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 in vitro model of the intestinal epithelial barrier, the Caco-2 cells were plated on Transwell filters (Corning, Inc., Corning, NY, USA) and regularly monitored visually using an inverted microscope (Olympus, Tokyo, Japan) and through epithelial resistance measurements.

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% (Fig. 1). Furthermore, the LDH assay indicated that alcohol did not increase the release of the cytosolic enzyme LDH into the media at concentrations of ≤10% (Fig. 2). Alcohol concentrations of >5% caused cell shedding but not cell fragmentation. At an alcohol concentration of 10%, the ratio of cell shedding was up to 51.67±3.36%. Therefore, subsequent experiments were performed using alcohol concentrations of 1–5%.

As shown in Fig. 3, 5% alcohol induced a significant time-dependent decrease in transepithelial electrical resistance (TEER), with the lowest value obtained after 60 min of alcohol treatment. A time-dependent increase in the fluorescent yellow flux rate as a result of alcohol treatment was observed, with the maximum flux rate occurring at 60 min (Fig. 4). The aforementioned results indicated that the decreased TEER value was associated with an increase in the fluorescent yellow flux rate.

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 Fig. 5, the expression levels of the tight junction-associated protein, ZO-1, exhibited a progressive decline following 20 min of incubation, reached a minimum level at 60 min and exhibited an increasing trend after 60 min of incubation. Significant differences in ZO-1 expression between the control and alcohol groups were observed at 60 min of incubation (P<0.05).

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 (Fig. 6). Significant differences in claudin-1 expression were identified among the control and alcohol-treated groups at 60 min (P<0.05).

The distribution of ZO-1 and claudin-1 was assessed using immunofluorescence microscopy (Fig. 7). Alcohol was shown to induce the discontinuous distribution of ZO-1 and claudin-1 at the cellular membrane.