Ferroptosis: An emerging therapeutic target in stroke

Abbreviations ACSL4 acyl-CoA synthetase long-chain family member 4 DMT1 divalent metal transporter 1 EBI early brain injury Fer-1 ferrostatin-1 Fpn ferroportin FTH1 ferritin heavy chain 1 FTL ferritin light chain GPX4 glutathione peroxidase 4 GSH glutathione GSSG oxidized glutathione LOXs lipoxygenases PBI primary brain injury PUFAs polyunsaturated fatty acids RNA-seq RNA sequencing ROS reactive oxygen species SAH subarachnoid hemorrhage SBI secondary brain injury STEAP3 six-transmembrane epithelial antigen of prostate 3 TF transferrin TFR1 transferrin receptor 1 TfRs transferrin receptors 1 INTRODUCTION

Stroke is the second leading cause of mortality and a major cause of disability worldwide, which imposes a subtantial burden on patients, families, and society. Its incidence continues to increase because of expanding population numbers and an aging population (Krishnamurthi et al., 2013). Thus, the development of innovative methods to prevent and treat stroke has come to the forefront of clinical research. The cause and onset of stroke determine the appropriate treatment plan, which primarily includes symptomatic treatments to maintain vital signs, as well as surgery or drug therapy to remove the source of the stroke. Although acute management can obtain an immediate effect to save lives and reduce disability rates, we often cannot stop or reverse neuronal damage. Thus, neuroprotection is a promising treatment strategy. Thorough research on the mechanism and metabolic changes of stroke revealed a series of pathophysiological processes after stroke, such as inflammation, excitotoxicity, and oxidative stress (Chamorro et al., 2016b; Esenwa & Elkind, 2016; Iadecola & Anrather, 2011). These pathophysiological processes severely damage neurons, glial cells, and endothelial cells, and also crosstalk and trigger each other to leading to a vicious cycle, ultimately leading to secondary neuronal cell death. Extensive efforts have been made to explore the therapeutic targets in these pathophysiological processes, which may prevent neuronal damage and promote repair of damaged nerves before the cell damage cascades are initiated (Chamorro et al., 2016a).

Ferroptosis, first described by Dixon et al. in 2012, is a form of cell death characterized by the accumulation of intracellular iron and cellular accumulation of lipid reactive oxygen species (ROS) (Dixon et al., 2012). The morphological characteristics of ferroptosis are the decreased mitochondrial size, increased mitochondrial membrane density, and reduced mitochondrial crista and mitochondrial outer-membrane rupture during ferroptosis, which may be caused by dysfunction of voltage-dependent anion channels (VDACs) and the alteration of mitochondrial membrane fluidity because of lipid peroxidation products (DeHart et al., 2018; Gao et al., 2019) (Table 1). In addition, ferroptosis can be reversed by lipophilic antioxidants (Ferr-1) or iron chelators (DFO) (Table 2), but cannot be prevented by inhibitors of apoptosis or autophagy (Cao & Dixon, 2016). Previous studies of ferroptosis have mostly focused on the field of cancer. In recent years, studies have found that ferroptosis also occurs in neurological diseases, including Parkinson's disease, Alzheimer's disease, and stroke. Inhibitors of ferroptosis, such as ferrostatins, are also protective in models of such neurological diseases (Ahmad et al., 2014; Fang, Gao, et al., 2020; Pichler et al., 2013; Tuo et al., 2017). Therefore, the optimization of existing inhibitors or the development of novel inhibitors to block ferroptosis could be a potential therapeutic target for these neurological diseases (Stockwell et al., 2017).

