Integrity Of Caco-2 Cell Monolayers Research Articles

Daidzein Protects Caco-2 Cells against Lipopolysaccharide-Induced Intestinal Epithelial Barrier Injury by Suppressing PI3K/AKT and P38 Pathways.

The intestinal epithelium provides an important barrier against bacterial endotoxin translocation, which can regulate the absorption of water and ions. The disruption of epithelial barrier function can result in water transport and tight junction damage, or further cause diarrhea. Therefore, reducing intestinal epithelial barrier injury plays an important role in diarrhea. Inflammatory response is an important cause of intestinal barrier defects. Daidzein improving the barrier integrity has been reported, but the effect on tight junction proteins and aquaporins is not well-described yet, and the underlying mechanism remains indistinct in the human intestinal epithelium. This study aimed to investigate the effects and mechanisms of daidzein on intestinal epithelial barrier injury induced by LPS, and a barrier injury model induced by LPS was established with human colorectal epithelial adenocarcinoma cell line Caco-2 cells. We found that daidzein protected the integrity of Caco-2 cell monolayers, reversed LPS-induced downregulation of ZO-1, occludin, claudin-1, and AQP3 expression, maintained intercellular junction of ZO-1, and suppressed NF-κB and the expression of inflammatory factors (TNF-α, IL-6). Furthermore, we found that daidzein suppressed the phosphorylation of the PI3K/AKT and P38 pathway-related proteins and the level of the related genes, and the PI3K/AKT and P38 pathway inhibitors increased ZO-1, occludin, claudin-1, and AQP3 expression. The study showed that daidzein could resist LPS-induced intestinal epithelial barrier injury, and the mechanism is related to suppressing the PI3K/AKT and P38 pathways. Therefore, daidzein could be a candidate as a dietary supplementation or drug to prevent or cure diarrhea.

Mast cells disrupt the duodenal mucosal integrity: Implications for the mechanisms of barrier dysfunction in functional dyspepsia

Background Functional dyspepsia (FD) is a common functional gastrointestinal (GI) disorder, but its pathophysiology is poorly understood. Mast cells (MCs) may play a critical role in the development of FD. Therefore, the aim of this study was to investigate the effect of MCs on barrier function, tight junction (TJ) proteins and related signaling pathways. Methods The expression of the TJ proteins claudin-8, ZO-1 and occludin in biopsy tissues from seven FD patients and five controls was assessed. Based on the in vivo results, we further investigated the effect of (1) MC degranulation in a coculture model of Caco-2/RBL-2H3 cells and tryptase in Caco-2 monolayers, (2) MC degranulation in the presence or absence of a PAR-2 antagonist and (3) MC degranulation in the presence or absence of an ERK1/2 signaling pathway inhibitor. The epithelial integrity of Caco-2 cell monolayers was assessed by measuring the transepithelial electrical resistance (TEER). The expression of TJ proteins was evaluated by western blotting, QT-PCR and immunostaining. Results Epithelial claudin-8, ZO-1 and occludin protein expression were significantly reduced in tissues from FD patients compared with controls. MC degranulation and tryptase decreased the TEER and reduced the expression of TJ proteins in Caco-2 cell monolayers. A PAR-2 antagonist and an ERK1/2 signaling pathway inhibitor significantly reduced the effect of MC degranulation on the TEER and TJ protein expression in Caco-2 cell monolayers. Conclusions MCs disrupt duodenal barrier function by modulating the levels of TJ proteins, and the PAR-2 and ERK1/2 signaling pathways may mediate the pathogenesis of FD.

Evolution of cranberry juice compounds during in vitro digestion and identification of the organic acid responsible for the disruption of in vitro intestinal cell barrier integrity.

