Intestinal Inflammatory Bowel Disease Research Articles

Intestinal epithelial Cldn-7 regulates intestinal inflammation by altering the gut microbiota

Background and aimTight junctions maintain gut homeostasis by forming a physical barrier that protects the gut from invasion by microbiota. Cldn-7 is an important component involved in this protection, but the relationship between Cldn-7, intestinal inflammation, and gut microbiota has not been clarified. Here, we hypothesize that Cldn-7 depletion affects intestinal inflammation by altering the gut microbiota. MethodsBased on the induced intestinal condition of Cldn-7 knockout mice (Cldn7fl/fl;villin-CreaERT2), we established the intestinal flora depletion model and colitis model by antibiotic drinking and feeding with dextran sodium sulfate (DSS). The environment of Cldn-7 gene deletion mice was changed by co-housing experiment. AB-PAS staining and Muc2 were used to detect the effect of co-housing and Cldn-7 deficiency on the mucus layer after flora depletion. qRT-PCR was used to detect the expression of intestinal inflammatory factors and AMPs in mice. Feces were collected and proportions of microbiota were analyzed by 16 S rRNA amplicon sequencing. ResultsMice in the co-housing experiment had altered intestinal microbiota, including diversity, composition, and functional prediction, compared to controls. Intestinal inflammation was restored to some extent following altered intestinal microbiota. The intestinal inflammation caused by Cldn-7 deficiency and susceptibility to DSS could be reduced after antibiotic administration compared to controls, in terms of phenotype, pathological changes, inflammatory factors, mucus barrier, and expression of AMPs. ConclusionsIn analyses of intestinal tissues, colitis induction, and gut microbiota in mice with intestinal disruption of Cldn-7, we found this protein to prevent intestinal inflammation by regulating the gut microbiota. Cldn-7might therefore be an important mediator of host–microbiome interactions. Our research has revealed that Cldn-7 plays an indispensable role in maintaining intestinal homeostasis by regulating the gut microbiota and impacting intestinal inflammation. These findings provide new insights into the pathogenesis of ulcerative colitis.

Saikosaponin A Ameliorates Metabolic Inflammation and Intestinal Barrier Damage in DIO Mice through the Nrf2/ARE Pathway.

Obesity is linked to impaired intestinal barrier function and inflammation. Saikosaponin A (SSA), a triterpene saponin from Bupleurum chinense, has shown beneficial effects on intestinal colitis in mice. However, the mechanisms underlying SSA's protective effects against obesity are not fully understood. To investigate the effects of SSA on body weight, metabolic disturbances, and intestinal health in diet-induced obese (DIO) mice, and to elucidate the potential mechanisms involved. In the in vivo study, DIO mice were supplemented with SSA. Body weight, fasting blood glucose, and metabolic parameters were measured. Intestinal barrier function and inflammation were assessed. In the in vitro study, intestinal epithelial cells were treated with palmitic acid and lipopolysaccharide to induce inflammation. SSA was then administered to evaluate its effects on cell barrier integrity and inflammatory responses. The role of the nuclear factor-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway was investigated by silencing Nrf2. SSA supplementation significantly (p < 0.05) decreased body weight and fasting blood glucose levels in DIO mice, and markedly improved metabolic disturbances. This treatment also enhanced intestinal barrier function and reduced metabolic inflammation, likely through increased antioxidant capacity of intestinal epithelial cells via activation of the Nrf2/ARE signaling pathway. In vitro, SSA maintained cell barrier integrity and reduced inflammatory responses by activating the Nrf2/ARE signaling pathway, decreasing intracellular reactive oxygen species content, and increasing transepithelial electrical resistance. However, silencing Nrf2 abolished SSA's protective effects. SSA enhances the antioxidant capacity of intestinal epithelial cells, maintains intestinal barrier integrity, and reduces intestinal inflammation in DIO mice through the activation of the Nrf2/ARE signaling pathway. These findings offer new insights into the protective role of SSA in obesity and metabolic diseases.

Effect of knocking out mouse Slc44a4 on colonic uptake of the microbiota-generated thiamine pyrophosphate and colon physiology.

