MUC1 mRNA Expression Research Articles

Impacts of sodium butyrate on intestinal mucosal barrier and intestinal microbial community in a weaned piglet model.

Butyrate is thought to enhance intestinal mucosal homeostasis, but the detailed mechanism remains unclear. Therefore, further investigation on the mechanism of butyrate regulation of intestinal mucosal homeostasis was performed. This study used weaned piglets with similar intestinal metabolic function to humans as a research model. The dietary supplemented 0.2% sodium butyrate group (0.2% S) and negative control group (CON) were established to detect the effects of butyrate on growth performance, intestinal tissue morphology, mucosal barrier function, and intestinal microbial community structure in weaned piglets. There was an increase in average daily gain (ADG) during three different experimental periods and a reduction in average daily feed intake (ADFI) and feed-to-gain ratio (F:G) during days 1-35 and days 15-35 in 0.2% S compared with CON (P > 0.05). Furthermore, villus height in the ileum and duodenum was increased, and crypt depths in the colon and jejunum were reduced in both groups (P < 0.05). Moreover, the ratio of villus height and crypt depth (V/C) in 0.2% S both in the ileum and jejunum was significantly increased (P < 0.05) compared with CON. The relative mRNA expression of PKC, MUC1, CLDN1, and ITGB1 was upregulated in the ileum of 0.2% S compared with CON (P < 0.05). The digesta samples of 0.2% S, both in the ileum (P < 0.05) and colon, contained greater intestinal bacterial abundance and diversity of probiotics, including Lactobacillus, Streptococcus, Megasphaera, and Blautia, which promoted amino acid metabolism and energy production and conversion in the colon and the synthesis of carbon-containing biomolecules in the ileum. In summary, dietary supplementation with 0.2% sodium butyrate was shown to have a tendency to improve the growth performance of weaned piglets and enhance intestinal mucosal barrier function via altering the gut microbiota.

Akkermansia deficiency and mucin depletion are implicated in intestinal barrier dysfunction as earlier event in the development of inflammation in interleukin-10-deficient mice.

Dysbiosis and mucin depletion are related with intestinal barrier dysfunction and seems to be an early pathophysiological event in inflammatory bowel disease (IBD). The objective of this work is to study these parameters in the natural history of colitis in IL-10 deficient mice (IL-10-/-). Wild type (WT) and IL-10-/-. mice were followed until sacrifice at 3, 5, 10, 20, 57, and 70 weeks. Body weight, colonic weight/length ratio and in vivo intestinal permeability were registered. Expression of inflammatory and adhesion molecules in the colon was explored by qPCR as Mucin-2 (MUC-2) and molecules involved in goblet cell maturation Interleukin-18 (IL-18) and WAP Four-Disulfide Core Domain 2 (WFDC2), the endoplasmic reticulum stress markers X-box-binding protein (Xbp-1) and Reticulon-4B (RTN-4B). Bacterial composition in feces and colonic mucosa was determined by massive sequencing of the V3-V4 regions of 16S rDNA gene. IL-10-/- mice showed histological inflammation at weeks 20 and 57, but most notably the intestinal permeability was significantly higher from week 10. Concordantly, the number of goblet cells and expression of MUC-2, IL-18, WFDC2 and Xbp-1 were significantly lower in KO from week 10. Nevertheless, no significant differences were found in the mRNA expression of MUC-2 or Xbp-1 between both groups-derived colon organoids. Significant bacterial differences began at week 5, being the Akkermansia deficiency in KO the most relevant result. Gut microbiota alterations and mucin depletion are associated with early intestinal barrier dysfunction and precede overt gut inflammation in this animal model of IBD.

Compound probiotics can improve intestinal health by affecting the gut microbiota of broilers.

Probiotics, as a widely used additive, have played a unique advantage in replacing antibiotic products. As a result, the probiotic effects on broiler development, intestinal flora, intestinal barrier, and immunity were assessed by this investigation. Four hundred and eighty 1-day-old Arbor Acres broilers were randomly allotted to 4 groups of 5 replicates with 24 broilers each. The control was fed only a basal corn-soybean meal diet. Probiotics I, probiotics II, and probiotics III were fed basal diet and 1, 5, and 10 g/kg compound probiotics (Lactobacillus casei: Lactobacillus acidophilus: Bifidobacterium = 1:1:2), respectively. We found that broilers in the compound probiotic group exhibited better growth performance and carcass characteristics compared with control, especially among probiotics III group. The intestinal barrier-related genes relative expression of Claudin, Occludin, MUC2, and ZO-1 mRNA in the probiotic group increased at 21 and 42 d compared with control, especially among probiotics III group (P < 0.05). The early gut immune-related genes (TLR2, TLR4, IL-1β, and IL-2) mRNA increased compared with control, while the trend at 42 d was completely opposite to that in the earlier stage (P < 0.05). Among them, probiotics III group showed the most significant changes compared to probiotics II group and probiotics I group. Select probiotics III group and control group for 16S rDNA amplicon sequencing analysis. The 16S rDNA amplicon sequencing results demonstrated that probiotics increased the relative abundance of beneficial microbes such as o_Bacteroidales, f_Rikenellaceae, and g_Alistipes and improved the cecum's gut microbiota of 42-day-old broilers. Additionally, adding the probiotics decreased the relative abundance of harmful microbes such as Proteobacteria. PICRUSt2 functional analysis revealed that most proteins were enriched in DNA replication, transcription, and glycolysis processes. Therefore, this study can provide theoretical reference value for probiotics to improve production performance, improve intestinal barrier, immunity, intestinal flora of broilers, and the application of probiotics.

