Expression Levels Of Occludin Research Articles

Relationship between uric acid and cerebral amyloid angiopathy

Purpose: To explored Relationship between uric acid and cerebral amyloid angiopathy; Materials and methods: ZO-1 and RAGE in HBMECs were detected by western blotting, and then, we analyzed ZO-1, occludin, and RAGE mRNA expression levels in different treatment groups using RTPCR. Cell counts and the relative αSMA fluorescence intensity were measured in order to evaluate the protective effect of uric acid against injury to HBVSMCs. Analysis of variance showed that LDH leakage rate was used to verify the uric acid protective effect on the injury induced by Aβ1–40. After that, the level of uric acid in serum and Aβ1–40 in brain tissue was analyzed by western blotting and immunohistochemistry to evaluate the protective effect of uric acid in the brain of APP23 mice. Meanwhile, Occludin, ZO-1, and RAGE protein levels were measured by western blotting; Results: Uric acid reduced the negative effects of Aβ on the vascular endothelium and smooth muscle cells and protected the vascular wall in vitro. In APP23 mice, Aβ1–40 and Aβ1–42 levels were significantly elevated in brain tissues and further increased after uric acid concentration was decreased. In APP23 mice, ZO-1 and occludin expression levels were both significantly lower than those in wild-type animals. After uric acid concentration was lowered in APP23 mice, ZO-1 and occludin expression levels were significantly lower than those in untreated animals; Conclusions: Uric acid in the blood protects the blood vessels from CAA damage to the blood vessel wall, and reduces the occurrence of cerebral hemorrhage.

Effects of norepinephrine on colonic tight junction protein expression during heat stress.

Stress induced by changes in the internal or external environment in humans and animals leads to intestinal epithelial damage, in a manner that is associated with impaired intestinal barrier function. However, the role of the stress hormone norepinephrine (NE) in impairments in barrier function remains poorly understood. In the present study, a rat heat-exposed model was used to observe changes in the tight junction proteins Occludin and zonula occludens-1 (ZO-1), in addition to those in protease-activated receptor 2 (PAR-2) and transient receptor potential ankyrin 1 channel (TRPA1) in colon. The levels of plasma NE were detected using an ELISA kit. Different concentrations of NE were used to culture the human colon cell line Caco-2 for 6 and 24 h to investigate the cell viability using Cell Counting Kit-8 assay, whilst the expression levels of Occludin, ZO-1, PAR-2 and TRPA1 were examined using western blotting and immunofluorescence in Caco-2 cells and immunohistrochemistry in rat colon tissues. Although there was no clear histological damage to the rat colonic mucosa, there were decreased expression levels of tight junction proteins Occludin and ZO-1 after heat exposure. In addition, PAR-2 expression was increased by heat exposure. It was found that TRPA1 expression was concentrated to the luminal surface of the colon in the heat exposed group compared with that in the control group. After the administration of increasing concentrations of NE for 6 h, treatment did not affect cell viability. Furthermore, after application of NE for 24 h, cell viability gradually increased as the NE concentration was elevated from 10 to 100 µM. However, no significant increase in viability was observed when the cells were treated with 120 and 160 µM NE. Occludin expression was decreased when 10 µM NE was applied for 6 or 24 h. By contrast, 60 µM NE significantly downregulated Occludin expression in the 6 h group, but caused an insignificant decrease in the 24 h group. It was found that ZO-1 expression was upregulated after treatment with 10 µM NE for 6 h, whilst downregulation was observed after treatment with 10 µM NE for 24 h. PAR-2 protein expression was increased after application of NE for both 6 and 24 h, but not after treatment with 60 µM NE. In addition, TRPA1 expression was not affected by the treatment of NE, but increased positive staining was observed on the luminal side of the mucosa, which appeared to be concentrated in the cells of the luminal side in the rat colon after heat exposure. Collectively, the present results suggested that expression of tight junction proteins Occludin and ZO-1, in addition to that of PAR-2, can be regulated by NE, which may contribute to impairments in barrier function observed during heat stress.

