Claudins, which are known as transmembrane proteins play an essential role in tight junctions (TJs) to form physical barriers and regulate paracellular transportation. To understand equine diseases, it is helpful to measure the tissue-specific expression of TJs in horses. Major equine diseases such as colic and West Nile cause damage to TJs. In this study, the expression level and distribution of claudin-1, -2, -4, and -5 in eight tissues were assessed by Western blotting and immunohistochemistry methods. Claudin-1 was primarily identified in the lung, duodenum, and uterus, claudin-2 was evenly observed in equine tissues, claudin-4 was abundantly detected in the liver, kidney and uterus, and claudin-5 was strongly expressed in the lung, duodenum, ovary, and uterus, as determined by Western blotting method. The localization of equine claudins was observed by immunohistochemistry methods. These findings provide knowledge regarding the expression patterns and localization of equine claudins, as well as valuable information to understand tight junction-related diseases according to tissue specificity and function of claudins in horses.
Animals ; Architectural Accessibility ; Blotting, Western ; Claudin-1* ; Claudin-2 ; Claudin-4 ; Claudin-5 ; Claudins ; Colic ; Duodenum ; Female ; Horse Diseases ; Horses ; Immunohistochemistry ; Kidney ; Liver ; Lung ; Methods ; Organ Specificity ; Ovary ; Tight Junctions ; Transportation ; Uterus

OBJECTIVETo investigate the role of cyclic adenosine monophosphate(cAMP) in the regulation of intestinal epithelial barrier function under hypoxia.

METHODSIntestinal epithelial barrier was established by Caco-2 monolayers. Cells were divided into four groups: normoxia (Nx), normoxia plus Forskolin(Nx+FSK), hypoxia(Hx), hypoxia plus SQ22536(Hx+SQ22536). cAMP concentrations of different groups were assessed by cAMP enzyme immunoassay kit. RT-PCR and Western blotting were used to detect the mRNA and protein expressions of claudin-1 and occludin under normoxic and hypoxic condition. Caco-2 monolayers were grown on Millicell filters, and transepithelial electrical resistance(TER) was measured using a Millipore electric resistance system.

RESULTSThe concentration of cAMP under hypoxic conditions(Hx group) was higher compared with Nx group [(6.30±0.50) pmol/L vs. (2.38±0.18) pmol/L, P<0.01]. At the same time, both mRNA and protein expressions of claudin-1 and occluding were lower in Hx group than those in Nx group(all P<0.05). TER decreased by 76.30±0.64(P<0.01). When the monolayers were exposed to hypoxia plus SQ22536 (Hx+SQ22536 group), the concentration of cAMP was(2.12±0.23) pmol/L, which was lower than that under hypoxic conditions(Hx group, P<0.01). Both mRNA and protein expressions of claudin-1 and occludin were higher compared to Hx group (all P<0.01). TER increased by 32.96±2.16 (P<0.05).

CONCLUSIONWhen Caco-2 cells are exposed to hypoxia, barrier function, claudin-1 and occludin expression are diminished in parallel with a high level of intracellular cAMP compared with the normoxic condition. Inhibition of the intracellular cAMP level under hypoxia can maintain the intestinal epithelial function through regulating the claudin-1 and occludin expression and attenuate the permeability of intestinal mucosa.

Adenosine Monophosphate ; Caco-2 Cells ; Claudin-1 ; metabolism ; Humans ; Intestinal Mucosa ; metabolism ; Intestines ; Occludin ; metabolism

