The various chemicals, antibodies, and other materials used in this experiment, along with their manufacturers, are listed below: angiotensin fragment 1–7, sodium pentobarbital, chloroquine diphosphate salt (C6628), and caerulein (CAE) were purchased from Sigma–Aldrich (Merck KGaA, Darmstadt, Germany); sodium taurocholate from Solarbio (Beijing, China); Rat Amylase (ab102523) and Lipase (ab102524) Activity Assay Kits, rabbit anti–rat microtubule-associated protein 1 light chain 3 A/B (LC3Ⅰ/Ⅱ) multiclonal antibody (ab62721), and rabbit anti–rat p62/SQSTM (p62) multiclonal antibody (ab91526) from Abcam (Cambridge, United Kingdom); rabbit anti–rat Akt (pan) antibody (#4691), rabbit anti–rat phospho-Akt (Ser473) antibody (#4060), rabbit anti–rat mTOR antibody (#2983), and rabbit anti–rat phospho-mTOR (Ser2448) antibody (#5536) from Cell Signaling Technology Inc. (Danvers, MA, USA); dactolisib (BEZ235, NVP-BEZ235) from Selleck Chemicals (Houston, TX, USA); mouse β–actin monoclonal antibody (TA-09), goat anti–rabbit and goat anti–mouse secondary antibodies, and biotinylated goat anti–rabbit IgG antibody from ZSJQ–BIO (Beijing, China); F-12K medium from American Type Culture Collection (ATCC; Manassas, VA, USA); fetal bovine serum from Gibco (Thermo Fisher Scientific Inc., Waltham, MA, USA); protease inhibitor, protein phosphatase inhibitor, phenylmethylsulfonyl fluoride (PMSF), and radioimmunoprecipitation assay (RIPA) lysis buffer from Applygen (Beijing, China); BCA Protein Assay Kit from Bioss (Beijing, China); polyvinylidene fluoride (PVDF) membranes (0.45 µm and 0.22 µm) and enhanced chemiluminescence (ECL) kits from Millipore (Darmstadt, Germany); and the Image Lab software from Bio–Rad (Hercules, CA, USA).
2.2 Animal models and grouping
Specific pathogen–free male Wistar rats (age, 8–10 weeks; weight, 200–250 g) were purchased from Charles River (Beijing, China). The animals were housed in a standard climate-controlled breeding room, with stable surroundings, an indoor temperature of 22℃ ± 1℃, and 12-h light-dark cycles, and fed on standard laboratory chow with water ad libitum. The rats acclimatized for 1 week before being randomly assigned to different groups.
Rats were randomly divided into the following groups: sham operation group (control), SAP groups (2, 12, 24, and 48 h, these time points refer to the time elapsed after the injection of sodium taurocholate), and Ang1-7 group. Each group included 8 rats. After a week of acclimatization, the rats were anesthetized using an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight). The biliopancreatic duct was occluded at the distal duodenum using a vascular clip. In the SAP groups, the canal was infused slowly with freshly prepared 3.75% sodium taurocholate (0.1 mL/100 g body weight) through a 1-mL syringe. Rats of the Ang1-7 group were immediately administered Ang1-7 (25 µg/kg weight), which was injected into a caudal vein, after the induction of SAP. The control group received an injection of the corresponding volume of normal saline. The time of death of the rats in each group was recorded for the calculation of mortality rate. After the rats were sacrificed, blood and pancreatic tissues were collected for the following experiments.
2.3 Cell culture and treatment
Rat pancreatic acinar AR42J cells from ATCC were cultured in F-12K medium supplemented with 20% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin (complete medium) at 37 °C in a humidified atmosphere containing 5% CO2. The cells were stimulated with 10− 6 mol/L CAE to induce AP in vitro. Chloroquine (10, 20, or 40 µmol/L) was used to impede the fusion of autophagosomes and lysosomes. Some cells were pretreated with different concentrations of Ang1-7 (0.1, 1, or 10 µmol/L) and/or BEZ235 (100 µmol/L). A vehicle containing 2.5% fetal bovine serum was used to starve the cells for 12 h before stimulation with drugs. At 24 h after drug administration, the cells were harvested for the subsequent experiments. The control-group cells were collected without any special treatment.
2.4 Enzyme-linked immunosorbent assay
Blood samples, after standing for 2 h, were centrifuged at 3000 rpm for 5 min to separate the serum. The serum α-amylase (AMYα) level was measured using the Amylase Assay Kit, and lipase activity was measured using the Lipase Activity Assay Kit, according to the manufacturer’s instructions.
2.5 Histopathological analysis
Transverse sections (4 µm) were incised from paraffin-embedded pancreatic tissues, and then, stained with hematoxylin-eosin for histopathological assessment. Histological scoring of the pancreas was performed using a blinded method, as described by Schmidt et al. . Morphometric documentation, including evaluation of bleeding, inflammation, necrosis, and edema, was obtained by mapping the surface of the pancreas into 10 geographic fields and evaluating each field independently. The histological scores (0 to 4 for each item, representing varying levels of severity) were used to evaluate the pathological severity of AP in each rat.
In brief, the paraffin-embedded sections were de-paraffinized, rehydrated using an ethanol gradient, blocked to avoid nonspecific antibody binding, and then incubated overnight with anti-LC3A/B (1:50) and anti-p62 (1:50) antibodies at 4℃. The following day, the sections were incubated with biotinylated goat anti–rabbit IgG antibody, followed by incubation with an avidin-biotin peroxidase complex. Localization of the peroxidase conjugates was achieved using the substrate diaminobenzidine tetrahydrochloride. The sections were counterstained with Mayer’s hematoxylin.
2.7 Western blotting analysis
Pancreatic tissue samples were lysed with a lysis buffer consisting of protease inhibitor, protein phosphatase inhibitor, PMSF, and RIPA lysis buffer. The total protein concentration was determined with a BCA Protein Assay kit. Then, the proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were first incubated with 5% non-fat dry milk for 2 h at room temperature on a shaking-table, and then incubated overnight with the primary antibodies (1:1000) at 4℃. All the antibodies were diluted in Tris-buffer saline/Tween 20 (TBST). After being washed three times with TBST, the membranes were incubated for 1–2 h with peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (1:5000 diluted in TBST). After another three washes with TBST, ECL was used to detect immune reactive bands, and densitometric analysis of the bands was performed using Image Lab software.
2.8 Statistical analysis
All data were expressed as mean ± standard deviation (SD). Statistical analysis was carried out using SPSS software (ver. 21.0; SPSS Inc., Chicago, IL, USA). One-way analysis of variance with either the Kruskal-Wallis or Friedman test was used for multiple comparisons among different groups. Differences were considered statistically significant at P < 0.05. Kaplan-Meier survival curves were generated for survival analyses using the log rank or Gehan-Breslow-Wilcoxon test.