Mucin Secretion Research Articles

A32 CATHELICIDIN REGULATES GOBLET CELL MUCUS SECRETION DURING CITROBACTER RODENTIUM-INDUCED COLITIS

Abstract Background Colonic goblet cells by secreting Muc2 mucin and specific proteins is critical for physically entrapping and expelling invading enteropathogens. Thus, is not surprising that Muc2-/- littermates exhibit increased susceptibility to attaching/effacing Citrobacter rodentium colonization. The colonic epithelium also secretes small cathelicidin peptide, which potentially interacts intimately with goblet cells and was presumed to accumulate within the sterile inner mucus layer as a simple antimicrobial peptide defense. Aims To determine the effects of cathelicidin on mucin secretion in goblet cells during C. rodentium-induced colitis and the impact on the mucus barrier defense. Methods We used cathelicidin-deficient (Camp-/-) mice, mouse colonoids and human colonic LS174T like-goblet epithelial cells to elucidate the mechanisms by which cathelicidin regulates goblet cell secretions. Results Camp -/- littermates infected with C. rodentium displayed increased fecal shedding and epithelial colonization. Camp-/- littermates at the peak of C. rodentium infection (7 dpi) showed a deficient mucin layer with fewer Alcian blue/PAS filled goblet cells and a reduction in fucose (UEA-1+) and N-acetylglucosamine (WGA+) glycoproteins. By transmission electron microscopy (TEM), goblet cells in Camp-/- colons were swollen and retained a large number of mucus granules during C. rodentium infection. C. rodentium infected Camp-/- littermates showed impaired reactive oxygen species (ROS) production and a transcriptomic profiling associated with decreased ROS biosynthesis and an increase in ROS negative regulators. In mucin producing LS174T colonic epithelial cells, human cathelicidin LL-37 promptly induced the secretion of goblet cell-associated TFF3 and RELMβ, via a ROS-dependent mechanism. Conclusions These findings revealed that mice lacking cathelicidin (Camp-/-) were more susceptible to C. rodentium colonization caused by defective goblet cell mucus and mucin-associated protein secretion via a ROS-dependent mechanism. Importantly, cathelicidin regulated mucus secretion revealing a non microbicidal action of this peptide with homeostatic properties on the colonic mucus barrier, critical in excluding luminal microbiota away from the epithelia to clear bacterial infections and restore gut homeostasis. Funding Agencies NSERC

A novel cysteine-rich adaptor protein is required for mucin packaging and secretory granule stability in vivo

Mucins are large, highly glycosylated extracellular matrix proteins that line and protect epithelia of the respiratory, digestive, and urogenital tracts. Previous work has shown that mucins form large, interconnected polymeric networks that mediate their biological functions once secreted. However, how these large matrix molecules are compacted and packaged into much smaller secretory granules within cells prior to secretion is largely unknown. Here, we demonstrate that a small cysteine-rich adaptor protein is essential for proper packaging of a secretory mucin in vivo. This adaptor acts via cysteine bonding between itself and the cysteine-rich domain of the mucin. Loss of this adaptor protein disrupts mucin packaging in secretory granules, alters the mobile fraction within granules, and results in granules that are larger, more circular, and more fragile. Understanding the factors and mechanisms by which mucins and other highly glycosylated matrix proteins are properly packaged and secreted may provide insight into diseases characterized by aberrant mucin secretion.

