Intestinal Caco-2 Research Articles

Antioxidant Activity and DPP-IV Inhibitory Effect of Fish Protein Hydrolysates Obtained from High-Pressure Pretreated Mixture of Rainbow Trout (Oncorhynchus mykiss) and Atlantic Salmon (Salmo salar) Rest Raw Material

The use of fish rest raw material for the production of fish protein hydrolysates (FPH) through enzymatic hydrolysis has received significant interest in recent decades. Peptides derived from fish proteins are known for their enhanced bioactivity which is mainly influenced by their molecular weight. Studies have shown that novel technologies, such as high-pressure processing (HPP), can effectively modify protein structures leading to increased biological activity. This study investigated the effect of various HPP conditions on the molecular weight distribution, antioxidant activity, and dipeptidyl-peptidase IV (DPP-IV) inhibitory effect of FPH derived from a mixture of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) rest raw material. Six different treatments were applied to the samples before enzymatic hydrolysis; 200 MPa × 4 min, 200 MPa × 8 min, 400 MPa × 4 min, 400 MPa × 8 min, 600 MPa × 4 min, and 600 MPa × 8 min. The antioxidant and DPP-IV inhibitory effects of the extracted FPH were measured both in vitro and at cellular level utilizing human intestinal Caco-2 cells. The results indicated that low and moderate pressures (200 and 400 MPa) increased the proportion of larger peptides (2–5 kDa) in the obtained FPH, while treatment at 600 MPa × 4 min resulted in a higher proportion of smaller peptides (1–2 kDa). Furthermore, HPP led to the formation of peptides that demonstrated increased antioxidant activity in Caco-2 cells compared to the control, whereas their potential antidiabetic activity remained unaffected.

Bacillus amyloliquefaciens Regulates the Keap1/Nrf2 Signaling Pathway to Improve the Intestinal (Caco-2 Cells and Chicken Jejunum) Oxidative Stress Response Induced by Lipopolysaccharide (LPS)

This article aims to investigate the mechanism by which Bacillus amyloliquefaciens alleviates lipopolysaccharide (LPS)-induced intestinal oxidative stress. The study involved two experimental subjects: human colorectal adenocarcinoma (Caco-2) cells and Arbor Acres broiler chickens. The experiment involving two samples was designed with the same treatment groups, specifically the control (CK) group, lipopolysaccharide (LPS) group, Bacillus amyloliquefaciens (JF) group, and JF+LPS group. In the Caco-2 experiment, we administered 2 μg/mL of LPS and 1 × 106 CFU/mL of JF to the LPS and JF groups, respectively. In the broiler experiment, the LPS group (19–21 d) received an abdominal injection of 0.5 mg/kg BW of LPS, whereas the JF group was fed 1 × 107 CFU/g of JF throughout the entire duration of the experiment (1–21 d). The results indicated the following: (1) JF significantly decreased the DPPH free radical clearance rate and hydrogen peroxide levels (p < 0.05). (2) JF significantly enhanced the total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GSH Px) activity in Caco-2 cells (p < 0.05), while concurrently reducing malondialdehyde (MDA) content (p < 0.05). (3) Compared to the CK group, JF significantly increased the mRNA expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), SOD, catalase (CAT), GSH-Px, interleukin-4 (IL-4), interleukin-10 (IL-10), Claudin, Occludin1, zonula occludens-1 (ZO-1), and mucin 2 (MUC2) in Caco-2 cells (p < 0.05), while concurrently reducing the mRNA expression of Kelch-like ECH-associated protein 1 (Keap1), tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-8 (IL-8) (p < 0.05). In comparison to the LPS group, the JF+LPS group demonstrated a significant increase in the mRNA expression of Nrf2, SOD, GSH-Px, and IL-4, as well as Occludin1, ZO-1, and MUC2 in Caco-2 cells (p < 0.05), alongside a decrease in the mRNA expression of Keap1, TNF-α, and IL-1β (p < 0.05). (4) In broiler chickens, the JF group significantly elevated the levels of T-AOC, CAT, and GSH-Px in the jejunum while reducing MDA content (p < 0.05). Furthermore, the CAT level in the JF+LPS group was significantly higher than that observed in the LPS group, and the levels of MDA, TNF-α, and IL-1β were significantly decreased (p < 0.05). (5) In comparison to the CK group, the JF group exhibited a significant increase in Nrf2 levels in the jejunum of broiler chickens (p < 0.05). Notably, the mRNA expression levels of IL-4, IL-10, Claudin, Occludin1, ZO-1, and MUC2 were reduced (p < 0.05), while the mRNA expression levels of Keap1, TNF-α, and IL-1β also showed a decrease (p < 0.05). Furthermore, the mRNA expression levels of Nrf2, Occludin1, ZO-1, and MUC2 in the JF+LPS group were significantly elevated compared to those in the LPS group (p < 0.05), whereas the mRNA expression levels of Keap1 and TNF-α were significantly diminished (p < 0.05). In summary, JF can enhance the intestinal oxidative stress response, improve antioxidant capacity and intestinal barrier function, and decrease the expression of inflammatory factors by regulating the Keap1/Nrf2 signaling pathway.

