Response Of Caco-2 Cells Research Articles

P0055 Impact of Indole-3-Propionic Acid on Bacterial Invasion Potential of Klebsiella Strains Isolated from Pediatric Ulcerative Colitis Patients

Abstract Background Indole-3-propionic acid (IPA), a tryptophan metabolite produced by microbes, has demonstrated beneficial effects, including reversing dysbiosis, balancing bacterial populations, and reducing inflammation.1,2,3 However, the mechanisms behind these effects and their impact on pathogenic microbes remain unclear. This study aimed to evaluate the effects of IPA on bacterial invasion in vitro, using potential pathobionts (Klebsiella variicola and Klebsiella quasipneumoniae) isolated from non-inflamed sections of the colon in pediatric ulcerative colitis (UC) patients. We hypothesized that IPA could reduce virulence traits, promoting a more homeostatic environment, which could lower inflammation in pediatric UC patients. Methods Caco-2 cells (colonic epithelial cell line) were infected with Klebsiella strains, with or without 0.5 mM IPA. Bacterial invasion was assessed via gentamicin protection assay, and qPCR measured Interleukin-8, IL-10, and TNF-α expression in infected cells. Transepithelial electrical resistance (TEER) assay evaluated barrier integrity, and qPCR analyzed the expression of bacterial virulence genes, including entB (siderophores), manC (capsule), and hcp1 (type VI secretion) in IPA-treated and untreated cultures. Additionally, siderophore production was quantified using a Chrome-Azurol Sulfonate (CAS) assay, and Anthony’s stain visualized the bacterial capsule. Results IPA-treated Caco-2 cells showed significantly reduced bacterial invasion, with further reductions when bacteria were also exposed to IPA. qPCR revealed a significant decrease in IL-8 expression in IPA-treated cells during infection. IL-10 levels decreased non-significantly, and TNF-α expression showed variable results. A trend toward improved tight junction integrity was observed in the TEER assay with IPA treatment, although no significant differences were detected. IPA treatment increased the expression of entB, manC, and hcp1 genes, except for K. variicola, where manC expression remained unchanged. IPA significantly reduced the capsule thickness of K. variicola and resulted in a significant reduction in K. quasipneumoniae siderophore production. Conclusion IPA reduces bacterial invasion and modulates immune responses in Caco-2 cells infected with Klebsiella pathobiont strains. It reduces the virulence of K. quasipneumoniae by targeting siderophore production and diminishes the virulence of K. variicola by reducing capsule formation, suggesting its potential as an anti-virulence agent against these pathobionts. These findings highlight IPA’s potential as an immunomodulatory agent for managing IBD. However, further research is needed into other Klebsiella virulence pathways and host-microbiota interactions.

Influences of Growth-Related Myopathies on Peptide Patterns of In Vitro Digested Cooked Chicken Breast and Stress-Related Responses in an Intestinal Caco-2 Cell Model.

The objective of this study was to determine the effects of growth-related myopathies, i.e., normal, wooden breast (WB), white striping (WS), and the combined lesions of WS and WB (WS + WB), on the molecular response of Caco-2 cells. A total of 24 cooked chicken breasts (n = 6 per myopathy) was subjected to an in vitro digestion using an enzymatic process mimicking human gastrointestinal digestion. Based on peptidomics, in vitro protein digestion of the abnormal samples, particularly WB meat, resulted in more peptides with lower molecular mass relative to those of normal samples. The cooked meat hydrolysates obtained at the end of the digestion were applied to a Caco-2 cell model for 4 h. The cell viability of treated normal and abnormal samples was not different (p ≥ 0.05). Absolute transcript abundances of genes associated with primary oxidative stress response, including nuclear factor erythroid 2 like 2, superoxide dismutase, and hypoxia-inducible factor 1 were determined using a droplet digital polymerase chain reaction. No significant differences in transcript abundance of those genes in Caco-2 cells were demonstrated between normal and the abnormal samples (p ≥ 0.05). Overall, the findings supported that, compared to normal meat, the cooked chicken meat with growth-related myopathies might be digested and absorbed to a greater extent. The cooked abnormal meat did not exert significant transcriptional impacts regarding oxidative stress on the human epithelial Caco-2 cells.

