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BACKGROUND/OBJECTIVES: Lactoferrin (Lf) is an iron-binding glycoprotein with multiple bioactivities, including promotion of cell proliferation and differentiation, immunomodulation, and antimicrobial activity. Lf, a basic glycoprotein, can bind to α-lactalbumin (α-Lac), an acidic whey protein. The current study aimed to evaluate whether Lf forms protein complexes with α-Lac and proteins/peptides from whey protein hydrolysate (WPH) and nonfat bovine milk powder (MP) and whether forming protein complexes influences resistance to gastrointestinal digestion and affects the bioactivities of Lf in human intestinal epithelial cells (HIECs and differentiated Caco-2 cells). METHODS: Lf was blended with α-Lac, WPH, or MP. Assays were conducted to evaluate the bioactivities of proteins (Lf, α-Lac, WPH, or MP) and Lf-protein blends on HIECs and Caco-2 cells. RESULTS: (1) Lf forms complexes with α-Lac and proteins/peptides from WPH and MP; (2) compared with Lf alone, complexed Lf shows greater resistance to in vitro digestion; (3) forming protein complexes does not affect Lfs binding to the Lf receptor or its uptake by HIECs; and (4) forming protein complexes does not impact Lfs bioactivities, including the promotion of cell proliferation and differentiation, reduction of cell permeability by upregulating tight-junction proteins, immune modulation through the regulation of IL-18, inhibition of enteropathogenic Escherichia coli growth, and modulation of immune responses to EPEC infection. CONCLUSIONS: Lf forms complexes with α-Lac and other milk proteins/peptides from WPH and MP in protein blends, and forming complexes does not affect the functionalities of Lf.

Intestinal permeabilization is central to the pathophysiology of chronic gut inflammation. This study investigated the efficacy of glucoraphanin (GR), prevalent in cruciferous vegetables, particularly broccoli, and its derivative sulforaphane (SF), in inhibiting tumor necrosis factor alpha (TNFα)-induced Caco-2 cell monolayers inflammation and permeabilization through the regulation of redox-sensitive events. TNFα binding to its receptor led to a rapid increase in oxidant production and subsequent elevation in the mRNA levels of NOX1, NOX4, and Duox2. GR and SF dose-dependently mitigated both these short- and long-term alterations in redox homeostasis. Downstream, GR and SF inhibited the activation of the redox-sensitive signaling cascades NF-κB (p65 and IKK) and MAPK ERK1/2, which contribute to inflammation and barrier permeabilization. GR (1 μM) and SF (0.5-1 μM) prevented TNFα-induced monolayer permeabilization and the associated reduction in the levels of the tight junction (TJ) proteins occludin and ZO-1. Both GR and SF also mitigated TNFα-induced increased mRNA levels of the myosin light chain kinase, which promotes TJ opening. Molecular docking suggests that although GR is mostly not absorbed, it could interact with extracellular and membrane sites in NOX1. Inhibition of NOX1 activity by GR would mitigate TNFα receptor downstream signaling and associated events. These findings support the concept that not only SF, but also GR, could exert systemic health benefits by protecting the intestinal barrier against inflammation-induced permeabilization, in part by regulating redox-sensitive pathways. GR has heretofore not been viewed as a biologically active molecule, but rather, the benign precursor of highly active SF. The consumption of GR and/or SF-rich vegetables or supplements in the diet may offer a means to mitigate the detrimental consequences of intestinal permeabilization, not only in disease states but also in conditions characterized by chronic inflammation of dietary and lifestyle origin.

The present study emphasized the potential antitumor activity of compound A; [Pd (Im)2Cl2], a nano‐sized coordination compound with a size of 23.76 nm. Characterization was performed using elemental analysis, molar conductance, magnetic measurement, FTIR, Ultraviolet–visible (UV–vis) spectroscopy, thermal analysis, mass spectrometry, and NMR. The complex showed stability up to 300°C. Furthermore, the compound's formation spontaneity was supported by activation parameters calculated from TGA. The di‐aqua form of the separated solid complex, compound B [Pd (Im)2(H2O)2]2+, was successfully prepared. The geometry optimization of imidazole and its complexes showed that both complexes were more stable than free imidazole. The cis di‐aqua form of [Pd (Im)2(H2O)2]2+ exhibits superior stability with a more potent IC50 value of 5.72±0.68 μg/mL. This form demonstrates greater inhibitory impacts on Caco‐2 cancer cell proliferation compared to [Pd (Im)2Cl2]. Moreover, the cytoxicity of [Pd (Im)2(H2O)2]2+ against Caco‐2 cells was confirmed by a significant increase in LDH levels in treated Caco‐2 cells compared to untreated ones. Treatment with [Pd (Im)2(H2O)2]2+ in Caco‐2 cells markedly raised the percentage of early and late apoptotic cells, accompanied by Go/G1 phase arrest with a more intense comet nucleus. Apoptosis induction in [Pd (Im)2(H2O)2]2+ treated cells was mediated through increased reactive oxygen species (ROS) production. Moreover, [Pd (Im)2(H2O)2]2+ markedly raised the expression levels of cleaved Caspase‐3, Bax, and P53 in treated Caco‐2 cells, while concurrently dropping the levels of Bcl‐2, cyclin D1, and CDK4. Overall, these findings indicate that [Pd (Im)2(H2O)2]2+ triggers significant cytotoxicity in Caco‐2 cancer cells in a dose‐dependent manner, primarily via ROS‐mediated cell death, possibly via the mitochondrial pathway. Additionally, the active species in selected proteins related to apoptosis and cell cycle arrest were explored and compared with theoretical molecular docking results. The outcomes also validated the safety of using [Pd (Im)2(H2O)2]2+ against healthy cells, suggesting it as a promising treatment option for colon cancer in humans. [ABSTRACT FROM AUTHOR]

