S2335

The H9c2 rat cardiomyocyte line was purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Cardiomyocytes were isolated from 1-to 3-day-old neonatal Sprague Dawley (SD) rats referring to previous experimental methods [24 (link), 25 (link), 26 (link)]. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) containing 4.5 g/L d-glucose, supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, and 100 U/mL streptomycin in a circumstance of 5% CO₂ at 37°C. When density is up to 70%, cells were starved with serum-free medium for 2 h before treatment. Cells were pretreated with oridonin at test concentrations (Selleck, S2335, dissolved in DMSO) 1 h before incubation with Ang II (1 μM, Aladdin, A107852) for 24 h. DMSO solution (1‰) was used as the vehicle control.

In the in vitro inhibiting experiment, LPS-primed PMs were pre-incubated with the following inhibitors: KCl (50 mM, PB0440, Sangon Biotech) to block K⁺ efflux, ATP receptor P2X7R inhibitor oxidized ATP (oATP, 500 μM, A6779, Sigma-Aldrich), cathepsin B inhibitor CA-074Me (10 μM, C5857, Calbiochem), Nlrp3 inhibitors including MCC950 (10 μM, S7809, Selleckchem), CY-09 (5 μM, S5774, Selleckchem) and Oridomin (20 μM, S2335, Selleckchem), and Caspase-1 inhibitor Z-YVAD-FMK (10 μM, A3707, Alexis Biochemicals), at the indicated concentrations for 1 h. Then, LPS-primed PMs were treated with the Stx2 for 16 h in vitro. All supernatants were collected and detected for IL-1β by ELISA and LDH with Cytotox 96 Kit.