Sinapic Acid Alleviated Inflammation-Induced Intestinal Epithelial Barrier Dysfunction in Lipopolysaccharide- (LPS-) Treated Caco-2 Cells.

The integrity and permeability of the intestinal epithelial barrier are important indicators of intestinal health. Impaired intestinal epithelial barrier function and increased intestinal permeability are closely linked to the onset and progression of various intestinal diseases. Sinapic acid (SA) is a phenolic acid that has anti-inflammatory, antihyperglycemic, and antioxidant activities; meanwhile, it is also effective in the protection of inflammatory bowel disease (IBD), but the specific mechanisms remain unclear. Here, we evaluated the anti-inflammatory of SA and investigated its potential therapeutic activity in LPS-induced intestinal epithelial barrier and tight junction (TJ) protein dysfunction. SA improved cell viability; attenuated epithelial permeability; restored the protein and mRNA expression of claudin-1, ZO-1, and occludin; and reversed the redistribution of the ZO-1 and claudin-1 proteins in LPS-treated Caco-2 cells. Moreover, SA reduced the inflammatory response by downregulating the activation of the TLR4/NF-κB pathway and attenuated LPS-induced intestinal barrier dysfunction by decreasing the activation of the MLCK/MLC pathway. This study demonstrated that SA has strong anti-inflammatory activity and can alleviate the occurrence of high intercellular permeability in Caco-2 cells exposed to LPS.

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.

Tight junction proteins in the small intestine and prefrontal cortex of female rats exposed to stress of chronic isolation starting early in life.

BACKGROUND: Simultaneous evaluation of barrier protein expression in the gut and the brain and their modulation under stress conditions have not been studied before now. As the permeability and function of the gut and blood-brain barrier are different and both express the MRs, we hypothesized that stress of post-weaning social isolation induces changes in tight junction protein expression in the gut which are (1) independent of changes in the brain and (2) are mediated via the mineralocorticoid receptor (MR). METHODS: First, using UPLC-MS/MS we have successfully validated and selected a dose (1.2 mg/rat/day) of the MR antagonist spironolactone to treat female rats exposed to stress of chronic isolation or control conditions from postnatal day 21 for 9 weeks. KEY RESULTS: Isolation stress caused an enhancement of gene expression of occludin and ZO-1 and a decrease in claudin-5 and MR expression in both the small intestine and prefrontal cortex. Isolation stress failed to decrease claudin-5 (small intestine) and MR (prefrontal cortex) gene expression in spironolactone-treated rats. MR blockade resulted in a decrease in claudin-15 expression in the small intestine. Anxiogenic effect of chronic stress, measured in elevated plus-maze test, was partly prevented by spironolactone treatment. CONCLUSIONS & INFERENCES: Claudins, the main regulators of intestinal barrier permeability responded to chronic stress of social isolation and/or simultaneous blockade of MR in female rats by alterations independent of changes in the brain cortex. The results suggest a physiological role of MR in the control of claudin expression in the small intestine, but not in the brain cortex.

Reduced occludin and claudin-7 expression is associated with urban locations and exposure to second-hand smoke in allergic rhinitis patients.

The breakdown of nasal epithelial barrier occurs in allergic rhinitis (AR) patients. Impairment of cell junction molecules including tight junctions (TJs) and desmosomes plays causative roles in the pathogenesis of AR. In this study, we investigated the transcript expression levels of TJs including occludin (OCLN), claudin-3 and -7 (CLDN3 and CLDN7), desmoglein 3 (DSG3) and thymic stromal lymphopoietin (TSLP) in AR patients (n = 30) and non-allergic controls (n = 30). Nasal epithelial cells of non-allergic controls and AR patients were collected to examine their mRNA expression levels, and to correlate with clinico-demographical and environmental parameters. We demonstrated that the expression of OCLN (p = 0.009), CLDN3 (p = 0.032) or CLDN7 (p = 0.004) transcript was significantly lower in AR patients compared with non-allergic controls. No significant difference was observed in the expression of DSG3 (p = 0.750) or TSLP (p = 0.991) transcript in AR patients compared with non-allergic controls. A significant association between urban locations and lower OCLN expression (p = 0.010), or exposure to second-hand smoke with lower CLDN7 expression (p = 0.042) was found in AR patients. Interestingly, none of the TJs expression was significantly associated with having pets, frequency of changing bedsheet and housekeeping. These results suggest that defective nasal epithelial barrier in AR patients is attributable to reduced expression of OCLN and CLDN7 associated with urban locations and exposure to second-hand smoke, supporting recent findings that air pollution represents one of the causes of AR.