Protective Effect of Irsogladine against Aspirin-Induced Mucosal Injury in Human Induced Pluripotent Stem Cell-Derived Small Intestine

Background and Objectives: Acetylsalicylic acid (ASA) is widely used for preventing cerebrovascular and cardiovascular diseases. Gastrointestinal (GI) tract injury is one of the major complications of aspirin use, potentially leading to severe GI bleeding. However, no drugs for preventing aspirin-induced small intestinal injury have been developed. The aim of this study was to establish a human experimental model for investigating aspirin-induced small intestinal mucosal injury. In addition, we evaluated the protective effect of Irsogladine against aspirin-induced small intestinal mucosal injury using human induced pluripotent stem cell-derived 2D monolayer crypt-villus structural small intestine (2D-hiPSC-SI). Materials and Methods: Human iPS cell-derived intestinal organoids were seeded and cultured in Air-liquid interface. The permeability of 2D-hiPSC-SI was evaluated using Lucifer yellow. Changes in structure and mucosal permeability of 2D-hiPSC-SI after addition of aspirin were confirmed over time, and changes in intestinal epithelium-related markers were evaluated by real-time qPCR and Immunofluorescence staining. The effect of Irsogladine on prevention of aspirin mucosal injury was examined by adding Irsogladine to the culture medium. Results: Cultured 2D-hiPSC-SI showed multi-lineage differentiation into small intestinal epithelium comprised of absorptive cells, goblet cells, enteroendocrine cells, and Paneth cells, which express CD10, MUC2, chromogranin A, and lysozyme, respectively. RNA in situ hybridization revealed intestinal stem cells that express Lgr5. ASA administration induced an increase in the mucosal permeability of 2D-hiPSC-SI. ASA-injured 2D-hiPSC-SI showed decreased mRNA expression of multi-lineage small intestinal cell markers as well as intestinal stem cell marker Lgr5. Administration of Irsogladine on the basal side of the 2D-hiPSC-SI resulted in significant increases in Mki67 and Muc2 mRNA expression by 2D-hiPSCs at 48 h compared with the control group. Administration of 400 µg/mL Irsogladine to the ASA-induced small intestinal injury model resulting in significantly decreased mucosal permeability of 2D-hiPSC-SI. In immunofluorescence staining, Irsogladine significantly increased the fluorescence intensity of MUC2 under normal conditions and administration of 400 µg/mL ASA. Conclusions: we established a novel ASA-induced small intestinal injury model using human iPSC-derived small intestine. Irsogladine maintains mucosal permeability and goblet cell differentiation against ASA-induced small intestinal injury.

Dietary supplementation with a microencapsulated complex of thymol, carvacrol, and cinnamaldehyde improves intestinal barrier function in weaning piglets.

The authors previously prepared a microencapsulated complex of thymol, carvacrol, and cinnamaldehyde (MEEO). This study aimed to evaluate the effect of MEEO on the intestinal mucosal barrier and homeostasis in weaning piglets. A comparison of the effect of MEEO versus chlortetracycline (CTC) was performed in this study. Piglets were divided into three groups - control (Con), MEEO, and CTC groups - and raised for 28 days. The results showed that MEEO significantly elevated the ratio of the villus height and the crypt depth in the jejunum and decreased the crypt depth in the ileum compared with the other groups (P < 0.05); it also upregulated the messenger ribonucleic acid (mRNA) expression of tight junction protein in the small intestine. Compared with the Con group, MEEO increased the concentration of secretory immunoglobulin A (sIgA), cathelicidin antimicrobial peptides (CAMP), and interleukin 10 (IL-10), while decreasing the interleukin 1 beta (IL-1β) concentration in both jejunal and ileal mucosa (P < 0.05). The mRNA expression of jejunal mucosal MUC1 and ileal mucosal MUC2 was increased in the MEEO group compared with the other groups (P < 0.05). Intestinal microbial analysis showed that dietary treatment had little impact on the ileal microbial structure. A significant rise in the genus Lactobacillus was, however, found in the MEEO group. There is a positive correlation between the Lactobacillus and sIgA, and between the Lactobacillus and CAMP, indicating that an improvement in the mucosal barrier function by the addition of MEEO may be associated with the proliferation of Lactobacillus. Dietary supplementation with MEEO improves intestinal barrier function in weaning piglets, the effect of which was superior to CTC. © 2022 Society of Chemical Industry.

