Model Of Intestinal Epithelium Research Articles

Mechanism of Resveratrol on LPS/ATP-induced pyroptosis and inflammatory response in HT29 cells

Pyroptosis plays an important role in maintenance of intestinal homeostasis, the abnormal activation of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome can promote the event and development of ulcerative colitis (UC). Its protective effects such as inhibiting pyroptosis in various inflammation-related diseases have been demonstrated, but whether resveratrol (RES) can also alleviate the progression of the disease by inhibiting pyroptosis in UC and the mechanism have rarely been studied. In this study, lipopolysaccharide (LPS) combined with adenosine triphosphate (ATP) was used to induce HT29 human colon cancer cells to construct an intestinal epithelial cell pyroptosis and inflammation model in vitro to investigate the anti-inflammatory effect of RES, reveal the regulatory mechanism of RES on pyroptosis, and provide a new theoretical basis for the treatment of UC. In vitro experiences, HT29 cells were dividing into control group, LPS/ATP group, RES low-dose group, RES high-dose group, NF-κB inhibitor pyrrolidine dithiocarbamate group (PDTC group), and LPS/ATP+PDTC group. The mRNA expressions of pyroptosis-related indicators such as NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), Caspase-1(CASP1), IL-18, IL-1β, and inflammatory factors such as TNF-α and IL-6 were detected by qRT-PCR. The protein expressions of pyroptosis-related indicators NLRP3, ASC, CASP1, IL-18, IL-1β, NF-κB-p65 in the nucleus, and IκBα and p-IκBα in the cytoplasm were detected by Western blot. Immunofluorescence saw the distribution and expression of NLRP3, ASC and NF-κB-p65 protein in each group. The morphology and degree of pyroptosis in each group were observed by transmission electron microscope. The results showed that compared with the control group, the pyroptosis-related proteins including NLRP3, ASC, CASP1, IL-18, IL-1β, and inflammatory factors including TNF-α and IL-6 in the LPS/ATP group were increased, and LPS/ATP activated the activity of NF-κB signaling pathway. Compared with the LPS/ATP group, RES downregulated the expression of pyroptosis-related proteins and inflammatory factors in HT29 cells, and inhibited the activation of the NF-κB signaling pathway in HT29 cells pyroptosis. RES down-regulates the pyroptosis of HT29 cells induced by LPS/ATP and the expression of pyroptosis-related indicators NLRP3, ASC, CASP1, IL-18, IL-1β and inflammatory factors TNF-α and IL-6 in the inflammatory response and inhibits the occurrence of pyroptosis. The mechanism is related to the inhibition of NF-κB pathway activity.

Non-invasive real-time investigation of colorectal cells tight junctions by Raman microspectroscopy analysis combined with machine learning algorithms for organ-on-chip applications

IntroductionColorectal cancer is the third most common malignancy in developed countries. Diagnosis strongly depends on the pathologist’s expertise and laboratory equipment, and patient survival is influenced by the cancer’s stage at detection. Non-invasive spectroscopic techniques can aid early diagnosis, monitor disease progression, and assess changes in physiological parameters in both heterogeneous samples and advanced platforms like Organ-on-Chip (OoC).MethodsIn this study, Raman microspectroscopy combined with Machine Learning was used to analyse structural and biochemical changes in a Caco-2 cell-based intestinal epithelial model before and after treatment with a calcium chelating agent.ResultsThe Machine Learning (ML) algorithm successfully classified different epithelium damage conditions, achieving an accuracy of 91.9% using only 7 features. Two data-splitting approaches, “sample-based” and “spectra-based,” were also compared. Further, Raman microspectroscopy results were confirmed by TEER measurements and immunofluorescence staining.DiscussionExperimental results demonstrate that this approach, combined with supervised Machine Learning, can investigate dynamic biomolecular changes in real-time with high spatial resolution. This represents a promising non-invasive alternative technique for characterizing cells and biological barriers in organoids and OoC platforms, with potential applications in cytology diagnostics, tumor monitoring, and drug efficacy analysis.