BACKGROUND/AIMS: Postoperative ileus (POI) is characterized by impaired propulsive function of the gastrointestinal tract after surgery. Although inflammation is considered to be an important pathogenesis of POI, significant data are lacking. We aim to correlate the recovery time of postoperative dysmotility with that of inflammation and mucosal permeability. METHODS: An experimental POI model of guinea pig was used. Contractile activity of the circular muscle of the stomach, jejunum, ileum, and proximal colon was measured through a tissue bath study. Inflammatory cells were counted, and the expression of calprotectin and tryptase were analyzed. The expression of protease-activated receptor 2 (PAR-2), claudin-1, and claudin-2 were analyzed with immunofluorescence. RESULTS: The small bowel and colon showed decreased contractile amplitude in the POI groups compared to control. In contrast to the colon, the contractile amplitude of the small bowel significantly recovered in the POI group at 6 hours after the operation compared to the control group. Inflammation was highly significant in the POI groups compared to the control and sham groups, especially in the colon. Immunofluorescence showed increased PAR-2 expression in the POI groups compared to sham. The decreased claudin-1 expression and increased claudin-2 expression may suggest increased mucosal permeability of the small bowel and colon in the POI groups. CONCLUSIONS: Increased inflammation and mucosal permeability may play an important role in the differential recovery stages in POI. These data may provide further insights into the pathophysiology and potential new therapeutic prospects of POI.
Animals ; Baths ; Claudin-1 ; Claudin-2 ; Colon ; Fluorescent Antibody Technique ; Gastrointestinal Tract ; Guinea Pigs ; Guinea ; Ileum ; Ileus ; Inflammation ; Jejunum ; Leukocyte L1 Antigen Complex ; Permeability ; Receptor, PAR-2 ; Stomach ; Tryptases

BACKGROUND/AIMS: Detailed characterization of the ultrastructural morphology of intercellular space in gastroesophageal reflux disease has not been fully studied. We aimed to investigate whether subtle alteration in intercellular space structure and tight junction proteins might differ among patients with gastroesophageal reflux disease. METHODS: Esophageal biopsies at 5 cm above the gastroesophageal junction were obtained from 6 asymptomatic controls, 10 patients with reflux symptoms but without erosions, and 18 patients with erosions. The biopsies were morphologically evaluated by transmission electron microscopy, and by using immunohistochemistry for tight junction proteins (claudin-1 and claudin-2 proteins). RESULTS: The expressions of tight junction proteins did not differ between asymptomatic controls and gastroesophageal reflux disease patients. In patients with gastroesophageal reflux disease, altered desmosomal junction morphology was only found in upper stratified squamous epithelium. Dilated intercellular space occurred only in upper stratified squamous epithelium and in patients with erosive esophagitis. CONCLUSIONS: This study suggests that dilated intercellular space may not be uniformly present inside the esophageal mucosa and predominantly it is located in upper squamous epithelium. Presence of desmosomal junction alterations is associated with increased severity of gastroesophageal reflux disease. Besides dilated intercellular space, subtle changes in ultrastructural morphology of intercellular space allow better identification of inflamed esophageal mucosa relevant to acid reflux.
Biopsy ; Claudin-2 ; Epithelium ; Esophagogastric Junction ; Esophagus ; Extracellular Space ; Gastroesophageal Reflux ; Humans ; Immunohistochemistry ; Intercellular Junctions ; Microscopy, Electron, Transmission ; Mucous Membrane ; Tight Junction Proteins ; Tight Junctions

OBJECTIVETo observe the effect of SIRT1 on intestinal barrier function of epithelial Caco-2 cells under hypoxia and investigate its mechanism.

METHODSCaco-2 cells were randomly divided into three groups: normoxia group (Nx), hypoxia group (Hx,1%O2 for 6 h) and hypoxia plus 40 μmol/L Resveratrol (agonist of SIRT1) group (Hx+Res). Transepithelial electrical resistance (TER) was determined. mRNA and protein expressions of SIRT1 and tight junctions (ZO-1, Occludin, Claudin-1) were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.

RESULTSBoth mRNA and protein expressions of SIRT1 were significantly reduced in Hx group as compared with Nx group (0.40±0.02 vs. 0.70±0.07, P=0.001; 0.37±0.03 vs. 0.76±0.03, P=0.001). The mRNA and protein expressions of SIRT1 were significantly increased in Hx+Res group as compared with Hx group(0.50±0.02 vs. 0.40±0.02, P=0.026; 0.54±0.02 vs. 0.37±0.03, P=0.011). The expression levels of ZO-1, Occludin and Claudin-1 in Hx group were lower than those in Nx group (P<0.05), however, pretreatment with Resveratrol could attenuate the decreased expression of above 3 molecules under hypoxia(P<0.05). TERs of Nx group, Hx group and Hx+Res group were (142±7) Ohm/cm(2), (94±3) Ohm/cm(2) and (119±7) Ohm/cm(2) respectively. Compare with the Nx group, the TER of Hx group was significantly decreased(P<0.05). TER of Hx+Res group was significantly increased compare with Hx group, but it was still significantly lower than that in Nx group(P<0.05).

