Mucin Expression Research Articles

Regional Conjunctival Differences in Glycocalyx Mucin Expression in Dry Eye and Normal Subjects.

To compare regional conjunctival expression of membrane-associated mucins (MAMs) MUC1, MUC4, and MUC16 in normal and dry eye (DE) subjects. Adults with and without signs and symptoms of DE were recruited. Impression cytology was performed to collect MAMs from four bulbar and upper eyelid palpebral conjunctival regions of both eyes. After protein extraction, samples from both eyes of a single subject were pooled by region, and expression was analyzed using a capillary electrophoresis nano-immunoassay system. The chemiluminescence intensity of each antigen binding signal was calculated after normalization to the total protein amount. Statistical analyses were conducted using GraphPad Prime 9. Samples from thirteen to sixteen DE and seven to eleven normal subjects were analyzed. In normal samples, MUC1 expression from the nasal bulbar conjunctiva was significantly greater than superior (P = 0.004) and inferior (P = 0.005). In DE samples, MUC1 expression was highest superiorly. Significant differences in MUC4 and MUC16 expression were not seen in normal samples. MUC4 and MUC16 expression was upregulated superiorly (P < 0.0001) and inferiorly (P < 0.0001) in DE compared with those regions in normal samples. Although MAMs form a hydrophilic barrier called the glycocalyx, each mucin may have unique functions that are currently unexplored. All MAMs were expressed in the upper palpebral conjunctiva. Increased MUC1 expression nasally in healthy subjects suggests a functional need for increased protection. When comparing DE with normal eyes, upregulation of MUC1 superiorly, and in both MUC4 and MUC16 both superiorly and inferiorly, may indicate a need to decrease eyelid friction during blinking, especially in DE.

Abnormalities in microbiota/butyrate/FFAR3 signaling in aging gut impair brain function.

Aging-related abnormalities in gut microbiota are associated with cognitive decline, depression, and anxiety, but underlying mechanisms remain unstudied. Here, our study demonstrated that transplanting old gut microbiota to young mice induced inflammation in the gut and brain coupled with cognitive decline, depression, and anxiety. We observed diminished mucin formation and increased gut permeability ("leaky gut") with a reduction in beneficial metabolites like butyrate because of decline in butyrate-producing bacteria in the aged gut microbiota. This led to suppressed expression of butyrate receptors, free fatty acid receptors 2 and 3 (FFAR2/3). Administering butyrate alleviated inflammation, restored mucin expression and gut barriers, and corrected brain dysfunction. Furthermore, young mice with intestine-specific loss of FFAR2/3 exhibited gut and brain abnormalities akin to those in older mice. Our results demonstrate that reduced butyrate-producing bacteria in aged gut microbiota result in low butyrate levels and reduced FFAR2/3 signaling, leading to suppressed mucin formation that increases gut permeability, inflammation, and brain abnormalities. These findings underscore the significance of butyrate-FFAR2/3 agonism as a potential strategy to mitigate aged gut microbiota-induced detrimental effects on gut and brain health in older adults.

Microbial interventions in yak colibacillosis: Lactobacillus-mediated regulation of intestinal barrier.

The etiology of Escherichia coli in yaks, along with its drug resistance, results in economic losses within the yak breeding industry. The utilization of lactic acid bacteria treatment has emerged as a viable alternative to antibiotics in managing colibacillosis. To elucidate the therapeutic mechanisms of Lactobacillus against Escherichia coli-induced intestinal barrier damage in yaks, we employed yak epithelial cells as the experimental model and established a monolayer epithelial barrier using Transwell. The study encompassed four groups: a control group, a model group (exposed to E. coli O78), a low-dose Lactobacillus group (E. coli O78 + 1 × 105CFU LAB), and a high-dose Lactobacillus group (E. coli O78 + 1 × 107CFU LAB). Various techniques, including transmembrane resistance measurement, CFU counting, RT-qPCR, and Western Blot, were employed to assess indicators related to cell barrier permeability and tight junction integrity. In the Model group, Escherichia coli O78 significantly compromised the permeability and tight junction integrity of the yak epithelial barrier. It resulted in decreased transmembrane resistance, elevated FD4 flux, and bacterial translocation. Furthermore, it downregulated the mRNA and protein expression of MUC2, Occludin, and ZO-1, while upregulating the mRNA expression and protein expression of FABP2 and Zonulin, thereby impairing intestinal barrier function. Contrastingly, Lactobacillus exhibited a remarkable protective effect. It substantially increased transmembrane resistance, mitigated FD4 flux, and reduced bacterial translocation. Moreover, it significantly upregulated the mRNA and protein expression of MUC2, Occludin, and ZO-1, while downregulating the mRNA and protein expression of FABP2 and Zonulin. Notably, high-dose LAB demonstrated superior regulatory effects compared to the low-dose LAB group. In conclusion, our findings suggest that Lactobacillus holds promise in treating yak colibacillosis by enhancing mucin and tight junction protein expression. Furthermore, we propose that Lactobacillus achieves these effects through the regulation of Zonulin.

