Abelmoschus manihot polysaccharide fortifies intestinal mucus barrier to alleviate intestinal inflammation by modulating Akkermansia muciniphila abundance.

The intestinal mucus barrier is an important line of defense against gut pathogens. Damage to this barrier brings bacteria into close contact with the epithelium, leading to intestinal inflammation. Therefore, its restoration is a promising strategy for alleviating intestinal inflammation. This study showed that Abelmoschus manihot polysaccharide (AMP) fortifies the intestinal mucus barrier by increasing mucus production, which plays a crucial role in the AMP-mediated amelioration of colitis. IL-10-deficient mouse models demonstrated that the effect of AMP on mucus production is dependent on IL-10. Moreover, bacterial depletion and replenishment confirmed that the effects of AMP on IL-10 secretion and mucus production were mediated by Akkermansia muciniphila. These findings suggest that plant polysaccharides fortify the intestinal mucus barrier by maintaining homeostasis in the gut microbiota. This demonstrates that targeting mucus barrier is a promising strategy for treating intestinal inflammation.

Progress on immuno-microenvironment and immune-related therapies in patients with pseudomyxoma peritonei.

Pseudomyxoma peritonei (PMP) is an indolent malignant syndrome. The standard treatment for PMP is cytoreductive surgery combined with intraperitoneal hyperthermic chemotherapy (CRS + HIPEC). However, the high recurrence rate and latent clinical symptoms and signs are major obstacles to further improving clinical outcomes. Moreover, patients in advanced stages receive little benefit from CRS + HIPEC due to widespread intraperitoneal metastases. Another challenge in PMP treatment involves the progressive sclerosis of PMP cell-secreted mucus, which is often increased due to activating mutations in the gene coding for guanine nucleotide-binding protein alpha subunit (GNAS). Consequently, the development of other PMP therapies is urgently needed. Several immune-related therapies have shown promise, including the use of bacterium-derived non-specific immunogenic agents, radio-immunotherapeutic agents, and tumor cell-derived neoantigens, but a well-recognized immunotherapy has not been established. In this review the roles of GNAS mutations in the promotion of mucin secretion and disease development are discussed. In addition, the immunologic features of the PMP microenvironment and immune-associated treatments are discussed to summarize the current understanding of key features of the disease and to facilitate the development of immunotherapies.

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.

Teleost Muc2 and Muc5ac: Key guardians of mucosal immunity in flounder (Paralichthys olivaceus).

Mucins secreted by mucous cells constitute a core part of the defense line against the invasion of pathogens. However, mucins' structure and immunological functions remain largely unknown in teleost fish. In this study, two typical mucins, Muc2 and Muc5ac of flounder (Paralichthys olivaceus), were cloned and their physicochemical properties, structure and conservation were analyzed. Notably, specific antibodies against flounder Muc2 and Muc5ac were developed. It was verified at the gene and protein level that Muc2 was expressed in the hindgut and gills but not in the skin, while Muc5ac was expressed in the skin and gills but not in the hindgut. After flounders were immunized by immersion with inactivated Edwardsiella tarda, Muc2 and Muc5ac were significantly upregulated at both the gene expression and protein levels, and Muc2+/Muc5ac+ mucous cells proliferated and increased secretion of Muc2 and Muc5ac. Moreover, Muc2 and Muc5ac exerted retention and clearance effects on E. tarda in a short period (within 1 dpi). These results revealed the characterization of fish mucins Muc2 and Muc5ac at the protein level and clarified the role of mucins as key guardians to maintain the mucus barrier, which advanced our understanding of teleost mucosal barrier.

Urolithin A-mediated augmentation of intestinal barrier function through elevated secretory mucin synthesis.

