Amino acid metabolism is tightly linked to the regulation of ferroptosis (Angeli et al., 2017). Upregulating GPX4 expression in the ICH model can inhibit ferroptosis and treat ICH (Peng et al., 2022). The GPX4 is currently recognized as a central repressor of ferroptosis, and its activity depends on antioxidant glutathione (GSH). The GSH is a tripeptide composed of glutamic acid, cysteine, and glycine. The three kinds of amino acids are from different pathways. The system xc- antiporter, comprised of SLC7A11 and SLC3A2, is responsible for the transmembrane import of extracellular cystine, which is reduced back to intracellular cysteine. Due to the limited concentration of cysteine in cells, cysteine is considered to be the rate-limiting precursor for GSH synthesis. Glutamate and glutamine are also important regulators of ferroptosis (Gao et al., 2015). Researchers have found in mice, rabbits, and patients with ICH that glutamate levels in brain tissue surrounding the hematoma were elevated (Li et al., 2017b; Epping et al., 2022). The addition of human glutamate to the culture medium of HT22 hippocampal neurons resulted in a significant increase in cell death (Chen et al., 2022a). Li et al. (2017b) found that the application of glutaminase inhibitors could inhibit the decomposition of glutamine into glutamate, and significantly reduce the number of degenerate nerve cells around hematoma. These all confirmed that poor clinical outcomes and increased volume of the residual cavity after ICH are associated with high concentrations of glutamate in blood within the first 24 h from symptom onset (Li et al., 2017a). The presence of large amounts of glutamate will be the rate-limiting precursor for GSH synthesis which is the key to ferroptosis (Leasure et al., 2021). Some study has also highlighted the role that selenium plays in modulating ferroptosis via its co-translational incorporation into selenocysteine in GPX4 (Ingold et al., 2018). A single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in an intracerebral hemorrhage model. These all findings give us some insights into the treatment of ICH by inhibiting ferroptosis based on amino acid metabolism.

Iron metabolism disorder is thought to be a key factor in ferroptosis. While lipid peroxidation causes ferroptosis, an increase in intracellular iron is a risk factor for ferroptosis, and the level of damage is greater when iron is raised. Iron, a putative neurotoxin, is a major product of lysed erythrocytes in hematoma after ICH. It can be engulfed by microglia and infiltrating macrophages in the perihematomal zone and metabolized into ferrous/ferric iron, which induces the formation of lethal ROS and lipid peroxidation contributing to ferroptosis and SBI (Zhang et al., 2022a). Since intraparenchymal hematomas and red blood cells are the main sources of free iron in the ICH brain, resolution of hematoma and the clearance and phagocytosis of red blood cells might reduce iron-induced ferroptosis. Further research is necessary to examine these methods for treating iron-induced ferroptosis. Deferasirox (DFR), a trivalent iron chelator, suppressed microglia/macrophage activation in peri-hematoma area at 3 days after ICH and significantly suppressed the intracellular Fe2+ accumulation and cell death caused by hemin exposure. It might be a useful therapeutic agent for the therapy of ICH (Imai et al., 2021). Excessive iron form highly toxic hydroxyl radicals and trigger ROS formation to attack DNA, proteins, and lipid membranes, thereby disrupting cellular functions and causing neuronal death (Magtanong and Dixon, 2018; Zhang et al., 2022c). Wu et al. have shown that iron ions overload occurs in the posterior brain of ICH which accumulates within 3 days after ICH causing brain edema and cell death (Urday et al., 2015; Selim, 2022). Mechanistically, following ICH, excessive ferric irons from red blood cells (RBC) bind to transferrin (TF) in serum transported into cells through receptor-mediated effects (Schwartz-Duval and Sokolov, 2022). Ferric irons are reduced to ferrous ions by divalent metal transporter 1 (DMT1) and accumulation in nerve cells. Ferrous ions induce excessive lethal ROS and lipid peroxide formation. In a previous study of ICH in mice, two kinds of iron chelators, deferoxamine (DFX) and VK28, reduced the number of nerve cell death, iron ion accumulation, microglia activation and improved the neural function of mice eventually (Li et al., 2017a). Iron-dependent Fenton chain reaction is likely the key to ferroptosis. When GPX4 is lacking, phospholipid hydroperoxides (PLOOHs) in the cell cannot be removed in time and will react with iron to trigger the Fenton chain reaction, generating more PLOOHs. This is also a hallmark of ferroptosis (Conrad and Pratt, 2019). This reaction not only damages lipids and proteins but also causes oxidative damage to DNA, including DNA base modifications and DNA strand breaks (Gu et al., 2022; Wu et al., 2022). While an iron overload does not always induce ferroptosis, it will enhance cell death when it occurs. The mechanisms of brain iron metabolism are still poorly understood, which greatly limits the development of therapeutic drugs targeting brain iron efflux after ICH. Therefore, elucidating the mechanisms underlying iron metabolism is crucial to developing effective therapeutic strategies to reduce iron accumulation in ICH (Bao et al., 2020).