Neuroprotective effect of mealworm protein hydrolysate-derived bioactive peptides in human microglial cells

Abstract The larva of Tenebrio molitor, better known as mealworm, is one of the most studied insects today, since it was recently classified as safe for human consumption. Specifically, its high protein content makes it a splendid candidate for the search for bioactive peptides, with properties ranging from anti-inflammatory to neuroprotective. In this study, the effect of a digested hydrolysate on microglia cells (which previously demonstrated high anti-inflammatory activity in intestinal CACO-2 cells) was analysed, specifically on their levels of gene expression of various cytokines, such as IL-6, IL-10, or IL-1β, among others. In addition, four chemically synthesised peptides, selected from this digested hydrolysate based on in silico analysis, including the estimation of the bioactive properties of these peptides, their physicochemical properties and their toxicokinetic, were evaluated in the microglia cells as well. In addition, molecular docking assays of the peptides were performed with two receptors relevant to inflammation. Relevant changes in the expression of IL-6, IL-1β, and BDNF were observed due to the action of the peptides, especially in the case of IYVDAVIN and SLPSLPEPV. These results provide an insight on how specific peptides found in Tenebrio molitor could be used as therapeutics agent in the immunomodulation of brain cells.

Targeted dual-receptor phage cocktail against Cronobacter sakazakii: insights into phage-host interactions and resistance mechanisms

IntroductionCronobacter sakazakii is a notorious foodborne pathogen, frequently contaminating powdered infant formula and causing life-threatening diseases in infants. The escalating emergence of antibiotics-resistant mutants has led to increased interest in using bacteriophage as an alternative antimicrobial agent.MethodsTwo phages, CR8 and S13, were isolated from feces and soil samples and their morphology, physiology, and genomics were characterized. Phage receptor was determined using deletion mutants lacking flgK, rfaC, fhuA, btuB, lamb, or ompC genes, followed by complementation. Phage-resistant mutants were analyzed for phenotypic changes and fitness trade-offs using motility assays and Caco-2 cell invasion models.ResultsCR8 and S13 were identified as members of Caudoviricetes. Phage CR8 and phage S13 utilize flagella and LPS, respectively, to adhere to host cells. Bacterial challenge assay demonstrated delayed emergence of the resistant mutant as well as stronger lytic activity of a phage cocktail consisting of CR8 and S13 than the single phage treatment. Phenotypic analysis of the phage cocktail resistant strain, designated as CSR strain, revealed that the resistance resulted from the impaired receptor proteins for phage, such as defects in motility and alteration in LPS structure. CSR strain exhibited significant attenuation in invading human intestinal epithelial Caco-2 cells compared to WT cells.ConclusionThis study demonstrates that the development of the phage cocktail targeting distinct host receptors can serve as a promising antimicrobial strategy to effectively control C. sakazakii.