TABLE 1. The characteristics of different cell deaths Cell death Morphology Ferroptosis Decreased mitochondrial size, increased mitochondrial membrane density, reduced mitochondrial crista, and mitochondrial outer-membrane rupture Necrosis Cell swelling, rupture of plasma membrane, release of cell contents, pyknosis, karyorrhexis, karyolysis Apoptosis Cell shrinkage, detached from surrounding cells, increased cytoplasm density, disappearance of the mitochondrial membrane potential, pyknosis, extensive plasma membrane blebbing, apoptotic bodies Pyroptosis Cell swelling, membrane blebbing, plasma membrane rupture, release of cell contents, unaffected mitochondrial integrity Necroptosis Cell swelling and rounding up, rupture of plasma membrane, release of cell contents, without chromatin condensation TABLE 2. Reagents that modulate ferroptosis in stroke Reagent Target Impact on ferroptosis Inducers Erastin System Xc− Prevents cystine import; causes GSH depletion RSL3 GPX4 Covalent inhibitor of GPX4 that causes accumulation of lipid hydroperoxides Inhibitors Ferrostatins Lipid peroxidation Blocks lipid peroxidation Liproxstatins Lipid peroxidation Blocks lipid peroxidation Trolox Lipid peroxidation Blocks propagation of lipid peroxidation; may inhibit lipoxygenases N-Acetylcysteine Lipid peroxidation Neutralizes toxic lipids generated by arachidonate-dependent ALOX5 activity Deferoxamine Iron Depletes iron and prevents iron-dependent lipid peroxidation Ceruloplasmin Iron Depletes iron and prevents iron-dependent lipid peroxidation Selenium Selenoproteins Increases abundance of selenoproteins

At present, studies have revealed that ferroptosis mediates the pathophysiological process of stroke and inhibition of ferroptosis may improve the stroke prognosis. Accumulating studies have suggested that ferroptosis may be a potent therapeutic target for stroke intervention in clinical treatment (Ratan, 2020; Weiland et al., 2019). Therefore, this present review systematically summarizes the current mechanism of ferroptosis and studies of ferroptosis in stroke. Furthermore, we highlight recent progress pertaining to ferroptosis in stroke according to multi-omics approaches (i.e., genomics, transcriptomics, proteomics, and metabolomics), which might provide innovative ideas for further stroke research.

2 MECHANISM OF FERROPTOSIS

According to current research, the general mechanism of ferroptosis involves the failure of glutathione peroxidase 4 (GPX4) and abnormal iron and amino acid metabolism, which subsequently causes the intracellular accumulation of ROS and programmed cell death. In addition, ferroptosis is tightly regulated by intracellular signaling pathways, such as the nuclear factor E2-related factor 2 (NRF2) signaling pathway and the Hippo pathway, which affect the activity of GPX4 directly or indirectly (Dodson et al., 2019; Wu et al., 2019) (Figure 1).

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Mechanism of ferroptosis. Ferric iron (Fe3+) is bound to transferrin (TF), imported into cells through the membrane protein transferrin receptor 1 (TFR1), and then stored in the endosome where Fe3+ is converted into Fe2+ by six-transmembrane epithelial antigen of prostate 3 (STEAP3). Fe2+ is transported by the divalent metal transporter 1 (DMT1) out of the endosome. Most of Fe2+ is stored in the plasma membrane ferritin (FTH1 and FTL), and the exportation of Fe2+ is mediated by the membrane protein ferroportin 1 (FPN1). Excess Fe2+ generates ROS through the Fenton reaction or in an iron-catalyzed enzymatic manner and participates in the synthesis of LOXs to catalyze the oxidation of PUFAs, which can induce ferroptosis. System Xc− transports cystine inside and glutamate outside. Cysteine is decomposed from cystine and then generates glutathione (GSH), which is utilized as a substrate for the lipid repair function of glutathione peroxidase 4 (GPX4). GPX4 reduces PUFA lipid peroxides (L-OOH) to lipid alcohols (L-OH). Lastly, ferroptosis could be induced by the failure of amino acid metabolism and accumulation of lipid peroxides

2.1 Iron metabolism

Iron is an essential metal for normal cellular function and participates in many physiological processes, such as oxygen transport, cellular respiration, DNA synthesis, myelinization, and neurotransmitter biosynthesis in the nervous system (DeGregorio-Rocasolano et al., 2019). However, the failure of uptake, transport, storage, and utilization of intracellular iron will cause excess intracellular free Fe2+ deposition and will initiate the Fenton reaction to generate ROS (Xu et al., 2020). Furthermore, ROS subsequently modify and interfere with proteins, lipids, and DNA, which induce cell death (Singh et al., 2014).