Maintaining the mucus layer is crucial for the innate immune system. Urolithin A (Uro A) is a gut microbiota-derived metabolite; however, its effect on mucin production as a physical barrier remains unclear. This study aimed to elucidate the protective effects of Uro A on mucin production in the colon. In vivo experiments employing wild-type mice, NF-E2-related factor 2 (Nrf2)-deficient mice, and wild-type mice treated with an aryl hydrocarbon receptor (AhR) antagonist were conducted to investigate the physiological role of Uro A. Additionally, in vitro assays using mucin-producing cells (LS174T) were conducted to assess mucus production following Uro A treatment. We found that Uro A thickened murine colonic mucus via enhanced mucin 2 expression facilitated by Nrf2 and AhR signaling without altering tight junctions. Uro A reduced mucosal permeability in fluorescein isothiocyanate-dextran experiments and alleviated dextran sulfate sodium-induced colitis. Uro A treatment increased short-chain fatty acid-producing bacteria and propionic acid concentration. LS174T cell studies confirmed that Uro A promotes mucus production through the AhR and Nrf2 pathways. In conclusion, the enhanced intestinal mucus secretion induced by Uro A is mediated through the actions of Nrf-2 and AhR, which help maintain intestinal barrier function.

Epigenetic programming mediates abnormal gut microbiota and disease susceptibility in offspring with prenatal dexamethasone exposure.

Prenatal dexamethasone exposure (PDE) can lead to increased susceptibility to various diseases in adult offspring, but its effect on gut microbiota composition and the relationship with disease susceptibility remains unclear. In this study, we find sex-differential changes in the gut microbiota of 6-month-old infants with prenatal dexamethasone therapy (PDT) that persisted in female infants up to 2.5 years of age with altered bile acid metabolism. PDE female offspring rats show abnormal colonization and composition of gut microbiota and increased susceptibility to cholestatic liver injury. The aberrant gut microbiota colonization in the PDE offspring can be attributed to the inhibited Muc2 expression caused by decreased CDX2 expression before and after birth. Integrating animal and cell experiments, we further confirm that dexamethasone could inhibit Muc2 expression by activating GR/HDAC11 signaling and regulating CDX2 epigenetic modification. This study interprets abnormal gut microbiota and disease susceptibility in PDT offspring from intrauterine intestinal dysplasia.

Early intestinal development of chickens divergently selected for high or low 8-wk body weight and a commercial broiler.

The early posthatch period is crucial to intestinal development, shaping long-term growth, metabolism, and health of the chick. The objective of this study was to determine the effect of genetic selection on morphological characteristics and gene expression during early intestinal development. Populations of White Plymouth Rocks have been selected for high weight (HWS) and low weight (LWS) for over 63 generations, and some LWS display symptoms of anorexia. Intestinal structure and function of these populations were compared to a commercial broiler Cobb 500 (Cobb) during the perihatch period. Egg weights, yolk-free embryo BW, yolk weights, and jejunal samples from HWS, LWS, and Cobb were collected on embryonic day (e) 17, e19, day of hatch, day (d) 3, d5, and d7 posthatch for histology and gene expression analysis. The RNAscope in-situ hybridization method was used to localize expression of the stem cell marker, olfactomedin 4 (Olfm4). Villus height (VH), crypt depth (CD), and VH/CD were measured from Olfm4 stained images using ImageJ. mRNA abundance for Olfm4, stem cell marker Lgr5, peptide transporter PepT1, goblet cell marker Muc2, marker of proliferation Ki67, and antimicrobial peptide LEAP2 were examined. Two-factor ANOVA was performed for measurements and Turkey's HSD was used for mean separation when appropriate. Cobb were heaviest and LWS the lightest (P < 0.01). at each timepoint. VH increased in Cobb and CD increased in HWS compared to LWS (P < 0.01). PepT1 mRNA was upregulated in LWS (P < 0.01), and Muc2 mRNA was decreased in both HWS and LWS compared to Cobb (P < 0.01). Selection for high or low 8-wk body weight has caused differences in intestinal gene expression and morphology when compared to a commercial broiler.

Upregulated Tß4 expression in inflammatory bowel disease impairs the intestinal mucus barrier by inhibiting autophagy in mice.

