E el 0060c

Blood samples (0.8 mL) were extracted at the endpoints of experiments and maintained at RT for 30 min, followed by centrifugation at 3000 rpm for 15 min at 4 °C to facilitate supernatant collection. After treatment, the AC16 cells were collected via centrifugation. The quantification of MDA (malonaldehyde; E-EL-0060c, Elabscience, China), CK-MB (creatine kinase isoenzyme MB; E-E-M0355c, Elabscience, China), GSH (glutathione; E-BC-K030-M, Elabscience, China), ATP (E-BC-K157-M, Elabscience, China), and LDH (lactate dehydrogenase; E-BC-K046-M, Elabscience, China) in mouse serum or AC16 cells of different groups was performed using an ELISA kit or activity assay kit following the manufacturers’ protocols. MitoTEMPO (Aladdin, 1,334,850–99-5, China) was used to verify whether the mitochondrial targeted ROS inhibitor causes damage to cardiomyocytes.

The activities of glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) were measured using GSH-Px, CAT, and SOD activity assay kits (Catalog No.: E-BC-K096-M, E-BC-K031-M, E-BC-K020-M; Elabscience, Wuhan, China), respectively. Malondialdehyde (MDA), interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α) levels were measured using MDA, IL-1β, IL-6, IL-8, and TNF-α ELISA kits (Catalog No.: E-EL-0060c, E-EL-H0149c, E-EL-H6156, E-EL-H6008, E-EL-H0109c, Elabscience). After cell culture at 24 h, the culture supernatant was collected and centrifuged at 1000 × g for 20 min to remove impurities and cellular debris. The supernatant was collected for further analysis. For the detection, 50 µl of the supernatant was taken and analyzed. Following completion of the experiment, the optical density (OD) of each well was measured using a Multiscan MK3 reader (Thermo Fisher Scientific). All reactions were repeated thrice.

The serum levels of NLRP3, pro-caspase-1, IL-1β, IL-18, and MDA were determined using mouse NLRP3 ELISA KIT (MM-0656M1, Meimian, Wuhan, China), mouse caspase-1 ELISA KIT (MM-0820M1, Meimian), mouse pro-IL-1β ELISA KIT (MM-0905M1, Meimian), mouse pro-IL-18 ELISA KIT (MM-0906M1, Meimian), mouse IL-1β ELISA KIT (MM-0905M1, Meimian), mouse IL-18 ELISA KIT (MM-0906M1, Meimian) and mouse MDA ELISA KIT (E-EL-0060c, Elabscience, Wuhan, China) according to the manufacturer’s protocol. The absorbance of wells was determined with a microplate reader (MuLTiSKAN MK3, Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm wavelength to analyze the sample concentration.