MUC1 mRNA Levels Research Articles

Relationship between pathogenic E.coli O78-induced intestinal epithelial barrier damage and Zonulin expression levels in yaks.

The Zonulin gene overexpression and knockout cell lines were successfully constructed, the TEER value of the barrier of Zonulin overexpression cell lines began to decrease at 1 h after the addition of E.coli O78 and reached the lowest value at 4 h, and the TEER value of Zonulin interference cell lines decreased within 1-4 h after the addition of E.coli O78. At 4 h, the FD4 passing capacity of Zonulin overexpression cell lines was significantly higher than that of interfering cell lines, reaching twice as much as siRNA-1. The amount of bacterial translocation in overexpressed cell lines increased rapidly within 1-4 h, and the concentration of E.coli in the lower chamber was significantly higher than that in the siRNA-1 group at 4 h, but there was no significant change in the siRNA-1 group in the 1-4 h. There was no significant change in the mRNA level of MUC1 in Zonulin overexpression and interference cell lines after the addition of E.coli O78. In the overexpression group, the mRNA levels of MUC2, Occludin, and ZO-1 were significantly decreased, and the mRNA level of FABP2 was increased considerably. These results suggest stimulate epithelial cells to secrete Zonulin protein. Many Zonulin proteins regulate the opening of tight junction structures, reduce the transmembrane resistance of the cell barrier, and improve the permeability of the cell barrier and the amount of bacterial translocation.

Anemoside B4 attenuates necrotic enteritis of laying hens induced by Clostridium perfringens via inhibiting NF-κB and PI3K/Akt/mTOR signalling pathways

Poultry necrotic enteritis is an important enteric disease which might be controlled by antibiotics. However, with the excessive use of antibiotics, the phenomenon of drug resistance of Clostridium perfringens is becoming increasingly prominent. Anemoside B4 exhibits important anti-inflammatory, antioxidant and immunomodulatory effects. This study was performed to estimate the effect of Anemoside B4 on chicken necrotic enteritis induced by C. perfringens in vivo and in vitro. In the in vivo experiment we investigated the efficacy of Anemoside B4 on the growth curve, biofilm formation, haemolytic activity, virulence-related gene expression and NF-κB and PI3K/AKT/mTOR activation in Caco-2 cells induced by C. perfringens. The results showed that 12.5–50 μg/mL Anemoside B4 had no antibacterial activity but could inhibit biofilm formation, attenuate haemolytic activity and virulence-related gene expression of C. perfringens and weaken NF-κB and PI3K/Akt/mTOR activation triggered by C. perfringens in Caco-2 cells. In the in vivo experiment, 60 17-day-old healthy White Leghorns were randomly divided into six groups. The growing laying hens of the control group were fed a basic diet, and those of the five challenged groups were fed a basic diet (infection group), added 0.43 g/kg Anemoside B4 (0.43 g/kg Ane group), 0.86 g/kg Anemoside B4 (0.86 g/kg Ane group), 1.72 g/kg Anemoside B4 (1.72 g/kg Ane group) and 40 mg/kg lincomycin (lincomycin group), respectively. All challenged laying hens were infected with 1 × 109 CFU C. perfringens from day 17–20. Blood and intestinal samples were obtained, and the data demonstrated that Anemoside B4 improved the blood biochemical parameters, attenuated jejunum tissue injury, increased the spleen, thymus, bursa of fabricius index, and decreased lesion scores of the jejunum and the ileum. In the jejunum, Anemoside B4 and lincomycin downregulated the expression of IL-1β, IL-6, IL-10, TNF-α and IFN-γ at mRNA levels. Moreover, Anemoside B4 significantly enhanced both mRNA and protein levels of tight junctions ZO-1, Claudin-1 and MUC-2 in the jejunum. Anemoside B4 weakened p-P65, p-PI3K, p-Akt and p-mTOR protein expression in the jejunum infected by C. perfringens. Diets supplemented with Anemoside B4 alleviated C. perfringens-induced necrotic enteritis in laying hens by inhibiting NF-κB and PI3K/Akt/mTOR signalling pathways and improving intestinal barrier functions.

Effect of chronic stress on gel-forming mucins in the small intestine of BALB/c mice.

