Human Intestinal Caco-2 Cell Model Research Articles

Anti-oxidative and anti-inflammatory effects of different lutein isomers in Caco-2 cell model

The purpose of this work was to investigate the differences in the anti-oxidative stress and anti-inflammatory effects between all-E (trans)-lutein and its 9Z (cis)- and 13Z-isomers using the human intestinal Caco-2 cell model. I–TiO2 catalyst was used to isomerize all-E-lutein to obtain 9Z-lutein and 13Z-lutein. In anti-oxidative stress studies, all three lutein isoforms could significantly reduce H2O2-induced interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) secretion (p < 0.05) by 50% or more at 4 μM. At lower concentration (2 μM), 9Z-lutein and 13Z-lutein showed higher protection against oxidative stress than all-E-lutein by significantly reducing IL-8 secretion by 48% and 40% (p < 0.05), respectively. In addition, three lutein isomers, especially 9Z-lutein could up-regulate the activity of antioxidant enzymes including catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and the content of endogenous antioxidant glutathione (GSH) at 2 μM. In the anti-inflammatory assay, 9Z-lutein and all-E-lutein exhibited higher anti-inflammatory effects than 13Z-lutein in reducing the secretion and expression of IL-8 in TNF-α induced inflammatory Caco-2 cells. Compared with the positive control, 9Z-lutein and all-E-lutein could reduce the expression of the IL-8 gene by 49% and 52%, respectively at 4 μM. This study provided a theoretical basis for the potential health benefits of Z-luteins.

Human intestinal Caco-2 cell model to evaluate the absorption of 7-ketophytosterols and their effects on cholesterol transport

7-Ketophytosterols are the major oxidation products of phytosterols in foods, which have been associated with atherosclerosis. However, their absorption mechanism remains unclear. The aim of our work was to investigate the absorption mechanism of 7-ketophytosterols and their effects on the cholesterol transport using Caco-2 cell model. The absorption percentage of 7-ketositosterol and 7-ketocampesterol was 1.16 %−1.68 % and 1.18 %−2.23 % respectively in the Caco-2 model, which is higher than that of their parent phytosterols, but lower than cholesterol-d7. The apparent permeability of 7-ketositosterol and 7-ketocampesterol at 30 μmol/L in the basolateral (BL)-to-apical (AP) direction were 0.42- and 0.55-fold of that in the AP-to-BL direction, indicating an active intake in the permeation mechanism of 7-ketophytosterols. Ezetimibe could significantly inhibit the transport of 7-ketophytosterols (P < 0.05), which means that their transport depends on niemann-pick c1-like 1 (NPC1L1) protein. The transport of cholesterol-d7 was significantly inhibited by 7-ketophytosterols (P < 0.05). Taken together, this study deepened our understanding of the absorption mechanism of common food-born 7-ketophytosterols and provides useful information on the inhibition of 7-ketophytosterols absorption.

Optimization of Supercritical Carbon Dioxide Extraction of Polyphenols from Black Rosehip and Their Bioaccessibility Using an In Vitro Digestion/Caco-2 Cell Model.

The fruits of Rosa pimpinellifolia are rich sources of (poly)phenols, however they are underutilized due to the limited information available. The influence of the pressure, temperature, and co-solvent concentration (aqueous ethanol) of the supercritical carbon dioxide extraction (SCO2-aqEtOH) on the extraction yield, total phenolic-, total anthocyanin-, catechin-, cyanidin-3-O-glucoside contents, and total antioxidant activity of black rosehip was investigated simultaneously. The maximum obtained total phenolic and total anthocyanin contents under the optimized extraction conditions (280 bar, 60 °C and 25% ethanol, v/v) were 76.58 ± 4.25 mg gallic acid equivalent and 10.89 ± 1.56 mg cyanidin-3-O-glucoside equivalent per g of the dry fruits, respectively. The optimal extract obtained by SCO2-aqEtOH was compared to two other extraction procedures: ultrasonication using ethanol as solvent (UA-EtOH) and pressurized hot water extraction (PH-H2O). The bioaccessibility and cellular metabolism of the phenolic compounds in the different black rosehip extracts were assessed using an in vitro digestion coupled with a human intestinal Caco-2 cell model. The in vitro digestive stability and cellular uptake of the phenolic compounds had no significant difference among the different extraction methods. The results of this study confirm the efficiency of SCO2-aqEtOH extraction for phenolic compounds and, in particular, for anthocyanins, and could be used to produce new functional food ingredients from black rosehip with high antioxidant power containing both hydrophilic and lipophilic compounds.