Surfactant protein D prevents mucin overproduction in airway goblet cells via SIRPα

Mucin overproduction is a common feature of chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), and exacerbates their underlying respiratory condition. Surfactant protein D (SP-D) protects against airway diseases through modulation of immune reactions, but whether it also exerts direct effects on airway epithelial cells has remained unclear. Therefore, we sought to investigate the inhibitory role of SP-D on mucin production in airway epithelial cells. We prepared air–liquid interface (ALI) cultures of human primary bronchial epithelial cells (HBECs), which recapitulated a well-differentiated human airway epithelium. Benzo(a)pyrene (BaP), a key toxicant in cigarette smoke, induced mucin 5AC (MUC5AC) production in ALI-cultured HBECs, airway secretory cell lines, and airway epithelia of mice. Then, the protective effects of SP-D against the BaP-induced mucin overproduction were examined. BaP increased MUC5AC production in ALI cultures of HBECs, and this effect was attenuated by SP-D. SP-D also suppressed the BaP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and MUC5AC expression in NCI-H292 goblet-like cells, but not in NCI-H441 club-like cells. Signal regulatory protein α (SIRPα) was found to be expressed in HBECs and NCI-H292 cells but absent in NCI-H441 cells. In NCI-H292 cells, SP-D activated SH2 domain-containing tyrosine phosphatase-1 (SHP-1), downstream of SIRPα, and knockdown of SIRPα abolished the suppressive effects of SP-D on BaP-induced ERK phosphorylation and MUC5AC production. Consistent with these in vitro findings, intratracheal instillation of SP-D prevented the BaP-induced phosphorylation of ERK and Muc5ac expression in airway epithelial cells in a mouse model. SP-D acts directly on airway epithelial cells to inhibit mucin secretion through ligation of SIRPα and SHP-1-mediated dephosphorylation of ERK. Targeting of SIRPα is therefore a potential new therapeutic approach to suppression of mucin hypersecretion in chronic airway diseases such as COPD and asthma.

Influences of probiotics and gut microbiota on immunomodulation for the treatment of patients with cancer: a systematic review

Introduction: Immunomodulation and immunostimulation are the main mechanisms of action of probiotics in the fight against cancer. Objective: It was to carry out a systematic review of the influences of probiotics and gut microbiota on immunomodulation for the treatment of cancer patients. Methods: The PRISMA Platform systematic review rules were followed. The research was carried out from September to October 2023 in the Web of Science, Scopus, PubMed, Science Direct, Scielo, and Google Scholar databases. The quality of the studies was based on the GRADE instrument and the risk of bias was analyzed according to the Cochrane instrument. Results and Conclusion: A total of 125 articles were found, and 35 articles were evaluated in full and 18 were included and developed in the present systematic review study. Considering the Cochrane tool for risk of bias, the overall assessment resulted in 45 studies with a high risk of bias and 15 studies that did not meet GRADE and AMSTAR-2. Most studies showed homogeneity in their results, with X2=55.4%>50%. It was concluded that probiotics suppress inflammation by inhibiting several signaling pathways and reducing the global activation of NF-κβ and the production of pro-inflammatory cytokines. Probiotics influence receptor antagonism, receptor expression, binding and expression of adapter proteins, expression of negative regulatory signal molecules, and mainly the induction of microRNAs. These microRNAs can modulate the gene expression of tumor cells together with regulatory T cells that have important functions in the tumor microenvironment, mainly in inducing immune evasion. Some modulatory effects of probiotics include the production of cytokines by epithelial cells, increased mucin secretion and phagocytosis and NK cell activity, activation of T and NKT cells, stimulation of IgA production, and decreased proliferation of T cells. The gut microbiota has a major impact on the systemic immune system. The specific microbiota controls cell differentiation in which CD4+ T cells (Th17 cells) secrete IL-17. The presence of Th17 cells and regulatory T cells is associated with the gut microbiota.

Congenital lung cyst: Report of two cases

Background: Bronchogenic cysts are lesions of congenital origin derived from the primitive foregut and are the most prevailing primary cysts of the mediastinum. Most commonly unilocular, they contain clear fluid or mucinous secretions or, less commonly, haemorrhagic secretions or air. They are lined by columnar ciliated epithelium, and their walls often contain cartilage and bronchial mucous glands. It is unusual for them to have a patent connection with the airway, but when present, such a communication may promote infection of the cyst by allowing bacterial entry. The first successful surgical excision of a bronchogenic cyst was reported by Maier in mid twentieth century leading to its classification based on Mailer postulation. No such reports have been described in South/South Nigeria.