Effect of Polydeoxyribonucleotide (PDRN) Treatment on Corneal Wound Healing in Zebrafish (Danio rerio).

This study aimed to develop a corneal epithelial injury model in zebrafish (Danio rerio) and investigate the effectiveness of polydeoxyribonucleotide (PDRN) treatment on in vivo corneal epithelial regeneration and wound healing. Chemical injury to zebrafish cornea was produced by placing a small cotton swab containing 3% acetic acid solution. PDRN treatment was performed by immersing corneal-injured zebrafish in water containing PDRN (2 mg/mL) for 10 min at 0, 24, 48, and 72 h post-injury (hpi). The level of corneal healing was evaluated by fluorescein staining, histological examination, transcriptional profiling, and immunoblotting techniques. Fluorescein staining results demonstrate that PDRN treatment significantly (p &lt; 0.05) reduced the wounded area of the zebrafish eye at 48 and 72 hpi, suggesting that PDRN may accelerate the corneal re-epithelialization. Histopathological evaluation revealed that injured corneal epithelial cells were re-organized at 72 hpi upon PDRN treatment with increased goblet cell density and size. Moreover, transcriptional analysis results demonstrate that PDRN treatment induced the mRNA expression of adora2ab (6.3-fold), pax6a (7.8-fold), pax6b (29.3-fold), klf4 (7.3-fold), and muc2.1 (5.0-fold) after the first treatment. Besides, tnf-α (2.0-fold) and heat-shock proteins (hsp70; 2.8-fold and hsp90ab1; 1.6-fold) have modulated the gene expression following the PDRN treatment. Immunoblotting results convincingly confirmed the modulation of Mmp-9, Hsp70, and Tnf-α expression levels upon PDRN treatment. Overall, our corneal injury model in zebrafish allows for understanding the morphological and molecular events of corneal epithelial healing, and ophthalmic responses for PDRN treatment following acid injury in zebrafish.

Mulberry leaf extract improves intestinal barrier function and displays beneficial effects on colonic microbiota and microbial metabolism in weaned piglets.

Mulberry leaf extract (MLE) extracted from mulberry leaves is rich in a variety of bioactive ingredients and can be used as feed additives of weaned piglets. The present study was conducted to evaluate the effects of dietary MLE supplementation on intestinal barrier function, colon microbial numbers and microbial metabolites of weaned piglets. MLE supplementation increased the villus height and the villus height/crypt depth ratio in jejunum and ileum (P < 0.05), increased the mRNA expression of ZO-1, Claudin-1 and MUC-2 in the ileal mucosa (P < 0.05), and decreased the serum level of lipopolysaccharide (P < 0.01). Meanwhile, MLE reduced the mRNA expression of tumor necrosis factor-α and interleukin-1β (P < 0.05) and increased secretory immunoglobulin A level in the ileal mucosa (P < 0.05). In addition, MLE increased the numbers of beneficial bacteria Bifidobacterium and Lactobacillus (P < 0.05) and decreased the number of potential pathogenic bacteria Escherichia coli (P < 0.05) in the colon. Correspondingly, MLE supplementation reduced the pH value of colonic digesta (P < 0.05) and altered the microbial fermentation pattern of the colon by increasing the concentrations of microbial metabolites derived from carbohydrates fermentation such as lactate, acetate, butyrate and total short-chain fatty acids (P < 0.05), and decreasing the concentrations of microbial metabolites derived from amino acid fermentation such as p-cresol, skatole, spermine, histamine and tryptamine (P < 0.05). MLE supplementation improved intestinal barrier function and displayed beneficial effects on colon microbes and microbial metabolism in weaned piglets. © 2022 Society of Chemical Industry.

Aberrant Mucin Expression Profiles Associate With Pediatric Inflammatory Bowel Disease Presentation and Activity.

