2.1 Chemicals and antibodies
Antibodies were purchased for immunoblot analysis,including anti-rabbit SLC7A11 (#26864-1-AP, Proteintech, China), anti-Mouse glutathione peroxidase 4 (GPX4) (#67763-1-Ig, Proteintech, China), anti-rabbit Ferritin Heavy Chain 1(FTH)1 (#10727-1-AP,Proteintech, China), anti-rabbit microtubule-associated protein1 light chain 3B (LC3B) (#14600-1-AP, Proteintech, China),anti-rabbit microtubule-associated P62/SQSTM1 Polyclonal antibody (#18420-1-AP, Proteintech, China) and GAPDH (#10494-1-AP, Proteintech,China). Autophagy inhibitor 3-Methyladenine (3-MA) (#S2767 , Selleck,USA). Ferroptosis specific inhibitor Fer-1 (#S6243,Selleck,USA).
2.2 Animals and animal model
The animal procedures used in this study were performed according to the National Institutes of Health guidelines for laboratory animals and were approved by the Animal Care and Experiment Committee of Lanzhou University (Lanzhou, China). All animals received humane care and all efforts were made to minimize suffering .28 adult male Sprague–Dawley rats (age, 8–10 weeks; weight, 250-290 g) were obtained from the animal center of Lanzhou University (Lanzhou, China). The rats were randomly assigned to five groups: the sham control group, the SAP group ,the Fer -1-treated SAP (SAP + Fer-1) group,the 3-MA-treated SAP (SAP + 3-MA) group. (each subgroup contained 7 rats).
Before the experiment, the rats fasted for 12 hours. After anesthesia with 3% sodium pentobarbital (20 mg/ kg), the SAP model was induced by standard pressure-controlled infusion of freshly prepared 5% sodium taurocholate (Sigma) solution (1 ml/kg) into the biliary pancreatic duct.[11]In the sham control group, the duct was infused with an equal quantity of sterile saline. In the SAP + Fer-1 group, Fer-1 (Selleck), a ferroptosis inhibitor, was administered i.p. at a concentration of 10 mg/kg 1 h before the establishment of the SAP model, following previous study protocols.[12, 13] In the SAP + 3-MA group,3-MA (Selleck), a autophagy inhibitor, was administered i.p. at a concentration of 20 mg/kg 1 h before the establishment of the SAP model, following previous study protocols.Mice in each group were fed standard granular food, free drinking water, and a sterile environment (25 ± 2 ° C; Light-dark cycle 12/12 h). All rats were killed 24 hours later and samples were collected for follow-up experiments.
Sample Collection
After severe acute pancreatitis was induced, the rats were anesthetized again 24 hours later. Blood samples were collected by puncture from the right ventricle. The blood samples were centrifuged at 3000 rpm at 4°C for 15 minutes after standing for 30min. The supernatant was subpacked into a group labeled, dried and sterilized EP centrifuge tube (200µl/ tube) and frozen in a -80℃ refrigerator for the detection of amylase, lipase activity, interleukin-6, and tumor necrosis factor α (TNF-α) levels. The pancreatic head, ileum, lung, and kidney tissues near the cecum were removed and fixed with 4% paraformaldehyde for sectionalization. The remaining tissues were stored at -80 °C for subsequent analysis.
Serum Assays
The activity of serum amylase (AMY) and lipase (LIPA) in blood samples was measured by the amylase kit. Serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were detected using the standard diagnostic kit (Elabscience,China) according to the manufacturer's instructions.
Iron Measurements
Pancreatic tissue was prepared into 10% tissue homogenate. Tissue iron determination reagent (#E-BC-K773-M,Elabscience,China) to determine the relative iron content in the kidney. After treatment according to the manufacturer's instructions, the absorbance is measured at 520 nm and the iron content is calculated by using the corresponding formula.
Assessment of MDA, GPX4 and GSH content
Fresh pancreatic tissue was analyzed for malondialdehyde (MDA) and glutathione (GSH) levels and GPX4 activity using a commercially available kit (Jiancheng Biotech).
Transmission Electron Microscopy (TEM)
Pancreatic tissue was cleaned with pre-cooled PBS (pH 7.4) and then fixed in phosphate buffered glutaraldehyde (2.5%) and osmium tetroxide (1%). The pancreas sample was then cut, stained whole with 2% uranyl acetate (UA), dehydrated in a graded ethanol series, and embedded in epoxy resin. The slices (70-90 nm) are then stained with UA and lead citrate. Ultrastructure images were captured using transmission electron microscopy (Hitachi HT 7700, Tokyo, Japan).
Tissue Histology and Immunofluorescence
For histological analysis, the rat pancreas was immobilized in 4% formalin followed by paraffin embedding. The specimen was cut into 4μm thick slices and stained with hematoxylin and eosin (H&E). The slices are then examined with an optical microscope. The pathological score was evaluated blind by two independent pathologists based on previously established scoring criteria.[14,15]
The expressions of ACSL4, FTH1 and LC3 were determined by immunofluorescence analysis. The low-temperature sections (4 μm thick) were fixed with 4% paraformaldehyde fixative at room temperature for 20 minutes, and then washed in PBS 3 times for 3 minutes. The slices were then incubated with a blocking solution (normal goat serum) at room temperature for 20 minutes. Next, the slices were incubated overnight with rabbit anti-ACSL 4 (Proteintech), rabbit anti-FTH1 (Proteintech), and rabbit anti-LC3 (Proteintech) primary antibodies at 4 °C. The slices were washed three times with PBS for 3 minutes each time, and incubated with secondary antibodies at room temperature for another 1 hour. Then the slices were washed again with PBS, covered with a cover glass, and observed and photographed under a fluorescence microscope (Olympus, Tokyo, Japan).
Western Bolt Analysis
The expressions of SLC7A11, GPX 4, LC3, p62 and FTH1 in pancreatic tissues were detected by Western blot. Total protein was extracted from rat pancreas and quantified by BCA protein assay kit (Solarbio, Beijing, China). The extracted proteins were separated on a 10 or 15% sodium dodecyl sulphate-polyacrylamide gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. Incubate the membrane overnight at 4 °C with the following primary antibodies: SLC7A11 (Proteintech), GPX 4 (Proteintech), LC3 (Proteintech), p62 (Proteintech), FTH 1 (Proteintech), GAPDH (Proteintech). Finally, the membrane was incubated with the corresponding secondary antibody at room temperature for 60 minutes, and the density of the blotted bands was measured using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
Statistical analysis
2.3 Statistical analysis
The data were presented as means ± standard deviation (S.D.),analyzed and visualized using ggplot2 in R version 3.6.3. Kaplan-Meier analysis was used to assess the survival rate, and the survival curves were plotted by GraphPad Prism 6.0 (San Diego, CA, USA). Two groups were compared by Student’s t-test and multiple groups were compared by one-way analysis (ANOVA) of variance followed by Tukey’s post hoctest. P values < 0.05 were considered statistically significant.