Disrupted intestinal barrier homeostasis is fundamental to inflammatory bowel disease. Thymosin ß4 (Tß4) improves inflammation and has beneficial effects in dry-eye diseases, but its effects on the intestinal mucus barrier remain unknown. Therefore, this study evaluated the underlying regulatory mechanisms and effects of Tß4 by examining Tß4 expression in a mouse model with dextran sodium sulfate (DSS)-induced colitis and colonic barrier damage. Additionally, we intraperitoneally injected C57BL/6 mice with Tß4 to assess barrier function, microtubule-associated protein 1 light chain 3 (LC3II) protein expression, and autophagy. Finally, normal human colon tissue and colon carcinoma cells (Caco2) were cultured to verify Tß4-induced barrier function and autophagy changes. Mucin2 levels decreased, microbial infiltration increased, and Tß4 expression increased in the colitis mouse model versus the control mice, indicating mucus barrier damage. Moreover, Tß4-treated C57BL/6 mice had damaged intestinal mucus barriers and decreased LC3II levels. Tß4 also inhibited colonic mucin2 production, disrupted tight junctions, and downregulated autophagy; these results were confirmed in Caco2 cells and normal human colon tissue. In summary, Tß4 may be implicated in colitis by compromising the integrity of the intestinal mucus barrier and inhibiting autophagy. Thus, Tß4 could be a new diagnostic marker for intestinal barrier defects.

An Increase in Mucin2 Expression Is Required for Colon Cancer Progression Mediated by L1.

An induction in the expression of the cell adhesion receptor L1, a Wnt target gene, is a characteristic feature of Wnt/ß-catenin activation in colon cancer cells at later stages of the disease. We investigated the proteins secreted following L1 expression in colon cancer cells and identified Mucin2 among the most abundant secreted proteins. We found that suppressing Mucin2 expression in L1-expressing colon cancer cells inhibits cell proliferation, motility, tumorigenesis, and liver metastasis. We detected several signaling pathways involved in Mucin2 induction in L1-expressing cells. In human colon cancer tissue, Mucin2 expression was significantly reduced or lost in the adenocarcinoma tissue, while in the mucinous subtype of colon cancer tissue, Mucin2 expression was increased. An increased signature of L1/Mucin2 expression reduced the survival rate of human colon cancer patients. Thus, induction of Mucin2 expression by L1 is required during mucinous colon cancer progression and can serve as a marker for diagnosis and a target for therapy.

Lactiplantibacillus plantarum OLL2712 Induces Autophagy via MYD88 and Strengthens Tight Junction Integrity to Promote the Barrier Function in Intestinal Epithelial Cells.

Autophagy is an important system conserved in eukaryotes that maintains homeostasis by degrading abnormal proteins. Autophagy incompetence in intestinal epithelial cells causes the abnormal function of intestinal stem cells and other cells and damages intestinal barrier function. The disruption of the intestinal barrier causes chronic inflammation throughout the body, followed by impaired glucose and lipid metabolism. Lactiplantibacillus plantarum OLL2712 (OLL2712) is a lactic acid bacterium that induces interleukin-10 production from immune cells, alleviates chronic inflammation, and improves glucose and lipid metabolism. In this study, we hypothesized that OLL2712 exerts anti-inflammatory effects by inducing autophagy and ameliorating intestinal barrier dysfunction, and we investigated its autophagy-inducing activities and functions. Caco-2 cells stimulated with OLL2712 for 24 h showed an increased number of autolysosomes per cell, compared with unstimulated cells. Therefore, the permeability of fluorescein isothiocyanate dextran 4000 (FD-4) was suppressed by inducing autophagy. In contrast, mucin secretion in HT-29-MTX-E12 cells was also increased by OLL2712 but not via autophagy induction. Finally, the signaling pathway involved in autophagy induction by OLL2712 was found to be mediated by myeloid differentiation factor 88 (MYD88). In conclusion, our findings suggest that OLL2712 induces autophagy in intestinal epithelial cells via MYD88, and that mucosal barrier function is strengthened by inducing autophagy.

Dietary iron regulates intestinal goblet cell function and alleviates Salmonella typhimurium invasion in mice.

Iron is an important micronutrient that plays a vital role in host defenses and bacterial pathogenicity. As iron treatments increase the risk of infection by stimulating the growth and virulence of bacterial pathogens, their roles in anti-infection immunity have frequently been underestimated. To estimate whether adequate dietary iron intake would help defend against pathogenic bacterial infection, mice were fed iron-deficient (2 mg kg-1 feed), iron-sufficient (35 mg kg-1 feed), or iron-enriched diet (350 mg kg-1 feed) for 12 weeks, followed by oral infection with Salmonella typhimurium. Our results revealed that dietary iron intake improved mucus layer function and decelerated the invasion of the pathogenic bacteria, Salmonella typhimurium. Positive correlations between serum iron and the number of goblet cells and mucin2 were found in response to total iron intake in mice. Unabsorbed iron in the intestinal tract affected the gut microbiota composition, and the abundance of Bacteroidales, family Muribaculaceae, was positively correlated with their mucin2 expression. However, the results from antibiotic-treated mice showed that the dietary iron-regulated mucin layer function was not microbial-dependent. Furthermore, in vitro studies revealed that ferric citrate directly induced mucin2 expression and promoted the proliferation of goblet cells in both ileal and colonic organoids. Thus, dietary iron intake improves serum iron levels, regulates goblet cell regeneration and mucin layer function, and plays a positive role in the prevention of pathogenic bacteria.