CONCLUSIONSExpression of SIRT1 is significantly reduced under hypoxia. Activation of SIRT1 can maintain the epithelial barrier function through regulating the expression of tight junctions under hypoxia.

Caco-2 Cells ; Cell Hypoxia ; Claudin-1 ; metabolism ; Epithelial Cells ; metabolism ; Humans ; Intestinal Mucosa ; cytology ; Occludin ; metabolism ; Sirtuin 1 ; metabolism ; Zonula Occludens-1 Protein ; metabolism

OBJECTIVETo examine changes in expression of tight junction protein claudin-2 in the renal tissues of children with acute kidney injury (AKI), and to investigate the relationship of claudin-2 expression with renal pathological lesion and renal functional lesion.

METHODSTwenty-four children who were diagnosed with AKI and had renal biopsies between December 2009 and December 2011 were included in the study. These patients were divided into mild AKI (n=7) and severe AKI groups (n=17). Children with isolated hematuria whose renal biopsy showed minor glomerular lesion were selected as the control group. Serum creatinine levels were measured by automatic biochemical analyzer. Tubulointerstitial damage was evaluated by renal pathological scores and expression of claudin-2 was examined by immunohistochemistry. The correlations of claudin-2 expression with renal pathological score and serum creatinine level were assessed by Pearson correlation analysis.

RESULTSThe mild and severe AKI groups had significantly higher serum creatinine levels than the control group (190 ± 68 μmol/L and 477 ± 128 μmol/L vs 29 ± 7 μmol/L, P<0.01), and the severe AKI group had a significantly higher serum creatinine level than the mild AKI group (P<0.01). The tubulointerstitial damage score was significantly lower in the mild AKI group than in the severe AKI group (10.4 ± 1.7 vs 14.0 ± 1.5; P<0.05). The mild and severe AKI groups had significantly smaller areas of claudin-2 expression than the control group (5.0 ± 0.5% and 3.7 ± 0.7% vs 8.0 ± 0.7%; P<0.01), and the severe AKI group had a significantly smaller area of claudin-2 expression than the mild AKI group (P<0.01). The area of claudin-2 expression was negatively correlated with serum creatinine level and tubulointerstitial damage score (r=-0.809 and -0.903; P<0.01).

CONCLUSIONSThere are changes in the distribution and expression of claudin-2 in proximal tubular epithelial cells among children with AKI, and claudin-2 expression is closely related to renal pathological lesion and renal functional lesion.

Acute Kidney Injury ; metabolism ; pathology ; physiopathology ; Child ; Child, Preschool ; Claudin-2 ; analysis ; Creatinine ; blood ; Female ; Humans ; Infant ; Kidney ; chemistry ; pathology ; Male