Targeted and non-targeted metabolic characteristics of 6,7-dihydroxy-2,4- dimethoxyphenanthrene during simulated gastrointestinal digestion

6,7-dihydroxy-2,4-dimethoxyphenanthrene (PC4), isolated and identified from Chinese yam, was one of the important characteristic active ingredients. The present study established simulated gastrointestinal digestion and caco-2 intestinal epithelial models to investigate the digestive and metabolic characteristics of PC4. The results indicated that PC4 was mainly digested and metabolized in the intestine, with a digestion rate of 94 %, producing active characteristic metabolites including 2,6-dimethoxy-4-phenanthrenol (MC1) and 1-methoxyphenanthrene (MC2). Molecular docking indicated that the COX-2 enzyme inhibitory activity of MC1 might be superior to that of the prototype PC4. During 24 h co-incubation of PC4 with caco-2 monolayer epithelial, the signal pathway related to lipid decomposition was up-regulated within 0–12 h, while the diabetes complications AGE-RAGE was inhibited just in 0–6 h period significantly. The research database provided scientific basis for the digestion and metabolism of PC4, and laid theoretical foundation for the scientific development of Chinese yam functional foods.

Regulatory effects of the p38 mitogen-activated protein kinase-myosin light chain kinase pathway on the intestinal epithelial mechanical barrier and the mechanism of modified Pulsatilla decoction in the treatment of ulcerative colitis.

To investigate the mechanism of the protective effect of modified Pulsatilla decoction (, MPD) on the mechanical barrier of the ulcerative colitis (UC) intestinal epithelium in vitro and in vivo. We established an intestinal epithelial crypt cell line-6 cell barrier injury model by using lipopolysaccharide (LPS). The model was then treated with p38 mitogen-activated protein kinase-myosin light chain kinase (p38MAPK-MLCK) pathway inhibitors, p38MAPK-MLCK pathway silencing genes (si-p38MAPK, si-NF-κB, and si-MLCK), and MPD respectively. Transepithelial electronic resistance (TEER) measurements and permeability assays were performed to assess barrier function. Immunofluorescence staining of tight junctions (TJ) was performed. In in vivo experiment, dextran sodium sulfate-induced colitis rat model was conducted to evaluate the effect of MPD and mesalazine on UC. The rats were scored using the disease activity index based on their clinical symptoms. Transmission electron microscopy and hematoxylin-eosin staining were used to examine morphological changes in UC rats. Western blotting and real-time quantitative polymerase chain reaction were performed to examine the gene and protein expression of significant differential molecules. In in vitro study, LPS-induced intestinal barrier dysfunction was inhibited by p38MAPK-MLCK pathway inhibitors and p38MAPK-MLCK pathway gene silencing. Silencing of p38MAPK-MLCK pathway genes decreased TJ expression. MPD treatment partly restored the LPS-induced decreased in TEER and increase in permeability. MPD increased the gene and protein expression of TJ, while down-regulated the LPS-induced high expression of p-p38MAPK and p-MLC. In UC model rats, MPD could ameliorate body weight loss and disease activity index, relieve colonic pathology, up-regulate TJ expression as well as decrease the expression of p-p38MAPK and p-MLC in UC rat colonic mucosal tissue. The p38MAPK-MLCK signaling pathway can affect mechanical barrier function and TJ expression in the intestinal epithelium. MPD restores TJ expression and attenuates intestinal epithelial barrier damage by suppressing the p38MAPK-MLCK pathway.

