3-n-Butylphthalide Protects SH-SY5Y Cells from Ferroptosis by Inhibiting ACSL4-Mediated Lipid Peroxidation.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, and its pathogenesis is closely associated with oxidative stress, mitochondrial dysfunction, and iron-dependent cell death. In our previous study, we showed that 3-n-butylphthalide (NBP) alleviates behavioral deficits in a PD mouse model. However, the underlying mechanisms remain unclear. Here, we found that NBP treatment significantly attenuated ferroptosis induced by Erastin or RSL3 in SH-SY5Y cells, as evidenced by improved cell viability, reduced reactive oxygen species (ROS) production, decreased mitochondrial oxidative stress, and lower levels of lipid peroxidation. Molecular docking analysis revealed that NBP interacts with ACSL4 at residues PRO-404, TYR-425, VAL-447, ILE-526, and LYS-649. A cellular thermal shift assay combined with site-directed mutagenesis indicated that PRO-404 and ILE-526 are critical for the interaction between ACSL4 and NBP. Furthermore, pulse-chase experiments showed that NBP enhances ACSL4 protein stability. Notably, ACSL4 overexpression abrogated the protective effects of NBP in Erastin- or RSL3-treated cells. Collectively, our data indicate that NBP protects SH-SY5Y cells from ferroptosis, likely through the suppression of ACSL4-mediated lipid peroxidation. These results highlight the therapeutic potential of NBP for treating ferroptosis-related neurodegenerative diseases such as PD.