E ck a301

To measure the effect of carpaine on H₂O₂-induced MMP in H9c2, 5,5,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1) dye was used to measure the state of mitochondrial polarization. JC-1 dye staining was conducted according to manufacturer instruction (E-CK-A301, Elabscience, Texas, USA). Briefly, following the incubation treatment, the culture medium was discarded, and the cells were washed with JC-1 buffer. JC-1 working solution was then added and the cells were incubated in a CO₂ incubator for 20 min. The cells were then washed with JC-1 buffer and culture medium was added. Immediately, the fluorescence was observed using a BX63 automated upright microscope (Olympus, Tokyo, Japan) and the relative ratio of red and green fluorescence was measured and analyzed using ImageJ software to represent the mitochondrial depolarization.

To determine the occurrence of ferroptosis, we measured levels of iron, the lipid peroxidation metabolite malondialdehyde (MDA) and glutathione (GSH), by using tissue and cellular ferrous iron assay kits (E-BC-K773-M and E-BC-K881-M, Elabscience, China), MDA assay kits (E-BC-K025-M and E-BC-K028-M, Elabscience, China) and GSH assay kits (RK05819, ABclonal, China). Mitochondrial membrane potential was measured using JC-1 mitochondrial membrane potential assay dye (E-CK-A301, Elabscience, China), following the manufacturer's instructions. The ultrastructural changes of ferroptosis in lung tissue were examined by transmission electron microscopy (Hitachi H-7800, Hitachi, Naka, Japan), which was performed by Hubei BIOSSCI Biotech Co., Ltd.