Ferroptosis and endoplasmic reticulum stress in ischemic stroke

Abstract

Ferroptosis is a form of non-apoptotic programmed cell death, and its mechanisms mainly involve the accumulation of lipid peroxides, imbalance in the amino acid antioxidant system, and disordered iron metabolism. The primary organelle responsible for coordinating external challenges and internal cell demands is the endoplasmic reticulum, and the progression of inflammatory diseases can trigger endoplasmic reticulum stress. Evidence has suggested that ferroptosis may share pathways or interact with endoplasmic reticulum stress in many diseases and plays a role in cell survival. Ferroptosis and endoplasmic reticulum stress may occur after ischemic stroke. However, there are few reports on the interactions of ferroptosis and endoplasmic reticulum stress with ischemic stroke. This review summarized the recent research on the relationships between ferroptosis and endoplasmic reticulum stress and ischemic stroke, aiming to provide a reference for developing treatments for ischemic stroke.

Introduction

Stroke, the most common clinical cerebrovascular disease, is the second leading cause of death and a major disabling disease that results in organic brain injury and death of neurons, commonly with the clinical characteristics of sudden onset and rapid localized or diffuse cerebral dysfunction. Stroke is categorized as ischemic or hemorrhagic, and ischemic stroke accounts for the majority of cases (Saini et al., 2021). The death of neurons caused by ischemic stroke makes full recovery difficult or impossible. Therefore, understanding the mechanisms of cell death following ischemic stroke is of vital importance for developing new treatment options and improving the prognosis following ischemic stroke. A previous study demonstrated that various cell death patterns—including pyroptosis, apoptosis, necrosis, autophagy, and others, separately or in combination—contribute to neuronal death and brain injury in ischemic stroke (Sekerdag et al., 2018).

In 2012, a novel type of cell death, ferroptosis, was discovered by Dixon et al. (2012). The process of ferroptosis is characterized by a dependency on iron and the generation of reactive oxygen species (ROS) during cell death. They found that the erastin, an oncogenic RAS-selective lethal small molecule, triggered a novel form of nonapoptotic cell death that was iron-dependent and differed from autophagy, necrosis, and apoptosis, and this was named ferroptosis. Morphologically, a unique feature of cells undergoing ferroptosis is that the mitochondria are smaller and have a higher membrane density than those of healthy cells, which is different from autophagy (formation of double membrane-enclosed vesicles), necrosis (swelling of the cytoplasm and organelles, rupture of the plasma membrane), and apoptosis (condensation and margination of chromatin) (Kroemer et al., 2009; Galluzzi et al., 2012; Li and Jia, 2023; Xiong et al., 2023). In the study by Dixon et al. (2012), the cell membranes were intact, there were no ruptures or projections, and nuclei were not significantly altered. Regarding bioenergetics, ferroptotic cells do not experience significant depletion of adenosine triphosphate, which makes ferroptosis different from necrosis, which is caused by bioenergetic failure. Six genes (ACSF2, ATP5G3, CS, IREB2, RPL8, and TTC35) were confirmed to be drawn into the regulation of ferroptosis through a customized array of short hairpin RNAs targeting 1087 mitochondrial genes and a rigid confirmation pipeline. Most importantly, in terms of biochemistry, ferroptosis is the iron-dependent excessive accumulation of lethal lipid ROS. Moreover, it can be inhibited by iron chelators or ferrostatin-1 (Fer-1), rather than other cell death inhibitors (Dixon et al., 2012).

