Hyperbaric oxygen protects HT22 cells and PC12 cells from damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of Nrf2/System Xc-/GPX4 axis-mediated ferroptosis

This study was to investigate the protective effect of hyperbaric oxygen (HBO) on HT22 and PC12 cell damage caused by oxygen-glucose deprivation/reperfusion-induced ferroptosis. A 2-h oxygen-glucose deprivation and 24-h reperfusion model on HT22 and PC12 cells was used to simulate cerebral ischemia-reperfusion injury. Cell viabilities were detected by Cell Counting Kit-8 (CCK-8) method. The levels of reactive oxygen species (ROS) and lipid reactive oxygen species (Lipid ROS) were detected by fluorescent probes Dihydroethidium (DHE) and C11 BODIPY 581/591. Iron Colorimetric Assay Kit, malondialdehyde (MDA) and glutathione (GSH) activity assay kits were used to detect intracellular iron ion, MDA and GSHcontent. Cell ferroptosis-related ultrastructures were visualized using transmission electron microscopy (TEM). Furthermore, PCR and Western blot analyses were used to detect the expressions of ferroptosis-related genes and proteins. After receiving oxygen-glucose deprivation/reperfusion, the viabilities of HT22 and PC12 cells were significantly decreased; ROS, Lipid ROS, iron ions and MDA accumulation occurred in the cells; GSH contents decreased; TEM showed that cells were ruptured and blebbed, mitochondria atrophied and became smaller, mitochondrial ridges were reduced or even disappeared, and apoptotic bodies appeared. And the expressions of Nrf2, SLC7A11 and GPX4 genes were reduced; the expressions of p-Nrf2/Nrf2, xCT and GPX4 proteins were reduced. Notably, these parameters were significantly reversed by HBO, indicating that HBO can protect HT22 cells and PC12 cells from damage caused by oxygen-glucosedeprivation/reperfusion via the inhibition of Nrf2/System Xc-/GPX4 axis-mediated ferroptosis.