Introduction

Inflammatory bowel disease (IBD) is a complex chronic and relapsing inflammatory disorder of the gastrointestinal tract with unclear pathogenesis. Accumulating evidence suggests food emulsifiers as a risk factor for the development of IBD. It is indicated that food emulsifiers carboxymethylcellulose (CMC) can alter gut microbiota and promote intestinal inflammation in IBD under normal diet. Since high-fat diet is the main life scenario for CMC use, little is known about the effect of CMC on colitis model in different high-fat diet.

Aims & Methods

C57/BL6 mice (3-5 weeks old) were randomly divided into three high-fat diet (HFD) groups, which only differed by fat composition (40 %), derived from corn oil (HCO, n-6 polyunsaturated fatty acids), olive oil (HOO, monounsaturated fatty acids) and milk fat (HMF, saturated fatty acids), and exposed to CMC (1.0% w/v) in drinking water for 4 weeks before the establishment of dextran sulfate sodium (DSS) induced acute colitis mice model. Colon inflammation severity was evaluated using survival rate, HE staining, colon length, body weight change and DAI score. Ki67 and TUNEL assays were performed to detect proliferation and apoptosis.

Results

Compared with the CMC+DSS group, the HMF+CMC+DSS group markedly increased survival rate (P=0.013), colon length (P=0.027) and body weight (day4, day5, day6 and day7, P<0.05, respectively), and relieved DAI score (day4 and day6, P<0.05, respectively) and histological damage (P<0.0001). The HOO+CMC+DSS group seemly had no significant difference from the CMC+DSS group. In contrast, the HCO+CMC+DSS group developed more aggressive colitis with increased DAI score (day5, P<0.01) and decreased body weight (day1, day2, day5 and day6, P<0.05, respectively). In terms of maintaining homeostasis of the intestinal barrier, the HMF+CMC+DSS group showed the highest Ki67 (P=0.012, P=0.0002 P=0.042, respectively) and TUNEL positive cells (P=0.324, P<0.0001, P<0.0001, respectively) compared with the CMC+DSS, HOO+CMC+DSS or HCO+CMC+DSS group. Between CMC+DSS and HOO+CMC+DSS or HCO+CMC+DSS group, there was no difference in Ki67 positive cells, while TUNEL positive cells significantly decreased (P=0.002, P=0.008, respectively).

Conclusion

The function of CMC on DSS colitis varies in different high-fat. Unexpectedly, CMC protects against the development of high-milk-fat related colitis by maintaining homeostasis of proliferation and apoptosis.

References

The function of CMC on DSS colitis varies in different high-fat. Unexpectedly, CMC protects against the development of high-milk-fat related colitis by maintaining homeostasis of proliferation and apoptosis.

Disclosure

none

Introduction

It has emerged that the energy metabolism of colonocytes shapes the gut microbiota, and loss of epithelial hypoxia accompanied by respiration-dependent dysbiosis is observed in experimental colitis^{1, 2}. The inhibitory effect of tacrolimus on inducible nitric oxide synthase (iNOS), a vital protein in nitrate respiration, was reported in human colonic epithelial cell lines³. Our previous study reported that tacrolimus significantly alleviated colitis in mice and induced beneficial alterations of the gut microbiome⁴. However, the mechanism of how tacrolimus improves the gut microbiota in colitis remains to be elucidated.

Aims & Methods

In this study, we aim to explore the effects of tacrolimus on colonocyte metabolism in colitis. Male C57BL/6J mice aged 8 to 10 weeks were employed. Solutions of 2.5% dextran sulfate sodium (DSS) were prepared in the drinking water to induce colitis for 7 consecutive days. Tacrolimus (Tacro) at a dose of 10 mg/kg was administered via gavage, while aminoguanidine hydrochloride (AG), the selective iNOS inhibitor, was given intraperitoneally at a dose of 100 mg/kg per day. The body weight was observed daily. For hypoxia detection, mice were treated with 60 mg/kg of pimonidazole HCl intraperitoneally 1 hour before euthanasia. Colon lengths were measured, and the colon samples were fixed in 4% paraformaldehyde for subsequent hematoxylin and eosin staining and hypoxia staining. Total RNA and protein were extracted from the isolated colonocytes for the measurement of iNOS expression. For the in vitro study, the human colorectal adenocarcinoma cells (Caco-2) were incubated with 10 ug/mL tacrolimus or 0.1% DMSO for 4 hours, then the oxygen consumption rates were measured on the Seahorse XFe24 Analyzers according to the manufacturer’s protocols.