Humans and mammals obtain vitamin B1 from dietary and gut microbiota sources. A considerable amount of the microbiota-generated vitamin exists in the form of thiamine pyrophosphate (TPP), and colonocytes are capable of absorbing TPP via a specific carrier-mediated process that involves the colonic TPP transporter (cTPPT encoded by SLC44A4). Little is known about the relative contribution of the SLC44A4 transporter toward total colonic carrier-mediated TPP uptake and its role in colon physiology. To address these issues, we generated an Slc44a4 knockout (KO) mouse model (by Cre-Lox recombination) and found a near-complete inhibition in colonic carrier-mediated [3H]TPP uptake in the Slc44a4 KO compared with wild-type (WT) littermates. We also observed a significant reduction in KO mice's body weight and a shortening of their colon compared with WT. Using RNAseq and Ingenuity pathway analysis (IPA) approaches, we found that knocking out the colonic Slc44a4 led to changes in the level of expression of many genes, including upregulation in those associated with intestinal inflammation and colitis. Finally, we found that the Slc44a4 KO mice were more susceptible to the effect of the colitogenic dextran sodium sulfate (DSS) compared with WT animals, a finding that lends support to the recent prediction by multiple genome-wide association studies (GWAS) that SLC44A4 is a possible colitis susceptibility gene. In summary, the results of these investigations show that Slc44a4 is the predominant or only transporter involved in the colonic uptake of TPP, that the transporter is important for colon physiology, and that its deletion increases susceptibility to inflammation.NEW & NOTEWORTHY This study shows that Slc44a4 is the predominant or only transport system involved in the uptake of the gut microbiota-generated thiamine pyrophosphate (TPP) in the colon and that its deletion affects colon physiology and increases its susceptibility to inflammation.

Exploring the molecular mechanisms and shared gene signatures between celiac disease and ulcerative colitis based on bulk RNA and single-cell sequencing: Experimental verification

Many immune-mediated diseases have the common genetic basis, as an autoimmune disorder, celiac disease (CeD) primarily affects the small intestine, and is caused by the ingestion of gluten in genetically susceptible individuals. As for ulcerative colitis (UC), which most likely involves a complex interplay between some components of the commensal microbiota and other environmental factors in its origin. These two autoimmune diseases share a specific target organ, the bowel. The etiology and immunopathogenesis of both conditions characterized by chronic intestinal inflammation, ulcerative colitis and celiac disease, are not completely understood. Both are complex diseases with genetics and the environmental factors contributing to dysregulation of innate and adaptive immune responses, leading to chronic inflammation and disease. This study is designed to further clarify the relationship between UC and CeD. The GEO database was used to download gene expression profiles for CeD (GSE112102) and UC (GSE75214). The GSEA KEGG pathway analysis revealed that immune-related pathways were significantly associated with both diseases. Further, we screened 187 shared differentially expressed genes (DEGs) of the two diseases. Gene Ontology (GO) and WikiPathways were carried out to perform the biological process and pathway enrichment analysis. Subsequently, based on the DEGs, the least absolute shrinkage and selection operator (LASSO) analysis was performed to screen for the diagnostic biomarkers of the diseases. Moreover, single-cell RNA-sequencing (RNA-seq) data from five colonic propria with UC showed that REG4 expression was present in Goblet cell, Enteroendocrine cell, and Epithelial. Finally, our work identified REG4 is the shared gene of UC and CeD via external data validation, cellular experiments, and immunohistochemistry. In conclusion, our study elucidated that abnormal immune response could be the common pathogenesis of UC and CeD, and REG4 might be a key potential biomarker and therapeutic target for the comorbidity of these two diseases.

Inhibitory Effect of Lactococcus and Enterococcus faecalis on Citrobacter Colitis in Mice.