Isongifolene Improves Crohn's Disease-Like Colitis in Mice by Reducing Apoptosis of Intestinal Epithelial Cells

To investigate the effect and molecular mechanism of isolongifolene (ISO) on the apoptosis of intestinal epithelial cells and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced Crohn's disease (CD)-like colitis in mice. In the animal experiments, mice were randomly assigned to the wild type (WT) group, TNBS group and TNBS+ISO group, with 8 mice in each group. Colitis models of mice were established in the TNBS group and the TNBS+ISO group by rectal injection of TNBS. After modeling, the mice in the TNBS+ISO group were given ISO intervention via intragastric gavage (10 mg/kg), and the other two groups were given the same amount of normal saline via intragastric gavage. The mice were sacrificed on the 7th day. The changes in body mass, disease activity scores (DAI), and the colon length of mice were measured, and transepithelial electrical resistance (TEER) of the colon tissues was determined. The score of colon inflammation was calculated according to HE staining. The levels of intestinal mucosal inflammatory factors, including tumor necrosis factor alpha (TNF-α), interferon (IFN)-γ, interleukin (IL)-1β, and IL-6, were measured by RT-PCR and ELISA. The apoptosis of colon tissue cells was determined by TUNEL assay. The expressions of apoptotic proteins (cleaved caspase-3/caspase-3 and Bax), an anti-apoptotic protein (Bcl-2), and tight junction proteins (ZO-1 and claudin-1) were detected by Western blot and immunofluorescence. In the cell experiment, TNF-α was used to induce intestinal epithelial cell Caco-2 apoptosis model, which was treated with ISO. Then, intervention with the AMPK inhibitor Compound C was given. TUNEL assay, Western blot assay, and immunofluorescence assay were performed to measure apoptosis and the expression of apoptosis proteins in the Caco-2 cells. Gene Ontology (GO) enrichment analysis was performed to predict the biological function of ISO. Then, the mechanism involved was verified by examination of the mice and Caco-2 cells. Western blot was performed to determine the expression levels of p-AMPK/AMPK and p-PGC1α in the colon tissues from the mice of different groups and Caco-2 cells. The apoptosis of the cells was determined by TUNEL assay. According to the results of the animal experiment, ISO could alleviate experimental colitis and intestinal barrier dysfunction, leading to improvements in body mass loss, colon length shortening, DAI score, inflammatory rating, and TEER values (all P<0.05) in mice. Furthermore, ISO decreased the expression of pro-inflammatory factors TNF-α, IFN-γ, IL-1β, and IL-6 and increased the expression of the tight junction proteins ZO-1 and claudin-1 (all P<0.05). In the cell experiment, in a TNF-α-induced intestinal epithelial cell model, ISO was also found to protect intestinal barrier against damage. ISO reduced the proportion of apoptotic intestinal epithelial cells, reduced the expression of cleaved-caspase-3/caspase-3 and Bax, and upregulated the level of Bcl-2 (all P<0.05). GO enrichment predictive analysis showed that the role of ISO in improving CD-like enteritis might be associated with the negative regulation of apoptosis. Verification of the mechanism showed that the expression of p-AMPK and p-PGC1α in the mice colon tissue was significantly upregulated after ISO intervention (P<0.05). In contrast, the AMPK inhibitor Compound C increased the apoptosis rate of ISO-treated Caco-2 cells and decreased the relative expression levels of ZO-1 and claudin-1 (P<0.05). ISO reduces intestinal epithelial cell apoptosis at least in part by activating AMPK/PGC1α signaling pathway, thereby alleviating TNBS-induced intestinal barrier dysfunction and CD-like colitis in mice.

Upgrading In Vitro Digestion Protocols with Absorption Models

Intestinal digestion and absorption are complex processes; thus, it is a challenge to imitate them realistically. There are numerous approaches available, with different disadvantages and advantages. The simplest methods to mimic absorption are the non-cell-based transport models but these lack important characteristics of enterocytes of the intestine. Therefore, the most often used method is to measure absorption through viable mammalian cells (most commonly Caco-2 cells, cultured on membrane insert plates), which not only assures the incorporation of brush border enzymes (responsible for the final digestion of peptides and disaccharides), it also simulates the absorption process. This means that influx/efflux transporter-facilitated transport, carrier-mediated transport, endocytosis, and transcytosis is also imitated besides passive diffusion. Still, these also lack the complexity of intestinal epithelium. Organoids or ex vivo models are a better approach if we want to attain precision but the highest accuracy can be achieved with microfluidic systems (gut-on-a-chip models). We propose that more research is necessary, and food absorption should also be studied on gut-on-a-chips, especially with fragmented organoids. Our review supports the choices of a proper intestinal epithelium model, which may have a key role in functional food development, nutrition studies, and toxicity assessment.

