Search Results (174)

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation in which excessive cell death and the release of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1) amplify mucosal injury. Although necrosis—particularly regulated forms including necroptosis and ferroptosis—has emerged as a contributor to IBD pathogenesis, the therapeutic potential of targeting necrotic cell death remains incompletely explored. We investigated whether MIT-001 (previously known as NecroX-7), a mitochondria-targeted reactive oxygen species (ROS) scavenger with anti-necrotic activity, ameliorates intestinal inflammation in an acute dextran sulfate sodium (DSS)-induced colitis model. In vitro, MIT-001 reduced hydrogen peroxide-induced necrotic cell death in IEC-18 intestinal epithelial cells and was associated with a qualitative reduction in the 55-kDa cleaved poly(ADP-ribose) polymerase-1 (PARP-1) fragment (a marker of necrosis), with no apparent change in the apoptosis-related 89-kDa fragment. In vivo, oral administration of MIT-001 to C57BL/6 mice with DSS-induced colitis was associated with preservation of colon length, reduced histological injury, and a marked decrease in HMGB1-positive cells in colonic tissue. Among pro-inflammatory cytokines, IL-1β expression was significantly reduced, while IL-12, monocyte chemoattractant protein-1 (MCP-1), and TNF-α showed non-significant downward trends. These findings indicate that MIT-001 ameliorates DSS-induced colitis in association with reduced HMGB1 and IL-1β expression, supporting further investigation of mitochondria-targeted anti-necrotic strategies as a potential adjunctive approach in IBD. Full article

The colonic epithelial barrier is a multilayered defense system comprising the mucus layer, intestinal epithelial cells (IECs), and the underlying lamina propria. These components collectively maintain mucosal homeostasis and restrict microbial translocation. Disruption of this barrier is a hallmark of chronic intestinal inflammation particularly in IBDs, and is primarily driven by pro-inflammatory cytokines, such as TNF-α, IFN-γ, IL-1β, and IL-6. TNF-α and IFN-γ synergistically induce epithelial cell apoptosis and tight junction disassembly through mechanisms involving TNFR2 upregulation, myosin light chain kinase (MLCK) activation, and adherens junction destabilization. IL-1β amplifies paracellular permeability via NF-κB-dependent MLCK induction and OCLN downregulation, while IL-6 promotes barrier leakiness by upregulating CLDN-2 and sustaining self-reinforcing inflammatory loops that maintain chronic inflammation and impede epithelial repair. This leads to persistent immune-cell infiltration, chronic tight junction remodeling, and failure of barrier replenishment. Consequently, leaky colon facilitates microbial and antigen translocation into the lamina propria, further activating immune cells and perpetuating pro-inflammatory signaling. This review synthesizes current evidence and studies on the cooperative and self-reinforcing roles of pro-inflammatory cytokines, providing insight into the mechanisms underlying chronic intestinal barrier dysfunction and highlighting the need for therapeutic strategies that simultaneously target multiple inflammatory axes to restore barrier integrity in inflammatory bowel disorders. Full article

Inflammatory bowel diseases (IBDs) are diseases of chronic inflammation and intestinal epithelial cell (IEC) death that affect an estimated 7 million people worldwide. Intestinal barrier restoration is the most important determinant of remission in IBD, yet there are very few existing therapies that protect IECs from damage or support epithelial repair. The goal of this study was to develop a model system and tools that can be used to identify therapeutics that promote IEC survival in IBD. We developed a Beclin-1 cleavage reporter (BICR) that detects calpain-mediated Beclin-1 cleavage and the switch from autophagy to programmed cell death. We modified BICR with the HIV Tat peptide (BICR-Tat) and tested it in a model of live bacterial stress using commensal E. coli and IEC. BICR sensitively and specifically detected calpain activity in cell-free assays, and BICR-Tat successfully detected Beclin-1 cleavage and autophagy failure in IEC. Achieving IEC survival in the microbe-challenged IBD gut would be an important advance toward intestinal barrier restoration in this intractable disease. The BICR-Tat reporter coupled with the model of microbial stress developed in this study could enable high-throughput screening approaches to identify therapeutics with the potential to achieve barrier healing and sustained remission in IBD. Full article