Mucin Expression Profiles in Ulcerative Colitis: New Insights on the Histological Mucosal Healing.

A structural weakness of the mucus barrier (MB) is thought to be a cause of ulcerative colitis (UC). This study aims to investigate the mucin (MUC) composition of MB in normal mucosa and UC. Ileocolonic biopsies were taken at disease onset and after treatment in 40 patients, including 20 with relapsing and 20 with remitting UC. Ileocolonic biopsies from 10 non-IBD patients were included as controls. Gut-specific MUC1, MUC2, MUC4, MUC5B, MUC12, MUC13, MUC15, and MUC17 were evaluated immunohistochemically. The promoters of mucin genes were also examined. Normal mucosa showed MUC2, MUC5B, and MUC13 in terminal ileum and colon, MUC17 in ileum, and MUC1, MUC4, MUC12, and MUC15 in colon. Membranous, cytoplasmic and vacuolar expressions were highlighted. Overall, the mucin expression was abnormal in UC. Derangements in MUC1, MUC4, and MUC5B were detected both at onset and after treatment. MUC2 and MUC13 were unaffected. Sequence analysis revealed glucocorticoid-responsive elements in the MUC1 promoter, retinoic-acid-responsive elements in the MUC4 promoter, and butyrate-responsive elements in the MUC5B promoter. In conclusion, MUCs exhibited distinct expression patterns in the gut. Their expression was disrupted in UC, regardless of the treatment protocols. Abnormal MUC1, MUC4, and MUC5B expression marked the barrier dysfunction in UC.

GROUP 3 INNATE LYMPHOID CELLS RELY ON ER STRESS RESPONSE FOR CYTOKINE PRODUCTION IN IBD

Abstract BACKGROUNDS Group 3 innate lymphoid cells (ILC3s) recently emerged as important regulators and potential drug targets for IBD. However, the response of ILC3s to environmental stimuli during intestinal inflammation remains elusive. IRE1a-XBP1 serves as the regulatory hub of the endoplasmic reticulum stress response that plays a vital role in intestinal inflammation. METHODS We generated conditional knockout Ire1aflo/flox,Rorc-Cre (Ire1aΔRorc) and Rag-/-Ire1aΔRorc mice with Ire1a deleted in ILC3s. Intestinal infections were induced by Citrobacter rodentium and Clostridium difficile. ILC3s-derived cytokines including IL-22 is crucial for host defense in the acute phase of C. rodentium and C. difficile infection. Dextran sodium sulfate (DSS) was given in drinking water to induce acute colitis. We recruited patients with active Crohn’s disease (CD) and collected ileal biopsies during routine colonoscopy before starting ustekinumab, a non-selective anti-IL-23 antibody. Mucosal ILC3s were isolated for analysis of ILC3s by flow cytometry. RESULTS In murine intestinal ILC3s, the activity of IRE1a-XBP1 pathway follows a robust 24h circadian rhythm which parallels that of clock genes and cytokines including IL-22. XBP1s is activated in ILC3s ex vivo by vasoactive intestinal peptide, which was previous shown to orchestrate the circadian expression of IL-22 by ILC3s. IRE1a-XBP1 in intestinal ILC3s is further activated in response to IL-23 and colitis in mice, as well as in inflamed IBD tissues compared to normal tissues from healthy individuals. We further showed that IRE1a-XBP1 activation in stimulated ILC3s requires mitochondrial reactive oxygen species (mtROS) (Fig 1). Using the Ire1aΔRorc mice, we demonstrated that IRE1a-XBP1 is critical for cytokine expression by ILC3s. Ire1aΔRorc mice are highly susceptible to acute C. rodentium and C. difficile infection as well as acute DSS colitis (similar vulnerabilities were found in Rag-/-Ire1aΔRorc mice compared to Rag-/-Ire1aflox/flox littermates). Ire1aΔRorc mice exhibit reduced IL-22 and IL-17A in ILC3s and impaired epithelial barrier function with diminished expression of mucins and antimicrobial peptides. The susceptibilities of Ire1aΔRorc and Rag-/-Ire1aΔRorc mice to colitis were rescued by administration of recombinant IL-22 (Fig 2). Moreover, the frequency of XBP1s+ ILC3s in ileal mucosa from CD patients before initiation of ustekinumab positively correlates with response to therapy. CONCLUSIONS We demonstrate that a non-canonical mtROS-IRE1a-XBP1 pathway augments cytokine production by intestinal ILC3s. Loss of IRE1a-XBP1 in ILC3s impairs the optimal and rhythmic expression of protective cytokines and renders the mice more susceptible to intestinal infections and colitis. We also identify XBP1+ ILC3s as a potential biomarker for predicting responsiveness to anti-IL-23 therapies in IBD.

