Ubiquitination of System Xc- and GPX4 in rat brain stimulates ferroptosis and inflammation in hippocampal neurons after subarachnoid hemorrhage

Background: Ferroptosis is a peroxidation-driven form of cell death. Glutathione peroxidase 4 (GPX4) and System Xc − are leading regulators of ferroptosis. This study investigated System Xc − and GPX4 ubiquitination in ferroptosis after subarachnoid hemorrhage (SAH). Methods: : SAH rat model was established by artery perforation operation. The mortality, neurological score, brain water content and blood brain barrier permeability were and analyzed. The morphology of mitochondria was observed, the content of reactive oxygen species and the pathology of hippocampal neurons were evaluated, and the iron deposition was observed. Levels of ferroptosis-related proteins and proinflammatory factors were detected. The protein interaction between System Xc − and GPX4 was verified. After ferroptosis was induced by FIN56 in neurons, the neurons were transfected with OTUB1 overexpression vector or ubiquitination inhibitors to verify the regulatory mechanism of System Xc − and GPX4 ubiquitination in ferroptosis. Results: : After SAH operation, GPX4 was downregulated, and induced iron deposition and ROS accumulation. In SAH rats, System Xc − function was lost, GSH decreased, MDA content and SLC3A2, SLC7A11 and GPX4 ubiquitination increased, and OTUB1 expression decreased. SLC3A2 and OTUB1, GPX4 and OTUB1 can interact with each other in protein. Overexpression of OTUB1 reduced the ubiquitination of SLC3A2, SLC7A11 and GPX4 in FIN56-induced ferroptosis in neurons. Ubiquitination inhibitor or OTUB1 increased GSH level in ferroptotic neurons, and reduced ROS accumulation and inflammatory damage. Conclusion: SAH induced the ubiquitination of System Xc − and GPX4, resulting in GSH depletion, increased lipid oxidative damage, and promoted ferroptosis in hippocampal neurons.