Colon Samples Research Articles

Therapeutic effects of genistein in experimentally induced ulcerative colitis in rats via affecting mitochondrial biogenesis.

Ulcerative colitis (UC) is an inflammatory bowel disease that affects the mucosa of the colon, resulting in severe inflammation and ulcers. Genistein isa polyphenolic isoflavone present in several vegetables, such as soybeans and fava beans. Therefore, we conducted the following study to determine the therapeutic effects of genistein on UC in rats by influencing antioxidant activity and mitochondrial biogenesis and the subsequent effects on the apoptotic pathway. UC was induced in rats by single intracolonic administration of 2ml of 4% acetic acid. Then, UC rats were treated with 25-mg/kg genistein. Colon samples were obtained to assess the gene and protein expression of nuclear factor erythroid 2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1), peroxisome proliferator-activated receptor-gamma coactivator (PGC-1), mitochondrial transcription factor A (TFAM), B-cell lymphoma 2 (BCL2), BCL2-associated X (BAX), caspase-3, caspase-8, and caspase-9. In addition, colon sections were stained with hematoxylin/eosin to investigate the cell structure. The microimages of UC rats revealed inflammatory cell infiltration, hemorrhage, and the destruction of intestinal glands, and these effects were improved by treatment with genistein. Finally, treatment with genistein significantly increased the expression of PGC-1, TFAM, Nrf2, HO-1, and BCL2 and reduced the expression of BAX, caspase-3, caspase-8, and caspase-9. In conclusion, genistein exerted therapeutic effects against UC in rats. This therapeutic activity involved enhancing antioxidant activity and increasing mitochondrial biogenesis, which reduced cell apoptosis.

Over-production of exopolysaccharide by Lacticaseibacillus rhamnosus CNCM I-3690 strain cutbacks its beneficial effect on the host

Most lactobacilli produce extracellular polysaccharides that are considered to contribute to the probiotic effect of many strains. Lacticaseibacillus rhamnosus CNCM I-3690 is an anti-inflammatory strain able to counterbalance gut barrier dysfunction. In this study ten spontaneous variants of CNCM I-3690 with different EPS-production were generated and characterized by their ropy phenotype, the quantification of the secreted EPS and genetic analysis. Amongst them, two were further analysed in vitro and in vivo: an EPS over-producer (7292) and a low-producer derivative of 7292 (7358, with similar EPS levels than the wild type (WT) strain). Our results showed that 7292 does not have anti-inflammatory profile in vitro, and lost the capacity to adhere to the colonic epithelial cells as well as the protective effect on the permeability. Finally, 7292 lost the protective effects of the WT strain in a murine model of gut dysfunction. Notably, strain 7292 was unable to stimulate goblet cell mucus production and colonic IL-10 production, all key features for the beneficial effect of the WT strain. Furthermore, transcriptome analysis of colonic samples from 7292-treated mice showed a down-regulation of anti-inflammatory genes. Altogether, our results point out that the increase of EPS production in CNCM I-3690 impairs its protective effects and highlight the importance of the correct EPS synthesis for the beneficial effects of this strain.

Defined Pig Microbiota with a Potential Protective Effect against Infection with Salmonella Typhimurium.

A balanced microbiota is a main prerequisite for the host's health. The aim of the present work was to develop defined pig microbiota (DPM) with the potential ability to protect piglets against infection with Salmonella Typhimurium, which causes enterocolitis. A total of 284 bacterial strains were isolated from the colon and fecal samples of wild and domestic pigs or piglets using selective and nonselective cultivation media. Isolates belonging to 47 species from 11 different genera were identified by MALDI-TOF mass spectrometry (MALDI-TOF MS). The bacterial strains for the DPM were selected for anti-Salmonella activity, ability to aggregate, adherence to epithelial cells, and to be bile and acid tolerant. The selected combination of 9 strains was identified by sequencing of the 16S rRNA gene as Bacillus sp., Bifidobacterium animalis subsp. lactis, B. porcinum, Clostridium sporogenes, Lactobacillus amylovorus, L. paracasei subsp. tolerans, Limosilactobacillus reuteri subsp. suis, and Limosilactobacillus reuteri (two strains) did not show mutual inhibition, and the mixture was stable under freezing for at least 6 months. Moreover, strains were classified as safe without pathogenic phenotype and resistance to antibiotics. Future experiments with Salmonella-infected piglets are needed to test the protective effect of the developed DPM.