Sea Cucumber Viscera Processed by Protease Hydrolysis Combined with Cordyceps militaris Fermentation Protect Caco-2 Cells against Oxidative Damage via Enhancing Antioxidant Capacity, Activating Nrf2/HO-1 Pathway and Improving Cell Metabolism.

Excessive reactive oxygen species (ROS) may lead to oxidative damage and metabolic disorder. The pathogenesis of human bowel inflammation is closely related to oxidative damage of intestinal epithelial cells caused by ROS. This study aimed to explore the high-value utilization of the byproducts of sea cucumber in antioxidant food for colitis prevention. The technology of protease hydrolysis combined with Cordyceps militaris fermentation was used to obtain fermented sea cucumber viscera protease hydrolysates (FSVHs). The results revealed that FSVH could enhance antioxidant capacity and alleviate oxidative damage and apoptosis by activating the Nrf2/HO-1 pathway and triggering the self-protection immune mechanisms. Moreover, the FSVH supplementation could upregulate antioxidant-related metabolic pathways of Caco-2 cells such as glutathione metabolism, confirming the enhanced antioxidant capacity of damaged cells. In summary, FSVH could exert protective effects on Caco-2 cells in response to oxidative damage, providing a promising prospect for sea cucumber resource utilization and colitis prevention.

Role of ethanolamine utilization and bacterial microcompartment formation in Listeria monocytogenes intracellular infection.

Ethanolamine (EA) affects the colonization and pathogenicity of certain human bacterial pathogens in the gastrointestinal tract. However, EA can also affect the intracellular survival and replication of host cell invasive bacteria such as Listeria monocytogenes (LMO) and Salmonella enterica serovar Typhimurium (S. Typhimurium). The EA utilization (eut) genes can be categorized as regulatory, enzymatic, or structural, and previous work in LMO showed that loss of genes encoding functions for the enzymatic breakdown of EA inhibited LMO intracellular replication. In this work, we sought to further characterize the role of EA utilization during LMO infection of host cells. Unlike what was previously observed for S. Typhimurium, in LMO, an EA regulator mutant (ΔeutV) was equally deficient in intracellular replication compared to an EA metabolism mutant (ΔeutB), and this was consistent across Caco-2, RAW 264.7, and THP-1 cell lines. The structural genes encode proteins that self-assemble into bacterial microcompartments (BMCs) that encase the enzymes necessary for EA metabolism. For the first time, native EUT BMCs were fluorescently tagged, and EUT BMC formation was observed in vitro and in vivo. Interestingly, BMC formation was observed in bacteria infecting Caco-2 cells, but not the macrophage cell lines. Finally, the cellular immune response of Caco-2 cells to infection with eut mutants was examined, and it was discovered that ΔeutB and ΔeutV mutants similarly elevated the expression of inflammatory cytokines. In conclusion, EA sensing and utilization during LMO intracellular infection are important for optimal LMO replication and immune evasion but are not always concomitant with BMC formation.IMPORTANCEListeria monocytogenes (LMO) is a bacterial pathogen that can cause severe disease in immunocompromised individuals when consumed in contaminated food. It can replicate inside of mammalian cells, escaping detection by the immune system. Therefore, understanding the features of this human pathogen that contribute to its infectiousness and intracellular lifestyle is important. In this work we demonstrate that genes encoding both regulators and enzymes of EA metabolism are important for optimal growth inside mammalian cells. Moreover, the formation of specialized compartments to enable EA metabolism were visualized by tagging with a fluorescent protein and found to form when LMO infects some mammalian cell types, but not others. Interestingly, the formation of the compartments was associated with features consistent with an early stage of the intracellular infection. By characterizing bacterial metabolic pathways that contribute to survival in host environments, we hope to positively impact knowledge and facilitate new treatment strategies.