Physiologically relevant in vitro intestinal models are essential for studying key physiological processes, including barrier function, drug screening and gut-microbiota interactions. However, conventional 2D culture systems often fail to recapitulate structural and functional complexity. Here, we aimed to validate a 3D bioelectronic transmembrane platform, previously used for monitoring human intestinal epithelium and vascular endothelium, for modeling the rat small intestinal barrier in vitro. The device integrates a poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) scaffold supporting co-cultures of rat intestinal epithelial cells (IEC-6) and rat fibroblasts (208F), enabling real-time monitoring of barrier formation through electrical measurements using electrochemical impedance spectroscopy (EIS). Barrier formation was monitored over 21 days and exhibited a time-dependent increase in barrier resistance. The 3D platform was compared with traditional 2D insert-based cultures and ex vivo rat tissue using an Ethylene Glycol Tetraacetic Acid (EGTA)-induced calcium switch assay to evaluate barrier disruption and recovery. EGTA treatment and removal induced reversible barrier disruption in the 3D in vitro and ex vivo models, whereas 2D in vitro cultures showed limited recovery. These findings demonstrate that the 3D platform more faithfully recapitulates native tissue architecture and function, closely paralleling ex vivo responses. Our study highlights the importance of validating advanced 3D in vitro models and establishes this bioelectronic platform as a robust tool for drug screening, barrier studies, and preclinical gastrointestinal research. Full article

Beta-2-microglobulin (B2M) is a key component protein in the processing and presentation of major histocompatibility complex (MHC)-I antigens and plays an important role in the immune system regulation. Previous studies have shown that B2M is significantly overexpressed in the intestinal tissues of sheep that are resistant to E. coli F17b infection (defined by milder clinical symptoms post-challenge) compared to those that are susceptible (exhibiting severe diarrhea). Based on this finding, this study aimed to investigate whether B2M influences the adhesion of E. coli F17b to sheep intestinal epithelial cells (IECs) and to assess its role in regulating IEC proliferation and migration. We tested this by overexpressing and knocking down B2M in IECs, and then measured bacterial adhesion through colony counts and fimbrial gene expression (RT-qPCR). Moreover, cell health was assessed using proliferation (CCK-8 and EdU) and migration (scratch) assays. The results showed that upregulation of B2M expression inhibited E. coli F17b adhesion and promoted IEC proliferation and migration. Silencing B2M increased bacterial adhesion and impaired cell function. In summary, B2M helps protect sheep IECs from E. coli F17b by strengthening the epithelial barrier through improved cell growth, proliferation, and migration. These findings elucidate part of the host defense mechanism against E. coli F17b, providing a basis for further research. Full article

This work conducted a transcriptome analysis of canine intestinal epithelial cells (cIECs) treated with nicotinamide mononucleotide (NMN), a physiologically active nucleotide with a pyridine base known for its anti-aging and anti-inflammatory effects. In our experiment, cIECs were cultured and segregated into a control group (Ctrl) and an NMN-treated group. The finding demonstrated that NMN significantly affects cell proliferation in cIECs in comparison to the Ctrl. The transcriptome analysis indicated a high enrichment of genes associated with the cell cycle, proliferation, cellular senescence, and inflammatory pathways in NMN-treated cIECs, showing that NMN has the capacity to modify these biological processes. Compared to the Ctrl group, NMN treatment significantly increased ATP, SOD, CAT and GSH levels and decreased the activities of ROS and MDA. NMN treatment also significantly increased the activity of the relative complex I, III and V enzymes compared to the Ctrl group. Furthermore, the expression of MAPK13, EDN1, TNFAIP6, TNFSF15 and SLC7A11 were decreased significantly, while ACOX2, CPT1C, CCNA1 and CCNE1 were increased significantly in NMN-5μM treatment compared to Ctrl. NMN-treated significantly decreased the expression of Hdac2, Hdac6 and Hdac8, while increasing the expression of Kdm5a, Kdm5b and Kdm5c compared to the Ctrl group. Additionally, ChIP-qPCR use discovered that NMN-treatment significantly downregulated the enrichment of EDN-1 at target loci of NR4A1, SRC1, P300, Pol II and Ser5- Pol II compared to the Ctrl group. Expression of the NR4A1 gene suggests that its exert in biological activities by inhibiting inflammatory responses and anti-aging pathways. Then, we detected the transcriptional activation linked histone markers and found that H3K23ac and H3K27ac were significantly downregulated, while H3K27me3 was significantly upregulated in the NMN-treatment compared to the Ctrl group. We conclude that NMN regulates EDN-1 expression in cIECs through mechanisms involving NR4A1 and histone modifications, highlighting its potential role in canine intestinal health. Full article