The endoplasmic reticulum (ER) is responsible for protein synthesis, folding, and structural maturation in cells (Anelli and Sitia, 2008; Homentcovschi and Higuchi-Sanabria, 2022). Cells can experience ER stress when genetic or environmental factors cause the ER protein folding machinery to work beyond its capability (Tabas and Ron, 2011). Ca²⁺ homeostasis disruption caused by cytosolic Ca²⁺ overload and Ca²⁺ depletion in the ER lumen triggers ER stress and protein misfolding. In response to ischemia, hypoxia, hypertension, and toxic stimuli, Ca²⁺ homeostasis may be dysregulated, leading to ER stress and protein misfolding (Walter and Ron, 2011; Marchant et al., 2022). In the presence of a critical amount of misfolded proteins in the ER, cells balance their protein folding capacity and the demand to correct the situation by initiating a signal transduction pathway called the unfolded protein response (UPR). If the insult exceeds the UPR’s regulatory capacity, ER stress will lead to cell death (Hetz et al., 2020). This article reviews the role of ferroptosis and ER stress in ischemic stroke and their potential relevance.

Search Strategy and Selection Criteria

Relevant articles published between January 1993 and January 2023 in the MEDLINE database were searched. The search terms were: Ferroptosis (MeSH Terms) AND Ischemic stroke (MeSH Terms), Endoplasmic reticulum stress (MeSH Terms) AND Ischemic stroke (MeSH Terms), Ferroptosis (MeSH Terms) AND Endoplasmic reticulum stress (MeSH Terms). After removing irrelevant and duplicate studies from the retrieved studies, the title and abstract of each article were read in a preliminary screening, and articles that were not highly relevant or were deemed unnecessary were deleted. Full texts were then read, and studies with a clear explanation of sample sizes, experimental methods, and procedures were included.

Ferroptosis in Ischemic Stroke

Ischemic stroke develops as a result of thrombus formation, embolisms, or atherosclerosis obstructing cerebral arteries. There are several cell death pathways that have been shown to play a role in ischemic stroke, including apoptosis, necroptosis, autophagy, parthanatos, and pyroptosis (Tuo et al., 2022a). Research on ferroptosis in hemorrhagic stroke has greatly progressed, with three major ferroptosis signaling pathways having been implicated in several studies (Li et al., 2017; Zheng et al., 2019; Bai et al., 2020; Diao et al., 2020). Researchers have been increasingly exploring whether ferroptosis is seen in ischemic stroke and what role it might play.

Changes in glucose metabolism and the mitochondrial respiratory chain play important roles in ischemia/reperfusion injury (IRI), which is also associated with ferroptosis (Yao et al., 2021). In simple terms, the nicotinamide adenine dinucleotide phosphate hydride (NADPH) produced by the pentose phosphate pathway enables the glutathione (GSH) reductase-mediated reduction from GSH disulfide to GSH while supporting cystine intake mediated by solute carrier family 7 member 11 (SLC7A11) and further conversion to cysteine for GSH synthesis (Ballatori et al., 2009; Liu et al., 2020). The ferroptosis inducers erastin and RAS-selective lethal 3 (RSL3) can reduce the glycolytic activity of tumor cells (DeHart et al., 2018; Wang et al., 2019). ROS are produced primarily by mitochondria in cells. An interruption of the mitochondrial electron transport chain reduces the levels of lipid ROS in cells, which significantly inhibits ferroptosis in tumor cells. The intermediates of the tricarboxylic acid cycle, its downstream products such as succinic acid and fumaric acid, and alpha-ketoglutaric acid can enhance ferroptosis caused by cysteine depletion (Gao et al., 2019; Shin et al., 2020). It is believed that ferroptosis contributes to cerebral IRI because of these factors.

Several studies have reported that ferroptosis is a primary driver of ischemic injury in mouse models assessing the liver, heart, and kidneys (Dixon et al., 2012; Friedmann Angeli et al., 2014; Linkermann et al., 2014; Skouta et al., 2014; Gao et al., 2015). Neuronal damage during reperfusion was shown to be exacerbated by iron accumulation both in animal models and clinical trials of ischemic stroke before ferroptosis was identified (Kondo et al., 1997; Lipscomb et al., 1998; Ding et al., 2011; Park et al., 2011; Fang et al., 2013), and this could be reduced by iron chelation in animals (Prass et al., 2002; Hanson et al., 2009). Speer et al. (2013) speculated that oxidative glutamate toxicity (likely a neuronal form of ferroptosis) might conduce ischemic neuronal death and that one target for the beneficial effects of iron chelators may be the hypoxia-inducible factor prolyl-hydroxylase domain pathway. However, only recently have researchers begun to explore the role of ferroptosis in ischemic stroke.