Characteristics of mucin hypersecretion in different inflammatory patterns based on endotypes of chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is usually accompanied by mucin hypersecretion that can lead to mucus accumulation and impair nasal mucociliary clearance, thus exacerbating airway inflammation. Abnormal mucin hypersecretion is regulated by different T helper (Th) cytokines, which are associated with different endotype-driven inflammatory responses. Therefore, it is of great significance to understand how these factors regulate mucin hypersecretion to provide precise treatment strategies for different endotypes of CRS. BODY: Thus far, the most common endotypes of CRS are classified as type 1, type 2, or type 3 immune responses based on innate and adaptive cell-mediated effector immunity, and the representative Th cytokines in these immune responses, such as IFN-γ, TNF-α, IL-4, IL-5, IL-13, IL-10, IL-17, and IL-22, play an important regulatory role in mucin secretion. We reviewed all the related literature in the PubMed database to determine the expression of these Th cytokines in CRS and the role they play in the regulation of mucin secretion. We believe that the main Th cytokines involved in specific endotypes of CRS play a key role in regulating abnormal mucin secretion, which contributes to better understanding of the pathogenesis of CRS and provides therapeutic targets for airway inflammatory diseases associated with mucin hypersecretion.

Eubacterium coprostanoligenes alleviates chemotherapy-induced intestinal mucositis by enhancing intestinal mucus barrier

Chemotherapy-induced mucositis represents a severe adverse outcome of cancer treatment, significantly curtailing the efficacy of these treatments and, in some cases, resulting in fatal consequences. Despite identifying intestinal epithelial cell damage as a key factor in chemotherapy-induced mucositis, the paucity of effective treatments for such damage is evident. In our study, we discovered that Eubacterium coprostanoligenes promotes mucin secretion by goblet cells, thereby fortifying the integrity of the intestinal mucus barrier. This enhanced barrier function serves to resist microbial invasion and subsequently reduces the inflammatory response. Importantly, this effect remains unobtrusive to the anti-tumor efficacy of chemotherapy drugs. Mechanistically, E. copr up-regulates the expression of AUF1, leading to the stabilization of Muc2 mRNA and an increase in mucin synthesis in goblet cells. An especially significant finding is that E. copr activates the AhR pathway, thereby promoting the expression of AUF1. In summary, our results strongly indicate that E. copr enhances the intestinal mucus barrier, effectively alleviating chemotherapy-induced intestinal mucositis by activating the AhR/AUF1 pathway, consequently enhancing Muc2 mRNA stability.

Differentiation of Pig Gastric Primary Cells into Mucus Producing Epithelial Cells.

There is a growing interest in the development of in vitro models that mimic the intrinsic characteristics of cells in vivo to replace and/or reduce the use of experimental animals. The stomach is lined with mucus secreting epithelial cells, creating a thick mucus layer that protects the underlying epithelial cells from acid, pathogens, and other harmful agents. Mucins are a main component of the mucus layer, and their secretion is an important protective feature of epithelial cells in vivo. Here, we present a method that differentiates pig gastric primary cells into mucin secreting epithelial cells by culturing the cells on polyester membranes under semi-wet interface for 14days, using differentiation medium containing the N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) in the basolateral compartment for the first 7days and subsequent 7-day culture in non-differentiation medium. The in vitro mucosal surfaces created by these cells are harvested 2weeks post confluence, and two preservation methods are described to fix the monolayers for further analysis.

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.