Colonic mucin-2 attenuates acute necrotizing pancreatitis in rats by modulating intestinal homeostasis.

Mucin-2 (MUC2) secreted by goblet cells participates in the intestinal barrier, but its mechanism in acute necrotizing pancreatitis (ANP) remains unclear. In acute pancreatitis (AP) patients, the functions of goblet cells (MUC2, FCGBP, CLCA1, and TFF3) decreased, and MUC2 was negatively correlated with AP severity. ANP rats treated with pilocarpine (PILO) (PILO+ANP rats) to deplete MUC2 showed more serious pancreatic and colonic injuries, goblet cell dysfunction, gut dysbiosis, and bacterial translocation than those of ANP rats. GC-MS analysis of feces showed that PILO+ANP rats had lower levels of butyric acid, isobutyric acid, isovaleric acid, and hexanoic acid than those of ANP rats. The expression of MUC2 was associated with colonic injury and gut dysbiosis. All these phenomena could be relieved, and goblet cell functions were also partially reversed by MUC2 supplementation in ANP rats. TNF-α-treated colonoids had exacerbated goblet cell dysfunction. MUC2 expression was negatively correlated with the levels of pro-inflammatory cytokines (IL-1ß and IL-6) (p < .05) and positively related to the expression of tight junction proteins (Claudin 1, Occludin, and ZO1) (p < .05). Downregulating MUC2 by siRNA increased the levels of the pro-inflammatory cytokines in colonoids. MUC2 might maintain intestinal homeostasis to alleviate ANP.

Mucin2 regulated by Ho1/p38/IL-10 axis plays a protective role in polystyrene nanoplastics-mediated intestinal toxicity.

Micro/nano-plastics (MPs/NPs) are a newly discovered environmental pollutant that can be ingested by humans through food and drinking water. In this study we evaluated the impact of MPs/NPs on the intestinal barrier and its mechanism. Doses of MPs/NPs were used to treat Caco-2/HT29-MTX in-vitro model and in-vivo model. In in-vitro model, 20 nm polystyrene nanoplastics (PS-NPs) had higher cytotoxicity than larger particles (200 nm and 2000 nm), and led to the increase of the permeability along with the decreased expression of tight junction proteins. Intriguingly, 20 nm PS-NPs elevated the expression of MUC2 simultaneously. Further studies revealed that PS-NPs increased the expression of HO1 through ROS generation, and then activated p38 to elevate IL-10 secretion in Caco-2 cell. The IL-10 secreted by Caco-2 cell promoted the expression of MUC2 in HT29-MTX cell through STAT1/3. Elevated MUC2 expression alleviates the cytotoxicity of PS-NPs. Besides, increased intestinal permeability and up-regulation of MUC2 through Ho1/p38/IL-10 pathway was also observed in 20 nm PS-NPs treated mouse model. In conclusion, PS-NPs can induce the intestinal toxicity and result in the increased adaptive expression of MUC2 to resist this adverse effect. People with inadequate mucin expression need to pay more attention to the toxicity of PS-NPs. This study provided a valuable insight for clarifying the mechanism and potential risk of intestinal toxicity induced by nanoplastics.

The role of high molecular weight hyaluronic acid in mucoadhesion on an ocular surface model.