Iron exists as both Fe2+ and Fe3+, while the circulating iron exists in the form of ferric iron (Fe3+) by binding to transferrin (TF) (DeGregorio-Rocasolano et al., 2019). Free Fe3+ is imported into cells through the membrane protein transferrin receptor 1 (TFR1) and then stored in the endosome where Fe3+ is converted into Fe2+ by six-transmembrane epithelial antigen of prostate 3 (STEAP3), an endosomal ferric reductase (Yan & Zhang, 2019). Subsequently, Fe2+ is transported by the divalent metal transporter 1 (DMT1) out of the endosome to cytoplasm (Xie et al., 2016). Generally, cytoplasmic Fe2+ is stored in the iron–storage protein complex, ferritin heavy chain 1 (FTH1), and ferritin light chain (FTL), which maintains the equilibrium of the labile iron pool and prevents the formation of ROS (Mao et al., 2020). Conversely, some Fe2+ is exported into the extracellular space via the membrane protein ferroportin 1 (FPN1) (Masaldan et al., 2019; Troadec et al., 2010).

Excess iron ions (Fe2+) can follow some pathways to induce the iron-dependent accumulation of lipid ROS in ferroptosis, and this part will be discussed in the section of ROS production.

2.2 Amino acid metabolism

Cystine/glutamate-related amino acid metabolism has an important role in ferroptosis. System Xc−, an antiporter on the cell membrane, transports cystine inside and glutamate outside in a 1:1 ratio. Cystine is transported to the cell and then reduced into cysteine, which combines with glutamic acid and glycine to generate glutathione (GSH). GSH, an important antioxidant that protects cells against oxidative damage, is utilized as a substrate for the lipid repair function of GPX4 (Dixon et al., 2012). GPX4 uses GSH as a cofactor to reduce peroxides to their corresponding alcohols. In this catalysis, GPX4 uses two molecules of GSH as substrates and produces one molecule of oxidized glutathione (GSSG). GSSG can be reduced back to GSH by GSH reductase in an NADPH-dependent manner (Magtanong & Dixon, 2018).

2.3 Lipid peroxidation and ROS production

ROS is a by-product of cell metabolism. ROS maintains body stability and participates in cell signaling during normal physiological metabolism. During pathological conditions, such as radiation exposure, chemical drug effects, and excessive transition metal ions, excess intracellular ROS accumulation results in cell death (Droge, 2002). ROS are formed by the partial reduction of molecular oxygen to superoxide (O2−), hydrogen peroxide (H2O2), lipid peroxides (ROOH), or the corresponding hydroxyl (HO•) and peroxyl radicals (ROO•). A growing body of work implicates lipid peroxides as key mediators of many pathological states including inflammation, cancer, and neurodegenerative disease (Gaschler & Stockwell, 2017). Lipid peroxidation is initiated by OH•, leading to the formation of lipid radicals and lipid peroxyl radicals, which react with PUFAs in a propagation reaction to generate lipid peroxides. Excessive lipid peroxidation is associated with the iron-dependent form of cell death known as ferroptosis (Stockwell et al., 2017).

As we mentioned, iron participates in the ROS accumulation via three pathways. Firstly, intracellular free Fe2+ leads to the generation of ROS through the Fenton reaction, an inorganic chemical reaction involving peroxides and Fe2+ to yield soluble hydroxyl (HO•) or lipid alkoxy (RO•) radicals (Dixon & Stockwell, 2014). Secondly, ROS is also generated in lipid autoxidation, an autocatalytic classic free radical chain reaction that can generate lipid hydroperoxides in the presence of iron (Shah et al., 2018). Lastly, iron is also an important component in the catalytic subunit of lipoxygenases (LOXs) that oxidize polyunsaturated fatty acids (PUFAs) to result in lipid peroxides (Dixon & Stockwell, 2014).

ROS can react with PUFAs in lipid membranes, particularly arachidonic acid and adrenic acid (Doll & Conrad, 2017; Yan & Zhang, 2019), to induce lipid peroxidation, which triggers ferroptosis in the cell (Friedmann Angeli et al., 2014; Skouta et al., 2014). The intracellular level and location of PUFAs determine the degree of lipid peroxidation production as well as the incidence of the ferroptosis (Wu et al., 2018).

Before lipid peroxidation occurs, PUFAs need to be esterified with membrane phospholipids by the catalysis of acyl-CoA synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) (Doll & Conrad, 2017; Kenny et al., 2019; Shindou & Shimizu, 2009). Then, LOXs, such as cyclooxygenases, cytochrome p450, and LOXs, participate in the process of iron-dependent lipid peroxidation and can catalyze PUFAs into lipid hydroperoxides (Doll & Conrad, 2017; Lei et al., 2019). Excess iron ions in the cytoplasm would cause free radical-induced lipid hydroperoxides, further leading to cell damage. Concurrently, these free radicals can transfer protons to initiate a new round of lipid oxidation reaction and cause more serious oxidative damage (Yang & Stockwell, 2016).