Intestinal homeostasis involves the collaboration of gut barrier components, such as goblet cells and IgA-microbiota complexes, that are under the control of stress that promotes inflammatory responses addressed primarily in the colon. The aim of this study was to evaluate the effect of stress on mucins, goblet cells, and proinflammatory parameters in the proximal and distal regions of the small intestine. A group (n = 6) of female 8-week-old BALB/c mice underwent board immobilization stress (2 h per day for 4 days) and were sacrificed with isoflurane. Samples from proximal and distal small segments were collected to analyze the following: 1) goblet cells stained with periodic acid-Schiff (PAS) and with alcian blue (AB) to visualize histologically neutral and acidic mucins, respectively; 2) IgA-microbiota complexes identified by flow cytometry in intestinal lavages; and 3) MUC2, MUC5AC, and IL-18 mRNA levels in whole mucosal scrapings by reverse transcription-qPCR. Regarding the unstressed group, in the proximal region of small intestine both PAS+ and AB+ goblet cells were unchanged; however, MUC5AC and IL-18 mRNA levels were increased, and the percentage of IgA-microbiota complexes was reduced. In the distal segment, the number of PAS+ goblet cells was increased, whereas the number of AB+ goblet cells was reduced and did not affect the remaining parameters. The data suggest that stress induces inflammation in the proximal small intestine; these findings may provide an experimental reference for human diseases that may affect the proximal small intestine, such as Crohn's disease, in which stress contributes to the progression of intestinal inflammation or relapse.

Hyaluronic Acid-Conjugated Thermoresponsive Polymer-Based Bioformulation Enhanced Wound Healing and Gut Barrier Repair of a TNBS-Induced Colitis Injury Ex Vivo Model in a Dynamic Perfusion Device.

Impairment of intestinal epithelium is a typical feature of inflammatory bowel disease (IBD) that causes leakage of bacteria and antigens from the intestinal lumen and thus results in persistent immune activation. Hence, healing and regeneration of the damaged gut mucosa is a promising therapeutic approach to achieve deep remission in IBD. Currently, available systemic therapies have moderate effects and are often associated with numerous side effects and malignancies. In this study, we aimed to develop a topical therapy by chemically conjugating a temperature-responsive polymer, i.e., poly(N-isopropylacrylamide), along with hyaluronic acid to obtain a sprayable therapeutic formulation that upon colon instillation adheres to the damaged gut mucosa due to its temperature-induced phase transition and mucoadhesive properties. An ex vivo adhesion experiment demonstrates that this therapeutic formulation forms a thin physical coating on the mucosal lining at a physiological temperature within 5 min. Physicochemical characterization of (P(NIPAM-co-NTBAM)-HA) established this formulation to be biocompatible, hemo-compatible, and non-immunogenic. Prednisolone was encapsulated within the polymer formulation to achieve maximum therapeutic efficacy in the case of IBD-like conditions as assessed in a custom-fabricated perfusion-based ex vivo model system. Histological analysis suggests that the prednisolone-encapsulated polymer formulation nearly restored the mucosal architecture after 2,4,6-trinitrobenzenesulfonic acid-induced damage. Furthermore, a significant (p ≤ 0.001) increase in mRNA levels of Muc-2 and ZO-1 in treated groups further confirmed the mucosal epithelial barrier restoration.

Essential oil from Artemisia argyi alleviated liver disease in zebrafish (Danio rerio) via the gut-liver axis.

The popularity of intensive fish farming has led to the emergence of fish diseases characterized by hepatobiliary syndrome. Artemisia argyi (A. argyi) essential oils have anti-inflammatory and anti-oxidant effects. However, their alleviating effects and mechanism on liver disease in fish are still unclear. Thus, adult zebrafish were used to construct an animal model to observe histopathological damages, determine biochemical parameters and expression of inflammatory cytokines and mRNAs in the PPAR-γ/NF-κB pathway, and conduct 16S sequencing of intestinal microbiota. The results found that after treatment with A. argyi essential oil, the histopathological damage caused by ethanol was relieved; the CAT, SOD, and GSH levels were remarkably elevated, while the MDA level was obviously lowered (P<0.05); the expression levels of IL-10 and IFN-γ mRNAs were enhanced, but the levels of IL-1β, IL-6, PPAR-γ, NF-κB, and TNF-α mRNAs were reduced (P<0.05) relative to the EtOH group. A. argyi essential oil remarkably attenuated the damage to intestinal tissue structure, and elevated the levels of Muc2, ZO-1, Claudin-1, and Occludin mRNA (P<0.05). Sequencing of the gut flora showed that A. argyi essential oil significantly altered the composition of gut microbes compared with the EtOH group. In addition, KEGG and COG analyses also showed significant (P<0.05) changes in acetate cycling metabolism in the EtOH group, catechol 2, 3-dioxygenase and nitroreductase were significantly increased (P<0.001), and lipid metabolism and terpenoid synthesis were significantly elevated (P<0.001) in A. argyi essential oil group. The results indicate that A. argyi essential oil could effectively relieve ethanol-caused histopathological damage of livers by modulating the composition of gut microbiota, thus inhibiting the level of IL-1β and mRNAs in the PPAR-γ/NF-κB pathway, increasing the IL-10 level, reducing the oxidative stress. This may offer a rationale for further research on the rationality of A. argyi as a substitute for feed antibiotics in aquaculture.

