Human Intestinal Caco-2 Cells Research Articles

Cholesterol uptake in the intestine is regulated by the LASP1-AKT-NPC1L1 signaling pathway.

Cholesterol is essential for the stability and architecture of the plasma membrane and a precursor of bile acids and steroid hormones in mammals. Excess dietary cholesterol uptake leads to hypercholesterolaemia, atherosclerosis and plays a role in cancer development. The role of actin-binding scaffolding protein LIM and SH3 protein 1 (LASP1) in cholesterol trafficking has not been investigated previously. Cholesterol levels, its uptake and excretion were studied in mice deficient for low density lipoprotein receptor and Lasp1 (Ldlr-/-Lasp1-/- mice) upon feeding a high fat diet, and in LASP1-knockdown, differentiated human intestinal epithelial Caco-2 cells. Compared to diet-fed Ldlr-/- control mice, Ldlr-/-Lasp1-/- mice displayed a reduction in serum cholesterol levels. Mechanistically, we identified a new role of LASP1 in controlling the translocation of the intestinal cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) to the apical cell surface, which was limited in LASP1-knockdwon human CaCo-2 enterocytes and in the intestine of Ldlr-/- Lasp1-/- compared to Ldlr-/-mice, linked to LASP1-pAKT signaling but not CDC42 activation. In line, a reduction in cholesterol reabsorption was noted in LASP1-knockdown CaCo-2 cells in vitro, and an enhanced cholesterol excretion via the feces was observed in Ldlr-/- Lasp1-/- mice. These data uncover a novel function of Lasp1 in cholesterol trafficking, promoting cholesterol reabsorption in the intestine. Targeting LASP1 locally could thus represent a novel targeting strategy to ameliorate hypercholesterolemia and associated diseases.

Antioxidant and anti-inflammatory function of Eupatorium adenophora Spreng leaves (EASL) on human intestinal Caco-2 cells treated with tert-butyl hydroperoxide

Chronic non-communicable diseases (CNCDs) pose a significant public health challenge. Addressing this issue, there has been a notable breakthrough in the prevention and mitigation of NCDs through the use of antioxidants and anti-inflammatory agents. In this study, we aim to explore the effectiveness of Eupatorium adenophora Spreng leaves (EASL) as an antioxidant and anti-inflammatory agent, and its potential applications. To construct a cellular model of oxidative damage and inflammation, Caco-2 cells were treated with tert-butyl hydroperoxide (t-BHP). The biocompatibility of EASL-AE with Caco-2 cells was assessed using the MTT assay, while compatibility was further verified by measuring LDH release and the protective effect against oxidative damage was also assessed using the MTT assay. Additionally, we measured intracellular oxidative stress indicators such as ROS and 8-OHdG, as well as inflammatory pathway signalling protein NFκB and inflammatory factors TNF-α and IL-1β using ELISA, to evaluate the antioxidant and anti-inflammatory capacity of EASL-AE. The scavenging capacity of EASL-AE against free radicals was determined through the DPPH Assay and ABTS Assay. Furthermore, we measured the total phenolic, total flavonoid, and total polysaccharide contents using common chemical methods. The chemical composition of EASL-AE was analyzed using the LC–MS/MS technique. Our findings demonstrate that EASL-AE is biocompatible with Caco-2 cells and non-toxic at experimental levels. Moreover, EASL-AE exhibits a significant protective effect on Caco-2 cells subjected to oxidative damage. The antioxidant effect of EASL-AE involves the scavenging of intracellular ROS, while its anti-inflammatory effect is achieved by down-regulation of the NFκB pathway. Which in turn reduces the release of inflammatory factors TNF-α and IL-1β. Through LC–MS/MS analysis, we identified 222 compounds in EASL-AE, among which gentianic acid, procaine and L-tyrosine were the compounds with high antioxidant capacity and may be the effective constituent for EASL-AE with antioxidant activity. These results suggest that EASL-AE is a natural and high-quality antioxidant and anti-inflammatory biomaterial that warrants further investigation. It holds great potential for applications in healthcare and other related fields.

TLR7-dependent eosinophil degranulation links psoriatic skin inflammation to small intestinal inflammatory changes in mice.

