Healthy 8-week-old male Bal B/C mice were obtained from Guangdong Medical Experimental Animal Center (Guangzhou, China). Mice were housed in an environment of constant temperature (25°C) and humidity in a 12–12 h light–dark cycle with free access to a specific pathogen-free laboratory diet and distilled water during all the experiments. After 1 week of acclimatization, weight-matched mice were randomly placed on a normal potassium (NK) mouse chow or a LK diet (Diet Research, New Brunswick, USA). All animals were killed on days 7, 14, 21 and 28 after treatment. Mice were intraperitoneally anesthetized with chloral hydrate and blood specimens were obtained by eye enucleation.
2.2 Influence of LK on intestinal movement in mice
Mice were administered activated carbon by gavage and the propulsion distance of activated carbon in the small intestine was detected after 30 min.
2.3 Plasma and tissue collection
Blood was obtained by eye enucleation. Luminal contents were removed from the ileum and cecum and flash frozen in liquid nitrogen. Samples of mesenteric lymph nodes (MLN), liver, spleen, kidney and intestine were rapidly harvested and processed. One half of the intestine was fixed in 4% buffered paraformaldehyde (pH 7.4) at 4°C overnight and embedded in paraffin for morphological staining and immunohistochemical analysis. Other sections were frozen in liquid nitrogen immediately for subsequent evaluation.
2.4 Measurement of intestinal permeability in mice
Microbiota DNA were extracted from ileal and cecal luminal contents using the QiAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). Amplification was performed on the V4 region of the 16S rRNA genes via polymerase chain reaction (PCR) using the FastStart Universal SYBR Green Master (ROX) Kit (Roche, Basel, Switzerland) with an ABI Prism 7900 H T Sequence Detection System (Life Technology, Carlsbad, CA, USA). The primer sequences for PCR analysis are shown in Supplementary Table 1.
2.5 Barrier function assessment
The paracellular and transcellular pathways were measured as the flux of 4 kDa fluorescein isothiocyanate-dextran (FD-4; Sigma–Aldrich, St. Louis, MO,USA). Mice were gavaged with FD-4 (1 ml/100 g) and blood samples were collected after 30 min. Concentration of FD-4 was measured via fluorescence at excitation 485 nm and emission 528 nm.
2.6 Plasma lipopolysaccharide-binding protein
Lipopolysaccharide-binding protein levels were detected in plasma samples by ELISA (Cusabio Life Sciences, College Park, MD, USA).
2.7 Cell cultures
Caco-2 cells were purchased from the American Type Culture Collection and maintained at 37°C in a culture medium composed of Dulbecco’s Modified Eagle’s medium with 4.5 mg/ml glucose, 50 U/ml penicillin, 50 U/ml streptomycin, 4 mM glutamine, 25 mM HEPES, and 10% fetal bovine serum. The cells were kept at 37°C in a 5% CO2 environment. Culture medium was changed every 2 days. Caco-2 cells were subcultured after partial digestion with 0.25% trypsin and 0.9 mM EDTA in Ca2+- and Mg2+-free phosphate-buffered saline (PBS).
The following antibodies were used: rabbit claudin-1 polyclonal antibody (Abcam, Cambridge, MA, USA), rabbit claudin-2 polyclonal antibody (Abcam), rabbit occludin polyclonal antibody (Abcam), mouse β-actin monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG, HRP-conjugated anti-rabbit IgG and Alexa-Fluor-488-conjugated anti-rabbit IgG were from Cell Signaling Technology.
2.9 Serum biochemistry
Serum was isolated from blood by centrifugation (1000 rpm at 4°C for 10 min). Serum potassium were measured by a Hitachi 7180 biochemistry autoanalyzer (Yokohama, Japan).
2.10 Intestinal histopathology
Paraffin-embedded intestines were cut into 2-µm sections and serial 6-µm sections. The 2-µm sections were stained with hematoxylin–eosin (HE). Immunofluorescence analysis was performed on the serial 6-µm sections. The HE-stained tissue sections were used to assess the morphological changes in the intestinal wall.
