Tight Junction Gene Expression Research Articles

149 Development of a leaky gut model: Impact of 24-h feed restriction on ileal expression of tight junction proteins in weaned pigs

Abstract Weanling barrows, 26 to 28 d old [n = 90; Duroc terminal line Fast genetics, initial body weight (BW)= 5.63 ± 0.43 kg] were used to evaluate the effect of 24-h complete feed restriction on growth and intestinal integrity. Upon arrival at the NutriQuest modeling center (Fergus Falls, MN), pigs were weighed, blocked by BW, and randomly assigned to 1 of 30 pens. Five treatments were randomly assigned to 6 pens: 1) Positive Control (PC), and 4 treatments that had 24-hr feed restriction (FR); 2) Negative control (NC), 3) Direct Fed Microbial (DFM) A, 4) DFM B, and 5) DFM C. Feed intake and BW were determined weekly and before and after FR. All pigs were fed a common diet. Fifteen pigs were removed prior to FR due to enteric disease. On d 18, feed was removed from pigs on treatments 2 to 5 for 24 h. After 24 h, 8 pigs per treatment were harvested for tissue collection. Feed access was restored to the remaining pigs who were harvested 3 d later. Ileal samples were collected and immediately frozen. Plasma and fecal samples were collected upon arrival, at FR, and 24 h and 4-d post-FR. Ileal expression of claudin-3, occludin, and tight-junction-1 genes was determined using QuantStudio 3 Real Time PCR system. Fold-change in gene expression compared with NC was calculated for each treatment. Plasma samples were analyzed for LPS binding protein (LBP) concentration, and fecal samples were analyzed for myeloperoxidase (MPO) concentration. Pig (n = 75) was the experimental unit for BW, LBP, MPO, and gene expression data. Pen (n = 30) was the experimental unit for feed intake. Data were analyzed using PROC GLIMMIX of SAS. Preplanned orthogonal contrasts were performed to determine the effect of PC vs. 4 FR treatments. To determine the relationship of tight junction gene expression and LBP and MPO, PROC REG of SAS was used. Data are described as PC vs. FR treatments to focus on model development. Final BW for pigs on PC was greater compared with the FR treatments (P < 0.05; Table 1). From 1 to 4 d post-FR, average daily gain (ADG) for FR treatments was greater than PC (P < 0.01). There was no effect of treatment on feed intake or feed efficiency. Plasma LBP and fecal MPO concentrations were greater 24 h post-FR for pigs on PC compared with FR treatments (P < 0.01). Fold change in occludin and claudin-3 expression was greater for PC compared with FR treatments (P ≤ 0.05). For pigs euthanized 24 h post-FR, there was a positive relationship between LBP concentrations and occludin expression (P < 0.02). Twenty-four hour FR in weaned pigs alters ileal tight junction gene expression and plasma LBP concentrations; therefore, a timed FR event may serve as a model for leaky gut.

150 Development of a leaky gut model: Meta-analysis of plasma and fecal markers of leaky gut

Abstract Intestinal integrity can be compromised in livestock and poultry species during stress events, including off-feed events. To develop a leaky gut model, weanling barrows 26 to 28 d old [n = 307; Duroc terminal line Fast genetics, initial body weight (BW) = 5.90 ± 0.47 kg), were used in a total of 6 randomized complete block experiments at the NutriQuest modeling center (Fergus Falls, MN). All trials included positive control (PC) and negative control (NC) treatments. The other treatments evaluated (n = 8) were various strains and levels of direct-fed microbial (DFM) products. With the exception of PC, all treatments had complete 24-h feed restriction (FR) approximately 18 d after arrival. Pigs assigned to PC had ad libitum access to feed. Pigs were housed 3 pigs per pen in 1 of 30 pens, and all pigs were fed a common high-quality basal diet. For 3 trials, pigs received Excede (Zoetis; Parsipanny, NJ) IM upon arrival and were provided gentamycin via water for 3 d post-arrival. For 3 trials, no prophylactic antibiotics were administered. Eighteen days post-arrival, fecal and plasma samples were collected from all pigs and feed was removed from FR pens. After 24 h, plasma and fecal samples were collected from all pigs, and one-half of the pigs per trial were harvested for tissue collection. Four days after FR, samples were collected similarly, and the remaining pigs were harvested. Plasma samples were analyzed for LPS binding protein concentrations (LBP), and fecal samples were analyzed for myeloperoxidase (MPO) concentrations. Data from the PC (n = 90) and NC (n = 92) treatments only were analyzed as Individual Participant Data meta-analysis using PROC MIXED of SAS. Data from all pigs (n = 307) were analyzed using PROC REG of SAS with forward step-wise selection to determine relationships between response variables. Plasma LBP concentrations did not differ between PC and NC 24 h after FR (P = 0.14). However, change in LBP concentrations and the ratio of LBP immediately before and after FR, differed between PC and NC (P < 0.001). For NC, LBP concentrations decreased, and for PC, LBP concentrations increased (Table 1). There was no effect of treatment on fecal MPO concentrations. Using the broader dataset (n = 307), variables that were predictive of plasma LBP concentrations immediately following FR were trial (P < 0.001), treatment (P < 0.001), and fecal MPO concentrations (P < 0.05). Variables that were predictive of change in LBP concentrations before and after FR were trial (P < 0.001), treatment (P < 0.001), and initial BW (P < 0.01). These data, taken with data presented in a companion abstract on ileal tight junction gene expression, suggest that plasma LBP concentrations immediately following a FR event are a useful marker of leaky gut. Fecal MPO concentrations do not consistently respond to FR.