MUCIN MRNA ISOFORM SIGNATURES AS POTENTIAL NOVEL BIOMARKERS TO EVALUATE DISEASE STATUS IN PATIENTS WITH INFLAMMATORY BOWEL DISEASES

Abstract Mucosal barrier dysfunction and aberrant mucin expression are major hallmarks in the pathophysiology of IBD. Mucins are highly polymorphic, and the presence of genetic differences can alter gene expression, resulting in several mRNA isoforms via alternative splicing. While most isoforms encode similar biological functions, others alter protein function, potentially resulting in progression towards disease. Currently, little attention has been given to the importance of mucin mRNA isoforms in IBD. The aim of our study is to investigate the potential of mucin mRNA isoforms as novel biomarkers for the evaluation of IBD activity and subtypes. To obtain this goal, RNA was extracted from colonic and terminal ileal biopsies of IBD patients that underwent an endoscopy at the Antwerp University Hospital (UZA). Additionally, patients without a history of IBD undergoing an endoscopy due to a positive iFOBT which show no endoscopic abnormalities, were included as controls. Library preparation was performed with the PacBio Iso-Seq multiplex protocol adapted for targeted transcriptome sequencing. Targeted capture was accomplished by using a custom-designed pool of probes, developed for the capture of all mucin gene transcripts. Samples were sequenced on the PacBio Sequel platform at the University of Antwerp. The data was analyzed by using the isoseq3-pipeline and additional filtering with SQANTI3 was performed. In total 106 biopsies were sequenced on the PacBio platform. The resulting intestinal mucin transcriptome was merged with the human reference transcriptome. On this combined mucin transcriptome Illumina bulk RNA sequencing data from over 2000 intestinal biopsies (GEO dataset GSE193677) were mapped to determine mucin isoform expression. An external dataset (GEO dataset GSE165512) was used for additional validation. A classification random forest was trained on this data to distinguish inflamed IBD from non-inflamed control patients based on the mucin isoform expression alone. The model performed well on train and test datasets but decreased in the external validation (Table 1). Dividing the samples based on disease phenotype greatly increases performance on the external validation dataset (Table 1). When only training on ileal biopsies, the model proved to be excellent in distinguishing Crohn’s disease patients from controls (Table 1). Classification of inflamed ulcerative colitis from control patients based on only the biopsies from the distal colon was similar to the latter (Table 1). Our machine learning model was able to distinguish Crohn’s disease from control patients and ulcerative colitis from control patients based on mucin mRNA isoform expression. In addition, our data suggests that the mucin isoform expression may vary between different regions within the colon. Table 1 Area under the receiver operator curve (AUCROC) from a random forest classifier trained on mucin isoform expression of inflamed biopsies of IBD and non-inflamed control patients.