The regulatory effects of Lonicera japonica flos on fecal microbiota from humans with type 2 diabetes in a SHIME model

IntroductionLonicera japonica flos, a well-known herbal medicine in Asian countries, has been widely used for the treatment of type 2 diabetes (T2D). The gut microbiota and relevant metabolites have been shown to be involved in the development of T2D. This study was designed to investigate the effects of Lonicera japonica flos on fecal microbiota from humans with T2D using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) system. MethodsAn aqueous extract of Lonicera japonica flos (LJE) was prepared. Colorimetry and high-performance liquid chromatography (HPLC) were used to analyze the chemical composition of LJE. Three colonic compartments of SHIME were inoculated with feces from T2D donors, and LJE was added to SHIME to simulate daily oral administration for 7 days. The microbial community and the concentrations of short-chain fatty acids (SCFAs) of colonic samples were analyzed by 16 S rRNA sequencing technology and gas chromatography, respectively. In addition, polyphenols in LJE before and after fermentation by fecal microbiota were measured using HPLC. ResultsLJE is rich in polyphenols and polysaccharides and alters the microbial community, as indicated by the decreased relative abundance of the detrimental genera Shigella and Lactobacillus and the increased abundance of SCFA-producing bacteria, including the genera Prevotella and Bacteroides. LJE significantly promoted SCFA production, including acetic, propionic, and butyric acids, in each colonic compartment. Furthermore, chlorogenic acid in LJE was biotransformed by fecal microbiota. ConclusionsLJE exerted beneficial regulation on the T2D fecal microbial community. The regulatory effects of gut microbiota may partly contribute to the therapeutic action of Lonicera japonica flos on T2D.

The protective effect of 999 XiaoErGanMao granules on the lungs and intestines of influenza A virus-infected mice

Context Gastrointestinal symptoms are a common complication of influenza virus infection in children, which the gut-lung axis become involved in its biological progress. The protective effect of 999 XiaoErGanMao granules (XEGMG) on multi-organ injury in viral pneumonia remains unclear. Objective To investigate the therapeutic effect of XEGMG on lungs and intestines injury in A/FM/1/47 (H1N1) influenza virus-infected mice. Materials and methods Male BALB/c mice were infected with the 2LD50 H1N1 influenza virus and then treated with XEGMG (6 or 12 g/kg) intragastrically once a day for 4 days. The lung and colon samples were then collected for pathological observation, and assays for inflammatory cytokines and intestinal barrier. Mouse feces were collected to evaluate the intestinal microbiota. Results Treating with XEGMG (12 g/kg) can mitigate body weight loss caused by 2LD50 H1N1 infection. It can also reduce lung index and pathological damage with the decreased inflammatory cytokines such as IL-6 and IL-1β. Furthermore, XEGMG (12 g/kg) can maintain the goblet cell number in the colons to protect the intestinal barrier and regulate the major flora such as Firmicutes, Bacteroidetes, and Muribaculaceae back to normal. Meanwhile, the expression of IL-17A in the colon tissues was significantly lower in the group of XEGMG (6, 12 g/kg) compared to H1N1 group. Discussion and conclusions XEGMG can protect against H1N1 invasion involved in gut-lung axis regulation. The results provide new evidence for the protective effect of XEGMG, which is beneficial to vulnerable children.

Evidence of Association between CTLA-4 Gene Polymorphisms and Colorectal Cancers in Saudi Patients.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) has been identified as an immunosuppressive molecule involved in the negative regulation of T cells. It is highly expressed in several types of autoimmune diseases and cancers including colorectal cancer (CRC). (1) Objective: To explore the association between CTLA-4 single nucleotide polymorphisms (SNP) and risk to (CRC) in the Saudi population. (2) Methods: In this case-control study, 100 patients with CRC and 100 matched healthy controls were genotyped for three CTLA-4 SNPs: rs11571317 (-658C > T), rs231775 (+49A > G) and rs3087243 (CT60 G > A), using TaqMan assay method. Associations were evaluated using odds ratios (ORs) and 95% confidence intervals (95% CIs) for five inheritance models (co-dominant, dominant, recessive, over-dominant and log-additive). Furthermore, CTLA-4 expression levels were evaluated using quantitative real-time PCR (Q-RT-PCR) in colon cancer and adjacent colon tissues. (3) Results: Our result showed a significant association of the G allele (OR = 2.337, p < 0.0001) and GG genotype of the missense SNP +49A > G with increased risk of developing CRC in codominant (OR = 8.93, p < 0.0001) and recessive (OR = 16.32, p < 0.0001) models. Inversely, the AG genotype was significantly associated with decreased risk to CRC in the codominant model (OR = 0.23, p < 0.0001). In addition, the CT60 G > A polymorphism exhibited a strong association with a high risk of developing CRC for the AA genotype in codominant (OR = 3.323, p = 0.0053) and in allele models (OR = 1.816, p = 0.005). No significant association was found between -658C > T and CRC. The haplotype analysis showed that the G-A-G haplotype of the rs11571317, rs231775 and rs3087243 was associated with high risk for CRC (OR = 57.66; p < 0.001). The CTLA-4 mRNA gene expression was found significantly higher in tumors compared to normal adjacent colon samples (p < 0.001). (4) Conclusions: Our findings support an association between the CTLA-4 rs231775 (+49A > G) and rs3087243 (CT60 G > A) polymorphisms and CRC risk in the Saudi population. Further validation in a larger cohort size is needed prior to utilizing these SNPs as a potential screening marker in the Saudi population.