Niaodukang mixture inhibits micro-inflammation in CKD rats by enhancing MiR-146a levels in enterogenous exosomes

Ethnopharmacological relevanceThe Niaodukang mixture (NDK) is a preparation known for its ability to lower serum creatine levels in individuals with chronic kidney disease (CKD) and is commonly administered at medical facilities like the Zhongshan Hospital of Traditional Chinese Medicine. The initial use of NDK was mainly to treat CKD. Our hospital frequently utilizes NDK, which consists of Rheum officinaleBaill., Salvia miltiorrhiza Bunge., Astragalus aaronii (Eig) Zohary., Carthamus tinctorius L., and Sanguisorba officinalis L., for treating patients with CKD-MBD. It has the effects of eliminating dampness and turbidity and dredging kidney collaterals. However, The impact and process of NDK in chronic kidney disease remain unknown. Aim of the studyTo determine whether microRNA-146a (miR-146a) is associated with CKD micro-inflammationand whether NDK protects against CKD micro-inflammation by modulating the miR-146a/nuclear factor kappa-B (NF-κB) signaling pathway. Materials and methods(1) An adenine-induced rat model of chronic kidney disease was created through the use of materials and methods. The levels of miR-146a in exosomes from plasma and ileum were determined by RT-PCR. (2) Human cloned colon adenocarcinoma (Caco-2)cellswere stimulated with lipopolysaccharide (LPS)and transfected with miR-146a mimic and inhibitor. Following that, the Western blot and RT-PCR techniques were used to measure the protein and mRNA quantities of Toll-like receptor 4 (TLR4), NF-κB, and TNF receptor-associated factor 6 (TRAF6). (3) Enzyme-linked immunosorbent assay (ELISA) was used to identify serum levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α). (4) Plasma exosomes were extracted, and the exosomes in intestinal tissues were extracted via ultrahigh-speed centrifugation.Negative staining electron microscopy was used to analyze the morphology of exosomes and the ultrastructure of intestinal tissue and exosomes. The particle size of the exosomes was measured using nanoparticle tracking analysis. ResultsThe pathological characteristics of CKD rats included those associated with systemic micro-inflammation, which may be associated with the release of exosomes in intestinal tissue. NDK suppressed the inflammatory response in Caco-2 cells and decreased the levels of IL-1β, IL-6, and TNF-α in rats with CKD. The expression of miR-146a, which regulates inflammation, differed between plasma-derived and enterogenous exosomes in CKD rats, which may be due to stimulation of ileal exosome release into the blood. NDK effectively reduced the levels of TRAF6, NF-κB, and TLR4 in the ileum tissue of CKD rats. ConclusionNDK can effectively improve micro-inflammation in CKD ratsby enhancing the release of enterogenous exosomes, thereby enhancing the release of exosome-associated miR-146a and inhibiting micro-inflammation.

Bacillus cereus CwpFM induces colonic tissue damage and inflammatory responses through oxidative stress and the NLRP3/NF-κB pathway

Bacillus cereus (B. cereus) from cow milk poses a threat to public health, causing food poisoning and gastrointestinal disorders in humans. We identified CwpFM, an enterotoxin from B. cereus, caused oxidative stress and inflammatory responses in mouse colon and colonic epithelial cells. Colon proteomics revealed that CwpFM elevated proteins associated with inflammation and oxidative stress. Notably, CwpFM induced activation of the NLRP3/NF-κB signaling, but suppressed antioxidant NFE2L2/HO-1 expression in the intestine and epithelial cells. Consistently, CwpFM exposure led to cytotoxicity and ROS accumulation in Caco-2 cells in a dose-dependent manner. Further, NAC (ROS inhibitor) treatment abolished NLRP3/NF-κB activation due to CwpFM. Moreover, overexpression of Nfe2l2 or activation of NFE2L2 by NK-252 reduced ROS production and inhibited activation of the NLRP3/NF-κB pathway. Inhibition of NF-κB by ADPC and/or suppression of NLRP3 by MCC950 attenuated CwpFM-induced inflammatory responses in Caco-2 cells. Collectively, CwpFM induced oxidative stress and NLRP3/NF-κB activation by inhibiting the NFE2L2/HO-1 signaling and ROS accumulation, leading to the development of intestinal inflammation. Our data elucidate the role of oxidative stress and innate immunity in CwpFM enterotoxicity and contribute to developing diagnostic and therapeutic products for B. cereus-related food safety issues.