DOP89 Unfolded protein response controls group 3 innate lymphoid cells in IBD

Abstract Background Group 3 innate lymphoid cells (ILC3s) have recently emerged as important regulators and potential drug targets for IBD. However, the response of ILC3s to environmental stimuli during intestinal inflammation remains elusive. IRE1a-XBP1 serves as the regulatory hub of endoplasmic reticulum stress and the unfolded protein response (UPR) that plays a vital role in intestinal inflammation. Methods We generated conditional knockout Ire1aflo/flox,Rorc-Cre (Ire1aΔRorc) and Rag-/-Ire1aΔRorc mice with Ire1a deleted in ILC3s. Intestinal infections were induced by Citrobacter rodentium and Clostridium difficile. ILC3s-derived cytokines including IL-22 is crucial for host defense in the acute phase of C. rodentium and C. difficile infection. Dextran sodium sulfate (DSS) was given in drinking water to induce acute colitis. We recruited patients with active Crohn’s disease (CD) and collected ileal biopsies during routine colonoscopy before starting ustekinumab, a non-selective anti-IL-23 antibody. Mucosal ILC3s were isolated for analysis of ILC3s by flow cytometry. Results In murine intestinal ILC3s, the activity of IRE1a-XBP1 pathway follows a robust 24h circadian rhythm which parallels that of clock genes and cytokines including IL-22. XBP1s is activated in ILC3s ex vivo by vasoactive intestinal peptide, which was previous shown to orchestrate the circadian expression of IL-22 by ILC3s. IRE1a-XBP1 in intestinal ILC3s is further activated in response to IL-23 and colitis in mice, as well as in inflamed IBD tissues compared to normal tissues from healthy individuals. We further showed that IRE1a-XBP1 activation in stimulated ILC3s requires mitochondrial reactive oxygen species (mtROS) (Fig 1). Using the Ire1aΔRorc mice, we demonstrated that IRE1a-XBP1 is critical for cytokine expression by ILC3s. Ire1aΔRorc mice are highly susceptible to acute C. rodentium and C. difficile infection as well as acute DSS colitis (similar vulnerabilities were found in Rag-/-Ire1aΔRorc mice compared to Rag-/-Ire1aflox/flox littermates). Ire1aΔRorc mice exhibit reduced IL-22 and IL-17A in ILC3s and impaired epithelial barrier function with diminished expression of mucins and antimicrobial peptides. The susceptibilities of Ire1aΔRorc and Rag-/-Ire1aΔRorc mice to colitis were rescued by administration of recombinant IL-22 (Fig 2). Moreover, the frequency of XBP1s+ ILC3s in ileal mucosa from CD patients before initiation of ustekinumab positively correlates with response to therapy. Conclusion We demonstrate that a non-canonical mtROS-IRE1a-XBP1 pathway augments cytokine production by intestinal ILC3s. We also identify XBP1+ ILC3s as a potential biomarker for predicting responsiveness to anti-IL-23 therapies in IBD.

Altered O-linked glycosylation in benign and malignant meningiomas.

Changes in protein glycosylation have been reported in various diseases, including cancer; however, the consequences of altered glycosylation in meningiomas remains undefined. We established two benign meningioma cell lines-SUT-MG12 and SUT-MG14, WHO grade I-and demonstrated the glycan and glycosyltransferase profiles of the mucin-type O-linked glycosylation in the primary benign meningioma cells compared with two malignant meningioma cell lines-HKBMM and IOMM-Lee, WHO grade III. Changes in O-linked glycosylation profiles in malignant meningiomas were proposed. Primary culture technique, morphological analysis, and immunocytochemistry were used to establish and characterize two benign meningioma cell lines. The glycan profiles of the primary benign and malignant meningiomas cell lines were then analyzed using lectin cytochemistry. The gene expression of O-linked glycosyltransferases, mucins, sialyltransferases, and fucosyltransferases were analyzed in benign and malignant meningioma using the GEO database (GEO series GSE16581) and quantitative-PCR (qPCR). Lectin cytochemistry revealed that the terminal galactose (Gal) and N-acetyl galactosamine (GalNAc) were highly expressed in primary benign meningioma cells (WHO grade I) compared to malignant meningioma cell lines (WHO grade III). The expression profile of mucin types O-glycosyltransferases in meningiomas were observed through the GEO database and gene expression experiment in meningioma cell lines. In the GEO database, C1GALT1-specific chaperone (COSMC) and mucin 1 (MUC1) were significantly increased in malignant meningiomas (Grade II and III) compared with benign meningiomas (Grade I). Meanwhile, in the cell lines, Core 2 β1,6-N-acetylglucosaminyltransferase-2 (C2GNT2) was highly expressed in malignant meningiomas. We then investigated the complex mucin-type O-glycans structures by determination of sialyltransferases and fucosyltransferases. We found ST3 β-galactoside α-2,3-sialyltransferase 4 (ST3GAL4) was significantly decreased in the GEO database, while ST3GAL1, ST3GAL3, α1,3 fucosyltransferases 1 and 8 (FUT1 and FUT8) were highly expressed in malignant meningioma cell lines-(HKBMM)-compared to primary benign meningioma cells-(SUT-MG12 and SUT-MG14). Our findings are the first to demonstrate the potential glycosylation changes in the O-linked glycans of malignant meningiomas compared with benign meningiomas, which may play an essential role in the progression, tumorigenesis, and malignancy of meningiomas.

