Hydrogen treatment attenuates ferroptosis and alleviates spinal cord ischemia-reperfusion injury by activating the Nrf2/HO-1 signaling pathway.

PURPOSE: Spinal cord ischemia-reperfusion injury (SCIRI) can lead to significant losses in sensory and motor functions. The precise role of hydrogen (H2) as an antioxidant in the process of ferroptosis is not fully determined.

MATERIALS AND

METHODS

This study used an abdominal aorta ligation technique to establish a SCIRI model in rats. Following oxygen-glucose deprivation/reoxygenation (OGD/R), HT22 cells were treated with H2 to assess its impact on ferroptosis. Hindlimb motor function was evaluated using the motor deficit index (MDI) and Basso, Beattie, Bresnahan (BBB) scoring, while neuronal damage was assessed via hematoxylin-eosin (HE) and Nissl staining. The DCFH-DA fluorescence probe was used for measuring reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) was assessed with JC-1 staining and Tetramethylrhodamine methyl ester (TMRM) staining. Levels of Fe2+, glutathione (GSH), and malondialdehyde (MDA) were quantified using specific assay kits. Protein expressions of ACSL4, GPX4, Nrf2, HO-1, and FTH1 were analyzed via Western blotting. Immunocytochemistry was used to detect Nrf2 and HO-1 expressions.

RESULTS

The administration of H2 significantly improved hindlimb motor function in SCIRI rats, concurrently reducing cellular ROS, Fe2+, MDA, and ACSL4 levels. Furthermore, there was an observed increase in FTH1, GSH, and GPX4 levels. Mechanistically, H2 treatment upregulated Nrf2 and HO-1 expression in SCIRI rat spinal cord tissues and in OGD/R-induced HT22 cells. These effects, however, were reversed upon administration of brusatol, an Nrf2 inhibitor.

CONCLUSIONS

In summary, these findings demonstrate that H2 confers neuroprotection in SCIRI through the activation of the Nrf2/HO-1 signaling pathway and the inhibition of ferroptosis.