Cranberry juice is increasingly consumed for its richness in polyphenols having a positive impact on human health. Unfortunately, when regularly consumed, its high concentration in organic acids may cause some intestinal discomforts. In the present study, its organic acid content was reduced of 41% by electrodialysis with bipolar membrane (EDBM), and the resulted deacidified juice was divided in five different juices readjusted or not with different concentrations of citric and/or malic acid(s) corresponding to the concentration of this/these acid(s) recovered during EDBM or at the titratable acidity (TA) of the non-deacidified cranberry juice. The evolution of the cranberry juice main interesting compounds (organic acids and polyphenols), according to the concentration and nature of the organic acids present, was studied for the first time at each specific stages of the digestion. After digestion, Caco-2 cells were exposed to all digested juices to identify the organic acid(s) responsible for the loss of integrity of the epithelial barrier. It appeared that organic acid contents did not change during the different steps of the digestion while polyphenolic compounds decreased starting from the gastric phase. Whatever the organic acid concentration or nature, the concentration of PACs significantly decreased between the salivary and the gastric steps but was different according to their structure when the concentration of most of anthocyanins significantly decreased at the gastric step. Also, to the best of our knowledge, it was the first time that citric acid was demonstrated as the organic acid responsible for the loss of integrity of Caco-2 cell monolayers.

Deacidification of cranberry juice protects against disruption of in-vitro intestinal cell barrier integrity

Cranberry juice is a well-known functional juice that has many beneficial effects on human health. However, it also has a high concentration of organic acids which may cause gastrointestinal discomfort. Hence, the organic acid content in cranberry juice was reduced to different levels of deacidification (0%, 19%, 37%, 50%, and 77%) by electrodialysis to study the impact of the deacidification rate on intestinal cell integrity. Before in vitro tests on Caco-2 cells, all samples underwent three steps of in vitro digestion: oral, gastric and intestinal. Digested and deacidified cranberry juices were applied to Caco-2 cells and the transepithelial electrical resistance (TEER) was measured after 24 hours of contact to evaluate the resulting cell integrity. In the presence of deacidified cranberry juice, the integrity of caco-2 cell monolayers measured by the ΔTEER was increased by 56% in comparison with raw cranberry juice, but a minimal deacidification rate of 37% was necessary to reach this level of protection.

Nanoscale Delivery of Resveratrol towards Enhancement of Supplements and Nutraceuticals.

Resveratrol was investigated in terms of its stability, biocompatibility and intestinal permeability across Caco-2 cell monolayers in its free form or encapsulated in solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs). SLNs and NLCs presented a mean diameter between 160 and 190 nm, high negative zeta potential of −30 mV and resveratrol entrapment efficiency of 80%, suggesting they are suitable for resveratrol oral delivery. Nanoencapsulation effectively protected resveratrol from photodegradation, and MTT assays demonstrated that neither resveratrol nor lipid nanoparticles adversely affected cell viability and integrity of Caco-2 cell monolayers. The in vitro intestinal permeability of resveratrol was significantly increased by NLCs, and SLNs did not impair the absorption of resveratrol. Resveratrol oral absorption can be enhanced during meals, since the intestinal permeability was increased in the presence of fed-state intestinal juices. SLNs and NLCs constitute carrier systems for resveratrol oral administration, for further use as supplements or nutraceuticals.

Permeability of plumbagin across human intestinal cell in vitro.

Plumbagin is the active compound isolated from plants used in traditional medicine for treatment of various diseases such as activities malaria, leishmaniasis, viral infections and cancers. The aim of the study was to investigate the permeability of plumbagin across Caco-2 (human epithelial colorectal adenocarcinoma) cell monolayer and its effects on the expression and function of P-glycoprotein. The integrity of Caco-2 cell monolayer was evaluated by measuring trans-epithelial electrical resistance and permeation (Papp) of Lucifer yellow across the cell monolayer. The effect of plumbagin on P-glycoprotein was detected by measuring its interference with the transport of the P-glycoprotein substrate (R123) and the effect on MDR-1 mRNA expression was detected by RT-PCR. The Papp of plumbagin (2-8 µM) for the apical to basolateral and basolateral to apical directions were 10.29-15.96 × 10(-6) and 7.40-9.02 × 10(-6) cm/s, respectively, with the efflux ratios of 0.57-0.73. Plumbagin is not either a substrate or inhibitor of P-glycoprotein. It did not interfere with the P-glycoprotein-mediated R123 transport across Caco-2 cell monolayer, as well as the function of P-glycoprotein and the expression of MDR-1 mRNA. Results suggest moderate permeability of plumbagin across the Caco-2 cell monolayer in both directions. The transport mechanism is likely to be a passive transport.