Recent evidence of gut microbiota dysbiosis in the context of psoriasis and the increased cooccurrence of inflammatory bowel disease and psoriasis suggest a close relationship between skin and gut immune responses. Using a mouse model of psoriasis induced by the Toll-like receptor (TLR) 7 ligand imiquimod, we found that psoriatic dermatitis was accompanied by inflammatory changes in the small intestine associated with eosinophil degranulation, which impaired intestinal barrier integrity. Inflammatory responses in the skin and small intestine were increased in mice prone to eosinophil degranulation. Caco-2 human intestinal epithelial cells were treated with media containing eosinophil granule proteins and exhibited signs of inflammation and damage. Imiquimod-induced skin and intestinal changes were attenuated in eosinophil-deficient mice, and this attenuation was counteracted by the transfer of eosinophils. Imiquimod levels and the distribution of eosinophils were positively correlated in the intestine. TLR7-deficient mice did not exhibit intestinal eosinophil degranulation but did exhibit attenuated inflammation in the skin and small intestine following imiquimod administration. These results suggest that TLR7-dependent bidirectional skin-to-gut communication occurs in psoriatic inflammation and that inflammatory changes in the intestine can accelerate psoriasis.

New Approach Methods to Assess the Enteropathogenic Potential of Strains of the Bacillus cereus Group, including Bacillus thuringiensis.

Bacillus cereus (Bc) is a wide group of Gram-positive and spore-forming bacteria, known to be the etiological agents of various human infections, primarily food poisoning. The Bc group includes enteropathogenic strains able to germinate in the digestive tract and to produce enterotoxins such as Nhe, Hbl, and CytK. One species of the group, Bacillus thuringiensis (Bt), has the unique feature of producing insecticidal crystals during sporulation, making it an important alternative to chemical pesticides to protect crops from insect pest larvae. Nevertheless, several studies have suggested a link between the ingestion of pesticide strains and human cases of food poisoning, calling their safety into question. Consequently, reliable tools for virulence assessment are worth developing to aid decision making in pesticide regulation. Here, we propose complementary approaches based on two biological models, the human intestinal Caco-2 cell line and the insect Drosophila melanogaster, to assess and rank the enteric virulence potency of Bt strains in comparison with other Bc group members. Using a dataset of 48 Bacillus spp. strains, we showed that some Bc group strains, including Bt, were able to induce cytotoxicity in Caco-2 cells with concomitant release of IL-8 cytokine, a landmark of pro-inflammatory response. In the D. melanogaster model, we were able to sort a panel of 39 strains into four different classes of virulence, ranging from no virulence to strong virulence. Importantly, for the most virulent strains, mortality was associated with a loss of intestinal barrier integrity. Interestingly, although strains can share a common toxinotype, they display different degrees of virulence, suggesting the existence of specific mechanisms of virulence expression in vivo in the intestine.

Anti-oral cancer properties of potential probiotic lactobacilli isolated from traditional milk, cheese, and yogurt

This study investigates the probiotic and anti-cancer effects of 21 isolated Lactobacillus strains from cheese, milk, and yogurt in Kermanshah, Iran, on oral cancer cell lines KB and OSCC. Four selected isolates (Y33, M45, C5, and C28) displayed good viability and resistance to specific antibiotics. Notably, strains C28 and Y33 exhibited the best results, showing susceptibility or semi-susceptibility to five antibiotics. Y33, with high cell surface hydrophobicity (62%), demonstrated significant anti-pathogenic activity, inhibiting the growth of tested pathogens and displaying strong adhesion to human intestinal Caco-2 cells (52%). Further assessments, including acridine orange/ethidium bromide staining and mRNA expression analysis, revealed four isolates (C5, C28, M45, and Y33) with promising probiotic properties. Particularly, Y33’s protein-based extract metabolites showed dose- and time-dependent inhibition of KB and OSCC cancer cell lines, inducing apoptosis without significant cytotoxic effects on normal cells. Y33 (Lactiplantibacillus plantarum) exhibited the strongest probiotic potential, surpassing conventional anti-cancer drugs, suggesting its therapeutic potential for preventing oral cancer cell proliferation and improving survival rates in oral cancer patients.