Gingerols synergize with anthocyanins to induce antioxidant activity in vitro.

Oxidative stress caused by free radicals contributes to the pathogenesis of multiple chronic health conditions. Phytochemicals protect against oxidative stress; however, low bioavailability from dietary sources limits their health benefits. This study aimed to assess the effects of anthocyanins and gingerols' combination on the cellular antioxidant response of Caco-2 cells against oxidative stress. A strong synergism was observed for anthocyanin-gingerol (Ac-G) w/w combined ratios of 8:1 and 2:1 (dosages of (1 + 0.125) and (1 + 0.5) μg/mL) in the cellular antioxidant activity (CAA) and cytoprotective effects, with synergistic effect indicator (SE) values of 1.41 and 1.61, respectively. The synergism of Ac-G combinations promoted cellular antioxidant defense systems and cytoprotective effects by reducing the induced GPx enzyme activity, protecting SOD enzyme activity, reducing cellular ROS generation, increasing glutathione content, and inhibiting lipid peroxidation. Thus, Ac-G combinations showed potential in supporting the endogenous antioxidant systems to protect cells from oxidation and restore physiological redox status. The Ac-G formulation is a promising healthy option that can be developed into functional foods or nutraceutical products. Furthermore, it could help address the low bioavailability of these phenolics, as higher effects were achieved when combining the same doses.

LncRNA-CBR3-AS1 promotes and enhances the malignancy of ulcerative colitis via targeting miRNA-145-5p/FN1.

Growing evidence suggested that long non-coding RNA (lncRNA) played a crucial role in the progression of ulcerative colitis (UC). Therefore, the purpose of this study is to understand how the lncRNA CBR3-AS1, which has been found to be up-regulated in UC, contributes to the bio-progression of the disease. To determine the concentration and relationship of the lncRNA CBR3-AS1, miRNA-145-5p, and FN1 in the LPS-induced Caco-2 model cells, qRT-PCR was employed in this study. Starbase was used to predict the target sites of the lncRNA CBR3-AS1 and the miRNA-145-5p, and Targetscan was used to predict the probable linking points of the FN1 and the miRNA-145-5p, which was confirmed by a twofold luciferase reporter test. The vitality of Caco-2 cells was determined using the CCK-8 and FCM tests. Using the ELISA kit, TNF, IFN, IL-6, and IL-17 were identified. The results of the experiment show that in Caco-2 cells treated with 10 ng/mL LPS, LncRNA CBR3-AS1 was up-regulated. Additionally, Caco-2 cells' LPS-induced apoptosis and inflammatory response were inhibited by lncRNA CBR3-AS1 inhibition. Dual-luciferase reporter experiments demonstrated that miRNA-145-5p and lncRNA CBR3-AS1 might connect. Moreover, miRNA-145-5p, which was shown to be poorly expressed in UC, was found to suppress inflammatory and apoptotic responses in Caco-2 cells activated by LPS. It's significant that FN1 was confirmed to be miRNA-145-5p's downstream target. Sh-CBR3-AS1's inhibitory effects were reversed by miRNA-145-5p knockdown, and the effects of the miRNA-145-5p inhibitor were reversed by sh-FN1. In conclusion, LncRNA CBR3-AS1 may offer a unique method for treating UC by suppressing the function of miRNA-145-5p, which is implicated in the development of UC.

Maternal Epidermal Growth Factor Promotes Neonatal Claudin-2 Dependent Increases in Small Intestinal Calcium Permeability.