Assessing the in vivo ameliorative effects of Lactobacillus acidophilus KLDS1.0901 for induced non-alcoholic fatty liver disease treatment.

Reputed as a significant metabolic disorder, non-alcoholic fatty liver disease (NAFLD) is characterized by high-fat deposits in the liver and causes substantial economic challenges to any country's workforce. Previous studies have indicated that some lactic acid bacteria may effectively prevent or treat NAFLD. Overall, L. acidophilus KLDS1.0901 protected against HFD-induced NAFLD by improving liver characteristics and modulating microbiota composition, and thus could be a candidate for improving NAFLD. This study aimed to assess the protective effects of L. acidophilus KLDS1.0901 on a high-fat diet(HFD)-induced NAFLD. First, hepatic lipid profile and histological alterations were determined to study whether L. acidophilus KLDS1.0901 could ameliorate NAFLD. Then, the intestinal permeability and gut barrier were explored. Finally, gut microbiota was analyzed to elucidate the mechanism from the insights of the gut-liver axis. The results showed that Lactobacillus KLDS1.0901 administration significantly decreased body weight, Lee's index body, fat rate, and liver index. L. acidophilus KLDS1.0901 administration significantly improved lipid profiles by decreasing the hepatic levels of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) and by increasing the high-density lipoprotein cholesterol (HDL-C) levels. A conspicuous decrease of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum was observed after L. acidophilus KLDS1.0901 administration. Meanwhile, the H&E and Oil Red O-stained staining showed that L. acidophilus KLDS1.0901 significantly reduced liver lipid accumulation of HFD-fed mice by decreasing the NAS score and lipid area per total area. Our results showed that L. acidophilus KLDS1.0901 administration decreased the interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α) concentrations accompanied by the increase of interleukin-10 (IL-10). L. acidophilus KLDS1.0901 administration could improve the intestinal barrier function by upregulating the mRNA levels of occludin, claudin-1, ZO-1, and Muc-2, which were coupled to the decreases of the concentration of LPS and D-lactic acid. Notably, L. acidophilus KLDS1.0901 administration modulated the gut microbiota to a near-normal pattern. Hence, our results suggested that L. acidophilus KLDS1.0901 can be used as a candidate to ameliorate NAFLD.

Pathological mechanism of intestinal mucosal barrier injury of large intestine dampness-heat syndrome rats and the protective effect of Yujin powder

Large intestine dampness-heat syndrome (LIDHS) is frequently-occurring in the inflammatory intestinal disease of animals and human. Yujin powder (YJP) is a classical prescription for treating LIDHS. To explore the pathological mechanism of intestinal mucosal barrier injury of LIDHS and the protection of YJP, the LIDHS rat model was established through imitating the inducing conditions of LIDHS and treated with YJP. The integrity of ileal and colonic mucosa was detected through histopathological examination. The serum DAO, D-LA and ET levels were detected by ELISA. The mRNA and protein expression levels of Occludin, ZO-1 and MUC2 in ileum and colon were detected using RT-PCR and immunohistochemistry methods, respectively. The results showed that the ileal and colonic epithelium of LIDHS rats were destroyed; the serum DAO, D-LA and ET levels were significantly increased; the mRNA and protein expression levels of Occludin, ZO-1 and MUC2 in ileum and colon were all abnormally expressed. After treatment with YJP, the mucosal integrity was restored; the levels of serum DAO, D-LA and ET, mRNA and protein levels of Occludin and ZO-1 in ileum and colon and MUC2 in ileum were back-regulated; however, MUC2 level in colon was further increased. The results demonstrated that the intestinal mucosal barrier was damaged in LIDHS rats and Occludin, ZO-1 and MUC2 were abnormally expressed, and YJP could repair the intestinal mucosal barrier through up-regulating the expression of Occludin and ZO-1 in ileum and colon as well as MUC2 in colon and down-regulating MUC2 in ileum.