Probiotics are beneficial for intestinal diseases. Research shows that probiotics can regulate intestinal microbiota and alleviate inflammation. Little research has been done on the effects of probiotics on colitis in mice. The purpose of this study was to investigate the inhibitory effect of the strains isolated and screened from the feces of healthy piglets on the enteritis of rocitrobacter. The compound ratio of isolated Lactobacillus L9 and Enterococcus faecalis L16 was determined, and the optimal compound ratio was selected according to acid production tests and bacteriostatic tests in vitro. The results showed that when the ratio of Lactobacillus L9 to Enterococcus faecalis L16 was 4:1, the pH value was the lowest, and the antibacterial diameter was the largest. Then, in animal experiments, flow cytometry was used to detect the number of T lymphocytes in the spleen and mesenteric lymph nodes of mice immunized with complex lactic acid bacteria. The results showed that the number of T lymphocytes in the spleen and mesenteric lymph nodes of mice immunized with complex lactic acid bacteria significantly increased, which could improve the cellular immunity of mice. The microbiota in mouse feces were sequenced and analyzed, and the results showed that compound lactic acid bacteria could increase the diversity of mouse microbiota. It stabilized the intestinal microbiota structure of mice and resisted the damage of pathogenic bacteria. The combination of lactic acid bacteria was determined to inhibit the intestinal colitis induced by Citrobacter, improve the cellular immune response of the body, and promote the growth of animals.

Enterococcus durans 98D alters gut microbial composition and function to improve DSS-induced colitis in mice

Enterococcus durans, is a potential functional strain with the capacity to regulate intestinal health and ameliorate colonic inflammation. However, the strain requires further investigation regarding its safety profile and potential mechanisms of colitis improvement. In this study, the safety of E. durans 98D (Ed) as a potential probiotic was studied using in vitro methods. Additionally, a dextran sulfate sodium (DSS)-induced murine colitis model was employed to investigate its impact on the intestinal microbiota and colitis. In vitro antimicrobial assays revealed Ed sensitivity to common antibiotics and its inhibitory effect on the growth of Escherichia coli O157, Streptococcus pneumoniae CCUG 37328, and Staphylococcus aureus ATCC 25923. To elucidate the functional properties of Ed, 24 weight-matched 6-week-old female C57BL/6J mice were randomly divided into three groups (n = 8): NC group, Con group (DSS), and Ed group (DSS + Ed). Ed administration demonstrated a protective effect on colitis mice, as evidenced by improvements in body weight, colonic length, reduced disease activity index, histological scores, diminished splenomegaly, and decreased goblet cell loss. Furthermore, Ed downregulated the expression of the pro-inflammatory cytokine genes (IL-6, IL-1β, and TNF-α) and upregulated the expression of the anti-inflammatory cytokine gene IL-10. The 16S rRNA gene sequencing revealed significant alterations in microbial α-diversity, with principal coordinate analysis indicating distinct differences in microbial composition among the three groups. At the phylum level, the relative abundance of Actinomycetota significantly increased in the Ed-treated group. At the genus level, Ed treatment markedly elevated the relative abundance of Paraprevotella, Rikenellaceae_RC9, and Odoribacter in DSS-induced colitis mice. In conclusion, Ed exhibits potential as a safe and effective therapeutic agent for DSS-induced colitis by reshaping the colonic microbiota.

Investigating the impact of antibiotic-induced dysbiosis on protection from Clostridium difficile colitis by mouse colonic innate lymphoid cells.

Innate lymphoid cells (ILCs) play a critical role in maintaining intestinal health in homeostatic and diseased conditions. During Clostridium difficile infection (CDI), IL-33 activates ILC2 to protect from colonic damage and mortality. The function of IL-33 and ILC is tightly regulated by the intestinal microbiota. We set out to determine the impact of antibiotic-induced disruption of the microbiome on ILC function. Our goal was to understand antibiotic-induced changes in ILC function on susceptibility to C. difficile colitis in a mouse model. We utilized high-throughput single-cell RNAseq to investigate the phenotypic features of colonic ILC at baseline, after antibiotic administration with or without IL-33 treatment. We identified a heterogeneous landscape of colonic ILCs with gene signatures of inflammatory, anti-inflammatory, migratory, progenitor, plastic, and antigen-presenting ILCs. Antibiotic treatment decreased ILC2 while coordinately increasing ILC1 and ILC3 phenotypes. Notably, Ifng+, Ccl5+, and Il23r+ ILC increased after antibiotics. IL-33 treatment counteracted the antibiotic effect by downregulating ILC1 and ILC3 and activating ILC2. In addition, IL-33 treatment markedly induced the expression of type 2 genes, including Areg and Il5. Finally, we identified amphiregulin, produced by ILC2, as protective during C. difficile infection. Together, our data expand our understanding of how antibiotics induce susceptibility to C. difficile colitis through their impact on ILC subsets and function.IMPORTANCEClostridium difficile infection (CDI) accounts for around 500,000 symptomatic cases and over 20,000 deaths annually in the United States alone. A major risk factor of CDI is antibiotic-induced dysbiosis of the gut. Microbiota-regulated IL-33 and innate lymphoid cells (ILCs) are important in determining the outcomes of C. difficile infection. Understanding how antibiotic and IL-33 treatment alter the phenotype of colon ILCs is important to identify potential therapeutics. Here, we performed single-cell RNAseq of mouse colon ILCs collected at baseline, after antibiotic treatment, and after IL-33 treatment. We identified heterogeneous subpopulations of all three ILC subtypes in the mouse colon. Our analysis revealed several potential pathways of antibiotic-mediated increased susceptibility to intestinal infection. Our discovery that Areg is abundantly expressed by ILCs, and the protection of mice from CDI by amphiregulin treatment, suggests that the amphiregulin-epidermal growth factor receptor pathway is a potential therapeutic target for treating intestinal colitis.