Neonatal Hyperoxia Downregulates Claudin-4, Occludin, and ZO-1 Expression in Rat Kidney Accompanied by Impaired Proximal Tubular Development.

Hyperoxia is essential to manage in preterm infants but causes injury to immature kidney. Previous study indicates that hyperoxia causes oxidative damage to neonatal kidney and impairs renal development. However, the underlying mechanisms by which neonatal hyperoxia effects on immature kidney still need to be elucidated. Tight junction, among which the representative proteins are claudin-4, occludin, and ZO-1, plays a crucial role in nephrogenesis and maintaining renal function. Inflammatory cytokines are involved in the pleiotropic regulation of tight junction proteins. Here, we investigated how neonatal hyperoxia affected the expression of key tight junction proteins and inflammatory factors (IL-6 and TNF-α) in the developing rat kidneys and elucidated their correlation with renal injury. We found claudin-4, occludin, and zonula occludens-1 (ZO-1) expression in proximal tubules was significantly downregulated after neonatal hyperoxia. The expression of these tight junction proteins was positively correlated with that of IL-6 and TNF-α, while claudin-4 expression was positively correlated with injury score of proximal tubules in mature kidneys. These findings indicated that impaired expression of tight junction proteins in kidney might be a potential mechanism of hyperoxia-induced nephrogenic disorders. It provides new insights to further study oxidative renal injury and development disorders and will be helpful for seeking potential therapeutics for hyperoxia-induced renal injury in the future.

Isoorientin Inhibits Inflammation in Macrophages and Endotoxemia Mice by Regulating Glycogen Synthase Kinase 3ß.

Isoorientin has anti-inflammatory effects; however, the mechanism remains unclear. We previously found isoorientin is an inhibitor of glycogen synthase kinase 3ß (GSK3ß) in vitro. Overactivation of GSK3ß is associated with inflammatory responses. GSK3ß is inactivated by phosphorylation at Ser9 (i.e., p-GSK3ß). Lithium chloride (LiCl) inhibits GSK3ß and also increases p-GSK3ß (Ser9). The present study investigated the anti-inflammatory effect and mechanism of isoorientin via GSK3ß regulation in lipopolysaccharide- (LPS-) induced RAW264.7 murine macrophage-like cells and endotoxemia mice. LiCl was used as a control. While AKT phosphorylates GSK3ß, MK-2206, a selective AKT inhibitor, was used to activate GSK3ß via AKT inhibition (i.e., not phosphorylate GSK3ß at Ser9). The proinflammatory cytokines TNF-α, IL-6, and IL-1ß were detected by ELISA or quantitative real-time PCR, while COX-2 by Western blotting. The p-GSK3ß and GSK3ß downstream signal molecules, including NF-κB, ERK, Nrf2, and HO-1, as well as the tight junction proteins ZO-1 and occludin were measured by Western blotting. The results showed that isoorientin decreased the production of TNF-α, IL-6, and IL-1ß and increased the expression of p-GSK3ß in vitro and in vivo, similar to LiCl. Coadministration of isoorientin and LiCl showed antagonistic effects. Isoorientin decreased the expression of COX-2, inhibited the activation of ERK and NF-κB, and increased the activation of Nrf2/HO-1 in LPS-induced RAW264.7 cells. Isoorientin increased the expressions of occludin and ZO-1 in the brain of endotoxemia mice. In summary, isoorientin can inhibit GSK3ß by increasing p-GSK3ß and regulate the downstream signal molecules to inhibit inflammation and protect the integrity of the blood-brain barrier and the homeostasis in the brain.

[Study on the damage of the tight junctions of nasal mucosal epithelial cells by artemisia annua pollen].

