Pro caspase 1

Alanine amiotransferase (ALT), triglyceride (TG) and total cholesterol (TC) assay kits were purchased from Nanjing Jiancheng Biotechnology (Nanjing, Jiangsu, China). RNAios Plus, Pri-meScript™ RT reagent kit, and TB Green® Pre-mix Ex Taq™ II were obtained from Takara Biotechnology (Tokyo, Japan). The Caspase-1 Activity Assay Kit and Hoechst 33342/Propidium Iodide (PI) Double Staining Kit were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). All other chemicals used were of analytical grade and commercially available.
The following primary antibodies were used: mouse anti-GAPDH (6C5) antibody from Beyotime (Shanghai, China), mouse anti-ASC/TMS1/PYCARD (F-9) antibody from Santa Cruz (Santa Cruz, CA), mouse anti-beta-actin (2D4H5) antibody from Proteintech (Wuhan, China), rabbit polyclonal antibody against GSDMD from Proteintech (Wuhan, China), and rabbit anti-cleaved N-terminal GSDMD (EPR20829-408) antibody from Abcam (Cambridge, UK). The rabbit polyclonal antibodies against NLRP3, pro caspase-1, caspase-1 p20, pro IL-1β, IL-1β, and IL-18 were obtained from Wanleibio (Shenyang, Liaoning, China).

Total proteins were extracted from lung tissue homogenates using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). The total protein concentrations were measured using an enhanced BCA protein assay kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Subsequently, the extracted protein samples were separated on 10–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes, and the PVDF membranes were blocked with 5% fat-free milk for 2 h. Then, the membranes were incubated with primary antibodies against NLRP3 (1:1,000, Abcam, Cambridge, United Kingdom), precursors of cysteinyl aspartate specific proteinase (procaspase)-1(1:500, Wanleibio, Shenyang, China) and cysteinyl aspartate specific proteinase (caspase-1) p20 (1:500, Wanleibio, Shenyang, China), gasdermin D (GSDMD), the N-terminal of GSDMD (GSDMD-N) (1:1,000, Cell Signaling Technology, United States), and β-actin (1:1,000, ZSGB-BIO, Beijing, China) at 4°C for 24 h. After three washes, the membranes were incubated with a peroxidase-conjugated secondary antibody (1:1,000, ZSGB-BIO, Beijing, China). Finally, the immune complexes were visualized with BeyoECL Plus (Beyotime Biotechnology, Shanghai, China), and the bands were quantified with ImageJ software.

Each frozen hippocampal and cortical tissue (weighing 30-50 mg) and precooled RIPA lysis buffer (Solarbio, China), added with protein phosphatase inhibitor complex (Biomed, China) and phenylmethylsulfonyl fluoride (PMSF, BestBio, China), were mixed in a proportion of 1 : 5, ground into homogenate, and centrifuged for liquid supernatant. The supernatant was then quantified by Pierce™ BCA protein assay (23225, Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were separated with 12% SDS-PAGE gel and transferred onto PVDF (Millipore, USA) membranes. The membranes were incubated with antibodies of NLRP3 (1 : 1000, Abcam, USA), pro-caspase-1 (1 : 500, Wanleibio, China), cleaved-caspase-1 (1 : 500, Wanleibio, China), ASC (1 : 1000, Abcam, USA), GSDMD (1 : 1000, Abcam, USA), CD63 (1 : 500, Wanleibio, China), CD9 (1 : 1000, Abcam, USA), and GAPDH (1 : 1000, Cell Signaling Technology, USA), respectively, overnight at 4°C. After washing by TBST, the membranes were incubated with horseradish enzyme labeling goat anti-rabbit IgG (1 : 5000, Boster, China) for 1 h at room temperature and subjected to ECL detection reagent (Millipore, USA). The protein bands were visualized by autoradiography and analyzed by ImageJ.