The purpose of this study was to characterise the effect of cranberry (Vaccinium macrocarpon) juice on unicellular and multicellular systems, specifically food spoilage yeasts (Wickerhamomyces anomalus, Dekkera bruxellensis and Rhodotorula mucilaginosa) and intestinal cells (IEC-6 and Caco-2 cells). The effects of both raw cranberry juice and juice digested in vitro were investigated. The juices were analysed for polyphenol content using high-performance liquid chromatography coupled with mass spectrometry. The cranberry juice was evaluated for its impact on yeast surface hydrophobicity and anti-adhesive action using the MATH test and luminometry/microscopy, respectively. We also assessed the effects of raw and digested cranberry juices on IEC-6 and Caco-2 cells by measuring cell viability, metabolic modulation, genotoxicity, and antioxidant activity. Chromatographic analysis of the raw cranberry juice revealed the presence of diverse bioactive compounds, identified as hydroxybenzoic and hydroxycinnamic acids, flavonols, and anthocyanins. After digestion, the cranberry juice remained a rich source of phenolic acids. The yeast strain R. mucilaginosa was characterised by the highest hydrophobicity and adhesive abilities, but cell adhesion in the presence of raw cranberry juice was several times lower for all the tested strains. Both tested cranberry juices reduced ROS levels and were well tolerated by intestinal epithelial cells, without significant cytotoxic or genotoxic effects. Our findings provide new insights into the safety of using cranberry juice across unicellular and multicellular systems. However, further validation in real-world settings is necessary before practical applications. Full article

Intestinal impairment plays a pivotal role in many chronic conditions, including chronic kidney disease (CKD), a progressive disorder affecting over 800 million people worldwide. CKD does not only affect the kidney, but it is recognized as a systemic condition characterized by chronic low-grade inflammation, that contributes to disease progression and associated complications. The intestine is one of the major sources of CKD-associated inflammation, also due to the production and accumulation of some uremic toxins, normally excreted by healthy kidneys, such as indoxyl sulfate (IS). IS is a pro-inflammatory and pro-oxidant protein-bound uremic toxin that increases intestinal epithelial permeability, promotes microbial translocation, and enhances inflammatory and oxidative responses. Although IS-induced intestinal damage has been documented, the underlying molecular mechanisms and effective therapeutic strategies to counteract its effects remain to be elucidated. Against this backdrop in the present study, we investigated the impact of plumericin, an iridoid spironolactone, on IS-induced intestinal impairment using IEC-6, an intestinal epithelial cells model. In IS-treated IEC-6, plumericin reduces apoptosis, inhibits inflammation and oxidative stress, and restores epithelial wound repair. In these conditions plumericin also promotes Nrf-2 and inhibits NF-kB and AhR activation induced by IS. Moreover, the same inhibitory effect of plumericin on inflammation and oxidative stress and in promoting wound repair is also observed in the presence of IS and pro-inflammatory stimuli, as occurs in CKD considering the associated systemic low-grade inflammation. These findings suggest that plumericin may represent a promising therapeutic candidate for intestinal impairment in CKD acting with an integrated mechanism. Full article

(1) Background: Humans and animals are exposed daily to numerous food-associated noxious molecules, including fungal toxins or mycotoxins. Effects of mycotoxins on the intestinal epithelial cells (IECs) are well characterized. However, their impact on the enteric nervous system (ENS), particularly on enteric glial cells (EGCs), has not been evaluated. (2) Methods: In the present work, the impact of major mycotoxins (eighteen mycotoxins in total, both regulated and non-regulated (including emerging ones) mycotoxins) on EGCs was evaluated in vitro in terms of antiproliferative and cytotoxic effects using rat EGCs as a model. Inhibitory concentrations on cell division and cell viability were determined using the resazurin assay, and biochemical analysis was performed to identify the mechanism(s) of action involved. (3) Results: Of the eighteen mycotoxins tested, twelve were found to be toxic; apicidin, deoxynivalenol, and cyclohexadepsipeptide mycotoxins (enniatins and beauvericin) were the most toxic, with active concentrations as low as 0.19 ± 0.07 µM for deoxynivalenol. Mechanistic studies revealed that toxicity occurs through the induction of oxidative stress, alteration of the membrane integrity, and/or induction of apoptosis. (4) Conclusions: As far as we know, the data presented here show for the first time that EGCs are targets of foodborne mycotoxins, even at low concentrations potentially achieved in cases of ingesting contaminated food. Full article

