2.1. Reagents and antibodies
L-Arg monohydrochloride, Dihydroethidium (DHE) dye and DAPI were from Sigma-Aldrich (St. Louis, MO, USA). GSDMD antibody was from Affinity (Shanghai, China), REDD1 Antibody (AF18) and p-NF-Kb (p-P65) were from Santa Cruz Biotechnology (CA, USA). Resveratrol was from Aladdin (Shanghai, China). HIF-1α was from Abcam (Boston, USA). Malondialdehyde (MDA) assay kit, catalase (CAT) assay kit, total glutathione peroxidase (GPX) assay kit with NADPH and anti-β-actin antibody were purchased from Beyotime Biotech (Shanghai, China). Mouse IL-1β elisa kit was purchased from Elabscience Biotechnology Co. Ltd (Wuhan, China). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Experimental animal
Healthy wild type C57BL/6 mice and healthy GSDMD−/− C57BL/6 mice (male, 8 weeks old, weighing 22–25 g) were used to establish the animal model in the experimental, all of which were separately purchased from animal feeding center of Sichuan University Health Science Center (Chengdu, China) and Shanghai Model Organisms (Shanghai, China). Under specific-pathogen-free (SPF) conditions, all mice were kept in individually ventilated cages with wooden blocks as a bed (5 mice per cage) at 25°C and 50% humidity at 12 hours of dark/light cycle. All mice were kept for 7 days before the experiment. Mice were provided standard diet and free tap water. The animal experiment procedure was carried out in accordance with the Guide of Laboratory Animal Care and Use from the United States National Institution of Health and was approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan University, China (20211363A).
2.3. Experimental model
AP models were induced by L-Arg as described previously with minor modification [19, 20]. Mice were injected with 3 g/kg of 14% L-Arg solution intraperitoneally every hour for 3 hours. The L-Arg solution was prepared in saline and filtered to sterilize. The mice in the control group were injected with sterile normal saline (0.85% sodium chloride). The mice were kept on the heating pad between injections and 4 hours after the last injection. The mice were euthanized 72 hours after the first injection of L-Arg.
2.4. Pancreatic primary acinar cells isolation
Mice pancreatic primary acinar cells were prepared by enzymatic digestion with collagenase IV as described previously . In short, the pancreas from mice were taken out and digested with collagenase IV (200 U·mL-1) at 37°C for 19 minutes. After incubating with Collagenase IV, the separated cells were mechanically broken and filtered through a 100 µm cell strainer, and then centrifuged at 700 rpm for 2 minutes to obtain cell pellets. The cells were resuspended in an extracellular solution, which were stored in extracellular solution. The components  of the extracellular solution are: 140 mM NaCl, 4.7 mM KCl, 1.13 mM MgCl2, 1.2 mM CaCl2, 10 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES], and 10 mM D-glucose at pH 7.35–7.45. The cells were then treated at room temperature and used within 4 hours after separation.
2.5. Endoplasmic reticulum protein extraction
ER proteins were prepared using the ER extraction kit (SIGMA, ER0100) according to the manufacturer's instructions.
2.6. Histopathological examination
Paraffin sections (5 µm) of pancreas tissues were stained with hematoxylin and eosin (H&E). The pancreatic histopathology score was blindly assessed by two pathologists for edema, inflammatory cell infiltration, and necrosis, from 0 to 3, assessed as previously described [18, 23]. Each slide was observed under an optical microscope (ZEISS, Jena, GMBH).
2.7. Immunofluorescence staining
Briefly, we prepared a paraffin section (3 µm) of the pancreas and dissolved blank goat serum in PBST containing 0.05% Tween 20 to make the concentration reach 5%, and then block for 0.5 hour. Slices were then incubated with anti-GSDMD (1:100 dilution) and anti-CALNEXIN (1:100 dilution) antibodies overnight at 4°C at the same time. Then slices were incubated with corresponding secondary antibodies (1 hour, 37°C) in dark. After washing, DAPI (4′,6-diamidino-2-phenylindole) (1:2000 dilution) was used for nuclear staining (10 minutes, 37°C) in dark. Stained specimens were visualized with a fluorescence microscope (LEICA LAS X, Germany). The staining of TXNIP (1:100), HIF-1α (1:100), REDD1 (1:100) and p-IRE-1α (1:300) was as described above.
Isolated primary acinar cells were incubated with L-Arg for 50 minutes, then fixed with 4% paraformaldehyde for 1 hour, next incubated with anti-GSDMD (1:100 dilution) and anti-CALNEXIN (1:100 dilution) antibodies overnight at 4°C in 24-well-plate. Then cells were incubated with corresponding secondary antibodies (1 hour, 37°C) in dark. Then counterstained with DAPI for 10 minutes. The results were showed with a fluorescence microscope (LEICA LAS X, Germany).
2.8. Necrotic cell death measurement
Freshly primary acinar cells were treated with L-Arg (60 mM) and incubated at room temperature for 50 minutes with or without various concentrations of Drug D (50 µM, 100 µM and 200 µM). Then Hoechst 33342 (50 µg/mL) and propidium iodide (PI, 1 µmol/mL) were used to stain total nuclei and necrotic primary acinar cells characterized by plasma membrane rupture , respectively. Automatic ZEISS AX10 imager A2/AX10 cam HRC (Jena GmbH, Germany) was used to record the images. The total number of acinar cells showing PI uptake was calculated from each condition, with a minimum of 1000 cells counted, to provide the percentage (necrosis %) with five isolates per condition.
2.9. Oxidative stress measurement
According to the instructions, the activities of glutathione (GSH), Catalase (CAT), Glutathione peroxidase (GPX) and malondialdehyde (MDA) in pancreas tissues were detected by a commercial biochemical kit (built in Nanjing, China). The results were showed with a fluorescence microscope (LEICA LAS X, Germany), and select a representative field of view for application.
2.10. IL-1β level measurement
According to the instructions, the level of IL-1β in pancreatic tissues was detected by a commercial biochemical kit.
2.11. Western blot analysis
Protein lysates were prepared from pancreatic tissues or isolated primary acinar cells by homogenizing in RIPA buffer containing protease and phosphatase inhibitors. Twenty micrograms of protein lysate were loaded on a 12% polyacrylamide gel. Use primary antibodies TXNIP (1:1000), REDD1 (1:1000), p-P65 (1:1000), p-IRE1-α (1:1000) and β-actin (1:1000). The image was detected by an enhanced chemiluminescence (ECL) detection system (Millipore, USA). β-actin was used as a loading control. Data were collected and analyzed from 3 independent samples.
2.12. Serum amylase, amylase and LDH secretion measurement
Blood samples were collected, centrifuged at 3000 rpm for 10 minutes, and 50 µL of serum was diluted to 200 µL. Serum lipase, amylase and LDH were measured by an automatic biochemical analyzer (Roche, Mannheim, Germany) according to the product specification.
2.13. Molecular docking studies
In silico investigation was carried out to rationalize the binding mechanism of Drug D with GSDMD (PDB ID: 6N9N). The chemical composition and protein crystal structure of Drug D and GSDMD were imported into Discovery Studio 2019 software. The docking process was done by using the docking optimization CDOCKER tool in the software.
2.14. Statistical analysis
The data and statistical analysis are in line with the recommendations of pharmacological experiment design and analysis [18, 25, 26]. Data are expressed as mean ± SEM and analyzed using one-way analysis of variance (ANOVA) or Student t-test. GraphPad Prism version 5.01 (GraphPad Prism Software Inc., San Diego, CA, USA) was used for statistical analyses and preparation of figures. A value of P < 0.05 was considered statistically significant.