A Gas Therapy Strategy for Intestinal Flora Regulation and Colitis Treatment by Nanogel-Based Multistage NO Delivery Microcapsules.

Current approaches to treating inflammatory bowel disease focus on the suppression of overactive immune responses, the removal of reactive intestinal oxygen species, and regulation of the intestinal flora. However, owing to the complex structure of the gastrointestinal tract and the influence of mucus, current small-molecule and biologic-based drugs for treating colitis cannot effectively act at the site of colon inflammation, and as a result, they tend to exhibit low efficacies and toxic side effects. In this study, nanogel-based multistage NO delivery microcapsules are developed to achieve NO release at the inflammation site by targeting the inflammatory tissues using the nanogel. Surprisingly, oral administration of the microcapsules suppresses the growth of pathogenic bacteria and increases the abundance of probiotic bacteria. Metabolomics further show that an increased abundance of intestinal probiotics promotes the production of metabolites, including short-chain fatty acids and indole derivatives, which modulate the intestinal immunity and restore the intestinal barrier via the interleukin-17 and PI3K-Akt signaling pathways. This work reveals that the developed gas therapy strategy based on multistage NO delivery microcapsules modulates the intestinal microbial balance, thereby reducing inflammation and promoting intestinal barrier repair, ultimately providing a new therapeutic approach for the clinical management of colitis.

Effects of myeloperoxidase on inflammatory responses with hypoxia in Citrobacter rodentium-infectious mice.

Myeloperoxidase (MPO)has been identified as a mediator in various inflammatory diseases. Bacterial infection of the intestine and hypoxia can both lead to inflammatory responses, but the role of MPO in these phenomena remains unclear. By building the MPO-/- mice, we evaluated relevant inflammatory factors and tissue damage in mice with intestinal Citrobacter rodentium infection and hypoxia. The body weight and excreted microorganisms were monitored. Intestinal tissues were collected 7 days after bacterial infection under hypoxia to undergo haematoxylin-eosin staining and assess the degree of pathological damage. ELISA assays were performed to quantify the serum levels of TNF-α, IFN-γ, IL-6, and IL-1β inflammatory cytokines. PCR, WB, and IF assays were conducted to determine the expression of chemokines MCP1, MIP2, and KC in the colon and spleen. The C. rodentium infection and hypoxia caused weight loss, intestinal colitis, and splenic inflammatory cells active proliferation in wild-type mice. MPO deficiency alleviated this phenomenon. MPO-/- mice also displayed a significant decline in bacteria clearing ability. The level of TNF-α in the serum and spleen was both lower in MPO-/- hypoxia C. rodentium-infected mice than that in wild-type mice. The chemokines expression levels of MIP2, KC,and MCP1 in the spleen and colon of each bacterial infected group were significantly increased (p < .05), while in hypoxia, the factors in the spleen and colon were decreased (p < .05). MPO deficiency was found to lower the levels of these chemokines compared with wild-type mice. MPO plays an important role of the inflammatory responses in infectious enteritis and hypoxia in mice, and the loss of MPO may greatly reduce the body's inflammatory responses to fight diseases.

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.

