A high-concentrate diet induces colonic inflammation and barrier damage in Hu sheep.

Long-term feeding of a high-concentrate diet can induce subacute ruminal acidosis (SARA) in ruminants, which further leads to systemic inflammatory response. However, few studies have examined the effects of feeding a high-concentrate diet on the hindgut of ruminants. The purpose of this study was to investigate the effects of a high-concentrate diet on the composition of gut microbiota in colonic contents, inflammatory response, and barrier damage in the colon tissue of ruminants. A total of 12 healthy multiparous lactating Hu sheep were randomly allotted into the following 2 groups: a high-concentrate (HC) group (concentrate:forage = 7:3) and a low-concentrate (LC) group (concentrate:forage = 3:7). All sheep were fitted with ruminal fistulas. The formal feeding experiment lasted for 8 wk. After the feeding experiment, rumen fluid, portal vein blood, hepatic vein blood, colonic contents, and colon tissue samples were collected. The results showed that feeding the HC diet induced SARA in Hu sheep and significantly reduced pH in the colonic contents. The abundances of Firmicutes, Verrucomicrobiota, and Actinobacteriota decreased significantly, whereas those of Bacteroidota, Spirochaetota, and Fibrobacterota significantly increased in colonic contents. At the genus level, the relative abundances of 29 genera were significantly altered depending on the different type of diets. Analysis of the 10 bacterial genera with high relative abundance revealed that feeding the HC diet significantly reduced the abundance of UCG-005, Christensenellaceae R-7 group, UCG-010-norank, Monoglobus, [Eubacterium] coprostanoligenes group_norank, and Alistipes, whereas the abundances of Rikenellaceae RC9 gut group, Treponema, Bacteroides, and Prevotella increased. Compared with the LC group, feeding the HC diet significantly increased the concentration of LPS in rumen fluid, portal vein blood, hepatic vein blood, and colonic contents, and significantly upregulated the mRNA expression levels of proinflammatory cytokines in colon tissue, including TNF-α, IL-1ß, IL-6, and IL-8, indicating the occurrence of inflammatory response in the colon tissue. In addition, the structure of colonic epithelial cells was loose, the intercellular space became larger, epithelial cells were exfoliated, and the mRNA and protein abundances of ZO-1, occludin, claudin-1, claudin-3, and claudin-4 were significantly decreased in the HC group, which was consistent with the results of immunohistochemistry. Furthermore, feeding the HC diet increased the ratios of DNA methylation and chromatin compaction in the promoter regions of occludin and claudin-1, which in turn inhibited their transcriptional expression. Therefore, the present study demonstrated that feeding an HC diet induced SARA in Hu sheep, altered the composition and structure of the microbial community in the colonic contents, induced an inflammatory response, and disrupted the intestinal mucosal barrier in the colonic tissue.

Tear film instability is associated with weakened colocalization between occludin and MUC5AC in scopolamine-induced dry eye disease (DED) rats.

PURPOSE: Dry eye disease (DED) is a disease with tear film instability because of multiple factors. This study was conducted to explore roles of occludin and MUC5AC in tear film instability in DED rat model. METHODS: A total of 20 SD rats were divided into DED group (n = 10) and normal control (NC) group (n = 10). DED rat model was established by subcutaneously injecting with scopolamine hydrobromide. Clinical examinations, including tear breakup time (tBUT), Schirmer's test and corneal fluorescein staining, were conducted to determine corneal functions. Transmission electron microscopy was used to measure the ultrastructures of corneal epithelial cells. Western blotting assay was used to identify occludin expression in corneal tissues of DED rats. Real-time PCR (RT-PCR) was performed to verify gene transcription of occludin and MUC5AC. Colocalization between occludin and MUC5AC was identified with confocal fluorescence microscopy. RESULTS: Tear breakup time was significantly shorter, and corneal fluorescein staining score was predominantly higher in DED rats compared to those in normal rats (P < 0.05). Normal rats showed a steady tear secretion throughout the whole experiments, while DED rats showed a dramatic reduction on day 14. DED rats demonstrated ultrastructural damage of Golgi apparatus and endoplasmic reticulum in corneal epithelial cells. Occludin and MUC5AC expressions were significantly downregulated in corneal tissue of DED rats compared with those of normal rats (P < 0.05). Percentage of occludin-MUC5AC-colocalized corneal epithelial cells in DED rats was significantly less compared with those in normal rats (P < 0.01). CONCLUSIONS: Tear film stability was damaged in scopolamine-induced DED rats because of the weakened colocalization between occludin and MUC5AC molecule. This study would provide a potential clue for the pathogenesis and a promising theoretical basis for clinical work of DED.