Xylitol attenuates diabetes induced intestinal permeability changes and inflammatory injury by improving intestinal tight junction protein expression and mucus secretion in rats

ABSTRACT The purpose of this study was to investigate the protective effect of xylitol on gut mucosal barrier injury in a type 2 diabetes rat model. Treatment with xylitol not only ameliorated diabetic-induced changes to small intestinal mucosa morphology and decreased diamine oxidase concentration in the serum of diabetic rats, but also modified the inflammatory infiltrates and improved the secretory IgA levels of the small intestinal epithelium. Increased expression of zo-1, occludin, mucin-2 and intestinal trefoil factor also was detected in small intestinal tissue. Intragastric administration of xylitol suppressed diabetes-induced increases of myeloperoxidase, eosinophil peroxidase, IL-6 and TNF-α. These results suggest that xylitol reduces mucosal inflammatory lesions and improves the intestinal barrier in diabetic rats by maintaining the structural integrity of the mucous layer, inhibiting the expression of inflammatory mediators and enhancing the expression of tight junction proteins associated with intestinal permeability.

Effects of Bacillus subtilis on the immune parameters, intestinal morphology and mucin gene expression in broilers exposed to Salmonella enterica challenge

The objective of this study was to evaluate the effect of the probiotic Bacillus subtilis supplementation on bacterial population, morphometry and mucin gene expression of intestine, and on immune response in Salmonella challenged broilers. Treatments were: 1) negative control (no probiotic- no challenging); 2) probiotic treated (no challenging); 3) positive control (no probiotic-salmonella challenging), and 4) Salmonella challenged chicks fed probiotic. Salmonella infection resulted in significant decrease in the relative weight of thymus. In challenged birds, dietary addition of probiotic increased (P&lt;0.0.5) the relative weights of bursa and thymus. Population of lactic acid bacteria was significantly higher in probiotic treated groups as compared to negative control or challenged chickens. Challenged chickens had the lowest count of goblet cells and those received probiotic had the highest goblet cells count. The expression of mucin 2 gene was higher in group received dietary probiotic as compared with other treatments. Difference for gene expression was not found in challenged chickens and the negative control (P&gt;0.05). In conclusion, the dietary inclusion of Bacillus subtilis could alleviate the negative effects of Salmonella infection on intestinal cells and bacterial population and could improve the growth and development of immune organs and function in infected broilers.&#x0D;

Alleviating effect of Nexrutine on mucosal inflammation in mice with ulcerative colitis: Involvement of the RELA suppression.

Nexrutine is an herbal extract derived from Phellodendron amurense, known for its anti-inflammatory, antidiarrheal, and hemostatic properties. However, its effect on ulcerative colitis (UC)remains unclear. A mouse model of UC was induced by 3% dextran sulfate sodium, while human colonic epithelial cells NCM-460were exposed to lipopolysaccharide. Both models were treated with Nexrutine at 300or 600 mg/kg, with Mesalazine applied as a positive control regimen. The disease activity index (DAI)of mice was calculated, and the pathological injury scores were assessed through hematoxylin and eosin staining. The viability of NCM-460 cells was determined using the CCK-8method. Inflammatory cytokines were detected using ELISA kits. Expression of mucin 3 (MUC3),Claudin-1, and tight junction protein (ZO-1)was detected to analyze mucosal barrier integrity. Target genes of Nexrutine were predicted using bioinformatics tools. Expression of RELA proto-oncogene (RELA)was analyzed using qPCR and western blot assays. The Nexrutine treatments significantly alleviated DAI of mice, mitigated pathological changes in their colon tissues, decreased the production of pro-inflammatory cytokines, enhanced the barrier integrity-related proteins, and increased NCM-460 cell viability in vitro. RELA, identified as a target gene of Nexrutine, showed elevated levels in UC models but was substantially suppressed by Nexrutine treatment. Adenovirus-mediated RELA upregulation in mice or the overexpression plasmid of RELA in cells counteracted the effects of Nexrutine treatments, exacerbating UC-related symptoms. This study demonstrates that Nexrutine alleviates inflammatory mucosal barrier damage in UC by suppressing RELA transcription.