The Potential Amoebicidal Effect of Some Milk forms as an Inhibitor for Entamoeba histolytica Viability Isolated from Diarrhea

Milk includes numerous compounds such as lacto-peroxidase, lactoferrin, immunoglobulin G, secretory immunoglobulin A, and Lysozymes The invasive trophozoites of E. histolytica are capable to inhabit host tissues and provoke diseases such as intestinal colitis. The objective of the study was to compare different forms of milk’s effect on E. histolytica trophopzoite by measuring protein concentration in vitro using a spectrophotometer and number of trophozoite by microscopy. For this purpose, E. histolytica trophozoites were exposed to several types of milk (human, sheep, cattle, goats, and formula) at concentrations of 6, 12, 18, and 24% for each type after 8, 16, and 24 hours of incubation. According to the findings, sheep and goat milk had lower total protein concentrations after 8 hours than other types of milk when compared to the control group. After 16 hours of incubation, raise the milk concentration to 24% leading to a reduction in the formula milk's total protein concentration to 5g/l. after 8 and 16 hours of incubation, sheep milk and cattle have significantly lower total protein concentrations (24% vs. 8%), respectively. The action of formula and human milk is increased by 24-hour incubation. Increase the trophozoite inhibition percentage compared to metronidazole in human milk, which was followed by sheep and cattle milk with respective values of 60.9%, 56.1%, and 46.3% respectively. Human milk, goat and formula milk had the greatest inhibitory activity after incubation 24hrs. at 24%.the extent of amoebicidal activity of milk was shown to depend on incubation period and concentration of milk.

29 Evaluation of Diet Composition on Early Post-Weaned Feed Intake and Intestinal Health

Abstract Weaning is an inevitable early life stressor in commercial production that can cause an immediate reduction in voluntary feed intake and growth during a critical period of gastrointestinal development. Further, the reduction in voluntary feed intake is strongly correlated with the risk of disease during that period. Therefore, our objective was to test the effect of different dietary interventions on early nursery pig feed intake, performance, fecal consistency, and histological parameters. Over 2 repetitions, a total of 90 newly weaned gilts (19-21 d of age, BW 5.62 ± 1.1 kg) were selected, individually penned and randomly assigned to 1 of 5 dietary treatments (n = 18 pigs/treatment) that included: 1) Control diet (CON), 2) CON + 5% sugar beet pulp (BP), 3) CON + 10% high fructose corn syrup (HFCS), 4) CON + 5% soyhulls (SH), or 5) CON + 3,000 ppm Zn and 200 ppm Cu (d 0-7), and 2,000 ppm Zn and 200 ppm Cu (d 8-14; ZC). Each repetition was conducted over a 14-d period consisting of 2 phases (Phase 1: d 0-7, phase 2: d 8-14). Pigs were all sourced from a natural endemic enteric pathogen positive nursery flow and confirmed positive for rotavirus, coccidia and F18 E. coli. Fecal consistency was scored and feed disappearance was recorded daily. On d 0, 7 and 14, pig BW were collected. Weekly feed disappearance and BW were used to calculate ADG, ADFI and feed efficiency (G:F) within phase and overall. On d 14, all pigs were euthanized, and fixed sections of ileum and colon were collected to assess histopathology including incidence of villus atrophy and colitis. Pig was the experimental unit, and performance and histology data were analyzed by dietary treatment. Daily measures of feed intake were analyzed by repeated measures with the fixed effects of diet, day, and their interaction. Starting BW did not differ across dietary treatments (P = 0.845). Over the 14-d test period, HFCS and ZC treatments tended to increase ADG (0.20 and 0.15 kg/d, respectively) compared with CON, BP and SH (0.09, 0.12, and 0.13 kg/d, respectively; P = 0.076). Further, the addition of a sweetener (HFCS) did not improve daily feed intake, while SH reduced daily feed intakes by 32% compared with all other treatments (P &amp;lt; 0.001). Although all pigs had some degree of diarrhea, fecal consistency did not differ between dietary treatments (P = 0.618). Histopathology scoring for intestinal atrophy (ileum) and colitis (colon) were not different (P = 0.645 and 0.440, respectively). In conclusion, the addition of BP, HFCS, SH and ZC had an effect on growth and feed intake but there was no benefit in atrophy, colitis, and fecal scores when encountering a natural enteric pathogen burden.