Clostridioides difficile is the leading cause of healthcare- and antibiotic-associated diarrhea. Surface layer protein A (SlpA), an essential component of the bacterium's outermost layer, contributes to colonization and inflammation. The peroxisome proliferator-activated receptor gamma (PPAR-γ) has been demonstrated to improve intestinal integrity and prevent inflammation in host cells. Here, we investigated the role of PPAR-γ in SlpA-mediated inflammation in Caco-2 cells and THP-1 derived macrophages. The extraction of SlpA was carried out for three toxigenic C. difficile clinical strains (RT126, RT001, RT084) and a non-toxigenic strain (ATCC 700057). The gene expression of tight junction (TJ) proteins and inflammatory markers was determined using RT-qPCR. The production of proinflammatory cytokines and nitric oxide was measured by ELISA and Griss reaction, respectively. Western blotting was performed to detect PPAR-γ level before and after adding its agonist, pioglitazone. SlpA of C. difficile strains enhanced the expression of TLR-4, NF-κB, MyD88, IL-17, MCP-1, IL-8, IL-6, TNF-α, IL-1β, whilst the gene expression level of JAM-A, claudin-1, occludin, PPAR-γ and its receptor (CD36) was decreased in both Caco-2 cells and THP-1 derived macrophages. Moreover, pioglitazone caused a notable elevation in the expression level of PPAR-γ, only following treatment with RT126 SlpA. Besides, pioglitazone pretreatment improved TJ impairment in Caco-2 cells and attenuated proinflammatory cytokine expression in both SlpA-treated cell lines. SlpA can attenuate PPAR-γ expression, trigger TJ disruption, and stimulate inflammatory response in host cells. Notably, these events could be reversed by pretreatment of cells with PPAR-γ agonist. Further experiments are required to corroborate the present findings. [ABSTRACT FROM AUTHOR]

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Electrospun nanofibers offer a highly promising platform for the delivery of vaginal lactobacilli, providing an innovative approach to preventing and treating vaginal infections. To advance the application of nanofibers for the delivery of lactobacilli, tools for studying their safety and efficacy in vitro need to be established. In this study, fluorescent (mCherry and GFP) and luminescent (NanoLuc luciferase) proteins were expressed in three vaginal lactobacilli (Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii) and a control Lactiplantibacillus plantarum with the aim to use this technology for close tracking of lactobacilli release from nanofibers and their adhesion on epithelial cells. The recombinant proteins influenced the growth of the bacteria, but not their ability to produce hydrogen peroxide. Survival of lactobacilli in nanofibers immediately after electrospinning varied among species. Bacteria retained fluorescence upon incorporation into PEO nanofibers, which was vital for evaluation of their rapid release. In addition, fluorescent labelling facilitated efficient tracking of bacterial adhesion to Caco-2 epithelial cells, while luminescence provided important quantitative insights into bacterial attachment, which varied from 0.5 to 50% depending on the species. The four lactobacilli in dispersion or in nanofibers were not detrimental for the viability of Caco-2 cells, and did not demonstrate hemolytic activity highlighting the safety profiles of both bacteria and PEO nanofibers. To summarize, this study contributes to the development of a promising delivery system, tailored for local administration of safe vaginal lactobacilli. [ABSTRACT FROM AUTHOR]