Objective: To investigate the damage and mechanism of artemisia annua pollen on tight junction of human nasal mucosa epithelial cells (HNEpC). Methods: HNEpC were cultured in vitro. Different concentrations of artemisia annua pollen (0, 20, 40, 80, 100, 160, 200 µg/ml) were used to intervene the cells for 24 h, and the cell proliferation activity was detected by the CCK-8 method. The expression and phosphorylation of p38MAPK signaling pathway were detected by Western Blot before and after the intervention of SB203580, a p38MAPK inhibitor in HNEpC. Immunofluorescence chemical staining, Western Blot and quantitative real-time PCR (qPCR) were used to observe the expression and distribution of tight junctions Occludin and Claudin-1. SPSS 21.1 software was used for statistical analysis. Results: CCK-8 results showed that, compared with the control group, the proliferation activity of HNEpC increased after 6 h intervention with different concentrations of artemisia annua pollen (all P<0.05). After 12 h of intervention, the proliferation activity of HNEpC in the 20, 40, 80, 100 and 160 µg/ml groups was not significantly changed (all P>0.05), while that in the 200 µg/ml group was decreased (P<0.05). After the intervention for 24 h, the proliferation activity of cells in the 20 and 40 µg/ml groups was not significantly changed (all P>0.05), while that in the 80, 100, 160 and 200 µg/ml groups was decreased (all P<0.05). Immunofluorescence staining showed that the Occludin and Claudin-1 proteins in the normal control group were localized on the cell membrane and expressed more and formed a ring structure around the cell membrane. However, under the intervention of high concentration artemisia annua pollen, its expression level decreased, appeared broken, fuzzy, and nonuniform distribution. Western Blot and qPCR results showed that after 24 h of intervention, the expression levels of HNEpC Claudin-1 protein and its mRNA in the pollen groups (40, 80, 100, 160, 200 µg/ml) of artemisia annua decreased compared with those of those of the control group (mRNA expression levels were 0.567±0.214, 0.443±0.109, 0.462±0.160, 0.497±0.134, 0.388±0.076 compared with 1.001±0.067, respectively, all P<0.05). However, the mRNA of Occludin protein and its mRNA only decreased in the 200 µg/ml treatment group (mRNA expression level was 0.631±0.109 compared with 1.016±0.026, P<0.05), while all the other treatment groups increased (mRNA expression levels were 1.258±0.134, 1.827±0.103, 2.429±0.077, 1.707±0.085, 1.477±0.066 compared with 1.016±0.026, respectively, all P<0.05). Western Blot showed that p-p38MAPK expression increased after intervention with 100, 160, 200 µg/ml artemisia annua pollen for 24 h. SB203580 could inhibit the decreasing expression of Occludin caused by artemisinin pollen (mRNA expression was 1.255±0.179 compared with 0.631±0.109, P<0.05), but had no effect on Claudin-1 protein expression. Conclusion: Pollen from artemisia annua may activate p38MAPK signaling pathway and destroy the close connection of HNEpC.

Arsenite-induced downregulation of occludin in mouse lungs and BEAS-2B cells via the ROS/ERK/ELK1/MLCK and ROS/p38 MAPK signaling pathways.

Occludin is an important tight junction (TJ) protein in pulmonary epithelial cells. In this study, we identified changes in occludin in arsenic-induced lung injury in vivo and in vitro. Upon intratracheal instillation with arsenic trioxide (As2O3) at a daily dose of 30 µg/kg for 1 week, levels of occludin mRNA and protein expression decreased significantly in mouse lung tissue. Levels of occludin mRNA and protein expression in BEAS-2B cells were reduced upon exposure to As2O3 in a concentration- and time-dependent manner. In addition, exposure to As2O3 significantly increased expression of p-p38, p-ERK1/2, p-ELK1, and MLCK in mouse lung tissue and BEAS-2B cells. Treatment with As2O3 induced oxidative stress in mouse lung tissue and BEAS-2B cells. In BEAS-2B cells, exposure to As2O3 reduced transepithelial resistance, which was partially restored with N-acetyl-cysteine (NAC) treatment. Reduced expression of occludin mRNA and protein induced by As2O3 was entirely restored with NAC and resveratrol. However, SB203580, PD98059, and ML-7 partially blocked As2O3-induced occludin reduction in BEAS-2B cells. These results indicate that As2O3 inhibits occludin expression in vivo and in vitro at least partially via the ROS/ERK/ELK1/MLCK and ROS/p38 MAPK signaling pathways.

Hyperglycemia and advanced glycation end products disrupt BBB and promote occludin and claudin-5 protein secretion on extracellular microvesicles.

