Fifty-eight SD rats weighing 250-320 g (Fudan University Medical Animal Center, Shanghai) were anesthetized with intraperitoneal injections of 2% saline-ketamin injection liquid (30 mg/g weight). The rats were fixed in a supine position and prepared in a sterile manner for catheterization and operation. A silastic catheter (0.6 mm in inner diameter, 1.0 mm in outer diameter) was inserted through the external jugular vein into the superior vena cava. The catheter was tunneled subcutaneously to the midscapular region and guarded by the flexible spring, and then hooked up to an infusion pump (B. Braun). The abdominal infection model was established by cecum perforation and ligation (CPL)[5], here one third of the cecum distal was ligated and perforated with holes through which a piece of rubber patch was placed and the pinhead hole (#12) fixed. A silastic catheter (1.2 mm in diameter) was placed by jejunostomy 30-40 cm away from the cecum, and the catheter was tunneled abdominally subcutaneously to the back. The rats were maintained in individual metabolic cages[6]. The silastic catheters of jejunum and neck vein were traversed together through the spring and linked to the infusion pump on the circumrotate equipment[7]. The PN and EN nutrient solution was infused at a constant infusion rate by 2 pumps for 5 d during the experimental period.

After establishment of model, all rats received intravenous 0.9% saline solution at 2.0 mL/h for 24 h. Rats were divided into three groups, PN group (n = 7): supported by PN; PN + EN group (EN group, n = 7): supported by PN + EN(peptide), EN was dripped through jejunum tube; PN + EN + probiotic group (probiotic group, n = 7): supported by PN + EN(peptide), the probiotics was infused by jejunostomy tube at about 10 mL/d. The 3 groups were isonitrogenic and isocaloric (Table 1) during 1-5 d. In the PN group, 100% calorie and nitrogen were supplied by the PN solution at the infusion speed of 3.3 mL/h and 80 mL/d. In EN groups, 80% calorie and nitrogen were supplied by the PN solution at an infusion speed of 2.6 mL/h, and 64 mL/d, and the other 20% were supported by EN (Pepti-2000) at 16 mL/d, which were diluted 2:1 to 24 mL with saline solution and the infusion speed maintained at 1 mL/h. In the probiotic group, the probiotics (supported by Shanghai Jiaotong University Limited Company) were added at 10 mL/d. The daily dose of amino acids was 2.5 g of nitrogen per kilogram, the amount of non-protein calories was 348 kJ/d and the total nitrogen was 504 mg/d. The rats were killed and samples taken in the morning of the 6th d.

Probiotics containing live lactobacillus plantarum (activity 1 × 10⁸ cfu/mL) was infused at about 10 mL/d by jejunostomy tube twice per day through 1-5 d.

The fecal sample (0.1 g) in cecum was placed in 9 mL Ringer dilution solution(which contained cysteine) and incubated anaerobically at 36 ± 1°C supplemented with brucella blood agar (2 d, total anaerobic CFU), bacteroides bile esculin agar (2 d), egg yolk agar (after equal volumes of 95% ethanol were added to the dilutions and the preparations stood for 30 min to select for clostridial spores), and Rogosa SL agar (Difco) (2 d for lactobacilli and 4 d for bifidobacteria, after Lactobacillus colonies were marked at 2 d). The dilutions were removed from the anaerobic glove box and inoculated (100 μL inocula) into plates which contained the following media: supplementary brucella blood agar (2 d, total aerobic CFU), MacConkey agar (Difco) (1 d, enterobacteria), bile esculin azide agar (Difco) (1 d, Cl Perfrigens) and were incubated aerobically at 37°C. To analyze the total Lactobacillus population, 10 colonies were picked randomly from a dilution agar plate containing about 100 colonies. The bacterial colonies were counted and identified by Microscan Autoscan-4 Machine (Dade Behringcom).