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

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

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

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

Biogenic selenium nanoparticles by Lactobacillus casei ATCC 393 alleviate the intestinal permeability, mitochondrial dysfunction and mitophagy induced by oxidative stress.

Selenium (Se) is an essential trace element. Nano-selenium has attracted great attention due to its various biological properties, especially strong antioxidant activity, high bioavailability, and low toxicity. Our previous studies demonstrated that the selenium nanoparticles (SeNPs) synthesized by Lactobacillus casei ATCC 393 (L. casei ATCC 393) alleviate hydrogen peroxide (H2O2)-induced intestinal epithelial barrier dysfunction via the mitochondrial pathway. However, the mechanism of SeNPs exerting antioxidant activity through the mitochondrial pathway remains unclear. This study was conducted to investigate the role of mitophagy in the protective effects of SeNPs on H2O2-induced porcine intestinal epithelial cells against oxidative damage. The results showed that the SeNPs synthesized by L. casei ATCC 393 had no cytotoxicity on IPEC-J2 cells and effectively antagonized the cytotoxicity of 500 μM H2O2 on IPEC-J2 cells. Moreover, SeNPs attenuated the H2O2-induced intestinal epithelial barrier dysfunction and ROS overproduction, as well as alleviated the adenosine triphosphate (ATP) level and the mitochondrial membrane potential (MMP) decrease. In addition, compared to the oxidative stress model group, pretreatment with biogenic SeNPs significantly up-regulated the expression levels of occludin and claudin-1. Moreover, when compared to the oxidative stress model group, SeNPs inhibited the phosphorylation level of the mammalian target of rapamycin (m-TOR), as well as the expression levels of Unc-51-like kinase 1(ULK1), light chain 3 (LC3)-II/LC3-I, PTEN-induced kinase 1 (PINK1) and Parkin proteins. The fluorescence colocalization images of mitochondria and lysosomes demonstrated that SeNPs significantly reduced the fusion of mitochondria and lysosomes when compared to the oxidative stress model group. These results demonstrate that the SeNPs synthesized by L. casei ATCC 393 can effectively alleviate the H2O2-induced intestinal epithelial barrier dysfunction through regulating mTOR/PINK1-mediated mitophagy.

Quercetin protects retina external barrier from oxidative stress injury by promoting autophagy

Purpose: This study aimed to investigate the protective effects of quercetin on the tight junction proteins of human retinal pigment epithelial cells (ARPE-19 cells) suffering from oxidative stress injury and explore the possible mechanism. Methods: H2O2 (300 μM) was used to establish an oxidative stress model of ARPE-19 cells. ARPE-19 cells were pretreated with different concentrations (0–80 μM) of quercetin before H2O2 exposure. The expression and distribution of tight junction proteins and autophagy-related proteins were detected by Western blot and immunostaining. ARPE-19 cells were pretreated with 5 mM 3-methyladenine (3-MA). Results: The cell viability weakened in the H2O2 group compared with the control group. However, it was preserved after pretreatment with quercetin. It was observed that the expression levels of occludin, claudin-1 were decreased in the H2O2 group. Quercetin treatment significantly enhanced the expression levels of them as compared to the H2O2 group. H2O2 alone strongly decreased the Zonula occludens protein 1 (ZO-1) expression in the cytomembrane. Quercetin supplementation enhanced the accumulation of ZO-1 in ARPE-19 cells. The expression levels of Beclin-1 and Microtubule associated protein light chain 3 II (LC-3II) increased, and that of P62 decreased in the quercetin protection group. The appearance of LC-3II, which examined by immunofluorescence experiments, enhanced in the quercetin protection group as compared with the control group. The expression levels of beclin-1 and LC-3II increased, and that of P62 increased in the autophagy-inhibited group compared with the quercetin protection group. The levels of occludin and claudin-1 also decreased. Conclusion: Quercetin prevents the loss of tight junction proteins by upregulating autophagy after oxidative stress in ARPE-19 cells.