Regulatory role of phosphoproteins in the development of bovine small intestine during early life.

The small intestine is the primary site of nutrient digestion and absorption, which plays a key role in the survival of neonatal calves. A comprehensive assessment of the phosphoproteomic changes in the small intestine of neonatal calves is unavailable; therefore, we used phosphopeptide enrichment coupled with liquid chromatography-tandem mass spectrometry to investigate the changes in the phosphoproteome profile in the bovine small intestine during the first 36 h of life. Twelve neonatal male calves were assigned to one of the following groups: (1) calves not fed colostrum and slaughtered approximately 2 h postpartum (n = 3), (2) calves fed colostrum at 1 to 2 h and slaughtered 8 h postpartum (n = 3), (3) calves fed 2 colostrum meals (at 1-2 and 10-12 h) and slaughtered 24 h postpartum (n = 3), (4) calves fed 3 colostrum meals (at 1-2, 10-12, and 22-24 h) and slaughtered 36 h postpartum (n = 3). Mid-duodenal, jejunal, and ileal samples of the calves were collected after slaughter. We identified 1,678 phosphoproteins with approximately 3,080 phosphosites, which were mainly Ser (89.9%), Thr (9.8%), and Tyr (0.3%) residues; they belonged to the prodirected (52.9%), basic (20.4%), acidic (16.6%), and Tyr-directed (1.7%) motif categories. The regional differentially expressed phosphoproteins included zonula occludens 2, sorting nexin 12, and protein kinase C, which are mainly associated with developmental processes, intracellular transport, vesicle-mediated transport, and immune system process. They are enriched in the endocytosis, tight junction, insulin signaling, and focal adhesion pathways. The temporal differentially expressed phosphoproteins included occludin, epsin 1, and bridging integrator 1, which were mainly associated with macromolecule metabolic process, cell adhesion, and growth. They were enriched in the spliceosomes, adherens junctions, and tight junctions. The observed changes in the phosphoproteins in the tissues of small intestine suggest the protein phosphorylation plays an important role in nutrient transport and immune response of calves during early life, which needs to be confirmed in a larger study.

Tissue Content and Pattern of Expression of Claudin-3 and Occludin in Normal and Neoplastic Tissues in Patients with Colorectal Cancer.

BACKGROUND: Metastasis is the worst prognostic variable of patients with colorectal cancer (CRC). For the development of metastases, it is necessary that cancer cells detach from the primary tumor, migrate into the angiolymphatic system, and invade the tissue where they will develop. The breakdown of the tight junctions (TJs) plays an important role in colorectal metastatic tumors. Claudin-3 and occludin are the main component proteins of TJs. AIM: To analyze the expression and tissue content of claudin-3 and occludin in normal and neoplastic tissues of patients with metastatic CRC. METHODS: Fifty-seven consecutive patients with stage III and IV CRC were included. Fragments of neoplastic tissue were collected from the tumor margins, and samples of the normal tissue were collected from the same patient in a standardized distance of 10 cm from the cranial margin of the tumor. Immunohistochemistry technique was used to identify the tissue staining of claudin-3 and occludin. To measure the content of both proteins in cellular membranes of normal and cancer cells, a validated immunoscore was used. RESULTS: Claudin-3 and occludin in normal tissues are in the apical and lateral membranes of cells, while in the neoplastic, in cytoplasm. The mean of the tissue content of claudin-3 in the normal tissue was 2.57 ± 0.16, while in the neoplastic tissue was 1.03 ± 0.13. The contents of occludin were 2.77 ± 0.1 in normal tissue, while in the neoplastic were 1.08 ± 0.14. CONCLUSION: There is a reduction in the content of the claudin-3 and occludin in the cell membranes of the neoplastic tissue in patients with CRC.

The Elk1/MMP-9 axis regulates E-cadherin and occludin in ventilator-induced lung injury.