Results

Hypoxia in the colonic epithelium was significantly disrupted in DSS-induced colitis mice, which was partially restored by tacrolimus treatment. The iNOS expression on both mRNA and protein levels was significantly upregulated by DSS and downregulated after tacrolimus administration, while the addition of AG enhanced the effects of tacrolimus. Compared with the individual tacrolimus intervention, the combined treatment of tacrolimus and AG achieved a better remission in weight loss, colonic shortening, and colonic pathological changes including the disruption of crypt-villus structure, goblet cell loss and immune cell infiltration. In vitro measurement of mitochondrial oxidative phosphorylation showed that tacrolimus significantly enhanced oxygen consumption, and promoted ATP production and spare respiratory capacity in Caco-2 cells.

Conclusion

Our results indicated that tacrolimus improved the oxidative metabolism of colonocytes in experimental colitis, which could be an important contributor to the alterations in the gut microbes induced by tacrolimus. The observed effect of tacrolimus in colonocytes may be associated with the inhibition of iNOS.

References

1. Litvak Y, Byndloss MX, Bäumler AJ. Colonocyte metabolism shapes the gut microbiota. Science 2018;362.
2. Cevallos SA, Lee JY, Velazquez EM, et al. 5-Aminosalicylic Acid Ameliorates Colitis and Checks Dysbiotic Escherichia coli Expansion by Activating PPAR-γ Signaling in the Intestinal Epithelium. mBio 2021;12.
3. Hämäläinen M, Lahti A, Moilanen E. Calcineurin inhibitors, cyclosporin A and tacrolimus inhibit expression of inducible nitric oxide synthase in colon epithelial and macrophage cell lines. Eur J Pharmacol 2002;448:239-44.
4. Lv W, Zhang D, He T, et al. Combination of Lactobacillus plantarum improves the effects of tacrolimus on colitis in a mouse model. 2023;13.

Introduction

Maintaining intestinal homeostasis relies on the interplay between individuals and their unique gut microbiota. Disruptions may result in chronic inflammation and Inflammatory Bowel Disease (IBD) [1]. This study delves into this interplay, emphasizing the characterization of diet-derived microbiome-associated metabolites and prioritizing them based on their effects on epithelial barrier integrity and inflammatory status.

Aims & Methods

Diet-derived microbiome-associated metabolites – epigallocatechin-gallate, gallic acid (GA), protocatechuic acid (PCA), ellagic acid (EA), and urolithins (Uro-A, B, C, D) – were selected through extensive literature research. In the commercial colon cell model (Caco-2) and 3D epithelial organoids from primary adult intestinal-epithelial stem cells, inflammation was induced with TNF-alpha and IFN-gamma (25ng/ml and each) to mimic IBD conditions. The expression of inflammatory cytokines genes (TNF-alpha, CXCL-9, MCP-1, IL-8) and epithelial barrier integrity markers (ZO-1 and OCLN) were evaluated by qRT-PCR. Epithelial barrier integrity was visualized using FITC-dextran assay.