The effect of phytic acid on tight junctions in the human intestinal Caco-2 cell line and its mechanism

This study investigated the effect of phytic acid (IP6), a potential absorption enhancer of flavonoid components, on tight junction (TJ) integrity in Caco-2 cell monolayers and its possible mechanisms. Transepithelial electrical resistance (TEER) across the monolayers decreased rapidly, and the flux of fluorescein sodium (a paracellular marker) increased after treating with IP6 in a concentration-dependent manner. Confocal microscopy results showed that IP6 produced a concentration-dependent attenuation in the distribution of occludin, ZO-1, and claudin-1. Immunoblot analysis revealed that IP6 could down-regulate the expression level of these TJ proteins, which resulted in the opening of TJ. Additionally, the divalent cations Ca2+ and Mg2+ influenced the IP6-induced distribution of occludin, ZO-1, and claudin-1 in different directions, which enhanced barrier function. In conclusion, IP6 can decrease the integrity of Caco-2 cell monolayers by modulating the TJ proteins' localization and down-regulating the expression levels of TJ proteins including claudin-1, occludin, and ZO-1; the reduction effects of divalent cations such as Ca2+ and Mg2+ on the regulation of TJ induced by IP6 should be addressed. The present work will offer some useful guidance for the application of IP6 in drug delivery area.

Glucose and calcium ions may modulate the efficiency of bovine β-casomorphin-7 permeability through a monolayer of Caco-2 cells

Milk and dairy products provide a lot of valuable nutritive elements. They are also sources of biologically active peptides, including β-casomorphins that manifest the properties of morphine. An activity of DPPIV seems to be most crucial factor decreasing the efficiency of the β-casomorphin-7 (BCM7) transport. The increase of BCM7 concentration in blood may intensify symptoms of apparent life threatening events (ALTE), autism, schizophrenia, and allergy. This study aimed at identifying the influence of several selected substances on a transport efficiency of bovine BCM7 through an intestinal monolayer in a Caco-2 cell model system. Applying the ELISA method, the permeability coefficient of BCM7 through the Caco-2 monolayer was calculated. TEER values were used to evaluate the integrity of Caco-2 cell monolayers. An increase of glucose and Ca2+ concentrations in the culture medium was accompanied by an increase of the BCM7 transport efficiency. The lowest permeability coefficients of BCM7 were observed for the membranes with high electrical resistances. The transport was enhanced in the presence of milk infant formulas, whereas no changes were observed when using μ-opioid receptor antagonist (casoxin-6). The results may be useful in understanding the pathogenesis of inflammation and food allergy in infants.

Lecithin in mixed micelles attenuates the cytotoxicity of bile salts in Caco-2 cells

This study was designed to investigate the cytotoxicity of bile salt–lecithin mixed micelles on the Caco-2 cell model. Cell viability and proliferation after mixed micelles treatments were evaluated with the MTT assay, and the integrity of Caco-2 cell monolayer was determined by quantitating the transepithelial electrical resistance and the flux of tracer, FITC-dextran 4400. The apoptosis induced by mixed micelles treatments was investigated with the annexin V/PI protocol. The particle size of mixed micelles was all smaller than 100nm. The mixed micelles with lower than 0.2mM sodium deoxycholate (SDC) had no significant effects on cell viability and proliferation. When the level of SDC was higher than 0.4mM and the lecithin/SDC ratio was lower than 2:1, the mixed micelles caused significant changes in cell viability and proliferation. Furthermore, the mixed micelles affected tight junctions in a composition-dependent manner. Specifically, the tight junctions were transiently opened rather than damaged by the mixed micelles with SDC of between 0.2 and 0.6mM. The mixed micelles with more lecithin also induced less apoptosis. These results demonstrate that relatively higher concentrations of mixed micelles are toxic to Caco-2 cells, while phospholipids can attenuate the toxicity of the bile salts.

