occludin mRNA positive rate between relatively normal gastric tissue and gastric cancer as well as between paracancerous gastric tissue and gastric cancer. The expression of occludin mRNA in moderately and poorly differentiated groups was gradually reduced when compared with well differentiated group, which suggests that there be a significant correlation between tumor differentiation and the expression of occludin mRNA. Furthermore, the positive signals of occludin mRNA distributed extensively in the cytoplasm of SGC7901/VCR cell, being vincristine resistant, derived from parental gastric cell line SGC7901. The positive signals of SGC7901/VCR were stronger than those of SGC7901 cells.

CONCLUSIONOccludin mRNA, being mainly located in epithelial cells and its expression correlated with tumor differentiation, may be involved in the development of multi-drug resistance in gastric cancer.

Drug Resistance, Multiple ; physiology ; Drug Resistance, Neoplasm ; physiology ; Humans ; In Situ Hybridization ; Membrane Proteins ; genetics ; metabolism ; Occludin ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; metabolism ; Tight Junctions ; metabolism

This paper is aimed to investigate the effect of fluid shear stress on the tight junction of laryngeal squamous carcinoma (Hep2) cells and to explore the potential molecular mechanism. Hep2 cells were selected and subjected to the fluid shear stress of 1.4 dyn/cm2 for different time, respectively. The morphological changes of Hep2 cells under shear stress were observed using inverted microscope. The cell-cell junctions were examined by transmission electron microscope (TEM). The expressions of tight junction proteins (including Occludin, Claudin-5 and ZO-1) and the distribution of Claudin-5 were examined by Western blot assay and laser scanning confocal microscope, respectively. The results indicated that Hep2 cells turned to spindle-like shapes after exposed to shear stress, and showed the trend of the recovering to original shapes when the shear stress was cancelled. The cell-cell junctions were tight under the shear flow condition, and the permeability was reduced under the condition of 1.4 dyn/cm shear flow. The expressions of tight junction proteins were enhanced with increased duration of shear flow, but reduced after removing shear flow. The result of Claudin-5 expression by immufluorescence assay was consistent with that by Western blot. The Claudin-5 mainly distributed in the cytoplasm under static condition, while it located at the intercellular after shear flow stimulation, and it appeared intercellular and cytoplasm after stopping shear flow stimulation. Therefore, it can be concluded that shear stress changes the morphology of laryngeal squamous carcinoma Hep2 cells, and upregulates the tight junction.
Blotting, Western ; Carcinoma, Squamous Cell ; pathology ; Claudin-5 ; metabolism ; Hep G2 Cells ; Humans ; Laryngeal Neoplasms ; pathology ; Occludin ; metabolism ; Stress, Mechanical ; Tight Junctions ; Zonula Occludens-1 Protein ; metabolism

OBJECTIVE: To observe the time-course expression of zonula occludens-1 (ZO-1) in cerebral cortex after traumatic brain injury (TBI). METHODS: The TBI model of mouse was established. The mice were divided in 1 h, 3 h, 6 h, 12 h, 24 h, 3 d, 7 d after TBI, sham and control groups. The permeability of the blood brain barrier was evaluated by measuring the extravasation of Evans blue (EB) dye. The expression of ZO-1 in cerebral cortex in the injured area was detected by Western blotting and immunohistochemistry. RESULTS: The extravasation of EB dye of injured cortex gradually increased from 1 h, peaked at 1-3 d and approximately decreased to normal at 7 d after TBI. Western blotting revealed that the expression of ZO-1 gradually decreased after 1 h, was at the lowest at 1-3 d, and then significantly increased after 7 d but was still lower than that of normal and sham groups. The result of immunohistochemistry showed that ZO-1 had strong expression in vessel of normal cortex, gradually decreased after TBI, and almost disappeared at 3 d after TBI and gradually recovered to normal level later. CONCLUSION The expression of ZO-1 in the injured cortex after TBI initially decreases and then increases. The negative correlation between ZO-1 expression and EB extravasation after TBI could be used as a new indicator for wound age estimation.
Animals ; Blood-Brain Barrier ; Blotting, Western ; Brain Injuries/physiopathology* ; Cerebral Cortex/metabolism* ; Immunohistochemistry ; Mice ; Permeability ; Tight Junctions/metabolism* ; Zonula Occludens-1 Protein/metabolism*