Effects of dietary dihydromyricetin supplementation on intestinal barrier and humoral immunity in growing-finishing pigs

In this study, we investigated the effect of dietary dihydromyricetin (DHM) supplementation on intestinal barrier and humoral immunity in growing-finishing pigs. The data showed that dietary DHM supplementation improved jejunal barrier function by upregulating the protein expressions of Occludin and Claudin-1 and the mRNA levels of MUC1 and MUC2. Dietary DHM supplementation increased the amylase, lipase, sucrase and maltase activities and the mRNA expression of nutrient transporter (SGLT1, GLUT2, PepT1) in the jejunum mucosa. Dietary DHM supplementation significantly reduced the E. coli population in the cecum and colon and increased the Lactobacillus population in the cecum. In addition, dietary DHM supplementation increased the contents of butyric acid and valeric acid in cecum and colon. In serum, dietary DHM supplementation reduced interleukin-1β (IL-1β) content and increased interleukin-10 (IL-10), Immunoglobulin M (IgM) and Immunoglobulin A (IgA) contents (p < 0.05). In addition, compared with the control group, dietary DHM supplementation improved secretory immunoglobulin A (sIgA) and interleukin-10 (IL-10) contents and down-regulated TNF-α protein expression in jejunum mucosa (p < 0.05). Together, this study demonstrated that dietary DHM supplementation improved intestinal barrier function, digestion and absorption capacity and immune function in growing-finishing pigs.

S100P Expression via DNA Hypomethylation Promotes Cell Growth in the Sessile Serrated Adenoma/Polyp-Cancer Sequence

Background/Aims: Sessile serrated adenomas/polyps (SSA/Ps) are a putative precursor lesion of colon cancer. Although the relevance of DNA hypermethylation in the SSA/P-cancer sequence is well documented, the role of DNA hypomethylation is unknown. We investigated the biological relevance of DNA hypomethylation in the SSA/P-cancer sequence by using 3-dimensional organoids of SSA/P. Methods: We first analyzed hypomethylated genes using datasets from our previous DNA methylation array analysis on 7 SSA/P and 2 cancer in SSA/P specimens. Expression levels of hypomethylated genes in SSA/P specimens were determined by RT-PCR and immunohistochemistry. We established 3-dimensional SSA/P organoids and performed knockdown experiments using a lentiviral shRNA vector. DNA hypomethylation at CpG sites of the gene was quantitated by MassARRAY analysis. Results: The mean number of hypomethylated genes in SSA/P and cancer in SSA/P was 41.6 ± 27.5 and 214 ± 19.8, respectively, showing a stepwise increment in hypomethylation during the SSA/P-cancer sequence. S100P, S100α2, PKP3, and MUC2 were most commonly hypomethylated in SSA/P specimens. The mRNA and protein expression levels of S100P, S100α2, and MUC2 were significantly elevated in SSA/P compared with normal colon tissues, as revealed by RT-PCR and immunohistochemistry, respectively. Among these, mRNA and protein levels were highest for S100P. Knockdown of the S100P gene using a lentiviral shRNA vector in 3-dimensional SSA/P organoids inhibited cell growth by >50% (p < 0.01). The mean diameter of SSA/P organoids with S100P gene knockdown was significantly smaller compared with control organoids. MassARRAY analysis of DNA hypomethylation in the S100P gene revealed significant hypomethylation at specific CpG sites in intron 1, exon 1, and the 5′-flanking promoter region. Conclusion: These results suggest that DNA hypomethylation, including S100P hypomethylation, is supposedly associated with the SSA/P-cancer sequence. S100P overexpression via DNA hypomethylation plays an important role in promoting cell growth in the SSA/P-cancer sequence.

Isolation, identification, and impact on intestinal barrier integrity of Lactiplantibacillus plantarum from fresh tea leaves (Camellia sinensis).

Lactic acid bacteria (LAB) are safe microorganisms that have been used in the processing of fermented food for centuries. The aim of this study was to isolate Lactobacillus from fresh tea leaves and examine the impact of an isolated strain on intestinal barrier integrity. First, the presence of Lactobacillus strains was investigated in fresh tea leaves from Kagoshima, Japan. Strains were isolated by growing on De Man, Rogosa and Sharpe (MRS) agar medium containing sodium carbonate, followed by the identification of one strain by polymerase chain reaction (PCR) and pheS sequence analysis, with the strain identified as Lactiplantibacillus plantarum and named L. plantarum LOC1. Second, the impact of strain LOC1 in its heat-inactivated form on intestinal barrier integrity was investigated. Strain LOC1, but not L. plantarum ATCC 14917T or L. plantarum ATCC 8014, significantly suppressed dextran sulfate sodium (DSS)-induced decreases in transepithelial electrical resistance values of Caco-2:HT29-MTX 100:0 and 90:10 co-cultures. Moreover, in Caco-2:HT29-MTX co-cultures (90:10 and 75:25), levels of occludin mRNA were significantly increased by strain LOC1 compared with untreated co-cultures, and strain LOC1 had higher mRNA levels of MUC2 and MUC4 mucins than L. plantarum ATCC 14917T and L. plantarum YT9. These results indicate that L. plantarum LOC1 may be used as a safe probiotic with beneficial effects on the intestinal barrier, suggesting that fresh tea leaves could be utilized as a safe source for isolating probiotics.