Intestinal safety of water-soluble beta-cyclodextrins in paediatric oral solutions of spironolactone: effects on human intestinal epithelial Caco-2 cells.

Effects of water-soluble beta-cyclodextrins (beta CDs) on intestinal epithelial integrity were investigated, to establish the safe use of these beta CDs as solubilizers of spironolactone in paediatric enteral solutions. Mannitol permeability and transepithelial resistance (TER) of human intestinal epithelial Caco-2 cell monolayers during exposure to dimethyl-beta-cyclodextrin (DM beta CD), hydroxypropyl-beta-cyclodextrin (HP beta CD) and sulphobutyl ether beta-cyclodextrin (SBE beta CD) were followed. Staining methods were used to discern cells with damaged membranes and to study the integrity of cytoskeletal actin and tight junctions. Cytotoxicity of the beta CDs was tested by effects on intracellular dehydrogenase activity. Exposure to HP beta CD and SBE beta CD solutions had only minor effects on the integrity of Caco-2 cell monolayers. In contrast, DM beta CD clearly increased the epithelial permeability for the hydrophilic marker [14C]mannitol across Caco-2 monolayers, decreased TER and showed a dose-dependent cytotoxicity. According to staining, DM beta CD increased the permeability of the apical cell membrane without discernable effects on cytoskeletal actin. HP beta CD and SBE beta CD appear to be safe additives for use in enteral spironolactone preparations with respect to their acute local effects on epithelial integrity.

A Moderate Interaction of Maltosyl-α-cyclodextrin with Caco-2 Cells in Comparison with the Parent Cyclodextrin

The cytotoxicity of maltosyl-alpha-cyclodextrin (G2-alpha-CyD) and maltosyl-beta-cyclodextrin (G2-beta-CyD) toward Caco-2 cells was compared with that of natural alpha-cyclodextrin (alpha-CyD), beta-cyclodextrin (beta-CyD) and gamma-cyclodextrin (gamma-CyD). The degree of increase in cytotoxicity was dependent on the CyD's type and the concentration: the cytotoxicity of CyDs at the same concentration increased in the order of gamma-CyD<G2-beta-CyD<G2-alpha-CyD<<alpha-CyD, although beta-CyD could not be compared to other CyDs because of low solubility in water. Alpha-CyD decreased transepithelial electrical resistance (TEER) and increased the apical-to-basolateral (AP-to-BL) transport of [3H]mannitol, a paracellular transport marker, in a concentration-dependent manner, suggesting that alpha-CyD decreased the integrity of Caco-2 cell monolayers. In addition, alpha-CyD increased the AP-to-BL transport of rhodamine 123, a transcellular transport marker, under the experimental conditions being independent of P-glycoprotein. In contrast, G2-alpha-CyD, G2-betaCyD and gamma-CyD had slight effect on both TEER and the transport of mannitol and rhodamine 123 even at relatively high concentrations up to 150 mM. The inability of G2-alpha-CyD and G2-beta-CyD to effect TEER and the transport of mannitol and rhodamine 123 could be explained by the findings that these maltosylated CyDs released only a small amount of membrane constituents from Caco-2 cell monolayers and interacted only weakly with monolayers composed of L-alpha-dipalmitoylphosphatidylcholine (DPPC) formed on water. These results indicate that G2-alpha-CyD has less cytotoxicity and less disturbing ability toward Caco-2 cell monolayers than alpha-CyD, and G2-beta-CyD has, at least, comparable cytotoxicity to beta-CyD toward them. Thus, from the safety point of view, highly water-soluble G2-alpha-CyD and G2-beta-CyD may be particularly useful in various pharmaceutical formulations.