BACKGROUND: Ventilator-induced lung injury (VILI) is a common complication in the treatment of respiratory diseases with high morbidity and mortality. ETS-domain containing protein (Elk1) and Matrix metalloproteinase (MMP) 9 are involved in VILI, but the roles have not been fully elucidated. This study examined the mechanisms of the activation of MMP-9 and Elk1 regulating barrier function in VILI in vitro and in vivo. METHODS: For the in vitro study, Mouse lung epithelial cells (MLE-12) were pre-treated with Elk1 siRNA or MMP-9 siRNA for 48 h prior to cyclic stretch at 20% for 4 h. For the in vivo study, C57BL/6 mice were pre-treated with Elk1 siRNA or MMP-9 siRNA for 72 h prior to 4 h of mechanical ventilation. The expressions of Elk1, MMP-9, Tissue inhibitor of metalloproteinase 1 (TIMP-1), E-cadherin, and occludin were measured by Western blotting. The intracellular distribution of E-cadherin and occludin was shown by immunofluorescence. The degree of pulmonary edema and lung injury were evaluated by Hematoxylin-eosin (HE) staining, lung injury scores, Wet/Dry (W/D) weight ratio, total cell counts, and Evans blue dye. RESULTS: 20% cyclic stretch and high tidal volume increases the expressions of Elk1, MMP-9, and TIMP-1, increases the ratio of MMP-9/TIMP-1, decreases the E-cadherin and occludin level. Elk1 siRNA or MMP-9 siRNA reverses the degradations of E-cadherin, occludin, and the ratio of MMP-9/TIMP-1 caused by cyclic stretch. Elk1 siRNA decreases the MMP-9 level with or not 20% cyclic stretch and high tidal volume. CONCLUSIONS: The results demonstrate mechanical stretch damages the tight junctions and aggravates the permeability in VILI, Elk1 plays an important role in affecting the tight junctions and permeability by regulating the balance of MMP-9 and TIMP-1, thus indicating the therapeutic potential of Elk1 to treat VILI.

Compromised barrier integrity of human feto-placental vessels from gestational diabetic pregnancies is related to downregulation of occludin expression.

AIMS/HYPOTHESIS: Reduced occupancy of junctional occludin is a feature of human placental vessels in the diabetic milieu. However, the functional consequence of this and whether this loss is due to differential expression of occludin splice variants is not known. Our study aimed to investigate the effects of gestational diabetes mellitus (GDM), and its treatment, on endothelial junctional integrity, gene and protein expression of occludin splice variants, and potential regulation of expression by microRNAs (miRNAs). METHODS: Term placentas were obtained from normal pregnancies (n = 21), and pregnancies complicated by GDM where glucose levels were controlled by diet (n = 11) or metformin (n = 6). Gene and microRNA (miRNA) expression were determined by quantitative real-time PCR; protein expression by immunoblotting; endothelial junctional occupancy by fluorescence microscopy and systematic sampling; and paracellular leakage by perfusion of placental microvascular beds with 76 Mr dextran. Transfection studies of miRNAs that target OCLN were performed in HUVECs, and the trans-endothelial electrical resistance and tracer permeability of the HUVECs were measured. RESULTS: All three predicted OCLN gene splice variants and two occludin protein isoforms were found in human placental samples. In placental samples from diet-controlled GDM (d-GDM) pregnancies we found a lower percentage of conduit vessels showing occludin immunoreactivity (12%, p < 0.01), decreased levels of the fully functional occludin isoform-A protein (29%), and differential gene expression of OCLN variant 2 (33% decrease), variant 3 (3.3-fold increase). These changes were not seen in samples from the group with metformin-controlled GDM. In d-GDM placentas, increased numbers of conduit microvessels demonstrated extravasation of 76 Mr dextran (2.0-fold). In d-GDM expression of one of the five potential miRNAs targeting OCLN, miR-181a-5p, expression was 2.1-fold that in normal pregnancies. Experimental overexpression of miR-181a-5p in HUVECs from normal pregnancies resulted in a highly significant downregulation of OCLN variant 1 (69%) and variant 2 (46%) gene expression, with decreased trans-endothelial resistance (78%) and increase in tracer permeability (1.3-fold). CONCLUSIONS/INTERPRETATION: Downregulation of expression of OCLN variant 2 and the fully functional occludin isoform-A protein are a feature of placentas in d-GDM pregnancies. These may be behind the loss of junctional occludin and the increased extravasation of exogenous dextran observed. miR-181a-5p was in part responsible for the downregulation of occludin in placentas from d-GDM pregnancies. Induced overexpression of miR-181a-5p compromised the integrity of the endothelial barrier. Our data suggest that, despite good glucose control, the adoption of lifestyle changes alone during a GDM pregnancy may not be enough to prevent an alteration in the expression of occludin and the subsequent functional consequences in placentas and impaired vascular barrier function in offspring. Graphical abstract.