OBJECTIVE: To investigate whether ferroptosis exists in sepsis induced intestinal injury, and to verify the association between ferroptosis in sepsis induced intestinal injury and intestinal inflammation and barrier function by stimulating and inhibiting the nuclear factor E2-related factor 2/glutathione peroxidase 4 (Nrf2/GPX4) pathway. METHODS: Forty-eight SPF grade male Sprague-Darvley (SD) rats with a body weight of 220-250 g were divided into sham operation group (Sham group), sepsis group (CLP group), sepsis+iron chelating agent deferoxamine (DFO) group (CLP+DFO group) and sepsis+ferroptosis inducer Erastin group (CLP+Erastin group) using a random number table method, with 12 rats in each group. The sepsis model was established by cecal ligation and puncture (CLP). The Sham group was only performed with abdominal opening and closing operations. After modeling, the CLP+DFO group received subcutaneous injection of 20 mg/kg of DFO, the CLP+Erastin group was intraperitoneally injected with 20 mg/kg of Erastin. Each group received subcutaneous injection of 50 mg/kg physiological saline for fluid resuscitation after surgery, and the survival status of the rats was observed 24 hours after surgery. At 24 hours after model establishment, 6 rats in each group were selected. First, live small intestine tissue was taken for observation of mitochondrial morphology in smooth muscle cells under transmission electron microscopy and determination of reactive oxygen species (ROS). Then, blood was collected from the abdominal aorta and euthanized. The remaining 6 rats were sacrificed after completing blood collection from the abdominal aorta, and then small intestine tissue was taken. Western blotting was used to detect the expression of intestinal injury markers such as Claudin-1 and ferroptosis related proteins GPX4 and Nrf2. Observe the pathological changes of small intestine tissue using hematoxylin-eosin (HE) staining and complete Chiu score; Detection of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, IL-6) levels in serum using enzyme-linked immunosorbent assay (ELISA). The levels of serum iron ions (Fe³⁺), malondialdehyde (MDA), and D-lactate dehydrogenase (D-LDH) were measured. RESULTS: (1) Compared with the Sham group, the 24-hour survival rate of rats in the CLP group and CLP+Erastin group significantly decreased (66.7%, 50.0% vs. 100%, both P < 0.05), while there was no significant difference in the CLP+DFO group (83.3% vs. 100%, P = 0.25). (2) Western blotting results showed that compared with the Sham group, the expressions of GPX4 and Claudin-1 in the small intestine tissue of the CLP group, CLP+DFO group, and CLP+Erastin group decreased significantly, while the expression of Nrf2 increased significantly (GPX4/β-actin: 0.56±0.02, 1.03±0.01, 0.32±0.01 vs. 1.57±0.01, Claudin-1/β-actin: 0.60±0.04, 0.96±0.07, 0.41±0.01 vs. 1.40±0.01, Nrf2/β-actin: 0.88±0.02, 0.72±0.01, 1.14±0.01 vs. 0.43±0.02, all P < 0.05). Compared with the CLP group, the expressions of GPX4 and Claudin-1 were significantly increased in the CLP+DFO group, while the expression of Nrf2 was significantly reduced. In the CLP+Erastin group, the expressions of GPX4 and Claudin-1 further decreased, while the expression of Nrf2 further increased (all P < 0.05). (3) Under the light microscope, compared with the Sham group, the CLP group, CLP+DFO group, and CLP+Erastin group showed structural disorder in the small intestinal mucosa and submucosal tissue, significant infiltration of inflammatory cells, and destruction of glandular and villous structures. The Chui score was significantly higher (3.25±0.46, 2.00±0.82, 4.50±0.55 vs. 1.25±0.45, all P < 0.05). (4) Under transmission electron microscopy, compared with the Sham group, the mitochondria in the other three groups of small intestinal smooth muscle cells showed varying degrees of volume reduction, increased membrane density, and reduced or disappeared cristae. The CLP+Erastin group showed the most significant changes, while the CLP+DFO group showed only slight changes in mitochondrial morphology. (5) Compared to the Sham group, the CLP group, CLP+DFO group, and CLP+Erastin group had serum levels of TNF-α, IL-1β, IL-6, MDA, D-LDH, and ROS in small intestine tissue were significantly increased, while the serum Fe³⁺ content was significantly reduced [TNF-α (ng/L): 21.49±1.41, 17.24±1.00, 28.66±2.72 vs. 14.17±1.24; IL-1β (ng/L): 108.40±3.09, 43.19±8.75, 145.70±11.00 vs. 24.50±5.55; IL-6 (ng/L): 112.50±9.76, 45.90±6.52, 151.80±9.38 vs. 12.89±6.11; MDA (μmol/L): 5.61±0.49, 3.89±0.28, 8.56±1.17 vs. 1.86±0.41; D-LDH (kU/L): 39.39±3.22, 25.38±2.34, 53.29±10.53 vs. 10.79±0.52; ROS (fluorescence intensity): 90 712±6 436, 73 278±4 775, 110 913±9 287 vs. 54 318±2 226; Fe³⁺ (μmol/L): 22.19±1.34, 34.05±1.94, 12.99±1.08 vs. 51.74±11.07; all P < 0.05]. Compared with CLP group, the levels of TNF-α, IL-1β, IL-6, MDA, D-LDH and ROS in CLP+Erastin group were further increased, and the content of Fe³⁺ was further decreased, the CLP+DFO group was the opposite (all P < 0.05). CONCLUSIONS Ferroptosis exists in the intestinal injury of septic rats, and stimulating or inhibiting ferroptosis through the Nrf2/GPX4 pathway can effectively intervene in the inflammatory state and intestinal mechanical barrier of the body.
Rats ; Male ; Animals ; NF-E2-Related Factor 2 ; Tumor Necrosis Factor-alpha ; Ferroptosis ; Reactive Oxygen Species ; Actins ; Claudin-1 ; Interleukin-6 ; Sepsis/metabolism* ; Iron