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in pigs. Virulence factors, such as colonization factors and enterotoxins, bind to specific receptors on intestinal epithelial cells (IECs), impairing the integrity of the IEC barrier by inducing cell death. Pyroptosis is a newly discovered form of programmed cell death (PCD), which is widely involved in the pathogenesis of multiple infectious gastrointestinal diseases. However, it is still unclear whether pyroptosis contributes to the ETEC-mediated damage of IECs. This study demonstrated that ETEC infection activated NLRP3 inflammasome and triggered gasdermin D (GSDMD)-executed pyroptosis of mouse IECs in vitro and in vivo. Mechanistically, ETEC infection triggered endoplasmic reticulum (ER) stress response to increase the expression of thioredoxin-interacting protein (TXNIP) by upregulation of C/EBP homologous protein (CHOP), which subsequently activated NLRP3 inflammasome. Removal of ER stress by tauroursodeoxycholic acid (TUDCA) alleviated the pyroptosis of IECs that was caused by ETEC infection. In addition, the induced ER stress impaired mitochondria and led to mitochondrial reactive oxygen species (mtROS) overproduction and cytosolic release of mitochondrial DNA (mtDNA), which activated STING, another factor that contributed to ETEC-triggered pyroptosis. Chemical inhibition of STING attenuated ETEC-induced pyroptosis of IECs. Collectively, this study demonstrated that the activation of the STING/ER stress/mitochondrial impairment/NLRP3 inflammasome axis is a critical pathway in the ETEC infection-derived pyroptosis of IECs. Hence, targeting ER stress response may serve as a promising therapeutic strategy to prevent ETEC infection caused damage to IECs. Full article

Colorectal cancer (CRC) is the fourth most common and the third mostly deadly cancer globally. Even with alternative therapies, some patients do not respond to treatment. Identifying modulations in the tumor microenvironment (TME) of CRC is a significant challenge due to the complex and dynamic nature of the TME. The intestinal epithelium comprises different types of secretory lineage cells, including goblet, tuft, Paneth, and enteroendocrine cells (EECs). Yet the relevance of each subtype of secretory intestinal epithelial cell (IEC) within the TME is still debated. This study investigated the involvement of IECs in CRC development through an integrative bioinformatics analysis. We used publicly available datasets from the National Center for Biotechnology Information, the Cancer Genome Atlas Program, and the National Cancer Institute’s Proteomics Tumor Analysis Consortium, encompassing both human and mouse CRC samples. Our findings reveal a CRC microenvironment characterized by elevated expression levels of genes associated with WNT pathway activity. Remarkably, there was increased expression of Paneth cell-associated markers and transcription factors, such as WISP1, LYZ, SOX9, and DEFA1. Conversely, EEC-specific gene markers, such as GCG (encoding glucagon-like peptide-1) and CHGA exhibited significant downregulation in CRC tissue compared with healthy tissue, partially due to Paneth cell activity. Gene ontology analysis showed species-conserved downregulation in hormone/peptide secretion-related pathways in both mouse and human CRC. Of note, lower levels of GCG and CHGA correlated with reduced overall survival and demonstrated a correlation with the cell cycle, apoptosis, and proliferation. These results suggest that the disruption of enteroendocrine cell signaling is a hallmark of CRC development and may hold prognostic and therapeutic value in treating CRC patients. Full article

Long-duration space missions expose astronauts to galactic cosmic radiation (GCR), a complex spectrum of high-charge, high-energy (HZE) ions that pose significant risks of chronic tissue injury. To model these effects, we examined intestinal outcomes in wild-type mice 5 months after low-dose (50 cGy) 33-ion mixed-field GCR simulation (GCRsim). GCRsim induced sustained DNA double-strand breaks (DSBs) and oxidative stress, as shown by elevated γH2AX foci and 4-HNE staining. Intestinal epithelial cells (IECs) exhibited pronounced senescence, marked by increased SA-β-gal activity, p16 upregulation, LaminB1 loss, and induction of senescence-associated secretory phenotype (SASP) cytokines (Cxcl10, IL-6, IL-1β, Icam1). GCRsim also elevated circulating LINE-1 DNA and reduced expression of DNA-degrading nucleases (DNase2, TREX1), indicating impaired extracellular DNA clearance. Targeted molecular study revealed persistent activation of the cGAS–STING pathway, with elevated cGAS, STING, pTBK1, pIKKα/β, and nuclear pIRF3, pIRF7, and p65, consistent with chronic innate immune signaling. Functionally, GCRsim altered nutrient absorption gene expression—upregulating glucose transporters (Slc2a2, Slc2a5, Slc5a1) and gut hormones (Cck, Gip), while downregulating cholesterol/fat transporters (Npc1, Npc1l1). Biochemical markers supported intestinal injury, with decreased serum citrulline and increased intestinal fatty acid-binding protein (I-FABP), indicating barrier compromise. Collectively, these findings demonstrate that GCRsim drives sustained intestinal dysfunction, highlighting the need for countermeasures to protect GI health during deep-space missions. Full article