Effects of magnolol on egg production, egg quality, antioxidant capacity, and intestinal health of laying hens in the late phase of the laying cycle

Magnolol is a multifunctional plant polyphenol. To evaluate the effects of magnolol on laying hens in the late laying period, 360 (50-week-old) laying hens were randomly assigned to 4 dietary treatments: a non-supplemented control diet (C), and control diets supplemented with 100, 200, and 300 mg/kg of magnolol (M100, M200, and M300), respectively. Each treatment had 6 replicates with 15 hens per replicate. Results showed that dietary supplementation of 200 and 300 mg/kg of magnolol increased the laying rate and the M200 group had a lower feed conversion ratio (P < 0.05). Magnolol supplementation (200 and 300 mg/kg) could linearly increase albumen height and Haugh unit of fresh eggs in the late phase of the laying cycle (P < 0.01). And magnolol linearly alleviated the decline of the albumen height and Haugh unit of eggs stored for 14 d (P < 0.01). The total superoxide dismutase activity in the ovaries of M100 group was greater than that in the other treatments (P < 0.05). As dietary magnolol levels increased, villus height of jejunum and ileum linearly increased (P < 0.01). M200 and M300 groups had higher expression level of occludin in the ileum compared with group C (P < 0.01). The level of nitric oxide production and inducible nitric oxide synthase expression in the ileum of M200 group were lower than that in the C group (P < 0.05). In conclusion, dietary supplementation of 200 and 300 mg/kg magnolol can improve hen performance, albumen quality of fresh and storage eggs, and hepatic lipid metabolism in the late laying cycle. Also, magnolol has a good effect on increasing villi and improving the intestinal mucosal mechanical barrier function.

Effects of dechlorane plus on intestinal barrier function and intestinal microbiota of Cyprinus carpio L

Dechlorane Plus (DP) is a typical polychlorinated flame retardant that has been emerged in chemical products. Due to its accumulation and amplification effect, the toxicity of DP has become a widespread environmental safety issue. However, whether DP can affect the intestinal tract of teleost fish remains largely unclear. To understand its effects on the intestinal barrier, morphological characteristics and intestinal microbiome of common carp, different concentrations (30, 60 and 120 μg/L) of DP were exposed to common carps for 4 weeks. The results indicated that DP evidently shortened the intestinal folds and damaged the intestinal epithelium layer. In addition, the mRNA expression levels of occludin, claudin-2 and zonula occludens-1 (ZO-1) were significantly decreased with increasing DP concentrations. Furthermore, the relative abundance of some microbiota species were also changed significantly. Our study first demonstrated that DP could cause damage to the intestinal epithelium and destroy the intestinal barrier and increase the relative abundance of pathogenic bacteria, thereby increasing the probability of contact between intestinal epithelium and pathogenic bacteria, which in turn lead to an increased susceptibility to various diseases and poor health. In summary, our findings reveal that chronic DP exposure can have a harmful effect on the intestinal flora balance and is potentially linked to human disease.

Expression of VEGF- and tight junction-related proteins in the neocortical microvasculature of patients with drug-resistant temporal lobe epilepsy

The blood-brain barrier (BBB) maintains the optimal microenvironment for brain function. Tight junctions (TJs) allow endothelial cells to adhere to each other, leading to the formation of a barrier that prevents the penetration of most molecules via transcellular routes. Evidence has indicated that seizure-induced vascular endothelial growth factor (VEGF) type 2 receptor (VEGFR-2) pathway activation weakens TJs, inducing vasodilatation and increasing vascular permeability and subsequent brain injury. The present study focused on investigating the expression levels of VEGF-related (VEGF-A and VEGFR-2) and TJ-related proteins (claudin-5, occludin and ZO-1) in the neocortical microvasculature of patients with drug-resistant temporal lobe epilepsy (TLE). The results obtained from hippocampal sclerosis TLE (HS-TLE) patients were compared with those obtained from patients with TLE secondary to lesions (lesion-TLE) and autopsy samples. The Western blotting and immunofluorescence results showed that VEGF-A and VEGFR-2 protein expression levels were increased in HS-TLE and lesion-TLE patients compared to autopsy group. On the other hand, claudin-5 expression was higher in HS-TLE patients and lesion-TLE patients than autopsies. The expression level of occludin and ZO-1 was decreased in HS-TLE patients. Our study described modifications to the integrity of the BBB that may contribute to the pathogenesis of TLE, in which the VEGF system may play an important role. We demonstrated that the same modifications were present in both HS-TLE and lesion-TLE patients, which suggests that seizures modify these systems and that they are not associated with the establishment of epilepsy.

