Il 1β

Lipopolysaccharide (Escherichia coli 055:B5) was purchased from Sigma-Aldrich (St. Louis, MO, United States). Mouse TNF-α, IL-1β, and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from ImmunoWay Biotechnology (Newark, DE, United States). The myeloperoxidase (MPO) determination kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb, NF-κB p65 (D14E12) XP Rabbit mAb, Phospho-IκBα (Ser32) (14D4) Rabbit mAb, IκBα (L35A5) Mouse mAb, Phospho-IKKβ (Ser176/180) (16A6) Rabbit mAb, IKKβ (D30C6) Rabbit mAb and TLR4, β-actin were provided by Cell Signaling Technology (Beverly, MA, United States). MyD88 (A0980) Rabbit pAb and TRAF6 (A0973) Rabbit pAb were purchased from ABclonal Biotechnology Co., Ltd. (Cambridge, MA, United States).

Proteins were collected from macrophage lysates or mouse aorta. Western blot procedures were conducted as standard protocol with specific antibodies against P22^phox (1:1000, Santa Cruz Biotechnology), P67^phox (1:1000, Santa Cruz Biotechnology), ASM (1:1000, Santa Cruz Biotechnology), GSDMD (1:1000, Abcam, Cambridge, UK), Caspase-1 (1:1000, Abclonal, Wuhan, China), IL-1β (1:1000, ImmunoWay, Plano, TX, USA), NLRP3 (1:1000, Adipogen, San Diego, CA, USA), and β-actin (1:1000, Cell Signaling Technology, Danvers, MA, USA). The blots were visualized using an ImageQuant Las4000mini instrument (Cytiva, Marlborough, MA, USA). Western blot images were analyzed, and quantification was performed using the Image J software (NIH, Bethesda, MD, USA).

Immunohistochemistry was performed with the Streptavidin-Biotin Complex (SABC) kit (Boster Bio, Pleasanton, CA, United States). The paraffin sections were first dewaxed and thermally repaired with a 3% citric acid repair solution. Then, 3% H₂O₂ was added dropwise and incubated at room temperature for 10 min. The sections were washed three times with PBS and blocked with goat serum at 37°C for 30 min. Then, the sections were incubated with diluted primary antibody [CCN1, hypoxia-inducible factor-1 α (HIF-1α), VEGF, caspase-3, Bcl-2, IL-1β, and TNF-α, 1:200, ImmunoWay, Plano, TX, United States] at 4°C overnight. On the second day, the sections were washed with PBS three times. Biotinylated goat anti-rabbit IgG was dropped and incubated at 37°C for 30 min, then washed with PBS three times. The SBC-POD reagent was added dropwise to the samples and incubated at 37°C for 30 min. For negative control groups, the primary antibody was replaced with PBS. Under the microscope, 3, 3′-diaminobenzidine was applied for 50 s and counterstained with hematoxylin, sealed with neutral gum, and then observed and photographed under the microscope. Brown cells were positive for the respective antibody, and ImageJ was used to calculate the cumulative optical density of each immunohistochemical section by the double-blind method.