Cognitive impairment is a well-known complication of diabetes mellitus (DM). Microvascular compromise was described one DM complication. Recently we showed blood brain barrier (BBB) permeability and memory loss are associated with diminution of tight junctions (TJ) in brain endothelium and pericyte coverage and inflammation in cerebral microvessels and brain tissue paralleling hyperglycemia in mice of both DM types. The current study demonstrates that exposure of brain microvessels to hyperglycemic conditions or advanced glycation end products (AGEs) ex vivo resulted in significant abnormalities in membranous distribution of TJ proteins. We found significant increase in the amount of extracellular vesicles (EVs) isolated from DM mice and enhanced presence of TJ proteins, occludin and claudin-5, on EVs. Exposure of BMVECs to high glucose and AGEs led to significant augmentation of ICAM and VCAM expression, elevated leukocyte adhesion to and migration across BMVEC monolayers, and increased BBB permeability in vitro. Pericytes exposed to hyperglycemia and AGEs displayed diminished expression of integrin α1, PDGF-R1ß and connexin-43. Our findings indicate BBB compromise in DM ex vivo, in vitro and in vivo models in association with BMVEC/pericyte dysfunction and inflammation. Prevention of BBB injury may be a new therapeutic approach to avert cognitive demise in DM.

Paracellular permeability changes induced by multi-walled carbon nanotubes in brain endothelial cells and associated roles of hemichannels.

Multi-walled carbon nanotubes (MWCNTs) have promising applications in neurology depending on their unique physicochemical properties. However, there is limited understanding of their impacts on brain microvascular endothelial cells, the cells lining the vessels and maintaining the low and selective permeability of the blood-brain barrier. In this study, we examined the influence of pristine MWCNT (p-MWCNT) and carboxylated MWCNT (c-MWCNT) on permeability and tight junction tightness of murine brain microvascular endothelial cells, and investigated the potential mechanisms in the sight of hemichannel activity. Treatment with p-MWCNT for 24 h at subtoxic concentration (20 µg/mL) decreased the protein expression of occludin, disrupted zonula occludens-1 continuity, and elevated monolayer permeability as quantified by transendothelial electrical resistance and paracellular flux of 4000 Da fluorescein isothiocyanate-dextran conjugates. Moreover, p-MWCNT exposure also increased hemichannel activity with upregulated protein expression and altered subcellular localization of connexin (Cx)43 and pannexin (Panx)1. p-MWCNT-induced elevation in endothelial permeability could be prevented by hemichannel inhibitor carbenoxolone and peptide blocker of Cx43 and Panx1, indicating the crucial role of activated Cx43 and Panx1 hemichannels. Furthermore, Cx43 and Panx1 hemichannel-mediated ATP release might be involved in p-MWCNT-induced rise in endothelial permeability. In contrast, the above effects caused by p-MWCNT were not observed in cells treated with c-MWCNT, the functionalized form with more stable dispersion and a lower tendency to aggregate. Our study contributes further understanding of the impact of MWCNTs on brain endothelial tightness and permeability, which may have important implications for the safety application of MWCNTs in nanomedicine.

Low OCEL1 expression is associated with poor prognosis in human non-small cell lung cancer.

BACKGROUND: Occludin/ELL domain containing 1 (OCEL1) is a novel discovered protein with its molecular functions remaining unknown and its role in lung cancer has not been directly explored. OBJECTIVES: This study focused on the role of OCEL1 in the progression and prognosis of non-small cell lung cancer (NSCLC). METHODS: A public database and tissue samples (80 NSCLC tissue samples and paired normal lung samples) were used to compare differences in OCEL1 expression and investigate its relationship with clinical characteristics and prognosis. RESULTS: Compared to adjacent normal lung tissue samples, OCEL1 expression was significantly down-regulated in tumor tissues. In addition, there was a negative correlation between OCEL1 and Ki67 expression levels. Low OCEL1 expression was significantly associated with lymph node metastasis, higher TNM stage, and poor prognosis. Importantly, multivariate analysis identified OCEL1 expression as an independent predictor for unfavorable NSCLC prognosis. CONCLUSIONS: These results indicated that OCEL1 protein may serve as a novel prognostic biomarker in NSCLC.

C1 Esterase Inhibitor Reduces BBB Leakage and Apoptosis in the Hypoxic Developing Mouse Brain.