Intestinal mucosal healing is nowadays preferred as the therapeutic endpoint in inflammatory bowel disease (IBD), but objective measurements at the molecular level are lacking. Because dysregulated mucin expression is suggested to be involved in mucosal barrier dysfunction in IBD, we investigated mucin expression in association with barrier mediators and clinical characteristics in colonic tissue of a pediatric IBD population. In this cross-sectional monocentric study, we quantified messenger RNA (mRNA) expression of mucins, intercellular junctions, and cell polarity complexes in inflamed and noninflamed colonic biopsies from pediatric IBD (n = 29) and non-IBD (n = 15) patients. We then validated mucin expression at protein level and correlated mucin mRNA expression with expression of barrier mediators and clinical data. The expression of MUC1, MUC3A, MUC4, and MUC13 was increased in the inflamed colon of pediatric IBD patients compared with the noninflamed colon of non-IBD control subjects. Especially MUC13 mRNA expression associated with the expression of barrier mediators, including CDH1, OCLN, and TJP2. MUC1 and MUC3B mRNA expression in combination with calprotectin levels most accurately discriminated IBD patients from non-IBD control subjects (90.6% area under the receiver-operating characteristic curve [AUCROC], 92.0% sensitivity, 73.7% specificity), whereas aberrant mRNA expression of MUC1, MUC3A, MUC4, and MUC13 was distinctive for ulcerative colitis and of MUC3B for Crohn's disease. Furthermore, expression of MUC3A, MUC3B, and MUC4 correlated with clinical disease activity (ie, Pediatric Ulcerative Colitis Activity Index and Pediatric Crohn's Disease Activity Index), and of MUC1, MUC2, MUC4, and MUC13 with endoscopic colitis severity in ulcerative colitis patients. Colonic mucin expression is disturbed in pediatric IBD patients and associates with disease activity and presentation, suggesting its use as molecular marker to aid in disease diagnosis and management.

In vivo evidence of the prevents DSS-induced colitis of Lactiplantibacillus plantarum L15.

Ulcerative colitis (UC) is challenging to treat and severely impacts patients and families. A previous study reported immunomodulatory and reduction of pro-inflammatory properties for the Lactiplantibacillus plantarum L15. This study aimed to analyze the preventive properties and mechanistic actions in an in vivo colitis model. The histopathological alteration, inflammation cytokines, and intestinal barrier function were analyzed. Subsequently, the cecal gut microbiota contents and products from different groups were detected. Finally, gene expressions related to the NF-κB signaling process were evaluated. L. plantarum L15 significantly decreased disease activity index (DAI), myeloperoxidase activity (MPO), pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) level, and increased weight change, colon length, and production of inflammation-suppressing cytokines. Furthermore, this strain supplementation substantially increased ZO-1, Occludin, and Claudin-1, and MUC2 mRNA expression levels with a corresponding decrease in serum lipopolysaccharide and D-lactic acid contents. In addition, L. plantarum L15 improved gut microbiota composition and increased short-chain fatty acid (SCFAs) in the colon content, which significantly reduced the transfer of NF-κB p65 to the nucleus. Our findings provide a theoretical basis for L. plantarum L15 as a preventive candidate for UC.

Effect of Dietary Lactose Supplementation on Growth Performance and Intestinal Epithelium Functions in Weaned Pigs Challenged by Rotavirus.

Simple SummaryLactose is a kind of carbohydrate that exists in mammal milk. It has some physiological functions, such as providing energy, regulating gut microbiota, and affecting immunity. Rotavirus (RV) is the main pathogen that induces severe diarrhea in piglets, which impairs their growth and development. In this study, we investigated whether different levels (4% and 6%) of dietary lactose supplementation alleviates RV-induced diarrhea in weaned piglets. The results showed that lactose administration relieved the negative effect of RV on growth, which was derived from the improvement of nutrient utilization, gut barrier function, and immunity. Moreover, supplementing 6% lactose in the diets had a tendency to alleviate diarrhea in RV-infected piglets. Thus, we suggest that the diet of weaned piglets should be supplemented with more than 4% lactose (especially in the early period of weaning) if the cost of feed can be afforded.The purpose of this study was to investigate whether dietary lactose supplementation relieves rotavirus (RV)-induced diarrhea and gut dysfunction. Thirty-six crossbred weaned piglets were randomly allocated into three groups and fed diets containing 0, 4%, and 6% lactose for 20 days. On Day 15, half of the piglets in each group were orally infused with RV. RV infection impaired growth performance; induced severe diarrhea; decreased serum D-xylose concentration and morphology and sIgA level of jejunal mucosa; downregulated MUC1, MUC2, occludin, Bcl-2, IL-4, pBD3, pBD2, and pBD1 mRNA expression of jejunal mucosa and/or mesenteric lymph nodes; upregulated Bax, caspase-3, IL-2, IFN-γ, and IFN-β mRNA expression of jejunal mucosa and/or mesenteric lymph nodes; and damaged microbiota and metabolites of cecal digesta in weaned piglets (p < 0.05). Dietary lactose supplementation improved nutrient digestibility and growth performance and relieved the negative influence of RV challenge on intestinal barrier function, mRNA expression of cytokines, and host defense peptides of jejunal mucosa and/or mesenteric lymph nodes in weaned piglets (p < 0.05). Dietary administration of 6% lactose tended to relieve diarrhea (p = 0.07). These results suggest that lactose in feed increases growth performance and has a tendency to alleviate RV-induced diarrhea, derived from the improvement of nutrient utilization, gut barrier function, and immunity.