Walnut milk residues (WMR) were investigated for the first time through their phenolic characterization including soluble (free, esterified, and etherified) phenolics and those released from their insoluble-bound form (insoluble-bound phenolic hydrolysates, IBPHs) and their antioxidant properties. Free phenolics were recovered and alkaline or acid hydrolysis were used to recover the remaining phenolic fractions. Total phenolic compounds (TPCs) and their antioxidant activity were analyzed by Folin–Ciocalteu, FRAP, and ORAC methods, respectively. Soluble phenolics (free + esterified + etherified fractions) showed a higher TPC (275.3 mg GAE 100 g−1 dw) and antioxidant activity (FRAP: 138.13 µmol TE g−1 dw; ORAC: 45.41 µmol TE g−1 dw) with respect to the IBPH. There was a significant correlation between TPC and FRAP and ORAC values regardless of the fraction and tested sample. Phenolic acids and flavonoids were identified and quantified by ultra-performance liquid chromatography–electrospray tandem mass spectrometry (UPLC-ESI-MS/MS). Gallic acid, mainly in the free form (3061.0 µg 100 g−1), was the most representative, followed by biochanin A, identified for the first time in a walnut product and mostly present in the fraction released from the esterified form (593.75 µg 100 g−1). No detrimental cytotoxic impact on Caco-2 cells was observed. Hence, WMR could be considered a potential source for the development of nutraceutical and/or antioxidant food additives. [ABSTRACT FROM AUTHOR]

Squalene, a triterpene found in extra virgin olive oil, has therapeutic properties in diseases related to oxidative stress, such as cancer. However, its hydrophobic nature and susceptibility to oxidation limit its bioavailability outside of olive oil. To expand its applications, alternative delivery methods are necessary. The objective of the present study was to examine the impact of squalene encapsulated in PLGA (poly(lactic-co-glycolic) acid) nanoparticles (PLGA + Sq) on the proliferation of human colon carcinoma Caco-2 cells, as well as its underlying mechanism of action. The findings demonstrated that PLGA + Sq exert no influence on differentiated cells; however, it is capable of reducing the proliferation of undifferentiated Caco-2 cells through apoptosis and cell cycle arrest in the G1 phase. This effect was initiated by the release of cytochrome c into the cytoplasm and the subsequent activation of caspase-3. Furthermore, squalene exhibited pro-oxidant activity, as evidenced by an increase in intracellular ROS (reactive oxygen species) levels. The results of the squalene effect on genes associated with cell death, inflammation, and the cell cycle indicate that its antiproliferative effect may be post-transcriptional. In conclusion, PLGA + Sq demonstrate an antiproliferative effect on Caco-2 cells through apoptosis by altering redox balance, suggesting squalene's potential as a functional food ingredient for colorectal cancer prevention. [ABSTRACT FROM AUTHOR]

Background and Objectives: The most common cause of severe foodborne salmonellosis is S. Typhimurium. Its interaction with intestinal epithelial cells is little known. Lactic acid bacteria (LAB) were recognized as a prominent probiotic gastrointestinal microbiota of humans and animals that confer health-promoting and protective effects. This study aims to determine the anti-invasion and antibacterial effects of heat-killed LAB (HK-LAB) isolates against S. Typhimurium towards human intestinal cells. Materials and Methods: 12 HK-LAB isolates from 3 sources of origin (stingless bee, plant, and food) were tested to determine the adhesion of HK-LAB to Caco-2 cells, anti-invasion and antibacterial activities against S. Typhimurium, the adhesion and invasion pattern of S. Typhimurium on intestinal epithelial cells (Caco-2) and assessing the effect of LAB on the S. Typhimurium-host cell interaction. Results: Tairu isolates from food have the highest adhesion rate with 19 ± 1.32/10 Caco-2 cells followed by HK-LAB R-isolate from plant 17 ± 0.70/10 Caco-2 cells, which is similar to the control (Lactobacillus casei). In the anti-invasion assay, the two HK-LAB isolates that had the strongest adherence to Caco-2 cells, Tairu-isolate inhibited at 78.1 ± 3.06% and R-isolate inhibited at 64.76 ± 9.02% compared to the positive control (63.81 ± 1.15%), which led to increased suppression of S. Typhimurium accordingly. Tairu and R isolates were tested for their antibacterial ability against S. Typhimurium. Both R and Tairu isolates displayed strong inhibition zones (27 ± 0.06 mm, 23 ± 0.06 mm) respectively. Conclusion: These findings suggest that the anti-invasion activities of HK-LAB R and Tairu may correlate to their bactericidal effects that serve to protect the host from infection. [ABSTRACT FROM AUTHOR]

BACKGROUND: The supplemental effect of zinc depends not only on adequate intake, but also on how efficiently it is absorbed in the small intestine. In the present study, weak hydrophobic peptides (WHP), strong hydrophobic peptides (SHP), positively charged peptides (PCP) and negatively charged peptides (NCP) were isolated from soybean peptides (SP). The peptide‐Zn complexes (PCP‐Zn, NCP‐Zn, WHP‐Zn, SHP‐Zn and SP‐Zn) were prepared to compare their promotion zinc absorption capacity in the Caco‐2 cells monolayers model. RESULTS: We found that the carboxyl, carbonyl and amino groups in peptide were the primary binding sites of Zn. Compared with zinc sulfate, the peptide‐Zn complexes with different charge and hydrophobic peptides could improve zinc solubility at different pH. NCP‐Zn had a lower Zn‐binding capacity but a higher zinc absorption capacity compared to that of PCP‐Zn in Caco‐2 cells. In addition, the capacity of PCP‐Zn to promote zinc absorption was lower than the control group (SP‐Zn). There were no significant differences in transport rates, retention rates and uptake rates of WHP‐Zn, SHP‐Zn and SP‐Zn. NCP‐Zn could improve the activity of Zn‐related enzymes, and the expression levels of PepT1 and ZnT1 were higher than other peptide‐Zn complexes. CONCLUSION: The promotion zinc absorption capacity of peptide‐Zn complexes was not completely dependent on the Zn‐binding capacity, but also depended on the charge and hydrophobicity of peptides. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