Effect of the Single and Combined Use of Curcumin and Piperine on Growth Performance, Intestinal Barrier Function, and Antioxidant Capacity of Weaned Wuzhishan Piglets.

This study was conducted to evaluate effects of the single and combined use of curcumin (CUR) and piperine (PIP) on performance, intestinal barrier function, and antioxidant capacity of weaned piglets. A total of 50 Wuzhishan piglets weaned at 35 days of age were randomly assigned to five groups receiving a corn–soybean basal diet (CON), the basal diet supplemented with 50 mg/kg piperine, 200 mg/kg curcumin (low-CUR), 200 mg/kg curcumin + 50 mg/kg piperine (PIP + CUR), and 300 mg/kg curcumin (high-CUR), respectively. The results showed that the feed/gain ratio (F/G) and plasma d-lactate and diamine oxidase activity (DAO) of the CUR + PIP and high-CUR groups were lower than those of the CON group (all P < 0.05), while the jejunum and ileum villus height, the villus height/crypt depth ratio, and the messenger RNA (mRNA) expression levels of occludin, claudin-1, and zonula occluden-1 in jejunal and ileal mucosa were higher in the CUR + PIP and high-CUR groups than in the CON group (all P < 0.05). Moreover, the piglets in the CUR + PIP and high-CUR groups had higher serum and intestinal mucosa activity of superoxide dismutase and glutathione peroxidase and lower malonaldehyde concentration than piglets in the CON group (all P < 0.05). The above parameters were not significantly different between the CUR + PIP and high-CUR groups (P > 0.05). In conclusion, the combination of CUR and PIP seemed to be as advantageous as high-CUR to piglets, but it was more effective than the single use of CUR and PIP. These data indicated that the basal diet supplemented with CUR + PIP or high-CUR could improve the intestinal permeability and suppress oxidative stress of weaned Wuzhishan piglets.

Tumor necrosis factor ligand-related molecule 1A affects the intestinal mucosal barrier function by promoting Th9/interleukin-9 expression.

ObjectivesTo investigate the effect of tumor necrosis factor ligand-related molecule 1A (TL1A) on the intestinal mucosal barrier in mice with chronic colitis.MethodsMale TL1A-overexpressing transgenic mice and male C57BL/6 wild-type mice were used to establish a dextran sodium sulfate (DSS)-induced colitis model. The expression of occludin and claudin-1 was observed. Bacterial distribution in the intestinal mucosa and Th9/interleukin (IL)-9 expression were detected. In vitro co-culture systems of naive CD4+ T cells and Caco-2 cells were established and TL1A was added. Changes in transepithelial electrical resistance and IL-9 expression were measured. CD4+IL-9 cells were detected by flow cytometry.ResultsDSS mice showed a significant down-regulation of occludin and claudin-1 compared with controls. Expression levels of occludin, zonulin-1, and claudin-1 in the Caco-2+TGF-β+IL-4+TL1A group were significantly lower than in the Caco-2+TGF-β+IL-4 group. Bacterial distribution was clearly disordered in the DSS group. Transmembrane resistance of the Caco-2+TGF-β+IL-4+TL1A group was significantly lower and IL-9 expression significantly higher than in the Caco-2+TGF-β+IL-4 group.ConclusionsTL1A overexpression promotes destruction of the intestinal mucosal barrier in mice with chronic colitis. The underlying mechanism may be associated with the promoting role of TL1A in Th9/IL-9 expression, which further destroys the mucosal barrier.

Effect of peritoneal dialysis solution with different pyruvate concentrations on intestinal injury.