Bacillus subtilis programs the differentiation of intestinal secretory lineages to inhibit Salmonella infection.

The role of intestinal microbiota on fate determination of intestinal epithelial cells has not been extensively examined. In this study, we explore the effect of Bacillus subtilis on programmed intestinal epithelial differentiation. We find that B.subtilis stimulates the differentiation of intestinal secretory cells. Moreover, B.subtilis inhibits the Notch pathway to reduce the expression of hairy and enhancer of split 1, thereby shifting intestinal stem cell differentiation toward a secretory cell fate. Moreover, we demonstrate that the programming effect of B.subtilis on intestinal differentiation is Toll-like receptor 2 pathway dependent. B.subtilis is associated with increased numbers of Paneth and goblet cells in the intestine. This results in the production of antimicrobial peptides to protect the intestinal mucosal barrier against Salmonella typhimurium. This study demonstrates that B.subtilis contributes to the differentiation of secretory cells by affecting Notch pathway signaling to maintain the intestinal barrier.

Zranb1-mutant mice display abnormal colonic mucus production and exacerbation of DSS-induced colitis

Expression of mucin MUC2, a component of the colonic mucus layer, plays a crucial role in intestinal homeostasis. Here, we describe a new regulator of MUC2 expression, the deubiquitinase ZRANB1 (Trabid). A ZRANB1 mutation changing cysteine to serine in amino acid position 443, affects ubiquitination. To analyze ZRANB1 function in the intestine, we generated Zranb1 C443S mutant knock-in (Zranb1C443S/C443S) mice using the CRISPR/Cas9 system. Zranb1C443S/C443S mice exhibited decreased mRNA expression and MUC2 production. Colonic organoids from Zranb1C443S/C443S mice displayed decreased Muc2 mRNA expression following differentiation into goblet cells. Finally, we analyzed dextran sulfate sodium-induced colitis to understand ZRANB1's role in intestinal inflammation. Zranb1C443S/C443S mice with colitis exhibited significant weight loss, reduced colon length, and worsening clinical and pathological scores, indicating that ZRANB1 contributes to intestinal homeostasis. Together, these results suggest that ZRANB1 regulates MUC2 expression and intestinal inflammation, which may help elucidating the pathogenesis of inflammatory bowel disease and developing new therapeutics targeting ZRANB1.

Expression of Mucin Family Proteins in Non-Small-Cell Lung Cancer and its Role in Evaluation of Prognosis.

Lung cancer is still the major contributor to cancer-related mortality. Over 85% of patients suffer from non-small-cell lung cancer (NSCLC). Mucins (MUCs) are large glycoproteins secreted or membrane-bound produced by epithelial cells in normal and malignant tissues. They are the major components of the mucous gel that covers the surface of the respiratory epithelium. Certain MUCs have been used or proposed to act as biomarkers for lung cancer. Nevertheless, the expression, messenger ribonucleic acid (mRNA) levels, and the prognostic value of MUCs in NSCLC are yet to be investigated systematically. In this research, the biological information of MUC proteins in patients with NSCLC was examined using a series of databases. The results based on gene expression profiling interactive analysis (GEPIA) illustrated that the expression of MUC3A, MUC4, MUC5B, MUC13, MUC16, and MUC21 mRNAs was remarkably upmodulated in lung adenocarcinoma (LUAD) patients, whereas the MUC1 expression was downregulated in lung squamous cell carcinoma (LUSC) patients. Kaplan–Meier plotter (KM Plotter) analysis revealed that elevated mRNA expression levels of MUC3A and MUC16 were linked to unfavourable overall survival (OS) in NSCLC, while increased mRNA expression of MUC1 and MUC15 was linked to good OS, especially in LUAD patients. In addition, differential expression of MUC1, MUC3A/3B, MUC8, MUC12, MUC15, and MUC16 mRNA was linked to the prognoses of NSCLC patients with varied clinical-pathological subtypes. Genetic alterations of MUCs in NSCLC primarily involved mutations, fusion, amplification, deep deletion, and multiple alterations according to cancer genomics (cBioPortal). Therefore, we propose that combinations of MUC proteins can act as prognostic biomarkers and demonstrate the therapeutic potential for NSCLC-related therapy.