Activation of goblet-cell stress sensor IRE1β is controlled by the mucin chaperone AGR2

Intestinal goblet cells are secretory cells specialized in the production of mucins, and as such are challenged by the need for efficient protein folding. Goblet cells express Inositol-Requiring Enzyme-1β (IRE1β), a unique sensor in the unfolded protein response (UPR), which is part of an adaptive mechanism that regulates the demands of mucin production and secretion. However, how IRE1β activity is tuned to mucus folding load remains unknown. We identified the disulfide isomerase and mucin chaperone AGR2 as a goblet cell-specific protein that crucially regulates IRE1β-, but not IRE1α-mediated signaling. AGR2 binding to IRE1β disrupts IRE1β oligomerization, thereby blocking its downstream endonuclease activity. Depletion of endogenous AGR2 from goblet cells induces spontaneous IRE1β activation, suggesting that alterations in AGR2 availability in the endoplasmic reticulum set the threshold for IRE1β activation. We found that AGR2 mutants lacking their catalytic cysteine, or displaying the disease-associated mutation H117Y, were no longer able to dampen IRE1β activity. Collectively, these results demonstrate that AGR2 is a central chaperone regulating the goblet cell UPR by acting as a rheostat of IRE1β endonuclease activity.

Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B.

Introduction: Club cells are precursors for mucus-producing goblet cells. Interleukin 1β (IL-1B) is an inflammatory mediator with pro-mucin activities that increases the number of mucus-producing goblet cells. IL-1B-mediated mucin production in alveolar adenocarcinoma cells requires activation of the cAMP response element-binding protein (CREB). Whether the pro-mucin activities of IL-1B require club cell CREB is unknown. Methods: We challenged male mice with conditional loss of club cell Creb1 and wild type littermates with intra-airway IL-1B or vehicle. Secondarily, we studied human "club cell-like" H322 cells. Results: IL-1B increased whole lung mRNA of secreted (Mucin 5ac, Mucin 5b) and tethered (Mucin 1, Mucin 4) mucins independent of genotype. However, loss of club cell Creb1 increased whole lung mRNA of member RAS oncogene family (Rab3D), decreased mRNA of the muscarinic receptor 3 (M3R) and prevented IL-1B mediated increases in purinergic receptor P2Y, (P2ry2) mRNA. IL-1B increased the density of goblet cells containing neutral mucins in wildtype mice but not in mice with loss of club cell Creb1. These findings suggested that club cell Creb1 regulated mucin secretion. Loss of club cell Creb1 also prevented IL-1B-mediated impairments in airway mechanics. Four days of pharmacologic CREB inhibition in H322 cells increased mRNA abundance of forkhead box A2 (FOXA2), a repressor of goblet cell expansion, and decreased mRNA expression of SAM pointed domain containing ETS transcription factor (SPDEF), a driver of goblet cell expansion. Chromatin immunoprecipitation demonstrated that CREB directly bound to the promoter region of FOXA2, but not to the promoter region of SPDEF. Treatment of H322 cells with IL-1B increased cAMP levels, providing a direct link between IL-1B and CREB signaling. Conclusion: Our findings suggest that club cell Creb1 regulates the pro-mucin properties of IL-1B through pathways likely involving FOXA2.

Laparoscopic Approach in the Surgical Management of McKittrick–Wheelock Syndrome

Mckittrick-Wheelock syndrome is a pathological entity that is represented by the presence of a large rectal tumour with histopathological findings of tubular-villous adenoma that associates important secretion of electrolyte-rich mucin.

Effects of diquafosol sodium in povidone iodine-induced dry eye model.