One of the leading causes of cancer-related deaths worldwide is colorectal cancer (CRC). Extracellular ATP (e-ATP) and purinergic receptors (P2R) play a central role in CRC proliferation and progression. Human antigen R (HuR) is becoming more and more understood to be essential for the expression of genes linked to cancer. The current study demonstrates that ATP can mediate CRC (Caco-2 cells) progression via induction of HuR nucleocytoplasmic shuttling and subsequent expression of cancer-related genes, a consequence mostly mediated via the P2R receptor. It was also noted that suppression of HuR activity by using dihydrotanshinone I (DHTS) prevents cancer-related gene expression and subsequent CRC (Caco-2 cells) progression induced by ATP. The expression of cyclin A2/cyclin-dependent kinase 2 (CDK2), Bcl-2, ProT-α, hypoxia‐inducible factor1-α (HIF1-α), vascular endothelial growth factor A (VEGF-A), transforming growth factor-β (TGF-β) and matrix metallopeptidase 9 (MMP-9) induced by ATP were highly reduced in the presence of either PPADS (non-selective P2R antagonist) or DHTS. In addition, e-ATP-induced Caco-2 cell proliferation as well as cell survival were highly reduced in the presence of either PPADS or DHTS or selective CDK-2 inhibitor (Roscovitine) or selective Bcl-2 inhibitor (ABT-263). Furthermore, it was found that MMP-9 is critical for Caco-2 cells migration induced by e-ATP as demonstrated by a clear reduction in cells migration in the presence of a selective MMP-9 inhibitor (Marimastat). Collectively, these data demonstrate that ATP through P2R activation can induce HuR nucleocytoplasmic shuttling that could be translated into an increase in cancer-related genes expression and subsequent, cell proliferation and progression. [ABSTRACT FROM AUTHOR]

Damage to intestinal epithelial cells is present in obesity and other diseases because of inflammatory and oxidative processes. This damage compromises the gastrointestinal barrier, killing enterocytes, altering intestinal permeability, and eliciting abnormal immune responses that promote chronic inflammation. Recent evidence shows that spirulina is a potent natural agent with antioxidant and anti-inflammatory properties. Objectives: This study was conducted to evaluate the effect of spirulina aqueous extract (SPAE) on the alterations of the intestinal epithelium induced by lipid micelles (LMs) and/or inflammation induced by lipopolysaccharides (LPSs) in the Caco-2 cell line. Methods: In the current research, we assessed the protective actions of SPAE against cytotoxicity, oxidative stress, inflammation, and epithelial barrier perturbation by using an in vitro model, the intestinal Caco-2 cells, treated with LPSs and/or LMs. We also performed an in silico molecular docking analysis with spirulina's bioactive compound, phycocyanobilin. Results: Our results showed that SPAE has no cytotoxic effect on Caco-2 cells. On the contrary, it improved cell viability and exhibited anti-inflammatory and antioxidant actions. SPAE also protected against endoplasmic reticulum stress and tight junction proteins, thus improving the epithelial barrier. The in silico study revealed a strong binding affinity of the phycocyanobilin compound with human SOD and NADPH oxidase and a good binding affinity towards COX-2 and iNOS. Conclusions: Taken together, these findings demonstrate the beneficial actions of SPAE on Caco-2 cells, suggesting it may be useful in preserving the epithelial intestinal barrier in human conditions involving oxidative stress and inflammation such as obesity. [ABSTRACT FROM AUTHOR]

The oral bioavailability of curcumin is inherently low, which significantly limits its application in food systems. The objective of this study was to evaluate the impact of high-pressure processing on the stability and bioaccessibility of curcumin within an emulsion gel during simulated gastrointestinal transit and to assess its cellular uptake. Our findings suggest that increasing pressure levels and high κ-carrageenan concentrations can enhance the stability of the curcumin delivery system. Elevated κ-CG concentrations were found to retard the action of proteases on dissociating protein molecules from the gel network. The emulsion gel effectively slowed the release of free fatty acids and reduced the curcumin release rate during the gastric phase. Scanning electron microscopy images revealed that higher pressures induced the formation of a more uniform and dense network structure in the gel. While the gel network structures were well-preserved after gastric digestion, they were disrupted into smaller particles following intestinal digestion, with particle size increasing with higher applied pressures. Cytotoxicity assays indicated that the digesta from the intestinal phase was highly toxic to Caco-2 cells. Among the tested samples, the emulsion gel prepared with 1.0% κ-CG at 600 MPa demonstrated the highest curcumin bioavailability, reaching 63.82 ± 7.10%. These findings underscore the potential of HPP-induced emulsion gels as a viable delivery system for enhancing curcumin bioaccessibility and cellular uptake. [ABSTRACT FROM AUTHOR]