Hyaluronic acid (HA) is frequently formulated in eye drops to improve the stability of the tear film by hydration and lubrication. Mucoadhesion is related to the ocular residence time and therefore to the effectiveness of the eye drops. The ocular residence time of the HA formulation is correlated with the ability of HA to create specific strong interactions in the ocular surface with the mucus layer, mainly composed of a mixture of secreted mucins (MUC; gel forming MUC5AC and MUC2) and shed membrane-bound soluble mucins (MUC1, MUC4, and MUC16). Dry eye disease (DED) is a multifactorial pathology of the preocular tear film with possible damage to the ocular surface classified in two types: (1) aqueous-deficient dry eye and (2) evaporative dry eye, caused by a decrease in goblet cell density that reduces MUC expression and/or by meibomian gland dysfunction, that results in a drop in the lipidic fraction of the tear film. In this work, the binding affinity between HA and MUC2 has been evaluated with three complementary approaches because the secreted MUCs play a pivotal role in the viscoelastic properties of the tear film: 1. Rheological analysis, measuring the mucoadhesive index and the complex viscosity in relation to MM (Molecular Mass) and concentration; 2. Fluorescence analysis, using a fluorescent hydrophobic probe, to investigate the conformational change of MUC2 during the interaction with the HA polymer; 3. Surface plasmon resonance analysis, used to measure the affinity between MUC2 (immobilized on the surface of a sensor chip) and the HA polymers that flowed on it at the molecular level. For all these tests, the mucoadhesive performance of the natural HA linearly increases with the MM, whereas cross-linked HA and other emollient and gelling agents (formulated in artificial tears) do not show the same mucoadhesive properties (with the exception of xanthan gum). The mucoadhesive performance of high MM HA has also been confirmed in conditions that simulate the pathological condition of the tear film during DED by decreasing the MUC2 or oleic acid concentration. Physico-chemical analysis of a series of marketed artificial tears confirms the linear correlation between the MM of the HA used in the products and the mucoadhesive index measured on the ocular surface model.

Intracellular Polysaccharides of Aspergillus cristatus from Fuzhuan Brick Tea Leverage the Gut Microbiota and Repair the Intestinal Barrier to Ameliorate DSS-Induced Colitis in Mice.

The intracellular polysaccharides of Aspergillus cristatus (IPSs) from Fuzhuan brick tea have been demonstrated to improve immune function linked to modulating the gut microbiota. Herein, to further investigate the efficacy of IPSs to maintain gut homeostasis, the protection of the purified fraction of IPSs (IPSs-2) on the mice with colitis induced by dextran sulfate sodium (DSS) and the underlying mechanisms were explored in this study. The results revealed that IPSs-2 alleviated the typical symptoms of colitis and suppressed the excessive inflammatory mediators, regulating the genes related to inflammatory responses in the colon at the mRNA level. Meanwhile, IPSs-2 treatment reinforced the intestinal barrier function by ameliorating the DSS-induced histological injury, facilitating the differentiation of goblet cells to enhance Mucin-2 generation, and enhancing the expression of tight junction proteins to alleviate colitis. In addition, IPSs protected against colitis by promoting the production of short-chain fatty acids (SCFAs), the activation of SCFAs receptors, and the leverage of the gut microbiota via the enrichment of Bacteroides, Parabacteroides, Faecalibacterium, Flavonifractor_plautii, and Butyricicoccus, linking with reducing inflammation and repairing intestinal barrier function. Overall, our research revealed the therapeutic potential of IPSs-2 as a prebiotic for attenuating inflammatory bowel disease and provided a rationale for future investigation.

Bacillus subtilis pretreatment alleviates ethanol-induced acute liver injury by regulating the Gut-liver axis in mice.

This study was designed to investigate the hepatoprotective effects of Bacillus subtilis, a commensal bacterial species in the human gut, on ethanol-induced acute liver damage and the underlying mechanisms in mice. Male ICR mice challenged with three doses of ethanol (5.5 g/kg BW) exhibited a significant increase in serum aminotransferase activities and TNF-α level, liver fat accumulation, and activation of NF-κB signaling and NLRP3 inflammasome, which was suppressed by pretreatment with Bacillus subtilis. Besides, Bacillus subtilis inhibited acute ethanol-induced intestinal villi shortening and epithelial loss, the decline of protein levels of intestinal tight junction protein ZO-1 and occludin, and elevation of serum LPS level. Furthermore, the upregulation of mucin-2 (MUC2) and the downregulation of anti-microbial Reg3B and Reg3G levels induced by ethanol were repressed by Bacillus subtilis. Lastly, Bacillus subtilis pretreatment significantly increased the abundance of the intestinal Bacillus, but had no effects on the binge drinking-induced increase of Prevotellaceae abundance. These results demonstrate that Bacillus subtilis supplementation could ameliorate binge drinking-induced liver injury, and thus may serve as a functional dietary supplement for binge drinkers.