HMGB1 impacts the intestinal epithelial barrier by initiating NETs to regulate macrophage polarization.

High mobility group box 1 (HMGB1) has the capability of activating the immune response and taking part in macrophage polarization. Despite this, there is significant scope for exploration into how HMGB1 regulates macrophage polarization phenotype and influences intestinal epithelial barrier function. Investigating the role of HMGB1 in the creation of neutrophil extracellular traps (NETs) and the mechanism of its impact on macrophages could provide novel insights into intervening in intestinal inflammation and barrier damage. Therefore, the research examined the relationship between the macrophage polarization phenotype and HMGB1. Additionally, we analyzed how cell proliferation and cytokines changed in CaCo-2 cells following co-culture with HMGB1-influenced macrophages and intestinal epithelial CaCo-2 cells. We discovered that up-regulation of HMGB1 expression enhanced the creation of NETs, whereas inhibition of NETs formation led macrophages to switch from the anti-inflammatory M2 phenotype to the pro-inflammatory M1 phenotype. Additionally, we observed that macrophages induced by NETs containing HMGB1 can prompt CaCo-2 cell apoptosis and exacerbate the inflammatory response. HMGB1-containing NETs hinder tight junction protein expression in CaCo-2 cells by inducing macrophage M1 polarization, thereby impairing intestinal epithelial barrier function. Therefore, our findings indicate that by inhibiting the expression of HMGB1, the formation of NETs can be inhibited. This, in turn, mediates macrophage polarization and offers potential new therapies for intestinal diseases.

Molecular Mechanisms of Skatole-Induced Inflammatory Responses in Intestinal Epithelial Caco-2 Cells: Implications for Colorectal Cancer and Inflammatory Bowel Disease.

Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in intestinal epithelial cells significantly contribute to inflammatory bowel disease (IBD) and colorectal cancer (CRC). Given our previous findings that TNF-α is upregulated in intestinal epithelial Caco-2 cells induced by skatole, a tryptophan-derived gut microbiota metabolite, the present study aimed to explore the relationship between skatole and IL-6, alongside TNF-α. Skatole elevated the promoter activity of IL-6 as well as TNF-α, and increased IL-6 mRNA expression and protein secretion. In addition to activating NF-κB, the NF-κB inhibitor BAY 11-7082 reduced skatole-induced cell survival and the mRNA expression of IL-6 and TNF-α. NF-κB activation was attenuated by the extracellular signal-regulated kinase (ERK) pathway inhibitor U0126 and the p38 inhibitor SB203580, but not by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. U126 and SB203580 also decreased the skatole-induced increase in IL-6 expression. When skatole-induced AhR activation was inhibited by CH223191, in addition to promoting NF-κB activation, IL-6 expression was enhanced in a manner similar to that previously reported for TNF-α. Taken together, these results suggest that skatole-elicited NF-κB activation induces IL-6 and TNF-α expression, although AhR activation partially suppresses this process. The ability of skatole to increase the expression of IL-6 and TNF-α may significantly affect the development and progression of these diseases. Moreover, the balance between NF-κB and AhR activation appears to govern the skatole-induced increases in IL-6 and TNF-α expression. Therefore, the present findings provide new insights into the mechanisms linking tryptophan-derived gut microbiota metabolites with colorectal disease.