A higher concentration of calcium in breast milk than blood favors paracellular calcium absorption enabling growth during postnatal development. We aimed to determine whether suckling animals have greater intestinal calcium permeability to maximize absorption and to identify the underlying molecular mechanism. We examined intestinal claudin expression at different ages in mice and in human intestinal epithelial (Caco-2) cells in response to hormones or human milk. We also measured intestinal calcium permeability in wildtype, Cldn2 and Cldn12 KO mice and Caco-2 cells in response to hormones or human milk. Bone mineralization in mice was assessed by μCT. Calcium permeability across the jejunum and ileum of mice were 2-fold greater at 2 wk than 2 mo postnatal age. At 2 wk, Cldn2 and Cldn12 expression were greater, but only Cldn2 KO mice had decreased calcium permeability compared to wildtype. This translated to decreased bone volume, cross-sectional thickness, and tissue mineral density of femurs. Weaning from breast milk led to a 50% decrease in Cldn2 expression in the jejunum and ileum. Epidermal growth factor (EGF) in breast milk specifically increased only CLDN2 expression and calcium permeability in Caco-2 cells. These data support intestinal permeability to calcium, conferred by claudin-2, being greater in suckling mice and being driven by EGF in breast milk. Loss of the CLDN2 pathway leads to suboptimal bone mineralization at 2 wk of life. Overall, EGF-mediated control of intestinal claudin-2 expression contributes to maximal intestinal calcium absorption in suckling animals.

The Gliadin Hydrolysis Capacity of B. longum, L. acidophilus, and L. plantarum and Their Protective Effects on Caco-2 Cells against Gliadin-Induced Inflammatory Responses.

Non-celiac wheat sensitivity (NCWS) is a poorly understood gluten-related disorder (GRD) and its prominent symptoms can be ameliorated by gluten avoidance. This study aimed to determine the effectiveness of a probiotic mixture in hydrolyzing gliadin peptides (toxic components of gluten) and suppressing gliadin-induced inflammatory responses in Caco-2 cells. Wheat dough was fermented with a probiotic mix for 0, 2, 4, and 6 h. The effect of the probiotic mix on gliadin degradation was monitored by SDS-PAGE. The expression levels of IL-6, IL-17A, INF-γ, IL-10, and TGF-β were evaluated using ELISA and qRT-PCR methods. According to our findings, fermenting wheat dough with a mix of B. longum, L. acidophilus, and L. plantarum for 6 h was effective in gliadin degradation. This process also reduced levels of IL-6 (p = 0.004), IL-17A (p = 0.004), and IFN-γ (p = 0.01) mRNA, as well as decreased IL-6 (p = 0.006) and IFN-γ (p = 0.0009) protein secretion. 4 h fermentation led to a significant decrease in IL-17A (p = 0.001) and IFN-γ (p = 0.003) mRNA, as well as reduced levels of IL-6 (p = 0.002) and IFN-γ (p < 0.0001) protein secretion. This process was also observed to increase the expression levels of IL-10 (p < 0.0001) and TGF-β (p < 0.0001) mRNA. 4 h fermentation of wheat flour with the proposed probiotic mix might be a good strategy to develop an affordable gluten-free wheat dough for NCWS and probably other GRD patients.

Screening of Eight Biofortified Rice Varieties Based on Total Content of Zinc and Iron as Well as Its Uptake by Caco-2 Cells: An In Vitro Cellular Model

Zinc (Zn) and iron (Fe) are two essential micronutrients that help the body to maintain its normal physiology and metabolism. Rice is the most important staple crop because it nourishes approximately half of the world’s population. The present study was intended to screen a set of eight rice varieties for total grain Zn and Fe content along with their uptake in differentiated Caco-2 cells. The total grain Zn and Fe content, as well as their bioaccessibility, was determined using inductively coupled plasma mass spectrometry (ICP-MS) techniques. The Caco-2 cell-based fluorescence assays and ferritin synthesis assays were used to determine Zn and Fe uptake, respectively. The transcriptional expression of micronutrient transporters has been analyzed in Caco-2 cells in response to the uptake of Zn and Fe from in vitro digested rice samples. The findings of this study revealed a comparative analysis of two prominent micronutrient contents in eight different varieties of polished rice. DRRDhan 48 (4.93 ± 0.10 mg/kg), Zinco Rice (4.65 ± 0.01 mg/kg), and Protozin (4.43 ± 0.03 mg/kg) were shown to have low phytate content, and high grain Zn content (32.49 ± 2.8, 27.9 ± 1.8, 26.7 ± 1.2 mg/kg, respectively), as well as significantly greater Zn uptake in differentiated Caco-2 cells. The outcome of this study provides an easy and convenient approach for the screening of food items or cereals based on micronutrient uptake.