Marked Changes in Blood-Brain Barrier Biomarkers After Direct Bypass Surgery for Moyamoya Angiopathy: Preliminary Study.

OBJECTIVE: The blood-brain barrier (BBB) of patients with moyamoya angiopathy (MMA) is unstable, which may contribute to transient neurologic symptoms (TNS) after direct bypass surgery. However, BBB-related proteins have never been investigated. The purpose of this study was to evaluate the perioperative serum levels of biomarkers representing BBB function in MMA patients based on the hypothesis that postoperative hemodynamic change may disrupt the BBB. METHODS: A total of 12 hemispheres in 11 patients with MMA were prospectively examined. Direct revascularization surgery was performed for all cases. The serum levels of tight junction (occludin and claudin 5), adherens junction (vascular endothelial-cadherin) proteins, and matrix metalloproteinase (MMP)-2 and MMP-9 were measured quantitatively 1 day before surgery and on postoperative days 1, 4, and 7. RESULTS: Successful patency of the direct bypass was achieved in all. The serum level of occludin was significantly increased on postoperative day 1, and the levels in 2 cases with TNS were markedly elevated over 10-fold higher than baseline. Furthermore, the postoperative MMP-9 levels were significantly elevated on each day. On the other hand, there was no significant fluctuation in claudin 5, vascular endothelial-cadherin, and MMP-2 level. CONCLUSIONS: Marked changes in biomarkers representing the tight junction of the BBB were observed. These preliminary results suggest that marked hemodynamic change and TNS in some patients are associated with disruption of the BBB after direct bypass surgery for MMA.

Brain endothelial cell junctions after cerebral hemorrhage: Changes, mechanisms and therapeutic targets.

Vascular disruption is the underlying cause of cerebral hemorrhage, including intracerebral, subarachnoid and intraventricular hemorrhage. The disease etiology also involves cerebral hemorrhage-induced blood-brain barrier (BBB) disruption, which contributes an important component to brain injury after the initial cerebral hemorrhage. BBB loss drives vasogenic edema, allows leukocyte extravasation and may lead to the entry of potentially neurotoxic and vasoactive compounds into brain. This review summarizes current information on changes in brain endothelial junction proteins in response to cerebral hemorrhage (and clot-related factors), the mechanisms underlying junction modification and potential therapeutic targets to limit BBB disruption and, potentially, hemorrhage occurrence. It also addresses advances in the tools that are now available for assessing changes in junctions after cerebral hemorrhage and the potential importance of such junction changes. Recent studies suggest post-translational modification, conformational change and intracellular trafficking of junctional proteins may alter barrier properties. Understanding how cerebral hemorrhage alters BBB properties beyond changes in tight junction protein loss may provide important therapeutic insights to prevent BBB dysfunction and restore normal function.

Vitamin A and Retinoic Acid Exhibit Protective Effects on Necrotizing Enterocolitis by Regulating Intestinal Flora and Enhancing the Intestinal Epithelial Barrier.