Gut Microbiota-Derived Trimethylamine Promotes Inflammation with a Potential Impact on Epigenetic and Mitochondrial Homeostasis in Caco-2 Cells.

Trimethylamine (TMA), a byproduct of gut microbiota metabolism from dietary precursors, is not only the precursor of trimethylamine-N-oxide (TMAO) but may also affect gut health. An in vitro model of intestinal epithelium of Caco-2 cells was used to evaluate the impact of TMA on inflammation, paracellular permeability, epigenetics and mitochondrial functions. The expression levels of pro-inflammatory cytokines (IL-6, IL-1β) increased significantly after 24 h exposure to TMA 1 mM. TMA exposure was associated with an upregulation of SIRT1 (TMA 1 mM, 400 μM, 10 μM) and DNMT1 (TMA 1 mM, 400 µM) genes, while DNMT3A expression decreased (TMA 1 mM). In a cell-free model, TMA (from 0.1 µM to 1 mM) induced a dose-dependent reduction in Sirtuin enzyme activity. In Caco-2 cells, TMA reduced total ATP levels and significantly downregulated ND6 expression (TMA 1 mM). TMA excess (1 mM) reduced intracellular mitochondrial DNA copy numbers and increased the methylation of the light-strand promoter in the D-loop area of mtDNA. Also, TMA (1 mM, 400 µM, 10 µM) increased the permeability of Caco-2 epithelium, as evidenced by the reduced transepithelial electrical resistance values. Based on our preliminary results, TMA excess might promote inflammation in intestinal cells and disturb epigenetic and mitochondrial homeostasis.

Naringenin inhibits the microsomal triglyceridetransfer protein/apolipoprotein B axis to inhibit intestinal metaplasia progression.

Intestinal metaplasia (IM) is a premalignant condition that increases the risk for subsequent gastric cancer (GC). Traditional Chinese medicine generally plays a role in the treatment of IM, and the phytochemical naringenin used in Chinese herbal medicine has shown therapeutic potential for the treatment of gastric diseases. However, naringenin's specific effect on IM is not yet clearly understood. Therefore, this study identified potential gene targets for the treatment of IM through bioinformatics analysis and experiment validation. Two genes (MTTP and APOB) were selected as potential targets after a comparison of RNA-seq results of clinical samples, the GEO dataset (GSE78523), and naringenin-related genes from the GeneCards database. The results of both cell and animal experiments suggested that naringenin can improve the changes in the intestinal epithelial metaplasia model via MTTP/APOB expression. In summary, naringenin likely inhibits the MTTP/APOB axis and therefore inhibits IM progression. These results support the development of naringenin as an anti-IM agent and may contribute to the discovery of novel IM therapeutic targets.

Structure-dependent capacity of procyanidin dimers to inhibit inflammation-induced barrier dysfunction in a cell model of intestinal epithelium

Diet is of major importance in modulating intestinal inflammation, as the gastrointestinal tract is directly exposed to high concentrations of dietary components. Procyanidins are flavan-3-ol oligomers abundant in fruits and vegetables. Although with limited or no intestinal absorption, they can have GI health benefits which can promote overall health. We previously observed that epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) dimers inhibit in vitro colorectal cancer cell proliferation and invasiveness. Inflammation-mediated intestinal barrier permeabilization can result in a chronic inflammatory condition and promote colorectal cancer onset/progression. Thus, this study investigated the structure-dependent capacity of ECG, EGCG and (−)-epicatechin (EC) dimers to inhibit tumor necrosis factor alpha (TNFα)-induced inflammation, oxidative stress, and loss of barrier integrity in Caco-2 cells differentiated into an intestinal epithelial cell monolayer. Cells were incubated with TNFα (10 ng/ml), in the absence/presence of ECG, EGCG and EC dimers. The three dimers inhibited TNFα-mediated Caco-2 cell monolayer permeabilization, modulating events involved in the loss of barrier function and inflammation, i.e. decreased tight junction protein levels; increased matrix metalloproteinases expression and activity; increased NADPH oxidase expression and oxidant production; activation of the NF-κB and ERK1/2 pathways and downstream events leading to tight junction opening. For some of these mechanisms, the galloylated ECG and EGCG dimers had stronger protective potency than the non-galloylated EC dimer. These differences could be due to differential membrane interactions as pointed out by molecular dynamics simulation of procyanidin dimers-cell membrane interactions and/or by differential interactions with NOX1. Results show that dimeric procyanidins, although poorly absorbed, can promote health by alleviating intestinal inflammation, oxidative stress and barrier permeabilization.