To investigate the effects of direct peritoneal resuscitation with pyruvate-peritoneal dialysis solution (Pyr-PDS) of different concentrations combined with intravenous resuscitation on acid–base imbalance and intestinal ischemia reperfusion injury in rats with hemorrhagic shock. Sixty rats were randomly assigned to group SHAM, group intravenous resuscitation, and four direct peritoneal resuscitation groups combined with intravenous resuscitation: group NS, LA, PY1, and PY2, that is, normal saline, lactate-PDS (Lac-PDS), lower concentration Pyr-PDS (Pyr-PDS1), and higher concentration Pyr-PDS (Pyr-PDS2), respectively. Two hours after hemorrhagic shock and resuscitation, the pH, oxygen partial pressure, carbon dioxide partial pressure (PCO2), base excess, and bicarbonate ion concentration (HCO3−) of the arterial blood were measured. The intestinal mucosal damage index and intercellular adhesion molecule 1 (ICAM-1), tumor necrosis factor-α, interleukin-6, zonula occludens-1, claudin-1, and occludin levels in intestinal issues were detected. Two hours after resuscitation, group PY2 had higher mean arterial pressure, pH, oxygen partial pressure, and base excess and lower PCO2of arterial blood than group PY1 ( P &lt; 0.05). Tumor necrosis factor-α and interleukin-6 levels in group PY2 were significantly lower than those in group PY1 ( P &lt; 0.05). Zonula occludens-1, claudin-1, and occludin expression levels were significantly higher in group PY2 than in group PY1 ( P &lt; 0.05). Direct peritoneal resuscitation with Pyr-PDS2 combined with intravenous resuscitation enhanced the hemodynamics, improved the acid–base balance, and alleviated intestinal ischemia reperfusion injury from hemorrhagic shock and resuscitation in rats. The mechanisms might include correction of acidosis, inhibition of inflammatory response, enhancement of systemic immune status, regulation of intestinal epithelial permeability, and maintenance of intestinal mucosal barrier function.Impact statementHemorrhagic shock is a life-threatening condition after trauma or during surgery. Acid–base imbalance and intestinal ischemia reperfusion injury are two significant causes in the pathogenetic process and multiple organ dysfunction. As a result, it is urgent and necessary to find an effective method of resuscitation in order to reverse the acid–base imbalance and protect organ function. This current study confirmed the protection against hypoxic acidosis and intestinal ischemia reperfusion injury by peritoneal resuscitation with pyruvate combined with intravenous resuscitation in rats with hemorrhagic shock. And the peritoneal dialysis solution with pyruvate of high concentration plays a crucial role in the process. It provided a new idea and possible direction of fluid resuscitation for alleviating organ injuries, protecting organ functions, and improving clinical prognosis after hemorrhagic shock and resuscitation.

The effects and combinational effects of Bacillus subtilis and montmorillonite on the intestinal health status in laying hens

This study was conducted to evaluate the effects and combinational effects of Bacillus subtilis (BS) and montmorillonite (MMT) on laying performance, gut mucosal oxidation status, and intestinal immunological and physical barrier functions of laying hens. Three hundred sixty laying hens (29-week-old) were randomly assigned to a 2 × 2 factorial arrangement of treatments (n = 6) for 10 wk as follows: (1) basal diet; (2) the basal diet plus 5 × 108 cfu BS/kg; (3) the basal diet plus 0.5 g MMT/kg; and (4) the basal diet plus 5 × 108 cfu BS/kg and 0.5 g MMT/kg. Dietary supplementation with BS increased egg production and egg mass, the activities of catalase (CAT) and total superoxide dismutase in the intestinal mucosa, and villus height and villus height-to-crypt depth ratio of the jejunum (P < 0.05) but downregulated the mRNA expression levels of toll-like receptor 4 and myeloid differentiation factor 88 (MyD88) in the duodenum and jejunum, interleukin 1 beta in the duodenum, and nuclear factor kappa B P65 (NF-κB P65) and tumor necrosis factor alpha in the jejunum (P < 0.05). Dietary supplementation with MMT increased egg production and egg mass, the concentration of secretory immunoglobulin A in the duodenum, and the occludin mRNA expression level in the jejunum (P < 0.05) but reduced feed conversion ratio, malondialdehyde concentration in the duodenum and jejunum, and the mRNA expression level of MyD88 in the jejunum (P < 0.05). In addition, there was an interaction effect between BS and MMT supplementation on the CAT activity and the MyD88 mRNA expression level in the duodenum and the mRNA expression level of occludin in the jejunum (P < 0.05). In conclusion, dietary BS and MMT and their combination may improve the intestinal health status of laying hens, which may contribute to the increase in hens' laying performance.