Expression Analysis of Genes Corresponding to Mucins and Their Glycans from Cervical Tissue Using RNA Sequencing.

In the female reproductive tract, mucin proteins are the main component of mucus secreted by cervical goblet cells and play an essential role in many biological functions. They act as a medium for lubrication and mainting a cervical mucosal barrier against ascending pathogens from the vagina while also allowing sperm migration. The expression of mucin genes as well as the levels of O-glycosylation changes across the oestrous cycle. Detection and characterization of mucins and their glycans is important to understand the interface between the external and the internal environment, as the cervical epithelium represents the first line of defense against infections of the upper reproductive tract. Advances in the field of molecular biology have made possible to study differences in mucin and glycan gene expression which can help to understand impeded sperm transport as well as variation in the susceptibility to infection. This chapter discusses procedures relevant for both animals and humans on how to recover cervical tissue and perform a gene expression analysis of genes corresponding to mucins and their glycans using RNA sequencing.

Recombinant Production of Glycoengineered Mucins in HEK293-F Cells.

Recombinant mucins are attractive polymeric building blocks for new biomaterials, biolubricants, and therapeutics. Advances in glycoengineered host cell systems now enable the recombinant production of mucins with tailored O-glycan side chains, offering new opportunities to tune the functionality of mucins and investigate the biology of specific O-glycan structures. Here, we provide a protocol for the scalable production of glycoengineered mucins and mucin-like glycoproteins in suspension-adapted HEK293-F cells. The protocol includes the preparation of engineered cell lines with homozygous knockout (KO) of glycosyltransferases using CRISPR/Cas9 and homology-directed repair (HDR) templates designed for efficient screening of clones. Strategies are provided for the stable introduction of mucin expression cassettes into the HEK293-F genome and the subsequent isolation of high-expressing cell populations. The high-titer production of recombinant mucins in conventional shaker flasks is described as an example production strategy using these cell lines.

인간 기도 상피세포에서 IKK/IκBα/NF-κB p65 신호전달 경로를 경유한 호흡기 MUC5AC 뮤신 유전자의 발현에 미치는 올레아놀산의 영향

In the present study, we investigated whether oleanolic acid, an anti-inflammatory natural product, significantly affects the phorbol 12-myristate 13-acetate (PMA)-induced gene expression of airway MUC5AC mucin. Confluent NCIH292 cells were pretreated with oleanolic acid for 30 min and then stimulated with PMA for 24 h or the indicated periods. The MUC5AC mucin mRNA expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA. To elucidate the action mechanism of oleanolic acid, effect of oleanolic acid on PMA-induced NF-B signaling pathway was investigated by western blot analysis. The results were as follows: (1) Oleanolic acid significantly inhibited the production of MUC5AC mucin protein and down-regulated the expression of MUC5AC mucin mRNA; (2) Oleanolic acid inhibited PMA-induced activation (phosphorylation) of inhibitory kappa B kinase (IKK), and thus phosphorylation and degradation of inhibitory kappa Ba (IB); (3) Oleanolic acid inhibited PMA-induced nuclear translocation of nuclear factor kappa B (NF-B) p65, led to the down-regulation of MUC5AC mucin gene expression in NCI-H292 cells. These results suggest that oleanolic acid can regulate the gene expression of mucin by acting on airway epithelial cells through regulation of NF-B signaling pathway.

Red Cabbage Juice-Mediated Gut Microbiota Modulation Improves Intestinal Epithelial Homeostasis and Ameliorates Colitis.

