A total of 15 male Bal b/c mice, 58-62 days of age, weighing 20-24 g, at the time of irradiation, were housed in conventional cages with free access to drinking water and standard chow. A pathogen-free environment with controlled humidity and temperature was maintained. These mice were randomly divided into 3 groups: one group received sham-irradiation (n = 5), the other two groups received single dose of 9.0 Gy (n = 5). The dose was chosen based on the data from our previous experiments. Whole-body irradiation was performed with a ⁶⁰Co-source at a dose rate of 0.78 Gy/min. The irradiated mice were killed 3 h and 72 h post-radiation, and intestines were removed and histological sections were made according to reference[15].

Mouse intestinal epithelial cells were isolated according to the method described by Bjerknes[16] with slight modifications. In brief, animals were sacrificed, and the small intestine was removed and perfused with 30 ml of cold PBS. Then it was gently everted using a glass rod and flushed by PBS with its two ends enveloped. The swollen intestine was transferred to a flask, and immersed into warm PBSE solution (PBS containing EDTA 1 mmol/L), and shaken at 150 cycles per min. After shaken for 5 min, the intestine was transferred to another clean flask with warm PBSE solution, and vibrated for another 5 min. The shed intestinal epithelial cells were centrifuged at 500 × g for 10 min, and prepared for protein extraction.

The cell pellet harvested as described above was then washed in ice-cold PBS 3 times. Cells were resuspended in cold PBS, and counted. Total proteins were extracted from 10⁷ cells with an appropriate volume (200 μl) of lysis buffer containing 7 mol/L urea, 2 mol/L thiourea, 65 mmol/L DTT, 2% CHAPS, 2 mmol/L PMSF, 0.5% IPG buffer, and protease inhibitor mixture. The extraction mixture was sonicated using a MSE 100 ultrasonic probe, and then centrifuged at 12000 × g for 20 min. After transferred to a clean tube, the supernatant was stored at -70 °C as aliquots. The protein concentration was determined using Bradford dye-binding assay with bovine serum albumin as the standard.

Two-dimensional electrophoresis was carried out by using the Mini-PROTEIN 2-D apparatus (Bio-Rad). For isoelectric focusing (IEF), precast IPG strips (Immobiline DryStrips, Amersham Pharmacia Biotech, Uppsala, Sweden) were used. Samples were applied via rehydration of IPG strips in sample solution for more than 12 h. Before application, the samples were diluted to a total volume of 350 μl with rehydration buffer (8 mol/L urea, 2% CHAPS, 0.5% IPG buffer, 0.3% DTT, and a trace of bromophenol blue). Protein sample (1000 μg) was loaded onto an 18 cm IPG strip (pH3-10, linear), and isoelectric focusing was run for 45 KVh at 20 °C. To improve the sample entry, low voltage (100V) was applied for 2 h at the beginning. After IEF separation, the IPG strips were immediately equilibrated for 2 × 15 min with buffer (50 mmol/L Tris-HCl, pH6.8, 6 mol/L urea, 30% glycerol, 2% SDS, and a trace of bromophenol blue). DTT (2%) was added in the first step, and iodoacetamide in the second step. For the second dimensional separation, the concentration of homogeneous SDS-polyacrylamide gels was 13%. The proteins in the equilibrated strips were run at a current of 30 mA/strip for about 5 h until the bromophenol dye reached the bottom of the gels. Molecular mass marker was run on the same gel to determine the relative molecular masses of the proteins.

After electrophoresis, the resolved proteins in 2-DE gels were fixed in ethanol/acetic acid/water (4/1/5) for at least 1 h, and then visualized by Coomassie blue R-250. The gels were scanned (Gel Doc 2000, Bio-Rad), and the images were processed with PDQuest software (Ver 7.0, Bio-Rad). To determine the variation, three gels were prepared for each sample. The computer analysis allowed automatic detection and quantification of protein spots, as well as matching between control gel and treatment. Protein spots that were new, absent, up- and down-regulated, were simultaneously displayed on the same image using the gels of sham-irradiated sample as the reference.

The differential protein spots were exactly excised from the gels. Gel pieces were destained with 50% acetonitrile for several times until Coomassie blue was invisible. Then gel pieces were dried in a vacuum centrifuge. The shrinking gel pieces were re-swollen with 5 μl of protease solution (trypsin at 0.05 μg/μl in digestion buffer of 25 mmol/L NH4HCO3). Additional 50 μl of digestion buffer was added into the gel piece. Digestion was performed overnight at 37 °C. Next, 50 μl of 5% TFA solution was added to the gel piece, followed by incubation for 60 min at 42 °C. The supernatant was collected and concentrated using ZipTip pipette tips (Millipore) according to the manufacturer’s instructions. The samples were mixed on the MALDI target with matrix DBA solution. Peptide mass maps were generated by applying Biosystems Voyager 6192 MALDI-TOF-Mass Spectrometry (ABI, USA). Database searching was performed using monoisotopic peptide masses obtained from MALDI-TOF-MS. The SWISS-PROT and TrEMBL database was searched with PeptIdent software (http://www.expasy.org/tools/pep-tident.html). Peptide masses were assumed to be monoisotopic masses and cystines were assumed to be iodoacetamided. The peptide mass tolerance was set to 0.2Da, and the maximum of missed cleavage site was set to 1. Species was set as mouse.

Total proteins were separated on 12% SDS-polyacrylamide gel, and then transferred to nitrocellulose membranes. After blocked for 1 h in the Tris/NaCl (50 mmol/L Tris-HCl, 200 mmol/L NaCl, 0.05% Tween-20, pH 7.4) containing 2% BSA, membranes were probed with the goat anti-mouse Enolase polyclonal antibody (Santa Cruz, USA). Immunoreactive bands were visualized with a solution containing 1 mg/ml DAB and 0.01% H₂O₂.