Effect of ultra-high pressure homogenization on the antirotaviral activity of bovine milk whey

In the present study, ultra-high pressure homogenization (UHPH, 100–250 MPa) was applied on bovine whey. The ability of UHPH-treated whey to neutralize the bovine rotavirus strain WC3 was evaluated in vitro using a model of human intestinal epithelium. Results showed that whey homogenized at 100 and 200 MPa inhibited the rotavirus infection in a dose-response way, exhibiting >95% neutralization at 1.5 mg mL−1 protein concentration. However, after homogenization of whey at 250 MPa, a clear neutralization pattern could not be observed. This could be attributed to aggregation and/or denaturation of some bioactive proteins, as it has been evidenced in the electrophoretic profile of that sample. Interestingly, the fat fraction obtained from all UHPH-treated wheys, showed almost complete neutralization at 1 mg mL−1 protein concentration. Therefore, whey fractions could be used as functional ingredients in novel non-thermal processed products for the control of viral infections. Industrial relevanceThis study provides useful information to increase the commercial value of sweet, skimmed whey as a functional ingredient and its recognition as a natural source of antivirals. The use of emerging processing technologies, such as UHPH, could favor the preservation of certain bioactivity within the dairy by-product.

Intestinal Epithelial Co-Culture Sensitivity to Pro-Inflammatory Stimuli and Polyphenols Is Medium-Independent.

The complexification of in vitro models requires the compatibility of cells with the same medium. Since immune cells are the most sensitive to growth conditions, growing intestinal epithelial cells in their usual medium seems to be necessary. This work was aimed at comparing the sensitivity of these epithelial cells to pro-inflammatory stimuli but also to dietary polyphenols in both DMEM and RPMI-1640 media. Co-cultures of Caco-2 and HT29-MTX cells were grown for 21 days in the two media before their stimulation with a cocktail of TNF-α (20 ng/mL), IL-1β (1 ng/mL), and IFN-γ (10 ng/mL) or with LPS (10 ng/mL) from E. coli (O111:B4). The role of catechins (15 µM), a dietary polyphenol, was evaluated after its incubation with the cells before their stimulation for 6 h. The RPMI-1640 medium did not alter the intensity of the inflammatory response observed with the cytokines. By contrast, LPS failed to stimulate the co-culture in inserts regardless of the medium used. Lastly, catechins were unable to prevent the pro-inflammatory response observed with the cytokines in the two media. The preservation of the response of this model of intestinal epithelium in RPMI-1640 medium is promising when considering its complexification to evaluate the complex cellular crosstalk leading to intestinal homeostasis.

Attenuated replication and damaging effects of SARS-CoV-2 Omicron variants in an intestinal epithelial barrier model.