With increasing recognition of the essential role of lipid peroxidation in ferroptosis execution, strategies are aimed at inhibiting lipid peroxidation. Additionally, attractive cytoprotective strategies are emerging, such as LOX inhibitors, radical-trapping antioxidants, vitamin E analogs, liproxstatins, ferrostatins, deuterated lipids, and ACSL4 inhibitors (Angeli et al., 2017). However, more research is needed.

3 FERROPTOSIS IN STROKE 3.1 Ferroptosis in acute ischemic stroke

Before ferroptosis was defined, disturbance of brain iron homeostasis was considered an important component of acute neuronal injury following ischemic stroke in both clinical and preclinical studies (Davalos et al., 1994; Kondo et al., 1995). Increased iron deposition was also found in the basal ganglia, thalami, and the periventricular and subcortical white matter of children following severe ischemic–hypoxic insult (Tuo et al., 2017). Furthermore, brain iron accumulation occurs with normal human aging, which coincides with aging as the most significant risk factor for ischemic stroke (Ayton et al., 2013).

Emerging evidence implicates ferroptosis as a mechanism of acute ischemic stroke in vivo. In rat models, pMCAO resulted in elevated iron levels in the lesioned hemisphere that correlated with reduced iron export. It was hypothesized that this iron accumulation could sensitize to, or perhaps induce, ferroptosis. Consistent with such a model, liproxstatin-1 or ferrostatin-1 treatment limited the infarct size resulting from pMCAO and provides neuroprotective effect, even when administered 6 hr after reperfusion (Tuo et al., 2017). Iron chelators (e.g., deferoxamine and 2,2'-dipyridyl) were shown to improve neurological outcome by reducing reperfusion damage after ischemic stroke in the tMCAO and pMCAO rat models (Demougeot et al., 2004; Hanson et al., 2009). Meanwhile, the levels of transferrin receptors (TfRs) and iron-loaded transferrin (holo-transferrin [HTf]) were also revealed to increase after ischemic stroke in the tMCAO rat model (DeGregorio-Rocasolano et al., 2018; Park et al., 2011). Additionally, antioxidant GSH was remarkably reduced with enhanced lipid peroxidation in ischemic brain lesions in the tMCAO rat model (Ahmad et al., 2014). Another study also demonstrated that selenium could activate transcription factors, TFAP2c and Sp1, to effectively enhance the expression of GPX4 and inhibit ferroptosis in the tMCAO model (Alim et al., 2019). LOX-mediated generation of lipid hydroperoxides has been suggested to participate in ferroptosis (Shintoku et al., 2017). Several LOX inhibitors have been found to reduce infarct size after ischemic stroke in the tMCAO rat model (Tuo et al., 2017; Yigitkanli et al., 2013).

3.2 Ferroptosis in intracerebral hemorrhage

Primary brain injury (PBI) and secondary brain injury (SBI) are the two stages of the post-intracerebral hemorrhage (ICH) pathological process. PBI is characterized by the initial mechanical injury caused by the blood. Clinical management at this stage includes medical treatment, such as addressing hypertension, coagulopathy, or even surgery (e.g., external ventricular drainage or hematoma evacuation). PBI is followed by SBI, which is also considered a devastating stage after ICH (Cordonnier et al., 2018; Mohammed Thangameeran et al., 2020). Evidence from preclinical and clinical studies suggests that the host immune response, release of thrombin and clot components (iron and heme), increased cytotoxicity, and inflammation may contribute to SBI after ICH (Aronowski & Zhao, 2011; Harukuni & Bhardwaj, 2006; Lou et al., 2009; Wasserman & Schlichter, 2007; Xiong et al., 2014). The intricate mechanisms, including related modes of cell deaths during SBI, have become the interest of the current topics. A better understanding of the post-ICH cell death cascade is essential for future preclinical studies.

