Il 18

Antibodies were as follows: GSDMD antibody (Lot: #E9S1X) was purchased from Cell Signaling Technology (Massachusetts, USA). Caspase-1 (Lot: #ab179515) and S100 antibodies (Lot: #ab52642) were purchased from Abcam (Cambridge, England, UK). NLRP3 (Lot: # T55651) and Rab32 (Lot: # TD9825) was purchased from Abmart (Shanghai, China).
Reagents were as follows: Rat IL-1β (Lot: #E-EL-R0012c) and IL-18 (Lot: #E-EL-R0567c) ELISA Kits were obtained from Elabscience (Shanghai, China). Lipopolysaccharides (LPS, Lot: #L4391-1MG) and adenosine triphosphate disodium (ATP, Lot: #A2383-5G) was purchased from Sigma-Aldrich. We obtained Mitoquinone mesylate (MitoQ, Lot: # HY-100116A) from MedChemExpress (Monmouth Junction, NJ, USA).

Proteins were extracted from TGs tissues with NP-40 buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined using the Bicinchoninic Acid Kit for Protein Determination (Cat. No: BCA1-1KT; Sigma-Aldrich). Protein (50 μg) for each sample was separated on a 10% SDS-PAGE gel and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., USA). After blocking with 5% non-fat dry milk at room temperature for 2 h, the membranes were incubated with the primary antibody of NLRP3 (Cat. No: ab214185; Abcam), IL-1β (Cat. No: sc-12742; Santa Cruz Biotechnology, USA), and IL-18 (Cat. No: ESAP10527; Elabscience Biotechnology) at room temperature for 2 h. β-actin (Cat. No: sc-130301; Santa Cruz Biotechnology) signals were used to correct for unequal loading. Following three washes with TBST, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Cat. No: sc-516102; dilution: 1:10,000; Santa Cruz Biotechnology) at room temperature for 2 h and visualized by chemiluminescence (Thermo Fisher Scientific, Inc.). Signals were analyzed with Quantity One¯ software version 4.5 (BioRad Laboratories, Inc., USA).

The rat blood serum and the supernatant of H9C2 cells were prepared and used for the examination of creatine kinase (CK). Lactate dehydrogenase (LDH; Nanjing Jiancheng Biological Engineering Institute, Nanjing, China; Cat# A020-2), interleukin (IL)-1β (Elabscience, Wuhan, China; Cat# E-EL-R0012c), and IL-18 (Elabscience; Cat# E-EL-R0567c) were detected using the commercial ELISA kits, according to the manufacturer's recommendation.

Plasma samples were extracted as described above. Enzyme immunoassay (commercially available) was employed to detect ET-1, IL-1β, and IL-18 (Elabscience Biotechnology Co. Ltd., Wuhan, China) in plasma and heart tissues. The absorbance was read at 450 nm by a microplate reader (Bio-Rad Laboratories, Hercules, America; Bio-Rad Model 3550 UV).