The zonula adherens matura redefines the apical junction of intestinal epithelia

Cell-cell apical junctions of epithelia consist of multiprotein complexes that organize as belts regulating cell-cell adhesion, permeability, and mechanical tension: the tight junction (zonula occludens), the zonula adherens (ZA), and the macula adherens. The prevailing dogma is that at the ZA, E-cadherin and catenins are lined with F-actin bundles that support and transmit mechanical tension between cells. Using super-resolution microscopy on human intestinal biopsies and Caco-2 cells, we show that two distinct multiprotein belts are basal of the tight junctions as the intestinal epithelia mature. The most apical is populated with nectins/afadin and lined with F-actin; the second is populated with E-cad/catenins. We name this dual-belt architecture the zonula adherens matura. We find that the apical contraction apparatus and the dual-belt organization rely on afadin expression. Our study provides a revised description of epithelial cell-cell junctions and identifies a module regulating the mechanics of epithelia.

The Main Targets of Okadaic Acid Toxin in Human Intestinal Caco-2 Cells: An Investigation of Biological Systems

Background: Okadaic acid (OA) is a toxin of polluted shellfish. Consuming the contaminated shellfish is accompanied by diarrhea and paralytic and amnesic disorders. There is a correlation between diarrhea and the consumed OA. Determining the critical targeted genes by OA was the aim of this study. Methods: The transcriptomic data about the effect of OA on human intestinal caco-2 cells were extracted from gene expression omnibus (GEO) and evaluated via the GEO2R program. The significant differentially expressed genes (DEGs) were included in a protein-protein interaction (PPI) network and the central nodes were enriched via gene ontology to find the crucial affected biological terms. Results: Among the 178 significant DEGs plus 50 added first neighbors, four hub-bottleneck genes (ALB, FOS, JUN, and MYC) were determined. Twenty-eight critical biological terms were identified as the dysregulated individuals in response to the presence of OA. “ERK1/2-activator protein-1 (AP-1) complex binds KDM6B promoter” was highlighted as the major class of biological terms. Conclusion: It can be concluded that down-regulation of ALB as a potent central gene leads to impairment of blood homeostasis in the presence of OA. Up-regulation of the other three central genes (JUN, FOS, and MYC) grossly affects the vital pathways in the human body.

Olive (Olea europaea L.) Seed as New Source of Cholesterol-Lowering Bioactive Peptides: Elucidation of Their Mechanism of Action in HepG2 Cells and Their Trans-Epithelial Transport in Differentiated Caco-2 Cells.

The production of olive oil has important economic repercussions in Mediterranean countries but also a considerable impact on the environment. This production generates enormous quantities of waste and by-products, which can be exploited as new raw materials to obtain innovative ingredients and therefore make the olive production more sustainable. In a previous study, we decided to foster olive seeds by generating two protein hydrolysates using food-grade enzymes, alcalase (AH) and papain (PH). These hydrolysates have shown, both in vitro and at the cellular level, antioxidant and antidiabetic activities, being able to inhibit the activity of the DPP-IV enzyme and modulate the secretion of GLP-1. Given the multifunctional behavior of peptides, both hydrolysates displayed dual hypocholesterolemic activity, inhibiting the activity of HMGCoAR and impairing the PPI of PCSK9/LDLR, with an IC50 equal to 0.61 mg/mL and 0.31 mg/mL for AH and PH, respectively. Furthermore, both samples restored LDLR protein levels on the membrane of human hepatic HepG2 cells, increasing the uptake of LDL from the extracellular environment. Since intestinal bioavailability is a key component of bioactive peptides, the second objective of this work is to evaluate the capacity of AH and PH peptides to be transported by differentiated human intestinal Caco-2 cells. The peptides transported by intestinal cells have been analyzed using mass spectrometry analysis, identifying a mixture of stable peptides that may represent new ingredients with multifunctional qualities for the development of nutraceuticals and functional foods to delay the onset of metabolic syndrome, promoting the principles of environmental sustainability.

