Expression Levels Of Claudin-1 Research Articles

Integrative metabolomics and gut microbiota analyses reveal the protective effects of DHA-enriched phosphatidylserine on bisphenol A-induced intestinal damage

The protective effects of docosahexaenoic acid-enriched phosphatidylserine (DHA-PS, 50 or 100 mg/kg) on bisphenol A (BPA)-induced intestinal damage were assessed. The biochemical indices, pathological examination, non-targeted metabolomics integrated with gut microbiota analysis, and immunofluorescence analysis were used to investigate the protective effects of DHA-PS and its underlying regulatory mechanism. The DHA-PS treatment improved the pathology of intestinal tract and increased the expression levels of Claudin-1, Occludin, and ZO-1. In addition, the DHA-PS treatment also notably decreased the levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, and increased the antioxidant enzymes activities. Moreover, DHA-PS treatment increased the relative abundances of Akkermansia, Alistipes, Butyricicoccus, Coriobacteriaceae_UCG-002, Enterorhabdus, and Lachnospiraceae_UCG-006. DHA-PS treatment alleviated BPA-induced intestinal damage by regulating the kynurenine pathway of tryptophan, lipid, and arachidonic acid metabolisms. Overall, this study suggested that DHA-PS could alleviate BPA-induced intestinal damage by enhancing the intestinal barrier integrity, improving gut microbial composition and metabolites, and inhibiting the TLR4/NF-κB pathways.

Dexmedetomidine alleviates blood-brain barrier disruption in rats after cerebral ischemia-reperfusion by suppressing JNK and p38 MAPK signaling.

Dexmedetomidine displays multiple mechanisms of neuroprotection in ameliorating ischemic brain injury. In this study, we explored the beneficial effects of dexmedetomidine on blood-brain barrier (BBB) integrity and neuroinflammation in cerebral ischemia/reperfusion injury. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1.5 h and reperfusion for 24 h to establish a rat model of cerebral ischemia/reperfusion injury. Dexmedetomidine (9 􀁐g/kg) was administered to rats 30 min after MCAO through intravenous injection, and SB203580 (a p38 MAPK inhibitor, 200 􀁐g/kg) was injected intraperitoneally 30 min before MCAO. Brain damages were evaluated by 2,3,5-triphenyltetrazolium chloride staining, hematoxylin-eosin staining, Nissl staining, and brain water content assessment. BBB permeability was examined by Evans blue staining. Expression levels of claudin-5, zonula occludens-1, occludin, and matrix metalloproteinase-9 (MMP-9) as well as M1/M2 phenotypes-associated markers were assessed using immunofluorescence, RT-qPCR, Western blotting, and gelatin zymography. Enzyme-linked immunosorbent assay was used to examine inflammatory cytokine levels. We found that dexmedetomidine or SB203580 attenuated infarct volume, brain edema, BBB permeability, and neuroinflammation, and promoted M2 microglial polarization after cerebral ischemia/reperfusion injury. Increased MMP-9 activity by ischemia/reperfusion injury was inhibited by dexmedetomidine or SB203580. Dexmedetomidine inhibited the activation of the ERK, JNK, and p38 MAPK pathways. Moreover, activation of JNK or p38 MAPK reversed the protective effects of dexmedetomidine against ischemic brain injury. Overall, dexmedetomidine ameliorated brain injury by alleviating BBB permeability and promoting M2 polarization in experimental cerebral ischemia/reperfusion injury model by inhibiting the activation of JNK and p38 MAPK pathways.

The effect of gut microbiota-derived carnosine on mucosal integrity and immunity in broiler chickens challenged with Eimeria maxima

In the first study, an in vitro culture system was developed to investigate the effects of carnosine on macrophage proinflammatory cytokine response using an established chicken macrophage cell line (CMC), gut integrity using a chicken intestinal epithelial cell line (IEC), muscle differentiation in quail muscle cells (QMCs) and primary chicken embryonic muscle cells (PMCs), and direct anti-parasitic effect against Eimeria maxima sporozoites. Cells to be tested were seeded in 24-well plates and treated with carnosine at 4 different concentrations (0.1, 1.0, and 10.0 µg). After 18 h of incubation, cells were harvested to measure gene expression of proinflammatory cytokines in CMC, tight junction (TJ) proteins in IECs, and muscle cell growth markers in QMCs and PMCs. In vivo trials were conducted to investigate the effect of dietary carnosine on disease parameters in broiler chickens challenged with E. maxima. One hundred and twenty male broiler chickens (0-day-old) were allocated into four treatment groups: (1) basal diet without infection (NC), (2) basal diet with E. maxima infection (PC), (3) carnosine at 10.0 mg/kg feed with PC (HCS), and (4) carnosine at 1.0 mg/kg feed with PC (LCS). All groups except NC were orally infected with E. maxima on day 14. Jejunal samples were collected for lesion scoring and jejunum gut tissues were used for transcriptomic analysis of cytokines and TJ proteins. In vitro, carnosine treatment significantly decreased IL-1β gene expression in CMC following LPS stimulation. In vivo feeding studies showed that dietary carnosine increased BW and ADG of chickens in E. maxima-infected groups and reduced the jejunal lesion score and fecal oocyst shedding in HCS group. Jejunal IL-1β, IL-8, and IFN-γ expression were suppressed in the HCS group compared to PC. The expression levels of claudin-1 and occludin in IECs were also increased in HCS following carnosine treatment. In conclusion, these findings highlight the beneficial effects of dietary carnosine supplementation on intestinal immune responses and gut barrier function in broiler chickens exposed to E. maxima infection.