Idebenone Protects against Spontaneous Chronic Murine Colitis by Alleviating Endoplasmic Reticulum Stress and Inflammatory Response.

Endoplasmic reticulum (ER) stress in intestinal secretory goblet cells has been linked to the development of ulcerative colitis (UC). Emerging evidence suggests that the short chain quinone drug idebenone displays anti-inflammatory activity in addition to its potent antioxidant and mitochondrial electron donor properties. This study evaluated the impact of idebenone in Winnie mice, that are characterized by spontaneous chronic intestinal inflammation and ER stress caused by a missense mutation in the mucin MUC2 gene. Idebenone (200 mg/kg) was orally administered daily to 5–6 weeks old Winnie mice over a period of 21 days. Idebenone treatment substantially improved body weight gain, disease activity index (DAI), colon length and histopathology score. Immunohistochemistry revealed increased expression of MUC2 protein in goblet cells, consistent with increased MUC2 mRNA levels. Furthermore, idebenone significantly reduced the expression of the ER stress markers C/EBP homologous protein (CHOP), activating transcription factor 6 (ATF6) and X-box binding protein-1 (XBP-1) at both mRNA and protein levels. Idebenone also effectively reduced pro-inflammatory cytokine levels in colonic explants. Taken together, these results indicate that idebenone could represent a potential therapeutic approach against human UC by its strong anti-inflammatory activity and its ability to reduce markers of ER stress.

Dairy Propionibacterium freudenreichii ameliorates acute colitis by stimulating MUC2 expression in intestinal goblet cell in a DSS-induced colitis rat model

An intact mucus layer is important in managing inflammatory bowel disease (IBD). Dairy Propionibacterium freudenreichii has probiotic potential, produces propionic acid and is known to promote health. The aim of this study was to evaluate the effects of P. freudenreichii on the improvement of colitis. LS 174T goblet cells and a dextran sodium sulfate (DSS)-induced colitis rat model were used to investigate the P. freudenreichii-induced stimulation of mucin production in vitro and in vivo, respectively. The mRNA and protein expression levels of MUC2, a main component of intestinal mucus, increased in the supernatant of P. freudenreichii culture (SPFC)-treated LS 174 cells. The SPFC and live P. freudenreichii (LPF) reduced the disease activity index (DAI) in the rats with DSS-induced colitis. After treatment with SPFC or LPF, the mRNA levels of typical pro-inflammatory cytokines decreased and the inflammatory state was histologically improved in the rats with DSS-induced colitis. The SPFC and LPF treatments increased the gene and protein expression levels of MUC2 in the rats with DSS-induced colitis compared with the expression levels in the negative control rats, and immunohistochemistry (IHC) showed an increase of the intestinal MUC2 level. In addition, SPFC and LPF augmented the level of propionate in the faeces of the rats with DSS-induced colitis. In conclusion, P. freudenreichii might improve acute colitis by restoring goblet cell number and stimulating the expression of MUC2 in intestinal goblet cells.