A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease.

IRAK4 is a critical mediator in NF-κB-regulated inflammatory signaling and has emerged as a promising therapeutic target for the treatment of autoimmune diseases; however, none of its inhibitors have received FDA approval. In this study, we identified a novel small-molecule IRAK4 kinase inhibitor, DW18134, with an IC50 value of 11.2 nM. DW18134 dose-dependently inhibited the phosphorylation of IRAK4 and IKK in primary peritoneal macrophages and RAW264.7 cells, inhibiting the secretion of TNF-α and IL-6 in both cell lines. The in vivo study demonstrated the efficacy of DW18134, significantly attenuating behavioral scores in an LPS-induced peritonitis model. Mechanistically, DW18134 reduced serum TNF-α and IL-6 levels and attenuated inflammatory tissue injury. By directly blocking IRAK4 activation, DW18134 diminished liver macrophage infiltration and the expression of related inflammatory cytokines in peritonitis mice. Additionally, in the DSS-induced colitis model, DW18134 significantly reduced the disease activity index (DAI) and normalized food and water intake and body weight. Furthermore, DW18134 restored intestinal damage and reduced inflammatory cytokine expression in mice by blocking the IRAK4 signaling pathway. Notably, DW18134 protected DSS-threatened intestinal barrier function by upregulating tight junction gene expression. In conclusion, our findings reported a novel IRAK4 inhibitor, DW18134, as a promising candidate for treating inflammatory diseases, including peritonitis and IBD.

Milk and Lacticaseibacillus paracasei BL23 effects on intestinal responses in a murine model of colitis.

Probiotic-containing fermented dairy foods have the potential to benefit human health, but the importance of the dairy matrix for efficacy remains unclear. We investigated the capacity of Lacticaseibacillus paracasei BL23 in phosphate-buffered saline (BL23-PBS), BL23-fermented milk (BL23-milk), and milk to modify intestinal and behavioral responses in a dextran sodium sulfate (DSS, 3% wt/vol) mouse model of colitis. Significant sex-dependent differences were found such that female mice exhibited more severe colitis, greater weight loss, and higher mortality rates. Sex differences were also found for ion transport ex vivo, colonic cytokine and tight junction gene expression, and fecal microbiota composition. Measurements of milk and BL23 effects showed BL23-PBS consumption improved weight recovery in females, whereas milk resulted in better body weight recovery in males. Occludin and Claudin-2 gene transcript levels indicated barrier function was impaired in males, but BL23-milk was still found to improve colonic ion transport in those mice. Proinflammatory and anti-inflammatory gene expression levels were increased in both male and female mice fed BL23, and to a more variable extent, milk, compared with controls. The female mouse fecal microbiota contained high proportions of Akkermansia (average of 18.1%) at baseline, and females exhibited more changes in gut microbiota composition following BL23 and milk intake. Male fecal microbiota harbored significantly more Parasutterella and less Blautia and Roseburia after DSS treatment, independent of BL23 or milk consumption. These findings show the complex interplay between dietary components and sex-dependent responses in mitigating inflammation in the digestive tract.NEW & NOTEWORTHY Sex-dependent responses to probiotic Lacticaseibacillus paracasei and milk and the potential of the dairy matrix to enhance probiotic protection against colitis in this context have not been previously explored. Female mice were more sensitive than males to colonic injury, and neither treatment effectively alleviated inflammation in both sexes. These sex-dependent responses may result from differences in the higher baseline proportions of Akkermansia in the gut microbiome of female mice.