Host physical, chemical, and immune responses constitute well-established defences against bacterial invasion. Recent studies have highlighted the critical role of cellular mechanisms, particularly the production of reactive oxygen species (ROS) in antibacterial defence. This review focuses on ROS generation by mammalian intestinal epithelial cells (IECs) and investigates whether ROS production is host-driven to eliminate bacteria or manipulated by bacteria to suppress or exploit ROS for enhanced internalisation. We examine the activation mechanisms of the NADPH oxidase (NOX) enzyme complex and the resulting ROS production in IECs, which, unlike professional phagocytes, lack the ability to engulf bacteria. The downstream effects of NOX-mediated ROS signalling are discussed in detail. Additionally, we explore the dynamic interplay between host and pathogen, with particular attention to how bacterial infection may disrupt or hijack host NOX-mediated ROS responses. The review concludes with key experimental considerations and outlines future directions in this evolving field. Overall, we present ROS as a double-edged sword, an essential antimicrobial effector that is also susceptible to bacterial subversion, highlighting its potential as a target in novel antimicrobial strategies. Full article

Intestinal fibrosis is a common complication of inflammatory bowel diseases (IBD), and, to date, effective and safe antifibrotic drugs are still lacking. Emerging evidence suggests that probiotics may provide novel strategies to counteract fibrotic processes. In this study, we evaluated the anti-fibrotic potential of a multi-strain probiotic formulation, OxxySlab^TM, using in vitro models of intestinal fibrosis and epithelial-to-mesenchymal transition (EMT). Human intestinal fibroblasts (CCD-18Co cell line) and epithelial cells (Caco-2 cell line, IECs) were stimulated with transforming growth factor-β1 (TGF-β1) to induce fibrotic and EMT phenotypes, respectively. Treatment with OxxySlab modulated cell proliferation and fibrosis-related markers, which we assessed through CCK-8 assay, Western blotting, and immunofluorescence. The probiotic lysate inhibited both canonical and non-canonical TGF-β1 signaling pathways, and it also reduced TGF-β1 gene expression in activated myofibroblasts, as shown by RT-qPCR. Furthermore, probiotic treatment reversed EMT features by restoring epithelial markers and downregulating mesenchymal markers. These findings highlight the beneficial effects of the multi-strain probiotic formulation as an adjunctive therapeutic agent targeting key pathways involved in intestinal fibrosis. Full article

Obesity is a complex chronic inflammatory condition that results from excess fat accumulation. It increases the risk of developing numerous co-morbidities such as Type 2 diabetes mellitus, cardiovascular disease, hypertension, and stroke. The adipose tissue is itself a vital endocrine organ that secretes numerous adipokines, cytokines, and exosomes, which are collectively known as the adipose-derived secretome (ADS). This ADS has been shown to influence and modulate many physiological processes. During obesity, the composition of ADS is altered, which may contribute to the development of obesity-associated diseases. Type-2 diabetes mellitus is one of the most common complications of obesity due to alterations in glucose homeostasis. Glucose absorption occurs via Na-glucose co-transport via SGLT1 at the brush border membrane (BBM) of small intestinal villus cells. This process of transepithelial glucose uptake is the primary method of glucose absorption from diet. However, how ADS mediates the function of SGLT1 is not yet known. This study aims to determine the mechanism of regulation of SGLT1 by ADS in intestinal epithelial cells. We show that ADS from OZR (but not LZR) stimulates SGLT1 in IEC-18 cells. OZR-ADS treatment diminished Na/K-ATPase activity in IEC-18 cells. Kinetic studies indicated that the mechanism of stimulation for SGLT1 during OZR-ADS treatment was secondary to an increase in the affinity (1/K_m) of the co-transporter for glucose without a change in co-transporter number. Western blot studies revealed that SGLT1 protein expression was unaltered in the two groups, confirming our kinetic studies. Immunoprecipitation demonstrated that an increase in the affinity of the SGLT1 protein was mediated by altered phosphorylation. In conclusion, during obesity, the adipose tissue secretome stimulates SGLT1 in intestinal epithelial cells, leading to an increase in affinity for glucose. The affinity change is due to alterations in SGLT1 phosphorylation. Together, these results may provide important insight into the mechanisms underlying altered glucose homeostasis in obesity and how this may lead to the development of Type 2 diabetes mellitus. Full article