A259 HIGH-FAT/HIGH-CHOLESTEROL DIETS PREDISPOSE THE HOST TO EXACERBATED ENTERIC INFECTIONS

Abstract Background High-fat/high-cholesterol diets are a well established risk factor for cardiovascular and metabolic diseases, given their propensity to trigger perturbations ranging from altering whole body lipid profile to the induction of intestinal dysbiosis. However much less is known about their effects on the host’s susceptibility to enteric infections. Aims To determine the effects of high-fat/high-cholesterol diets over the host’s susceptibility to enteric bacterial infections and identify the underlying molecular mechanisms. Methods C57BL/6 mice were given two different high-fat/high-cholesterol diets; HFHC (40% kcal fat, 1.25% cholesterol) or HFHCC (40% kcal fat, 1.25% cholesterol, 0.5% sodium cholate) and a control, normal diet (ND, 10% kcal fat, 0% cholesterol, 0% sodium cholate). After four weeks of administration, animals were euthanized and colonic tissue samples taken for histology, immunofluorescence, gene expression analyses, total protein lysates and microbiome sequencing (16S). A separated group of animals was gavaged with FITC-dextran to measure intestinal permeability. Mice fed with the diets for three weeks were infected with ~5x108 cfu of Citrobacter rodentium DBS100/StrpR by oral gavage, and kept on the corresponding diets after the infections. Bacterial shedding in the feces was followed for up to 30 days after infection. Results Administration of the HFHC and HFHCC diets caused an increase in intestinal permeability. Colonic sections stained with H&amp;E and alcian blue evidenced a decreased in the number of mucin-filled goblet cells and a thinner mucus layer, suggesting a defect in the assembly and/or stability of the mucus layer. Expression analyses revealed a drop in the mRNA levels of Muc1 and Muc2, suggesting reduced mucin production. The concentration of IgA was slightly reduced in colon lysates and the transcript levels of the antimicrobial peptide genes Ang4, Leap2 and Cramp were also significantly reduced. Immunofluorescent microscopy showed that goblet cell granules of HFHC- and HFHCC-fed mice were devoid of Relmβ and Tff3, indicating defective production of those two factors critical for intestinal epithelial defense and homeostasis. Collectively, our results suggest that HFHC and HFHCC diets induce differentiation and functional defects in goblet cells. Fecal shedding of C. rodentium showed an increased bacterial burden in HFHC- and HFHCC-fed animals, indicating a more aggressive bacterial infection, accompanied by increased epithelial damage. Conclusions Consumption of high-fat/high cholesterol diets perturb the colonic homeostasis and alter intestinal defenses and the integrity of the intestinal barrier, predisposing the host to a higher susceptibility to enteric infections. Funding Agencies CIHRNSERC

Gegen Qinlian decoction maintains colonic mucosal homeostasis in acute/chronic ulcerative colitis via bidirectionally modulating dysregulated Notch signaling

BackgroundGegen Qinlian decoction (GQ) is a well-known traditional Chinese medicine that has been clinically proven to be effective in treating ulcerative colitis (UC). However, its therapeutic mechanism has not been fully elucidated. Notch signaling plays an essential role in the regeneration of the intestinal epithelium. PurposeThis study was designed to ascertain the mechanism by which GQ participates in the recovery of the colonic mucosa by regulating Notch signaling in acute and chronic UC models. MethodsAcute and chronic UC mice (C57BL/6) were established with 3 and 2% dextran sulfate sodium (DSS), respectively, and treated with oral administration of GQ. The expression of the Notch target gene Hes1 and the Notch-related proteins RBP-J, MAML and Math1 was analyzed by western blotting. PTEN mRNA levels were detected by qRT-PCR. Mucin production that is characteristic of goblet cells was determined by Alcian blue/periodic acid-Schiff staining and verified by examining MUC2 mRNA levels by qRT-PCR. Cell proliferation was assayed by immunohistochemistry analysis of Ki67. HT-29 and FHC cells and Toll-like receptor 4 knockout (TLR4−/−) acute UC mice were also used in this study. ResultsGQ restored the injured colonic mucosa in both acute and chronic UC models. We found that Notch signaling was hyperactive in acute UC mice and hypoactive in chronic UC mice. GQ downregulated Hes1, RBP-J and MAML proteins and augmented goblet cells in the acute UC models, whereas GQ upregulated Hes1, RBP-J and MAML proteins in chronic UC mice, reducing goblet cell differentiation and promoting crypt base columnar (CBC) stem cell proliferation. Hes1 mRNA was suppressed in TLR4−/− UC mice, and GQ treatment reversed this effect. In vitro, GQ reduced Hes1 protein in Notch-activated HT29 and FHC cells but increased Hes1 protein in Notch-inhibited cells. ConclusionsGQ restored the colonic epithelium by maintaining mucosal homeostasis via bidirectional regulation of Notch signaling in acute/chronic UC models.

Occupational effect of sugarcane biomass burning on the conjunctival mucin profile of harvest workers and residents of an adjacent town - A Brazilian panel study

Pre-harvest burning of sugarcane fields produces large amounts of air pollutants which are known to cause health problems, including ocular surface abnormalities. In this study, we evaluated the effect of biomass burning on mucus quality and mucin gene expression (MUC1, MUC5AC, MUC16) in the conjunctiva of sugarcane workers (SWs) and residents of an adjacent town (RTs). Impression cytology samples of the inferior tarsal and bulbar conjunctiva of 78 SWs and 32 RTs were collected before (T1) and immediately after (T2) a 6-month harvest period. The neutral, acid and total mucus content of goblet cells was determined by PAS and AB staining. The levels of MUC5AC, MUC1 and MUC16 mRNA in the conjunctiva were measured by real-time PCR. Compared to RTs, SWs had higher levels of bulbar acid mucus and MUC16 mRNA and tarsal MUC5AC mRNA at T2 and lower levels of neutral mucus at T1 and T2. In the SW group, MUC1 mRNA levels were higher at T2 than at T1, but the levels of neutral and acid mucus were similar. In the RT group, acid mucus decreased and neutral mucus increased in the bulbar and tarsal conjunctiva at T2. In conclusion, our findings show that sugarcane harvesting is associated with abnormalities in mucus quality and content and changes in mucin mRNA levels on the ocular surface. This may help explain the ocular inflammatory signs and symptoms observed in subjects exposed to air pollutants and high temperatures from sugarcane biomass burning.