Chemical depolymerisation of chondroitin sulfate glycosaminoglycan improves both antioxidant capacity and Fe uptake in a human intestinal Caco-2 cell model

Chondroitin sulfate (CS) is a high molecular weight sulfated polysaccharide that has been reported to improve joint health. A recent study has demonstrated the ability of enzymatically produced CS-oligosaccharides to improve non-heme iron bioavailability. In this research, CS was chemically hydrolyzed under acid, alkaline, and combined (acid & alkaline) conditions at 60 °C for different times (0–60 h) to understand if the oligosaccharides obtained can improve iron uptake in human intestinal Caco-2 cell model. The acidic hydrolyses produced a collection of desulfated and low molecular weight CS crude oligosaccharides (CS crude oligos), while under alkaline condition a high degree of deacetylation was detected with limited depolymerisation and desulfation. The combined treated CS was first degraded and desulfated in acid solution followed by alkali deacetylation. CS crude oligos produced by the combined treatment possessed the superior antioxidant capacity measured by ferric reducing and DPPH scavenging activities. All chemically hydrolyzed CS samples showed greater (p < 0.05) ferritin formation in Caco-2 cells compared to the negative control, where the greatest ferritin level was achieved by the combined treated CS. The depolymerisation of CS followed by the deacetylation created CS structural changes resulting in the increase of antioxidant and Fe-absorption enhancing capacities.

Elucidation of the Intestinal Absorption Mechanism of Loganin in the Human Intestinal Caco-2 Cell Model.

Loganin, iridoid glycosides, is the main bioactive ingredients in the plant Strychnos nux-vomica L. and demonstrates various pharmacological effects, though poor oral bioavailability in rats. In this study, the intestinal absorption mechanism of loganin was investigated using the human intestinal Caco-2 cell monolayer model in both the apical-to-basolateral (A-B) and the basolateral-to-apical (B-A) direction; additionally, transport characteristics were systematically investigated at different concentrations, pHs, temperatures, and potential transporters. The absorption permeability (PappAB) of loganin, which ranged from 12.17 to 14.78 × 10−6cm/s, was high at four tested concentrations (5, 20, 40, and 80μM), while the major permeation mechanism of loganin was found to be passive diffusion with active efflux mediated by multidrug resistance-associated protein (MRP) and breast cancer resistance protein (BCRP). In addition, it was found that loganin was not the substrate of efflux transporter P-glycoprotein (P-gp) since the selective inhibitor (verapamil) of the efflux transporter exhibited little effects on the transport of loganin in the human intestinal Caco-2 cells. Meanwhile, transport from the apical to the basolateral side increased 2.09-fold after addition of a MRP inhibitor and 2.32-fold after addition of a BCRP inhibitor. In summary, our results clearly demonstrate, for the first time, a good permeability of loganin in the human intestinal Caco-2 cell model and elucidate, in detail, the intestinal absorption mechanism and the effects of transporters on iridoid glycosides compounds.

Characterization of loxoprofen transport in Caco-2 cells: the involvement of a proton-dependent transport system in the intestinal transport of loxoprofen.