Total RNAs were isolated from mouse intestinal epithelia with TriPure isolation reagent (Roche, USA) according to the manufacturer’s descriptions. Peroxiredoxin I, Q72VC8, and GAPDH transcripts were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA PCR kit (Takara, China). The primers for peroxiredoxin I are 5’GTTCTCACGGCTCTTTCTGTTT-3’ and 5’CTTCTGGCTGCTCAATGCTG-3’, Q8VC72 5’TGGACAAAGCCTTCATAGCA-3’ and 5’CCTGGCAGAAACCACAGTAGA-3’, GAPDH 5’ACCACAGTCCATGCCATCAC-3’ and 5’TCCACCACCCTGTTGCTGTA-3’. The amplification was performed with one denaturing cycle at 94 °C for 5 min, then 25-27 cycles at 94 °C for 40 s, at 55 °C for 40 s, at 72 °C for 40 s, and one final extension at 72 °C for 5 min. RT-PCR products were run on 1.2% agarose gel.

Mice after 9.0 Gy irradiation died within 4-6 days post-radiation, with the intestines severely injured. As shown in Figure 1, the histological changes after irradiation were in agreement with our previous experiments. Though the height of villi and crypts remained unchanged 3 h post-radiation, cell division of intestinal epithelia ceased, and the intestines were severely injured morphologically. Moreover, the height of intestinal crypts was obviously increased 72 h post-radiation, with villi decreased. This indicated that many survival epithelial cells proliferated.

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Figure 1 Histological changes in crypts following irradiation. A: Unirradiated controls (×100). B: 3 hours after a single dose of 9.0 Gy (×100). Villi and crypts remained unchanged, but cell division of intestinal epithelia ceased. C: 3 days after a single dose of 9.0 Gy (×100).

To study the different proteins involved in ionizing radiation, we applied 2-DE to analyze the proteomic alteration of mouse intestinal epithelia. The 2-D patterns were highly reproducible since each experiment was performed in triplicate and produced similar results. Figure 2 showed the representative 2-D maps of the mouse intestinal epithelia. Image analysis of 2-D gels revealed that averages of 638 ± 39, 566 ± 32 and 591 ± 29 protein spots were detected in 3 groups respectively, and the majority of these protein spots were matched. About 360 protein spots were matched between normal group and 3 h irradiation group, and the correlation coefficient was 0.78 by correlation analysis of gels. Three hundred and twelve protein spots were matched between normal group and 72 h irradiation group, and 282 protein spots between 3 h and 72 h irradiation groups. The unmatched spots represented those induced by irradiation as new or absent proteins, which were the main alteration of proteome.

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Figure 2 Two-dimensional electrophoresis maps. A: normal intestinal epithelial cells, B: 3 h irradiated epithelial cells, C: 72 h post-radiation. Number indicated proteins were summarized in Table 2.

Out of a total of 28 spots excised from the gels, 25 spots yielded nearly perfect MALDI spectra. A total of 19 spots were preliminarily identified by peptide mass fingerprinting. Figure 3 showed the typical peptide mass fingerprinting of Spot No. 11, whose peptides matched the mouse peroxiredoxin I. This spot was up-regulated by ionizing radiation. The identified proteins were summarized in Table 1. These proteins were involved in cellular process of anti-oxidation, metabolism, and protein post-translational processes. Other proteins, such as cellular structural proteins and hypothetic proteins derived from nucleic acid sequences, were also altered after irradiation. We also found, 6 spots with good MALDI spectra were returned with inconclusive match results, suggesting that they might be unknown proteins.

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Figure 3 MALDI-TOF-MS spectrum of spot P35700 in 2-DE map. MS spectrum of peptide mixture was obtained from a typical in-gel digestion of the 2-DE separated protein.

A protein spot was preliminarily identified as Enolase by MALDI-TOF-MS (Table 1), which was significantly increased in 3 h radiation group. Immunoblotting showed that Enolase was significantly up-regulated (2.1 fold more than the normal control, P < 0.01, Figure 4). This confirmed our data of 2-D electrophoresis.

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Figure 4 Western blot analysis of Enolase after radiation. A: Protein samples, obtained at indicated time after radiation, were separated by SDS-PAGE, and immunoblotted with corresponding antibody. Equal amounts of total proteins were applied to each lane. B: Relative protein level of Enolase was determined by quantitating the intensity of Enolase bands with a densitometer.

Semi-quantitative RT-PCR was used to detect the gene expression of peroxiredoxin I and Q8VC72. Total RNAs were isolated from normal and irradiated mouse intestine. The expression of peroxiredoxin I was markedly induced by radiation (Figure 5), using GAPDH as standard. However, the expression of Q8VC72 remained unchanged after radiation (Figure 6). This indicated that Q8VC72 might be a false-positive result.

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Figure 5 Semi-quantitative RT-PCR analysis of peroxiredoxin I. A: PCR product run on an agarose gel. The upper band repre-sented peroxiredoxin I (750 bp), and the lower band represented GAPDH (450 bp). B: Relative abundance of the mRNA of peroxiredoxin I was normalized by GAPDH.

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Figure 6 Semi-quantitative RT-PCR analysis of Q8VC72. RNA samples were prepared from mouse intestinal epithelia at indi-cated time. Left: The band represents Q8VC72 (461 bp). Right: The band represents GAPDH (450 bp). The expression of Q8VC72 remained unchanged after radiation.