A novel Lactiplantibacillus plantarum strain: probiotic properties and optimization of the growth conditions by response surface methodology

The objective of this study is to explore the probiotic properties and optimal growth conditions of Lactiplantibacillus plantarum BG24. L. plantarum BG24 exhibited a remarkable ability to utilize lactose, and to grow under acidic conditions and in the presence of high levels of bile salts. The strain showed the highest antibacterial activity against L. monocytogenes Scott A (zone of inhibition: 26 mm). L. plantarum BG24 was found to be resistant to 8 of the tested 19 antibiotics using the disc diffusion method.and its multiple antibiotic resistance (MAR) index was calculated as 0.421. The adhesion rate to human intestinal epithelial Caco-2 cells was determined as 37.51%. The enzyme profile of L. plantarum BG24 was investigated using API ZYM test kit and the highest enzymatic activities were found for Leucine arylamidase, β-glucosidase, Valine arylamidase, β-galactosidase and N-acetyl-β-glucosaminidase. L. plantarum BG24 strain showed higher microbial growth under static conditions (6.60 OD600) compared to 100 rpm (5.73 OD600) and 200 rpm (5.02 OD600) shaking speed due to its facultative anaerobic characteristic. However, different inoculation rates and glucose addition did not make a statistically significant difference on biomass formation (p > 0.05). The specific growth rate of L. plantarum BG24 was 0.416 h−1, the doubling time was 1.67 h, and the biomass productivity value was 0.14 gL−1 h−1 in the original MRS broth (pH 5.7) while higher values were found as 0.483 h−1, 1.43 h and 0.17 gL−1 h−1, respectively, in MRS broth (pH 6.5) medium enriched with 5 g/L yeast extract. The stirred tank bioreactor was used to optimise the growth of BG24 strain. The process variables was optimized at 0.05 vvm of aeration rate, 479 rpm of agitation speed, 3% of inoculation rate and 18 h of incubation time. The maximum biomass (g/L) production was obtained as 3.84 g/L at the optimized conditions.

27-Hydroxycholesterol activates the GSK-3β/β-catenin signaling pathway resulting in intestinal fibrosis by inducing oxidative stress: effect of dietary interventions.

Intestinal fibrosis, a common and serious complication of inflammatory bowel disease (IBD), results from chronic inflammation. A high-cholesterol diet may be a risk factor for IBD and 27-hydroxylcholesterol (27HC) is the main human cholesterol metabolite. This study investigated whether 27HC can induce intestinal fibrosis. The effects of cholesterol and 27HC on intestinal fibrosis were assessed in zebrafish and human intestinal epithelial Caco-2 cells. Cholesterol and 27HC induced intestinal inflammation and collagen deposition, inhibited E-cadherin (E-ca) expression in the intestinal epithelium, and promoted nuclear translocation of β-catenin in zebrafish. Cholesterol and 27HC up-regulated expression of COL-1, α-SMA, CTGF, TIMP1, N-cadherin, vimentin, glycogen synthesis kinase-3β (GSK-3β) and β-catenin, but inhibited E-ca, in Caco-2 cells. The expression of these proteins was inhibited by CYP27A1 knockdown and β-catenin knockdown. 27HC-induced nuclear translocation of β-catenin occurs in Caco-2 cells. p38, ERK, and AKT activate β-catenin and thereby participate in 27HC-induced epithelia-mesenchymal transition (EMT) and fibrosis. 27HC-increased oxidative stress and the fibrosis and EMT markers, the nuclear translocation of β-catenin, and the up-regulation of p-cell kinase proteins promoted by 27HC were inhibited by N-acetyl-L-cysteine (NAC). Folic acid (FA), resveratrol (RES), and NAC all ameliorated the 27HC-induced effects in Caco-2 cells and zebrafish. A high-cholesterol diet caused intestinal fibrosis in zebrafish, mediated by a major cholesterol metabolite, 27HC. 27HC increased oxidative stress and activated p38, ERK, AKT, and β-catenin, leading to EMT of epithelial cells and intestinal fibrosis. FA and RES both ameliorated intestinal fibrosis by restraining 27HC-induced β-catenin activation.