Many COVID-19 patients suffer from gastrointestinal symptoms and impaired intestinal barrier function is thought to play a key role in Long COVID. Despite its importance, the impact ofsevere acute respiratory syndrome coronavirus 2(SARS-CoV-2) on intestinal epithelia is poorly understood. To address this, we established an intestinal barrier model integrating epithelial Caco-2 cells, mucus-secreting HT29 cells and Raji cells. This gut epithelial model allows efficient differentiation of Caco-2 cells into microfold-like cells, faithfully mimics intestinal barrier function, and is highly permissive to SARS-CoV-2 infection. Early strains of SARS-CoV-2 and the Delta variant replicated with high efficiency, severely disrupted barrier function, and depleted tight junction proteins, such as claudin-1, occludin, and ZO-1. In comparison, Omicron subvariants also depleted ZO-1 from tight junctions but had fewer damaging effects on mucosal integrity and barrier function. Remdesivir, the fusion inhibitor EK1 and the transmembrane serine protease 2 inhibitor Camostat inhibited SARS-CoV-2 replication and thus epithelial barrier damage, while the Cathepsin inhibitor E64d was ineffective. Our results support that SARS-CoV-2 disrupts intestinal barrier function but further suggest that circulating Omicron variants are less damaging than earlier viral strains.

Multilevel Proteomic Profiling of Colorectal Adenocarcinoma Caco-2 Cell Differentiation to Characterize an Intestinal Epithelial Model.

Emergent advancements on the role of the intestinal microbiome for human health and disease necessitate well-defined intestinal cellular models to study and rapidly assess host, microbiome, and drug interactions. Differentiated Caco-2 cell line is commonly utilized as an epithelial model for drug permeability studies and has more recently been utilized for investigating host-microbiome interactions. However, its suitability to study such interactions remains to be characterized. Here, we employed multilevel proteomics to demonstrate that both spontaneous and butyrate-induced Caco-2 differentiations displayed similar protein and pathway changes, including the downregulation of proteins related to translation and proliferation and upregulation of functions implicated in host-microbiome interactions, such as cell adhesion, tight junction, extracellular vesicles, and responses to stimuli. Lysine acetylomics revealed that histone protein acetylation levels were decreased along with cell differentiation, while the acetylation in proteins associated with mitochondrial functions was increased. This study also demonstrates that, compared to spontaneous differentiation methods, butyrate-containing medium accelerates Caco-2 differentiation, with earlier upregulation of proteins related to host-microbiome interactions, suggesting its superiority for assay development using this intestinal model. Altogether, this multiomics study emphasizes the controlled progression of Caco-2 differentiation toward a specialized intestinal epithelial-like cell and establishes its suitability for investigating the host-microbiome interactions.

#794 LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axis

Abstract Background and Aims Long non-coding RNA(LncRNA) has been reported to be associated with intestinal barrier damage. The aim of this study was to explore the mechanism of lncRNA Nuclear enriched abundant transcript 1 (NEAT1) in uremic toxin-induced intestinal epithelial barrier injury. Method Human colon cancer cells (Caco-2) were used to establish intestinal epithelial injury models with the urea treatment in different conditions. Cell Counting Kit-8 (CCK-8) and Western blot screening the best concentration and time. The expressions of lncRNA NEAT1 and miR-122-5p were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot and immunofluorescence were used to detect the expression of tight junction proteins Occludin, ZO-1 and Claudin-1. Sodium fluorescein was used to detect the paracellular permeability of intestinal epithelial injury models. The binding of miR-122-5p to lncRNA NEAT1 and Occludin was determined by bioinformatics analysis and dual luciferase reporter assay. Results The best condition for the injury model was urea treatment in 144 mg/dl for 48 hours. With the increase of urea intervention time and concentration, the damage degree of intestinal epithelial cells is aggravated. Based on the qRT-PCR results, lncRNA NEAT1 was significantly down-regulated in the model group. Meanwhile, the tight junction proteins Occludin, ZO-1 and Claudin-1 were significantly reduced. The permeability of sodium fluorescein was significantly increased in the model group. Overexpression of lncRNA NEAT1 can alleviate the above performances. As the target gene of lncRNA NEAT1, miR-122-5p is significantly up-regulated in the model group. The dual luciferase reporter assay proved that miR-122-5p was targets to Occludin. The protective effect of overexpression lncRNA NEAT1 on intestinal epithelial barrier function is reversed by miR-122-5p mimics. Conclusion LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axis.

Assessment of Ingested Micro- and Nanoplastic (MNP)-Mediated Genotoxicity in an In Vitro Model of the Small Intestinal Epithelium (SIE).