Micronization of wholewheat flour increases iron bioavailability from hydrothermally processed wheat flour dough

Cereal products contribute significantly to dietary intake of essential minerals. In wheat, iron and zinc are stored in specific grain structures including the aleurone, scutellum and embryo. Wheat cell walls are resistant to digestion in the human gastrointestinal tract and therefore this study investigated the hypothesis that physical disruption of the cell walls would increase the bioaccessibility and bioavailability of iron and zinc from wheat-based foods. Flour was micronized using a combination of roller milling and a micro-mill and this reduced median particle size by two-thirds. Hydrothermally processed wheat flour doughs were subjected to in vitro digestion to determine mineral bioaccessibility. Mineral bioavailability from food digests was measured using human intestinal Caco-2 cells. Iron (but not zinc) bioavailability from wheat foods made using the micronized flour (2.5 ± 0.5 nmol/mg cell protein) was increased significantly compared with foods produced from standard milled flour (1.3 ± 0.1 nmol/mg cell protein; P = 0.031). Micronization of wheat flour has the potential to increase the absorption of the endogenous iron present in cereal foods and this might have health benefits for population groups with poor iron status.

Cytotoxic impacts of seven alternative bisphenols on human in vitro cellular models

Bisphenols (BPs), common in plastics, coatings, and resins, are under scrutiny for potential endocrine disruption. Despite banning bisphenol A (BPA), its perceived safer alternatives may still pose health risks, urging thorough studies on their toxicity mechanisms. This study aimed to investigate the cellular toxicity of the top seven most commonly used BPs, bisphenol S (BPS), bisphenol F (BPF), bisphenol AF (BPAF), bisphenol P (BPP), bisphenol AP (BPAP), bisphenol B (BPB), bisphenol E (BPE) in eight different relevant human in vitro cell models: liver (HepaRG), intestinal (Caco-2), breast (T47D), brain (HMC-3), lungs (MRC-5), kidney (HEK293), endothelial (HMEC-1), and skin (HEK-001) cell lines. BPE manifested the highest cytotoxicity in Caco-2 cells, presenting an EC50 value of roughly 0.2 μM (95% confidence interval). In contrast, HEK293 and HepaRG cells demonstrated significant resilience to BPS (EC50 > 1000 μM). BPAF, BPP, and BPAP had consistently low EC50 values across cell lines (6–27.9 μM, 0.6–134.7 μM, and 3.6–178.8 μM), indicating elevated toxicity. After 24 h, all bisphenols adhered to nominal concentrations except BPAF, BPP, and BPS. BPP's concentration notably decreased (30.82 ± 5.53% of nominal value). The results revealed diverse effects of bisphenol analogs on different cell types. These findings emphasized the considerable cytotoxic potential of specific bisphenol analogs across various human cell models, underlining the necessity for a re-evaluation of their safety and regulatory standards.

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.

Klebsiella pneumoniae increases invasion inintestinal epithelial cells by disrupting thecytoskeleton.

Klebsiella pneumoniae is an opportunistic pathogen and it can cause human mucosal lesions through the intestine, leading to bacteremia and abscess formation in liver and spleen. Previous studies have shown that K. pneumoniae can enter or cross cells through the intestinal epithelium, but the mechanism is unknown. In this study, we treated the intestinal epithelial cell line Caco-2 with KP1195, a clinically isolated strain with high adhesion and invasion of intestinal epithelial cells. The results showed that thetreatment of K. pneumoniae could increase the expression of integrin gene and further disrupt the changes of cytoskeleton. Treating Caco-2 with cytoskeletal inhibitor cytorelaxin D can significantly increase the efficiency of K. pneumoniae invading Caco-2 cells. These data suggest that disruption of the cytoskeleton through integrins may be one of the mechanisms by which K. pneumoniae increases intracellular invasion. This study provides a theoretical basis for further understanding of the mechanism of K. pneumoniae entering intestinal epithelial cells.