Ibrutinib disrupts blood-tumor barrier integrity and prolongs survival in rodent glioma model

In malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-tumor barrier (BTB). Ibrutinib, FDA-approved lymphoma agent, inhibits Bruton tyrosine kinase (BTK) and has previously been shown to independently impair aortic endothelial adhesion and increase rodent glioma model survival in combination with cytotoxic therapy. Yet additional research is required to understand ibrutinib’s effect on BTB function. In this study, we detail baseline BTK expression in glioma cells and its surrounding vasculature, then measure endothelial junctional expression/function changes with varied ibrutinib doses in vitro. Rat glioma cells and rodent glioma models were treated with ibrutinib alone (1–10 µM and 25 mg/kg) and in combination with doxil (10–100 µM and 3 mg/kg) to assess additive effects on viability, drug concentrations, tumor volume, endothelial junctional expression and survival. We found that ibrutinib, in a dose-dependent manner, decreased brain endothelial cell–cell adhesion over 24 h, without affecting endothelial cell viability (p < 0.005). Expression of tight junction gene and protein expression was decreased maximally 4 h after administration, along with inhibition of efflux transporter, ABCB1, activity. We demonstrated an additive effect of ibrutinib with doxil on rat glioma cells, as seen by a significant reduction in cell viability (p < 0.001) and increased CNS doxil concentration in the brain (56 ng/mL doxil alone vs. 74.6 ng/mL combination, p < 0.05). Finally, Ibrutinib, combined with doxil, prolonged median survival in rodent glioma models (27 vs. 16 days, p < 0.0001) with brain imaging showing a − 53% versus − 75% volume change with doxil alone versus combination therapy (p < 0.05). These findings indicate ibrutinib’s ability to increase brain endothelial permeability via junctional disruption and efflux inhibition, to increase BTB drug entry and prolong rodent glioma model survival. Our results motivate the need to identify other BTB modifiers, all with the intent of improving survival and reducing systemic toxicities.

The effects of Pediococcus acidilactici MA18/5M on growth performance, gut integrity, and immune response using in vitro and in vivo Pacific salmonid models.

Microbial management is central to aquaculture's efficiency. Pediococcus acidilactici MA18/5M has shown promising results promoting growth, modulation of the immune response, and disease resistance in many fishes. However, the mechanisms through which this strain confers health benefits in fish are poorly understood, particularly in Pacific salmonid models. Briefly, the aims of this study were to i) assess the protective effects of P. acidilactici MA18/5M by examining gut barrier function and the expression of tight junction (TJ) and immune genes in vitro and in vivo, and ii) to determine the protective effects of this strain against a common saltwater pathogen, Vibrio anguillarum J382. An in vitro model of the salmonid gut was employed utilizing the cell line RTgutGC. Barrier formation and integrity assessed by TEER measurements in RTgutGC, showed a significant decrease in resistance in cells exposed only to V. anguillarum J382 for 24 h, but pre-treatment with P. acidilactici MA18/5M for 48 h mitigated these effects. While P. acidilactici MA18/5M did not significantly upregulate tight junction and immune molecules, pre-treatment with this strain protected against pathogen-induced insults to the gut barrier. In particular, the expression of ocldn was significantly induced by V. anguillarum J382, suggesting that this molecule might play a role in the host response against this pathogen. To corroborate these observations in live fish, the effects of P. acidilactici MA18/5M was evaluated in Chinook salmon reared in real aquaculture conditions. Supplementation with P. acidilactici MA18/5M had no effect on Chinook salmon growth parameters after 10 weeks. Interestingly, histopathological results did not show alterations associated with P. acidilactici MA18/5M supplementation, indicating that this strain is safe to be used in the industry. Finally, the expression pattern of transcripts encoding TJ and immune genes in all the treatments suggest that variation in expression is more likely to be due to developmental processes rather than P. acidilactici MA18/5M supplementation. Overall, our results showed that P. acidilactici MA18/5M is a safe strain for use in fish production, however, to assess the effects on growth and immune response previously observed in other salmonid species, an assessment in adult fish is needed.