Loxoprofen, a propionate non-steroidal anti-inflammatory drug (NSAID), is used widely in East Asian countries. However, little is known about the transport mechanisms contributing to its intestinal absorption. The objectives of this study were to characterize the intestinal transport of loxoprofen using the human intestinal Caco-2 cell model. The transport of loxoprofen was investigated in cellular uptake studies. The uptake of loxoprofen into Caco-2 cells was pH- and concentration-dependent, and was described by a Michaelis-Menten equation with passive diffusion (Km : 4.8mm, Vmax : 142nmol/mg protein/30s, and Kd : 2.2μl/mg protein/30s). Moreover, the uptake of loxoprofen was inhibited by a typical monocarboxylate transporter (MCT) inhibitor as well as by various monocarboxylates. The uptake of [14 C] l-lactic acid, a typical MCT substrate, in Caco-2 cells was saturable with relatively high affinity for MCT. Because loxoprofen inhibited the uptake of [14 C] l-lactic acid in a noncompetitive manner, it was unlikely that loxoprofen uptake was mediated by high-affinity MCT(s). Our results suggest that transport of loxoprofen in Caco-2 cells is, at least in part, mediated by a proton-dependent transport system. Copyright © 2016 John Wiley & Sons, Ltd.

Effect of Microfluidization on in Vitro Micellization and Intestinal Cell Uptake of Lutein from Chlorella vulgaris

Chlorella is a nutrient-rich microalga that contains protein, lipid, minerals, vitamins, and high levels of lutein. This study evaluated the bioavailability of lutein from Chlorella vulgaris using a coupled in vitro digestion and human intestinal Caco-2 cell model. Lutein bioaccessibility was low, and approximately 75% of total C. vulgaris lutein was not micellized during the digestion process but remained in the insoluble digestate. Microfluidization improved lutein micellization efficiency during C. vulgaris digestion. C. vulgaris was microfluidized at a pressure exceeding 10000 psi, and the cell surface disruption was visualized by scanning electron microscopy. The mean C. vulgaris particle size was reduced from 3.56 to 0.35 μm with the microfluidization treatment. C. vulgaris microfluidization at 20000 psi was three times more efficient for aqueous lutein micelles production as compared with untreated C. vulgaris, and the final lutein content accumulated by intestinal Caco-2 cells was also higher with microfluidization. C. vulgaris lutein stability was not affected by microfluidization. These results indicate that microfluidization may be useful for improving lutein bioaccessibility from C. vulgaris during food processing.

Chemical Form of Selenium Affects Its Uptake, Transport, and Glutathione Peroxidase Activity in the Human Intestinal Caco-2 Cell Model

Determining the effect of selenium (Se) chemical form on uptake, transport, and glutathione peroxidase activity in human intestinal cells is critical to assess Se bioavailability at nutritional doses. In this study, we found that two sources of L-selenomethionine (SeMet) and Se-enriched yeast each increased intracellular Se content more effectively than selenite or methylselenocysteine (SeMSC) in the human intestinal Caco-2 cell model. Interestingly, SeMSC, SeMet, and digested Se-enriched yeast were transported at comparable efficacy from the apical to basolateral sides, each being about 3-fold that of selenite. In addition, these forms of Se, whether before or after traversing from apical side to basolateral side, did not change the potential to support glutathione peroxidase (GPx) activity. Although selenoprotein P has been postulated to be a key Se transport protein, its intracellular expression did not differ when selenite, SeMSC, SeMet, or digested Se-enriched yeast was added to serum-contained media. Taken together, our data show, for the first time, that the chemical form of Se at nutritional doses can affect the absorptive (apical to basolateral side) efficacy and retention of Se by intestinal cells; but that, these effects are not directly correlated to the potential to support GPx activity.