Electroacupuncture can Modify Stress, Low-Grade Inflammation in the Duodenum, and Damage to the Intestinal Barrier in Rats with Functional Dyspepsia through the CRF Signaling Pathway

In the domain of functional gastrointestinal disorders, Functional Dyspepsia (FD) stands out due to its widespread occurrence internationally. Historically, electroacupuncture (EA) has been employed as a therapeutic modality for FD, demonstrating notable clinical efficacy. This research aimed to delve into the impact of EA on stress responses, minor duodenal inflammatory processes, and the integrity of the intestinal barrier within FD-affected rodent models while also elucidating the underlying mechanisms. Thirty-six male Wistar rats were evenly distributed into three cohorts: a normal, a modeled FD, and an EA treatment group. The FD condition in the rats, barring those in the normal, was induced through a series of multifactorial procedures. For the EA cohort, the rats received electroacupuncture at the acupoints RN12 (Zhongwan) and ST36 (Zusanli) for 20 minutes daily over a span of one week. The gastric residue rate (GRR), intestinal propulsion rate (IPR), and changes in emotional state were measured in each group of rats. Additionally, serum levels of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and corticosterone (CORT) were detected, and the duodenal inflammatory condition and intestinal mucosal barrier status were observed through staining and fluorescence. The expression levels of Claudin-1, Junctional Adhesion Molecule 1 (JAM-1), Corticotropin-Releasing Factor (CRF), and Corticotropin-Releasing Factor Receptor 1 (CRF-R1) were also detected. The study demonstrated that EA had a positive effect on body weight and food intake, GRR, and IPR in FD rats. Additionally, the EA group showed a decrease in serum levels of CRH, ACTH, and CORT, as well as a decrease in the number of duodenal mast cells and tryptase content. Furthermore, the expression of tight junction proteins Claudin-1 and JAM-1 was increased in the EA group compared to the model group. EA also reduced the levels of CRF and CRF-R1 in the hypothalamus and duodenum. EA has been shown to improve the stress state of FD rats, inhibit the activation of mast cells in the duodenum, and reduce low-grade inflammatory response and damage to the intestinal mucosal barrier. It is believed that EA achieves these effects by modulating the expression of CRF and its receptors in the brain-gut interaction pathway through the CRF signaling pathway. This provides a new approach to treating FD.

Application of direct electric current to the corneal and conjunctival epithelia regulates the tight junctional assembly for ocular iontophoretic drug delivery

Objectives: In this study, we determined how iontophoresis (IP) affects tight junctions (TJs) in isolated rabbit corneas and conjunctiva.
 Methods: Direct electric current in the range of 0.5–2.0 and 0.5–10 mA/cm2 were applied to the cornea and conjunctiva, respectively, for 30 min. The localization and expression levels of TJ-associated proteins were assessed before and after the application of the electric currents using immunostaining and western blotting.
 Results: In both corneal and conjunctival epithelia, the localization of proteins, such as claudin-1, claudin-4, occludin, and ZO-1, was temporarily altered by anodal and cathodal IP; however, the protein relocalization was slower at higher currents. Additionally, in both anodal and cathodal IP, the expression levels of claudin-1 and occludin in the cornea and conjunctiva remained unchanged after the application of the electric currents compared with those before.
 Conclusion: Our results indicated that the application of a direct electric current temporarily regulated TJ assemblies without altering the levels of TJ-associated proteins in both the cornea and conjunctiva. This temporary weakening of the paracellular barrier by the current may be responsible for the enhanced drug transport across the cornea and conjunctiva induced by ocular IP.
 Keywords: ocular drug delivery, iontophoresis, electric current, cornea, conjunctiva, tight junction

Mechanobiological Mechanisms Involved in the Regualation of the Blood-Brain Barrier by Fluid Shear Force