Dietary L-Methionine modulates the gut microbiota and improves the expression of tight junctions in an in vitro model of the chicken gastrointestinal tract

BackgroundThe poultry industry encounters a number of factors that affect growth performance and productivity; nutrition is essential for sustaining physiological status and protecting against stressors such as heat, density, and disease. The addition of vitamins, minerals, and amino acids to the diet can help restore productivity and support the body’s defense mechanisms against stress. Methionine (Met) is indispensable for poultry’s energy metabolism, physiology, performance, and feed utilization capacity. Through this study, we aimed to examine the physiological effects of methionine supplementation on poultry as well as alterations of intestinal microbiome.MethodsWe utilized the DL- and L- form of methionine on Caenorhabditis elegans and the FIMM (Fermentor for intestine microbiota model) in-vitro digesting system. A genomic-analysis of the transcriptome confirmed that methionine supplementation can modulate growth-related physiological metabolic pathways and immune responses in the host poultry. The C. elegans model was used to assess the general health benefits of a methionine supplement for the host.ResultsRegardless of the type or concentration of methionine, supplementation with methionine significantly increased the lifespan of C. elegans. Feed grade L-Methionine 95%, exhibited the highest lifespan performance in C. elegans. Methionine supplementation increased the expression of tight junction genes in the primary intestinal cells of both broiler and laying hens, which is directly related to immunity. Feed grade L-Methionine 95% performed similarly or even better than DL-Methionine or L-Methionine treatments with upper doses in terms of enhancing intestinal integrity. In vitro microbial cultures of healthy broilers and laying hens fed methionine revealed changes in intestinal microflora, including increased Clostridium, Bacteroides, and Oscillospira compositions. When laying hens were given feed grade L-Methionine 95% and 100%, pathogenic Campylobacter at the genus level was decreased, while commensal bacteria were increased.ConclusionsSupplementation of feed grade L-Methionine, particularly L-Methionine 95%, was more beneficial to the host poultry than supplementing other source of methionine for maintaining intestinal integrity and healthy microbiome.

Effects of dietary binder sources on growth, digestive enzymes activity, and gut barrier function and microbial composition of larval largemouth bass (Micropterus salmoides)

A 4-week feeding trail was conducted to explore the impacts of dietary binder sources on growth performance, digestive enzymes activities, intestinal microbiota, and expression of genes involved in tight junctions and nutrient sensing pathway of larval largemouth bass with initial body weight of 8.43 ± 0.01 mg (13 day after hatching). Five isolipidic micro-diets were formulated with different binder sources including sodium alginate (SA), carrageenan (CG), hydroxypropyl methyl cellulose (HPMC), gelatin (GT), and starch (SC). The results revealed that the highest growth performance was observed in the CG group, and meanwhile, the promotion effects of SA and GT was statistically higher than that of HPMC and SC groups. SA, CG and GT had positive effects on digestive enzymes activity compared to other groups. Gene expression results showed that SA, CG and GT groups up-regulated the expression of tight junction genes, zo-1, claudin-4 and claudin-7, and activated the genes involved in the TOR signaling pathway, including tor, akt1, s6kβ1 and s6. Among all the groups, the CG had the most significant activation effect on TOR signaling pathway. Bacterial 16S rRNA V3–4 region analysis showed that the addition of different dietary binders influenced the diversity of intestinal microbial communities. The inclusion of SA and CG groups reduced the relative abundance of Proteobacteria and Actinobacteria at the phylum level, while at the genus level, the relative abundance of Rhodococcus and Achromobacter increased and the abundance of Plesiomonas was inhibited. In summary, dietary binder sources affected the growth, digestive function, gut barrier function and microbial composition, and nutrient sensing, and SA and CG could be suggested as the optimal dietary binders for formulation of micro-diets for largemouth bass larvae.