The fecal samples in cecum from each subject were also examined by PCR-denaturing gradient gel electrophoresis (DGGE) profiles. To extract bacterial DNA, 1 mL of fecal homogenate in pH 7.0 phosphate buffer (the buffer used for the azoreductase assay) was centrifuged at 14 600 g for 5 min (5°C). DNA was extracted from the resulting pellet with a Fast DNA kit (BIO 101, Vista, CA) by using CLS-TC (a cell lysis solution used for animal tissues and bacteria). The V2-V3 region of the 16S rDNA gene (positions from 339 to 539 in the Escherichia coli gene) of bacteria in the fecal samples was amplified by using primers bacteria ITS PS2 (5’-TG(C/T) ACA CACCGC CCG T-3’), PL2 (5’-GGG T (G/C/T) CCC CAT TC(A/G)G-3’). PCR was performed with 0.2-mL tubes by using a PCR Express thermal cycler (Hybaid, Teddington, UK). Each reaction mixture (50 μL) contained reaction buffer (10 mmol/L [final concentration] Tris-HCl, 2.5 mmol/L [final concentration] MgCl₂, 50 mmol/L [final concentration] KCl [pH 8.3]), each deoxynucleoside triphosphate at a concentration of 200 μmol/L, 20 pmoL of each primer, 1 μL of fecal DNA, and 2.5 U of Taq DNA polymerase (Boehringer, Mannheim, Germany). The following amplification program was used: 94°C for 3 min, followed by 30 cycles consisting of 94°C for 30 s, 56°C for 30 s, and 68°C for 60 s, and finally 7 min at 68°C. DGGE was performed by using a DCode universal mutation detection system (Bio-Rad, Richmond, CA) and gels that were 16 cm × 16 cm × 1 mm; 6% polyacrylamide gels were prepared and electrophoresed with 1 × TAE buffer prepared from 50 × TAE buffer (2 mol/L Tris base, 1 mol/L glacial acetic acid, 50 mmol/L EDTA). The denaturing gradient was formed by using two 6% acrylamide (acrylamide/bisacrylamide ratio, 37.5:1) stock solutions (Bio-Rad). The gels contained a 22%-55% gradient of urea and formamide that increased in the direction of electrophoresis. A 100% denaturing solution contained 400 mL/L formamide and 7.0 mol/L urea. Electrophoresis was performed at 130 V (constant voltage) and 60°C for about 4.5 h. Electrophoresis was stopped when a xylene cyanol dye marker reached the bottom of a gel. The gels were stained with an ethidium bromide solution (5 μg/mL) for 20 min, washed with deionized water, and reviewed by UV transillumination[8,9].

The distal ileum and colon tissue were taken to determine the occludin and IgA expression by indirect-immune histochemical fluorescence.

Occludin: The tissue was embedded into the frozen acetone at -20°C.Then it was sliced, and routinely dehydrated. To remove the endogenous peroxidase enzyme activity, the slides were treated with 0.1%-1% H₂O₂ at 37°C for 5-10 min. Then they were washed with PBS twice, and incubated at room temperature overnight with primary antibody 1:300 (goat anti-rat occludin antibody, Santa Cruz). And then they were washed with PBS 3 times and incubated with the second antibody 1:200 (anti-goat IgG, Santa Cruz) for 30 min, then washed again with PBS 3 times and the formula AB enzyme reagent was added. Then they were washed with PBS again and incubated with peroxidase enzyme for 30 s-10 min. After that, the slides were washed by H₂O₂, dehydrated and mounted.

IgA: The tissue was embedded into paraffin and sliced, then routinely dewaxed. The slides were washed with PBS 3 times, incubated with 3.3% H₂O₂ at room temperature for 10-15 min to block the endogenous peroxidase enzyme, then washed with PBS again, and albumin interdiction solution was added for 5 min to reduce the nonspecific background staining. Then slides were washed with PBS, and the first antibody 1:500 (mouse anti-rat IgA antibody, Sigma) was added at 4°C refrigerator overnight. After that slides were washed with PBS again, and second antibody 1:100(anti-mouse antibody, DAKO) labeled with biotin was added. Slides were then washed with PBS again, and visualised with DBA enzyme-substrate illustration system, then dyed again and developed.

The data were analyzed by HPIAS1000 high definition color image manipulation system. Under the same expanding multiple (400), the occludin density was determined per field of vision by light-densimeter (at least five fields per slide).

The gut tissues about 0.5 cm in length were taken from distal ileum and colon 5 cm away from the valva ileocaecalis. One fixation procedure was used for conventional thin-section electron microscopy. The fixation procedure included incubation with OsO₄ alone (1% or 2% in phosphate buffer) at 0°C for 30 min. After fixation, the ileum and cecum were washed extensively in Veronal acetate buffer (90 mmol/L, pH 6.0), stained by incubation at 0°C for 60 min in uranyl-magnesium acetate (0.5%) in the same buffer, washed again, dehydrated, and embedded. Thin sections were doubly stained with uranyl acetate and lead nitrate and the change of tight junction and microvilli were observed (2 × 10⁴) using a Philip EM 400 electron microscope.

Blood from vena cava (1 mL), mesenteric lymphatic tissue (5 g), liver and lung tissue, were collected, homogenized and placed into a tube containing cardio-cerebral leachate，incubated at 35°C. After 18-24 h samples were stained with methylene blue-eosin method, and continued to be cultured at 35°C. 18-24 h later, the number of bacterium was counted by Microscan Autoscan-4 machine.

Blood from the portal vein (1-2 mL) was taken into heparin lithium anticoagulant centrifuged at 500 r/min for 5 min. The supernatants were mixed with the limuluslysate reagent to examine the endotoxin level.