A novel tumour enhancer function of Insulin-like growth factor II mRNA-binding protein 3 in colorectal cancer

CRC cells evolve a variety of strategies to limit or circumvent apoptosis cell death. RNA binding proteins (RBPs) regulate many of the molecular mechanisms that underlie the development of cancer. The insulin-like growth factor II mRNA-binding proteins (IMP) family are oncofoetal RBPs, consisting of IMP1, IMP2 and IMP3, which have an important role in RNA metabolism. IMP3 is highly expressed in colorectal cancer (CRC) tissue, where its expression often correlates with poor prognosis. However, the role of IMP3 in CRC is not fully understood. IMP3 expression was analysed using a public database and by Western blotting and immunohistochemistry in human colon samples derived from patients with sporadic CRC and healthy subjects. To address whether IMP3 controls cancer cell survival, we analysed cell death pathways in in vitro and in vivo experiments after IMP3 downregulation by siRNA or an antisense oligonucleotide. IMP3 was highly expressed in CRC samples compared to normal control tissues. The knockdown of IMP3 enhanced a caspase-independent cell death in CRC cell lines. Furthermore, the treatment of CRC cells with IMP3 siRNA did not alter the expression of GSDMD, GPX-4 and the activated form of RIP3, three key molecules that govern pyroptosis, ferroptosis and necroptosis, respectively. Abrogation of IMP3 in CRC significantly reduced Bcl-2 and Bcl-xL mRNA and was associated with an altered mitochondrial membrane potential that allowed the nuclear migration of the apoptosis-inducing factor (AIF). Moreover, specific immunoprecipitation experiments on CRC human cell lines indicated that IMP3 binds Bcl-2 and Bcl-xL mRNA, suggesting that IMP3 acts as a regulator of the intrinsic apoptotic pathway through the surveillance of anti-apoptotic Bcl mRNA metabolism. Finally, we showed that IMP3 block inhibited the growth of CRC cell lines in vivo after transplantation into immunodeficient mice. Altogether, these data support a novel role for IMP3 in controlling the intrinsic caspase-independent apoptotic pathway in CRC.

Abstract 534: PRMT5 inhibition as a therapeutic strategy for KRAS mutant CRC: downstream mediators of the PRMT5-KRAS crosstalk

