Abstract: BACKGROUND: Embolization of cerebral arteries leads to cerebral tissue ischemia and hypoxia. Restoration of blood flow and reoxygenation usually results in cerebral ischemia-reperfusion injury. However, current research on the interaction between ferroptosis and endoplasmic reticulum stress in cerebral ischemia-reperfusion injury is relatively limited.
OBJECTIVE: To investigate the roles and mechanisms of endoplasmic reticulum stress and ferroptosis in cerebral ischemia-reperfusion injury.
METHODS: The HT-22 cell line was used and divided into four groups. An oxygen glucose deprivation/reperfusion (OGD/R) model was established in the model group. Cells were treated with 1.25 mmol/L 4-PBA (an endoplasmic reticulum stress inhibitor) for 2 hours prior to OGD/R, forming the 4-PBA group. Cells were treated with 1.25 mmol/L 4-PBA and 5 μmol/L CCT020312 (a protein kinase RNA-like ER kinase activator) for 2 hours before OGD/R, forming the 4-PBA+CCT group. Cell viability was detected by cell counting kit-8 experiment; western blot was used to detect the expression of ferroptosis-, endoplasmic reticulum stress, and protein kinase RNA-like ER kinase/transcription activating factor 4-related proteins; flow cytometry was used to detect cell apoptosis; and a reagent kit was used to detect the concentration of Fe2+.
RESULTS AND CONCLUSION: Compared with the control group, OGD/R treatment significantly suppressed neuronal cell proliferation activity by inducing ferroptosis, endoplasmic reticulum stress and apoptosis mechanisms. Treatment with the endoplasmic reticulum stress inhibitor 4-PBA effectively alleviated ferroptosis, apoptosis and inhibition of cell viability caused by OGD/R. In addition, 4-PBA significantly reduced the expression levels of protein kinase RNA-like ER kinase and transcription activating factor 4 induced by OGD/R. The protein kinase RNA-like ER kinase activator CCT020312 reversed the inhibitory effects of 4-PBA on endoplasmic reticulum stress, ferroptosis, apoptosis and cell activity in the OGD/R model. To conclude, endoplasmic reticulum stress promotes ferroptosis through the protein kinase RNA-like ER kinase/transcription activating factor 4 signaling pathway, thereby exacerbating cerebral ischemia-reperfusion injury. 

Key words: ferroptosis, endoplasmic reticulum stress, acute ischemic stroke, oxygen glucose deprivation/reperfusion, cell viability, apoptosis, protein kinase RNA-like ER kinase (PERK), transcription activating factor 4 (ATF4)