Analysis of data was performed by χ² and Dunnette t test with statistical program SPSS10.0, the experimental data were expressed as mean ± SD of the samples between 3 groups. The level of significance was set at P < 0.05 or P < 0.01.

The CPL model was established successfully in 58 SD rats; 37 rats died, with the total death rate during 6 d of 63.8%. The death rate in PN group (78.7%) was higher than that in probiotic group (41.6%, χ² = 5.658, P = 0.0174 < 0.05, Table 2) and EN group (46.1%, χ² = 4.691, P = 0.030 < 0.05, Table 3). Interloop abscess (70%), liver abscess (35%) and lung abscess (20%) were observed in the surviving rats, and the positive rate of the blood culture reached 37%.

In the cecum, the amount of all genus (Enterobacillus, t = -0.276, P = 0.018 < 0.05, Bifidobacteria, t = -2.749, P = 0.017 < 0.05, Lactobacillus, t = -8.033, P = 0.002 < 0.01, Cl Perfrigens, t = -2.317, P = 0.031 < 0.05) of the EN group were more than that of the PN group. In the probiotic group the amount of Enterobacilus (t = -2.30, P = 0.390 < 0.05), lactobacillus (t = -9.935, P = 0.001 < 0.01) and bifidobacteria (t = -5.644, P = 0.002 < 0.01) increased, and the amount of Cl Perfrigens (t = -3.144, P = 0.384 < 0.05) decreased as compared with PN group.

The profiles of DNA sequence in PN group on electro-phoresis (lanes 3-9) were less than that in the normal group (lanes 1-2), EN group (lanes 10-16), and probiotic group (lanes 17-23); lanes 3, 6, 7 presented with a new 16S rDNA sequence in the profile (labeled in Figure 1). The sequence profiles of expression in probiotics and EN groups were similar to that in the normal group (lanes 9-10).

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Figure 1 Gut bacterium colony DNA fingerprint profiles. Lane M: Marker; lanes 1-2: normal group; lanes 3-9: PN group; lanes 10-16: EN group; lanes 10-16: Probiotic group.

There was more occludin expression in the probiotic group and EN group on the surface, intracellular and inter-cell spaces of the gut epithelial cells as compared with PN group (Figures 2A-F). The occludin positive expression area per measured-window in EN group (intestine, t = -4.955, P = 0.034 < 0.05, cecum, t = -5.407, P = 0.019 < 0.05, Table 4) and probiotics group were higher than that in PN group (intestine, t = -5.426, P = 0.002 < 0.01, cecum, t = -6.112, P = 0.006 < 0.01), and that in the probiotic group was higher than that in EN group(intestine, t = -2.645, P = 0.023 < 0.05, cecum, t = -2.307, P = 0.042 < 0.05).

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Figure 2 Intestinal and colon epithelial occludin expression. A: Intestinal epithelium occludin expression in PN group (200 ×); B: decreased epithelium occludin expression in colon in PN group (200 ×); C: Intestinal epithelium occludin expression in probiotics group (200 ×); D: Colon epithelium occludin expression in probiotics group (200 ×); E: Intestinal epithelium occludin expression in probiotics group (200 ×); F: Colon epithelium occludin expression in probiotics group (200 ×).

The expression of IgA in EN group (intestine, t = -4.995, P = 0.003 < 0.01, cecum, t = -2.850, P = 0.014 < 0.05) and probiotic group (intestine, t = -3.575, P = 0.024 < 0.05, cecum, t = -4.946, P = 0.004 < 0.01) was higher than that in PN group, especially the small intestine of the EN group and the cecum of the probiotic group.

There were more integrated tight junctions, less mito-chondrion endoplasm and microvilli brushed in EN group and probiotics group as compared with PN group (Figures 3A-F). The readability of the tight junction in probiotics group was clearer than that in PN group and EN group.

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Figure 3 Ultra-structure of tight junction of the intestine and colon. A: Tight junction of intestine in PN group (5000 ×); B: Tight junction of colon junction in PN group (5000  ×); C: Tight junction of intestine in EN group (5000 ×); D: Tight junction of the colon in EN group (5000 ×); E: Tight junction of intestine in probiotics group (5000 ×); F: Tight junction of the colon in probiotics group (5000 ×).

Bacterium translocation rate (BTR) in PN group (60.7%) was higher than that in probiotic group (28.6%, χ² = 5.853, P = 0.016 < 0.05, Table 5) and EN group (32.1%, χ² = 4.595, P = 0.032 < 0.05, Table 5), whereas there was no marked difference of the BTR between the probiotics group and EN group (χ² = 0.0845, P = 0.7713 > 0.05)

The endotoxin level of the PN group was higher than that in EN group (t = 3.954, P = 0.002 < 0.01 ) and in probiotic group (t = 4.615, P = 0.001 < 0.01).