The present study aimed to investigate the effects of alcohol on intestinal epithelial barrier permeability and expression of the tight junction-associated proteins, zonula occludens-1 (ZO-1) and claudin-1. An alcohol-treated Caco-2 intestinal epithelial cell monolayer
Alcoholic liver disease (ALD) is a major cause of liver-associated morbidity and mortality worldwide (
The intestinal epithelial barrier is mainly composed of a monolayer of cells with intercellular tight junctions, a complex three-dimensional structure and a thick mucosal gel layer secreted by the mucous membrane; it also provides a dynamic and regulated barrier to the extracellular flux of the lumina (
The human colon adenocarcinoma Caco-2 cell line was obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Caco-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Grand Island, NY, USA) supplemented with 0.1 mmol/l non-essential amino acids, 10 mmol/l HEPES, 4.5 mg/ml glucose, 100 U/ml penicillin, 100 U/ml streptomycin, 4 mM glutamine and 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), and incubated in a humidified atmosphere (95% air, 5% CO2) at 37°C.
To establish an
Firstly, the abdomen was carefully incised from the midline and a 1- to 1.5-cm sample of the small intestine was dissected out. Then, an exposed area of 0.126 cm2 was utilized to mount the sample vertically in Ussing chambers. Following that, Krebs-Ringer bicarbonate solution (in mM: 128 NaCl, 5.1 KCl, 1.4 CaCl2, 1.3 MgCl2, 21 NaHCO3, 1.3 KH2PO4 and 10 NaH2PO4, pH 7.4) gassed with 95% O2–5% CO2 was used to bath the tissues. Following a 15-min equilibration period, an EVC 4000 Precision V/I clamp device (World Precision Instruments, Sarasota, FL, USA) was used for the measurement of the transepithelial electrical voltage and current per 5 min until 30 min. The calculation of epithelial resistance was achieved using Ohm’s law (R=V/I). The experiment was repeated at least three times using a different tissue sample each time.
The Caco-2 cells were seeded on flat-bottomed, 96-well tissue culture plates. Following incubation with alcohol (1, 2.5, 5, 7.5 and 10%) for 4 h, the cells were incubated with 100 μl MTT [5 mg/ml solution in phosphate-buffered saline (PBS); Sigma-Aldrich, Taufkirchen, Germany] for 1 h at 37°C. The culture media was removed, and 150 μl dimethylsulfoxide was added to each well. The absorbance of the resulting colored solution was measured at 570 nm with a microplate reader (Wallac Victor 2; Perkin-Elmer, Waltham, MA, USA). In this assay, the yellow MTT solution is converted into a blue formazan dye within the mitochondria and deposited intracellularly. The intensity of the blue stain is then quantitatively assessed by spectrometry and used as a measure of cell viability.
Cell viability was also assessed by measuring the release of cytosolic enzymes. The electrical resistance of Caco-2 cell monolayers cultured on Transwell filters was assessed using a Millicell-ERS instrument (Millipore, Bedford, MA, USA). The electrical resistance was expressed in units of Ω•cm2 using the surface area of the Transwell insert.
Fluorescent yellow (40 μg/ml; Sigma-Aldrich) in serum-free DMEM was added to the upper chamber of the Transwell system. Following incubation with alcohol for 0, 20, 40, 60, 120 or 180 min, the media from the lower chamber was collected. The absorbance was assessed using a fluorescence spectrophotometer (excitation wavelength, 427 nm; emission wavelength, 536 nm), and the concentration of fluorescent yellow was calculated based on the standard curve. The fluorescent yellow flux rate (%) was equal to the fluorescent yellow concentration in the lower chamber/the fluorescent yellow concentration in the upper chamber. Lactate dehydrogenase (LDH) was added into the medium. The Caco-2 cells were incubated with the varying concentrations of alcohol for 4 h. LDH assessment was performed in 250 μl-aliquots using an LDH kit (Doles reagents, Goiânia, GO, Brazil).
Transepithelial electrical resistance (TEER) and the fluorescent yellow flux rate were assessed to estimate the effects of alcohol on the paracellular permeability in the Caco-2 cell monolayers. The electrical resistance of the Caco-2 cell monolayers cultured on Transwell filters was assessed using a Millicell-ERS instrument (Millipore, Bedford, MA, USA). The electrical resistance was expressed in units of Ω•cm2 using the surface area of the Transwell insert. Fluorescent yellow (40 μg/ml; Sigma-Aldrich) in serum-free DMEM was added to the upper chamber of the Transwell system. Following incubation with alcohol for the varying times indicated, the media from the lower chamber was collected. The absorbance was assessed using a fluorescence spectrophotometer (excitation wavelength, 427 nm; emission wavelength, 536 nm), and the fluorescent yellow concentration was calculated based on the standard curve. The fluorescent yellow flux rate (%) was equal to the fluorescent yellow concentration in the lower chamber/the fluorescent yellow concentration in the upper chamber.