2.11 Measurement of transepithelial electrical resistance and paracellular permeability in vitro
Caco-2 cells were seeded and grown on collagen-coated polycarbonate membrane Transwell inserts with 0.4-µm pore size (Corning, NY, USA). Cells were allowed to grow until a transepithelial electrical resistance (TER) of > 350 Ω·cm2 developed, usually after 19–21 days, and monolayers were exposed to LK medium. An epithelial voltohmeter (Millipore, Bedford, MA, USA) was used for measurements of the TER of the filter-grown Caco-2 intestinal monolayers. To obtain the TER values, the background resistance value of a blank filter without cells was subtracted from the measured values and then the values were normalized to the area of the filter. Confluent monolayers of Caco-2 cells with TER values ≥ 350 Ω·cm2 were chosen for these studies. Cells in culture medium, treated with control or LK conditions, were assessed for paracellular diffusion of fluorescent Escherichia coli (K-12 strain; Invitrogen, Carlsbad, CA, USA) and Lucifer yellow (LY; Sigma–Aldrich). LY with concentrations of 100 µg/ml and fluorescent E. coli with multiplicity of infection of 20:1 were added to the apical side of cells and incubated for 2 and 6 h at 37°C, respectively. Monolayer permeability was assessed by measuring the fluorescence in the basal medium compartment of LY spectrophotometrically using SpectraMax M5 spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA) at excitation and emission spectra of 427 nm and 536 nm, and data were reported as relative fluorescent units. The number of fluorescent E. coli per cross-section field was determined by inverted microscopy (Olympus, Japan).
2.12 Immunofluorescence analysis
The paraffin-embedded intestine sections (6 µm) were dewaxed and rehydrated. After overnight antigen retrieval, the sections were incubated with block buffer (5% bovine serum albumin in PBS) for 1 h at room temperature. The sections were stained with anti-claudin-1 (1:100) or anti-occludin (1:100) antibody at 4°C overnight, followed by Alexa-Fluor-488-conjugated anti-rabbit IgG (1:2000) antibody. To identify nuclei, tissues were counterstained with the fluorescent dye 4′,6-diamidino-2-phenylindole for 5 min. In all cases, antibody-negative controls were evaluated to ensure that the results were not a consequence of cross reactivity or nonspecific binding of the secondary antibodies. All images were measured using a laser scanning confocal microscope (Zeiss LSM 510 META; Carl Zeiss, Oberkochen, Germany).
2.13 Western blotting
The frozen intestine (~ 100 mg) was pulverized in liquid nitrogen and suspended in 400 ml cell lysis buffer. The homogenates were sonicated for 15 s. The tissue lysate supernatants were extracted after centrifugation for 15 min at 13,500 × g at 4°C. Cells were harvested and lysed in the lysis buffer. The lysates were centrifuged at 12,000 × g, for 5 min at 4°C. Protein concentration was measured using the Bradford protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts of total protein were loaded and electrophoresed through SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore) for 2 h at 4°C. After blocking in 5% nonfat milk for 1 h at room temperature, the membranes were treated with anti-claudin-1 (1:1000 dilution), anti-occludin (1:1000 dilution), anti-claudin-2 (1:1000 dilution), or mouse anti-β-actin (1:1000 dilution) antibody at 4°C overnight. Secondary antibodies were HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology) or goat anti-mouse IgG (Cell Signaling Technology). Densitometric analysis was performed using an image analysis program (FluorChem8900; Alpha Innotech Corp., San Leandro, CA, USA).
2.14 Quantification of gene expression using real-time PCR
Total RNA from tissue samples or cultured cells was extracted using the Transcriptor First Strand cDNA Synthesis Kit (Roche). Real-time PCR for mRNA expression of TJ proteins, including claudin-1, claudin-2, and occludin, was performed using the FastStart Universal SYBR Green Master (ROX) Kit (Roche) with an ABI Prism 7900 H T Sequence Detection System (Life Technology). The primer sequences for real-time PCR analysis are shown in Supplementary Table 1. All reactions were conducted in triplicate. β-Actin was used as an internal control. Fold changes in gene expression were calculated using the 2−ΔΔCt method.