Pectin supplementation ameliorates intestinal epithelial barrier function damage by modulating intestinal microbiota in lipopolysaccharide-challenged piglets

During weaning, infants and young animals are susceptible to severe enteric infections, thus inducing intestinal microbiota dysbiosis, intestinal inflammation, and impaired intestinal barrier function. Pectin (PEC), a prebiotic polysaccharide, enhances intestinal health with the potential for a therapeutic effect on intestinal diseases. One 21-d study was conducted to investigate the protective effect of pectin against intestinal injury induced by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) in a piglet model. A total of 24 piglets (6.77±0.92 kg BW; Duroc × Landrace × Large White; barrows; 21 d of age) were randomly assigned into three groups: control group, LPS-challenged group, and PEC + LPS group. Piglets were administrated with LPS or saline on d14 and d21 of the experiment. All piglets were slaughtered and intestinal samples were collected after 3 h administration on d21. Pectin supplementation ameliorated the LPS-induced inflammation response and damage to the ileal morphology. Meanwhile, pectin also improved intestinal mucin barrier function, increased the mRNA expression of MUC2, and improved intestinal mucus glycosylation. LPS challenge reduced the diversity of intestinal microbiota and enriched the relative abundance of Helicobacter. Pectin restored alpha diversity and improved the structure of the gut microbiota by enriching anti-inflammatory bacteria and short-chain fatty acids (SCFAs)-producing bacteria, and increased the concentrations of acetate. In addition, Spearman rank correlation analysis also revealed the potential relationship between intestinal microbiota and intestinal morphology, intestinal inflammation, and intestinal glycosylation in piglets. Taken together, these results indicate that pectin enhances intestinal integrity and barrier function by altering intestinal microbiota composition and their metabolites, which subsequently alleviates intestinal injury and finally improves the growth performance of piglets.

Role of Short Chain Fatty Acids to Counteract Inflammatory Stress and Mucus Production in Human Intestinal HT29-MTX-E12 Cells.

Short chain fatty acids (SCFAs), especially butyrate (BUT), are known to promote intestinal health, but their role in the protection of intestinal barrier integrity is poorly characterized. The aim of the study was to set up an in vitro model of human colon epithelium using HT29-MTX-E12 cells to delineate the potential role of SCFAs under stress conditions. Accordingly, the HT29-MTX-E12 cells were differentiated for 42 days and subsequently exposed to dextran sulphate sodium (DSS). Further, the effects of BUT or its mixture with acetate and propionate (SCFAs-MIX) were tested to study proliferation, epithelial integrity and mucus production. The results showed that the concentration of 10% DSS for 24 h decreased the TEER about 50% compared to the control in HT29-MTX-E12 cells. The pre-treatment on HT29-MTX-E12 cells with BUT or SCFAs-MIX at specific concentrations significantly (p < 0.05) reduced the DSS-induced damage on epithelial cell integrity and permeability. Further, the treatment with specific concentrations of BUT and SCFAs-MIX for 24 h significantly promoted ZO-1, MUC2 and MUC5AC mRNA expression (p < 0.005). The present study demonstrated the suitability of HT29-MTX-E12 cells treated with DSS as an in vitro stress model of inflammatory bowel disease, which enabled us to understand the effect of bioactive SCFAs on the intestinal barrier.

The Effect of Oral Administration of the Antibacterial Peptide MPX on Intestinal Inflammation of Mice in Experimental Infection with Escherichia Coli Strain O157: H7

Escherichia coli is a gram-negative bacterium, an intestinal pathogen that can cause intestinal inflammation. Antimicrobial peptides are a class of small molecule peptides, which has good antibacterial activity against a variety of gram-positive and negative bacteria. In this regard, the authors aimed to study the effect of the antimicrobial peptide MPX, which was administered orally, on the intestinal wall of mice infected with the intestinal barrier function, which were infected with E. coli. Synthesis and purification of the antimicrobial peptide MPX (H-INWKGIAAMAKKLL-NH2) was performed by Jier Sheng Hua (Shanghai, China). Mass spectrometry and liquid chromatography (HPLC) were used for this purpose. Escherichia Coli (O157: H7 ATCC43889) was isolated from human faeces in which haemolytic uremic syndrome was reported. Solid LB agar (Solarbio, China) was used to isolate enterohemorrhagic E. coli. The culture was obtained from the Chinese Institute of Veterinary Drug Control (Beijing, China). The results of necropsy found that using of an orally administered MPX could alleviate the damage of E. coli to the liver, spleen, and the lungs were less affected. According to H&amp;E results in case of an orally administered MPX group considerably relieved duodenum and organs on day 7 and day 28. qRT-PCR results showed orally administered MPX could reduce the inflammation-related factors in the mRNA expression of IL-2 and IL-6 and TNF-α on day 7 and day 28. In addition, orally administered MPX could significantly increase them RNA expression of tight junction proteins Occludin and Zo-1 on day 7 and day 28. The results of immune histochemistry further showed that an orally administered MPX could increase the mRNA expression of MUC2 in jejunum. The above results showed that orally administered MPX could alleviate the attack of E. coli on the intestinal tract of mice, relieve intestinal inflammation, and improve the intestinal barrier function. This study lays a theoretical foundation for adding antimicrobial peptides to food. In orally administered MPX authors can see reducing the mRNA expression of inflammation-related factors, thereby alleviating the intestinal inflammation caused by E. coli infection in mice. Authors can add that orally administered MPX shows an increase in mRNA expression of tight junction protein in intestines and improves the intestinal barrier function. This study lays the foundation for adding antimicrobial peptides to food to relieve inflammation and improve barrier function in clinical practices