To investigate the effects of diquafosol sodium (DQS) for dry eye model induced with povidone-iodine (PI) solution. Ten Sprague Dawley rats as the control group. Thirty Sprague Dawley rats were used to establish the dry eye model with stimulation of 10 g/L PI for 14d, then divided rats into three groups: dry eye group with no treatment (DED group, n=10); phosphate buffer saline treated group (PBS group, n=10); diquafosol treated group (DQS group, n=10). Clinical changes were observed by tear production test, fluorescein staining, tear break-up time (TBUT) test, corneal confocal microscope and ocular surface comprehensive analyzer. Eyeballs were collected on day 10 of treatment for hematoxylin-eosin (HE), periodic acid-Schiff (PAS), and alcian blue staining. TUNEL assay, polymorphonuclear (PMN) and mucin 1 (MUC1) immunofluorescence were performed and corneal ultrastructural changes were detected by electron microscopy. Compared with DED and PBS groups, tear production (7.26±0.440 vs 4.07±0.474 mm; 7.26±0.440 vs 3.74±0.280 mm; all P<0.01) and TBUT (7.37±0.383s vs 1.49±0.260s; 7.37±0.383s vs 1.42±0.437s; all P<0.01) were significantly increased in DQS group. HE, PAS, and alcian blue staining and MUC1 immunofluorescence showed mucins and conjunctival goblet cells density (8.45±0.718 vs 5.21±0.813 cells/0.1 mm2; 8.45±0.718 vs 5.36±0.615 cells/0.1 mm2; all P<0.01) increased in DQS group. Confocal microscopy, PMN immunofluorescence and TUNEL staining showed inflammatory infiltration and corneal epithelial cells apoptosis decreased in DQS group. The increased number of microvilli in corneal epithelial and the recovered cell junction were observed in DQS group. PI instillation can induce goblet cells and mucin loss, epithelial cell apoptosis and inflammation, which are consistent with the pathological manifestations of dry eye. Diquafosol can repair the ocular surface damage caused by PI, reduce corneal inflammation, inhibit corneal epithelial cell apoptosis, promote mucin secretion and maintain tear film stability.

Rhubarb extract rebuilding the mucus homeostasis and regulating mucin-associated flora to relieve constipation.

In clinical trials, rhubarb extract (Rb) was demonstrated to efficiently alleviate constipation. We would like to find out the underlying mechanism of rhubarb relieving constipation. However, there are few studies on the effects of rhubarb on colonic mucus secretion and constipation. The aim of this study was to investigate the effects of rhubarb on colonic mucus secretion and its underlying mechanism. The mice were randomly divided into four groups. Group I was the control group and Group II was the rhubarb control group, with Rb (24 g/kg body weight [b.w.]) administered through intragastric administration for three days. Group III mice were given diphenoxylate (20 mg/kg b.w.) for five days via gavage to induce constipation. Group IV received diphenoxylate lasting five days before undergoing Rb administration for three days. The condition of the colon was evaluated using an endoscope. Particularly, the diameter of blood vessels in the colonic mucosa expanded considerably in constipation mice along with diminishing mucus output, which was in line with the observation via scanning electron microscope (SEM) and transmission electron microscope (TEM). We also performed metagenomic analysis to reveal the microbiome related to mucin gene expression level referring to mucin secretion. In conclusion, Rb relieves constipation by rebuilding mucus homeostasis and regulating the microbiome.

Improvement effect of Shegan Mahuang Decoction on rats with cold-induced asthma based on TRPV1/NRF-1/mtTFA pathway