BACKGROUND: Exaggerated inflammation that characterizes necrotizing enterocolitis (NEC) is caused by the invasion of pathogens through an immature intestinal barrier. Vitamin A (VA) and retinoic acid (RA) play important roles in the growth of epithelial tissue and in modulating immune function. OBJECTIVE: To investigate the roles of VA and RA in the development of NEC. METHODS: Levels of serum retinol in patients and in a NEC mouse model were detected with high-performance liquid chromatography. Bacterial communities of NEC mice treated with VA or PBS were detected by high-throughput sequencing. In vitro and in vivo, levels of inflammatory factors were measured by ELISA and RT-PCR, and expression levels of claudin-1, occludin, and ZO-1 were detected by Western blotting. Transepithelial electrical resistance (TEER) was measured in Caco-2 cell monolayers. RESULTS: The level of VA in the NEC patients was lower than in the control patients. In the NEC mice that were treated with VA versus PBS, the proportion of Escherichia-Shigella was lower, while the abundance of Bacteroides was markedly higher. Both in vivo and in vitro, the levels of inflammatory factors were significantly reduced, while the expression levels of claudin-1, occludin, and ZO-1 were increased, after the VA and RA treatments. Meanwhile, TEER was increased and lipopolysaccharide-induced damage was reduced in Caco-2 cell monolayers after RA treatment. CONCLUSIONS: These results suggest that VA may regulate intestinal flora, alleviate inflammatory reactions, and enhance the intestinal epithelial barrier in NEC. Thus, VA may be an effective drug for providing protection against NEC in newborns.

Roles of elevated 20­HETE in the breakdown of blood brain barrier and the severity of brain edema in experimental traumatic brain injury.

Breakdown of the blood brain barrier (BBB) is a secondary injury following traumatic brain injury (TBI) and can lead to the development of brain edema. However, the factors that contribute to the disruption of the BBB and increase the severity of brain edema in TBI remain to be elucidated. 20­hydroxyeicosatetraenoic acid (20­HETE) is a metabolite of arachidonic acid. The inhibition of 20­HETEsynthesis by HET0016 has been suggested as a strategy to decrease brain edema. The present study aimed to investigate whether the elevated production of 20­HETE in cerebral tissue may contribute to BBB breakdown and increase the severity of brain edema in rats with TBI. BBB permeability was quantified using dynamic contrast­enhanced magnetic resonance imaging and brain edema was measured according to brain water content. Superoxide production in injured tissue was also assessed. Liquid chromatography­mass spectrometry was used to evaluate 20­HETE production in injured tissue. Western blot analysis was used to assess the expression of occludin, zonula occludens (ZO)­1, matrix metalloproteinase (MMP)­9, and proteins of the c­Jun N­terminal kinase (JNK) pathway. A total of 3, 24 and 72 h following the induction of TBI, 20­HETE levels, BBB permeability and brain edema were identified to be increased, accompanied by an increase in superoxide production. Conversely, superoxide dismutase levels, in addition to the total antioxidative capability were decreased. In addition, the expression of MMP­9 and proteins of the JNK pathway was upregulated, whereas the expression of occludin and ZO­1 was observed to be suppressed. These results suggested that 20­HETE may aggravate BBB disruption following TBI, via enhancing the expression of MMP­9 and tight junction proteins. Furthermore, oxidative stress and the JNK signaling pathway may be involved in BBB dysregulation. In conclusion, the results of the present demonstrated that the production of 20­HETE was increased in cerebral tissue following traumatic injury, thus suggesting that it may contribute to the compromise of BBB integrity and the development of brain edema.

Quorum-sensing molecule dihydroxy-2,3-pentanedione and its analogs as regulators of epithelial integrity.

BACKGROUND AND OBJECTIVE: Quorum-sensing molecules regulate the behavior of bacteria within biofilms and at the same time elicit an immune response in host tissues. Our aim was to investigate the regulatory role of dihydroxy-2,3-pentanedione (DPD), the precursor of universal autoinducer-2 (AI-2), and its analogs (ethyl-DPD, butyl-DPD and isobutyl-DPD) in the integrity of gingival epithelial cells. MATERIAL AND METHODS: Human gingival keratinocytes were incubated with four concentrations (10 µmol L-1 , 1 µmol L-1 , 100 nmol L-1 and 10 nmol L-1 ) of DPD and its analogs for 24 hours. The numbers of viable cells were determined using a proliferation kit, matrix metalloproteinase (MMP)-2 and -9 activities were determined by gelatin zymography, and expression of occludin protein and occludin mRNA were determined by western blotting and RT-qPCR, respectively. RESULTS: Increased cell proliferation was observed in gingival keratinocytes incubated with 100 nmol L-1 of butyl-DPD. MMP-9 activity was elevated in cells incubated with 10 µmol L-1 of ethyl-DPD. On the other hand, MMP-2 activity did not show any significant change when gingival keratinocytes were incubated with or without DPD or analogs. Western blot analyses demonstrated five forms (105, 61, 52.2, 44 and 37 kDa) of occludin. Incubation with 1 µmol L-1 and 100 nmol L-1 of DPD and with 10 nmol L-1 of ethyl-DPD increased dimeric (105 kDa) forms of occludin, while incubation with 100 nmol L-1 of isobutyl-DPD increased monomeric (61 kDa) forms. DPD and ethyl-DPD decreased, and 100 nmol L-1 of isobutyl-DPD and 10 nmol L-1 of butyl-DPD increased, the monomeric (52.2 kDa and 44 kDa) forms of occludin, whereas ethyl-DPD decreased and isobutyl-DPD increased, the low-molecular-weight (37 kDa) forms. According to RT-qPCR analysis, the exposure of gingival keratinocytes to 10 µmol L-1 of isobutyl-DPD up-regulated expression of occludin. CONCLUSION: The results indicate that isobutyl-DPD has the potential to enhance the integrity of the epithelium by stimulating the formation of occluding, without affecting the proliferation or gelatinolytic enzyme activities of the exposed cells. The modulatory effect of an AI-2 analog on the epithelial cell response is shown for the first time.