Orchestration of MUC2 - The key regulatory target of gut barrier and homeostasis: A review.

The gut mucosa of human is covered by mucus, functioning as a crucial defense line for the intestine against external stimuli and pathogens. Mucin2 (MUC2) is a subtype of secretory mucins generated by goblet cells and is the major macromolecular component of mucus. Currently, there is an increasing interest on the investigations of MUC2, noting that its function is far beyond a maintainer of the mucus barrier. Moreover, numerous gut diseases are associated with dysregulated MUC2 production. Appropriate production level of MUC2 and mucus contributes to gut barrier function and homeostasis. The production of MUC2 is regulated by a series of physiological processes, which are orchestrated by various bioactive molecules, signaling pathways and gut microbiota, etc., forming a complex regulatory network. Incorporating the latest findings, this review provided a comprehensive summary of MUC2, including its structure, significance and secretory process. Furthermore, we also summarized the molecular mechanisms of the regulation of MUC2 production aiming to provide developmental directions for future researches on MUC2, which can act as a potential prognostic indicator and targeted therapeutic manipulation for diseases. Collectively, we elucidated the micro-level mechanisms underlying MUC2-related phenotypes, hoping to offer some constructive guidance for intestinal and overall health of mankind.

Effect of phytase and xylanase enzymes on growth performance and Mucin2 gene expression in broiler chickens.

BACKGROUND: Development of exogenous enzymes is one of the most important discoveries in animal nutrition. The supplementation of exogenous enzymes in broiler diets allows for supplying nutrient deficiencies and to decrease endogenous losses. OBJECTIVES: The effects of phytase (Hostazym and Phyzyme) and xylanase (Ronozyme) enzymes were investigated on growth performance and Mucin2 gene expression in broilers. METHODS: A completely randomized design was applied, including 7 treatments, 4 replicates and 25 birds per replicates. A total of 700 male Ross (308) broiler chickens were fed with similar diets supplemented by Hostazym and Phyzyme (500 and 1000 FTU/kg) and Ronozyme (100 and 200 EXU/kg). Weight gain (WG), feed intake (FI) and feed conversion ratio (FCR) were determined for three phases and entire rearing period. On 42 days of age, four birds per replicate were slaughtered. Total RNA was extracted from jejunum samples, and Mucin2 gene expression was measured by real-time PCR. RESULTS: Phytase and xylanase enzymes had a significant effect (p < 0.05) on traits (WG and FCR) in grower and finisher phases and whole rearing period, but FI was not affected by enzymes (p > 0.05). Carcass (74.13 g) and breast (27.76 g) weights by Hostazym (1000 FTU/kg) were higher than other treatments (p < 0.05). Weight of liver, bursa and spleen were significantly influenced by enzymes (p < 0.05). Likewise, bursa and spleen weights in Hostazym (1000 FTU/kg feed) and Ronozyme (200 EXU/kg feed) were significantly higher than other treatments (p < 0.05). Mucin2 gene expression was affected by enzymes in whole treatments. The lowest amount of Mucin2 gene expression belonged to Ronozyme (200 and 100 EXU/kg), and the highest was belonging to Hostazym (1000 FTU/kg). CONCLUSIONS: Phytase enzymes have higher effect on broiler performance and Mucin2 gene expression compared to xylanase. High doses of Hostazym (1000 FTU/kg feed) could be supplemented in broiler chicken diets to improve optimum growth and feed efficiency.

Activation of goblet cell Piezo1 alleviates mucus barrier damage in mice exposed to WAS by inhibiting H3K9me3 modification.