Abstract Despite decades of research, there are still no effective targeted treatments for the nearly 45% of colorectal cancer (CRC) patients harboring a mutation in their KRAS gene. Protein Arginine Methyltransferase 5 (PRMT5) is an epigenetic regulator undergoing clinical trials as a potential therapeutic target for cancer. Our group has previously reported that PRMT5 is overexpressed in KRAS mutant CRC when compared to KRAS wild-type (WT) CRC, and that PRMT5 inhibition exhibited greater therapeutic efficacy in KRAS mutant CRC when compared to KRAS WT CRC. We therefore proposed that PRMT5 may be a strong therapeutic target for KRAS mutant CRC, and that PRMT5 and KRAS may crosstalk. In this study, we investigated several key downstream signal transduction proteins that may be involved in mediating the potential crosstalk between PRMT5 and KRAS. This is important, as these key intermediate proteins may be strong therapeutic targets for inhibition, as well as valuable therapeutic targets for combination therapy along with PRMT5 inhibitors. We first conducted a literature review to determine which key downstream proteins may be involved in mediating the potential crosstalk between PRMT5 and KRAS. Our initial findings indicated that MYC, p65/RELA, TP53, P21/CDKN1A, ELF1, E2F1, and EIF4E may be key downstream proteins involved in mediating the potential crosstalk between PRMT5 and KRAS. We next used the STRING database to determine which of the aforementioned proteins have demonstrated interactions with PRMT5 and KRAS. We found that PRMT5 and KRAS both interact with MYC, p65/RELA, TP53, P21/CDKN1A, E2F1, and EIF4E with an interaction score &amp;gt; 0.150. We then used the Gene Expression Profiling Interactive Analysis (GEPIA) database to analyze the RNA-Seq data of CRC patients from The Cancer Genome Atlas (TCGA) database. We first used the GEPIA database to determine which proteins are overexpressed in CRC patient tumor samples compared to normal colon and rectum patient samples. We observed that MYC, TP53, E2F1, and EIF4E are all over 2-fold overexpressed in CRC patient tumor samples compared to normal colon and rectum patient samples (q &amp;lt; 0.01). We next used the GEPIA database to determine which proteins have an expressional correlation with PRMT5 and KRAS in CRC patient tumor samples. We found that MYC, E2F1, and EIF4E are all positively correlated with PRMT5 and KRAS with an R-Value &amp;gt; 0.70 and a P-Value &amp;lt; 0.01 in CRC patient tumor samples. Our study thus showcases the key downstream signal transduction proteins that may be involved in mediating the potential crosstalk between PRMT5 and KRAS. These findings are significant, as these key intermediate proteins may be strong therapeutic targets for inhibition, as well as strong therapeutic targets for a PRMT5 combination therapy. Additional research is currently underway to outline the molecular processes behind MYC, E2F1, and EIF4E’s interactions with both PRMT5 and KRAS. Citation Format: David Shifteh, Tzuriel Sapir, Moshe Pahmer, Sanjay Goel, Radhashree Maitra. PRMT5 inhibition as a therapeutic strategy for KRAS mutant CRC: downstream mediators of the PRMT5-KRAS crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 534.

Abstract 2268: Single cell RNA sequencing defines dynamic immune cell subsets in serial colon and peripheral blood samples in a patient with checkpoint inhibitor-induced colitis

Abstract Background: Immune checkpoint inhibition is an increasingly popular strategy for the management of numerous malignancies. Unfortunately, severe inflammatory toxicities affect up to 60% of patients, often leading to treatment discontinuation in settings where few alternative therapies are available to control disease. Checkpoint inhibitor induced colitis is a common toxicity for which the underlying mechanisms are poorly defined. Methods: To better understand the changing colon-specific and peripheral immune environments over the course of disease progression and treatment, we collected blood and colon tissue from a Merkel Cell carcinoma patient who developed colitis after treatment with pembrolizumab. We performed 10X Genomics single cell RNA sequencing on samples collected before, during and after checkpoint inhibition and various interventions to mitigate toxicity. Results: We identified both shared and colon-exclusive clonal expansion of T cells defined by distinct transcriptional programs, including cytotoxicity, memory, and proliferation markers, at various stages of the disease process. These clones decreased dramatically in the colon after infliximab and vedolizumab therapies. Conclusions: We identified several distinct T cell subsets as potential drivers of colitis in the colon. T cell clones that potentially recognize colonic tissue with a high degree of specificity can be harnessed for therapeutic intervention. Our findings emphasize the need for a more detailed understanding of the immune environment in the colon and suggest the potential for peripheral blood as a readout for immune activity during CPI treatment. Citation Format: Jacqueline E. Mann, Liliana Lucca, Matthew Austin, Ross D. Merkin, Lilach Aizenbud, Kevan Herold, Marie Robert, Harriet M. Kluger. Single cell RNA sequencing defines dynamic immune cell subsets in serial colon and peripheral blood samples in a patient with checkpoint inhibitor-induced colitis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2268.

Abstract 149: An organoid model for understanding the pathogenesis of Lynch syndrome