Effects of Acupuncture on 1-chloro-2,4-dinitrochlorobenzene-induced Allergic Contact Dermatitis in Mice.

Allergic contact dermatitis (ACD) is a chronic inflammatory skin disease. Topical corticosteroids are the first-line therapy for ACD despite their significant adverse effects. Acupuncture has been widely used in the treatment of various skin diseases, but its underlying mechanism remains unrevealed. In this study, we investigated the characteristics of acupuncture treatment based on effectiveness and mechanism. BALB/c mice received 1-chloro-2,4-dinitrobenzene (DNCB) application to build AD-like model. Results showed that acupuncture was an effective treatment method in inhibiting inflammatory conditions, serum IgE levels, and expression of proinflammatory cytokine Th2 (IL-4, IL-6), and Th2 (IL-1ß, TNF-α) mRNA compared with DNCB treatment. Acupuncture treatment also inhibited nuclear factor-κB p65, phosphorylation of IκBα, and phosphorylation of occludin proteins expression. Furthermore, it could improve the expression of epidermal growth factor in both mRNA and protein levels. These results suggest that acupuncture, as an alternative therapy treatment for its no significant side effects, was effective in alleviating ACD by reducing proinflammatory cytokines and changing proteins' expression.

Claudin-3 and occludin content in the glands of colonic mucosa devoid from fecal stream submitted to topical intervention with oil extract of Curcuma longa.

PURPOSE:: To evaluate the inflammatory intensity and measure the tissue content of the proteins claudin-3 and occludin in the colonic mucosa without fecal stream submit to intervention with curcumin. METHODS:: Thirty-six rats were submitted to a proximal colostomy and a distal mucous fistula and divided into two groups according to sacrifice to be performed two or four weeks. Each group was divided into three subgroups according daily application of enemas containing saline, curcumin at 50 mg/kg/day or 200 mg/kg/day. Colitis was diagnosed by histological analysis. Claudin-3 and occludin were determined by immunohistochemistry. The tissue content of claudin-3 and occludin were quantified by computer-assisted image analysis. Mann-Whitney, Student t and ANOVA tests were used to analyze the results establishing the level of significance of 5% for both (p<0.05). RESULTS:: Curcumin at both concentrations reduces the inflammation and preserves the tissue content of the proteins claudin-3 and occludin, which was related to the concentration used and to the time of the intervention. CONCLUSION:: The application of enemas with curcumin reduces inflammation and preserves the tissue content of the proteins claudin-3 and occludin in the colonic mucosa devoid from the fecal stream.