To explore the mechanobiological mechanism of fluid shear force (FSF) on the protection, injury, and destruction of the structure and function of the blood-brain barrier (BBB) under normal physiological conditions, ischemic hypoperfusion, and postoperative hyperperfusion conditions. BBB is mainly composed of brain microvascular endothelial cells. Rat brain microvascular endothelial cells (rBMECs) were used as model cells to conduct the investigation. rBMECs were seeded at a density of 1×105 cells/cm2 and incubated for 48 h. FSF was applied to the rBMECs at 0.5, 2, and 20 dyn/cm2, respectively, simulating the stress BBB incurs under low perfusion, normal physiological conditions, and high FSF after bypass grafting when there is cerebral vascular stenosis. In addition, a rBMECs static culture group was set up as the control (no force was applied). Light microscope, scanning electron microscope (SEM), and laser confocal microscope (LSCM) were used to observe the changes in cell morphology and cytoskeleton. Transmission electron microscope (TEM) was used to observe the tight junctions. Immunofluorescence assay was performed to determine changes in the distribution of tight junction-associated proteins claudin-5, occludin, and ZO-1 and adherens junction-associated proteins VE-cadherin and PECAM-1. Western blot was performed to determine the expression levels of tight junction-associated proteins claudin-5, ZO-1, and JAM4, adherens junction-associated protein VE-cadherin, and key proteins in Rho GTPases signaling (Rac1, Cdc42, and RhoA) under FSF at different intensities. Microscopic observation showed that the cytoskeleton exhibited disorderly arrangement and irregular orientation under static culture and low shear force (0.5 dyn/cm2). Under normal physiological shear force (2 dyn/cm2), the cytoskeleton was rearranged in the orientation of the FSF and an effective tight junction structure was observed between cells. Under high shear force (20 dyn/cm2), the intercellular space was enlarged and no effective tight junction structure was observed. Immunofluorescence results showed that, under low shear force, the gap between the cells decreased, but there was also decreased distribution of tight junction-associated proteins and adherens junction-associated proteins at the intercellular junctions. Under normal physiological conditions, the cells were tightly connected and most of the tight junction-associated proteins were concentrated at the intercellular junctions. Under high shear force, the gap between the cells increased significantly and the tight junction and adherens junction structures were disrupted. According to the Western blot results, under low shear force, the expression levels of claudin-5, ZO-1, and VE-cadherin were significantly up-regulated compared with those of the control group (P<0.05). Under normal physiological shear force, claudin-5, ZO-1, JAM4, and VE-cadherin were highly expressed compared with those of the control group (P<0.05). Under high shear force, the expressions of claudin-5, ZO-1, JAM4, and VE-cadherin were significantly down-regulated compared with those of the normal physiological shear force group (P<0.05). Under normal physiological shear force, intercellular expressions of Rho GTPases proteins (Rac1, Cdc42, and RhoA) were up-regulated and were higher than those of the other experimental groups (P<0.05). The expressions of Rho GTPases under low and high shear forces were down-regulated compared with that of the normal physiological shear force group (P<0.05). Under normal physiological conditions, FSF helps maintain the integrity of the BBB structure, while low or high shear force can damage or destroy the BBB structure. The regulation of BBB by FSF is closely related to the expression and distribution of tight junction-associated proteins and adherens junction-associated proteins.

Effects of puerarin as a feed additive on the laying performance, egg quality, endocrine hormones, antioxidant capacity, and intestinal morphology of aged laying hens

The aim of this study was to investigate the effects of puerarin (Pue), a phytoestrogen, on the production performance, egg quality, endocrine hormones, antioxidant capacity, and intestinal morphology in aged laying hens. A total of 180 Hy-Line Brown hens aged 480 d were randomly divided into 4 groups (n = 45 per group) and fed 0, 200, 400, and 800 mg/kg of Pue (Con, L-Pue, M-Pue, and H-Pue, respectively) during a 42-d experiment. Compared with the Con treatment, supplementation with H-Pue improved laying performance and egg quality by significantly increasing egg production, average egg weight, albumen height, yolk weight, and Haugh unit (P < 0.05) while decreasing the feed conversion ratio (P < 0.05). A diet supplemented with H-Pue significantly decreasing serum total triglycerides, total cholesterol, and low-density lipoprotein cholesterol, alanine aminotransferase (P < 0.05), and significantly increasing serum levels of follicle-stimulating hormone, luteinizing hormone and progesterone (P < 0.05). Antioxidant activity was improved by significantly increasing the activity of total antioxidant capacity, glutathione peroxidase and catalase but decreasing malondialdehyde levels in serum, jejunum, and ileum (P < 0.05), and superoxide dismutase activity exhibited a significantly increase in the jejunum and ileum (P < 0.05). Villus height and the ratio of villus height to crypt depth (P < 0.05) were significantly increased in the jejunum and ileum. In the jejunal and ileal mucosa, the three treatment groups increased the mRNA expression levels of Claudin-1 and Claudin-2 compared with Con (P < 0.05), and no significant effect was observed on the expression of Occludin and ZO-1. The results showed that dietary supplementation with Pue could improve the laying performance, egg quality, antioxidant capacity, hormonal profile, and intestinal morphology of aged laying hens.