Abstract Lynch syndrome (LS) is an inherited predisposition for developing cancer caused by germline variants in mismatch repair (MMR) genes. It is estimated that LS is responsible for 1 out of every 35 colorectal cancers and 1 out of every 56 endometrial cancers. Loss of MMR is also seen in many sporadic cancers. Still, the mechanism of tumorigenesis in these patients is unknown. Loss of MMR increases genomic mutation rate, but it is unclear if increased mutational burden alone leads to tumorigenesis. Recent studies of LS patient colon samples show fields of morphologically normal tissue in which cells have lost expression of an MMR protein, suggesting that loss of MMR in these cells is sufficient to confer an advantage over heterozygous cells still proficient in MMR. However, whether MMR loss provides a direct advantage to cell types associated with cancer in LS has not been directly tested. We hypothesize that loss of MMR is sufficient to confer a selective advantage to colonic stem cells and is the first step towards tumorigenesis in LS patients. To test this, we use a human colonic organoid model to examine competition between MMR-deficient (dMMR) and MMR-proficient (pMMR) cells over time. The colonic organoids are differentiated from human embryonic stem cells. To track cell populations, dMMR stem cells are labeled with mCherry and pMMR stem cells are labeled with GFP. We have initially observed that dMMR stem cells are much more efficient at generating colonic organoids. Thus, the cells are mixed so that resulting organoids initially contain a small population of dMMR cells in order to mimic the spontaneous loss of heterozygosity that occurs in LS patients’ colonic crypts. Live fluorescent imaging of individual organoids is used to quantify changes in cell population over time. Preliminary data indicate that dMMR cells can outgrow pMMR cells resulting in a shift in the ratio of green to red cells over a two-week time period. These results suggest that loss of MMR function imparts an immediate selective advantage. Furthermore, this assay could be used to find conditions or compounds that decelerate population shifts with the ultimate goal of finding a way to reduce dMMR fields in LS patients. In these ways, this assay can be leveraged for gaining new insights into MMR function, pathogenesis of LS, and potential therapies for LS. Citation Format: Caroline Guild, Kirby Madden-Hennessey, Chris Heinen. An organoid model for understanding the pathogenesis of Lynch syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 149.

The Robogut: A Bioreactor Model of the Human Colon for Evaluation of Gut Microbial Community Ecology and Function

The human colon is inhabited by a complex community of microbes. These microbes are integral to host health and physiology. Understanding how and when the microbiome causally influences host health will require microbiome models that can be tightly controlled and manipulated. While in vivo models are unrivalled in their ability to study host-microbial interplay, in vitro models are gaining in popularity as methods to study the ecology and function of the gut microbiota, and benefit from tight controllability and reproducibility, as well as reduced ethical constraints. In this set of protocols, we describe the Robogut, a single-stage bioreactor system designed to replicate the conditions of the distal human colon, to culture whole microbial communities derived from stool and/or colonic biopsy samples, with consideration of methods to create culture medium formulations and to build, run, and sample the bioreactor apparatus. Cleaning and maintenance of the bioreactor system are also described. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth medium preparation Support Protocol 1: Preparing medium supplements Basic Protocol 2: Preparing the bioreactor vessels Support Protocol 2: Making acid and base bottles Support Protocol 3: Preparing the effluent bottles Support Protocol 4: Making acid solution Support Protocol 5: Making base solution Basic Protocol 3: Preparing inoculum and inoculating bioreactors Alternate Protocol 1: Preparing inoculum less than 0.5% (w/v) of vessel volume Alternate Protocol 2: Preparing synthetic community aliquots and inoculation via the septum Alternate Protocol 3: Preparing inoculum from a tissue sample Basic Protocol 4: Sampling the bioreactor vessel Basic Protocol 5: Harvesting bioreactor vessel contents at end of experiment Support Protocol 6: Cleaning and sterilizing sampling needles Basic Protocol 6: Cleaning the bioreactor vessel Support Protocol 7: Cleaning bioreactor support bottles.

WITHDRAWN: Disturbances of purine and lipid metabolism in the microbiota-gut-brain axis in male adolescent nonhuman primates with depressive-like behaviors

IntroductionMajor depressive disorder (MDD) in adolescents is a widespread and growing global public health concern with unique characteristics and pathophysiological mechanisms that are distinct from MDD in adults. ObjectiveThe purpose of our work was to address this knowledge gap about the unique characteristics and pathophysiological mechanisms of adolescent depression from a microbiota-gut-brain (MGB) axis perspective. MethodTen healthy male cynomolgus macaques (Macaca fascicularis) were paired into five pairs based on age and body weight, and two cynomolgus macaques from each pair were randomly allocated to chronic unpredictable mild stress group, or unstressed control group. At endpoint, microbe composition from cecum, ascending colon, transverse colon, and descending colon were analyzed by metagenome sequencing, and the metabolite profiles of MGB axis including central (prefrontal cortex, hippocampus and amygdala) and peripheral (plasma, gut and feces of cecum, ascending colon, transverse colon and descending colon) samples were analyzed by metabolomic profiling. Then, we compare the gut microbiome and metabolic signatures in MGB axis between adolescent and adult depressed macaques. ResultsThe microbial composition and gut-brain metabolic signatures were widely divergent between adolescent and adult depressed macaques, though the phylum Firmicutes and lipid metabolism pathways were persistently altered in both populations. Purine and arginine biosynthesis metabolism were a specific hallmark of adolescent depressed macaques, while fatty acyl metabolism was specially altered in adult. These differential metabolic pathways in adolescent and adult depressed macaques were mainly mapped into the prefrontal cortex and hippocampus, respectively. Notably, the genus Clostridium and Haemophilus, characteristically disturbed in adolescent depressed macaques but not in adult, were also significantly associated with the majority of purine metabolites in MGB axis. ConclusionThese findings provide a new framework describing divergent pathophysiological mechanisms between adolescent and adult depression, and may open new windows for more effective treatment strategies of adolescent depression.