Claudin-3 and occludin content in the glands of colonic mucosa devoid from fecal stream submitted to topical intervention with oil extract of Curcuma longa

Abstract Purpose: To evaluate the inflammatory intensity and measure the tissue content of the proteins claudin-3 and occludin in the colonic mucosa without fecal stream submit to intervention with curcumin. Methods: Thirty-six rats were submitted to a proximal colostomy and a distal mucous fistula and divided into two groups according to sacrifice to be performed two or four weeks. Each group was divided into three subgroups according daily application of enemas containing saline, curcumin at 50 mg/kg/day or 200 mg/kg/day. Colitis was diagnosed by histological analysis. Claudin-3 and occludin were determined by immunohistochemistry. The tissue content of claudin-3 and occludin were quantified by computer-assisted image analysis. Mann-Whitney, Student t and ANOVA tests were used to analyze the results establishing the level of significance of 5% for both (p<0.05). Results: Curcumin at both concentrations reduces the inflammation and preserves the tissue content of the proteins claudin-3 and occludin, which was related to the concentration used and to the time of the intervention. Conclusion: The application of enemas with curcumin reduces inflammation and preserves the tissue content of the proteins claudin-3 and occludin in the colonic mucosa devoid from the fecal stream.

Effects of melatonin on spinal cord injury-induced oxidative damage in mice testis.

This study evaluated the effects of melatonin on spinal cord injury (SCI)-induced oxidative damage in testes. Adult male C57BL/6 mice were randomly divided into sham-, SCI- or melatonin (10 mg/kg, i.p.)-treated SCI groups. To induce SCI, a standard weight-drop method that induced a contusion injury at T10 was used. After 1 week, testicular blood flow velocity was measured using the Laser Doppler Line Scanner. Malondialdehyde (MDA), glutathione (GSH), oxidised glutathione (GSSG) and myeloperoxidase (MPO) were measured in testis homogenates. Microvascular permeability of the testes to Evan's Blue was examined by spectrophotometric and fluorescence microscopic quantitation. The tight junction protein zonula occludens-1 (ZO-1) and occludin in testes were assessed by immunoblot analysis. Melatonin increased the reduced blood flow and decreased SCI-induced permeability of capillaries. MDA levels and MPO activity were elevated in the SCI group compared with shams, which was reversed by melatonin. In contrast, SCI-induced reductions in GSH/GSSG ratio were restored by melatonin. Decreased expression of ZO-1 and occludin was observed, which was attenuated by melatonin. Overall, melatonin treatment protects the testes against oxidative stress damage caused by SCI.

Synergistic Effect of TNF-α and Dengue Virus Infection on Adhesion Molecule Reorganization in Human Endothelial Cells.

Dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) is a severe pathological manifestation of dengue virus (DENV) infection. Enhanced production of cytokines in dengue patients is proposed to induce endothelial barrier instability resulting in increased vascular leakage. Tumor necrosis factor (TNF)-α is an inflammatory cytokine that activates endothelial cells and enhances vascular permeability and plasma leakage in DHF/DSS. The present study investigated the in vitro effect of TNF-α and DENV infection on the expression of adherence junction proteins, tight junction proteins, and membrane integrity of human endothelial cell lines. Immunofluorescence staining and western blot analysis demonstrated platelet endothelial cell adhesion molecule-1 (PECAM-1) reorganization and decreased levels of the tight junction protein occludin in human endothelial cells treated with TNF-α and DENV, compared to mock, DENV, or TNF-α-treated cells. Permeability assessed by FITC-dextran as a transport molecule was increased and correlated with the unusual reorganization of PECAM-1. The altered distribution of PECAM-1 and low occludin protein levels in human endothelial cells treated with TNF-α and DENV correlated with increased permeability. In conclusion, the synergistic effect of TNF-α and DENV induced permeability changes in endothelial cells. These results contribute to the understanding of the mechanisms underlying enhanced vascular permeability in DENV infection.

iTRAQ-Based Membrane Proteomics Reveals Plasma Membrane Proteins Change During HepaRG Cell Differentiation.

HepaRG cell, a stabilized bipotent liver progenitor cell line, exhibits hepatocyte functions only after differentiation. However, the mechanism of transition from nondifferentiated to differentiated states, accompanied by proliferation migration and differentiation, remains poorly understood, particularly those proteins residing in the plasma membrane. In this study, the membrane protein expression change of HepaRG cell during differentiation were systematically analyzed using an iTRAQ labeled quantitative membrane proteomics approach. A total of 70 membrane proteins were identified to be differentially expressed among 849 quantified membrane proteins. Function and disease clustering analysis proved that 11 of these proteins are involved in proliferation, migration, and differentiation. Two key factors (MMP-14 and OCLN) were validated by qRT-PCR and Western blot. Blockade of MMP-14 further demonstrated its important function during tumor cell migration. The data sets have been uploaded to ProteomeXchange with the identifier PXD004752.