A29 INVESTIGATING THE IMPACT OF PARKINSON’S DISEASE-ASSOCIATED GENES ON INTESTINAL HOMEOSTASIS

Abstract Background Intestinal epithelial cells (IECs) provide an essential physical barrier between harsh luminal contents and underlying host tissue. The maintenance of intestinal homeostasis must be intricately regulated through the proliferation and differentiation of intestinal stem cells (ISCs). Dysregulation of this system results in the loss of barrier function, causing pathologies in both intestinal and extra-intestinal diseases. While Parkinson’s Disease (PD) is primarily a neurodegenerative disorder, there is increasing evidence linking PD progression and gastrointestinal (GI) dysfunction. Constipation and increased bowel permeability are often observed years prior to development of motor dysfunction in PD, and people with inflammatory bowel disease are more likely to develop PD. Our group developed a model to investigate the role of the gut in PD, demonstrating that mice with genetic ablation of the PD-associated gene Pink1 exhibited motor phenotypes only when previously infected with Gram-negative Citrobacter rodentium intestinal bacteria. As Pink1 and other PD-associated genes are expressed in IECs, we hypothesize that PD-associated gene mutations directly affect the epithelium and impact early PD pathophysiology. Aims 1. Determine how Pink1 mutation affects the transcriptional profiles in IECs at steady state and in vivo C. rodentium infection. 2. Use colonoid systems to study the effect of Pink1 mutation on epithelial activity. Methods ScRNAseq was performed on IECs isolated from uninfected and infected Pink1 WT and KO mice, sacrificed at day 7 and 14 (early and peak infection) to elucidate transcriptional differences between epithelial lineages of each genotype. Additionally, colonoids were derived from primary mouse tissue and treated with lipopolysaccharide (LPS) to determine how Pink1 KO affects the inflammatory response of the epithelium. Results Our data revealed that Pink1 KO profoundly affected several epithelial lineages. ISCs from infected Pink1 KO mice had dysregulated cell cycle genes, transit amplifying cells showed dysregulated expression of tight junction genes, and enterocytes displayed dysregulation in oxidative damage and apoptotic genes. Preliminary data from colonoids showed that Pink1 KO mice, when stimulated with LPS, had altered pro-inflammatory cytokine gene expression. We are also evaluating colon motility differences between genotypes through transit time assays, counting fecal pellets per hour, and fecal water content. Conclusions In Pink1 KO IECs, there is indeed an altered cellular response upon infection, but more information is needed to decern the mechanistic role of IECs in PD. By investigating the role of PD genes in the GI tract, these studies carry important implications for understanding the initiation and progression of PD. Funding Agencies CIHR

Intestinal Permeability, Gut Inflammation, and Gut Immune System Response Are Linked to Aging-Related Changes in Gut Microbiota Composition: A Study in Female Mice.

Aging entails changes at the cellular level that increase the risk of various pathologies. An association between gut microbiota and age-related diseases has also been attributed. This study aims to analyze changes in fecal microbiota composition and their association with genes related to immune response, gut inflammation, and intestinal barrier impairment. Fecal samples of female mice at different ages (2 months, 6 months, 12 months, and 18 months) and gene expression in colon tissue were analyzed. Results showed that the older mice group had a more diverse microbiota than the younger group. Additionally, the abundance of Cyanobacteria, Proteobacteria, Flavobacteriaceae, Bacteroides, Parabacteroides, Prevotellaceae_UCG-001, Akkermansia, and Parabacteroides goldsteinii increased with age. In contrast, there was a notable decline in Clostridiaceae, Lactobacillaceae, Monoglobaceae, Ligilactobacillus, Limosilactobacillus, Mucispirillum, and Bacteroides faecichinchillae. These bacteria imbalances were positively correlated with increased inflammation markers in the colon, including Tnf-α, Ccl2, and Ccl12, and negatively with the expression of tight junction genes (Jam2, Tjp1, and Tjp2), as well as immune response genes (Cd4, Cd72, Tlr7, Tlr12, and Lbp). In conclusion, high levels of diversity did not result in improved health in older mice; however, the imbalance in bacteria abundance that occurs with aging might contribute to immune senescence, inflammation, and leaky gut disease.

Effect of Millet Polyphenol-Rich Extracts on Human Fecal Bacteria and Dextran Sulfate Sodium-Induced Human Intestinal Epithelial Cell Derangements

ABSTRACT There is a rejuvenated interest in functional food research on polyphenols due to their gut microbial modulation and capacity to alleviate clinical conditions. In this study, polyphenol-rich extracts (PREs) from Finger millet (FM) and Kodo millet (KM) were assessed for modulating effects on selected human gut bacteria during fecal batch fermentation along with the protective effects on Dextran Sulfate Sodium (DSS)-induced colonic aberrations in vitro. Fecal batch fermentation of FM-PRE and KM-PRE enhanced the abundance of selected beneficial bacteria, such as Bifidobacterium spp. Lactobacillus spp. and the production of SCFAs. Catechin and naringin were the main phenolic fractions of fermented FM-PRE and KM-PRE. Moreover, the millet polyphenols protected the intestinal cells against DSS-induced intestinal barrier dysfunction by reducing inflammatory cytokines, TEER by ~40%, FITC-dextran fluorescence and upregulating the expression of tight junction genes, signifying improved gut functions. The study also imply that FM and KM polyphenols may inhibit intestinal inflammation.

Effects of pectin's degree of methyl esterification on TLR2-mediated IL-8 secretion and tight junction gene expression in intestinal epithelial cells: influence of soluble TLR2.