Colonic MUC2 Mucin Is Stabilized By FCGBP To Resist Salmonella Enterica Invasion In Goblet Cells

MUC2 mucin produced by goblet cells form the colonic mucus bilayer as the first line of innate defense against pathogen invasion by forming a physical barrier between the lumen and the underlying single layer of mucosal epithelial cells. Goblet cells also produce a variety of proteins that are associated with the mucus layer. Of these proteins, FCGBP is of significant interest due to its structural similarities to MUC2 mucin with unknown functions. In this study, we elucidated the putative functions of FCGBP embedded in MUC2 mucin in response to Salmonella enterica adherence and invasion in human colonic goblet cells. We hypothesis that FCGBP is coordinately produced with MUC2 mucin to play an integral role in forming the protective mucus layer in innate host defense. The aims of the study are: 1) to determine if FCGBP alters the structural integrity of the mucus layer and 2) to quantify structural alterations of the mucus layer in S. enterica adherence and invasion of goblet cells.To investigate whether FCGBP impaired mucus barrier functions, two cell types were investigated: Wildtype LS174T (WT) MUC2 mucus‐producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP MS). To determine if FCGBP MS led to loss in barrier function in the mucus layer, S. enterica adherence and invasion, and 0.2, 1, and 2 µM fluorescent bead penetrability inoculated on WT and FCGBP MS monolayers were quantified. MUC2 and FCGBP mRNA and protein expression induced by S. enterica in WT and FCGBP MS goblet cells were analysed by RT‐PCR and Western blotting. Cell killing by S. enterica was determined by LDH assay. S. enterica induced pro‐inflammatory cytokines responses were analyzed by RT‐PCR and human focus 15‐plex cytokine and chemokine array.FCGBP MS cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetrability further towards the epithelial cell surface temporally as compared to WT cells independent of bead size. Adherence of live S. enterica was significantly increased in FCGBP MS but not WT cells and induced robust MUC2 mRNA expression and mucus secretion in a time‐dependent manner. Similarly, S. enterica elicited high expression of pro‐inflammatory cytokine mRNA and protein release in FCGBP MS as compared to WT cells. FCGBP MS were readily invaded by S. enterica that resulted in increased cell death as compared to WT cells.More S. enterica adhered, invaded, and killed FCGBP MS as compared to WT cells. Fluorescent beads penetrated the mucus layer closer to the cell surface in FCGBP MS cells. In WT goblet cells, FCGBP and MUC2 were coordinately upregulated in response to S. enterica. The concurrent increase in pro‐inflammatory cytokine expression in FCGBP MS cells in response to S. enterica suggests that bacteria directly interacted with the cell surface suggesting an impaired protective mucus layer. The overall trend for increased bacterial invasion, pro‐inflammatory response, and cell death in FCGBP MS demonstrates that FCGBP was critical in providing structural integrity of the mucus layer.