BACKGROUND: Our recent studies found that intestinal mechanical signals can regulate mucus synthesis and secretion of intestinal goblet cells through piezo type mechanosensitive ion channel component 1 (Piezo1), but the detailed molecular mechanisms remain to be investigated. Previous studies using a water avoidance stress (WAS) model reported decreased intestinal mucus accompanied by abnormal intestinal motility. It has also been reported that the expression of mucin2 was negatively correlated with histone H3 lysine 9 trimethylation (H3K9me3), a key regulator of histone methylation, and that mechanical stimulation can affect methylation. In this study, we aimed to determine whether and how Piezo1 expressed on goblet cells regulates mucus barrier function through methylation modification. METHODS: A murine WAS model was established and treated with Yoda1 (Piezo1 agonist), and specific Piezo1 flox-mucin2 Cre mice were also tested. The mucus layer thickness and mucus secretion rate of mouse colonic mucosa were detected by a homemade horizontal Ussing chamber, intestinal peristaltic contraction was detected by the ink propulsion test and organ bath, goblet cells and mucus layer morphology were assessed by HE and Alcian blue staining, mucus permeability was detected by FISH, and the expression levels of Piezo1, H3K9me3 and related molecules were measured by Western blots and immunofluorescence. LS174T cells were cultured on a shaker board in vitro to simulate mechanical stimulation. Piezo1 and H3K9me3 were inhibited, and changes in mucin2 and methylation-related pathways were detected by ELISAs and Western blots. ChIP-PCR assays were used to detect the binding of H3K9me3 and mucin2 promoters under mechanical stimulation. RESULTS: Compared with those of the controls, the mucus layer thickness and mucus secretion rate of the mice exposed to WAS were significantly decreased, the mucus permeability increased, the number of goblet cells decreased, and the intestinal contraction and peristalsis were also downregulated and disordered. Intraperitoneal injection of Yoda1 improved mucus barrier function and intestinal contraction. In the colonic mucosa of mice exposed to WAS, Piezo1 was decreased, and histone H3 lysine 9 trimethylation (H3K9me3) and methyltransferase suppressor of variegation 3-9 homolog 1 (SUV39h1) were increased, but activating Piezo1 alleviated these effects of WAS. Piezo1 flox-mucin2 Cre mice showed decreased mucus expression and increased methylation compared to wild-type mice. Cell experiments showed that mechanical stimulation induced the activation of Piezo1, decreased H3K9me3 and SUV39h1, and upregulated mucin2 expression. Inhibition of Piezo1 or H3K9me3 blocked the promoting effect of mechanical stimulation on LS174T mucin2 expression. The binding of H3K9me3 to the mucin2 promoter decreased significantly under mechanical stimulation, but this could be blocked by the Piezo1 inhibitor GsMTx4. CONCLUSION: Piezo1 mediates mechanical stimulation to inhibit SUV39h1, thereby reducing H3K9me3 production and its binding to the mucin2 promoter, ultimately promoting mucin2 expression in goblet cells. This study further confirmed that piezo1 on goblet cells could regulate mucus barrier function through methylation.

Si-Ni-San improves experimental colitis by favoring Akkermensia colonization.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC) is widely believed to be a leading risk factor of colorectal cancer. Gut microbiota is a known vital player in the progression of UC. Si-Ni-San (SNS) has been considered to effectively treat colitis in clinical practice during thousands of years, yet whether SNS ameliorated acute colitis mouse model by modulating intestinal flora has not been distinctly elucidated. AIM OF THE STUDY: Our study aimed to elucidate the effect of SNS against acute murine colitis and focused on the underlying mechanisms of SNS targeting gut microbiota. MATERIALS AND METHODS: 16S RNA sequencing, molecular biological analysis, and fecal microbiota transplants (FMT) were conducted to reveal the mechanisms of SNS in regulating gut microbiota. RESULTS: In our study, SNS dramatically inhibited DSS-induced acute inflammatory responses by improving gut microbiota dysbiosis, as evidenced by decreased abundance proinflammatory species, upregulated abundance of anti-inflammatory species and potentially altered microbiota metabolite metabolism. Additionally, intestinal flora knockout and FMT experiments confirmed that the therapeutic effect of SNS on colitis was dependent on gut microbiota, and specifically on favoring the growth of potential probiotics, Akkermansia genus. Furthermore, we found that SNS alone and SNS combined with Akkermansia muciniphila (A. muciniphila) increased Mucin 2 (MUC2) production, thus enhancing the competitive edge of A. muciniphila among pathogenic gut microbiota. CONCLUSION: Our study shed lights on the underlying mechanism of SNS in attenuating acute murine colitis from the perspective of intestinal flora and provides novel insights into the discovery of adjacent therapeutic strategy against colitis based on SNS and probiotics. CLASSIFICATION: Gastro-intestinal system.