This study investigates the anti-inflammatory effects of pectins with different degrees of methyl esterification (DM) on intestinal epithelial cells (IECs) expressing low and high levels of TLR2. It also studies the influence of soluble TLR2 (sTLR2) which may be enhanced in patients with inflammatory bowel syndrome on the inflammation-attenuating effects of pectins. Also, it examines the impact of pectins on tight junction gene expression in IECs. Lemon pectins with DM18 and DM88 were characterized, and their effects on TLR2-1-induced IL8 gene expression and secretion were investigated in low-TLR2 expressing Caco-2 and high-TLR2 expressing DLD-1 cells. The results demonstrate that both DM18 and DM88 pectins can counteract TLR2-1-induced IL-8 expression and secretion, with more pronounced effects observed in DLD-1 cells expressing high levels of TLR2. Furthermore, the presence of sTLR2 does not interfere with the attenuating effects of low DM18 pectin and may even support its anti-inflammatory effects in Caco-2 cells. The impact of pectins and sTLR2 on tight junction gene expression also demonstrates cell-type-dependent effects. Overall, these findings suggest that low DM pectins possess potent anti-inflammatory properties and may influence tight junction gene expression in IECs, thereby contributing to the maintenance of gut homeostasis.

In vitro faecal fermentation metabolites of 2′-fucosyllactose protect against intestinal epithelial injury: Infant enterotype effects

In this study, we conducted in vitro fermentation with infant faecal inocula dominated by Bifidobacterium longum, Bifidobacterium breve, and Bacteroides enterotypes, and assessed the efficacy of the metabolome associated with 2′-fucosyllactose (2′-FL) fermentation on lipopolysaccharide-induced epithelial cell barrier damage on Caco-2 cells. The results revealed that propionate and butyrate were more prominent in the Bacteroides-dominated group, whereas the Bifidobacterium-dominated group was associated with an increased molar fraction of lactate (∼16 mm). Additionally, our study demonstrated that 2′-FL faecal metabolites (FMs) decreased the production of inflammatory cytokines, and promoted tight junction gene expression by inhibiting the TLR4/NF-κB/MLCK signalling pathway and activating the Nrf2/HO-1 pathway. The 2′-FL FMs from Bacteroides-dominated enterotypes had a unique advantage in regulating the TLR4/NF-κB/MLCK signalling pathway, which can be attributed to the effects of propionate and butyrate.

Methionine sources and genotype affect embryonic intestinal development, antioxidants, tight junctions, and growth-related gene expression in chickens

Methionine (Met) is an essential and first limiting amino acid in the poultry diet that plays a significant role in chicken embryonic development and growth. The present study examined the effect of in ovo injection of DL-Met and L-Met sources and genotypes on chicken embryonic-intestinal development and health. Fertilized eggs of the two genotypes, TETRA-SL layer hybrid (TSL) — commercial layer hybrid and Hungarian Partridge colored hen breed (HPC) — a native genotype, were randomly distributed into four treatments for each genotype. The treatment groups include the following: 1) control non-injected eggs (NoIn); 2) saline-injected (SaIn); 3) DL-Met injected (DLM); and 4) L-Met injected (LM). The in ovo injection was carried out on 17.5 d of embryonic development; after hatching, eight chicks per group were sacrificed, and the jejunum was extracted for analysis. The results showed that both DLM and LM groups had enhanced intestinal development as evidenced by increased villus width, villus height, and villus area (P < 0.05) compared to the control. The DLM group had significantly reduced crypt depth, glutathione (GSH) content, glutathione S-transferase 3 alpha (GST3), occludin (OCLN) gene expression and increased villus height to crypt depth ratio in the TSL genotype than the LM group (P < 0.05). The HPC genotype has overexpressed insulin-like growth factor 1 (IGF1) gene, tricellulin (MD2), occludin (OCLN), superoxide dismutase 1 (SOD1), and GST3 genes than the TSL genotype (P < 0.05). In conclusion, these findings showed that in ovo injection of Met enhanced intestinal development, and function, with genotypes responding differently under normal conditions. Genotypes also influenced the expression of intestinal antioxidants, tight junction, and growth-related genes.

Tight Junction Claudins and Occludin Are Differentially Regulated and Expressed in Genomically Defined Subsets of Colon Cancer.