High-Fat Diet Enriched with Bilberry Modifies Colonic Mucus Dynamics and Restores Marked Alterations of Gut Microbiome in Rats.

Emerging evidence suggests that high-fat diet (HFD) is associated with gut microbiome dysbiosis and related disorders. Bilberry is a prebiotic food component with known health benefits. Herein, the dynamics of the colonic mucus layer and microbiome during HFD and bilberry supplementation are addressed. The effects on colonic mucus thickness in vivo and gut microbiota composition (Illumina sequencing, quantitative real-time PCR) are investigated in young rats fed a low-fat diet or HFD with or without bilberries for 8 weeks (n = 8). HFD induced significant local colonic effects, despite no observed weight gain or systemic inflammation, as HFD causes epithelial upregulation of inducible nitric oxide synthase, which is counteracted by bilberry. The firmly adherent mucus layer becomes thicker and the mRNA levels of Muc2 and Tff3 are increased by HFD with or without bilberry. In parallel, HFD reduced the colonic abundance of mucolytic bacterial species Akkermansia muciniphila and Bacteroides spp. Finally, bilberry prevents HFD-induced microbiota dysbiosis, including expansion of pathobionts, for example, Enterobacteriaceae. HFD expand firmly adherent mucus thickness and reduce mucus-foraging bacteria populations in the colon prior to obesity. Enriching HFD with bilberry protects against intestinal inflammation and marked microbiota encroachment.

Expression of inositol-requiring enzyme 1β is downregulated in azoxymethane/dextran sulfate sodium-induced mouse colonic tumors.

Inflammatory bowel disease (IBD) is a risk factor in colon cancer. Endoplasmic reticulum (ER) stress is associated with IBD and cancer. In the current study an azoxymethane (AOM) and dextran sulfate sodium (DSS)-induced mouse colonic tumor model was established to analyze the expression of ER stress chaperone molecules. Female C57BL/6 mice were intraperitoneally injected with 12 mg/kg AOM. On the 7th day following AOM injection, mice were treated with 1% DSS supplemented to the drinking water for 7 days, then followed by 14 days of normal drinking water. The cycle of 7 days DSS plus 14 days normal water was repeated twice and colonic tumors were evaluated for their number and size. Mice in the control group were injected with saline and received normal drinking water for the course of the experiment. mRNA levels of cytokines, inositol-requiring enzyme (IRE)1α and 1β, their downstream targets X-box binding protein (XBP)1u, XBP1s and mucin (MUC) 2 and interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α were detected by reverse transcription-quantitative polymerase chain reaction. IRE1α, IRE1β and MUC2 protein expression was evaluated by immunohistochemistry, and IRE1α and IRE1β levels were further assessed by western blot analysis. It was observed that tumors developed in the distal colon of mice treated with AOM/DSS. IL-6, IL-8 and TNF-α mRNA levels were significantly increased in mice of the tumor group compared with mice of the control group. There were no significant differences in IRE1α mRNA and protein expression between the two groups and XBP1s mRNA levels were increased in the tumor compared with the control group. IRE1β and MUC2 mRNA levels were significantly decreased in the tumor compared with the control group (decreased by 42 and 30%, respectively). IRE1β and MUC2 proteins were predominately expressed in colonic epithelial cells and expression was decreased in the tumor compared with the control group. In conclusion, the downregulation of IRE1β and MUC2 may reduce the ability of colon tissues to resist inflammation, thus promoting the occurrence and development of colonic tumors.

Metabolism of Caprine Milk Carbohydrates by Probiotic Bacteria and Caco-2:HT29⁻MTX Epithelial Co-Cultures and Their Impact on Intestinal Barrier Integrity.