Iron toxicity plays a vital role in SBI (Hua et al., 2007). Due to the excessive hemoglobin release after cell lysis in the post-ICH phase, activated microglia and infiltrating macrophages at the injury site engulf and degrade hemoglobin (Qin et al., 2019), further releasing ferrous iron (Liu et al., 2019). This ferrous iron accumulates in the neurons and acts as the catalyst to form hydroxyl radicals through the Fenton reaction to drive ROS production. Lipid peroxidation and ferroptosis occur when GPX4 activity is inhibited (Duan et al., 2016; Mohammed Thangameeran et al., 2020). Using transmission electron microscopy, researchers observed shrunken mitochondria in the brains of ICH mice, providing powerful evidence of ferroptosis (Wan et al., 2019).

In organotypic hippocampal slice cultures (OHSCs), it was found that administration of ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, prevented neuronal death and reduced iron deposition induced by hemoglobin (Li et al., 2017). In an in vivo experiment, mice treated with Fer-1 after ICH had better evaluation scores when assessing neurological function after ICH. In addition, Fer-1 also reduced lipid ROS production and attenuated the increased expression level of prostaglandin-endoperoxide synthase 2 (PTGS2) and its gene product, cyclooxygenase-2 (COX-2), in OHSCs, as well as in the collagenase-induced ICH model in mice (Li et al., 2017; Zille et al., 2017). Notably, COX-2 is highly expressed in neurons after ICH, but COX-2 inhibition reduces ICH-induced SBI (Chu et al., 2004). A study showed that GPX4 expression was significantly reduced as early as 12 hr post-ICH and reached the lowest point at 24 hr after ICH. Overexpression of GPX4 by transfection with recombinant adenoviruses expressing GPX4 was able to rescue neurons from ferroptotic death and attenuated brain injury, further improving outcomes in rats after ICH (Zhang et al., 2018).

There are many types of cell death during ICH, and researchers found that Fer-1, in conjunction with other inhibitors that target various forms of cell death, prevented hemoglobin-induced cell death in OHSCs and human-induced pluripotent stem cell-derived neurons better than any inhibitor alone (Li et al., 2017). Therefore, it should be further determined whether a combination of inhibitors can improve ICH outcomes in animals better than one drug and whether this strategy can be effective in clinical trials (Wan et al., 2019).

3.3 Ferroptosis in subarachnoid hemorrhage

Subarachnoid hemorrhage (SAH), a devastating cerebrovascular disease with high mortality, is mainly induced by elevated intracranial pressure and subsequent transient brain ischemia due to rupture of an intracranial aneurysm and an increase of subarachnoid blood degradation products (Guo et al., 2019; Nishikawa & Suzuki, 2017). Although SAH accounts for only 5% of all strokes, it has high rates of mortality and disability, and poor clinical outcomes place a heavy burden on society and families (Fang, Shao, et al., 2020; Venti, 2012). Traditionally, vasospasm was considered the leading cause of poor outcome after SAH, and considerable efforts were invested in targeting it. However, many experimental studies targeting vasospasms did not improve the prognosis of SAH patients (Chen et al., 2014). Recent studies have indicated that early brain injury (EBI), leading to secondary brain injuries occurring within 72 h after SAH, may be the primary cause of poor outcome after SAH (Al-Mufti et al., 2017; Cahill et al., 2006; Suzuki, 2015). Thus, it is suggested that more efforts should be made to explore beyond anti-vasospasm treatment,and target other coexisting factors involved in the pathogenesis of EBI, which would be the new direction to improve outcome in patients after SAH (Vergouwen et al., 2011).

Before ferroptosis was discovered in SAH, accumulating evidence suggested that subarachnoid blood degradation products could activate an oxidation reaction and then cause lipid peroxidation, but some antioxidants show significant neuroprotection after SAH in several studies (Kamezaki et al., 2002; Sercombe et al., 2002; Yang et al., 2017). Additionally, iron content in the cerebrospinal fluid and cortex was increased after SAH in a rat model (Li et al., 2020; Suzuki et al., 2006). Moreover, some studies suggested that Fe2+ potently generates ROS by catalyzing H2O2 decomposition followed by hydroxyl radical production, membrane lipid peroxidation, and induced cell death after SAH (Horky et al., 1998; Mori et al., 2001).