Protective Effects of Danmu Extract Syrup on Acute Lung Injury Induced by Lipopolysaccharide in Mice through Endothelial Barrier Repair.

To investigate the effects of Danmu Extract Syrup (DMS) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and explore the mechanism. Seventy-two male Balb/C mice were randomly divided into 6 groups according to a random number table (n=12), including control (normal saline), LPS (5 mg/kg), LPS+DMS 2.5 mL/kg, LPS+DMS 5 mL/kg, LPS+DMS 10 mL/kg, and LPS+Dexamethasone (DXM, 5 mg/kg) groups. After pretreatment with DMS and DXM, the ALI mice model was induced by LPS, and the bronchoalveolar lavage fluid (BALF) were collected to determine protein concentration, cell counts and inflammatory cytokines. The lung tissues of mice were stained with hematoxylin-eosin, and the wet/dry weight ratio (W/D) of lung tissue was calculated. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1 β in BALF of mice were detected by enzyme linked immunosorbent assay. The expression levels of Claudin-5, vascular endothelial (VE)-cadherin, vascular endothelial growth factor (VEGF), phospho-protein kinase B (p-Akt) and Akt were detected by Western blot analysis. DMS pre-treatment significantly ameliorated lung histopathological changes. Compared with the LPS group, the W/D ratio and protein contents in BALF were obviously reduced after DMS pretreatment (P<0.05 or P<0.01). The number of cells in BALF and myeloperoxidase (MPO) activity decreased significantly after DMS pretreatment (P<0.05 or P<0.01). DMS pre-treatment decreased the levels of TNF-α, IL-6 and IL-1 β (P<0.01). Meanwhile, DMS activated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway and reversed the expressions of Claudin-5, VE-cadherin and VEGF (P<0.01). DMS attenuated LPS-induced ALI in mice through repairing endothelial barrier. It might be a potential therapeutic drug for LPS-induced lung injury.

A novel Sagittaria sagittifolia L. polysaccharides mitigate DSS-induced colitis via modulation of gut microbiota and MAPK/NF-κB signaling pathways

Sagittaria sagittifolia L. polysaccharides possess anti-inflammatory, antioxidant, and immune-modulatory properties. In this study, we identified a novel S. sagittifolia L. polysaccharide, named PSSP-1, and evaluated its potential in alleviating dextran sulfate sodium (DSS)-induced colitis in a mouse model. The results demonstrated that administration of PSSP-1 at doses of 100, 200, and 400 mg/kg·bw significantly reduced the disease activity index (DAI) and suppressed the expression of inflammatory cytokines in UC mice. Furthermore, PSSP-1 treatment upregulated the expression levels of claudin-1, occludin, and ZO-1, and promoted the diversity and abundance of beneficial gut microbiota, including Lactobacillus and Candidatus_Saccharimonas, while reducing the levels of Bacteroidetes and Verrucomicrobiota. Particularly, the Lactobacillus_johnsonii species may play a potentially significant role in modulating colitis. Subsequently, there was a significant increase in the levels of short-chain fatty acids (SCFAs). Additionally, the correlation analyses revealed positive associations between PSSP-1 supplementation and Nitrosospira and Dialister, which are implicated in gut inflammation. Mechanistically, PSSP-1 intervention inhibited the protein phosphorylation of key molecules in the MAPK and NF-κB signaling pathways. Collectively, these findings suggest that PSSP-1 mitigates colitis symptoms by repairing the intestinal barrier, promoting microbial metabolism, and regulating the gut microbiota-MAPK/NF-κB signaling pathways.

Dietary methionine sources and levels modulate the intestinal health status of broiler chickens

Given the key role of methionine in biological processes, adequate methionine should be provided to meet the nutritional requirements. DL-2-hydroxy-4-(methylthio)-butanoic acid (DL-HMTBA) has been considered as an important source of methionine. However, the effects of different sources and levels of methionine on the intestinal health status have not been clarified yet. An experiment was carried out to investigate the effects of different dietary sources and levels of methionine on the intestinal epithelial barrier, inflammatory cytokines expression, ileal morphology, microbiota composition, and cecal short chain fatty acids (SCFA) profiles. For this purpose, 720 male Arbor Acre broiler chicks at 1 d old were randomly assigned to a 2 × 3 factorial arrangement with 2 methionine sources (DL-methionine and DL-HMTBA) and 3 total sulfur amino acids (TSAA) levels (80%, 100%, and 120% of Arbor Acre recommendation). The results showed that DL-HMTBA supplementation promoted intestinal physical barrier at both gene expression level of claudin-1 and serum diamine oxidase level (P < 0.05), and the inflammatory cytokine IL-6 mRNA expression was down-regulated by dietary DL-HMTBA supplementation compared with the DL-methionine group (P < 0.05). Meanwhile, an upregulated gene expression of claudin-1 and zonula occluden-1 (ZO-1) were observed in the low-TSAA treatment on d 14 (P < 0.05), whereas this treatment increased the expression of IL-1β and IL-6 (P < 0.05). Villus height to crypt depth ratio was high (P < 0.05) in the middle-level TSAA group. Furthermore, DL-HMTBA supplementation optimized the microbiota of the ileum especially the relative abundance of Lactobacillus, where the digestion and absorption were completed, and elevated the concentrations of SCFA (acetate, propionate, and butyrate) in the cecal content on d 21 (P < 0.01). In conclusion, dietary DL-HMTBA supplementation improved the intestinal barrier function, immune homeostasis and optimized the microbiota to promote intestinal health status in broiler chickens.