Analysis of the passage of the marine biotoxin okadaic acid through an in vitro human gut barrier

The marine biotoxin okadaic acid (OA), produced by dinoflagellates, can accumulate in various bivalve molluscs. In humans, oral consumption of shellfish contaminated with OA induces acute toxic effects like diarrhea, nausea, vomiting and abdominal pain. However, tumorigenic and embryotoxic effects of OA have been also described. Current toxicokinetic studies with mice were performed with high cytotoxic oral doses leading presumably to a paracellular passage of OA through the gastrointestinal barrier. There are no studies available analyzing the absorption at low concentrations, which represent a realistic dietary exposure, making a reliable risk assessment difficult. Therefore, we performed a low-dose study using the human intestinal Caco-2 cell model to simulate the intestinal barrier. Low level exposure of 20–200 nM OA to the cell monolayer allows an only limited passage from the “luminal” to the “blood side”. Furthermore, we could detect a significant efflux of OA, which led to the suggestion that active transport mechanisms are involved in the elimination process of OA. In conclusion, our results indicate that besides the well known defense mechanisms of humans against this marine biotoxin – vomiting and diarrhea – further detoxification mechanisms are available to limit the absorption of toxic OA.

Highly Variable Contents of Phenolics in St. John’s Wort Products Affect Their Transport in the Human Intestinal Caco-2 Cell Model: Pharmaceutical and Biopharmaceutical Rationale for Product Standardization

The purposes of this study were to determine content uniformity of phenolics in the St. John's wort (SJW) supplements and to demonstrate how variations in the product matrices affect their absorption and efflux. LC and LC-MS/MS methods were used to determine the phenolic contents of 12 different products purchased locally or from the Internet. Three representative extracts were further submitted to Caco-2 cell transport experiment, and transport of rutin, hyperoside, and isoquercitrin was evaluated. The results indicated that the 12 different products displayed 12 different HPLC fingerprints, but all products contained the following major compounds: rutin, hyperoside, isoquercitrin, quercitrin, quercetin, and amentoflavone. The content uniformity of these major compounds was poor across products, with the smallest difference in the amounts of amentoflavone (3.6-fold) and largest difference in that of isoquercitrin (28.8-fold). The Caco-2 experiments indicated transport of rutin in products was vectorial, with the permeabilities varied about 3.6-fold in both directions of transport. The vectorial permeabilities of hyperoside and isoquercitrin were similarly different. Use of efflux transporter inhibitor studies suggested that MRP2 was involved in isoquercitrin's efflux and the product matrix affected the extent of its efflux. In conclusion, different SJW supplements had highly variable contents of phenolics, and the variability in product matrix and phytochemical compositions affected the permeabilities of key phenolics across the Caco-2 monolayers, which may further affect their bioavailabilities. Therefore, standardization will be necessary to ensure safe and efficacious using of supplements such as SJW.

Bioavailability of β-carotene isomers from raw and cooked carrots using an in vitro digestion model coupled with a human intestinal Caco-2 cell model

Not only is there limited information in the literature regarding the β-carotene (BC) isomer profile of micelles from digested foods; few studies have looked at their subsequent uptake and transport by human intestinal Caco-2 cells. Therefore, the aims of the present study were, first, to assess the profile of BC isomers in micelles from digested raw and cooked carrots; and, second, to determine their cellular uptake and transport. Greater amounts of all-trans-, 13-cis- and 15-cis-BC isomers were present in the micelles of cooked carrots compared with raw carrots. Furthermore, micelle fractions obtained from the most highly processed (pureed) carrots had greater (P < 0.05) amounts of all-trans-, 13-cis- and 15-cis-BC compared with those derived from raw and boiled carrots. A similar trend was seen with BC isomer uptake and transport. Our data suggest that the food matrix and degree of processing play important roles on carotenoid isomerization and bioavailability.

Bioaccessibility, Uptake, and Transport of Carotenoids from Peppers (Capsicum Spp.) Using the Coupled in Vitro Digestion and Human Intestinal Caco-2 Cell Model

Spanish bell peppers (Capsicum annuum L.) and chili peppers sourced from Kenya and Turkey were analyzed for their carotenoid content, bioaccessibility, and bioavailability. The order of total carotenoid content in peppers and their respective micelles was red > green > yellow. In terms of cellular carotenoid transport as a percentage of original food and micelle content, the order was yellow peppers > green > red; however, the opposite trend was seen for the actual amount of total carotenoids transported by Caco-2 cells. Although lutein was generally the most abundant carotenoid in the micelles (496.3-1565.7 microg 100 g(-1)), cellular uptake and transport of beta-carotene were the highest, 8.3-31.6 and 16.8-42.7%, respectively. Hence, the actual amount of carotenoids present in the original food and respective micelles seems to reflect the amount transported by Caco-2 cells. Therefore, color influenced the carotenoid profile, bioaccessibility, and bioavailability of carotenoids rather than pepper type.