Polystyrene Microplastics of Varying Sizes and Shapes Induce Distinct Redox and Mitochondrial Stress Responses in a Caco-2 Monolayer.

Currently, we lack crucial knowledge on how the physicochemical properties of particles affect cellular health, resulting in an important gap in our understanding of the human toxicity of microplastics (MPs). Our aim was to evaluate the impact of the size and the shape of MPs on uptake and the intracellular effects in a human epithelial colorectal adenocarcinoma (Caco-2) cell line. Spherical (200 nm and 2 µm) and fibre-/fragment-shaped (8.9 ± 10.1 µm by 1.14 ± 0.97 µm) polystyrene microplastics (PS-MPs) were used to study their uptake and the potential to induce redox and mitochondrial stress responses after 24 h of exposure. We demonstrated the cellular uptake of both spherical and fibre-/fragment-shaped MPs in a size-dependent manner. In response to 2 µm spheres, we observed differential expressions of redox-related genes, including HMOX1, CAT, and GPX1. All PS-MPs decreased the intracellular H2O2 levels, which can be attributed to mitochondrial stress responses, such as increased mitochondrial DNA content, footprint, and morphology. Altogether, we demonstrated uptakes and changes in redox and mitochondrial parameters for all PS-MPs, with the 200 nm spheres showing the most profound effects. This suggests that the induction of defensive responses in Caco-2 cells mainly correlates with the number of particles taken up.

In vitro antibacterial activity of Bacillus coagulans T242 on Caco-2 cells infected with Salmonella Typhimurium

This study aimed to investigate the antibacterial activity of Bacillus coagulans (B. coagulans) T242 and its ability to alleviate the infection of Caco-2 cells with Salmonella Typhimurium (S. Typhimurium) SL1344. B. coagulans T242 has a broad spectrum of the antibacterial activity. The cell-free supernatants of B. coagulans T242 inhibited the growth of S. Typhimurium SL1344 and changed its cell morphology. B. coagulans T242 inhibited the adhesion and invasion of S. Typhimurium SL1344 to Caco-2 cells by adhering to Caco-2 cells, significantly reduced the production of interleukin-6 (IL-6), Interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) which induced by S. Typhimurium SL1344, also increased interleukin-10 (IL-10) production, thereby alleviating the inflammatory response of Caco-2 cells. B. coagulans T242 vegetative cells and spores could alleviate the decreased expression of tight junction proteins (Claudin-1, Occludin and ZO-1) caused by S. Typhimurium SL1344 infection in Caco-2 cells by increasing the expression of Claudin-1, Occludin and ZO-1. This study demonstrated that B. coagulans T242 could protect Caco-2 cells by adhesion, reducing inflammatory response and enhancing intestinal barrier function. Therefore, B. coagulans T242 will hopefully be an effective alternative to antibiotics, and this study has important implications for food safety issues.

Bioactive peptides identified in pea and faba bean after in vitro digestion with human gastrointestinal enzymes

We investigated the digestive peptide profiles of pea and faba bean proteins to evaluate possible bioactive peptides (BAPs), and to test immune-modulating properties of selected peptides using in vitro cell model. More than 400 unique peptides were released upon gastrointestinal digestion of pea and faba bean proteins and identified by high-resolution mass spectrometry (HPLC-ESI-MS/MS). Among these, 24 peptides were potentially bioactive as predicted by in silico analysis. Also, up to 275 recognized BAPs and eight known allergens were identified within the peptide sequences of the digests. Four selected peptides significantly decreased the IL-8 response in Caco-2 cells, with a specific pea peptide, DKPWWPK, reducing the IL-8 response up to 40 % during IL-1β induction. Combined with other nutritional benefits, the immune-modulatory properties of legume proteins point to a regular introduction of legumes in the diet, as they represent a still scarcely exploited food category, especially in the Nordic countries.