Lack of effects of polystyrene micro- and nanoplastics on activity and expression of human drug transporters

Micro- and nanoplastics (MPs/NPs) constitute emerging and widely-distributed environmental contaminants to which humans are highly exposed. They possibly represent a threat for human health. In order to identify cellular/molecular targets for these plastic particles, we have analysed the effects of exposure to manufactured polystyrene (PS) MPs and NPs on in vitro activity and expression of human membrane drug transporters, known to interact with chemical pollutants. PS MPs and NPs, used at various concentrations (1, 10 or 100 µg/mL), failed to inhibit efflux activities of the ATP-binding cassette (ABC) transporters P-glycoprotein, MRPs and BCRP in ABC transporter-expressing cells. Furthermore, PS particles did not impair the transport of P-glycoprotein or BCRP substrates across intestinal Caco-2 cell monolayers. Uptake activities of solute carriers (SLCs) such as OCT1 and OCT2 (handling organic cations) or OATP1B1, OATP1B3, OATP2B1, OAT1 and OAT3 (handling organic anions) were additionally not altered by PS MPs/NPs in HEK-293 cells overexpressing these SLCs. mRNA expression of ABC transporters and of the SLCs OCT1 and OATP2B1 in Caco-2 cells and human hepatic HepaRG cells were finally not impaired by a 48-h exposure to MPs/NPs. Altogether, these data indicate that human drug transporters are unlikely to be direct and univocal targets for synthetic PS MPs/NPs.

Effect of ultrasound treatment on quality parameters and health promoting activity of fish protein hydrolysates extracted from side streams of Atlantic mackerel (Scomber scombrus).

Fish protein hydrolysates (FPH) obtained by enzymatic hydrolysis allows for smart valorization of fish side streams. However, further treatments are normally needed to enhance bioactive and functional properties of the obtained FPH. At present, the commonly used methods to improve functional properties of FPH include chemical and enzymatic modification. Chemical treatments often cause environmental problems, while the enzymatic modification method requires the use of quite expensive enzymes. In recent years, emerging technologies such as ultrasound treatment (US-treatment) have shown great potential in protein modification with high efficiency and safety, low energy consumption, and low nutritional destructiveness. In this study, high-power ultrasound treatments were applied to fish protein hydrolysates (FPH) extracted from Atlantic mackerel (Scomber scombrus) side streams to improve their quality parameters. The effect of three different treatments of 300 W, 450 W and 600 W at the operating frequency of 20 kHz for 10 min on the physicochemical, structural, and functional characteristics of FPH, were examined. The results have shown that with an increase in ultrasound power, the protein solubility of FPH increased linearly, and the changes were significant for all US-treated samples compared to control (untreated) samples. US-treatment significantly increased the degree of hydrolysis of FPH samples treated with 450 W and 600 W compared to control samples. The carbonyl content of FPH increased (significantly for 450 W and 600 W), while thiol groups decreased (significantly for 300 W and 450 W). This indicated that some US-treatments induced oxidation of FPH, however the values of the protein oxidation were low. Amino acid composition of FPH revealed that US-treatment increased the proportion of essential amino acids in the sample treated with 300 W and 450 W, but the increase was not significant. After the US-treatment, all FPH samples became lighter and less yellowish and reddish, which suggest potentially higher attractiveness to consumers. In addition, the in vitro antioxidant activity was assessed using the DPPH, FRAP, and ABTS assays and the cell-free dipeptidyl peptidase IV (DPP-IV) inhibitory activity was also measured. Moreover, these biological activities were measured at cellular level utilizing human intestinal Caco-2 cells. Specifically, the FPH capacity to lower H2O2-induced reactive oxygen species (ROS) and lipid peroxidation levels was used to measure its antioxidant activity. The findings suggest that Scomber scombrus hydrolysates could find use as ingredients for promoting health.

Assessment of drug-drug interaction of dapagliflozin with LCZ696 based on an LC-MS/MS method.