Effects of stimbiotic supplementation on gut health, immune response, and intestinal microbiota in weaned piglets challenged with E. coli.

In order to make piglet diets more effective, it is necessary to investigate effective methods for breaking down xylan in cereal. The objective of this study was to determine the effects of dietary stimbiotic (STB) supplementation on growth performance, intestinal morphology, immune response and intestinal microbiota in weaned piglets. A total of 24 (Duroc × Yorkshire × Landrace) weaned pigs (initial body weight of 8.01 ± 0.38 kg and 28 ± 3 d old), were assigned to 4 treatments with 6 replicates per treatment. Pigs were housed in individual pens for 17 days, including 5 days adaption period and 12 days after the first Escherichia coli (E. coli) challenge. The experiment was conducted in a 2 × 2 factorial arrangement of treatments consisting of two levels of challenge (challenge and non-challenge) and two levels of STB (0 and 0.5 g/kg diet). Supplementations of STB 0.5 g/kg improved the gain to feed ratio (G:F) (P < 0.05) in piglets challenged with shiga toxigenic E. coli (STEC). STB supplementation decreased (P < 0.05) white blood cells, neutrophils, lymphocytes, and expression levels of tumor necrosis factor-alpha and interleukin-6. Supplementation of STB improved (P < 0.05) the lymphocytes and neutrophils in piglets challenged with STEC on 12 dpi. Supplementation of STB also improved (P < 0.05) the villus height to-crypt depth ratio of ileum in piglets challenged with STEC. Supplementation of STB increased (P < 0.05) the expression levels of claudin-1 of ileum. In genus level, supplementation of STB increased (P < 0.001) the abundance of Prevotella compared to non-supplementation of STB groups in pre-inoculation period. Also, supplementation of STB decreased (P < 0.05) the abundance of Faecalibacterium and Eubacterium_coprostanoligenes_group compared to non-supplementation of STB groups in post-inoculation period. In phylum level, supplementation of STB increased (P < 0.05) the abundance of Desulfobacterota and Fibrobacterota in pre-inoculation period. E. coli challenge increased the abundance of Fibrobacterota compared to non-challenged group in post-inoculation period. In conclusion, these findings indicated that STB supplementation could alleviate a decrease of the performance, immune response, and inflammatory response in piglets induced by the STEC challenge.

Effect of fermented rapeseed meal on growth performance, nutrient digestibility, and intestinal health in growing pigs

To explore the effects of fermented rapeseed meal (FRSM) on growth performance and intestinal health, a total of 30 growing pigs were randomly allotted to three treatments consisting of corn-soybean meal diet (CSD), rapeseed meal diet (RSD), and fermented rapeseed meal diet (FRSD). Results showed that compared with RSD, FRSD feeding increased the average daily gain and final body weight in pigs (P < 0.01). Compared with RSD feeding, FRSD feeding elevated the apparent digestibility of crude protein, acid detergent fiber, and ether extract in pigs (P < 0.01). Moreover, the FRSD group exhibited greater apparent ileal digestibility of His, Thr, Lys, and Ser than the RSD group (P < 0.01). The digestible energy, metabolic energy, and nitrogen utilization were higher in the FRSD and CSD groups than in the RSD group (P < 0.01). As compared to the RSD, FRSD feeding decreased the serum concentration of leptin but significantly increased the concentrations of immunoglobulin (Ig) A, IgG, ghrelin, and enzyme activities of amylase, lipase, and trypsin in the pancreas (P < 0.05). Interestingly, the villus height, the ratio of villus height to crypt depth, and the activities of brush border enzymes (e.g., maltase and sucrase) in the small intestine were higher in the CSD and FRSD groups than in the RSD group (P < 0.05). As compared to the RSD, the FRSD feeding not only increased the expression level of the occludin in the small intestinal epithelium (P < 0.05) but also elevated the expression levels of claudin-1, MUC1, and PepT1 genes in the duodenum, and elevated the expression levels of SGLT1 and CAT1 genes in the jejunum (P < 0.05). Importantly, FRSD feeding significantly decreased the abundance of Escherichia coli, but increased the abundance of Lactobacillus and the content of butyrate in the cecum and colon (P < 0.05). These results indicated that compared with rapeseed meal, fermented rapeseed meal exhibited a positive effect on improving the growth performance and intestinal health in growing pigs, and the results may also help develop novel protein sources for animal nutrition and the feed industry.