Effects of okadaic acid, azaspiracid-1, yessotoxin and their binary mixtures on human intestinal Caco-2 cells.

Phycotoxins are responsible for foodborne intoxications. Symptoms depend on the ingested toxins but mostly imply gastro-intestinal and neurological disorders. Importantly, humans are exposed to combinations of several phycotoxins, resulting in possible mixture effects. Most previous studies, however, have been focused on single toxin effects. Thus, the aim of this study was to examine the effects of binary mixtures of three main phycotoxins, okadaic acid (OA), azaspiracid-1 (AZA1) and yessotoxin (YTX), on human intestinal Caco-2 cells. The focus was placed on cell viability studies and inflammation responses using a multi-parametric approach to assess cell population (nuclei staining), cell metabolism/viability (reductase activity and lysosomal integrity), and release of inflammation markers (e.g., interleukins). Mixture effects were evaluated using the concentration addition (CA) and independent action (IA) models. Our assays show that none of the toxins had an impact on the cell population in the tested concentration range. Only OA modulated reductase activity, while all three toxins had strong effects on lysosomal integrity. Furthermore, all toxins triggered the release of interleukin 8 (IL-8), with OA being most potent. Mixture effect analysis showed additivity in most cases. However, supra-additivity was observed in regards to IL-6 and IL-8 release for combinations implying high concentrations of OA. This study extends the knowledge on mixture effects of phycotoxins in human cells.

Ligand-based magnetic extraction and safety assessment of zinc oxide nanoparticles in food products

Zinc oxide (ZnO) is a zinc supplement widely used in health products and is approved by the FDA as Generally Regarded as Safe (GRAS). However, concerns have arisen regarding the potential health effects of nanoscale ZnO, as its reactivity differs from that of its bulk form. This has led to the need for an efficient method to extract ZnO from food products without altering its physicochemical properties, where conventional methods have proven to be inadequate. This study introduces an innovative approach using starch magnetic particles (SMPs) functionalized with a 12-amino acid peptide modified with five lysines (ZBP), that has specific affinity to ZnO. ZBP@SMPs effectively and rapidly extract intact ZnO from food products, achieving recovery efficiencies ranging from 60% to 90%, all while maintaining its morphology and crystallinity. The diameter of ZnO particles recovered from six commercial food products ranged from 25 to 500 nm, with 33% falling below 100 nm, highlighting the need for a size-dependent toxicity study. However, cytotoxicity assessment on human intestinal Caco-2 cells shows all ZnO samples affects cell proliferation and membrane integrity in a dose-dependent manner due to partial dissolution. This study contributes to understanding the safety of ZnO-containing food products and highlights potential health implications associated with their consumption.

Fish oil-based microemulsion can efficiently deliver oral peptide blocking PD-1/PD-L1 and simultaneously induce ferroptosis for cancer immunotherapy

Peptide immune checkpoint inhibitors in cancer immunotherapy have attracted great attention recently, but oral delivery of these peptides remains a huge challenge due to the harsh gastrointestinal environment, large molecular size, high hydrophilic, and poor transmembrane permeability. Here, for the first time, a fish oil-based microemulsion was developed for oral delivery of programmed death-1/programmed cell death-ligand 1 (PD-1/PD-L1) blocking model peptide, OPBP-1. The delivery system was characterized, in vitro and in vivo studies were conducted to evaluate its overall implication. As a result, this nutraceutical microemulsion was easily formed without the need of co-surfactants, and it appeared light yellow, transparent, good flowability with a particle size of 152 ± 0.73 nm, with a sustained drug release manner of 56.45 ± 0.36% over 24 h and a great stability within the harsh intestinal environment. It enhanced intestinal drug uptake and transportation over human intestinal epithelial Caco-2 cells, and drastically elevated the oral peptide bioavailability of 4.1-fold higher than that of OPBP-1 solution. Meanwhile, the mechanism of these dietary droplets permeated over the intestinal enterocytic membrane was found via clathrin and caveolae-mediated endocytic pathways. From the in vivo studies, the microemulsion facilitated the infiltration of CD8+ T lymphocytes in tumors, with increased interferon-γ (IFN-γ) secretion. Thus, it manifested a promising immune anti-tumor effect and significantly inhibited the growth of murine colonic carcinoma (CT26). Furthermore, it was found that the fish oil could induce ferroptosis in tumor cells and exhibited synergistic effect with OPBP-1 for cancer immunotherapy. In conclusion, this fish oil-based formulation demonstrated great potential for oral delivery of peptides with its natural property in reactive oxygen species (ROS)-related ferroptosis of tumor cells, which provides a great platform for functional green oral delivery system in cancer immunotherapy.

