The mice and the protocol involved in the study had been approved by Institutional Animal Care and Use Committee (IACUC). Approval ID: I07-038-3. All the mice were housed under standard conditions per protocols of IACUC and Hebei medical university vivarium in a barrier facility (GB 14925-2001).
C57BL/6 mice were purchased from Vital River Laboratory Animal Technology Co. Ltd for in vivo studies [License No. SCXK (Beijing) 2006-0009]. Acute colitis was induced by administration of dextran sodium sulfate (DSS; 40 000-50 000 MW; Sigma) drinking water. Male mice of 8 weeks received either regular drinking water (control) or 2% (w/v) DSS drinking water (model) ad libitum for 7 days, after which the mice were resumed on water for the remainder of the experiment. A total of 30 mice were randomly assigned to control, model and 1,25(OH)2D3−treated group (each group = 10). The mice in the 1,25(OH)2D3−treated group received 1,25(OH)2D3 (Sigma) daily (0.2 ug/25 g/d) by intragastric administration for 14 days , and the mice in control and model group were given normal saline without DSS.
Mice were executed at the end of the experiment. Serum was obtained, and Lipopolysaccharide (LPS) kit was purchased from Sigma, and Calcium (587-A) kit was purchased from Sigma. Serum calcium was measured according to the manufacturer’s instructions. Vitamin D deficiency was monitored by serum calcium analysis and normal serum calcium levels for mice are 2.00–2.75 mmol/L . LPS was measured by Limulus quantitative azo color (LQAC) test.
Assessment of disease activity
Rachmilewitz DAI was assessed by an investigator blinded to the protocol according to a standard scoring system. The combined score composes of the extent of body weight (BW) loss, stool consistency and detection of occult blood (OB) in the stool, and they are defined as follows. Loss in BW is scored as: no weight loss is scored as 0, weight loss of 1–5% from baseline as 1, 5–10% as 2, 10–20% as 3, and more than 20% as 4. For stool consistency, a score of 0 is assigned for wellformed pellets, 2 points for pasty and semiformed stools that do not adhere to the anus, and 4 points for liquid stools that were adhere to the anus. For OB, a score of 0 point is assigned for no blood, 2 points for positive hemoccult, and 4 points for gross bleeding. These scores are added together and divided by three, resulting in DAI ranging from 0 (healthy) to 4 (maximal activity of colitis)  and measured by area under curve (AUC).
Assessment of colonic injury and inflammation
Postmortem, the colon from the cecum to the anus was removed, and the entire colon length was measured as a marker of inflammation. On macroscopic examination, the degree of colon damage was scored as follows: 0, normal colon tissue; 1, minimal colon wall thickening without congestion; 2, moderate colon wall thickening with congestion; 3, moderate colon wall thickening, rigidity, and congestion; and 4, marked colon wall thickening, rigidity, and congestion. A segment of the proximal colon was fixed in 10% formalin, and embedded in paraffin; 4 um-thick sections of this tissue were stained with hematoxylin and eosin (H&E). Histologic evaluation was performed by two investigators blinded to the animal groups and the inflammation was graded as follows: severity of inflammation (0–3: none, slight, moderate, severe), extent of injury (0–3: none, mucosal, mucosal and submucosal, transmural), and crypt damage (0–4: none, basal 1/3 damaged, basal 2/3 damaged, only surface epithelium intact, entire crypt and epithelium lost). Then each score was multiplied by a equivalent with the percentage of tissue involved (×1: 0–25%, ×2: 26–50%, ×3: 51–75%, ×4: 76–100%).
Determination of myeloperoxidase (MPO) activity
MPO activity in homogenates of the colon was determined in this way: equal weights (100 mg wet weight) of the colon from each group were suspended in 1 mL buffer (0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer, pH 6.0) and sonicated at 30 cycles, twice for 30 s on ice. Homogenates were centrifuged, 2000 g, 4°C, and the supernatants were stored at 80°C. The samples were incubated with a substrate of odianisidine hydrochloride and the reaction was carried out in a 96-well plate by adding to 290 μL 50 mM phosphate buffer, 3 μL substrate solution (containing 20 mg/mL odianisidine hydrochloride), and 3 μL H2O2 (20 mM). The samples (10 μL each well) were added to each well to start the reaction. The reaction was stopped by adding 3 μL sodium azide (30%), and the plates were read for the assay at light absorbance of 460 nm. MPO activity was determined by the curve obtained from the standard MPO .