Results

The induction of inflammation significantly increased in key inflammatory markers by an average 7.5-fold change (±1.8SD) in Caco-2 and 3D IBD organoid models. Metabolite analysis in Caco-2 cells showed that GA exhibited a 1.24-fold increase in TNF-alpha (p=0.036) and a -7.22-fold decrease in MCP-1 (p=0.049) gene expressions. EA decreased the expression of TNF-alpha and MCP-1 genes by -1.59-fold (p=0.045) and -3.59-fold (p=0.036) respectively, whereas IL-8 was reduced by -7.62-fold (p=0.01). Uro-A uniquely decreased MCP-1 gene expression by -7.16-fold (p=0.004) but increased IL-8 by 8-fold (p=0.01). PCA demonstrated a statistically significant decrease in all tested inflammatory markers (p<0.05). Analysis of 3D IBD organoids highlighted unique effects in restoring the epithelial barrier and expression of inflammatory marker genes.

Conclusion

Our findings demonstrate the anti-inflammatory properties of gallic acid, ellagic acid, protocatechuic acid, and urolithin-A in both Caco-2 and 3D organoid models. This research elucidates the complex interrelationships within the nutrition-microbiome-metabolome axis and its consequential impact on gut health.

References

1. Zheng, D., Liwinski, T. & Elinav, E. Interaction between microbiota and immunity in health and disease. Cell Res 30, 492–506 (2020). https://doi.org/10.1038/s41422-020-0332-7

Disclosure

No conflict of interest to declare.

Introduction

Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract, with its etiology involving genetic, microbial and environmental factors. Dysregulated autophagy, high-fat (HF) diet and abnormal colonization of adherent-invasive E. coli (AIEC) have been identified as risk factors for CD. Physical activity (PA) represents a fundamental intervention that offers significant health benefits and has been associated with a reduced risk of developing various diseases, including CD. Although PA has been shown to induce autophagy in different tissues, exerting beneficial effects for the organism, its impact on autophagy in the intestine and its ability to suppress the deleterious effects of HF diet via the modulation of autophagy have not been investigated.

Aims & Methods

This study aims to (i) explore the influence of PA on the autophagy process in intestinal tissue, and (ii) assess the potential of PA to restore intestinal homeostasis and decrease inflammation caused by HF consumption. Mice deficient for the autophagy-related gene Atg16l1 specifically in intestinal epithelial cells (Atg16l1^flox/floxCreVillin, called by Atg16l1^∆IEC mice) or wild-type mice (WT or Atg16l1^flox/flox) were fed either a control or HF diet for 12 weeks, and then engaged in spontaneous PA on running wheels (active group) or not (sedentary control group) for the same duration. The travelled distance (km), and the speed (m/min) on the wheels were recorded continuously. At the end of the experiment, total fat mass and fat-free mass were measured with echoMRI. The weight of total mesenteric and epididymal adipose tissues were also determined post-mortem. Levels of autophagy markers were assessed by Western blot and qRT-PCR. Intestinal inflammation was assessed by quantification of fecal lipocalin-2 and intestinal pro-inflammatory cytokine levels.

Results

Under HF diet, both Atg16l1^∆IEC and WT mice exhibited substantial weight gain compared to the groups fed the control diet. Spontaneous PA notably attenuated weight gain, particularly in the HF diet-fed groups. Spontaneous PA also diminished the increase in total fat mass and the weight of mesenteric adipose tissue induced by HF consumption, while concurrently augmenting total fat-free mass. HF diet-fed mice exhibited a lower total running distance over the 12 weeks compared to those fed the control diet. Importantly, HF diet consumption led to increased fecal lipocalin-2 level compared to control diet, with a more pronounced effect in Atg16l1^∆IEC versus WT mice, and this level was reduced with spontaneous PA. In the absence of PA, HF diet-fed Atg16l1^∆IEC mice exhibited increased E. coli abundance compared to other mouse groups, and this was not observed in the active groups. Compared to control diet, HF diet led to dysregulation of expression levels of several autophagy markers and autophagy flux in intestinal tissues, and this was corrected by PA. In particular, there was a positive correlation between the total travelled distance and levels of several autophagy-related mRNAs.

Conclusion

Spontaneous PA can correct the dysregulation of autophagy and mitigate intestinal inflammation induced by the HF diet. This suggests that combining spontaneous PA with other therapeutic interventions may be a valuable strategy for CD patients with dysregulated autophagy.