Influences of wheat bran fiber on growth performance, nutrient digestibility, and intestinal epithelium functions in Xiangcun pigs

Dietary fiber (DF) has long been looked as an essential “nutrients” both for animals and humans as it can promote the intestinal tract development and modulate the intestinal epithelium functions and the gut microbiota. This study was conducted to investigate the influences of wheat bran fiber (WBF) on growth performance and intestinal epithelium functions in Xiangcun pigs. Twenty Xiangcun pigs with 60 days of age were divided to two groups and exposed to a basal diet (BD) or BD containing 4.3% wheat bran fiber (WFD). WFD improved the average daily gain (ADG) and feed-to-gain ratio (F:G) (p < 0.01). Moreover, WFD lowered the serum triglyceride (TC), d-lactate, and malonicdialdehyde (MDA) concentrations, but significantly improved the glutathione (GSH) activity and total antioxidant capacity (T-AOC) (p < 0.05). Interestingly, WFD observably improved the villus height (VH) and the villus height to crypt depth ratio (V/C) in the small intestine (p < 0.05). The jejunal sucrase and ileal maltase activities were higher in the WFD group (p < 0.05). WFD markedly elevated the tight junction protein ZO-1 and claudin-1 expression levels in the jejunum and ileum (p < 0.05). The sodium/glucose co-transporter 1 (SGLT1), glucose transporter 2 (GLUT2), and fatty acid transport proteins 4 (FATP-4) expression levels in jejunum and ileum were also elevated under WFD (p < 0.05). WFD decreased the IL-6 impression level in the duodenum and ileum, but significantly increased the IL-10 expression levels in jejunum and ileum (p < 0.05). Moreover, WFD reduced the abundance of E. coli, but elevated the abundances of beneficial microorganisms (e.g. Lactobacillus and Bacillus) and the production microbial metabolites (e.g. propionic acid and butyrate acid) in the cecum (p < 0.05).

Anti-Inflammatory and Transepithelial Transport Activities of Rapeseed (Brassica napus) Napin-Derived Dipeptide Thr-Leu in Caco-2 and RAW264.7 Cocultures

This study aimed to investigate the anti-inflammatory molecular activity of rapeseed napin-derived dipeptide Thr-Leu (TL) using Caco-2/RAW264.7 cell cocultures. This in vitro coculture intestinal inflammation model was used to assess the absorption, evolution, and anti-inflammatory effects of peptides. TL was absorbed by the intestinal epithelial cells with an apparent permeability of (2.48 ± 0.18) × 10-6 cm/s, primarily through the PepT1 pathway. TL treatment exerted anti-inflammatory and restorative effects on the impaired intestinal barrier function by enhancing the expression levels of occludin and ZO-1 in lipopolysaccharide (LPS)-induced Caco-2 cells. No significant change (P < 0.05) was detected in claudin-1 expression levels; however, the occludin expression levels were upregulated through the protein kinase C (PKC) signaling pathway. Compared with the LPS-induced group, TL (2.0 mM) reduced the levels of intracellular inflammation-related enzymes (iNOS: by 50.84%; COX-2: by 49.64%) on the coculture cell model. In addition, the interleukin (IL)-1β, IL-6, and TNF-α levels in RAW264.7 cells were significantly (P < 0.05) downregulated following TL treatment (2.0 mM) due to the suppression of the phosphorylation of the JNK-independent pathway on the basolateral side of the coculture cell model. These findings highlight the potential use of TL in functional foods or nutraceuticals to prevent intestinal inflammation.

Improving the response to oxaliplatin by targeting chemotherapy-induced CLDN1 in resistant metastatic colorectal cancer cells

BackgroundTumor resistance is a frequent cause of therapy failure and remains a major challenge for the long-term management of colorectal cancer (CRC). The aim of this study was to determine the implication of the tight junctional protein claudin 1 (CLDN1) in the acquired resistance to chemotherapy.MethodsImmunohistochemistry was used to determine CLDN1 expression in post-chemotherapy liver metastases from 58 CRC patients. The effects of oxaliplatin on membrane CLDN1 expression were evaluated by flow cytometry, immunofluorescence and western blotting experiments in vitro and in vivo. Phosphoproteome analyses, proximity ligation and luciferase reporter assays were used to unravel the mechanism of CLDN1 induction. RNAseq experiments were performed on oxaliplatin-resistant cell lines to investigate the role of CLDN1 in chemoresistance. The “one-two punch” sequential combination of oxaliplatin followed by an anti-CLDN1 antibody-drug conjugate (ADC) was tested in both CRC cell lines and murine models.ResultsWe found a significant correlation between CLDN1 expression level and histologic response to chemotherapy, CLDN1 expression being the highest in resistant metastatic residual cells of patients showing minor responses. Moreover, in both murine xenograft model and CRC cell lines, CLDN1 expression was upregulated after exposure to conventional chemotherapies used in CRC treatment. CLDN1 overexpression was, at least in part, functionally related to the activation of the MAPKp38/GSK3β/Wnt/β-catenin pathway. Overexpression of CLDN1 was also observed in oxaliplatin-resistant CRC cell lines and was associated with resistance to apoptosis, suggesting an anti-apoptotic role for CLDN1. Finally, we demonstrated that the sequential treatment with oxaliplatin followed by an anti-CLDN1 ADC displayed a synergistic effect in vitro and in in vivo.ConclusionOur study identifies CLDN1 as a new biomarker of acquired resistance to chemotherapy in CRC patients and suggests that a “one-two punch” approach targeting chemotherapy-induced CLDN1 expression may represent a therapeutic opportunity to circumvent resistance and to improve the outcome of patients with advanced CRC.

