Animals
Male Sprague-Dawley rats (6-8 weeks old), weighing 250–300 g, were used. Rats were acclimated for 1 week to a temperature-controlled room on a 12-h light/dark cycle and the ad libitum chow and water. Thirty-eight rats were randomly assigned to 3 groups: sham-operated group (SO group, n=8), SAP group receiving total parental nutrition (TPN group, n=15), and SAP group receiving early enteral nutrition (EEN group, n=15).
Animal model of SAP and nutrition support
SAP was induced in rats by infusion of 4% of sodium taurocholate as our previous study described by Kang et al. [13] Briefly, the rats were fasted overnight but allowed free access to water until 4 h before laparotomy. Animals were anesthetized by 4% isoflurane gas in table top animal anesthesia machine (RWD Life Science, Shenzhen, China). After the pancreatic duct was catheterized with a needle, the 4% sodium taurocholate was slowly injected from the tail of the pancreas. Approximately 2 min, the pancreas exhibited edema, exudation, and local hemorrhage, which indicated that the SAP was successfully induced. Rats in the SO group received the same surgical procedure without injection of sodium taurocholate. In all rats, one tube (1 mm epidural catheter) was placed at jejunum. Then this tube was tunneled subcutaneously and exited at the midpoint of back. The other tube (1 mm epidural catheter) was inserted into the right jugular vein. Then this tube was tunneled subcutaneously and exited at the midpoint of neck. After surgery, all rats were administered 4ml 0.9% saline subcutaneously to replace the fluid loss during surgery.
In EEN group, the rats received the EEN solution at 12h after surgery. The EEN solution contains 3.8% protein, 13.8% carbohydrates, 3.4% lipids, electrolytes, and multivitamins, with the calorie/volumn ratio of 1:1 (1 kcal/L). In TPN group, the rats received the TPN solution at 12h after surgery. The TPN solution contains 4.1% protein, 14.0% carbohydrates, 3.0% lipids, electrolytes, and multivitamins, with the calorie/volumn ratio of 1:1 (1 kcal/L). The EEN and TPN solutions were almost isocaloric and isonitrogenous to ensure the rats had the same nutritional intake. Each rat in both groups received the energy of 125 kcal/kg in the first day and 250 kcal/kg/d later. The samples were obtained after the treatment of nutrition 72 hours.
Enzyme-linked immunosorbent assay (ELISA)
We utilized the commercial ELISA kits (Jianglai biotechnology, Shanghai, China) to determine the serum amylase, diamine oxidase and endotoxin contents of abdominal aorta. The procedure was in accordance with the manufacturer’s instructions.
Hematoxylin eosin (HE) Staining
The tissues (2 cm) specimens obtained during the rat experiment were immediately fixed in 10% paraformaldehyde and incubated overnight at room temperature. Next, tissue samples were embedded in paraffin and 5µm sections were cut. Sections were deparaffinized in xylene and rehydrated in graded ethanol to distilled water and stained with hematoxylin and eosin for histological analysis. The pathology of pancreas was evaluated by improved Schmidt score method [14]. The pathology of lung was evaluated by Smith score method [15]. The pathology of ileum was evaluated by Chiu score method [16].
Immunohistochemistry assay
For immunohistochemistry, sections were deparaffinized in xylene and rehydrated in graded ethanol to distilled water. Subsequently, the sections were processed using the DAB Detection Kit (ZSJQ, Beijing, China) according to the manufacturer’s protocol. The IHC features shown in the figures are representative of all tissue samples studied. The primary antibodies are as following: Occludin Polyclonal Antibody (Invitrogen; 1:100 dilution); ZO-1 Polyclonal Antibody (Invitrogen; 1:100 dilution); Claudin-1 Polyclonal Antibody (MH25) (Invitrogen; 1:100 dilution).
Immunofluorescent assay
Frozen sections were cut at 15µm, and mounted on the slides. The nonspecific background was blocked by incubation with 3% bovine serum albumin in PBS for 1h at room temperature. The sections were incubated with Anti-ZO-1 (clone R40.76) (Merck&Millipore, 1:200 dilution), Occludin Rabbit Polyclonal Antibody (Proteintech, 1:50 dilution), Anti-Claudin 1 antibody (Abcam, 1:100 dilution), the mixture of Anti-mouse Monoclonal Anti-TCRγδ antibody (Abcam, 1:50 dilution) and Anti-LAMININ antibody (BOSTER, 1:50 dilution), and the mixture of Anti-CD8 antibody [OX-8] (Abcam, 1:50 dilution) and Anti-LAMININ antibody (BOSTER, 1:50 dilution) at 4℃ overnight. The sections were probed with Anti-rabbit IgG (H+L) Alexa Fluor 488 (Cell Signaling Technology, 1:1000 dilution) and Goat Anti-Rat IgG H&L Alexa Fluor 594 (Abcam, 1:100 dilution), the mixture of Anti-rabbit IgG (H+L) Alexa Fluor 488 (Cell Signaling Technology, 1:1000 dilution) and Anti-mouse IgG (H+L) Alexa Fluor 594 (Cell Signaling Technology, 1:1000 dilution), and the mixture of Anti-rabbit IgG (H+L) Alexa Fluor 488 (Cell Signaling Technology, 1:1000 dilution) and Goat Anti-Rat IgG H&L Alexa Fluor 594 (Abcam, 1:100 dilution). The nuclei were counterstained with proLong Gold Antifade Reagent with DAPI (4, 6-diamidino-2-phenylindole, Cell Signaling Technology, Beverly, MA, USA). Slides incubated without any primary antibody were used as negative controls.
Western blot
Proteins were separated by SDS gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked with 3% bovine serum albumin prepared in TBST (Tris-buffered saline, pH 7.5 containing 0.05% Tween-20) for 1 h and then incubated with antibodies overnight at 4℃: Occludin Polyclonal Antibody (Invitrogen; 1:125 dilution); ZO-1 Polyclonal Antibody (Invitrogen; 1:125 dilution); Claudin-1 Polyclonal Antibody (MH25) (Invitrogen; 1:125 dilution) and Anti-beta Actin Antibody (Abcam; 1:1000 dilution). The membranes were then washed four times in TBST and incubated with secondary antibody at room temperature for 1 h. An enhanced chemiluminescence reagent, WesternBright ECL HRP substrate (Advansta), was used to make the labeled protein bands detectable with Image System Minichemi (Tanon, Shanghai, China).
Statistical analysis
Statistical analyses were performed using SPSS 20.0 software. Quantitative data are expressed as means ± standard deviation. Levene’s tests were used to test homogeneity of variance. For data with equal variance, one-way analysis of variance (ANOVA) was used to compare the difference among groups. Categorical data were analyzed using chi square tests. Probability values less than 0.05 were considered statistically significant.