Isolation of mesenteric lymph nodes cells (MLNs) and lamina propria mononuclear cells (LPMCs)
Mesenteric lymph nodes (MLNs) were placed in a petri dish with 2 mL ice-cold RPMI 1640. Two prewet sterile glass slides were used to smash MLNs, and then passed through a 70 μm nylon cell strainer into a 50 mL Falcon tube. Petri dish and glass slides were rinsed with an additional 3 mL RPMI 1640, centrifuged, removed the supernatant, and resuspended the pellet in 1.0 mL RPMI 1640 with 10% FCS. The cells were then counted. Isolation of lamina propria mononuclear cells (LPMCs) was described as follows: The dissected gut was opened longitudinally, transferred onto HBSS/5 mM EDTA and incubated for 20 min at 37°C under slow rotation (40 g) in a thermal incubator. Gut tissues were then transferred onto a new petri dish with 1–2 mL collagenase solution (100 mL of RPMI 1640 with 10% FCS : Hyaluronidase, 0.25 g; Collagenase type II, 0.15 g; DNase I, 0.025 g) and minced into about 1 mm-square pieces. The minced tissues were then passed through a 70 um cell strainer, then centrifuged, resuspended the pellet in 6 ml of 45% Percoll solution, overlaid the cell suspension on top of 3 mL of the 72% Percoll solution, centrifuged, carefully collected the cells, centrifuged again, aspirated and resuspended the cells immediately in 500 μL RPMI 1640 with 10% FCS, and at last counted cells one by one .
For immunohistochemistry staining, antigens were retrieved by 10 min boiling in 10 mM citrate (pH 6.0). The slides were stained with mouse monoclonal anti-claudin-1 antibody, anti-occludin antibody (Santa Cruez), and rabbit polyclonal anti-zo-1 antibody (Invitrogen), rabbit polyclonal anti-IFN-γ antibody (Santa Cruez) and mouse monoclonal anti-TNF-α antibody (sigma). After incubation with peroxidase-conjugated secondary antibody, signals were visualized with a diaminobenzidine (DAB) peroxidase substrate kit (Vector Laboratories). For TJ immunofluorescence staining, Caco-2 cell monolayer (fixed in 95% ethanol) or colon sections were incubated with mouse monoclonal anti-claudin-1 antibody, anti-occludin antibody (Santa Cruez) and rabbit polyclonal anti-zo-1 antibodies (Invitrogen), then with an FITC-conjugated secondary antibody or Cy3-conjugated secondary antibody. Slides were examined with a Leica DMIRE2 scanning laser confocal microscope.
In vivo permeability
In vivo permeability assay was performed to assess barrier function and performed by fluorescein isothiocyanate dextran (FITC-D). Briefly, food and water were withdrawn for 4 h, and mice were gavaged with permeability tracer (60 mg/100 g BW of FITC-D (MW 4 000; Sigma). Serum was collected retroorbitally 4 h after FITC-D gavaged, and fluorescence intensity was measured (excitation, 492 nm; emission, 525 nm; Cytofluor 2 300 nm; Millipore), and FITC-D concentrations were determined from standard curves generated by serial dilution of FITC-D . Permeability was calculated by linear regression of sample fluorescence (Excel 5.0, Microsoft Office). Detection of viable bacteria in MLNs represented bacterial translocation from the lumen to the MLNs. The MLNs of left colonic regions were removed aseptically and dissected free of fat. A 0.1 ml aliquot of each homogenate was plated into blood agar, incubated at 37°C for 48 h, and then counted the number of colonies. The ratio of bacterial translocation was presented for percentage.
Specimens were fixed, washed in acetone, critical point dried, coated with gold by a sputter coater, and observed under scanning electron microscope (SEM) fitted with a lanthanum hexaboride cathode using an accelerating voltage of 10 kV. Cell monolayer samples were fixed in 2% glutaraldehyde and postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, 1 h, 37°C. The tissues were dehydrated in an ascending series of ethanol, infiltrated with eponate 12 resin and then embedded and polymerized, 70°C, 24 h. Resin-embedded blocks were sectioned at 70 nm and collected on 200 mesh, formvar-coated copper grids. Grids teue were stained with uranyl acetate and lead citrate and examined with a JEOL 1200 EX II transmission electron microscope (TEM).
Caco-2 cell culture
The Human Colon Carcinoma cell Line (Caco-2) was purchased from Shanghai Institutes of Biochemistry and Cell Biology for in vitro studies, and cultured in medium supplemented with 10% fetal bovine serum (FBS) and penicillin (50 U/ml)/streptomycin (50 μg/mL) in a 5% CO2 atmosphere at 37°C. Caco-2 cell monolayer was incubated with or without 2% DSS in the absence or presence of 10−9, 10−8, 10−7 M of 1,25(OH)2D3 (administered 2 h prior to DSS) for 48 h.
In vitro permeability
Paracellular permeability was determined by measuring the apical to the basolateral flux of FITC-D using a modification of previously described method . Briefly, confluent epithelial monolayer on a 0.33 cm2, 0.4 μm pore size permeable support was washed twice with Hanks’ balanced salt solution containing calcium chloride and magnesium sulfate (HBSS) and maintained at 37°C on a shaking warm plate. FITC-D, 1 mg/mL, was added apically at time 0, and 50 μL samples were removed from the basolateral compartment at 30 min intervals from 0–360 min, inclusively. Fluorescence intensity of each sample was measured (excitation, 492 nm; emission, 525 nm; Cytofluor 2 300 nm; Millipore Corp, Waters Chromatography, Bedford, MA), and FITC-D concentrations were determined from standard curves generated by serial dilution of FITC-D. Paracellular flux was calculated by linear regression of sample fluorescence (Excel 5.0, Microsoft WA, Power Macintosh 7200).
