Animals
Clean grade healthy Sprague-Dawley (SD) rats (male, 6–7 weeks, 250–300g) were purchased from Zhejiang Vital River Laboratory Animal Technology Company Limited. All rats were housed in an air-conditioned environment (ambient temperature controlled at 25±0.5℃, humidity controlled at 50%–60%) with a 12/12-h light/dark cycle in animal experiment center of Ruijin Hospital. Food and water were freely accessible by rats. The study was performed in accordance with the Principles of Laboratory Animal Care (NIH publication no.85Y23, revised 1996) and in compliance with the ARRIVE guidelines. All experiments were approved by the Animal Ethics Committee of Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine and carried out according to the institutional guidelines.
SAP modeling and experimental design
In the first stage, twenty-four rats were randomly assigned into four groups (n=6 in each group) to observe the expression of DC-DIGN in colon tissue: sham group, SAP 6h group, SAP 12h group and SAP 24h group. After screening out the time point with the highest expression of DC-SIGN in colon tissues, another twenty-four rats were divided into four groups according to the random number table, namely sham group, non-fluid resuscitation (NFR) group, IVFR group and FRVC group.
SAP model was established according to the method described by Aho et al.35. The rats were anesthetized by using isoflurane (RWD Life Science, Shenzhen, China). After the pancreatic duct at the tail of the pancreas was blocked by the vascular clip, closed venous indwelling needle (BD Company, Shanghai, China) was retrogradely penetrated into the pancreatic duct through the duodenal intestinal wall. 5% sodium taurocholate solution (0.1 mL/100 g body weight, Sigma, United States) was injected into the pancreatic duct with a microinjection pump. Before pulling out the needle, maintaining the pressure in the pancreatic duct for 5 minutes.
IVFR and FRVC operation of SAP rats were performed according to the previous study8 after the SAP models were completed. In IVFR model of SAP rats, normal saline was continuously infused at a rate of 4ml/kg/h for 12 hours with a microinjection pump through a Y-type trocar implanted in the right femoral vein. The FRVC operation method was to insert a Swan-Ganz floating catheter from the anus, inject about 0.5ml of air into the balloon to fix the catheter, and inject saline at the same speed as the IVFR model. In the sham group, the abdominal cavity of rat was opened and closed without other operation. The serum samples and tissues were immediately isolated and stored at -80°C until analysis.
Western blot
The protein concentration was measured using a BCA protein assay kit (Servicebio, Wuhan, China).20 μg of the protein samples were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel for electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Temecula, Calif). After being blocked with 5% skim milk at room temperature for 1 h, membranes were incubated overnight at 4℃ with rabbit primary antibodies of DC-SIGN (1:2000, Thermo Fisher scientific, Shanghai, China) and with HRP-conjugated secondary antibodies for 1 hour at room temperature. The blots were detected by chemiluminescence using the ECL reagent (Servicebio, Wuhan, China). The results were visualized using darkroom development techniques. The bands were analyzed with the AlphaEaseFC software and compared with GAPDH.
Immunohistochemical
For immunohistochemical detection of DC-SIGN expression, tissue sections on glass slides were placed in the citrate antigen retrieval solution (PH 6.0) retrieve the antigen. After being treated with endogenous peroxidase and nonspecific protein blocking, the sections were overnight incubated with DC-SIGN primary antibody (1:100, A01025-2, Boster Biological Technology Co., Ltd., Calif) at 4℃ and then washed three times for 5 min each with PBS. After being incubated with biotinylated secondary antibody (1:200, GB23303, Servicebio, Wuhan, China) for 1 h at room temperature, the sections were stained by diaminobenzidine for microscopic examination.
Histological analysis
The pancreas, colon, lung, liver, and kidney tissues were fixed in 4% paraformaldehyde for 24 hours. The target tissues were dehydrated and waxed. Then, the tissues were embedded and cut into 4-μm thick slices for hematoxylin and eosin (HE) staining. Two senior pathologists separately made evaluation on the tissues through an optical microscope. All of the histopathology evaluations were performed on six fields per section under 100× magnification and scored according to the previous study36-38.
Enzyme-linked immunosorbent assays
Enzyme-linked immunosorbent assays (ELISAs) were performed with the serum samples of rats using commercial rat-specific kits for tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and Lipopolysaccharide (LPS) (Multi Sciences Biotech Co., Ltd. Hangzhou, China) according to the product specifications.
Statistical Analysis
The clinical data were analyzed by SPSS 19.0 statistical software (SPSS, Inc., Chicago, IL) and GraphPad Prism 6 (GraphPad Software, San Diego, Calif). Differences between groups were analyzed using a one-way ANOVA and Mann-Whitney test. Data are expressed as mean ± standard error of mean (SEM). P<0.05 was considered statistically significant.