Inhibition of ferroptosis could reduce neuronal death and improve SAH prognosis. The inhibitor Fer-1 provided neuroprotection against EBI by alleviating ferroptosis, and the potential protective mechanism could be a result of lipid peroxidation suppression (Li et al., 2020). Furthermore, ceruloplasmin and ferroportin (Fpn) were downregulated, while DMT1 and TfR1 were upregulated after SAH, which increased intracellular iron levels and induced ferroptosis (Geng et al., 2018; Morris et al., 2018). Researchers found that administration of Fer-1 upregulates Fpn, decreases iron content, and then improves the lipid peroxidation and EBI after SAH (Li et al., 2020). Beclin 1 is a well-known regulator of autophagy that is involved in the generation of the PtdIns3K complex involved in activating autophagy. Recent studies have suggested that BECN1 inhibits System Xc− activity, but not System Xc− expression, by increasing the formation of the BECN1–System Xc− complex. Inhibition of BECN1 suppresses the accumulation of lipid peroxidation by increasing System Xc− activity in EBI after SAH (Guo et al., 2019; Kang et al., 2018; Song et al., 2018).

4 MULTI-OMICS APPROACHES

As mentioned above, a number of correlations between stroke and ferroptosis have been found, but how to quantify the ferroptosis in stroke remains a problem that creates uncertainty in experiments and clinical application. For now, there are some approaches to define ferroptosis during the process of stroke: (a) observing morphology of the mitochondria under electron microscope; (b) analysis of mitochondrial membrane potential (MMP) (JC-1); (c) using flow cytometry for cell cycle analysis; (d) measurement of ROS (H2DCFH-DA); and (e) testing malondialdehyde, free iron, and GSH.

With the continuous emergence of new technologies in omics, the development of omics research in the direction of quantification and high-throughput strategy has been accelerated. Through the integrated analysis of multi-omics data (e.g., genomics, transcriptomics, proteomics, and metabolomics), it will become a new direction to explore and analyze mechanisms(Table 3).

TABLE 3. Genes involved in ferroptosis of stroke Species Gene Name Homo sapiens CBS Cystathionine beta-synthase PARK7 Parkinsonism associated deglycase TP53 Tumor protein p53 GPX4 Glutathione peroxidase 4 CDKN2A Cyclin-dependent kinase inhibitor 2A HMOX1 Heme oxygenase 1 SOCS1 Suppressor of cytokine signaling 1 MIR9−1 MicroRNA 9–1 MAPK1 Mitogen-activated protein kinase 1 HSPB1 Heat shock protein family B (small) member 1 ALOX15 Arachidonate 15-lipoxygenase ATG7 Autophagy-related 7 [Homo sapiens] TNFAIP3 TNF alpha-induced protein 3 Mus musculus Alox15 Arachidonate 15-lipoxygenase Tlr4 Toll-like receptor 4 Trp53 Transformation-related protein 53 Nfe2l2 Nuclear factor, erythroid derived 2, like 2 Elavl1 ELAV (embryonic lethal, abnormal vision)-like 1 (Hu antigen R) Rattus norvegicus Mtor Mechanistic target of rapamycin kinase

The researchers conducted a transcriptome analysis of the protective effect of sodium tanshinone IIA sulfonate (STS) against atorvastatin (Ator)-induced cerebral hemorrhage in zebrafish using RNA sequencing (RNA-seq) technology and enrichment analysis to analyze potential genes. The results of that analysis indicated that the mechanisms underlying the protective effect of STS against Ator-induced zebrafish cerebral hemorrhage were involved in iron ion binding and ferroptosis (Zhou et al., 2020).

In another study, RNA-seq analysis of neurons exposed to the protective dose of Se showed robust upregulation and downregulation of many genes (238 differentially expressed genes). They found that expression of the GPX4 transcript that includes exon 1a (mitochondrial form of GPX4) was also significantly induced in the RNA-seq analysis by selenium. In addition to mitochondrial GPX4, analysis of exon 1b (nuclear form of GPX4) showed that it was also significantly induced by Se alone or Se paired with a ferroptotic stimulus. To organize the RNA-seq data into biologically coherent networks, they applied a supervised weighted gene co-expression network analysis to define networks of genes that are co-regulated with the significantly induced mitochondrial form of GPX4 (exon 1a). These data support the notion that nuclear or mitochondrial GPX4 could mediate protection from ferroptosis induced by pharmacological selenium (Alim et al., 2019).

High-throughput sequencing assists researchers in detecting the variation of the key points that upregulate or downregulate markedly and critically in the process of disease and treatment. Although this technique is widely used by many researchers in other fields, few studies use it to make deeper understanding regarding the significance of ferroptosis in stroke. We hope that the combination of bioinformatic analysis and traditional detection could produce more comprehensive and reliable results in the near future.