In the physiological state, the dynamic balance between oxidation and antioxidant reactions helps to keep the body operating normally. Polyunsaturated fatty acids (PUFAs), an integral component of the plasma membrane, may be oxidized in vivo enzymatically. Excess oxidized PUFA is converted by GPX4 to a non-toxic form. PUFAs can also be generated with fenton chemistry but a functional GPX4/GSH axis should be able to maintain homeostasis. One of the features of ferroptosis is the accumulation of LPO which causes a variety of damage to the structure and function of cells and membranes (Dixon et al., 2012). LPO is a lipid with a peroxide group formed after the reaction of unsaturated fatty acid chains with free radicals or ROS. Under normal conditions, the level of LPO is extremely low, but in pathological conditions, increased lipid peroxidation can lead to an increase. It has been illuminated as a clear mechanism to produce highly LPO with ROS up-regulation through the fenton chemistry (Liang et al., 2019; Lei et al., 2020). After ICH, ferrous ions overload, GPX4 deficiency, and PLOOHs cannot be cleared in time pointing to the susceptibility of the fenton chemistry. Overload ROS that exceeds the antioxidant capacity of cells leads to an enhanced oxidative stress response, which directly or indirectly damages proteins, nucleic acids, lipids, and other macromolecular substances (Tan et al., 2022). Finally, the membrane is damaged and the cell collapses and dies due to the lipid peroxidation inside the phospholipid of the cell membrane. In addition, GPX4, a selenoprotein, implies that selenium availability impacts the sensitivity to ferroptosis. It functions to reduce PLOOHs to lipid alcohols (L-OH) and to reduce H₂O₂ to H₂O then reduce the damage to membrane function (Tang et al., 2019). Delivery of selenium to cells or animals to upgrade GPX4 level can suppress ferroptosis, including in a mouse model of ICH (Friedmann Angeli and Conrad, 2018; Ingold et al., 2018; Alim et al., 2019). However, abnormal amino acid metabolism after ICH results in GPX4 deficiency as mentioned above. Other research has shown that the suppression of GPX4 is related to cyclooxygenase-2 (COX-2) and the increased expression of 15-lipoxygenase (ALOX15) (Meng et al., 2022). Li et al. (2017b) observed in a collagenase-induced ICH model that COX-2, encoded by-product cyclooxygenase-2 (PTGDS-2), was highly expressed in post-ICH neurons. High expression of COX-2 contributes to ferroptosis by inhibiting the antioxidant effect of GPX4. In addition, ALOX15 participates in the programmed degradation of organelles by binding to the membranes of various organelles in cells. In vitro ALOX15 is found to bind to mitochondria leading to membrane disintegration and ROS production (Choudhary et al., 2022). Currently, increased ALOX was observed after ICH in both humans and mice (Karuppagounder et al., 2018). Lipoxygenases (LOXs) have been also implicated as central players in ferroptosis (Shah et al., 2018). 5-lipoxygenase (5-LOX) inhibitor Zileuton could inhibit ferroptosis and play a protective role in nerve cells through the reduction of lipid peroxides (LPO) production (Gao et al., 2015; Shah et al., 2018). Therefore, the regulation of enzymes in lipid metabolism and enhancement of cellular antioxidant effects are other potential targets for inhibiting ferroptosis.

Apoptosis is an active process that is subject to strict gene activation, expression, and regulation, demarcated by permeabilization of the mitochondrial outer membrane and promoted by executioner caspases (Sekerdag et al., 2018). Studies on the regulation of apoptosis after ICH have been conducted earlier. In Young et al. (1989) reported that leukocytes infiltrating the brain in ICH can release harmful substances, such as proteolytic and oxidizing agents as well as cytokines, which can injure or kill cells through caspase-dependent or independent pathways (Zhang et al., 2022c). In a rabbit ICH model, the levels of active caspase-3, Fas, FasL, and active caspase-8 were upregulated in neurons near the hematoma driving neuronal apoptosis after ICH (Deng et al., 2018). Wang et al. (2022) reported that histone deacetylase 6 (HDAC6) inhibition protects against brain injury post-ICH by reducing neuron apoptosis and apoptosis-related protein expression levels by acetylation of malate dehydrogenase 1 (MDH1). Studies of tumors have shown that the ferroptosis inducer erastin activates the p53-dependent CHOP/PUMA axis and increases sensitivity to apoptosis induced by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (Hong et al., 2018). Ferroptosis has been shown to inhibit apoptosis through the JNK signaling pathway activity (Liu et al., 2012). Thus, there are some crosstalk between ferroptosis and apoptosis.