Metastatic colon cancer remains incurable despite improvements in survival outcomes. New therapies based on the discovery of colon cancer genomic subsets could improve outcomes. Colon cancers from genomic studies with publicly available data were examined to define the expression and regulation of the major tight junction proteins claudins and occludin in genomic groups. Putative regulations of the promoters of tight junction genes by colon-cancer-deregulated pathways were evaluated in silico. The effect of claudin mRNA expression levels on survival of colon cancer patients was examined. Common mutations in colon-cancer-related genes showed variable prevalence in genomically identified groups. Claudin genes were rarely mutated in colon cancer patients. Genomically identified groups of colon cancer displayed distinct regulation of claudins and occludin at the mRNA level. Claudin gene promoters possessed clustered sites of binding sequences for transcription factors TCF4 and SMADs, consistent with a key regulatory role of the WNT and TGFβ pathways in their expression. Although an effect of claudin mRNA expression on survival of colon cancer patients as a whole was not prominent, survival of genomic subsets was significantly influenced by claudin mRNA expression. mRNA expression of the main tight junction genes showed differential regulation in various genomically defined subgroups of colon cancer. These data pinpoint a distinct role of claudins and pathways that regulate them in these subgroups and suggest that subgroups of colon cancer should be considered in future efforts to therapeutically target claudins.

Effect of Echinacea purpurea (L.) Moench and its extracts on the immunization outcome of avian influenza vaccine in broilers

Ethnopharmacological relevanceEchinacea purpurea (L.) Moench (EP) is a perennial herbaceous flowering plant with immunomodulatory effects. However, the immunomodulatory effects of EP on broilers after vaccination are still unclear. Aim of the studyThe aim is to study the effect of EP and Echinacea purpurea (L.) Moench extracts(EE) on avian influenza virus (AIV) immunity, and further explore the potential mechanism of immune regulation. Materials and methodsBroilers were fed with feed additives containing 2% EP or 0.5% EE, and vaccinated against avian influenza. The samples were collected on the 7th, 21st, and 35th day after vaccination, and the feed conversion ratio (FCR) was calculated. Blood antibody titer, jejunal sIgA content, tight junction protein, gene and protein expression of TLR4-MAPK signaling pathway were also detected. ResultsThe results showed that vaccination could cause immune stress, weight loss, increase sIgA content, and up-regulate the expression of tight junction proteins, including zonula occludens-1 (ZO-1), Occludin, and Claudin-1, as well as the genes of Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), receptor-associated factor 6 (TRAF6), activator protein 1 (AP-1) protein gene expression on TLR4-mitogen-activated protein kinase (MAPK) signaling pathway, and the protein expression of MyD88, extracellular regulated protein kinases (ERK), and c-Jun N-terminal kinase (JNK). EP and EE could increase the body weight of broilers, further improve antibody titers, decrease FCR, increase sIgA levels, up-regulate the expression of tight junction proteins, including ZO-1, Occludin, and Claudin-1, as well as the genes of TLR4, MyD88, TRAF6, and AP-1 and the protein expression of MyD88, ERK, and JNK in the TLR4-MAPK signaling pathway. ConclusionIn conclusion, EP and EE can increase the broiler's production performance and improve vaccine immune effect through the TLR4-MAPK signaling pathway.

Estrogenic regulation of claudin 5 and tight junction protein 1 gene expression in zebrafish: A role on blood-brain barrier?

The blood-brain barrier (BBB) is a physical interface between the blood and the brain parenchyma, playing key roles in brain homeostasis. In mammals, the BBB is established thanks to tight junctions between cerebral endothelial cells, involving claudin, occludin, and zonula occludens proteins. Estrogens have been documented to modulate BBB permeability. Interestingly, in the brain of zebrafish, the estrogen-synthesizing activity is strong due to the high expression of Aromatase B protein, encoded by the cyp19a1b gene, in radial glial cells (neural stem cells). Given the roles of estrogens in BBB function, we investigated their impact on the expression of genes involved in BBB tight junctions. We treated zebrafish embryos and adult males with 17β-estradiol and observed an increased cerebral expression of tight junction and claudin 5 genes in adult males only. In females, treatment with the nuclear estrogen receptor antagonist (ICI182,780 ) had no impact. Interestingly, telencephalic injuries performed in males decreased tight junction gene expression that was partially reversed with 17β-estradiol. This was further confirmed by extravasation experiments of Evans blue showing that estrogenic treatment limits BBB leakage. We also highlighted the intimate links between endothelial cells and neural stem cells, suggesting that cholesterol and peripheral steroids could be taken up by endothelial cells and used as precursors for estrogen synthesis by neural stem cells. Together, our results show that zebrafish provides an alternative model to further investigate the role of steroids on the expression of genes involved in BBB integrity, both in constitutive and regenerative physiological conditions. The link we described between capillaries endothelial cells and steroidogenic neural cells encourages the use of this model in understanding the mechanisms by which peripheral steroids get into neural tissue and modulate neurogenic activity.