Protective role of circRNA CCND1 in ulcerative colitis via miR-142-5p/NCOA3 axis

BackgroundIncreasing research indicates that circular RNAs (circRNAs) play critical roles in the development of ulcerative colitis (UC). This study aimed to determine the role of circRNA CCND1 in UC bio-progression, which has been shown to be downregulated in UC tissues.MethodsReverse transcription quantitative polymerase chain reaction was used to determine the levels of circRNA CCND1, miR-142-5p, and nuclear receptor coactivator-3 (NCOA3) in UC tissues and in lipopolysaccharide (LPS)-induced Caco-2 cells. Target sites of circRNA CCND1 and miR-142-5p were predicted using StarBase, and TargetScan to forecast potential linkage points of NCOA3 and miR-142-5p, which were confirmed by a double luciferase reporter-gene assay. Cell Counting Kit 8 and flow cytometry assays were performed to assess Caco-2 cell viability and apoptosis. TNF-α, IL-1β, IL-6, and IL-8 were detected using Enzyme-Linked Immunosorbent Assay kits.ResultsCircRNA CCND1 was downregulated in UC clinical samples and LPS-induced Caco-2 cells. In addition, circRNA CCND1 overexpression suppressed LPS-induced apoptosis and inflammatory responses in Caco-2 cells. Dual-luciferase reporter-gene assays showed that miR-142-5p could be linked to circRNA CCND1. Moreover, miR-142-5p was found to be highly expressed in UC, and its silencing inhibited LPS-stimulated Caco-2 cell apoptosis and inflammatory responses. Importantly, NCOA3 was found downstream of miR-142-5p. Overexpression of miR-142-5p reversed the inhibitory effect of circRNA CCND1-plasmid on LPS-stimulated Caco-2 cells, and the effects of miR-142-5p inhibitor were reversed by si-NCOA3.ConclusionCircRNA CCND1 is involved in UC development by dampening miR-142-5p function, and may represent a novel approach for treating UC patients.

Chemoproteomics reveals Sofalcone inhibits the inflammatory response of Caco-2 cells by covalently targeting HMGB1.

Sofalcone (Sof), a synthetic analog of sophoradin, is a type of natural phenol derived from the traditional medicinal herb Sophora subprostrata, with potent anti-inflammatory activity. However, the mechanisms of action of Sof for treating intestinal-associated inflammation are not well known. In this work, we identified high mobility group box 1 (HMGB1) as the key covalent target of Sof for the anti-inflammatory activity in the human colonic epithelial cells through quantitative chemoproteomics profiling.

Transcriptome responses of intestinal epithelial cells induced by membrane vesicles of Listeriamonocytogenes

Membrane vesicles (MVs) serve as an essential virulence factor in several pathogenic bacteria. The release of MVs by Listeria monocytogenes is only recently recognized; still, the enigmatic role of MVs in pathogenesis is yet to be established. We report the transcriptome response of Caco-2 cells upon exposure to MVs and the L. monocytogenes that leads to observe the up-regulation of autophagy-related genes in the early phase of exposure to MVs. Transcription of inflammatory cytokines is to the peak at the fourth hour of exposure. An array of differentially expressed genes was associated with actin cytoskeleton rearrangement, autophagy, cell cycle arrest, and induction of oxidative stress. At a later time point, transcriptional programs are generated upon interaction with MVs to evade innate immune signals, by modulating the expression of anti-inflammatory genes. KEGG pathway analysis is palpably confirming that MVs appear principally responsible for the induction of immune signaling pathways. Besides, MVs induced the expression of cell cycle regulatory genes, likely responsible for the ability to prolong host cell survival, thus protecting the replicative niche for L. monocytogenes. Notably, we identified several non-coding RNAs (ncRNAs), possibly involved in the regulation of early manipulation of the host gene expression, essential for the persistence of L. monocytogenes.

TRPA1 protects mice from pathogenic Citrobacter rodentium infection via maintaining the colonic epithelial barrier function.