A198 COLONIC MUC2 MUCIN IS STABILIZED BY FCGBP TO RESIST SALMONELLA TYPHIMURIUM INVASION IN GOBLET CELLS

BackgroundThe colonic mucus bilayer is the first line of innate defense against pathogen invasion in the gut by forming a physical barrier between the lumen and the underlying single layer of mucosal epithelial cells. While MUC2 mucin is the primary component of the mucus layer, it also contains several mucus associated proteins of which FCGBP is of significant interest due to its structural similarities to MUC2 with unknown functions. Here we elucidated the functions of FCGBP in MUC2 mucin in response to S. typhimurium ( St) adherence and invasion in human colonic goblet cells.AimsHypothesis: FCGBP is coordinately produced with MUC2 mucin and plays an important role in innate host defense. The specific aims are:1. To determine if FCGBP alters the structural integrity of the mucus layer2. To determine the role of FCGBP in St adherence and invasion of goblet cellsMethodsTo investigate whether FCGBP impaired mucus barrier functions, wildtype LS174T (WT) MUC2 mucus-producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP MS) were used. To determine if FCGBP MS led to loss in barrier function, St adherence and invasion, and 0.2, 1, and 2 μM fluorescent bead penetration through the mucus monolayers were quantified. MUC2 and FCGBP mRNA and protein expression induced by St were analysed by RT-PCR and Western blotting. St-induced pro-inflammatory cytokines responses were analyzed by RT-PCR and human focus 15-plex cytokine and chemokine array. Cell killing was enumerated by LDH assay.ResultsFCGBP MS cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetration closer towards the epithelial cell surface temporally as compared to WT cells independent of bead size. Adherence of St was significantly increased in FCGBP MS cells and induced robust MUC2 mRNA and protein expressions in a time-dependent manner. Similarly, St elicited robust expression of pro-inflammatory cytokine mRNA and protein release in FCGBP MS as compared to WT cells. FCGBP MS were readily invaded by St that resulted in increased cell death as compared to WT cells.ConclusionsLoss of function by FCGBP MS, but not in WT cells, showed increased penetration of fluorescent beads through the mucus layer that resulted in increased St adherence, invasion, and cell death. In WT cells, FCGBP and MUC2 were coordinately upregulated in response to St. The concurrent increase in pro-inflammatory cytokine expression in FCGBP MS cells in response to St suggests that bacteria directly interacted with the cell surface indicative of an impaired mucus layer. The overall trend for increased bacterial invasion, pro-inflammatory response and cell death in FCGBP MS demonstrates that FCGBP was critical in providing structural integrity and protective functions of the mucus layer.Funding AgenciesCIHR

Fermented Corn-Soybean Meal Mixed Feed Modulates Intestinal Morphology, Barrier Functions and Cecal Microbiota in Laying Hens.

Simple SummaryFermented feed has been of wide concern in livestock and poultry production because of its many advantages. In this study, the nutritional quality of the feed before and after fermentation was assessed, and four supplemental levels of fermented feed were used to replace unfermented feed to study the influence of fermented feed on the gut health of the laying hens during the laying peak period. The results suggest that fermented feed can improve the intestinal morphology and barrier functions of laying hens, possibly by altering the cecal microbiome.This study aimed to evaluate the effects of fermented corn–soybean meal mixed feed on intestinal barrier function and cecal microbiota in laying hens. A total of 360 Jingfen No.6 laying hens (22 wk-old) were assigned to 4 dietary treatments, which were offered basal diets (without antibiotics) containing 0, 4, 6 and 8% of fermented mixed feed respectively. The results showed that the pH value and anti-nutritional factor concentrations in fermented mixed feed were lower than those in unfermented feed (p < 0.05). Moreover, fermentation in the feed significantly increased the crude protein content (p < 0.05). Supplementation with fermented feed significantly reduced the crypt depth and increased the villi height:crypt depth ratio of duodenum and jejunum (p < 0.05). Meanwhile, fermented feed increased the secretory immunoglobulin A content and MUC2 mRNA expression of jejunum (p < 0.05). These beneficial effects were exhibited at the addition level ≥6% and microbial composition of caeca in the control, and so 6% fermented feed groups were analyzed. The structure of the gut microbiota was remarkably altered by additions, characterized by increased abundances of some health-promoting bacteria, such as Parasutterella, Butyricicoccus and Erysipelotrichaceae (p < 0.05). In summary, fermented mixed feed modulated cecal flora, subsequently contributing to improvements in intestinal morphology and barrier functions in laying hens.

Assessment of the oxidative status and goblet cell response during eimeriosis and after treatment of mice with magnesium oxide nanoparticles

Magnesium nanoparticles have been the focus of study over the past few years because of its functionality in the body. Assessment of the impact of magnesium oxide nanoparticles (MgNPs) on eimeriosis has yet to be conducted. The goal of this study was to see how MgNPs affected the parasite Eimeria papillata infected jejunum. To induce eimeriosis, mice were infected with sporulated oocysts. For treatment, 5 mg / Kg MgNPs was used for 5 consecutive days. The infection reduced the number of intestinal goblet cells and their associated genes MUC2 and MUC4, as well as increasing oxidative damage in the jejunum. MgNPs significantly reduced the oocyst production in the feces by about 77 %. After treatment, the number of goblet cells per villus increased from 4.17% to 7.40.6%. Moreover, the MgNPs were able to upregulate the expression of MUC2 and MUC4-mRNA. MgNPs significantly increased the activity of catalase and superoxide dismutase, as well as the extent of glutathione, by day 5 after infection with the parasite. On contrary, MgNPs decreased the level of malondialdehyde and nitric oxide. The findings suggested that MgNPs could be an effective anti-eimeriosis agent due to their anti-eimerial and anti-oxidant roles, as well as the regulatory effect on the goblet cell mucin genes in the jejunum of mice.