The digestion and absorption properties of 1,3-dipalmitoyl-2-oleoyl glycerol (POP)-rich lipids was evaluated using in vitro gastrointestinal digestion and a Caco-2 cell-mediated coupled model. Caco-2 cell viability and monolayer integrity were assessed by an MTT assay and transepithelial electrical resistance. The IC50 for bile salts, pancreatin, and free fatty acid (FFA) were 0.22 mM, 0.22 mg/mL, and 1.47 mM, respectively, and no cytotoxicity was observed for bovine serum albumin (0.01–0.20 mM) or triacylglycerol (1.00–10.00 mM). The in vitro-digested POP-rich lipid containing FFA > 2.95 mM caused the disruption of monolayer tight junctions in Caco-2 cells. The major triacylglycerols (TAG) of POP-rich lipids were POP (50.8%), POO (17.8%), POL/OPL/PLO (7.6%), PPO (7.1%), and PLP (6.8%). Following digestion and uptake into Caco-2 cells, the resynthesized TAGs included PPO (20.6%), PPP (15.9%), POO (14.0%), POL/OPL/PLO (12.2%), POP (10.9%), OOO (7.5%), OPO (7.0%), OOL/OLO (6.7%), PLP (3.1%), and PPL (2.2%). The secreted major TAGs were POL/OPL/PLO (50.8%), PPP (11.1%), and OOL/OLO (8.4%), indicating a diverse TAG profile in newly synthesized lipids. This study provides a coupled model for lowering cytotoxicity and maintaining the monolayer in Caco-2 cells, and for evaluating the digestion and absorption properties of functional lipids containing specific fatty acids incorporated into TAG. [ABSTRACT FROM AUTHOR]

Infection with human immunodeficiency virus (HIV) increases risk for maladies of the gut barrier, which promotes sustained systemic inflammation even in virally controlled patients. We previously revealed morphological disorganization of colon epithelial barrier proteins in HIV-1 transgenic (Tg) rats. The current study evaluated mechanisms that may underlie gut barrier pathology induced by toxic HIV-1 proteins. Methamphetamine (meth) use is prevalent among HIV-infected individuals, and meth can exaggerate morbidity of HIV infection. Thus, we determined whether meth exposure worsened HIV-associated gut pathology using colon samples from HIV-1 Tg and non-Tg rats that self-administered meth 2 h/day for 21 days. Immunoblotting was conducted for occludin (a gut barrier protein) and matrix metalloproteinase-9 (MMP-9; a proteinase regulator of occludin). Colon levels of occludin were decreased, and MMP-9 levels and activity were increased in HIV-1 Tg rats. A Pearson correlation revealed an inverse relationship between occludin levels and MMP-9 activity. Doses of meth that were self-administered by Tg rats were lower than other rat models. Meth-induced trends in non-Tg rats were not significant, and meth did not exaggerate effects seen in Tg rats. Accordingly, only the HIV-effects on epithelial function were explored further. Transepithelial resistance (TER) across a monolayer of human colon epithelial cells (Caco-2) was used to examine treatments with the HIV-1 toxic protein, Tat, and the ability of pioglitazone, a PPARγ agonist that inhibits MMP-9, to mitigate Tat-induced changes. Exposure to Tat for 24 h decreased TER, which co-occurred with decreases in levels of barrier tight junction proteins (occludin, claudin-1, and zonula occludens-1) and with increases in the level and activity of MMP-9. Pretreatment or post-treatment with pioglitazone respectively prevented and restored Tat-induced impairments of Caco-2 barrier. Thus, while low doses of meth did not alter barrier proteins in the current study, exposure to HIV-1 proteins disrupted the gut barrier, and this action involved a dysregulation of MMP-9. [ABSTRACT FROM AUTHOR]