Impact of In Vitro Digestion on the Digestibility, Amino Acid Release, and Antioxidant Activity of Amaranth (Amaranthus cruentus L.) and Cañihua (Chenopodium pallidicaule Aellen) Proteins in Caco-2 and HepG2 Cells.

This study evaluated the impact of in vitro gastrointestinal digestion on the digestibility, amino acid release, and antioxidant activity of proteins from amaranth (Amarantus cruentus L.) and cañihua (Chenopodium pallidicaule Aellen). Antioxidant activity was assessed using ORAC, ABTS, DPPH, and cellular antioxidant activity (CAA) assays in human intestinal Caco-2 and hepatic Hep-G2 cell lines. The results showed that amaranth had higher protein digestibility (79.19%) than cañihua (71.22%). In addition, intestinal digestion promoted the release of essential amino acids, such as leucine, lysine, and phenylalanine, in both protein concentrates. Concentrations of amaranth and cañihua proteins, ranging from 0.125 to 1.0 mg mL-1, were non-cytotoxic in both cell lines. At a concentration of 0.750 mg mL-1, simulated gastrointestinal digestion enhanced cellular antioxidant activity. Intestinal digest fractions containing peptides >5 kDa were the principal contributors to CAA in both cell lines. Notably, cañihua proteins exhibited high CAA, reaching values of 85.55% and 82.57% in Caco-2 and HepG2 cells, respectively, compared to amaranth proteins, which reached 84.68% in Caco-2 and 81.06% in HepG2 cells. In conclusion, both amaranth and cañihua proteins, after simulated gastrointestinal digestion, showcased high digestibility and released peptides and amino acids with potent antioxidant properties, underscoring their potential health benefits.

Bio-Based Decontamination and Detoxification of Total Petroleum Hydrocarbon-Contaminated Dredged Sediments: Perspectives to Produce Constructed Technosols in the Frame of the Circular Economy

To accelerate the depletion of total petroleum hydrocarbons, a hydrocarburoclastic ascomycetes, Lambertella sp. MUT 5852, was bioaugmented to dredged sediments co-composting with a lignocellulosic matrix. After only 28 days of incubation, a complete depletion of the contamination was observed. The 16S rDNA metabarcoding of the bacterial community and a predictive functional metagenomic analysis were adopted to evaluate potential bacterial degrading and detoxifying functions. A combination of toxicological assays on two eukaryotic models, the root tips of Vicia faba and the human intestinal epithelial Caco-2 cells, was adopted to assess the robustness of the process not only for the decontamination but also for the detoxification of the dredged sediments. Bacterial taxa, such as Kocuria and Sphingobacterium sps., resulted to be involved in both the decontamination and detoxification of the co-composting dredged sediments by potential activation of diverse oxidative processes. At the same time, the Kocuria sp. showed plant growth-promoting activity by the potential expression of the 1-aminocyclopropane-1-carboxylate deaminase activity, providing functional traits of interest for a technosol in terms of sustaining primary producer growth and development.