Single-cell transcriptomic analysis of normal and pathological tissues from the same patient uncovers colon cancer progression

The aim of the present study was to elucidate the evolutionary trajectory of colon cells from normal colon mucosa, to adenoma, then to carcinoma in the same microenvironment. Normal colon, adenoma and carcinoma tissues from the same patient were analyzed by single-cell sequencing, which perfectly simulated the process of time-dependent colon cancer due to the same microenvironment. A total of 22 cell types were identified. Results suggest the presence of dominant clones of same cells including C2 goblet cell, epithelial cell subtype 1 (Epi1), enterocyte cell subset 0 (Entero0), and Entero5 in carcinoma. Epi1 and Entero0 were Co-enriched in antibacterial and IL-17 signaling, Entero5 was enriched in immune response and mucin-type O-glycan biosynthesis. We discovered new colon cancer related genes including AC007952.4, NEK8, CHRM3, ANO7, B3GNT6, NEURL1, ODC1 and KCNMA1. The function of TBC1D4, LTB, C2CD4A, AND GBP4/5 in T cells needs to be clarified. We used colon samples from the same person, which provide new information for colon cancer therapy.

Evaluation of desmoplasia in pancreatic and colon tumors by morphometric analysis

Abstract: Objectives: This study aims to quantify collagen deposition in the desmoplasia of pancreatic and colonial tumors. Methods: Samples of colon and pancreas tumors were selected from the Hospital das Clínicas de Pernambuco and submitted to trichomy with Gomori's Trichrome. Photomicrographic analysis was performed using the integrated Panthera imaging analysis system L. Results: It was identified that 31 colon tumors and 22 pancreatic tumors presented desmoplasia with the visualization of fibrillar collagen distributed irregularly around the tumor. There was a statistically significant difference (p = 0.0031) between collagen fiber deposition in desmoplastic and non-desmoplastic pancreatic tumors, as well as for colonic tumors (p &lt; 0.001). Conclusion: It was observed that more than half of the tumors analyzed presented desmoplasia with a difference of density of collagen fibrils statistically significant.

A202 INVESTIGATION OF THE DIRECT EFFECT OF HEMIN-HEMOPEXIN AND HAPTOGLOBIN STIMULATION IN INFLAMMATORY BOWEL DISEASE

Abstract Background Inflammatory bowel disease (IBD) is a global health issue defined by persistent intestinal epithelial inflammation resulting in bleeding, and it is one of the major risk factors of colorectal cancer (CRC). Hemin is an activator of heme-oxygenase and has anti-inflammatory, cytoprotective, and antiapoptotic properties by stimulating hemopexin and haptoglobin production. Hemopexin and haptoglobin are scavenging proteins with high affinity for hemin and hemoglobin, and are activated by interleukin (IL)-22. Purpose To evaluate the protective role of hemin through the stimulation of hemopexin and haptoglobin in experimental acute colitis. Method Mice were preconditioned with hemin by intraperitoneal injection with 5mg/kg body weight of hemin for three days while control mice were treated with PBS. Colitis was induced by giving 2.5-3% dextran sulfate sodium (DSS) in drinking water for 12 days. The disease activity index (DAI) was determined by daily monitoring of body weight, fecal bleeding and stool consistency. At the end of the experiment, liver, feces, serum and colon samples were harvested for the evaluation of fecal levels of hemopexin, fecal haptoglobin, IL-22, IL-22BP, IL-6 and TNF-α. Fecal microbiota composition will be determined using 16S rRNA sequencing. Result(s) We found that hemin treatment resulted in the presence of hemopexin and haptoglobin in fecal samples, where they may exert a protective role and influence the growth of pathobionts able to acquire iron using heme and hemoglobin as sources. Conclusion(s) The findings from this study will contribute to the understanding of the possible protective role of hemopexin and haptoglobin in ulcerative colitis. Please acknowledge all funding agencies by checking the applicable boxes below CIHR Disclosure of Interest None Declared