Transient receptor potential ankyrin 1 (TRPA1) is expressed in gastrointestinal tract and plays important roles in intestinal motility and visceral hypersensitivity. However, the potential role of TRPA1 in host defense, particularly against intestinal pathogens, is unknown. Here, we show that Trpa1 knockout mice exhibited increased susceptibility to Citrobacter rodentium infection, associated with the increased severity of diarrhea and intestinal permeability associated with the disrupted tight junctions (TJs) in colonic epithelia. We further demonstrated the expression of TRPA1 in murine colonic epithelial cells (CECs) and human epithelial Caco-2 cells both at protein level and transcription level. Using calcium imaging, TRPA1 agonists allyl isothiocyanates (AITC) and hydrogen peroxide were observed to induce a transient Ca2+ response in Caco-2 cells, respectively. Moreover, TRPA1 knockdown in Caco-2 cells resulted in the decreased expression of TJ proteins, ZO-1 and Occludin, and in the increased paracellular permeabilities and the reduced TEER values of Caco-2 monolayers in vitro. Furthermore, inhibition of TRPA1 by HC-030031 in the confluent Caco-2 cells caused the altered distribution and expression of TJ proteins, ZO-1, Occludin, and Claudin-3, and exacerbated the bacterial endotoxin lipopolysaccharide (LPS)-induced damage to these TJ proteins and actin cytoskeleton. By contrast, AITC pretreatment restored the distribution and expression of these TJ proteins in the confluent Caco-2 cells upon LPS challenge. Our results identify an unrecognized protective role of TRPA1 in host defense against an enteric bacterial pathogen by maintaining colonic epithelium barrier function, at least in part, via preserving the distribution and expression of TJ proteins in CECs.

Transcriptome Analysis Reveals Immunomodulatory Effect of Spore-Displayed p75 on Human Intestinal Epithelial Caco-2 Cells

Lacticaseibacillus rhamnosus GG (LGG) can promote intestinal health by modulating the immune responses of the gastrointestinal tract. However, knowledge about the immunomodulatory action of LGG-derived soluble factors is limited. In our previous study, we have displayed LGG-derived p75 protein on the spore surface of Bacillus subtilis. The objective of this study was to determine the effect of spore-displayed p75 (CotG-p75) on immune system by investigating transcriptional response of Caco-2 cells stimulated by CotG-p75 through RNA-sequencing (RNA-seq). RNA-seq results showed that CotG-p75 mainly stimulated genes involved in biological processes, such as response to stimulus, immune regulation, and chemotaxis. KEGG pathway analysis suggested that many genes activated by CotG-p75 were involved in NF-ĸB signaling and chemokine signaling pathways. CotG-p75 increased cytokines and chemokines such as CXCL1, CXCL2, CXCL3, CXCL8, CXCL10, CCL20, CCL22, and IL1B essential for the immune system. In particular, CotG-p75 increased the expression levels of NF-ĸB-related genes such as NFKBIA, TNFAIP3, BIRC3, NFKB2, and RELB involved in immune and inflammatory responses. This study provides genes and pathways involved in immune responses influenced by CotG-p75. These comprehensive transcriptome profiling could be used to elucidate the immunomodulatory action of CotG-p75.

Caco-2 Cell Response Induced by Peptides Released after Digestion of Heat-Treated Egg White Proteins.

The heat treatment of food proteins induces structural modifications that influence their interaction with human fluids and cells. We aimed to evaluate the Caco-2 cell response induced by peptides produced after digestion of heat-treated egg white proteins. In vitro digestion of ovalbumin (OVA), ovomucoid (OM), and lysozyme (LYS), untreated or previously heated, was performed. The digestibility of proteins and the response of Caco-2 cells exposed to peptides (&lt;10 kDa) generated during digestion were evaluated. Intact OVA and LYS persisted after the digestion of native proteins, whereas OM was completely hydrolysed. A heat treatment at 65 °C for 30 min did not alter the digestibility of OVA, whereas at 90 °C for 3 min, protein degradation was favoured. The digestibility of OM and LYS was not affected by heat treatment. Peptides derived from OVA and OM digestion induced IL-6 and IL-8 production. OVA and LYS digestion promoted the expression of Tslp, and Il6 and Il33, respectively. A heat treatment prior to OVA digestion reduced IL-6 production and Tslp expression. It was concluded that heat treatments can reduce the release of OVA-derived peptides, but not OM and LYS, with proinflammatory activity during digestion.