High concentrate diet induced inflammatory response and tight junction disruption in the mammary gland of dairy cows.

This study aimed to investigate the effect and mechanism of a high concentrate (HC) diet on the inflammatory response and cellular tight junctions (TJs) in the mammary gland of dairy cows. Twelve lactating Holstein dairy cows were randomly assigned into low concentrate (LC) and HC groups (n=6), which were fed with LC diet and HC diet respectively for 3 weeks. The HC diet lead to subacute ruminant acidosis with a rumen pH<5.6 more than 3h daily. The HC diet triggered an inflammatory response with increased levels of inflammatory cytokines in the lacteal vein, upregulated expression of inflammation-related genes, elevated activity of myeloperoxidase, and inflammatory cells infiltration in the mammary gland. Furthermore, the HC diet induced the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways with enhanced phosphorylation ratios of NF-κB P65, inhibitor of NF-κB (IκB), P38 and extracellular signal-regulated kinase 1/2 (ERK1/2) as well as decreased ratios of DNA methylation and chromatin compaction of genes coding for proinflammatory cytokines, which contributed to the upregulation of proinflammatory cytokine expression. The HC diet also destroyed the integrity of TJ with discontinuous and decreased expression levels of zonula occludens-1, Occludin, Claudin-4 and increased expression level of Claudin-1 in the mammary epithelial cells compared with LC group. Conclusively, the HC diet induced the activation of NF-κB and MAPK signaling pathways and epigenetic modifications, promoted the transcription of proinflammatory cytokines, and finally caused inflammatory response and TJ disruption in the mammary gland of dairy cows.

Evaluating the Correlation of Hedgehog Pathway with Claudin-1 Expression in Invasive Breast Cancer.

Being a heterogeneous disease, breast cancer illustrates different biological and phenotypic features which make its diagnosis and treatment challenging. This study aimed to evaluate the expression levels of crucial components of the Hedgehog signaling pathway, the correlation between the signal transducer Smo, and clinicopathologic features (lymph node metastasis and metastasis stage) in invasive breast carcinoma. Besides, the inverse correlation was considered between expression levels of Smo and Claudin-1. For this purpose, in a case-control study, we evaluated 72 tumor and adjacent normal tissue specimens obtained from invasive ductal breast cancer patients. The expression levels of Hedgehog signaling components (Smo, Gli1, and Ptch), Claudin-1, E-cadherin, and MMP2 were measured by qRT-PCR. The correlations between Smo expressions with some clinicopathologic parameters were also analyzed. Compared to normal adjacent tissues, the results showed up-regulation of Hedgehog signaling in invasive breast carcinoma samples. Upregulation of the signal transducer Smo correlated with tumor stages and lymph node metastasis of the breast tumors. This correlation was affected by the expression of Her2. A significant correlation existed between expression levels of the signal transducer Smo and Claudin-1, E-cadherin as an epithelial cell marker, and MMP2 as a metastasis-related gene in advanced metastatic tumor samples. Based on the obtained results, a new layer of molecular complexity was found, which should be considered in managing patients with invasive breast carcinoma. The results suggested a key role for Hedgehog signaling in invasive breast carcinoma. In terms of the inverse correlation between expression levels of Claudin-1 and Hedgehog signaling, Claudin-1 could serve as a candidate gene in diagnostic studies. Thus, its clinical significance should be further clarified.

Subacute Ruminal Acidosis as a Potential Factor that Induces Endometrium Injury in Sheep.