The co-administration of dapagliflozin (DPF) and sacubitril/valsartan (LCZ696) has emerged as a promising therapeutic approach for managing heart failure. Given that DPF and LCZ696 are substrates for P-glycoprotein, there is a plausible potential for drug-drug interactions when administered concomitantly. To investigate the pharmacokinetic changes when these drugs are co-administered, we have established and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of simultaneously detecting DPF, LBQ657 (the active metabolite of sacubitril) and valsartan in rat plasma. This method has demonstrated selectivity, sensitivity, and accuracy. Drug-drug interactions were examined by the LC-MS/MS method. The mechanisms were investigated using everted intestinal sac models and Caco-2 cells. The results showed that DPF significantly increased the area under the curve (AUC(0-t)) (3,563.3 ± 651.7 vs. 7,146.5 ± 1,714.9h μg/L) of LBQ657 (the active metabolite of sacubitril) and the AUC(0-t) (24,022.4 ± 6,774.3 vs. 55,728.3 ± 32,446.3h μg/L) of valsartan after oral co-administration. Dapagliflozin significantly increased the amount of LBQ657 and valsartan in intestinal sacs by 1- and 1.25-fold at 2.25 h. Caco-2 cell uptake studies confirmed that P-glycoprotein is the transporter involved in this interaction. This finding enhances the understanding of drug-drug interactions in the treatment of heart failure and provides a guidence for clinical therapy.

P06-17 Assessment of genome-wide methylation changes caused by TiO2 nanoparticles on human intestinal caco-2 cells

P06-17 Assessment of genome-wide methylation changes caused by TiO2 nanoparticles on human intestinal caco-2 cells

Apoptosis and Oxidative Stress in Human Intestinal Epithelial Caco-2 Cells Caused by Marine Phycotoxin Azaspiracid-2.

When humans consume seafood contaminated by lipophilic polyether phycotoxins, such as azaspiracids (AZAs), the toxins are mainly leached and absorbed in the small intestine, potentially causing intestinal damage. In this study, human intestinal epithelial Caco-2 cells were used to investigate the adverse effects of azaspiracid-2 (AZA-2) on human intestinal epithelial cells. Cell viability, apoptosis, oxidative damage and mitochondrial ultrastructure were investigated, and ribonucleic acid sequence (RNA-seq) analysis was applied to explore the potential mechanisms of AZA-2 toxicity to Caco-2 cells. Results showed that AZA-2 significantly reduced the proliferation of Caco-2 cells in a concentration-dependent response, and the 48 h EC50 of AZA-2 was 12.65 nmol L-1. AZA-2 can induce apoptosis in Caco-2 cells in a dose-dependent manner. Visible mitochondrial swelling, cristae disintegration, membrane rupture and autophagy were observed in Caco-2 cells exposed to AZA-2. Reactive oxygen species (ROS) and malondialdehyde (MDA) content were significantly increased in Caco-2 cells after 48 h of exposure to 1 and 10 nmol L-1 of AZA-2. Transcriptome analysis showed that KEGG pathways related to cellular oxidative damage and lipid metabolism were affected, mainly including mitophagy, oxidative phosphorylation, cholesterol metabolism, vitamin digestion and absorption, bile secretion and the peroxisome proliferator-activated receptor signaling pathway. The cytotoxic effects of AZA-2 on Caco-2 cells may be associated with ROS-mediated autophagy and apoptosis in mitochondrial cells. Results of this study improve understanding of the cytotoxicity and molecular mechanisms of AZA-2 on Caco-2 cells, which is significant for protecting human health.

Transport of perfluoroalkyl substances across human induced pluripotent stem cell-derived intestinal epithelial cells in comparison with primary human intestinal epithelial cells and Caco-2 cells