Inflammatory pathways involved in blood-brain barrier (BBB) vulnerability and hypoxic brain oedema in models of perinatal brain injury seem to provide putative therapeutic targets. To investigate impacts of C1-esterase inhibitor (C1-INH; 7.5-30 IU/kg, i.p.) on functional BBB properties in the hypoxic developing mouse brain (P7; 8% O2 for 6 h), expression of pro-apoptotic genes (BNIP3, DUSP1), inflammatory markers (IL-1ß, TNF-alpha, IL-6, MMP), and tight junction proteins (ZO-1, occludin, claudin-1, -5), and S100b protein concentrations were analysed after a regeneration period of 24 h. Apoptotic cell death was quantified by CC3 immunohistochemistry and TUNEL staining. In addition to increased apoptosis in the parietal cortex, hippocampus, and subventricular zone, hypoxia significantly enhanced the brain-to-plasma albumin ratio, the cerebral S100b protein levels, BNIP3 and DUSP1 mRNA concentrations as well as mRNA expression of pro-inflammatory cytokines (IL-1ß, TNF-alpha). In response to C1-INH, albumin ratio and S100b concentrations were similar to those of controls. However, the mRNA expression of BNIP3 and DUSP1 and pro-inflammatory cytokines as well as the degree of apoptosis were significantly decreased compared to non-treated controls. In addition, occludin mRNA levels were elevated in response to C1-INH (p < 0.01). Here, we demonstrate for the first time that C1-INH significantly decreased hypoxia-induced BBB leakage and apoptosis in the developing mouse brain, indicating its significance as a promising target for neuroprotective therapy.

DNA methylation, microRNA expression profiles and their relationships with transcriptome in grass-fed and grain-fed Angus cattle rumen tissue.

Rumen is an organ for supplying nutrients for the growth and production of bovine, which might function differently under grass-fed and grain-fed regimens considering the association of gene expression, DNA methylation, and microRNA expression. The objective of this study was to explore the potential mechanism influencing rumen function of grass-fed and grain-fed animals. Methylated DNA binding domain sequencing (MBD-Seq) and microRNA-Seq were respectively utilized to detect the DNA methylation and microRNA expression in rumen tissue of grass-fed and grain-fed Angus cattle. Combined analysis revealed that the expression of the differentially expressed genes ADAMTS3 and ENPP3 was correlated with the methylation abundance of the corresponding differentially methylated regions (DMRs) inside these two genes, and these two genes were reported to be respectively involved in biosynthesis and regulation of glycosyltransferase activity; the differentially expressed microRNA bta-mir-122 was predicted to possibly target the differentially expressed genes OCLN and RBM47, potentially affecting the rumen function; the microRNA bta-mir-655 was exclusively detected in grain-fed group; its targets were significantly enriched in insulin and TGF-beta signaling pathways, which might worked together to regulate the function of rumen, resulting in different characteristics between grass-fed and grain-fed cattle. Collectively, our results provided insights into understanding the mechanisms determining rumen function and unraveled the biological basis underlying the economic traits to improve the productivity of animals.

Disruption of blood-brain barrier by an Escherichia coli isolated from canine septicemia and meningoencephalitis.

Escherichia coli (E. coli) is one of the common pathogenic bacteria in veterinary clinical infection. As an opportunistic microorganism, E. coli normally does not cause diseases. However, it causes infections under certain circumstance to domesticated animal and poultry, resulting in severe diarrhea, septicemia, and respiratory infections. Although there are increasing reports regarding the infections of E. coli to domestic animals and poultry, the infection of E. coli in dogs is relatively less reported, especially on septicemia and meningoencephalitis. Here, we reported the isolation and identification of an E. coli isolate named CEC-GZL17 from dogs characterized by septicemia and sudden death, and found that CEC-GZL17 is able to cause meningoencephalitis. Exploration on the potential mechanism underlying meningoencephalitis demonstrated that CEC-GZL17 infection significantly increases TNF-α expression and inhibits ZO-1 and occludin expressions in brain tissue, indicating that the E coli likely use the mechanism to penetrate the blood-brain barrier via disrupting tight junction architecture, thus leading to the invasion to brain tissue.

Newly synthesized claudins but not occludin are added to the basal side of the tight junction.