The development and maturation of the neonatal intestine is generally influenced by diet and commensal bacteria, the composition of which, in turn, can be influenced by the diet. Colonisation of the neonatal intestine by probiotic Lactobacillus strains can strengthen, preserve, and improve barrier integrity, and adherence of probiotics to the intestinal epithelium can be influenced by the available carbon sources. The goal of the present study was to examine the role of probiotic lactobacilli strains alone or together with a carbohydrate fraction (CF) from caprine milk on barrier integrity of a co-culture model of the small intestinal epithelium. Barrier integrity (as measured by trans epithelial electrical resistance (TEER)), was enhanced by three bacteria/CF combinations (Lactobacillus rhamnosus HN001, L. plantarum 299v, and L. casei Shirota) to a greater extent than CF or bacteria alone. Levels of occludin mRNA were increased for all treatments compared to untreated co-cultures, and L. plantarum 299v in combination with CF had increased mRNA levels of MUC4, MUC2 and MUC5AC mucins and MUC4 protein abundance. These results indicate that three out of the four probiotic bacteria tested, in combination with CF, were able to elicit a greater increase in barrier integrity of a co-culture model of the small intestinal epithelium compared to that for either component alone. This study provides additional insight into the individual or combined roles of microbe–diet interactions in the small intestine and their beneficial contribution to the intestinal barrier.

Quercetin Increases MUC2 and MUC5AC Gene Expression and Secretion in Intestinal Goblet Cell-Like LS174T via PLC/PKCα/ERK1-2 Pathway.

The main dietary flavonoid quercetin, is known to preserve the integrity of gastrointestinal barrier and to have anti-inflammatory, anti-cancer, anti-fibrotic, and other beneficial properties. Many of the biological effects of quercetin appear to be associated to the modulation of cell signaling pathways, rather than to its antioxidant activity. In spite of the large number of data available on the molecular and cellular mechanisms by which quercetin exerts its biological effects, including protection of intestinal barrier function, there is a lack of data about the role of this substance on the expression and/or the secretion of mucins released by intestinal goblet cells. Here we investigated the effects of quercetin on the secretion and the gene expression of the main intestinal gel-forming mucins, MUC2 and MUC5AC, and the signaling mechanisms underlined, in human intestinal goblet cell-like LS174T. We found that quercetin increases intracellular Ca2+ levels and induces MUC2 and MUC5AC secretion in a Ca2+-dependent manner. Quercetin also induces mRNA levels of both secretory mucins. Quercetin stimulation of LS174T cells increases phosphorylation levels of extracellular signal regulated kinase (ERK)1-2 and protein kinase C (PKC) α and the induction of MUC2 and MUC5AC secretion and mRNA relies on phospholipase C (PLC), PKC, and ERK1-2 signaling pathways since the PLC inhibitor U73122, the PKC inhibitor bisindolylmaleimide (BIM) and the ERK1-2 pathway inhibitor PD98059, all revert the stimulatory effects of quercetin. We also demonstrated that the induction of mucin gene expression by quercetin is not limited to goblet cells. Indeed, quercetin induces mRNA levels of MUC2 and MUC5AC via PKCα/ERK1-2 pathway also in the human intestinal epithelial Caco-2 cells. These data highlight a novel mechanism thereby quercetin, regulating the secretory function of intestinal goblet cells and mucin levels in enterocytes may exert its protective effects on intestinal mucosal barrier.

Effect of Zinc Supplementation on Growth Performance, Intestinal Development, and Intestinal Barrier-Related Gene Expression in Pekin Ducks.

The current study was conducted to investigate the effect of zinc supplementation on the growth performance, intestinal morphology, and the transcription of the barrier function related genes in Pekin ducks. Seven-hundred and sixty-eight 1-day-old Pekin ducks were randomly assigned into six dietary treatments. Each treatment had eight replicates with 16 ducks per replicates. The ducks were fed either a corn-soybean meal basal diet or basal diets supplemented with 15, 30, 60, 120, and 240mg zinc/kg from zinc sulfate. This experiment lasted for 5weeks, and the jejunum sample were harvested at 14 and 35days of age. Results have shown that diets supplemented with zinc significantly increased the duck body weight, average daily gain, and average daily feed intake in different period of experiment (P<0.05); feed to gain ratio was decreased as the zinc level increased (P<0.05). Zinc supplementation increased the villus height and decreased the crypt depth in jejunum of ducks (P<0.05) at 14 and 35days of age. The transcription of tight junction protein CLDN1, OCND, ZO-1, and ZO-3 in jejunum were increased (P<0.05), and the messenger RNA (mRNA) levels of leak protein CLDN2 were decreased as the dietary zinc level increased (P<0.05) at 14 and 35days of age. The mRNA levels of chemical barrier-related genes MUC2 and TFF-2 in jejunum at 14 and 35days of age were increased (P<0.05) by zinc supplementation, and so did the transcription of immunological barrier-related genes lgA, pIgR, LYZ, and AvBD2 (P<0.05). In conclusion, dietary zinc supplementation exhibited growth-promoting effect on Pekin duck, improved intestinal morphology, and enhanced the intestinal barrier integrity.