Stromal BMP signaling regulates mucin production in the large intestine via interleukin-1/17.

Bone morphogenic protein (BMP) signaling is critical for intestinal development, homeostasis, and function performance. Although the function of BMP signaling in the intestinal epithelium is well appreciated, the direct effect of BMP on intestinal stromal cells is poorly understood. Here, we show that disruption of BMP signaling by genetic ablation of Alk3 or Smad4 expands the stromal cell pool, the mucosa tumefaction, and colonic polyposis in the large intestine. Interleukin (IL) secretion by stromal cells is notably increased, including IL-1, IL-11, and IL-17. Specifically, IL-1 and IL-17a hyperactivate the mucin production by goblet cells through nuclear factor κB signaling, and abnormal mucin accumulation results in the morphological changes, epithelial barrier destruction, and polyposis development. Together, our results provide an insight into the role of BMP signaling in intestinal stromal cells to regulate epithelium function. This study further highlights the role of mucin-producing goblet cells in intestinal homeostasis and colitis development.

Baizhu-Baishao herb pair ameliorates functional constipation and intestinal microflora disorder in rats.

In China, Rhizoma atractylodis macrocephalae-Paeonia lactiflora Pall (Biazhu-Baishao, BZBS) is a classic herb pair used to treat intestinal stress syndrome, ulcerative colitis and other diseases. However, the mechanism of BZBS in the treatment of functional constipation (FC) has been little studied and remains unclear. In this study, a behavioral investigation, colon tissue morphology, enzyme-linked immunosorbent assay (Elisa) and intestinal microflora analysis have been used to illuminate the potential mechanism of the effects of BZBS on FC in a rat model. A FC rat model was constructed and BZBS was given as treatment. Observations and recordings were made of the fecal moisture content, the defecation time of the first black stool, and the rate of intestinal propulsion. Elisa was used to detect the expression levels of substance P (SP), vasoactive intestinal peptide (VIP), 5-hydroxytryptamine (5-HT) in the colon. To ascertain the composition of the microbial community, a high throughput 16S ribosomal RNA (16S rRNA) gene sequencing technique was employed. Oral administration of BZBS significantly ameliorated several key excretion parameters, including the time to first black stool defecation, stool water content, and the propulsion rate in the small intestine in FC rats. It increased the expression of SP, VIP and 5-HT in the colon. 16S rRNA gene sequencing results showed that BZBS changed the microbial community structure, decreased the Bacteroidetes/Firmicutes ratio, increased the relative abundance of Blautia and Fusicatenibacter, and decreased the relative abundance of Ruminococcus and Roseburia. BZBS effectively alleviates FC and improves dysbacteriosis.

Defects in microvillus crosslinking sensitize to colitis and inflammatory bowel disease.

Intestinal epithelial cells are covered by the brush border, which consists of densely packed microvilli. The Intermicrovillar Adhesion Complex (IMAC) links the microvilli and is required for proper brush border organization. Whether microvillus crosslinking is involved in the intestinal barrier function or colitis is currently unknown. We investigate the role of microvillus crosslinking in colitis in mice with deletion of the IMAC component CDHR5. Electron microscopy shows pronounced brush border defects in CDHR5-deficient mice. The defects result in severe mucosal damage after exposure to the colitis-inducing agent DSS. DSS increases the permeability of the mucus layer and brings bacteria in direct contact with the disorganized brush border of CDHR5-deficient mice. This correlates with bacterial invasion into the epithelial cell layer which precedes epithelial apoptosis and inflammation. Single-cell RNA sequencing data of patients with ulcerative colitis reveals downregulation of CDHR5 in enterocytes of diseased areas. Our results provide experimental evidence that a combination of microvillus crosslinking defects with increased permeability of the mucus layer sensitizes to inflammatory bowel disease.