The Caco-2 cells were washed with Dulbecco’s PBS (D-PBS) containing 0.1 mM ethylenediamine tetraacetic acid (EDTA) three times without calcium and magnesium. The Caco-2 cells were then homogenized in 1 ml lysis buffer A [(Shanghai Pik-day, Shanghai, China) 2 mM EDTA, 10 mM ethylene glycol tetraacetic acid, 0.4% NaF, 20 mM Tris-HCl, protease inhibitor cocktail, phosphatase inhibitor and 1% Triton X-100 (pH 7.5)] at 4°C. Samples were centrifuged at 14,000 × g for 30 min, and the supernatant was transferred to a separate tube and collected as the soluble fraction. Buffer A (150 μl) with 1% sodium dodecyl sulfate (SDS) at 4°C was then added to the pellet. The pellet’s structure was disrupted with an ultrasonic crusher. The samples were then centrifuged at 14,000 × g for 30 min at 4°C. The supernatant was collected as the insoluble fraction. Equal amounts of protein (40–50 μg) were separated by SDS-PAGE and processed for immunoblotting with antibodies for ZO-1 (diluted 1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and claudin-1 (diluted 1:100; Santa Cruz Biotechnology, Inc.). The protein bands were scanned using ChemiImager 5500 V2.03 software (Alpha US Inc., Miami, FL, USA), and the integrated density values (IDVs) were calculated using a computerized image analysis system (Fluor Chen 2.0; Olympus, Yokohama, Japan) and normalized to that of β-actin.
The Caco-2 cell monolayers grown on glass coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Following blocking with 2% bovine serum albumin in PBS, the cells were incubated with rabbit anti-ZO-1 (diluted 1:50; Santa Cruz Biotechnology, Inc.) and rabbit anti-claudin-1 (diluted 1:100; Zymed, South San Francisco, CA) for visualization of the distribution of ZO-1 and claudin-1. The glass slides were analyzed using immunofluorescence microscopy (Olympus, Tokyo, Japan).
Experiments were repeated at least three times. Continuous variables are expressed as the mean ± standard deviation. Categorical data are presented as frequencies and percentages. Differences between the groups were compared using the two-tailed, non-paired Student’s t-test or one-way analysis of variance for continuous variables, where appropriate. Comparisons of categorical variables between the groups were performed using the χ2 test. All tests of statistical significance were two-sided, with P<0.05 being considered to indicate a statistically significant difference. The statistical analyses were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA).
The Caco-2 cells were initially treated for 3 h with varying concentrations (1, 2.5, 5, 7.5 and 10%) of alcohol, and cell viability was evaluated using the MTT and LDH assays. The MTT assay results showed that the cell viability was not altered at alcohol concentrations of <5% (
As shown in
The Caco-2 cells in the alcohol treatment groups were incubated with 5% alcohol for 0, 20, 40, 60, 120 and 180 min. As shown in
Expression of the claudin-1 protein in the alcohol-treated Caco-2 cells showed a progressive increase following 60 min of incubation, reached its maximum level at 60 min and then showed a decreasing trend following 60 min of incubation (
The distribution of ZO-1 and claudin-1 was assessed using immunofluorescence microscopy (
Experimental and clinical studies have revealed evidence that damaged intestinal epithelial integrity and intestinal barrier dysfunction are the two fundamental causes of increased intestinal permeability (
The Caco-2 cell line, originally derived from a human colon adenocarcinoma, resembles small intestinal enterocytes and spontaneously undergoes typical enterocytic differentiation (
Smaller molecules penetrate enterocytes through a transcellular route via a large number of transmembrane channels and carriers, whereas larger molecules are transported across enterocytes through a paracellular pathway via intercellular tight junctions (
In conclusion, the results of the present study indicated that alcohol increases the permeability of the intestinal epithelial barrier in a dose- and time-dependent manner. Alcohol induces a change in the expression of the tight junction-associated proteins, ZO-1 and claudin-1, which are two major sites of alcohol action, thus increasing intestinal epithelial barrier permeability.
This study was funded by the Scientific Research of the First Hospital of China Medical University (fsfh1313).
alcoholic liver disease
zonula occludens-1
transepithelial electrical resistance
lactate dehydrogenase
Effect of alcohol on Caco-2 cell viability. The MTT assay results showed that the cell viability was not altered at alcohol concentrations of <5%.
Effect of alcohol on the concentration of the cytosolic enzyme, LDH, in Caco-2 cells. The LDH assay indicated that alcohol did not increase the release of the cytosolic enzyme LDH into the media at concentrations of ≤10%. LDH, lactate dehydrogenase.
Effect of alcohol on changes in the TEER of the Caco-2 cells. Caco-2 cells in the 5% alcohol-treated groups exhibited a significant time-dependent decrease in TEER. *P<0.05 compared with control group. TEER, transepithelial electrical resistance.
Effect of alcohol on the changes in the fluorescent yellow flux rate of the Caco-2 cells. Caco-2 cells in the 5% alcohol-treated groups exhibited an increase in fluorescent yellow flux rate compared with the control cells.
Effect of 5% alcohol on ZO-1 expression in the Caco-2 cells. Alterations in the expression of the ZO-l protein revealed trends consistent with changes in the TEER value and the fluorescent yellow transmittance rate in the Caco-2 cells. *P<0.05 compared with control group; #P<0.05 compared with 0 min. ZO-1, zonula occludens-1.
Effect of 5% alcohol on claudin-1 expression in the Caco-2 cells. Alterations in the expression of the claudin-1 protein revealed trends consistent with changes in the TEER value and the fluorescent yellow transmittance rate in the Caco-2 cells.*P<0.05 compared with control group; #P<0.05 compared with 0 min.
Immunofluoresence localization (magnification, ×400) of ZO-1 and claudin-1 proteins in the Caco-2 cells. (A) Control; (B) Caco-2 cells treated with ethanol (5%) for 20 min; (C) Caco-2 cells treated with ethanol (5%) for 60 min. Alcohol was shown to induce the discontinuous distribution of ZO-1 and claudin-1 at the cellular membrane. ZO-1, zonula occludens-1.