Gut microbiota plays a crucial role in inflammatory bowel diseases (IBD) and can potentially prevent IBD through microbial-derived metabolites, making it a promising therapeutic avenue. Recent evidence suggests that despite an unclear underlying mechanism, red cabbage juice (RCJ) alleviates Dextran Sodium Sulfate (DSS)-induced colitis in mice. Thus, the study aims to unravel the molecular mechanism by which RCJ modulates the gut microbiota to alleviate DSS-induced colitis in mice. Using C57BL/6J mice, we evaluated RCJ's protective role in DSS-induced colitis through two cycles of 3% DSS. Mice were daily gavaged with PBS or RCJ until the endpoint, and gut microbiota composition was analyzed via shotgun metagenomics. RCJ treatment significantly improved body weight (p ≤ 0.001), survival in mice (p < 0.001) and reduced disease activity index (DAI) scores. Further, RCJ improved colonic barrier integrity by enhancing the expression of protective colonic mucins (p < 0.001) and tight junction proteins (p ≤ 0.01) in RCJ + DSS-treated mice compared to the DSS group. Shotgun metagenomic analysis revealed an enrichment of short-chain fatty acids (SCFAs)-producing bacteria (p < 0.05), leading to increased Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ) activation (p ≤ 0.001). This, in turn, resulted in repression of the nuclear factor κB (NFκB) signaling pathway, causing decreased production of inflammatory cytokines and chemokines. Our study demonstrates colitis remission in a DSS-induced mouse model, showcasing RCJ as a potential modulator for gut microbiota and metabolites, with promising implications for IBD prevention and treatment.

Changes in gene expression in the intestinal mucus of broilers with woody breast myopathy

Previous work has shown that dietary treatments affect woody breast (WB) incidence differently, which indicates that gut conditions such as gut barrier function, inflammation, and oxidative stress are likely related to WB. In this study, dietary supplementation with antibiotics (bacitracin) or probiotics (Bacillus subtilis) was investigated for their effects on the expression of transcripts related to gut barrier function, inflammation, and oxidative stress in the mucus lining of the jejunum from broilers with or without WB. A split-plot experimental design was used in this study. The dietary treatments served as the main plot factor and the breast muscle condition was the subplot factor. On d 41, jejunum mucus was collected from 1 bird from each of 3 replicate pens in each 3 dietary treatment groups that exhibited WB and an additional bird that contained a normal breast (3 biological replicates/treatment/phenotype; 3 × 3 × 2, total N = 18). Total RNA was extracted using a commercial RNA extraction kit. The expression levels of CLDN1, MUC6, TLR2A, TLR2B, TLR4, IFN-γ, IL-1β, IL-8L1, IL-10, NOS2, and SOD were determined using 2-step RT-qPCR analysis. The gene expression difference in ΔCt values was determined after normalizing with the chicken 18S rRNA gene. When the significant differences occurred between treatments, the relative fold change was calculated using the ΔΔCt method and the significance level was calculated. The PROC GLM procedure of SAS 9.4 was used, and the level of significance was set at P ≤ 0.05. There were no significant interactive effects between diet and the breast muscle condition on the expression of any of the genes tested. However, birds with WB exhibited higher MUC6 (P < 0.0001) gene expression levels than birds with normal breast muscles. In addition, the expression of SOD decreased in birds that were fed the antibiotic diet when compared to birds that were fed the probiotic diet (P = 0.014). In conclusion, WB identified in broilers tested in the current study is attributed to increased expression of mucin, indicating a correlation between WB incidence and gel-forming mucin secretion and pathogen signaling.

Lixisenatide ameliorated lipopolysaccharide (LPS)-induced expression of mucin and inflammation in bronchial epithelial cells.

Chronic obstructive pulmonary disease (COPD) induces serious social and economic burdens due to its high disability and mortality, the pathogenesis of which is highly involved with inflammation, oxidative stress (OS), and mechanism of mucin 5AC (MUC5AC) secretion. Lixisenatide is a selective glucagon-like peptide 1 receptor agonist recently reported to have anti-inflammatory properties. Our study will focus on the potential impact of lixisenatide on lipopolysaccharide (LPS)-induced mucin secretion and inflammation in 16 human bronchial epithelial (16HBE) cells to check its potential function in COPD. 16HBE cells were treated with LPS, with or without lixisenatide (10 and 20 nM) for 1 day. Remarkably declined cell viability, enhanced lactate dehydrogenase release, activated OS, and elevated release of inflammatory cytokines were observed in LPS-treated 16HBE cells, accompanied by the activation of nuclear factor-κB signaling, all of which were signally reversed by lixisenatide. Moreover, elevated expression and release of MUC5AC were observed in LPS-treated 16HBE cells but were markedly repressed by lixisenatide. Furthermore, the repressed nuclear factor erythroid 2-related factor 2 (Nrf2) level in LPS-treated 16HBE cells was notably rescued by lixisenatide. Lastly, following the knockdown of Nrf2, the protective function of lixisenatide on LPS-triggered MUC5AC release in 16HBE cells was significantly abrogated. Collectively, lixisenatide ameliorated LPS-induced expression of mucin and inflammation in bronchial epithelial cells by regulating Nrf2.