β(2 → 1)-β(2 → 6) and β(2 → 1) fructans protect from impairment of intestinal tight junction's gene expression and attenuate human dendritic cell responses in a fructan-dependent fashion

β(2 → 1)-β(2 → 6) branched graminan-type fructans (GTFs) and β(2 → 1) linear fructans (ITFs) possess immunomodulatory properties and protect human intestinal barrier function, however the mechanisms underlying these effects are not well studied. Herein, GTFs and ITFs effects with different degree of polymerization (DP) values on tight junctions (TJs) genes CLDN-1, -2 and -3, CDH1, OCLN and TJP1 were studied in Caco-2 gut epithelial cells, under homeostatic and inflammatory conditions. Also, cytokine production in dendritic cells (DCs) was studied. Higher DP fructans decreased the expression of the pore forming CLDN-2. Higher DP GTFs enhanced CLDN-3, OCLN, and TJP-1. Fructans prevented mRNA dysregulation of CLDN-1, -2 and -3 induced by the barrier disruptors A23187 and deoxynivalenol in a fructan-type dependent fashion. The production of pro-inflammatory cytokines MCP-1/CCL2, MIP-1α/CCL3 and TNFα by DCs was also attenuated in a fructan-type dependent manner and was strongly attenuated by DCs cultured with medium of Caco-2 cells which were pre-exposed to fructans. Our data show that specific fructans have TJs and DCs modulating effects and contribute to gut homeostasis. This might serve to design effective dietary means to prevent intestinal inflammation.

Panaxynol Alleviates Colorectal Cancer in a Murine Model via Suppressing Macrophages and Inflammation.

Currently available colorectal cancer (CRC) therapies have limited efficacy and severe adverse effects that may be overcome with the alternative use of natural compounds. We previously reported that panaxynol (PA), a bioactive component in American Ginseng, possesses anti-cancer properties in vitro and suppresses murine colitis through its pro-apoptotic and anti-inflammatory properties. Because colitis is a predisposing factor of CRC and inflammation is a major driver of CRC, we sought to evaluate the therapeutic potential of PA in CRC. AOM/DSS mice (C57BL/6) were administered 2.5mg/kg PA or vehicle 3x/week via oral gavage over 12 weeks. PA improved clinical symptoms (p≤0.05) and reduced tumorigenesis (p≤0.05). This improvement may be reflective of PA's restorative effect on intestinal barrier function; PA upregulated the expression of essential tight junction and mucin genes (p≤0.05) and increased abundance of mucin-producing goblet cells (p≤0.05). Given that macrophages play a substantial role in the pathogenesis of CRC and that we previously demonstrated that PA targets macrophages in colitis, we next assessed macrophages. We show that PA reduces the relative abundance of colonic macrophages within the lamina propria (p≤0.05), and this was consistent with a reduction in the expression of important markers of macrophages and inflammation (p≤0.05). We further confirmed PA's inhibitory effects on macrophages in vitro under CRC conditions (p≤0.05). These results suggest that PA is a promising therapeutic compound to treat CRC and improve clinical symptoms given its ability to inhibit macrophages and modulate the inflammatory environment in the colon.

Effects of lactic acid bacteria isolated from Tibetan chickens on the growth performance and gut microbiota of broiler.

Lactic acid bacteria (LAB) are organic supplements that have several advantages for the health of the host. Tibetan chickens are an ancient breed, which evolve unique gut microbiota due to their adaptation to the hypoxic environment of high altitude. However, knowledge of LAB isolated from Tibetan chickens is very limited. Thus, the purpose of this study was to assess the probiotic properties of Lactobacillus Plantarum (LP1), Weissella criteria (WT1), and Pediococcus pentosaceus (PT2) isolated from Tibetan chickens and investigate their effects on growth performance, immunoregulation and intestinal microbiome in broiler chickens. Growth performance, serum biochemical analysis, real-time PCR, and 16S rRNA sequencing were performed to study the probiotic effects of LP1, WT1, and PT2 in broiler chickens. Results showed that LP1, WT1 and PT2 were excellent inhibitors against Escherichia coli (E. coli ATCC25922), meanwhile, LP1, WT1, and PT2 significantly increased weekly weight gain, villus height, antioxidant ability and gut microbiota diversity indexes in broilers. In addition, LP1 and PT2 increased the relative abundance of Lactobacillus and decreased Desulfovibrio in comparison with T1 (control group). Additionally, oral LAB can reduce cholesterol and regulate the expression of tight junction genes in broiler chickens, suggesting that LAB can improve the integrity of the cecal barrier and immune response. In conclusion, LAB improved the growth performance, gut barrier health, intestinal flora balance and immune protection of broiler chickens. Our findings revealed the uniqueness of LAB isolated from Tibetan chickens and its potential as a probiotic additive in poultry field.