A54 THE IMPACT OF NON-INVASIVE TRANSCUTANEOUS AURICULAR VAGUS NERVE STIMULATION (NITAVNS) ON THE DEVELOPMENT OF EXPERIMENTAL COLITIS IN MICE

Abstract Background The anti-inflammatory impacts of stimulating the vagus nerve as a part of the gut-brain axis on intestinal macrophage activity and inflammatory cytokine production have been documented. While vagus nerve stimulation until now has been chiefly invasive, non-invasive auricular vagus nerve stimulation (NitaVNS) has no adverse effects associated with invasive stimulation. The NitaVNS-mediated activation of brainstem nuclei can exert anti-inflammatory effects directly on the colon or indirectly through the spleen via a macrophage-acetylcholine-dependent pathway. Purpose To examine the effects of NitaVNS on the development of acute colitis in mice using a preclinical model. Method Thirty C57BL/6 mice aged 11 to 12 weeks were divided into four groups: control (CTRL)-NitaVNS, CTRL-Sham, dextran sulphate sodium (DSS)-NitaVNS and DSS-Sham. Pre-set stimulation parameters (10 minutes duration, 10 V voltage, 500 s pulse width, and 20 Hz frequency) were applied for NitaVNS or sham-paw stimulation. 5% DSS was administered to induce colitis for five consecutive days, whereas CTRL mice received regular water. Weight loss, stool consistency, blood in the feces, and disease activity index were evaluated daily. Rectal bleeding, colon bleeding, and fecal consistency were determined as a composite macroscopic score upon sacrifice. Collected colon and blood samples were assessed for pro-inflammatory parameters such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, C-reactive protein (CRP), matrix metalloproteinase (MMP)-2, macrophage inflammatory protein (Mip)-1α and β, and anti-inflammatory cytokine tumor growth factor (TGF)-β using qRT-PCR and ELISA. Result(s) Compared to colitic-sham mice, colitic NitaVNS mice demonstrated a delayed onset and a significantly lower clinical disease severity. Colitis induced significant weight loss, rectal bleeding, and stool consistency loss, and NitaVNS significantly abolished these effects. In colitic conditions, NitaVNS decreased the disease activity index significantly compared to the colitic sham group. Colitis increased the macroscopic scores, and NitaVNS decreased them significantly. Conversely, colon length was significantly reduced in colitic conditions, but NitaVNS abrogated that deleterious effect. mRNA expression levels of IL-1β and TNF-α were significantly increased in colitic mice and NitaVNS decreased them significantly. Whereas Mip-1α and 1β were not modified in colitic conditions, NitaVNS significantly decreased these markers. MMP2 mRNA expression and colonic IL-6 and IL-1β protein levels were not altered in all conditions. However, colitis induced a significant decrease in colonic TGF-β levels, but NitaVNS did not amplify that effect. Interestingly, in the serum of colitic mice, NitaVNS significantly increased and decreased TGF-β and CRP levels, respectively. Conclusion(s) These findings suggest that NitaVNS possess some spatial anti-inflammatory effects in acute colitis through the up or downregulation of anti- or pro-inflammatory markers. Disclosure of Interest F. Hesampour: None Declared, D. Tshikudi: None Declared, A. Veysel Özden Employee of: Company providing the stimulator, C. N. Bernstein: None Declared, J.-E. Ghia: None Declared

A211 CAN AN EXPERIMENTAL MOUSE MODEL BE USED TO CHARACTERIZE BEHAVIOURAL COMORBIDITIES ASSOCIATED WITH INFLAMMATORY BOWEL DISEASES?