A network of claudin strands creates continuous cell-cell contacts to form the intercellular tight junction barrier; a second protein, occludin, is associated along these strands. The physiological barrier remains stable despite protein turnover, which involves removal and replacement of claudins both in the steady state and during junction remodeling. Here we use a pulse-block-pulse labeling protocol with fluorescent ligands to label SNAP/CLIP-tags fused to claudins and occludin to identify their spatial trafficking pathways and kinetics in Madin-Darby canine kidney monolayers. We find that claudins are first delivered to the lateral membrane and, over time, enter the junction strand network from the basal side; this is followed by slow replacement of older claudins in the strands. In contrast, even at early times, newly synthesized occludin is found throughout the network. Taking the results together with our previous documentation of the mechanism for claudin strand assembly in a fibroblast model, we speculate that newly synthesized claudins are added at strand breaks and free ends; these are most common in the basalmost edge of the junction. In contrast, occludin can be added directly within the strand network. We further demonstrate that claudin trafficking and half-life depend on carboxy-terminal sequences and that different claudins compete for tight junction localization.

MicroRNA-21 increases the expression level of occludin through regulating ROCK1 in prevention of intestinal barrier dysfunction.

OBJECTIVE: The aim of this study is to investigate the role of molecular mechanism of microRNA (miR)-21 on tight junction (TJ)-proteins and its protective effects on the intestinal barrier. METHODS: TJ proteins and target genes expression were analyzed in miR-21 inhibition and overexpression NCM460 cell lines. To further verify the role of miR-21, the mmu-miR-21 intestinal epithelial conditional knockout (IKO) mice model was established. MiR-21 expression was detected in clinical specimens of acute stercoral obstruction patients. RESULTS: Rho-associated protein kinase 1 (ROCK1) were identified as target genes of miR-21. There is a negative correlation between miR-21 expression level and TJ proteins levels. TJ protein and ROCK1 were significantly decreased in miR-21 IKO mice, which presented intestinal inflammation response and intestinal barrier dysfunction (both P < 0.05). Determination of clinical samples showed consistent results with NCM460 cell line and miR-21 IKO mice. CONCLUSIONS: MiR-21 could be a protective factor of intestinal barrier dysfunction, which promoting the expression of TJ protein by targeting ROCK1 in vivo and in vitro.

Enterococcus faecium HDRsEf1 elevates the intestinal barrier defense against enterotoxigenic Escherichia coli and regulates occludin expression via activation of TLR-2 and PI3K signalling pathways.

Previously, we isolated a novel probiotic strain, designated HDRsEf1. In this study, we investigated the effects of this probiotic strain on intestinal barrier function and how it regulates the tight junction protein occludin in vitro. We used an ETEC-infected mouse model for the in vivo experiment. Briefly, 40 ICR mice were randomly divided into four groups: control group, assigned to saline gavage; prevention group, given HDRsEf1 before and saline after infection with ETEC; infection group, given saline both before and after infection with ETEC; treatment group, given saline before and HDRsEf1 after infection with ETEC. The weight loss was alleviated both in the prevention and treatment groups. The ETEC-induced intestinal inflammation was alleviated and the occludin mRNA expression levels in the jejuna of infected mice were increased in the prevention group. We explored the mechanism by which HDRsEf1 regulates occludin expression in vitro and found that HDRsEf1 prevented the downregulation of occludin expression in the prevention group. Simultaneously, we found that toll-like receptor-2 (TLR-2) and phosphoinositide 3-kinase (PI3K) play an important role in maintaining occludin expression. Therefore, we concluded that HDRsEf1 can prevent ETEC-induced infection by enhancing the intestinal barrier function and increasing the expression levels of occludin. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterotoxigenic Escherichia coli (ETEC) is a major cause of infectious diarrhoea in children, and porcine ETEC has been the leading cause of post-weaning diarrhoea (PWD) in pigs. In our present study, we demonstrated for the first time that HDRsEf1 protects occludin from ETEC-induced suppression. Moreover, HDRsEf1 was found to regulate occludin expression via TLR-2 activation and the PI3K pathway. The results provide insights into the mechanism by which HDRsEf1 protects cells against ETEC infection and a rationale for the use of HDRsEf1 as a therapeutic and preventative agent.

Glucagon-Like Peptide-2 Improve Intestinal Mucosal Barrier Function in Aged Rats.