This study aimed to analyze the contributions of multiple transport mechanisms to the intestinal uptake of serotonin (5-HT) by employing a variety of in vitro experimental techniques, focusing on organic cation transporters expressed in the gastrointestinal (GI) tract, such as SERT, PMAT, THTR2, OCT3, and OCTN2. Analysis of the concentration dependence of 5-HT uptake by Caco-2 cells revealed multi-affinity kinetics with high-affinity and low-affinity components, suggesting that multiple transporters are involved in the intestinal 5-HT uptake. Comparative analysis of transporters using K m values obtained in Xenopus oocyte expression systems suggested that SERT is responsible for the high-affinity transport, while PMAT, THTR2, and OCT3 contribute to the low-affinity transport. Further analysis indicated that the relative contributions of SERT and PMAT to the intestinal 5-HT uptake (0.01 µM) are approximately 94.9% and 1.1%, respectively. Interestingly, at the concentration of 10 µM, the reported steady-state concentration of 5-HT in the human colon, the contributions of SERT, PMAT, THTR2, and OCT3 were estimated to be approximately 37.0%, 1.0%, 18.2%, and 20.5%, respectively. In conclusion, the present study indicated that the contributions of multiple transporters to 5-HT uptake in the GI tract are dependent upon the colon luminal concentration of 5-HT. [ABSTRACT FROM AUTHOR]

Caco-2 cells, a human colorectal adenocarcinoma cell line, are widely used to model small intestinal epithelial cells in the drug development process because they can predict drug absorption with high accuracy. However, Caco-2 cells have several issues. First, Caco-2 cells have little expression of cytochrome P450 3A4 (CYP3A4), which is a major drug-metabolizing enzyme in the human intestine. We previously developed Caco-2 cells whose expression of CYP3A4 can be controlled using doxycycline (Dox) (CYP3A4-Caco-2 cells) (Ichikawa et al., Sci. Rep, 2021). However, since the Tet-On system was used to regulate CYP3A4 expression in these cells, there was concern about drug-drug interactions. The second issue is that in the human small intestine, carboxylesterase 2 (CES2) is more highly expressed than carboxylesterase 1 (CES1), while in Caco-2 cells CES1 is more highly expressed. The third issue is the low level expression of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), a phase II drug-metabolizing enzyme. In this study, we used genome-editing technology to establish CES1 -knockout Caco-2 cells whose CYP3A4 and UGT1A1 expression can be regulated by the Tet-Off system. These cell lines would be useful in pharmaceutical researches, including intestinal toxicological studies, as an in vitro model for orally administered drugs. [Display omitted] • CES1-KO/CYP3A4 Caco-2 cells and CES1-KO/CYP3A4-UGT1A1 Caco-2 cells are established. • CES1-KO/CYP3A4 Caco-2 cells can be used to predict absorption of prodrugs metabolized by CES. • CES1-KO/CYP3A4 Caco-2 cells have the potential to predict the metabolism of orally administered drugs involving in CYP3A4 in the small intestine. • CES1-KO/CYP3A4-UGT1A1 Caco-2 cells have the potential to predict the metabolism of orally administered drugs involving in both CYP3A4 and UGT1A1 in the small intestine. [ABSTRACT FROM AUTHOR]

Soybean (Glycine max [L.] Merr.), one of the top 10 crops produced worldwide, is a significant dietary source of isoflavones. Understanding the bioaccessibility and bioavailability of soy isoflavones (SIs) is important fgor comprehending their various bioactivities in the human body. In vitro approaches can provide valuable insights into the bioavailability of SIs across food matrices and their step-by-step digestive mechanisms. This review focused on evaluating reports that employ invitro methods to study the bioavailability of SIs, using human-simulated gastrointestinal digestive system models and the Transwell system with Caco-2 cell monolayers. Specifically, this review discusses how the molecular structures of SIs influence their bioaccessibility and bioavailability. The differences in bioaccessibility and bioavailability of SI aglycones and their conjugated SIs indicate that the molecular structure is a critical factor influencing the bioactivities of SIs. This review provides important information for evaluating and understanding the physiological effects of soybean or SI-containing foods. [ABSTRACT FROM AUTHOR]

The increasing incidence of colorectal cancer and inflammatory diseases poses a major health concern, with oxidative stress playing a significant role in the onset of these pathologies. Factors such as excessive consumption of sugar-rich and fatty foods, synthetic food additives, pesticides, alcohol, and tobacco contribute to oxidative stress and disrupt intestinal homeostasis. Functional foods arise as a potential tool to regulate redox balance in the intestinal tract. Herbs (such as Thymus spp.) have long been screened for their antioxidant properties, but their use as antioxidants for medicinal purposes requires validation in biological models. In this study, we addressed the potential antioxidant protection and preventive effects of extracts from two thyme species at the intestinal level, as well as their molecular mechanisms of action. Caco-2 cells were pre-exposed (4 h) to aqueous (AD) and hydroethanolic (HE) extracts of Thymus carnosus and Thymus capitellatus, followed by a recovery period in culture medium (16 h), and then treated with tert-butyl-hydroperoxide (TBHP; 4 h), before analyzing cell viability. The effect of the extracts' main components was also analysed. Cellular oxidative stress, cell-death markers, and the expression of antioxidant-related proteins were evaluated using flow cytometry on cells pre-exposed to the AD extracts and salvianolic acid A (SAA). Results showed that pre-exposure to AD extracts or SAA reduced TBHP-induced oxidative stress and cell death, mediated by increased levels of nuclear factor erythroid 2-related factor 2 (Nrf2) protein. The protective activity of T. capitellatus AD extract was shown to be dependent on NAD(P)H quinone dehydrogenase 1 (NQO1) protein expression and on increased glutathione (GSH) content. Furthermore, ursolic acid induced cytotoxicity and low cellular antioxidant activity, and thus the presence of this triterpenoid impaired the antioxidant effect of HE extracts. Thus, AD extracts show high potential as prophylactic dietary agents, while HE extracts arise as a source of nutraceuticals with antioxidant potential. [ABSTRACT FROM AUTHOR]