Euphorbia factor L1 inhibited transport channel and energy metabolism in human colon adenocarcinoma cell line Caco-2

Euphorbia factor L1 (EFL1) is a kind of lathyrane-type diterpenoid and is isolated from the medical herb Euphorbia lathyris L. (Euphorbiaceae); it has been reported with the toxicity that causes intestinal irritation, but the underlying mechanisms are still obscure. The objective of this study was to assess the EFL1-induced intestinal cytotoxicity in human colon adenocarcinoma Caco-2 cells. The Caco-2 cells were treated with EFL1, and the intracellular calcium ion concentration, mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (mPTP), adenosine 5′-triphosphate (ATP) content, ATPase activities, TGF-β1 concentration, and transepithelial electrical resistance (TEER) were detected. The interaction between EFL1 and the tight junction proteins Occludin, Claudin-4, Tricellulin, ZO-1, JAM-1, and E-cadherin was simulated by molecular docking. The expression of proteins involved in the energy metabolism, the ion transporters and aquaporins, the tight junction, and the F-actin cytoskeleton were detected by Western blotting and cell immunofluorescence. As a result, EFL1 decreased the intracellular Ca2+, MMP, mPTP, ATP content, and ATPase activities in the Caco-2 cells. The AMPK/SIRT1/PGC-1α signaling pathway, which regulates the energy metabolism, was inhibited. The ion transporters NEH and CFTR, as well as the aquaporins in the Caco-2 cells, were decreased. The tight junction proteins were down-regulated, and the integrity of the intestinal barrier was injured; TGF-β1 was compensatively increased; so, the intestinal permeability was increased and was characterized by decreased TEER. The morphology of the F-actin cytoskeleton was destroyed. These findings indicated that EFL1 caused cytotoxicity in the human intestinal Caco-2 cells through mitochondrial damage, inhibition of the energy metabolism, and suppression of the ion and water molecule transporters, as well as the down-regulation tight junction and cytoskeleton protiens.

PSIII-29 A Novel Direct-Fed Microbial Supports in Vitro Intestinal Integrity Under Inflamed Conditions

Abstract Ruminants are often exposed to pathogenic and non-pathogenic challenges during daily handling procedures and management activities. These responses, in turn, may trigger the release of pro-inflammatory compounds that can have negative effects on the host. Based on this rationale, we hypothesized that 1) pro-inflammatory cytokines (TNF-α and IFN-γ) would compromise the in vitro gut barrier integrity, and 2) a novel direct-fed microbial (DFM) formulation would support the gut barrier integrity under inflammatory conditions. Therefore, our objective was to evaluate the ability of a novel DFM mixture to support barrier integrity of intestinal epithelial cell monolayers in the absence or presence of TNF-α and IFN-γ by measuring transepithelial electrical resistance (TEER). Human cancer-derived epithelial intestinal Caco-2 cell monolayers were seeded on transwells for approximately 20 days. A DFM mixture containing Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus licheniformis, and B. subtilis (BOVAMINE DEFEND Plus; Chr. Hansen A/S, Hørsholm, Denmark) at 1.0 × 108 CFU/transwell was then administered to the apical side of the cell monolayers. Then, two hours later a cocktail containing TNF-α / IFN-γ at a 10:1 ratio (100:10 ng/mL, respectively) was added to the basolateral side after which TEER was measured over a 16-h period and used to calculate the area under the curve (AUC). Following the administration of the pro-inflammatory challenge, TEER was decreased, reaching its least concentrations at 5.5 h post-challenge, whereas overall AUC decreased by 21.4% compared with non-challenged control cells. The DFM mixture by itself was able to support the integrity of the intestinal cells by stimulating TEER increase over time and increasing overall AUC vs. non-treated cells (P < 0.01). Moreover, when cells receiving the DFM were challenged with the pro-inflammatory cocktail, TEER concentrations were still above 100% and no decrease was observed over the evaluation period (16 h). Moreover, when compared with non-DFM-treated but challenged cells, AUC was greater for DFM vs. challenged control cells (P < 0.05). In conclusion, these data support our hypothesis that a DFM mixture containing L. animalis, P. freudenreichii, B. licheniformis, and B. subtilis (BOVAMINE DEFEND Plus) supports the gut barrier integrity with or without the pro-inflammatory challenge.