For transepithelial electric resistance (TEER) measurement, Caco-2 cells were cultured on collagen-coated transwell polycarbonate membrane filter inserts (Corning). The cells were seeded at a density of 1 × 105 cell/mL, the medium was changed every 1 or 2 days. The integrity of the monolayer was observed to accomplish after culturing for 21 days, and evaluated by measuring TEER with Millicell-Electrical Resistance System (ERS) equipment (Millipore). Monolayer showing TEER values of 130–200 ohm/cm2 were used for the experiments. The monolayer cells were gently rinsed three times with HBSS and equilibrated in the same solution for 30 min at 37°C (inside volume; 400 μL, outside volume; 600 μL). An aliquot (40 μL) of the apical solution was replaced by the same volume of sample solution, containing each compound at concentrations of 100–800 μg/mL, and the TEER value of the monolayer was monitored for up to 360 min after adding to the sample solution .
Real-time fluorescent quantitation PCR (real time Q-PCR)
The expressions of the gene zo-1, occludin, claudin-1, TNF-α and IFN-γ were characterized by real time Q-PCR. Briefly, Total RNA was extracted from colons or Caco-2 cells using Trizol (Gibco) reversely transcripted into cDNA according to manufacture’s protocol. The primers for the zo-1, occludin and claudin-1 genes were as follows: zo-1-Forward 5′-TCATCCCAAATAAGAACAGAGC-3′, zo-1-Reverse 5′-GAAGAACAACCCTTTCATAAGC-3′, (198 bp amplicon); occludin-Forward 5′-CTTTGGCTACGGAGGTGGCTAT-3′occludin-Reverse 5′-CTTTGGCTGCTCTTGGGTCTG-3′, (86 bp amplicon); Claudin-1-Forward 5′-GCTGGGT-TTCATCCTGGCTTCT-3′, Claudin-1-Reverse 5′-CCTGAGCGGTCACGATGTTGTC-3′, (110 bp amplicon); GAPDH-Forward 5′-GAGACCTTCAACACCCCAGC-3′, GAPDH-Reverse 5′-ATGTCACGCACGATTTCCC-3′, (263 bp amplicon); IFN-γ-Forward 5′-ATGAACGCTACACACTGCATCTT-3′IFN-γ-Reverse 5′-TTTCTTCCACATCTATGCCACTT3′ (139 bp amplicon); TNF-α-Forward5′-GGTTCTGTCCCTTTCACTCACT-3′TNF-α-Reverse5′-GAGAAGAGGCTGAGACATAGGC-3′ (169 bp amplicon). Reaction system: 10 μL 2.5 × Real master Mix, 1.25 μL 20 × SYBR solution, 0.5 μL upstream primer, 0.5 μL downstream primer and 2 μL DNA template was brought up to 25 μL with purified water. The amplification was performed with Quantitect™ SYBR® Green PCR Mastermix (Qiagen), using the following time and temperature profile: 95°C for 5 min, 45 cycles of 1 min at 95°C, 10 s at 60°C. Fluorescent quantitative analysis was performed with the thermal cycler’s software package to calculate the △Ct value. The levels of zo-1, occludin, claudin-1, TNF-α and IFN-γ were calculated by the 2-△△Ct analysis. The 2-△△Ct was presented as the relative expression of the gene expression.
Western blot analysis
The concentration of proteins of colon tissue and caco-2 cells were determined using coomassie brilliant blue assay, the extracts containing equal quantities of proteins (100 μg) were electrophoresed in 8% polyacrylamide gel. Subsequently, the separated proteins were transferred onto a nitrocellulose membrane. The membrane was blocked for non-specific binding for 30 min (5% skimmed milk in PBS), and then incubated overnight at 4°C with rabbit anti-zo-1 polyclonal antibody (1:100), mouse anti-claudin-1 monoclonal antibody (1:200), anti-occludin antibody (1:200), rabbit polyclonal anti-IFN-γ antibody (1:200), mouse monoclonal anti-TNF-α antibody (1:200) and rabbit anti-GAPDH monoclonal antibody (1:100). The membrane was subsequently incubated at room temperature for 2 h with goat anti-rabbit IgG (1:2 000)/anti-mouse IgG (1:2 000). Blots were developed with enhanced chemiluminescence detection reagents (Santa Cruz Biotechnology Inc), exposed on Kodak Xdmat blue XB-1 film and quantified by Bandscan 5.0 software using GAPDH as internal control. Densitometry is reported using the integral optical density value (IOD). The results were represented in the form of IOD ratio of the target protein to GAPDH.
Data were expressed as mean ± standard deviation (mean ± SD) and analyzed with SPSS 13.0 software. The comparison of mean variability among all the groups was conducted by one-way ANOVA analysis and two group comparison with LSD test. A Student’s t-test was carried out for independent samples. Statistical significance was considered at P < 0.05.