Objective: To optimize the preparation method of Hericium erinaceus-derived extracellular nanovesicles and investigate their antioxidant activity and protective effect against oxidative damage in Caco-2 cells. Methods: Hericium erinaceus-derived extracellular nanovesicles (HEDENVs) were isolated from fresh Hericium erinaceus by ultracentrifugation and sucrose gradient ultracentrifugation. The optimal extraction process of HEDENVs was optimized by one-way test and response surface test, while the particle size, Zeta potential, and morphology of HEDENVs were characterized by a laser particle size instrument and transmission electron microscope. Lipidomics and proteomics analysis of HEDENVs was performed by liquid chromatography-mass spectrometry. The in vitro antioxidant activity of HEDENVs was evaluated by determining the DPPH, ABTS+, and O2− radical scavenging capacity. A Caco-2 cell oxidative stress damage model was created using H2O2, and changes in ROS content in cells were measured using laser confocal microscopy. Results: The optimal extraction process of HEDENVs was the 25% interface of the sucrose concentration, centrifugation time was 2 h, and centrifugal speed was 86000 r/min, at which the concentration of HEDENVs was 402.24 μg/mL. HEDENVs were nanovesicles with a lipid bilayer membrane, the particle size was 110.7±16.9 nm, PDI=0.551, and the Zeta potential was −14.2 mV. Based on the analysis of lipidomics and proteomics, 346 lipids species and 52 proteins were identified in HEDENVs. The results of in vitro antioxidant test showed that the scavenging rate of 400 μg/mL HEDENVs for DPPH free radical was 94.79%, the scavenging rate for ABTS+ free radical was 96.67%, and the scavenging rate for O2− free radical was 96.83%. An oxidative damage model in Caco-2 cells was established by H2O2, HEDENVs could significantly reduce the production of ROS in damaged cells. Conclusion: It showed that HEDENVs had certain antioxidant activity in various antioxidant experiment.

Abstract Epithelial tissues in vitro undergo dynamic changes while differentiating heterogeneously on the culture substrate. This gives rise to diverse cellular arrangements which are undistinguished by conventional analysis approaches, such as transepithelial electrical resistance measurement or permeability assays. In this context, solid substrate-based systems with integrated electrodes and electrochemical impedance monitoring capability can address the limited spatiotemporal resolution of traditional porous membrane-based methods. This label-free technique facilitates local, continuous, long-term analysis of tissue barrier properties for organ-on-chip applications. Increasing spatial resolution requires small electrodes arranged in a dense array, known as high-density microelectrode arrays (HD-MEAs). Integrated with Complementary Metal Oxide Semiconductor (CMOS) technology for multiplexing and rapid impedance measurements, HD-MEAs can enable high spatiotemporal resolution assessments of epithelial tissues. Here, we used 16,384 CMOS-integrated HD-MEA chip with subcellular-sized electrodes (8 μm diameter, 15 μm pitch, patterned in 16 clusters each consisting of 1024 electrodes in a 32 × 32 matrix) and impedance sensing capability to monitor dynamic evolution of Caco-2 cells, such as their proliferation, barrier formation, and 3D structure development on the chip. Changes in impedance at the selected frequency of 1 kHz (|Z| 1kHz ) enabled monitoring and analyzing the life cycle of Caco-2 cells grown on the HD-MEA chips (up to + 453% change after 7 days in culture). The |Z| 1kHz maps of proliferating Caco-2 cells and the differentiating epithelial tissue developing 3D domes aligned with the corresponding optical images at cellular resolution, which demonstrates the capability of the chip in tracking the dynamic heterogeneity of Caco-2 tissues in a label free and real-time fashion. Importantly, |Z| 1kHz maps acquired during chemically induced barrier disruption showed electrodes covered with 3D cell domes experienced a stronger decrease in impedance than those covered with adherent cells (-41% ± sd 10% against -16% ± sd 10%, respectively). This method could thus, in principle, enable detection of tissue barrier disrupting and modifying agents with higher specificity. Epithelial barrier function assays benefit from using HD-MEA impedance sensors due to their increased informativity and resolution, which will be of great value in future organ-on-chip platforms.

