Animals and PS exposure
The animals were fed and treated as described [8, 9]. Thirty male Sprague-Dawley (SD) rats (Shanghai-BK Co., Ltd. Shanghai, China), weighing 120 ± 5 g, were housed individually in cages at a temperature of 24 ± 1°C, a humidity of 55 ± 5% in a 12-h light/dark cycle, and were given normal chow and free access to water. The iron content in diet was 35 mg/kg. After 7 d adaption, the rats were divided into two groups randomly: control group and PS group. Each group was subdivided into three subgroups: 1 d group, 3 d group and 7 d group (5 rats in every subgroup). Each animal was used only once in the experiment. All animal treatments were strictly in accordance with international ethical guidelines and the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals, and the experiments were carried out with the approval of the Committee of Experimental Animal Administration of the University.
PS model was created in rats as described previously [8]. Briefly, a communication box was divided into Room A and Room B with a transparent acrylic board. Room A included 10 little rooms with a plastic board-covered floor and Room B included 10 little rooms with a metal grid-exposed floor for electric insulation. Rats in Room B were randomly given electrical shock (0.6 mA for 1 s) for 30 min (60 times) through the floor and exhibited nociceptive stimulation-evoked responses, such as jumping up, defecation and crying; rats in Room A were only exposed to the responses of rats in Room B to establish PS model. PS was given to rats for 30 min every morning (10:00-10:30) for 7 days. At the end of the exposure, the rats were kept in the cages for another 4 min before they were taken out. Animals in the control group were only kept in the cages for 4 min without receiving any stress.
At the end of PS exposure all rats were deeply anesthetized by intraperitoneal (i.p.) injection of 7% chloral hydrate [8, 9]. Blood samples were collected from the heart followed by centrifuging at 3 000 g for 15 min, and the supernatants were obtained and stored at -80°C for futher determination. Then the rats were perfused through the left cardiac ventricle with ice-cold phosphate buffered saline (PBS; pH 7.4) to flush out the plasma. Hypothalamus and duodenum were quickly removed and snap frozen in liquid nitrogen, and kept in a -80°C freezer till use. Perfused duodenum were sectioned at 30 μm on a sliding microtome into free-floating tissue sections.
Contents of noradrenalin (NE) in hypothalamus, corticosterone (CORT) and adrenocorticotropic hormone (ACTH) in serum were analyzed using commercially available ELISA kits (R&D Systems, Inc., USA). Coefficient of variation (CV) values for NE, CORT and ACTH were 13%, 15% and 11% separately.
Iron absorption measurement
The fecal samples were collected, dried and weighted daily. Urine was not collected or analyzed for iron content, because iron excretion in urine was assumed to be negligible. The diets were changed daily. Samples were stored in polypropylene vials and kept at -20°C before analysis.
To insure accurate collection of feces at day 1, day 3 and day 7 during PS, rat was put into a plastic cage independently to prevent any loss of feces. Before food intake, animals were given carmine red by intragastric administration. Feces were collected between two red feces (not including the second). Apparent absorption of iron was expressed as (iron content in food - iron content in feces)/iron content in food × 100%.
Iron contents in the feces were measured using inductively coupled plasma mass spectrometry (ICP-MS). Before analysis, fecal samples were weighted and then desiccated in an oven at 90°C. The dried samples (0.2 g) were dissolved in 10 ml of 18 N HNO3 by incubation at 90°C for 24 h and then acid was evaporated at 58°C. Samples were resuspended in 5 ml of 2 N HNO3 and analyzed by ICP-MS using 2 N HNO3 as the blank.
Membrane and soluble protein preparation
The frozen duodenum samples were homogenized in 5 ml HEPES-EDTA buffer [20 mmol Hepes/L, pH 7.4; 1 mmol EDTA/L; 250 mmol sucrose/L; a protease inhibitor mixture containing 4-(2-aminoethyl)benzenesulfonyl fluoride, trans-epoxysuccinyl-L-leucylsmido(4-guanidino)butane, bestatin, leupeptin, aprotinin, and sodium EDTA (Sigma)].. The homogenate was centrifuged at 500 g for 5 min at 4°C. The supernatant fluid was then centrifuged at 100 000 g for 30 min at 4°C. The supernatant fluid (soluble protein) was collected for ferritin protein determination, and the crude membrane fraction was resuspended in 0.3 ml homogenization buffer for DMT1 and FPN1 protein determination. Samples were stored at -70°C till analysis. Protein concentrations of the soluble and membrane fractions were quantified by the method of Lowry [10].
Western blotting analysis of DMT1, FPN1 and ferritin expressions
Aliquots of the lysates containing 50 μg of protein were diluted in 2× sample buffer [50 mM Tris, pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.1% bromophenol blue, and 5% μ-mercaptoethanol] and heated for 5 min at 95°C before SDS-polyacrylamide gel electrophoresis (PAGE) on a 10% gel and subsequently transferred to a pure nitrocellulose membrane under conditions 200 mA during 120 min. The blots were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20 (TBS-T; 20 mM Tris-Cl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) for 2 h at room temperature. Proteins were incubated overnight at 4°C with a primary antibody against DMT1 (1:1000, ADI), FPN1 (1:1000, ADI), ferritin (1:5000, Sigma), or β-actin (1:10000, Sigma). The immunoreactive bands were detected by goat polyclonal anti-rabbit-HRP antibody (Santa Cruz, CA). The blots were developed by incubation in ECL chemiluminescence reagent (Amersham Life Science, Arlington Heights, IL, USA) and subsequently exposed to BioMax Light Film (Eastman Kodak Co., USA). Processed blots were quantified by using the BandScan 5.0 software.
Perl's staining
For Perl's staining, sections were processed through a series of graded alcohols, into xylene, and rehydrated back to water. Sections were incubated in a 1:1 solution of 2% HCl and potassium ferrocyanide (Sigma) for 30 min and rinsed in water. Sections were counterstained with Neutral Red, dehydrated in increasing concentrations of ethanol, cleared in xylene, and mounted on slides.
Statistical analysis
All results were expressed as means ± SD. Statistical analysis was carried out by using SPSS 11.0. One-way ANOVA, correcting for differences in sample variance, was used to determine whether differences were statistically significant in groups. A P value less than 0.05 was a considered statistically significant difference.