Glucagon-like peptide-2 (GLP-2) plays a major role in repairing impaired intestinal mucosa, but its mechanism in the improvement of intestinal barrier function during the aging process remains unclear. In this study, 26-month-old male Sprague-Dawley rats were randomized to control group and GLP-2 group treated with a dose of 250 µg•kg-1•d-1 by intraperitoneal injection. After 14 days of treatment, intestinal mucosal morphometric changes were observed by light microscopy and transmission electron microscopy (TEM). Small intestinal permeability was evaluated by fluorescein isothiocyanate (FITC)-labeled dextran. The mRNA and protein expression of Zonula Occludens-1 (ZO-1), occludin, claudin-1 and the GLP-2 receptor (GLP-2R) were detected by Real-time PCR and Western blot. Our results showed that GLP-2 administration significantly improved the age-related atrophy of intestinal mucosa and villi and increased small intestinal permeability. The mRNA and protein expression of ZO-1and occludin in ileum were up regulated in the GLP-2-treated old rats. In addition, the serum GLP-2 levels were negatively correlated with small intestinal permeability measured by FITC-dextran levels (r=-0.610, P<0.01). Taking all these data together, it is concluded that GLP-2 improved small intestinal epithelial barrier function in aged rats mainly by facilitating intestinal mucosa growth, alleviating the increased small intestinal permeability and increasing ZO-1 and occludin expression. Our observations provide evidence for the clinical significance of GLP-2 in preventing the intestinal epithelial barrier dysfunction during aging.

Magnolol treatment attenuates dextran sulphate sodium-induced murine experimental colitis by regulating inflammation and mucosal damage.

Magnolol, the main and active ingredient of the Magnolia officinalis, has been widely used in traditional prescription to the human disorders. Magnolol has been proved to have several pharmacological properties including anti-bacterial, anti-oxidant and anti-inflammatory activities. However, the effects of magnolol on ulcerative colitis (UC) have not been reported. The aim of this study was to investigate the protective effects and mechanisms of magnolol on dextran sulphate sodium (DSS)-induced colitis in mice. The results showed that magnolol significantly alleviated DSS-induced body weight loss, disease activities index (DAI), colon length shortening and colonic pathological damage. In addition, magnolol restrained the expression of TNF-α, IL-1ß and IL-12 via the regulation of nuclear factor-κB (NF-κB) and Peroxisome proliferator-activated receptor-γ (PPAR-γ) pathways. Magnolol also enhanced the expression of ZO-1 and occludin in DSS-induced mice colonic tissues. These results showed that magnolol played protective effects on DSS-induced colitis and may be an alternative therapeutic reagent for colitis treatment.

MicroRNA-130a regulates cerebral ischemia-induced blood-brain barrier permeability by targeting Homeobox A5.

Blood-brain barrier (BBB) disruption plays a critical role in brain injury induced by cerebral ischemia, and preserving BBB integrity during ischemia could alleviate cerebral injury. We examined the role of miR-130a in ischemic BBB disruption by using models of rat middle cerebral artery occlusion and cell oxygen-glucose deprivation. We found that ischemia significantly increased microRNA-130a (miR-130a) level and that miR-130a was predominantly from brain microvascular endothelial cells. Antagomir-130a, an antagonist of miR-130a, could attenuate brain edema, lower BBB permeability, reduce infarct volume, and improve neurologic function. MiR-130a overexpression induced by miR-130a mimic increased monolayer permeability, and intercellular inhibition of miR-130a by a miR-130a inhibitor suppressed oxygen-glucose deprivation-induced increase in monolayer permeability. Moreover, dual luciferase reporter system showed that Homeobox A5 was the direct target of miR-130a. MiR-130a, by inhibiting Homeobox A5 expression, could down-regulate occludin, thereby increasing BBB permeability. Our results suggested that miR-130a might be implicated in ischemia-induced BBB dysfunction and serve as a target for the treatment of ischemic stroke.-Wang, Y., Wang, M.-D., Xia, Y.-P., Gao, Y., Zhu, Y.-Y., Chen, S.-C., Mao, L., He, Q.-W., Yue, Z.-Y., Hu, B. MicroRNA-130a regulates cerebral ischemia-induced blood-brain barrier permeability by targeting Homeobox A5.