Introduction

The intestinal microbiome is now broadly accepted as a key driver of Ulcerative Colitis (UC), but how the gut flora impacts key disease pathologies, such as epithelial barrier dysfunction, is poorly understood. MB310 is a live biotherapeutic product for the treatment of UC comprising 8 gut commensal bacteria. These were selected based on a strong association with clinical response in UC patients when engrafted from the donor in a fecal microbiota transplantation (FMT) clinical study. To understand how the MB310 strains drive clinical benefit, we tested their impact on gut barrier integrity and the epithelial inflammatory response.

Aims & Methods

Effects on barrier function were assessed by measuring electric impedance of Caco-2 cells in the presence or absence of lipopolysaccharide (LPS). Furthermore, effects on epithelial permeability were assessed by measuring the translocation of fluorescein isothiocyanate (FITC)-labelled dextran through a 2-dimensional human colonic organoid-derived monolayer, following treatment with the barrier disruptor dextran sodium sulfate (DSS). To gain a mechanistic insight, transcriptomic analysis of 3-dimensional human colonic organoids was performed, following micro-injection with the MB310 consortium in the presence of inflammatory cytokines (Tumor Necrosis Factor [TNF] and Interferon [IFN]γ).

Results

First, the impact of the MB310 consortium on barrier integrity was explored by co-culturing the strains individually or as a consortium with a monolayer of the gut epithelial cell line, Caco-2. Five MB310 strains, as well as the MB310 consortium, significantly increased the integrity of a gut epithelial cell monolayer. Next, the strains were tested on a Caco-2 monolayer that had been damaged by an inflammatory insult, namely LPS, to reflect active UC. 2 strains enhanced repair of the monolayer after LPS challenge, leading to increased integrity.
Human colonic organoids can be plated out to form a tight monolayer that is impervious to large molecules, such as FITC-dextran. DSS treatment disrupts the monolayer leading to translocation of FITC-dextran. Overnight treatment with the MB310 consortium almost completely reversed this effect, which again demonstrates an ability of MB310 to repair gut epithelium following insult.
Finally, the impact of MB310 on the inflammatory response of the gut epithelium was assessed, by stimulating organoids with inflammatory cytokines to mimic UC conditions. Transcriptomics analysis revealed that the cytokines upregulated several inflammatory genes and pathways. MB310 treatment reversed the activation of many of the inflammatory genes and pathways, including many pathways associated with UC.

Conclusion

The accumulated data suggest that MB310 interacts with the host epithelial barrier leading to improvement of its integrity, repair after damage and reduction of leakiness. Moreover, transcriptomic analysis demonstrated the ability of MB310 to rewire epithelial cells to an anti-inflammatory programme, under inflammatory conditions. COMPOSER-1 is an ongoing phase 1b trial testing MB310 in UC patients.

Introduction

Inflammatory bowel disease (IBD) constitutes a group of chronic and relapsing idiopathic inflammatory disorder of the intestine that includes Crohn’s disease (CD) and ulcerative colitis (UC). Both diseases characterized by a cyclical nature alternating between active and quiescent states, that markedly diminish physical functioning and quality of life in patients. Our previous study have indicated that exercise of moderate intensity can be recommended as the alternative therapy in prevention and healing of experimental colitis in obese mice.

