2.1 Disorder of iron metabolism leads to ferroptosis
Iron is an important participant and regulator in the normal physiological activity of the brain and is abundant in brain tissue15,16. Physiologically, the brain is protected from whole-body iron fluctuations due to the protection provided by the blood-brain barrier (BBB) 17. Under normal circumstances, BBB endothelial cells through the transferrin receptor - transferrin (TFR - TF) compounds mediated endocytosis of inactive iron intake (Fe3+)18, then through the Six-transmembrane epithelial antigen of prostate 3 (STEAP3) convert Fe3+ to free iron (Fe2+). Fe2+ is transported from endosomes by divalent metal transporter 1 (DMT1) and exported to the extracellular space of the brain by active transport or exocytosis mediated by ferroportin (FPN)19,20, and can also be stored in ferritin or the labile iron pool (LIP)21, which is involved in lipid ROS production. Studies have shown that BBB capillary endothelial cells can store and release iron in a regulated manner, thus acting as an iron reservoir for the brain22. Under acute ischemic conditions, microglia are overactivated by ischemic stimuli, resulting in disruption of the BBB23, and free iron and ferritin enter the brain parenchyma when the BBB is disrupted. Studies have shown that the expressions of TFR1 and DMT1 can be significantly increased, and the expression level of FPN can be decreased in the middle cerebral artery ischemia model of MCAO rats24,25, indicating that the ability of intracellular iron uptake is enhanced and the ability of iron efflux is weakened after cerebral ischemia, which will eventually lead to intracellular iron overload. In addition, in the MCAO rat model, the assembly of cytoplasmic NCOA4 protein in neurons is significantly increased to recognize ferritin and transport it to lysosome for degradation, resulting in a significant increase in the concentration of free iron in neurons26. Knockdown of the NCOA4gene can reduce the uptake of ferritin, avoid the accumulation of free iron and eliminate the accumulation of reactive oxygen species, thereby interfering with ferroptosis 27. This suggests that NCOA4-mediated ferritin autophagy is one of the additional key mechanisms mediating ferroptosis in AIS. Therefore, any form of induced iron transport imbalance, autophagic degradation of ferritin, and impairment of the blood-brain barrier will lead to iron overload in the brain. Excessive intracellular Fe2+ can not only generate ROS through the Fenton reaction but also participate in the synthesis of lipoxygenase and then catalyze lipid peroxidation28,29. Lethal accumulation of intracellular lipid peroxides as well as reactive oxygen species promotes nuclear, protein, and membrane damage that ultimately mediates cell death. Studies have shown that the increase of intracellular free iron is positively correlated with the degree of neuronal damage after ischemic stroke30, which directly affects the recovery of cerebral nerve function. In the early stages of ischemia-reperfusion, iron overload increases the risk of bleeding transformation after early tPA administration, accelerates ischemia-induced elevation of serum matrix metalloproteinase-9, and enhances lipid peroxidation, thereby increasing the likelihood of adverse outcomes31. A clinical trial reports that the use of the iron-chelating agent deferoxamine to reduce systemic iron levels within 1-3 days of ischemic stroke may be beneficial for short-term outcomes in patients with acute ischemic stroke10. It has also been proposed that the use of ferroptosis inhibitors liproxstatin-1 and ferrostatin-1 can reduce neurological dysfunction and cerebral infarct size in MCAO mice32. Therefore, rational application of ferroptosis inhibitors such as deferoxamine to reduce the iron content in the brain after AIS can reduce neuronal death and promote the recovery of neurological function after ischemic stroke.