PURPOSE: The purpose of the present study was to assess the biological effects of TNF-alpha in Caco-2 well-differentiated colon adenocarcinoma cells and to determine radiation sensitivity in order to develop TNF-alpha into a cancer therapeutic agent. MATERIALS AND METHODS: A cell viability test was conducted via a colorimetric and colony forming assay after 1 day and 3 days of incubation with TNF-alpha. Western blotting analysis and immunofluorescence staining were conducted to explore TNF-alpha-induced morphological and molecular changes in the adhesion molecules, E-cadherin and claudin-4. The effects of gamma-irradiation at a dose of 2 Gy on cell survival were evaluated by a clonogenic assay. The molecular changes in apoptosis-regulatory proteins were assessed by Western blotting. RESULTS: Caco-2 cells were highly resistant to TNF alpha-induced cell death and 2 Gy of gamma-irradiation. However, we observed the downregulation of the adherens junctional protein, E-cadherin and translocation of tight junctional protein, claudin-4 from the membrane to the cytosol induced by TNF-alpha treatment which would indicate cell-cell junction disruptions. These alterations of junctional proteins influenced the regulation of cell death in response to 2 Gy of gamma-irradiation. The combined treatment of TNF-alpha with 2 Gy of gamma-irradiation reduced the survival of Caco-2 cells by down-regulating bcl-xl and activating JNK pathways. CONCLUSION: These results suggest that TNF-alpha might be potentially applied as a therapeutic agent in order to enhance sensitivity to 2 Gy of gamma-irradiation administered in radiotherapy for the treatment of human colon cancer.
Adenocarcinoma ; Blotting, Western ; Caco-2 Cells ; Cadherins ; Cell Death ; Cell Survival ; Claudin-4 ; Colon ; Colonic Neoplasms ; Cytosol ; Down-Regulation ; Fluorescent Antibody Technique ; Humans ; MAP Kinase Signaling System ; Membranes ; Proteins ; Radiation Tolerance ; Tumor Necrosis Factor-alpha

OBJECTIVETo study the effect of hypoxia on Slingshot protein expression in human intestinal epithelial cell and its relation with changes in barrier function of the cells.

METHODSThe human intestinal epithelial cell line Caco-2 was used to reproduce monolayer-cells. One portion of the monolayer-cell specimens were divided into six parts according to the random number table, and they were respectively exposed to hypoxia for 0 (without hypoxia), 1, 2, 6, 12, and 24 h. Transepithelial electrical resistance (TER) was determined with an ohmmeter. Another portion of the monolayer-cell specimens were exposed to hypoxia as above. Western blotting was used to detect the protein expressions of zonula occludens 1 (ZO-1), occludin, claudin-1, Slingshot-1, Slingshot-2, and Slingshot-3. The remaining portion of the monolayer-cell specimens were also exposed to hypoxia as above. The content of fibrous actin (F-actin) and globular actin (G-actin) was determined by fluorescence method. The sample number of above-mentioned 3 experiments was respectively 10, 10, and 18 at each time point. Data were processed with one-way analysis of variance and Dunnett test.