This study investigated the therapeutic effect of Shegan Mahuang Decoction(SGMHD) on cold-induced asthma in rats and explored its underlying mechanism. Seventy-two healthy male SD rats of specific pathogen free(SPF) grade were randomly divided into a blank group, a model group, a positive control group(dexamethasone, 0.4 mg·kg~(-1)), and low-, medium-, and high-dose SGMHD groups(3.2, 6.4, and 12.8 g·kg~(-1)). The blank group received saline, while the other groups were sensitized by intraperitoneal injection of ovalbumin(OVA) solution. Subsequently, the rats were placed in a cold chamber adjustable to 0-2 ℃, and OVA solution was ultrasonically nebulized to induce cold-induced asthma in rats. After three weeks of treatment, the general behaviors of rats were observed. Hematoxylin-eosin(HE) staining was used to evaluate pathological changes in lung tissues, periodic acid-Schiff(PAS) staining assessed mucin changes, and Masson staining was performed to examine collagen deposition. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of the inflammatory factors interleukin-4(IL-4) and vascular endothelial growth factor(VEGF) in serum and bronchoalveolar lavage fluid(BALF). Real-time quantitative polymerase chain reaction(RT-PCR) was employed to assess the mRNA expression levels of transient receptor potential vanilloid subfamily member 1(TRPV1), nuclear respiratory factor 1(NRF-1), and mitochondrial transcription factor A(mtTFA) in lung tissues. Western blot was used to measure the protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues. Compared with the blank group, the model group exhibited signs of rapid respiration, increased frequency of defecation with looser stools, and disheveled and dull fur. Pathological results showed significant infiltration of inflammatory cells in lung tissues, narrowing of bronchial lumens, increased mucin secretion, and enhanced collagen deposition in the model group. Additionally, the levels of IL-4 and VEGF in serum and BALF were significantly elevated, and the mRNA and protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues were significantly increased. Compared with the model group, SGMHD improved the behaviors of rats, alleviated pathological changes in lung tissues, mucin production, and collagen deposition, significantly decreased the levels of IL-4 and VEGF in serum and BALF, and reduced the mRNA expression levels of TRPV1, NRF-1, and mtTFA in lung tissues, with the medium-dose SGMHD group showing the most significant effect. Moreover, the protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues were also reduced, with the medium-dose SGMHD group exhibiting the most significant effect. In conclusion, this study demonstrates that SGMHD can alleviate airway inflammation and inhibit airway remodeling in cold-induced asthma rats. These effects may be associated with the modulation of the TRPV1/NRF-1/mtTFA signaling pathway.

Generation of human gastric assembloids from primary fetal organoids

PurposeUnderstanding human gastric epithelium homeostasis remains partial, motivating the exploration of innovative in vitro models. Recent literature showcases the potential of fetal stem cell-derived organoids in developmental and disease modelling and translational therapies. To scale the complexity of the model, we propose to generate assembloids, aiming to increase gastric maturation to provide new structural and functional insights.MethodsHuman fetal gastric organoids (fGOs) were expanded in 3D Matrigel cultures. Confluent organoid cultures were released from the Matrigel dome and resuspended in a collagen I hydrogel. Subsequently, the organoid mixture was seeded in a ring shape within a 24-well plate and allowed to gelate. The structure was lifted in the medium and cultured in floating conditions, allowing for organoid self-assembling into a gastric assembloid. After 10 days of maturation, the assembloids were characterized by immunostaining and RT-PCR, comparing different fetal developmental stages.ResultsSuccessful generation of human fetal gastric assembloids (fGAs) was achieved using spontaneous self-aggregation within the collagen I hydrogel. Immunostaining analysis of early and late fGAs showed the establishment of apico-basal cell polarity, secretion of gastric mucins, and the presence of chromogranin A in both samples. Transcriptional markers analysis revealed distinct disparities in markers associated with mature cell types between late and early fetal stages.ConclusionsfGOs can reliably be generated from human fetal samples. This pioneering assembloid approach paves the way for advancing our comprehension of human gastric epithelium homeostasis and its perturbation, offering a better in vitro platform for the study of gastric epithelial development and therapeutic translation.

PSIV-30 Exploring the Prebiotic Activities of Proanthocyanidins on a Platform Using the Three-Dimensionally (3D)-Cultured Organoids

