H₂S protects hippocampal HT22 neurons against rotenone-induced ferroptosis by upregulating Isthmin 1

Hydrogen sulfide (H₂S) has been recognized for its potent neuroprotective effects in Parkinson's disease (PD) models, yet the underlying cellular and molecular mechanisms remain to be fully elucidated. Ferroptosis, an iron-dependent form of regulated cell death, is increasingly implicated in the pathogenesis of PD. Isthmin 1 (ISM1) is a secreted protein predominantly expressed in the brain with emerging roles in neuroprotection and metabolic regulation. This study aimed to investigate whether H₂S protects against rotenone (ROT)-induced neuronal injury by modulating ferroptosis and to explore the potential mediating role of ISM1 in this process.

HT22 cells were exposed to ROT with or without the H₂S donor NaHS. Cell viability was assessed using the CCK-8 assay, and morphology was evaluated by transmission electron microscopy (TEM). Intracellular Fe²⁺ was measured using the Ferrous Ion Content Assay Kit. Malondialdehyde (MDA) and glutathione (GSH) were measured using ELISA Kits. The production of cytosolic and lipid reactive oxygen species (ROS) was measured using Flow cytometry. Protein levels of Glutathione Peroxidase 4 (GPX4), Ferritin Heavy Polypeptide 1 (FTH1), and Acyl-CoA Synthetase Long-chain Family Member 4 (ACSL4) were analyzed by Western blot.

NaHS markedly attenuated ROT-induced ferroptosis in HT22 cells. This was confirmed by suppression of mitochondrial injury, reduction in Fe²⁺ accumulation and lipid peroxidation, upregulation of the ferroptosis-related proteins GPX4 and FTH1, and downregulation of ACSL4. NaHS also increased ISM1 expression. Importantly, overexpression of ISM1 was sufficient to inhibit ROT-induced ferroptosis and lipid peroxidation.

These results indicate that H₂S counteracts ROT-induced ferroptosis in hippocampal neurons, at least in part, by upregulating ISM1, revealing a novel H₂S-ISM1-ferroptosis axis in neuroprotection.