RESULTS(1) Compared with that of cells exposed to hypoxia for 0 h, TER of cells exposed to hypoxia for 1 to 24 h was significantly reduced (P values below 0.01). (2) Compared with those of cells exposed to hypoxia for 0 h (all were 1.00), the protein expressions of ZO-1, occludin, and claudin-1 of cells exposed to hypoxia for 1 to 24 h were generally lower, especially those of cells exposed to hypoxia for 12 h or 24 h (respectively 0.69 ± 0.20, 0.47 ± 0.15, and 0.47 ± 0.22, P<0.05 or P<0.01). Compared with those of cells exposed to hypoxia for 0 h, the protein expressions of Slingshot-1 and Slingshot-3 of cells exposed to hypoxia for 1 to 24 h were not obviously changed (P values above 0.05). The protein expression of Slingshot-2 of cells was decreased at first and then gradually increased from hypoxia hour 1 to 24. The protein expression of Slingshot-2 of cells exposed to hypoxia for 24 h (1.54 ± 0.57) was significantly higher than that of cells exposed to hypoxia for 0 h (1.00, P<0.05). (3) Compared with those of cells exposed to hypoxia for 0 h, the content of F-actin of cells exposed to hypoxia for 1, 6, 12, and 24 h was significantly decreased, whereas the content of G-actin of cells exposed to hypoxia for 6-24 h was significantly increased, P<0.05 or P<0.01; the content of F-actin and G-actin of cells exposed to hypoxia for the other time points was not obviously changed (P values above 0.05).

CONCLUSIONSHypoxia may cause cofilin activation after dephosphorylation and the depolymerization of F-actin by inducing Slingshot-2 protein expression, which in turn affects the tight junction of human intestinal epithelial cells, thus leading to deterioration of barrier function of these cells.

Actins ; metabolism ; Blotting, Western ; Caco-2 Cells ; Cell Hypoxia ; Claudin-1 ; metabolism ; Epithelial Cells ; cytology ; metabolism ; Humans ; Intestines ; cytology ; Occludin ; metabolism ; Phosphoprotein Phosphatases ; metabolism ; Tight Junctions ; metabolism ; Zonula Occludens-1 Protein ; metabolism

PURPOSE: Protease-activated receptor 2 (PAR2) reportedly triggers the immune response in allergic asthma. We aimed to investigate the mechanism on allergic inflammation mediated by PAR2. METHODS: Human lung epithelial cells (A549 cells) were used for in vitro, and the German cockroach extract (GCE)-induced mouse model was developed for in vivo studies. RESULTS: In A549 cells, the levels of reactive oxygen species (ROS) and thymic stromal lymphopoietin (TSLP) were significantly increased by GCE treatment, but were suppressed by PAR2-antagonist (PAR2-ant) or N-acetylcysteine (NAC) treatment. Claudin-1 was degraded by GCE, and was restored by PAR2-ant or NAC in the cells. In the mouse model, the clinical appearance including bronchial hyperresponsiveness, bronchoalveolar lavage fluid analysis and total immunoglobulin E were significantly suppressed by PAR2-ant or NAC. Moreover, TSLP levels in the lung were suppressed by the same treatments in the lung. Claudin-1 was also degraded by GCE, and was restored by PAR2-ant or NAC. CONCLUSIONS: ROS generation and epidermal tight junction degradation are triggered by protease, followed by the induction of TSLP in allergic asthma. Our findings could suggest that PAR2-ant or anti-oxidants could be considered for allergic diseases as preventive alternatives.
Acetylcysteine ; Animals ; Asthma ; Blattellidae ; Bronchoalveolar Lavage Fluid ; Claudin-1 ; Epithelial Cells ; Humans ; Immunoglobulin E ; Immunoglobulins ; In Vitro Techniques ; Inflammation ; Lung ; Mice ; Oxygen ; Reactive Oxygen Species ; Receptor, PAR-2 ; Receptors, Proteinase-Activated ; Tight Junctions