The mRMECs (cat. no. CD-1065) were purchased from Cell Biologics, Inc. (Chicago, IL, USA). The mRMECs were cultured in a 100 mm collagen-coated culture dish containing 10 ml supplemented medium (RPMI-1640; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) with 20% fetal bovine serum (16000044; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and maintained at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. The culture medium was replaced every 2 days. The cells used in the present study were between passages 3 and 4. When the cells had grown to 70% confluence, they were cultured under hypoxic conditions (93% N₂, 5% CO₂, 2% O₂) at 37°C for 24 h. Then cells were then washed twice with phosphate-buffered saline (PBS) and then exposed to FK506 (F4679; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at different concentrations (0.1, 1 and 10 µM) for 24 and 48 h, respectively. FK506 was dissolved in dimethyl sulfoxide (<0.1%), which caused no deleterious effect on the viability of the mRMECs in preliminary experiments.

The permeability of the mRMEC monolayers was assessed via TEER measurement. The TEER across the endothelial cell layer was calculated using a Millicell-ERS Voltohmmeter (EMD Millipore, Bedford, MA, USA). Briefly, the mRMECs were seeded onto a membrane at a density of 3×10⁵ cells/cm². The TEER measurements were implemented following treatment of the cells with FK506 at different concentrations (0.1, 1 and 10 µM). The TEER value of the blank Transwell membrane was used as a blank value, and was subtracted from the sample value. The TEER values were calculated as Ω × cm² and presented as the mean ± standard error of mean of three independent experiments. Each cell monolayer with a TEER value >100 Ω × cm² was considered to contain tight junctions.

The mRMECs were plated in 12 mm diameter Transwell polyethylene terephthalate membrane inserts (0.4 µm pore size) at a density of 2×10⁵ per insert. The permeability was measured using FITC-dextran, with 1 mg/ml FITC-dextran added to the apical compartment 4 h prior to the assessment. The samples were collected (25 µl) from the lower compartment for assessment, and PBS was added to 250 µl. The fluorescence was measured on a fluorescence luminometer at wavelengths of 492 nm (excitation) and 520 nm (emission).

The cells were lysed on ice for 20 min using RIPA lysis buffer with protease inhibitor, and were centrifuged at 13,400 × g at 4°C for 10 min. The supernatant was collected and total protein concentration was measured by BCA protein assay (Beyotime Institute of Biotechnology, Shanghai, China). The proteins were then boiled in sodium dodecyl sulfate (SDS) sample buffer for 10 min and then equal amounts of lysate protein (10 µl/lane) were added to 10% SDS-polyacrylamide gel electrophoresis gels. Following electrophoresis, the proteins were transferred onto a polyvinylidene fluoride membrane and the membranes were blocked in 5% skim milk in Tris-HCl buffer salt solution-Tween (TBST) for 2 h at room temperature. The membranes were then incubated with the following primary antibodies overnight at 4°C: Anti-zonula occludens-1 (ZO-1; 61-7300; 1:500; Thermo Fisher Scientific, Inc.), anti-p-NFATc1 (SC32979; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-t-NFATc1 (SC13033; 1:500; Santa Cruz Biotechnology, Inc.). β-actin (3700S; 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA) was used as a loading control. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G secondary antibody (A21010; 1:5,000) and HRP-conjugated goat anti-rabbit IgG secondary antibody (A21020; 1:5,000; both from Abbkine Scientific Co., Ltd., Wuhan, China) for 2 h at room temperature. The immunoreactive bands were visualized with enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The mRMECs were plated on glass slides in a 12-well plate at a density of 1×10⁵ per well and pretreated with hypoxia and FK506 (10 µM). Following washing with PBS, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were then incubated with polyclonal rabbit primary antibodies against ZO-1 (1:500; Cell Signaling Technology, Inc.) in 5% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) at 4°C overnight, followed by blocking with 5% BSA for 2 h at room temperature. The next day, following washing in PBS, the slides were incubated with Alexa Fluor 555 conjugated anti-rabbit IgG secondary antibody (4413S; 1:1,000; Cell Signaling Technology, Inc.) for 2 h at room temperature, and the nuclei were labeled with 4′, 6-diamidino-2-phenylindole for 10 min. Images were captured under a confocal microscope (Zeiss 510; Carl Zeiss AG, Oberkochen, Germany).