Humans can be exposed to per- and polyfluoroalkyl substances (PFASs) via many exposure routes, including diet, which may lead to several adverse health effects. So far, little is known about PFAS transport across the human intestinal barrier. In the current study, we aimed to assess the transport of 5 PFASs (PFOS, PFOA, PFNA, PFHxS and HFPO-DA) in a human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cell (IEC) model. This model was extensively characterized and compared with the widely applied human colonic adenocarcinoma cell line Caco-2 and a human primary IEC-based model, described to most closely resemble in vivo tissue. The hiPSC-derived IEC layers demonstrated polarized monolayers with tight junctions and a mucus layer. The monolayers consisted of enterocytes, stem cells, goblet cells, enteroendocrine cells, and Paneth cells that are also present in native tissue. Transcriptomics analysis revealed distinct differences in gene expression profiles, where the hiPSC-derived IECs showed the highest expression of intestinal tissue-specific genes relative to the primary IEC-based model and the Caco-2 cells clustered closer to the primary IEC-based model than the hiPSC-derived IECs. The order of PFAS transport was largely similar between the models and the apparent permeability (Papp) values of PFAS in apical to basolateral direction in the hiPSC-derived IEC model were in the following order: PFHxS > PFOA > HFPO-DA > PFNA > PFOS. In conclusion, the hiPSC-derived IEC model highly resembles human intestinal physiology and is therefore a promising novel in vitro model to study transport of chemicals across the intestinal barrier for risk assessment of chemicals.

Effect of docosahexaenoic acid as an anti-inflammatory for Caco-2 cells and modulating agent for gut microbiota in children with obesity (the DAMOCLE study).

Docosahexaenoic acid (DHA) is a long-chain omega-3 polyunsaturated fatty acid. We investigated the dual health ability of DHA to modulate gut microbiota in children with obesity and to exert anti-inflammatory activity on human intestinal Caco-2 cells. In a pilot study involving 18 obese children (8-14years), participants received a daily DHA supplement (500mg/day) and dietary intervention from baseline (T0) to 4months (T1), followed by dietary intervention alone from 4months (T1) to 8months (T2). Fecal samples, anthropometry, biochemicals and dietary assessment were collected at each timepoint. At preclinical level, we evaluated DHA's antioxidant and anti-inflammatory effects on Caco-2 cells stimulated with Hydrogen peroxide (H2O2) and Lipopolysaccharides (LPS), by measuring also Inducible nitric oxide synthase(iNOS) levels and cytokines, respectively. Ten children were included in final analysis. No major changes were observed for anthropometric and biochemical parameters, and participants showed a low dietary compliance at T1 and T2. DHA supplementation restored the Firmicutes/Bacteroidetes ratio that was conserved also after the DHA discontinuation at T2. DHA supplementation drove a depletion in Ruminococcaceae and Dialisteraceae, and enrichment in Bacteroidaceae, Oscillospiraceae, and Akkermansiaceae. At genus level, Allisonella was the most decreased by DHA supplementation. In Caco-2 cells, DHA decreased H2O2-induced reactive oxygen species (ROS) and nitric oxide (NO) production via iNOS pathway modulation. Additionally, DHA modulated proinflammatory (IL-1β, IL-6, IFN-γ, TNF-α) and anti-inflammatory (IL-10) cytokine production in LPS-stimulated Caco-2 cells. An improvement in gut dysbiosis of children with obesity seems to be triggered by DHA and to continue after discontinuation. The ability to modulate gut microbiota, matches also with an anti-inflammatory effect of DHA on Caco-2 cells.

Phosphatidylcholine Surface Hydration-Dependent Adsorption to Mucin Enhances Intestinal Mucus Barrier Function.

