H9c2 cells

H9c2 cells were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). H9c2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin mixed solution (Beyotime, Shanghai, China). The cells were maintained at 37 °C in a humidified 5% CO₂ atmosphere. Acrolein (95%) was obtained from Sinopharm Chemical Reagent Company (Nanjing, China). Hydroxytyrosol (98%) was purchased from Aladdin Biological Reagent Company (Shanghai, China). Oleuropein (95%) was purchased from Solarbio Science and Technology Company (Beijing, China). Olive leaf extract (25%) was provided from Sinolife United Biotech Company (Nanjing, China). ISO was purchased from Yi Feixue Bio (Nanjing, China) for inducing MI in rat.

Embryonic rat heart‐derived cardiomyocyte‐like H9C2 cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China), and cultured in Dulbecco's modified Eagle medium (DMEM) medium (Gibco, Eggenstein, Germany) containing 1000 mg L⁻¹ of D‐glucose. The media was supplemented with 10% fetal bovine serum, and 100 U mL⁻¹ of penicillin and streptomycin. Where indicated, media containing HG comprised of 33 mmol L⁻¹ glucose. Previous study already excluded the osmotic effect of HG using 33 × 10⁻³ m mannitol.^[4b]Primary cultures of cardiac myocytes and fibroblasts were prepared from the ventricles of neonatal Sprague–Dawley rats (Wenzhou Medical University Animal Centre, Wenzhou, China) as described previously.^[30] Human embryonic kidney (HEK) 293T cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology and cultured in DMEM medium containing 1000 mg L⁻¹ of D‐glucose.

Rat embryonic cardiac myoblasts H9C2 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and maintained at 37°C (5% CO₂ and 95% O₂) in Dulbecco's modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) (LONESERA, Uruguay in South America). DAP was dissolved in pure water and used at a final concentration of 10 µM. The dosage of Dap was selected based on previous studies (Huang et al., 2019 (link); Arow et al., 2020 (link)), and our preliminary experiment showed that 10 µM Dap has the sufficient in vitro antioxidant capacity.
H9C2 cells were put in suitable plates and treated as follows for 36 h, namely the normal glucose (5.5 mM, Con) group, the normal glucose with 10 µM Dap (Con + Dap) group, the mannitol (33.0 mM, Man) group, the high glucose (33.0 mM, HG) group, and the high glucose with 10 µM Dap (HG + Dap) group. The Man group was used to estimate the influence of osmotic pressure.