Specific role of the tight junction proteins occludin and claudin-5 on the blood–brain barrier during Listeria monocytogenes infection

To investigate the blood-brain barrier (BBB) permeability of mice after Listeria monocytogenes infection for further study on the mechanism of L. monocytogenes crossing the BBB, a mouse model was established and Evans blue assay was performed to assess the BBB disruption. Using relative quantitative real-time PCR, the RNA expression of Zonula occludens-1 (ZO-1), occludin and claudin-5 were detected. In addition, the protein expression level of ZO-1, occludin and claudin-5 were detected by immunohistochemistry and western blot. The extravasation of Evans blue dye was significantly different between 24 h and 96 h (P &lt; 0.05). The mRNA expression of occludin and claudin-5 were down-regulated than that of the control group at each sampling point (P &lt; 0.05) and ZO-1 showed a significant change at 96 h (P &lt; 0.05). In addition, the protein expression level of occludin and claudin-5 decreased significantly at 48 h and 96 h (P &lt; 0.05) by immunohistochemistry and western blot, compared with the control, while ZO-1 was almost unchanged (P &gt; 0.05). All results indicating that the tight junction integrity of endothelial cells was destroyed and BBB permeability was enhanced in the process of L. monocytogenes infection, and this change was related to the decrease of the expression occludin and claudin-5.

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

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

Biogenic selenium nanoparticles synthesized by Lactobacillus casei ATCC 393 alleviate diquat-induced intestinal barrier dysfunction in C57BL/6 mice through their antioxidant activity.

Selenium (Se) as an essential micronutrient plays a crucial role in human health. Biogenic selenium nanoparticles (SeNPs) possess attractive biological properties, biocompatibility, stability and low-toxicity. This study was aimed to investigate the protective effect of biogenic SeNPs of size 50-80 nm synthesized by Lactobacillus casei ATCC 393 (L. casei ATCC 393) on diquat-induced intestinal barrier dysfunction in C57BL/6 mice and the intrinsic mechanisms. Our results showed that oral administration of SeNPs significantly inhibited the increase of the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), diamine oxidase (DAO) and d-lactic acid (d-LA) levels induced by diquat, and increased the total superoxide dismutase (T-SOD), thioredoxin reductase (TrxR) and glutathione peroxidase (GSH-Px) activities in serum and jejunum. Moreover, SeNPs increased the number of goblet cells, decreased the production of reactive oxygen species (ROS), maintained the mitochondrial functions, and improved the expression levels of occludin and claudin-1 in jejunum compared to the diquat-induced oxidative stress model group. In addition, SeNPs activated the nuclear factor (erythroid-derived-2)-like 2 (Nrf2), and improved the protein levels of heme oxygenase (HO)-1 and NADPH dehydrogenase (NQO)-1 compared to other treatment groups. These results suggested that biogenic SeNPs synthesized by L. casei ATCC 393 can protect the intestinal barrier function against oxidative damage via Nrf2-mediated signaling pathway.