Bioenergetic effects of pristine and ultraviolet-weathered polydisperse polyethylene terephthalate and polystyrene nanoplastics on human intestinal Caco-2 cells

The ubiquitous human exposure to nanoplastics (NPs) increasingly raises concerns regarding impact on our health. However, little is known on the biological effects of complex mixtures of weathered NPs with heterogenous size and irregular shape present in the environment. In this study, the bioenergetic effects of four such NPs mixtures on human intestinal Caco-2 cells were investigated. To this aim, Caco-2 cells were exposed to polydisperse nanoPET (<800 nm) and nanoPS (mixture of 100 and 750 nm) samples with and without ultraviolet (UV) weathering at low concentration range (102–107 particles/mL) for 48 h. Mitochondrial respiration, glycolytic functions and ATP production rates of exposed cells were measured by Seahorse XFe96 Analyzer. Among four NPs samples, polydisperse nanoPET with irregular shapes induced significant stimulation of mitochondrial respiration, glycolysis and ATP production rates in Caco-2 cells. Spherical nanoPS caused significant stimulation on glycolytic functions of Caco-2 cells at the highest concentration used (106 particles/mL). ATR-FTIR spectra and carbonyl index indicated formation of carbonyl groups in nanoPET and nanoPS after UV weathering. UV weathering could alleviate bioenergetic stress caused by NPs in Caco-2 cells and even shifted the energy pathways from mitochondrial respiration to glycolysis due to electrostatic repulsion between negatively charged UV-aged NPs and cell membranes. This research is the first to study in-vitro bioenergetic responses of NPs samples with multidimensional features (polymer type, irregular shape, heterogenous size, UV-weathering) on human health. It highlights that effects between pristine and weathered NPs are different at a bioenergetic level, which has important implications for the risk assessment of NPs on human health.

Organic Synthesis of New Secosteroids from Fucosterol, Its Intestinal Absorption by Caco-2 Cells, and Simulation of the Biological Activities of Vitamin D.

We previously examined the cellular uptake of six types of vitamin D in human intestinal Caco-2 cells. Since vitamins D5-D7 were commercially unavailable, we synthesized these compounds organically before studying them. This process led us to understand that new secosteroids could be generated as vitamin D candidates, depending on the sterol used as the starting material. We obtained two new secosteroids-compounds 3 and 4-from fucosterol in the current study. We investigated the intestinal absorption of these compounds using Caco-2 cells cultured in Transwells and compared the results with vitamin D3, a representative secosteroid. The intestinal absorption of compound 4 was comparable to that of vitamin D3. Compound 3 showed similar uptake levels but transported about half as much as vitamin D3. These compounds demonstrated intestinal absorption at the cellular level. Vitamin D is known for its diverse biological activities manifest after intestinal absorption. Using PASS online simulation, we estimated the biological activity of compound 3's activated form. In several items indicated by PASS, compound 3 exhibited stronger biological activity than vitamins D2-D7 and was also predicted to have unique biological activities.

Apoptosis and oxidative stress of mouse breast carcinoma 4T1 and human intestinal epithelial Caco-2 cell lines caused by the phycotoxin gymnodimine-A

Gymnodimine-A (GYM-A) is a cyclic imine phycotoxin produced by some marine dinoflagellates. It can cause rapid death of mice via intraperitoneal administration and frequently accumulate in shellfish potentially threatening human health. In this study, four different cell lines were exposed to GYM-A for the viability assessment. Results showed that GYM-A was cytotoxic with concentration-dependent pattern to each cell type, with mean IC50 values ranging from 1.39 to 2.79 μmol L−1. Results suggested that the loss of cell viability of 4T1 and Caco-2 cells was attributed to apoptosis. Furthermore, the collapse of mitochondrial membrane potential and caspases activation were observed in the GYM-A-treated cells. Reactive oxygen species (ROS) and lipid peroxides (LPO) levels were markedly increased in 4T1 and Caco-2 cells exposed to GYM-A at 2 μmol L−1, and the oxidative stress in 4T1 cells was more obvious than that in Caco-2 cells. Additionally, unusual ultrastructure impairment on mitochondria and mitophagosomes occurred in the GYM-A-treated cells. These results suggested that an ROS-mediated mitochondrial pathway for apoptosis and mitophagy was implicated in the cytotoxic effects induced by GYM-A. This is the first report to explore the cytotoxic mechanisms of GYM-A through apoptosis and oxidative stress, and it will provide theoretical foundations for the potential therapeutic applications of GYM-A.