The demand for economic benefits has led to an increase in the proportion of high-concentrate (HC) feed in the ruminant diet, resulting in an increased incidence of subacute ruminal acidosis (SARA). During SARA, a high concentration of lipopolysaccharide (LPS) translocated in the rumen induces a systemic inflammatory response. Inflammatory diseases, such as endometritis and mastitis, are often associated with SARA; however, in sheep, the mechanism of the effect of SARA on the endometrium has rarely been reported. Therefore, the aim of this study was to investigate, for the first time, the influence of LPS translocation on endometrial tight junctions (TJs) during SARA in sheep. The results showed that LPS and TNFα levels in the ruminal fluid, serum, and endometrial tissue supernatant during SARA increased, transcription levels of TLR4, NFκB, and TNFα in the endometrium increased, the protein expression level of claudin-1 in the endometrium increased, and the protein expression level of occludin decreased. 17β-estradiol (E2) inhibits claudin-1 protein expression and promotes occludin expression, and progesterone (P4) promotes claudin-1 protein expression and inhibits occludin protein expression. E2 and P4 regulate claudin-1 and occludin protein expression through their receptor pathways. Here, we found that LPS hindered the regulatory effect of E2 and P4 on endometrial TJs by inhibiting their receptor expression. The results of this study indicate that HC feeding can cause SARA-induced LPS translocation in sheep, increase susceptibility to systemic inflammation, induce the endometrial inflammatory response, and cause endometrial epithelial TJ damage directly and/or by obstructing E2 and P4 function. LPS translocation caused by SARA has also been suggested to induce an endometrial inflammatory response, resulting in endometrial epithelial barrier damage and physiological dysfunction, which seriously affects ruminant production. Therefore, this study provides new evidence that SARA is a potential factor that induces systemic inflammation in ruminants. It provides theoretical support for research on the prevention of endometritis in ruminants.

Clinicopathological digital image analyses before and after thermal stimulation subdivide acquired idiopathic generalized anhidrosis into inflammatory and non-inflammatory type

BackgroundAcquired idiopathic generalized anhidrosis (AIGA) manifests varying degrees of syringotropic inflammation. ObjectiveTo better understand the basis of inflammation in AIGA. MethodsChanges in the extent of cell infiltration around the sweat gland/duct and the difference in the expression level of immune privilege (IP)-related/sweat gland markers before and after thermal stimulation were assessed in AIGA. We also adopted a semi-quantitative digital image analysis of sweating as detected by the starch-iodine method. The changes in sweating before and after treatment was defined as the improvement index. ResultsNine AIGA cases were analyzed. Two cases with minimal inflammation were defined as non-inflammatory type (non-inf)AIGA, while others with evident cell infiltration were defined as inflammatory type (inf)AIGA. MHC class I was significantly upregulated with downregulation of macrophage migration inhibitory factor and alpha-melanocyte-stimulating hormone exclusively in the sweat duct of infAIGA after thermal stimulation (p < 0.05). Furthermore, the extent of inflammation and the ductal dermcidin expression prior to thermal stimulation were inversely correlated (r = − 0.807), while that and the ductal claudin-1 expression after thermal stimulation was positively correlated (r = 0.875). The improvement index positively correlated with the degree of inflammation after thermal stimulation implying possible contribution of inflammation in AIGA pathophysiology. In addition, interferon-induced protein 10 and claudin-1 expression level in the sweat duct before thermal stimulation respectively correlated with the improvement index (r = 0.750, and 0.762). ConclusionThe pathophysiology of AIGA may be subcategorized into two groups: IP-collapse possibly play some roles in infAIGA, while ductal dysfunction may exist in non-infAIGA.

Inchinkoto, the Traditional Japanese Kampo Medicine, Enhances Intestinal Epithelial Barrier Function In Vitro

Inchinkoto (ICKT), a traditional herbal medicine that is often used as a hepatoprotective drug in Japan, has pharmacological properties that include antioxidant, anti-inflammatory, and choleretic actions. Genipin is a metabolite of geniposide and the most abundant ingredient of ICKT; furthermore, it is considered to be the active substance responsible for its pharmacological properties in the liver. Drugs with such pharmacological characteristics are expected to prevent intestinal barrier dysfunction, which causes inflammatory bowel diseases (IBDs). However, no studies have investigated the effects of ICKT on the intestinal epithelial barrier. Therefore, we investigated the activity of ICKT in intestinal tight junctions by using cultured Caco-2 cell monolayers. The action of the compound on tight junctions was examined by measuring transepithelial electrical resistance (TEER) and sodium fluorescein (Na-F) permeability in the presence or absence of lipopolysaccharide (LPS). Moreover, the expression of the tight junction protein claudin-1 was assessed by using immunofluorescent staining. ICKT and genipin increased TEER and decreased Na-F permeability, which was suggestive of enhanced intestinal epithelial barrier function. Moreover, they prevented the LPS-induced destruction of the barrier, i.e., a decrease in TEER and an increase in Na-F permeability. Immunofluorescence staining revealed a high claudin-1 expression level on the cell surface, whereas exposure to LPS downregulated claudin-1. In turn, ICKT and genipin prevented the LPS-mediated reduction of claudin-1. These results suggest that ICKT enhances intestinal epithelial barrier function by upregulating claudin-1. Furthermore, genipin contributed to these effects. ICKT may be a promising medicine for the prevention and treatment of diseases associated with intestinal barrier disruption, such as IBD, obesity, and metabolic disorders.