Zingerone attenuates intestinal injury and colitis caused by a high-fat diet through Nrf2 signaling regulation

This study examined the protective effect of Zingerone against a high-fat diet (HFD)-induced intestinal damage. Control and HFD rats were treated with the vehicle or Zingerone (100 mg/kg, orally) (n = 8 rats/groups). An extra group, HFD + Zingerone + brusatol (an Nrf2 inhibitor). This study treatment lasted four weeks. Zingerone reduced the nuclear levels of NF-B p65 in control and HFD-fed rats while increasing SOD, CAT, GSH, levels of mRNA, cytoplasmic levels, and Nrf2 nuclear levels. Zingerone treatment attenuated the duodenal epithelial damage and maintained the mucosal barrier by reducing plasma FITC-DX and serum LPS in rats fed with HFD. Concomitantly, it lowered the duodenal MDA, TNF-α, IL-6, and IL-1β levels. These impacts included changes in body weight, duodenal lipid levels, and Keap-1 expression, a natural Nrf2 inhibitor. We concluded that Zingerone reduces HFD-induced duodenal injury. These findings support Zingerone's clinical applicability against various inflammatory diseases of the intestine.

Commensal bacteria promote azathioprine therapy failure in inflammatory bowel disease via decreasing 6-mercaptopurine bioavailability

Azathioprine (AZA) therapy failure, though not the primary cause, contributes to disease relapse and progression in inflammatory bowel disease (IBD). However, the role of gut microbiota in AZA therapy failure remains poorly understood. We found a high prevalence of Blautia wexlerae in patients with IBD with AZA therapy failure, associated with shorter disease flare survival time. Colonization of B.wexlerae increased inflammatory macrophages and compromised AZA's therapeutic efficacy in mice with intestinal colitis. B.wexlerae colonization reduced 6-mercaptopurine (6-MP) bioavailability by enhancing selenium-dependent xanthine dehydrogenase (sd-XDH) activity. The enzyme sd-XDH converts 6-MP into its inactive metabolite, 6-thioxanthine (6-TX), thereby impairing its ability to inhibit inflammation in mice. Supplementation with Bacillus (B.) subtilis enriched in hypoxanthine phosphoribosyltransferase (HPRT) effectively mitigated B.wexlerae-induced AZA treatment failure in mice with intestinal colitis. These findings emphasize the need for tailored management strategies based on B.wexlerae levels in patients with IBD.

Intestinal Epithelial Axin1 Deficiency Protects Against Colitis via Altered Gut Microbiota

Intestinal homeostasis is maintained by specialized host cells and the gut microbiota. Wnt/β-catenin signaling is essential for gastrointestinal development and homeostasis, and its dysregulation has been implicated in inflammation and colorectal cancer. Axin1 negatively regulates activated Wnt/β-catenin signaling, but little is known regarding its role in regulating host–microbial interactions in health and disease. Here, we aim to demonstrate that intestinal Axin1 determines gut homeostasis and host response to inflammation. Axin1 expression was analyzed in human inflammatory bowel disease datasets. To explore the effects and mechanism of intestinal Axin1 in regulating intestinal homeostasis and colitis, we generated new mouse models with Axin1 conditional knockout in intestinal epithelial cell (IEC; Axin1ΔIEC) and Paneth cell (PC; Axin1ΔPC) to compare with control (Axin1LoxP; LoxP: locus of X-over, P1) mice. We found increased Axin1 expression in the colonic epithelium of human inflammatory bowel disease (IBD). Axin1ΔIEC mice exhibited altered goblet cell spatial distribution, PC morphology, reduced lysozyme expression, and enriched Akkermansia muciniphila (A. muciniphila). The absence of intestinal epithelial and PC Axin1 decreased susceptibility to dextran sulfate sodium (DSS)-induced colitis in vivo. Axin1ΔIEC and Axin1ΔPC mice became more susceptible to DSS-colitis after cohousing with control mice. Treatment with A. muciniphila reduced DSS-colitis severity. Antibiotic treatment did not change the IEC proliferation in the Axin1Loxp mice. However, the intestinal proliferative cells in Axin1ΔIEC mice with antibiotic treatment were reduced compared with those in Axin1ΔIEC mice without treatment. These data suggest non-colitogenic effects driven by the gut microbiome. In conclusion, we found that the loss of intestinal Axin1 protects against colitis, likely driven by epithelial Axin1 and Axin1-associated A. muciniphila. Our study demonstrates a novel role of Axin1 in mediating intestinal homeostasis and the microbiota. Further mechanistic studies using specific Axin1 mutations elucidating how Axin1 modulates the microbiome and host inflammatory response will provide new therapeutic strategies for human IBD.