Necroptosis combines both necrosis and apoptosis, hence the term necroptosis (Vanden Berghe et al., 2014). It is a regulated form of necrotic cell death mediated by receptor-interacting kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and mixed lineage kinase domain-like (MLKL) (Conrad et al., 2016; Cao et al., 2022). RIPK1 activates RIPK3 and thereby recruits MLKL at the cell membrane, which causes membrane rupture and eventually triggers necroptosis (Samson et al., 2021; Gupta et al., 2022). Necroptosis is involved in cell death associated with ICH (Galluzzi et al., 2018). Neurovascular injury and related hemolysis of extravasated erythrocytes post-ICH producing hemoglobin degradation metabolites may trigger the neuroinflammatory response of surrounding astroglia resulting in activation of the necroptotic pathway. Meanwhile, Necrostatin-1, a specific RIPK1 inhibitor, has been shown to reduce cell death, hematoma volume, and neurobehavioral outcomes in a mouse model of ICH (Gupta et al., 2022). Numerous reports have suggested that ferroptosis is always accompanied by necroptosis (Lv et al., 2021). The major ultrastructural characteristics of hemin-induced neuron death are related to ferroptosis and not necroptosis. In contrast, molecular marker levels of both ferroptosis (ferric iron, GSH, and GPX4) and necroptosis (MLKL and RIPK3) may increase after ICH. NADPH might be a link between ferroptosis and necroptosis (Hou et al., 2019). However, such studies regarding ICH are lacking (Lin et al., 2016; Newton et al., 2016; Minagawa et al., 2020; Chen et al., 2022b).

Some research has revealed the important role of autophagy in ferroptosis, especially selective types of autophagy (e.g., ferritinophagy, lipophagy, clockophagy, and chaperone-mediated autophagy) (Liu et al., 2020). Ferritinophagy is the process of autophagic degradation of the iron storage protein ferritin, which is critical for the regulation of cellular iron levels. ferritinophagy promotes ferroptosis by releasing free iron from ferritin. Inhibition of Ferritinophagy inhibits ferritin degradation and therefore reduces free iron levels and thus limits subsequent oxidative injury during ferroptosis (Gao et al., 2016; Hou et al., 2016). Moreover, deficient ferritinophagy may increase the activity of iron-responsive element binding protein 2 (IREB2/IRP2) to promote ferroptosis (Dixon et al., 2012). According to Gao et al. (2016) autophagy regulates intracellular iron homeostasis and ROS synthesis to promote ferroptosis. In vitro experiments showed that Erastin, a synthetic small-molecule compound, which induces ferroptosis and activates autophagy, led to intracellular ferritin degradation to further increase the level of intracellular iron ions through autophagy, resulting in rapid accumulation of intracellular ROS, which promote ferroptosis. Hou et al. (2016) also demonstrated that the activation of autophagy further promoted ferroptosis by degrading ferritin in tumor cells. Suppressing autophagy is one of the ways to inhibit ferroptosis. Lipophagy, the autophagic digestion of lipid droplets can release free fatty acids. The level of lipid droplets is negatively related to oxidative stress-induced ferroptosis (Cho et al., 2022). Increased lipid droplet formation suppresses RSL3-induced ferroptosis in hepatocytes (Cho et al., 2022). In contrast, increased lipophagy promotes lipid droplet degradation and therefore increases lipid peroxidation-mediated ferroptosis (Cho et al., 2022). Chaperone-mediated autophagy (CMA) is a type of selective autophagy that uses molecular chaperones to deliver certain cytosolic proteins to lysosomes for degradation based on the recognition of specific amino acid sequences. ER stress-associated molecular chaperone, can limit erastin-induced GPX4 degradation and therefore protects against ferroptosis in pancreatic cancer cells (Zhu et al., 2017). These findings establish a model of interaction between CMA and autophagy to determine GPX4 protein stability in ferroptosis. In brief, ferroptosis and autophagy are inseparable and both contribute to neuronal death in ICH. Understanding the mechanism of autophagy and inhibiting it is one of the ways to inhibit neuronal ferroptosis.