Micro- and nanoplastics (MNPs) have become ubiquitous contaminants of water and foods, resulting in high levels of human ingestion exposure. MNPs have been found in human blood and multiple tissues, suggesting that they are readily absorbed by the gastrointestinal tract (GIT) and widely distributed. Growing toxicological evidence suggests that ingested MNPs may pose a serious health threat. The potential genotoxicity of MNPs, however, remains largely unknown. In this study, genotoxicity of primary and environmentally relevant secondary MNPs was assessed in a triculture small intestinal epithelium (SIE) model using the CometChip assay. Aqueous suspensions of 25 and 1000 nm carboxylated polystyrene spheres (PS25C and PS1KC), and incinerated polyethylene (PEI PM0.1) were subjected to simulated GIT digestion to create physiologically relevant exposures (digestas), which were applied to the SIE model at final MNP concentrations of 1, 5, and 20 μg/mL for 24 or 48 h. PS25C and PS1KC induced DNA damage in a time- and concentration-dependent manner. To our knowledge, this is one of the first assessment of MNP genotoxicity in an integrated in vitro ingestion platform including simulated GIT digestion and a triculture SIE model. These findings suggest that ingestion of high concentrations of carboxylated PS MNPs could have serious genotoxic consequences in the SIE.

Mechanisms of ingested polystyrene micro-nanoplastics (MNPs) uptake and translocation in an in vitro tri-culture small intestinal epithelium

Micro and nanoplastics (MNPs) are now ubiquitous contaminants of food and water. Many cellular and animal studies have shown that ingested MNPs can breach the intestinal barrier to reach the circulation. To date however, the cellular mechanisms involved in intestinal absorption of MNPs have not been investigated with physiologically relevant models, and thus remain unknown. We employed in vitro simulated digestion, a tri-culture small intestinal epithelium model, and a panel of inhibitors to assess the contributions of the possible mechanisms to absorption of 26 nm carboxylated polystyrene (PS26C) MNPs. Inhibition of ATP synthesis reduced translocation by only 35 %, suggesting uptake by both active endocytic pathways and passive diffusion. Translocation was also decreased by inhibition of dynamin and clathrin, suggesting involvement of clathrin mediated endocytosis (CME) and fast endophilin-mediated endocytosis (FEME). Inhibition of actin polymerization also significantly reduced translocation, suggesting involvement of macropinocytosis or phagocytosis. However, inhibition of the Na+-H+ exchanger had no effect on translocation, thus ruling out macropinocytosis. Together these results suggest uptake by passive diffusion as well as by active phagocytosis, CME, and FEME pathways. Further studies are needed to assess uptake mechanisms for other environmentally relevant MNPs as a function of polymer, surface chemistry, and size.

MTOR signaling pathway regulation HIF-1 α effects on LPS induced intestinal mucosal epithelial model damage

BackgroundSepsis-induced small-intestinal injury is associated with increased morbidity and mortality. Our previous study and other papers have shown that HIF-1α has a protective effect on intestinal mucosal injury in septic rats. The purpose of this study is to further verify the protective effect of HIF-1α on intestinal mucosa and its molecular mechanism in vitro experiments.MethodsCaco-2 cells were selected and experiment was divided into 2 parts. Part I: HIF-1α activator and inhibitor were used to treat lipopolysacchrides (LPS)-stimulated Caco-2 cells respectively, to explore the effect of HIF-1α on LPS induced Caco-2 cell epithelial model; Part II: mTOR activator or inhibitor combined with or without HIF-1α activator, inhibitor to treat LPS-stimulated Caco-2 cells respectively, and then the molecular mechanism of HIF-1α reducing LPS induced Caco-2 cell epithelial model damage was detected.ResultsThe results showed that HIF-1α activator decreased the permeability and up regulated tight junction (TJ) expression, while HIF-1α inhibitor had the opposite effect with the HIF-1α activator. mTOR activation increased, while mTOR inhibition decreased HIF-1α protein and expression of its downstream target molecules, which can be attenuated by HIF-1α activator or inhibitor.ConclusionThis study once again confirmed that HIF-1α alleviates LPS-induced mucosal epithelial model damage through P70S6K signalling pathway. It is of great value to explore whether HIF-2α plays crucial roles in the regulation of mucosal epithelial model functions in the future.