5 CONCLUSIONS AND PERSPECTIVES

In this review article, we have outlined our understanding of the ferroptosis pathways, mechanism of ferroptosis, and related regulatory mechanisms. We also summarized the recent advances in ferroptosis in ischemic stroke, post-ICH EBI, and SAH. Furthermore, we highlight recent research pertaining to ferroptosis in stroke according to the multi-omics approaches (e.g., genomics, transcriptomics, proteomics, and metabolomics), which might provide new ideas in future studies of stroke.

However, there are still many questions to be completed. Compared with cancer, it could be worth considering whether same mechanism prevents neurological diseases by inhibiting ferroptosis. Although there are indeed many similarities between cancer and neurological diseases, there is evidence from cancer cell models that not all ferroptotic triggers cause the same type of lipid oxidation damage and the same is true in different cells of different systems (Yang et al. 2016). Recent studies have shown that the iron-rich microenvironment that often characterizes malignancies supports rapid proliferation and contributes to carcinogenesis, which means that cancer cells could exist in high iron levels and places cancer under persistent oxidative stress (Toyokuni et al. 2017; Toyokuni 2009; Toyokuni et al. 1995). For neurons, dysregulation in iron homeostasis might be a central driver for such neurological diseases (Faux et al. 2014; Gangania et al. 2017). Moreover, histone deacetylase (HDAC) inhibitors selectively protect neurons while augmenting ferroptosis in cancer cells. It is possible that ferroptosis will exhibit greater regulatory complexity in brain cells or interact with other brain-specific cell death pathways in a way that would not be anticipated from results obtained in cancer cell studies of this process. Therefore, greater mechanical clarity in two related areas is required (Zille et al. 2019).

Meanwhile, we need to discover and integrate channels to fill up the mechanism network. Animal studies investigating the link between ferroptosis and neurological diseases typically apply inhibitors to block cell death, but have not yet clarify the specific signals within the complex environment of the brain that actually cause the induction of ferroptosis; in many cases, even the specific class of brain cells that is spared by ferroptosis inhibitors remains unknown (Magtanong & Dixon, 2018). The widely recognized neuroprotective effect of iron chelators is contributed by their ability to prevent ROS generation via the Fenton reaction. An additional neuroprotective mechanism of iron-chelating compounds is to regulate the transcriptional activator hypoxia-inducible factor 1α (HIF-1α), which may serve as important therapeutic targets (Guo et al., 2015). And we also need to complete the exact function of iron in ferroptosis. Except that iron participates in Fenton chemistry and the action of iron-dependent oxidases, the requirement for iron for ferroptosis may reflect the role of various metabolic enzymes in ROS generation, for which iron functions as a cofactor. Moreover, the molecular events that occur downstream of lipid oxidation are relatively unclear; thus, further study is required to determine the terminal executor(s) for ferroptotic cell death (Hirschhorn & Stockwell, 2019).

Studies of apoptosis have benefited from the ability to detect caspase-3 (Porter & Janicke, 1999). Analogous protein-based molecular markers do not found for ferroptosis. The optimal marker of ferroptosis in vivo would be linked to the unique features of this cell death, but it is not known if these products are formed during ferroptosis in vivo, and, even if they are, whether they are specific for this process. The best candidate in this connection may be increased levels of PUFA oxidation and membrane lipid ROS (Ji et al., 2012). However, associating the pattern of lipid oxidation to a specific cell type is currently beyond the technical reach of this method and likely not practical for most investigators to implement independently or routinely.

As the mechanism of ferroptosis becomes relatively clearer, the current research on the intervention effects of ferroptosis in stroke is far from enough. It is time for doctors and scientists to conduct ferroptosis research about interventions.

Moreover, many questions remain unanswered: (a) the exact mechanism and other pathways of ferroptosis after stroke; (b) key targets in ferroptosis; (c) the relationship between other forms of cell death, such as apoptosis, necroptosis, and pyroptosis; (d) whether the drugs that target ferroptosis can play an important role in the clinical treatment of stroke; and (e) what is the marker (key) protein of ferroptosis. Although there is much to be accomplished before this can be translated into clinical treatment, we believe that ferroptosis is one of the most important forms of cell death in brain diseases and that in-depth studies of ferroptosis will provide new opportunities for diagnosis and therapeutic intervention.

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