Cellular transport of lutein is greater from uncooked rather than cooked spinach irrespective of whether it is fresh, frozen, or canned

Lutein, a carotenoid found in significant levels in spinach, has attracted a great deal of attention owing to its reported function as a shield against the photooxidative effects of blue light. Therefore, the rationale of this study was to examine the effects of various processing and cooking methods on lutein bioavailability from spinach (Spinacia oleracea) using an in vitro digestion procedure coupled with the use of a human intestinal Caco-2 cell model. Fresh, frozen, and canned spinach were analyzed uncooked and after boiling or microwave cooking. Lutein content from the uncooked and cooked digested food (digestate) and appropriate micelles was determined. Micellarized lutein from the spinach samples was adjusted to 0.1 micromol/L and added to Caco-2 cells. Cellular uptake and secretion (cellular transport) of lutein were determined. Our results showed that digestate obtained from uncooked canned spinach had greater lutein content (P < .05) than uncooked fresh or frozen spinach. Microwave cooking, but not boiling, significantly lowered the lutein content of canned spinach digestate and micelles compared with their uncooked counterparts. Interestingly, there were no differences in the micellarization of lutein between the cooking and processing methods. Cellular transport of lutein was greater from uncooked spinach micelles compared with boiled or microwave-cooked spinach. To conclude, although the lutein content of digesta and micelles may have been modified, its micellarization was not significantly affected by any of the cooking or processing methods tested. In general, cellular transport of lutein was greatest in uncooked spinach irrespective of whether the spinach was fresh, frozen, or canned.

Transcriptional control of intestinal cytochrome P-4503A by 1alpha,25-dihydroxy vitamin D3.

It was previously shown that CYP3A4 is induced in the human intestinal Caco-2 cell model by treatment with 1alpha,25-dihydroxy vitamin D3 (1,25-D3). We demonstrate the vitamin D analog, 19-nor-1alpha,25-dihydroxy vitamin D2, is also an effective inducer of CYP3A4 in Caco-2 cells, but with half the potency of 1,25-D3. We report that treatment of LS180 cells, a human intestinal cell line, with 1 to 10 nM 1,25-D3 dose dependently increased CYP3A4 protein and CYP3A4 mRNA expression. CYP3A4- and CYP3A23-promoter-Luciferase reporter constructs transiently transfected into LS180 cells were transcriptionally activated in a dose-dependent manner by 1,25-D3, whereas mutation of the nuclear hormone receptor binding motif (ER6) in the CYP3A4 promoter abrogated 1,25-D3 activation of CYP3A4. Although the CYP3A4 ER6 promoter element has been shown to bind the pregnane X receptor (PXR), this receptor does not mediate 1,25-D3 induction of CYP3A4 because a) PXR is not expressed in Caco-2 cells; b) PXR mRNA expression is not induced by 1,25-D3 treatment of LS180 cells; and c) the ligand binding domain of human PXR was not activated by 1,25-D3. 1,25-D3 uses the vitamin D receptor to induce CYP3A4 because a) the vitamin D receptor (VDR)-retinoid X receptor (RXR) heterodimer binds specifically to the CYP3A4 ER6; b) selective mutation of the CYP3A4 ER6 disrupted the binding of VDR-RXR; and c) reporter constructs containing only three copies of the CYP3A4 ER6 linked to a TK-CAT reporter were activated by 1,25-D3 only in cells cotransfected with a human VDR expression plasmid. These data support the hypothesis that 1,25-D3 and VDR induce expression of intestinal CYP3A by binding of the activated VDR-RXR heterodimer to the CYP3A PXR response element and promoting gene transcription.