Exploring host-commensal-pathogen dynamics in cell line and organotypic human intestinal epithelial models

Host and microbiome intricately interact in the ecosystem of the human digestive tract, playing a crucial role in our health. These interactions can initiate immune responses in the epithelial cells, which, in turn, activate downstream responses in other immune cells. Here, we used a CaCo-2 and a human intestinal enteroid (HIE) model to explore epithelial responses to both commensal and pathogenic bacteria, individually and combined. CaCo-2 cells were co-cultured with peripheral blood mononuclear cells, revealing downstream activation of immune cells. While both systems showed comparable cytokine profiles, they differed in their responses to the different bacteria, with the organoid system being more representative of responses observed in humans. We provide evidence of the pro-inflammatory responses associated with these bacteria. These models contribute to a deeper understanding of the interactions between the microbiota, intestinal epithelium, and immune cells in the gut, promoting advances in the field of host-microbe interactions.

Selection of Fermentation Supernatant from Probiotic Strains Exhibiting Intestinal Epithelial Barrier Protective Ability and Evaluation of Their Effects on Colitis Mouse and Weaned Piglet Models.

The intestinal epithelial barrier can prevent the invasion of pathogenic microorganisms and food antigens to maintain a consistent intestinal homeostasis. However, an imbalance in this barrier can result in various diseases, such as inflammatory bowel disease, malnutrition, and metabolic disease. Thus, the aim of this study was to select probiotic strains with epithelial barrier-enhancing ability in cell-based model and further investigate them for their improving effects on colitis mouse and weaned piglet models. The results showed that selected specific cell-free fermentation supernatants (CFSs) from Ligilactobacillus salivarius P1, Lactobacillus gasseri P12, and Limosilactobacillus reuteri G7 promoted intestinal epithelial cell growth and proliferation, strengthening the intestinal barrier in an intestinal epithelial cell line Caco-2 model. Further, the administration of CFSs of L. salivarius P1, L. gasseri P12, and L. reuteri G7 were found to ameliorate DSS-induced colitis in mice. Additionally, spray-dried powders of CFS from the three strains were examined in a weaned piglet model, only CFS powder of L. reuteri G7 could ameliorate the feed/gain ratio and serum levels of D-lactate and endotoxin. In conclusion, a new potential probiotic strain, L. reuteri G7, was selected and showed ameliorating effects in both colitis mouse and weaned piglet models.

Mathematical model explains differences in Omicron and Delta SARS-CoV-2 dynamics in Caco-2 and Calu-3 cells.

Within-host infection dynamics of Omicron dramatically differs from previous variants of SARS-CoV-2. However, little is still known about which parameters of virus-cell interplay contribute to the observed attenuated replication and pathogenicity of Omicron. Mathematical models, often expressed as systems of differential equations, are frequently employed to study the infection dynamics of various viruses. Adopting such models for results of in vitro experiments can be beneficial in a number of aspects, such as model simplification (e.g., the absence of adaptive immune response and innate immunity cells), better measurement accuracy, and the possibility to measure additional data types in comparison with in vivo case. In this study, we consider a refinement of our previously developed and validated model based on a system of integro-differential equations. We fit the model to the experimental data of Omicron and Delta infections in Caco-2 (human intestinal epithelium model) and Calu-3 (lung epithelium model) cell lines. The data include known information on initial conditions, infectious virus titers, and intracellular viral RNA measurements at several time points post-infection. The model accurately explains the experimental data for both variants in both cell lines using only three variant- and cell-line-specific parameters. Namely, the cell entry rate is significantly lower for Omicron, and Omicron triggers a stronger cytokine production rate (i.e., innate immune response) in infected cells, ultimately making uninfected cells resistant to the virus. Notably, differences in only a single parameter (e.g., cell entry rate) are insufficient to obtain a reliable model fit for the experimental data.