The crucial role of zwitterionic phosphatidylcholines (PC) within mucus gel is essential for maintaining intestinal homeostasis, while the underlying mechanism remains incompletely understood. Herein, we compared the dynamic interfacial adsorption behavior of saturated dipalmitoylphosphatidylcholine (DPPC) and unsaturated dioleoylphosphatidylcholine (DOPC) to intestinal mucin and their impact on the intestinal mucus barrier function. Results of quartz crystal microbalance with dissipation showed that the highly surface-hydrated DPPC vesicles exhibited significantly faster and more extensive adsorption to purified intestinal mucin than the slightly surface-hydrated DOPC vesicles. Utilizing an intestinal Caco-2/HT29-MTX coculture model, we observed that DPPC vesicles adsorbed much more to the mucus gel compared to DOPC vesicles. Additionally, DPPC vesicle adsorption displayed increased wetting, and converse for DOPC vesicles. Interestingly, both of them exhibited nearly the same protective effects against cell injury induced by peptic-tryptic digests of gliadin (PTG). The partial mechanism involved the binding of PTG to DPPC and DOPC within the mucus gel, thereby restricting PTG contact with the underlying epithelial cells. These findings shed light on the intricate interfacial dynamics of PC adsorption to mucin and their implications for maintaining the integrity of the intestinal mucus barrier.

Capsaicin Reduces Obesity by Reducing Chronic Low-Grade Inflammation.

Chronic low-grade inflammation (CLGI) is associated with obesity and is one of its pathogenetic mechanisms. Lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls, is the principal cause of CLGI. Studies have found that capsaicin significantly reduces the relative abundance of LPS-producing bacteria. In the present study, TRPV1-knockout (TRPV1-/-) C57BL/6J mice and the intestinal epithelial cell line Caco-2 (TRPV1-/-) were used as models to determine the effect of capsaicin on CLGI and elucidate the mechanism by which it mediates weight loss in vivo and in vitro. We found that the intragastric administration of capsaicin significantly blunted increases in body weight, food intake, blood lipid, and blood glucose in TRPV1-/- mice fed a high-fat diet, suggesting an anti-obesity effect of capsaicin. Capsaicin reduced LPS levels in the intestine by reducing the relative abundance of Proteobacteria such as Helicobacter, Desulfovibrio, and Sutterella. Toll-like receptor 4 (TLR4) levels decreased following decreases in LPS levels. Then, the local inflammation of the intestine was reduced by reducing the expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 mediated by TLR4. Attenuating local intestinal inflammation led to the increased expression of tight junction proteins zonula occludens 1 (ZO-1) and occludin and the restoration of the intestinal barrier function. Capsaicin increased the expression of ZO-1 and occludin at the transcriptional and translational levels, thereby increasing trans-endothelial electrical resistance and restoring intestinal barrier function. The restoration of intestinal barrier function decreases intestinal permeability, which reduces the concentration of LPS entering the circulation, and reduced endotoxemia leads to decreased serum concentrations of inflammatory cytokines such as TNF-α and IL-6, thereby attenuating CLGI. This study sheds light on the anti-obesity effect of capsaicin and its mechanism by reducing CLGI, increasing our understanding of the anti-obesity effects of capsaicin. It has been confirmed that capsaicin can stimulate the expression of intestinal transmembrane protein ZO-1 and cytoplasmic protein occludin, increase the trans-epithelial electrical resistance value, and repair intestinal barrier function.

Effect of pasteurization and storage on the bioavailability of milk proteins during in vitro intestinal digestion and Caco-2 cell transport

This study fills a specific knowledge gap by comprehensively investigating the intestinal digestive dynamics and transport kinetics of pasteurized milk proteins during cold storage, utilizing dynamic in vitro digestion models, human colorectal adenocarcinoma cell line (Caco-2) transport simulations, peptideomics and amino acid analysis. The content of free amino acids during intestinal digestion and their transport capacity (excluding lysine) were highest in pasteurized milk stored for 7 days (PAST-D7), intermediate in pasteurized milk stored for 0 days (PAST-D0) and lowest in raw milk (RM). Conversely, the quantity and intensity of bioactive peptides during digestion decreased with pasteurization and storage. Additionally, pasteurization and storage reduced the intensity of bioactive peptides transported through Caco-2 cells and the types and intensity of potential allergenic peptides. These findings indicate that while pasteurization and storage may diminish some physiological benefits of milk, they may also enhance protein bioavailability and reduce allergenicity.