Pachymic acid protects against Crohn's disease-like intestinal barrier injury and colitis in miceby suppressingintestinal epithelial cell apoptosis via inhibiting PI3K/AKT signaling

To investigate the effect of pachymic acid (PA) against TNBS-induced Crohn's disease (CD)-like colitis in mice and explore the possible mechanism. Twenty-four C57BL/6J mice were randomized equally into control group, TNBS-induced colitis model group and PA treatment group. PA treatment was administered via intraperitoneal injection at the daily dose of 5 mg/kg for 7 days, and the mice in the control and model groups were treated with saline. After the treatments, the mice were euthanized for examination of the disease activity index (DAI) of colitis, body weight changes, colon length, intestinal inflammation, intestinal barrier function and apoptosis of intestinal epithelial cells, and the expressions of TNF-α, IL-6 and IL-1β in the colonic mucosa were detected using ELISA. The possible treatment targets of PA in CD were predicted by network pharmacology. String platform and Cytoscape 3.7.2 software were used to construct the protein-protein interaction (PPI) network. David database was used to analyze the GO function and KEGG pathway; The phosphorylation of PI3K/AKT in the colonic mucosal was detected with Western blotting. PA significantly alleviated colitis in TNBS-treated mice as shown by improvements in the DAI, body weight loss, colon length, and histological inflammation score and lowered levels of TNF-α, IL-6 and IL-1β. PA treatment also significantly improved FITC-dextran permeability, serum I-FABP level and colonic transepithelial electrical resistance, and inhibited apoptosis of the intestinal epithelial cells in TNBS-treated mice. A total of 248 intersection targets were identified between PA and CD, and the core targets included EGFR, HRAS, SRC, MMP9, STAT3, AKT1, CASP3, ALB, HSP90AA1 and HIF1A. GO and KEGG analysis showed that PA negatively regulated apoptosis in close relation with PI3K/AKT signaling. Molecular docking showed that PA had a strong binding ability with AKT1, ALB, EGFR, HSP90AA1, SRC and STAT3. In TNBS-treated mice, PA significantly decreased p-PI3K and p-AKT expressions in the colonic mucosa. PA ameliorates TNBS-induced intestinal barrier injury in mice by antagonizing apoptosis of intestinal epithelial cells possibly by inhibiting PI3K/AKT signaling.

Grimace scale assessment during Citrobacter rodentium inflammation and colitis development in laboratory mice.

Bacterial infections and chronic intestinal inflammations triggered by genetic susceptibility, environment or an imbalance in the intestinal microbiome are usually long-lasting and painful diseases in which the development and maintenance of these various intestinal inflammations is not yet fully understood, research is still needed. This still requires the use of animal models and is subject to the refinement principle of the 3Rs, to minimize suffering or pain perceived by the animals. With regard to this, the present study aimed at the recognition of pain using the mouse grimace scale (MGS) during chronic intestinal colitis due to dextran sodium sulfate (DSS) treatment or after infection with Citrobacter rodentium. In this study 56 animals were included which were divided into 2 experimental groups: 1. chronic intestinal inflammation (n = 9) and 2. acute intestinal inflammation (with (n = 23) and without (n = 24) C. rodentium infection). Before the induction of intestinal inflammation in one of the animal models, mice underwent an abdominal surgery and the live MGS from the cage side and a clinical score were assessed before (bsl) and after 2, 4, 6, 8, 24, and 48 hours. The highest clinical score as well as the highest live MGS was detected 2 hours after surgery and almost no sign of pain or severity were detected after 24 and 48 hours. Eight weeks after abdominal surgery B6-Il4/Il10-/- mice were treated with DSS to trigger chronic intestinal colitis. During the acute phase as well as the chronic phase of the experiment, the live MGS and a clinical score were evaluated. The clinical score increased after DSS administration due to weight loss of the animals but no change of the live MGS was observed. In the second C57BL/6J mouse model, after infection with C. rodentium the clinical score increased but again, no increased score values in the live MGS was detectable. In conclusion, the live MGS detected post-operative pain, but indicated no pain during DSS-induced colitis or C. rodentium infection. In contrast, clinical scoring and here especially the weight loss revealed a decreased wellbeing due to surgery and intestinal inflammation.