Neurons respond to ferroptosis stimuli by induction of selenoproteins, including antioxidant GPX4. A single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in an ICH model. According to Tuo et al. (2021), certain selenocompounds are selective anti-ferroptotic medications that can cross the blood-brain barrier and prevent neuronal death in ischemic stroke. Recent studies demonstrated that selenium can drive protective transcriptional responses, including the transcriptional activators TFAP2c and Sp1, to upregulate GPX4 and suppress ferroptosis (Alim et al., 2019). Pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptosis. The inhibition of ferroptosis and neuronal protection of selenium via transcriptional regulation have been verified in mouse models of ICH and ischemic stroke (Alim et al., 2019). In vitro and in vivo results highlight the potential of the pharmacological administration of selenium for the treatment of both hemorrhagic and ischemic stroke. It is also noteworthy that the Tat SelPep (a peptide that can increase GPX4 expression in the brain) can overcome the narrow therapeutic window of direct intracerebroventricular injections of sodium selenite, providing a novel strategy to deliver selenium with minimal toxicity (Alim et al., 2019).

Intracerebral hemorrhage leads to iron overload and the upregulation of iron-handling proteins, resulting in a brain injury that can be reduced by DFX, an iron chelator, indicating that iron imbalance is an essential initiator of ferroptosis and can provide new insights into the neuroprotective activity of iron chelators (Xue et al., 2022). DFX inhibits the overactivation of microglia by forming an iron amine chelate with iron ions around the hematoma, preventing iron ions from providing electrons to oxygen to form ROS (Dixon and Stockwell, 2014; Li et al., 2017b). This processing alleviates cerebral edema, neurological deficit, and brain atrophy after ICH in rats (Selim et al., 2019). Several iron chelators have been developed. DFX was approved by the FDA in 1968 as an iron chelator that concentrates in the brain following subcutaneous injection. By sequestering nonheme iron, DFX effectively diminishes hydroxyl radical formation and reduces brain damage after subarachnoid hemorrhage (SAH) (Pandya et al., 2021). In addition to SAH, studies have reported favorable effects of DFX in various hemorrhage models, including reduced iron overload, attenuated brain–blood barrier (BBB) disruption, reduced dendritic and white matter damage, improved neurological behavior, and lower rates of mortality (Selim et al., 2019). However, according to Selim et al. (2019), the Intracerebral Hemorrhage Deferoxamine (i-DEF) trial failed to demonstrate that using DFX to treat ICH patients was sufficient enough and further research is needed to determine its effectiveness. Compared with DFX, VK-28 has a greater advantage in that it can penetrate the intact BBB which is a more effective and safer advantage. Therefore, VK-28 may act at lower concentrations in the brain, making it more suitable for clinical (Li et al., 2017a). Deferiprone, an iron chelator that can cross the blood-brain barrier, is utilized for transfusion-dependent thalassemia as well as in Parkinson’s disease clinical trials (Devos et al., 2022). These results may stimulate further development of iron chelators for ICH treatment.

Lipoxygenases inhibitors with radical trapping can function as terminators of the radical chain reactions of lipid autoxidation to inhibit ferroptosis (Poon et al., 2021). For LOX inhibitors that lack radical trapping ability, those targeting 15-LOX-1, exhibit a degree of anti-ferroptosis activity (Kagan et al., 2017). 15-LOX-1 has been regarded as a potent target for stroke treatment. Among the six LOXs isoforms (15-LOX-1, 15-LOX-2, 12S-LOX, 12R-LOX, eLOX3, and 5-LOX), 15-LOX-1 levels increase under pathological conditions in both human and mice following stroke (Yigitkanli et al., 2013; Watanabe et al., 2022). Moreover, 15-LOX-1-KO mice exhibited a protective ability against ischemic injury in several experimental stroke models (Shen et al., 2020a), highlighting the benefits of inhibiting 15-LOX-1 during stroke treatment. Targeting 15-LOX-1 during both ischemic and hemorrhagic stroke treatment showed effective and potent neuroprotective activity in several mouse models (Yigitkanli et al., 2013; Shen et al., 2020b). 15-LOX-1 inhibitors lacking radical-trapping activity might block ferroptosis by directly inhibiting the complexes. This provides a novel direction for the future development of such inhibitors.

Divalent metal transporter 1 is a divalent metal ion transporter and is the only protein that transports ferrous iron from endosomes into the cytosol (Gao et al., 2015). In endosomes, upon release of ferric iron from transferrin following acidification, ferric iron is reduced to ferrous iron by a specific reductase and then ferrous iron is pumped in the cytosol by DMT1. After ICH, the expression of DMT1 is significantly increased. Ferrous ions induce the formation of excessive ROS and LPO, which are important factors causing ferroptosis in nerve cells (Nogueira et al., 2021). Pretreatment with the DMT1 inhibitor, ebselen, significantly reduced the iron ion transport activity of DMT1 and inhibited the production of ROS (Cheng et al., 2022). Research demonstrated that ebselen further attenuated DMT1 by inhibiting ferroptosis of neuronal cells after SAH in rats. At present, it is necessary to further strengthen the study of ebselen in cerebral hemorrhage.