Hydrogen treatment prevents lipopolysaccharide-induced pulmonary endothelial cell dysfunction through RhoA inhibition

BackgroundPulmonary microvascular endothelial cells (PMVECs) are initial targets of sepsis-induced acute lung injury (ALI). During the apoptosis of PMVECs, tight junctions (TJ) and adherens junctions (AJ) are firstly damaged. Previous studies have suggested hydrogen treatment can protect lung microvasculature of mice from sepsis-induced endothelial dysfunction and maintain the coherence of pulmonary endothelium, but the underlying mechanism remains unclear. MethodsWe investigated the role of hydrogen-rich medium on regulating intercellular junction proteins under lipopolysaccharide (LPS) treatment which mimicked sepsis in vitro. Changes of cytoskeleton regulatory protein ROCK and RhoA as well as PMVEC apoptotic rate were examined. ResultsLPS treatment reduced the expression levels of occludin and VE-cadherin in PMVECs, while hydrogen-rich medium can recover these changes. Furthermore, H2 can significantly ameliorate the excessive expression of ROCK and RhoA under sepsis-mimicking condition. The application of RhoA activator U-46619 resulted in a more significant elevation in cell apoptotic rate as well as reduction in the expression of junctional proteins. Using H2 can almost completely inhibit the effects of RhoA activator. ConclusionsOur findings suggest that RhoA is a crucial protein in the signaling pathway of LPS-induced endothelial cell dysfunction. Hydrogen treatment can prevent LPS-induced junctional injury and cell death by inhibiting the activity of RhoA.

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

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

Low Expression of Occludin in the Melanoma Patient.

Background & Objective:Malignant melanoma is the fatal cutaneous neoplasm which is curable by the early diagnosis. The expression of occludin protein which is an integral membrane protein is altered in an epithelial-to-mesenchymal transition. Although, recent studies provide sufficient evidence supporting the functional importance of occludin in cancer, the prognostic significance of occludin expression levels in melanoma remains obscure. The aim of this study was to determine occludin expression level and its correlation with clinicopathological features of the patients with melanoma. Methods:The occludin mRNA level was compared between paraffin-embedded tissues of 40 patients with melanoma and 10 subjects with normal skin. The quality and quantity of the RNA was determined and occludin expression level was measured using Real-time PCR and ∆∆CT computational technique.Results:The occludin mRNA level reduced five-fold in the melanoma patients compared to the control group (P=0.000). No significant difference was observed between male and female cases (P=0.533). No significant correlation was observed between occludin mRNA level, mitotic count (P=0.252), and Breslow levels (P=0.171) Conclusion:We can conclude that down-regulation of occludin expression in the patients with melanoma is a hallmark of cancer progression and it might be used as a prognostic factor. No significant correlation was found between occludin gene expression and clinicopathological characteristics including Clark level, Breslow staging, mitotic count, age and gender (P<0.05).

Complex TNF-α B cell epitope MAP vaccine alleviates murine ulcerative colitis.

The present study aimed to develop a tumor necrosis factor‑α (TNF‑α) B‑cell epitope/IL‑1β helper T lymphocyte epitope complex MAP vaccine for the alleviation of ulcerative colitis (UC) in mice. The B cell epitopes of murine TNF‑α (mTNF‑α) were predicted in silico and coupled with the universal interleukin 1β (IL‑1β) helper T‑cell epitope peptide VQGEESNDK to synthesize the eight‑branched MAP vaccine. Then, the immunological effects of the MAP vaccine were assessed in vitro and in vivo, as well as its impacts on DAI index, serum DAO levels, colon tissue tight junction protein amounts, ultrastructural changes, and MPO activity in BALB/c mice with UC. The amino acids LTLRSSSQNSSDKPV at positions 78‑92 of mTNF‑α may constitute the dominant B cell epitope. Based on this finding, an eight‑branched peptide structure, the TNF‑α B‑cell epitope/IL‑1β helper T‑cell epitope complex MAP vaccine, was synthesized. Indirect ELISA confirmed that MAP had a high affinity with commercialized mTNF‑α antibodies. Meanwhile, MAP induced high specific antibody titers in vivo, reduced the DAI score, serum MPO activity, colorectal lymph node colony count, ultrastructural injuries, colon tissue histological index score and MPO activity in UC mice, while increasing the expression levels of occludin, claudin1 and ZO1 in colon tissues. The synthetic complex MAP vaccine has good antigenicity and immunogenicity, and can alleviate UC in mouse models.