Aims & Methods

The intestinal alkaline phosphatase (IAP) is an important apical brush border enzyme, released from cells walls during stressful events maintains tight junction and barrier integrity It remains unknown, whether IAP treatment besides its protective action against colitis, can influence the gene expression of proinflammatory genes in adipose tissue, liver and skeletal muscle of obese mice forced to exercise on treadmill. In this study two major series of C57BL/6 male mice were fed ad libitum high fat diet (HFD, 70% energy from fat, series A) or standard diet (SD,10% energy from fat, series B) (Altromin, Lage, Germany) for 12 weeks and subjected to forced treadmill exercise (15 min/day, 6 wks., Panlab, Harvard Apparatus, MA, USA) with or without the intragastric treatment with AP (200 U/day) and trinitrobenzene sulfonic acid (TNBS) colitis was induced. Following colitis, the colonic blood flow (CBF) was examined by Laser Doppler flowmetry, the disease activity index (DAI) as well as histology of intestinal mucosa were determined. The biopsy samples of abdominal fat, liver and skeletal muscle were excised, and shock frozen in liquid nitrogen for determination of the gene expression of antioxidative enzymes SOD, GPx and fat biomarkers leptin and adiponectin as well as proinflammatory biomarkers IL-1β, IL-6, TNF-α, HIF-1α and iNOS by qPCR.

Results

In obese mice the DAI was significantly increased and a significant fall in CBF as compared with sedentary SD fed mice (P<0.05). Treadmill exercise exacerbated the DAI activity in obese mice and decreased CBF vs. SD fed mice (P< 0.05), and this effect was significantly inhibited by treatment with IAP (P< 0.05). In fat tissue and liver of obese mice, the adiponectin mRNA expression was downregulated, while the mRNA leptin expression being upregulated (p<0.05), and this ratio in expression of these factors was restored by IAP. In the skeletal muscle, the mRNA expression ratio of adiponectin to leptin remained unchanged. The SOD and GPx mRNA expression was unchanged in the liver, while the mRNA expression of proinflammatory markers HIF-1α, TNF-α and IL-1β was significantly increased (P< 0.05) in all tissues collected from the high fat diet fed mice forced to exercise as compared to SD animals. The treatment with IAP significantly reduced the expression of these markers in liver, skeletal muscle, and adipose abdominal tissue.(p<0.05).

Conclusion

We conclude that forced treadmill exercise not only exacerbates the severity of colonic damage and diminishes the colonic blood flow in obese mice but also exerts deleterious effect as manifested by the increased expression of local proinflammatory biomarkers in fat, liver, and skeletal muscle. IAP downregulates gene expression of proinflammatory biomarkers and deserves an attention as may exert the multi-organ protective activity in the treatment of IBD in humans. Supported by National Science Centre (NCN) within the framework of project grant (UMO-2015/19/B/NZ4/03130) and grant from UJCM, No.: N41/DBS/000783.

Introduction

Ulcerative colitis (UC) represents the widespread inflammatory bowel disease (IBD) affecting millions of patients worldwide. UC usually begins at the onset of adulthood and progresses across the patient’s lifetime. Snail mucus has many hidden activities; many of them have been discovered in recent years. Scientific researchers have demonstrated that mucus-derivate drugs can be used in many therapies. For example, it is used in creams to ease skin abrasions and scars, to cure respiratory diseases and heartburn, and at last, scientists discovered unexpected and previously unknown properties. At the beginning of the twentieth century, a new interest in gastropods has grown up for their possible employment in medicine and industries.

Aims & Methods

This study aimed to investigate the possible therapeutic effect of mucus extracted from helix pomica snail in an experimental ulcerative colitis model induced by dextran sulphate sodium. Twenty-four mice were included and categorized into four groups. The 1^st group acted as a normal control group, whereas the 2^nd group acted as a pathological control group and ingested dextran sulphate sodium in drinking water. The 3^rd Group was treated orally with sulfasalazine and then ingested dextran sulphate sodium in drinking water. The 4^th group was treated orally with helix pomatia mucus and then ingested dextran sulphate sodium in drinking water. After seven days, the animals were euthanized, and the intestinal specimens were collected for pathological and biochemical assessment.

Results

Our results showed an improvement in histopathology with regression of the intestinal lesions in the 4th group compared to the pathological control group. The levels of nuclear-kappa B, and IL-1B decreased significantly after using helix pomatia mucus.

Conclusion

Helix pomatia mucus could be a potential therapy to protect against experimentally induced ulcerative colitis.