Abstract Using antibiotics, the antimicrobial substance active against bacteria and promoting growth efficiency, in feedstuffs and food supply has been attracting great attention due to its side effects and contribution to antibiotic resistance. Therefore, exploring talternatives to antibiotics is of great significance and urgent need for human health and animal industries with a sustainable high efficiency. The objective of this study is to evaluate the biological and medicinal activities of the monomers of proanthocyanidins (PAs), such as epicatechin, epigallocatechin gallate, and flavanols such as quercetin, isoquercetrin, and rutin generated from engineered plants. The evaluations were performed in intestinal organoids isolated from ileum of neonatal piglets. The organoids after expended in vitro were incubated with or without the candidate compounds for 24 hours, and then treated with or without polyinosinic-polycytidylic acid [Poly (I:C), 10 µg/mL] for another 24 hours, a synthetic analog of double-stranded RNA (can induce a molecular pattern associated with viral infections). Cell proliferation, barrier function, toll-like receptor pathway and apoptosis as well as organic cation transporters were assessed by measuring the abundance of the corresponding genes. Concentration gradient (0 to 200 µM) measurements showed that isoquercetrin and epigallocatechin gallate might stimulate the intestinal cell proliferation, promote the uptake of L-carnitine, and increase barrier function and mucin secretion (P &amp;lt; 0.05). Epigallocatechin gallate and epicatechin might affect the inflammation responses via modifying expression of Interleukins but not induce apoptosis. Interactions between the examined these compounds and Poly (I: C) were not observed (P &amp;gt; 0.05), but the influence on the challenged pattern were tested for some of the measured genes (P &amp;lt; 0.05). Based on the data collected from this in vitro study, we conclude that epigallocatechin gallate is a potential PA monomer with all prebiotic characteristics and its applicable values in feedstuff and food supply should be studied in vivo studies in domestic (food) animals with and without antigen challenges. Supported by NC Biotechnology Center project (1107)2022-3082 and the North Carolina Agricultural Research Hatch Projects 02780.

Mechanism Underlying the Regulation of Mucin Secretion in the Uterus during Pregnancy

The function of endometrial epithelial cells is to secrete various substances that are rich in growth factors and nutrients. These substances support both embryo implantation and its subsequent development into a fetus. A vast number of mucins are expressed in endometrial epithelial cells, and they play an important role in regulating the processes of embryo implantation, pregnancy, and parturition. Previous studies have shown that mucin forms a mucus layer covering endometrial epithelial cells, which helps resist damage from foreign bacteria and their toxins. Therefore, this article aims to investigate the location of mucins in the endometrium, the mechanism of mucin secretion by the endometrium, and the regulation of mucins in the uterine epithelium by reproductive hormones, as well as the role of mucins in the protection of the epithelium’s structure. This research aims to provide a foundational understanding for future studies on the role and mechanism of endometrial mucins throughout the pregnancy cycle.

Complement C3-Deficiency-Induced Constipation in FVB/N-C3em1Hlee/Korl Knockout Mice Was Significantly Relieved by Uridine and Liriope platyphylla L. Extracts.

Complement component 3 (C3) deficiency has recently been known as a cause of constipation, without studies on the therapeutic efficacy. To evaluate the therapeutic agents against C3-deficiency-induced constipation, improvements in the constipation-related parameters and the associated molecular mechanisms were examined in FVB/N-C3em1Hlee/Korl knockout (C3 KO) mice treated with uridine (Urd) and the aqueous extract of Liriope platyphylla L. (AEtLP) with laxative activity. The stool parameters and gastrointestinal (GI) transit were increased in Urd- and AEtLP-treated C3 KO mice compared with the vehicle (Veh)-treated C3 KO mice. Urd and AEtLP treatment improved the histological structure, junctional complexes of the intestinal epithelial barrier (IEB), mucin secretion ability, and water retention capacity. Also, an improvement in the composition of neuronal cells, the regulation of excitatory function mediated via the 5-hydroxytryptamine (5-HT) receptors and muscarinic acetylcholine receptors (mAChRs), and the regulation of the inhibitory function mediated via the neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) were detected in the enteric nervous system (ENS) of Urd- and AEtLP-treated C3 KO mice. Therefore, the results of the present study suggest that C3-deficiency-induced constipation can improve with treatment with Urd and AEtLP via the regulation of the mucin secretion ability, water retention capacity, and ENS function.