Lead exposure aggravates glucose metabolism disorders through gut microbiota dysbiosis and intestinal barrier damage in high-fat diet-fed mice.

Lead (Pb) is an ancient toxic metal and is still a major public health issue. Our previous study found that Pb exposure promotes metabolic disorders in obese mice, but the molecular mechanisms remain unclear. The present study explored the effects of Pb exposure on glucose homeostasis in mice fed a normal diet (ND) and high-fat diet (HFD) from the perspective of gut microbiota. Pb exposure had little effect on glucose metabolism in ND mice, but exacerbated hyperglycemia and insulin resistance, and impaired glucose tolerance in HFD mice. Pb exposure impaired intestinal tight junctions and mucin expression in HFD mice, increasing intestinal permeability and inflammation. Moreover, Pb exposure altered the composition and structure of the gut microbiota and decreased short-chain fatty acids (SCFAs) levels in HFD mice. Correlation analysis revealed that the gut microbiota and SCFAs were significantly correlated with the gut barrier and glucose homeostasis. Furthermore, the fecal microbiota transplantation from Pb-exposed HFD mice resulted in glucose homeostasis imbalance, intestinal mucosal structural damage and inflammation in recipient mice. However, Pb did not exacerbate the metabolic toxicity in HFD mice under depleted gut microbiota. The findings of the present study suggest that Pb induces impairment of glucose metabolism in HFD mice by perturbing the gut microbiota. Our study offers new perspectives on the mechanisms of metabolic toxicity of heavy metals and demonstrates that the gut microbiota may be a target of action for heavy metal exposure. © 2023 Society of Chemical Industry.

In vivo dynamics of pro-inflammatory factors, mucins, and polymorph nuclear neutrophils in the bovine oviduct during the follicular and luteal phase

Dynamic functional changes in the oviductal microenvironment are the prerequisite for the establishment of pregnancy. The objective of this study was to gain the first insights into oestrous cycle-dependent dynamics of polymorph nuclear neutrophils (PMN) and the mRNA abundance of selected genes and their correlations in the oviduct of living cows. Mini-cytobrush samples were taken from the oviducts of healthy heifers (n = 6) and cows (n = 7) during the follicular (FOL) and luteal phase (LUT) by transvaginal endoscopy. Total RNA was isolated from the samples and subjected to reverse transcription-quantitative PCR for selected pro-inflammatory factors, glycoproteins, and a metabolic marker. The percentage of PMN was determined by cytological examination. The mean PMN percentage was 2.8-fold greater during LUT than FOL. During LUT, significantly greater mRNA abundance of the pro-inflammatory factors IL1B, CXCL1, CXCL3, and CXCL8 was observed. The OVGP1 mRNA abundance was twice as high during FOL than in LUT. Pearson correlation, principal component analysis and heatmap analyses indicated characteristic functional patterns with strong correlations among investigated factors. Using this novel approach, we illustrate complex physiological dynamics and interactions of the mRNA expression of pro-inflammatory factors, mucins, OVGP1, and PMN in the oviduct during the oestrous cycle.

BLV-miR-B1-5p Promotes Staphylococcus aureus Adhesion to Mammary Epithelial Cells by Targeting MUC1.

Bovine leukemia virus (BLV) is widely prevalent worldwide and can persistently infect mammary epithelial cells in dairy cows, leading to reduced cellular antimicrobial capacity. BLV-encoded microRNAs (BLV-miRNAs) can modify host genes and promote BLV replication. We previously showed that BLV-miR-B1-5p significantly promoted Staphylococcus aureus (S. aureus) adhesion to bovine mammary epithelial (MAC-T) cells; however, the pathway responsible for this effect remained unclear. This study aims to examine how BLV-miR-B1-5p promotes S. aureus adhesion to MAC-T cells via miRNA target gene prediction and validation. Target site prediction showed that BLV-miR-B1-5p could target the mucin family gene mucin 1 (MUC1). Real-time polymerase chain reaction, immunofluorescence, and dual luciferase reporter assay further confirmed that BLV-miR-B1-5p could target and inhibit the expression of MUC1 in bovine MAC-T cells while interfering with the expression of MUC1 promoted S. aureus adhesion to MAC-T cells. These results indicate that BLV-miR-B1-5p promotes S. aureus adhesion to mammary epithelial cells by targeting MUC1.