Expression of claudins, occludin, junction adhesion molecule A and zona occludens 1 in canine organs.

Tight junctions are the outermost structures of intercellular junctions and are classified as transmembrane proteins. These factors form selective permeability barriers between cells, act as paracellular transporters and regulate structural and functional polarity of cells. Although tight junctions have been previously studied, comparison of the transcriptional­translational levels of these molecules in canine organs remains to be investigated. In the present study, organ­specific expression of the tight junction proteins, claudin, occludin, junction adhesion molecule A and zona occludens 1 was examined in the canine duodenum, lung, liver and kidney. Results of immunohistochemistry analysis demonstrated that the tight junctions were localized in intestinal villi and glands of the duodenum, bronchiolar epithelia and alveolar walls of the lung, endometrium and myometrium of the hepatocytes, and the distal tubules and glomeruli of the kidney. These results suggest that tight junctions are differently expressed in organs, and therefore may be involved in organ­specific functions to maintain physiological homeostasis.

Protective effect of quercetin on pig intestinal integrity after transport stress is associated with regulation oxidative status and inflammation.

This experiment was conducted to evaluate the effects of quercetin supplementation on intestinal integrity, intestinal reactive oxygen species (ROS) levels and intestinal inflammation in pigs under transport stress. A total of 170 finishing pigs were randomly assigned into two groups. Animals in the control group consumed a basal diet, while those in the treatment group consumed the same diet supplemented with 25 mg quercetin per kg feed. After a 4-week period, pigs were transported for 5 hr. The quercetin-supplemented pigs showed decreased serum levels of endotoxin (P<0.05), increased height of jejunum villi (P<0.05), and increased occludin and zonula occudens-1 (ZO-1) mRNA expression in the jejunum (P<0.05). These parameters are associated with intestinal health and were markedly improved by quercetin supplementation. Pigs consuming the quercetin-supplemented diet had lower intestinal levels of ROS and malondialdehyde (MDA) compared with the control group (P<0.05). This finding coincided with greater inhibition of the innate immune system (P<0.05), including mitogen-activated protein kinase (MAPK), protein kinase B (Akt) and nuclear factor κB (NF-κB) signaling pathways, as well as decreased expression of inflammatory cytokines in the jejunum. These results indicate that quercetin alleviates intestinal injury in pigs during transport, probably through modulation of intestinal oxidative status and inflammation.

Intestinal Conditioning After Cardiac Arrest: The Use of Normothermic Extracorporeal Membrane Oxygenation in the Non-Heart-Beating Animal Model.

The effect of normothermic extracorporeal membrane oxygenation (NECMO) on small bowel preservation in a clinically relevant large animal model of expected donation after cardiac death (eDCD) was evaluated. Thirty domestic crossbred donor pigs were divided into five groups. The first group served as the live donation (LD) group, the second group served as the donation after cardiac death (DCD) group, and the remaining were further assigned into three subgroups: E1 group (1 h NECMO support), E3 group (3 h NECMO support), and E5 group (5 h NECMO support). Pathology, electron microscopy, energy metabolism, cell apoptosis, and tight junction (TJ) protein expression level of intestinal mucosa and the level of plasma d-lactic acid were evaluated in normal, cardiac death and at the end of extracorporeal support, respectively. The mean arterial pressure and PaO2 were maintained over 60 and 267 mm Hg during NECMO support, respectively. One hour of extracorporeal support could improve the energy status in intestines of the DCD group. Although the histologic damage and apoptosis of the E1 group had no significant difference with those of the LD and DCD groups (P > 0.05), the levels of intestinal mucosa TJ protein decreased (P < 0.05), and plasma d-lactic acid increased progressively (P < 0.05). With the extension of extracorporeal support, the degree of intestinal mucosa damage and intestinal permeability gradually increased, as well as the content of adenosine triphosphate in intestinal mucosa. The normothermic extracorporeal support for 1 h in DCD is beneficial for improving the energy status and viability of the bowel. However, the integrity of intestinal mucosa was destroyed gradually as extracorporeal support time went by. And the activation of intestinal epithelial apoptosis and hyperoxia might be the factors that lead to intestinal mucosa injury.