Total RNA were extracted from the mRMECs using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The purity and concentration of RNA samples were confirmed by the ratio of optical densities at 260 and 280 nm. Then, 1 µl RNA was reverse-transcribed in a reaction mixture containing 1 U RNase inhibitor, 500 ng random primers, 3 mM MgCl₂, 0.5 mM dNTP, 1X RT buffer and 10 U reverse transcriptase (Promega, Madison, WI, USA). Ths synthesized cDNA was used as a template for PCR reaction using GoTaq polymerase (Promega Corporation, Madison, WI, USA). The RT-qPCR analysis was performed using SYBR-Green qRT-PCR Master mix, according to the manufacturer's protocol (Biotool, Houston, TX, USA). qRT-PCR was performed for 40 cycles of denaturation at 94°C for 50 sec, annealing at 55°C for 50 sec and extension at 72°C for 50 sec. The quantification cycle values were normalized against β-actin and the 2^−∆∆Cq method (13) was used to calculate target gene expression. The results determined as the relative value compared with that of the control. The primers for target genes were obtained from the NCBI GeneBank database (http://www.ncbi.nlm.nih.gov/genbank/). The primers for COX-2, inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 (MCP-1) and β-actin were as follows: COX-2 forward, 5′-CCAGATGATATCTTTGGGGAGAC-3′ and reverse, 5′-CTTGCATTGATGGTGGCTG-3′; iNOS forward, 5′-ACAACAGGAACCTACCAGCTCA-3′ and reverse, 5′-GATGTTGTAGCGCTGTGTGTCA-3′; MCP-1 forward, 5′-ACTGAAGCCAGCTCTCTCTTCCTC-3′ and reverse, 5′-TTCCTTCTTGGGGTCAGCACAGAC-3′; β-actin forward, 5′-GGCGGACTATGACTTAGTTG-3′ and reverse, 5′-AAACAACAATGTGCAATCAA-3′. The samples were examined in triplicate and the experiment was repeated twice.

The supernatants of the mRMEC culture media were harvested 48 h following FK506 treatment and stored at −80°C until assayed. The levels of IL-6, ICAM-1 and VCAM-1 in the supernatants were measured using an ELISA kit. A 96-well plate containing 100 µl of captured antibody per well was incubated overnight at room temperature. Following incubation, the plate was blocked with 1% BSA in PBS for 1 h at room temperature. The samples were added (100 µl) and incubated for 2 h at room temperature. Following three washes with 0.05% Tween-20 in PBS, 100 µl of monoclonal antibodies were added to each well for 2 h at room temperature. Streptavidin-HRP was added to each well and incubated for 20 min at room temperature. The color reagent A (H₂O₂) and color reagent B (tetramethylbenzidine) substrate solution reactions were terminated at 20 min with 2 NH₂SO₄. Each well was immediately read on a microplate reader at 450 nm. The measurements were performed in triplicate.

All data are expressed as the mean ± standard error of the mean of three independent experiments. Differences in mean values among groups were subjected to one-way factorial analysis of variance followed by the Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS 20.0 software (IBM SPSS, Armonk, NY, USA).

At 24 and 48 h, the TEER values of the hypoxia groups were markedly lower, compared with the TEER in the control group. Compared with the hypoxia group, the TEER values of the FK506 groups (1 and 10 µM) were significantly higher, with 10 µM exhibiting a more marked effective, compared with 1 µM (Fig. 1).

Compared with the control group, the cell permeability of the hypoxia group was increased at 24 and 48 h. However, compared with the hypoxia group, the permeability of cells in the groups treated with FK506 at concentrations of 1 and 10 µM were significantly decreased. The reduction in cell permeability in the 10 µM group was more marked, compared with that in the 1 µM group (Fig. 2).

ZO-1 is a protein associated with tight junctions, which is involved in the epithelial function and barrier system. The results of the western blot analysis showed that the expression of ZO-1 was decreased in the mRMECs exposed to hypoxia. However, the protein expression levels of ZO-1 in the 1 and 10 µM FK506 groups were increased significantly, with higher expression in the 10 µM FK506 group (Fig. 3A and B).

The results of the immunofluorescence analysis were similar. Compared with the control group, the expression of ZO-1 in the hypoxia group was lower. Following treatment with FK506 at a concentration of 10 µM, the protein expression of ZO-1 was elevated (Fig. 3C). These results showed that FK506 suppressed the damage to tight junctions in hypoxia-induced mRMECs.

RT-qPCR analysis was performed to examine the mRNA expression levels of COX-2, iNOS and MCP-1 in the control, hypoxia and FK506 groups. As shown in Fig. 4A-C, hypoxia significantly increased the mRNA expression levels of COX-2, iNOS and MCP-1. No significant differences were found between the hypoxia group and the 0.1 µM FK506 group. However, compared with the hypoxia group, the mRNA expression levels were significantly decreased in the 1 and 10 µM FK506 group, and was more marked in the 10 µM group.

ELISA was performed to determine the concentrations of IL-6, ICAM-1 and VCAM-1 in the supernatants. It was found that the concentrations of IL-6, ICAM-1 and VCAM-1 in the hypoxia groups were significantly higher, compared with those in the control groups. The concentrations were decreased following treatment with FK506 (1 and 10 µM), which was more marked in the 10 µM group (Fig. 4D-F).

Western blot analysis was performed to examine the protein expression of NFATc1. As shown in Fig. 5, hypoxia significantly increased the protein expression of total (t-)NFATc1, whereas the expression of phosphorylated (p-)NFATc1 was downregulated, compared with the control group. Following treatment with FK506 at concentrations of 1 and 10 µM, the expression of t-NFATc1 was significantly decreased, whereas the expression of p-NFATc1 was significantly increased, compared with that in the hypoxia group. These changes in protein expression were more marked in the 10 µM group.