BACKGROUND & AIMS: Lacticaseibacillus rhamnosus GG (LGG) is the worlds most consumed probiotic but its mechanism of action on intestinal permeability and differentiation along with its interactions with an essential source of signaling metabolites, dietary tryptophan (trp), are unclear. METHODS: Untargeted metabolomic and transcriptomic analyses were performed in LGG monocolonized germ-free mice fed trp-free or -sufficient diets. LGG-derived metabolites were profiled in vitro under anaerobic and aerobic conditions. Multiomic correlations using a newly developed algorithm discovered novel metabolites tightly linked to tight junction and cell differentiation genes whose abundances were regulated by LGG and dietary trp. Barrier-modulation by these metabolites were functionally tested in Caco2 cells, mouse enteroids, and dextran sulfate sodium experimental colitis. The contribution of these metabolites to barrier protection is delineated at specific tight junction proteins and enterocyte-promoting factors with gain and loss of function approaches. RESULTS: LGG, strictly with dietary trp, promotes the enterocyte program and expression of tight junction genes, particularly Ocln. Functional evaluations of fecal and serum metabolites synergistically stimulated by LGG and trp revealed a novel vitamin B3 metabolism pathway, with methylnicotinamide (MNA) unexpectedly being the most robust barrier-protective metabolite in vitro and in vivo. Reduced serum MNA is significantly associated with increased disease activity in patients with inflammatory bowel disease. Exogenous MNA enhances gut barrier in homeostasis and robustly promotes colonic healing in dextran sulfate sodium colitis. MNA is sufficient to promote intestinal epithelial Ocln and RNF43, a master inhibitor of Wnt. Blocking trp or vitamin B3 absorption abolishes barrier recovery in vivo. CONCLUSIONS: Our study uncovers a novel LGG-regulated dietary trp-dependent production of MNA that protects the gut barrier against colitis.

In this study, the effect of Bangia fusco-purpurea polysaccharide (BFP) on Escherichia coli adhesion to Caco-2 monolayers and its potential mechanism was studied using an in vitro human colon adenocarcinoma cell (Caco-2) monolayer model. The effect of BFP on the adhesion of E. coli to Caco-2 monolayers was analyzed by fluorescence labeling with carboxyfluorescein diacetate and succinimidyl ester (CFDA-SE), and its effect on the gene expression of integrin β1 in Caco-2 cells, the adhesin FimH in E. coli, and inflammatory factors (IL-1β, IL-8 and TNF-α) induced by E. coli adhesion to Caco-2 cells and the tight junction proteins zonula occludens 1 (ZO-1) and occludin were analyzed using real-time polymerase chain reaction (real-time PCR). Also, the protein expression of ZO-1 and occludin in Caco-2 monolayers was detected using Western Blot analysis. Our results showed that BFP at concentrations of 400–800 μg/mL significantly inhibited the adhesion of E. coli to Caco-2 monolayers mainly by down-regulating the gene expression of integrin β1 in Caco-2 cells and FimH in E. coli. In addition, BFP significantly inhibited the up-regulation of inflammatory cytokine gene expression and the down-regulation of ZO-1 and occludin protein and gene expression induced by in Caco-2 cells induced by E. coli and its culture supernatant. In conclusion, BFP inhibited the adhesion of E. coli to Caco-2 cell monolayers, which will lay a scientific basis for the development of novel antimicrobial products and the high-value utilization and deep processing of BFP.

Objective: We tested the effect of 27-hydroxycholesterol (27OHC) on the expression and synthesis of two membrane transporters involved in sterols extrusion from the intestinal epithelium into the gut lumen: ATP-binding cassette A1 (ABCA1) and G8 (ABCG8). Special attention was given to ABCG8, a key player in the intestinal cell discharge of plant sterols. Methods: Differentiated CaCo-2 intestinal cells were supplemented with 27OHC, and added to the cell incubation medium at a final concentration of 1 or 5 μM. These 27OHC externally added amounts were proven to reach intracellular oxysterol levels within the range of those normally recovered in the human peripheral blood. Results: An up-regulation of the ABCA1 and ABCG8 mRNAs was observed in the CaCo-2 cells supplemented with 27OHC. Moreover, both 1 μM and 5 μM 27OHC induced a net, and steady, statistically significant, increase of both ABCA1 and ABCG8 protein levels. Of interest, the cellular pre-treatment with diphenylene iodonium, a selective inhibitor of NADPH oxidase, i.e. a major intracellular source of reactive oxygen species, fully inhibited the 27OHC enhancement of both ABCA1 and ABCG8 protein synthesis. Conclusion: This in vitro study shows for the first time that the addition of 27OHC to intestinal epithelial cells up-regulates ABCG8, the transporter discharging plant sterols into the gut lumen, besides confirming to induce ABCA1 as well. Importantly, the 27OHC-dependent up-regulation of the two transporters appears to involve a redox mechanism rather than the canonical liver-X-receptors-dependent pathway. Significance statement The 27OHC introduced with the diet might modulate the plant sterol extrusion in the gut, in parallel with that of cholesterol.