Abstract Background Inflammatory Bowel Diseases (IBD) are increasing globally with westernized countries experiencing a dramatic increase in cases of ulcerative colitis and Crohn’s disease. The corresponding rise in the comorbidities of anxiety and depression are linked to inflammation and microbiome dysbiosis; impacting the gut-brain axis (GBA). Another facet of the GBA are the tryptophan metabolites serotonin (5HT) and kynurenine (Kyn). 5HT and Kyn are metabolites implicated in anxious behaviours and memory deficiencies with dysbiosis influencing metabolite production. In inflammatory conditions, upregulation of Kyn impacts brain homeostasis as Kyn has both positive and negative brain metabolites. As disease outcomes are tied to the severity of depression/anxiety, characterization of the behavioural and tryptophan metabolite profile of an IBD model can allow us to develop better IBD therapies. Purpose To characterize the anxiety and memory phenotype with corresponding assessment of tryptophan metabolites 5HT and Kyn across tissues of interest in an spontaneous colitis model. Method Mucin 2 knockout mice (Muc2-/-) lack the protective mucin 2 layer in the distal colon, allowing for direct contact between the gut contents and the gut epithelial layer. Over time this contact results in the development of spontaneous colitis. Muc2-/- n=12 per sex were compared to C57Bl/6 mice for behaviour and tryptophan metabolites across brain, colon and serum. Open Field Test and Light/Dark tests were used to assess anxiety levels, Novel Object and Radial Arm maze were performed to assess memory deficits. Mice underwent one anxiety and one memory test and were euthanized following their last maze. Enzyme-linked immunosorbent assay (ELISA) assessment of 5HT and Kyn were performed on brain, colon and serum samples. T-tests were performed and graphs created using PRISM (v9.4.1). Result(s) Muc2-/- mice had decreased anxiety (p=0.0113) compared to C57Bl/6 wild type mice in the light/dark maze. Muc2-/- displayed no significant differences in novel object preferences or the number of memory errors in novel object recognition test and radial arm maze respectively. Serum kynurenine was found to be significantly elevated (p=0.0444) in Muc2-/- mice. The sex factor has to be mentioned here in the results not in the conclusions. Conclusion(s) IBD is increasing globally and problems with mental health is often comorbid. Within our model we were able to show that behavioural and biochemical differences do exist between our Muc2-/-and wild-type mice with depressed anxiety responses and heightened serum kynurenine, however the implications of these findings require further investigation. Understanding how inflammation is driving behavioural alterations can provide pathways to ameliorate the behavioural comorbidities in IBD patients suffering from anxiety of memory deficits. Disclosure of Interest None Declared

Stiffness Restricts the Stemness of the Intestinal Stem Cells and Skews Their Differentiation Toward Goblet Cells

Fibrosis and tissue stiffening are hallmarks of inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs invivo. We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+-proliferating cells. Conversely, cells expressing the stem cell marker, olfactomedin-4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of olfactomedin-4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs toward goblet cells. Furthermore, analysis of colon samples from murine colitis models and patients with IBD demonstrated cellular and molecular remodeling reminiscent of those observed invitro. Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.

AMPK activation alleviated DSS-induced colitis by inhibiting ferroptosis.

Ferroptosis is a newly discovered cell death mode that has been confirmed to occur in ulcerative colitis (UC) intestinal epithelial cells. Therefore, it is particularly important to find key targets to block ferroptosis. Human colon samples and animal models were used in this study. The ferroptosis related protein in human colon tissue was detected by immunohistochemistry. The clinical symptoms, iron abundance and lipid peroxidation level of experimental mice were measured to evaluate the role of AMPK activation in reversing ferroptosis. We verified the expression difference of ferroptosis-related genes and proteins between UC patients and healthy people. Besides, we also confirmed the occurrence of colonic ferroptosis in a DSS-induced mouse model. Activation of AMPK inhibits ferroptosis in the colon, improves symptoms and prolongs lifespan in DSS-induced colitis mice. Collectively, our results illustrated that AMPK may be an important target for the inhibition of ferroptosis, which provides a potential therapeutic strategy for colitis.

Probiotic Supplementation Attenuates Chemotherapy-Induced Intestinal Mucositis in an Experimental Colorectal Cancer Liver Metastasis Rat Model

The use of chemotherapeutic agents is of paramount importance when treating colorectal cancer (CRC). Unfortunately, one of the most frequent chemotherapy (CTx) side effects is intestinal mucositis (IM), which may present with several clinical symptoms such as nausea, bloating, vomiting, pain, and diarrhea and even can result in life-threatening complications. There is a focused scientific effort towards developing new therapies to prevent and treat IM. The aim of this study was to assess the outcomes of probiotic supplementation on CTx-induced IM in a CRC liver metastasis rat model. Six-week-old male Wistar rats received either a multispecies probiotic or placebo mixture. On the 28th experiment day, rats received FOLFOX CTx, and afterwards, the severity of diarrhea was evaluated twice daily. Stool samples were collected for further microbiome analysis. Additionally, immunohistochemical stainings of ileum and colon samples with were performed with MPO, Ki67, and Caspase-3 antibodies. Probiotic supplementation alleviates the severity and length of CTx-induced diarrhea. Additionally, probiotics significantly reduced FOLFOX-induced weight and blood albumin loss. Furthermore, probiotic supplementation mitigated CTx-induced histological changes in the gut and promoted intestinal cell regeneration. This study shows that multispecies probiotic supplementation attenuates FOLFOX-induced IM symptoms by inhibiting apoptosis and promoting intestinal cell proliferation.