Iron metabolism in ferroptosis after ischemic stroke

Iron is one of the essential trace elements in the human body, with deficiency accounting for anemia and abnormal iron-related enzyme levels. Iron accumulation can damage tissues and increase the risk of diseases such as cancer (Toyokuni, 2016). The Fenton reaction, where ferrous ions in the redox cycle react with H₂O₂ to produce hydroxyl radicals, which can cause harmful oxidative damage to membrane lipids, proteins, and DNA (Doll and Conrad, 2017) and, ultimately, leads to ferroptosis. Iron and iron derivatives, such as heme and iron-sulfur clusters, are incorporated into the enzymes that synthesize ROS and are critical to the function of these enzymes, including lipoxygenases (LOXs), NADPH, cytochrome P450 enzymes, xanthine oxidase, and subunits of the mitochondrial electron transport chain. Iron is also present at the active sites of the peroxisomes that destroy H₂O₂ (Abeysinghe et al., 1996; Dixon and Stockwell, 2014).

Physiologically, the brain is immune to systemic iron fluctuations, even under experimentally induced conditions of systemic iron overload (Castellanos et al., 2002; Millerot et al., 2005). However, in the case of ischemic brain injury, the blood-brain barrier permeability to blood cells and metal is increased (Garcia et al., 1994; Gidday et al., 2005). Dysregulation of neuronal iron homeostasis via upregulation of the transferrin receptor (TFR) triggers the formation of free radicals after transient forebrain ischemia, which can be inhibited by deferiprone (DFO) (Park et al., 2011). Experimental stroke increases the levels of circulating iron-loaded transferrin in ischemic brain tissue, increasing oxygen-glucose deprivation (OGD)-induced neuronal death by enhancing ROS production (DeGregorio-Rocasolano et al., 2018). Clinically, iron overload at admission, or high levels of ferritin in the blood, is also associated with poor outcomes in patients with ischemic stroke (Dávalos et al., 2000; Millan et al., 2007). A randomized clinical trial evaluating the potential benefits of intravenous DFO in patients with ischemic stroke found that DFO reduced systemic iron levels within 1–3 days of administration and may have long-term efficacy in patients with ischemic stroke (Millán et al., 2021). The loss of tau protein may lead to neurotoxic iron accumulation and ferroptosis after abolishing amyloid precursor protein trafficking to ferroportin (Lei et al., 2012). As a result, Tuo et al. (2017) investigated the role of tau in mediating iron export and ferroptosis in ischemic stroke. The researchers first verified that IRI acutely inhibited tau protein, and tau-related iron export failure led to the increase of iron in ischemic stroke. Moreover, promoting iron efflux, with ceruloplasmin and amyloid precursor protein was able to attenuate the infarct. All ferroptosis inhibitors significantly reduced functional deficits induced by middle cerebral artery occlusion (MCAO) and improved neurological scores. The study also explored the role of age in this pathway. In mice with tau knocked out, the iron content in the brains of young mice did not increase due to IRI because there was no tau, thus reducing the infarct volume, while in elderly mice, the prognosis after MCAO was much worse than that of wild-type mice due to excessive accumulation of iron due to tau knocked out over time. In another study, tau knockout mice lost homeostasis prematurely as they aged, causing brain iron accumulation (Lei et al., 2012). These results underline that research into age and its relationship to potential stroke interventions is necessary. The level of nuclear receptor coactivator 4 (NCOA4), a ferritinophagy regulatory protein, increased via ubiquitin specific peptidase 14 regulation after ischemic stroke both in vivo and in vitro. Knockout of NCOA4 in vivo and in vitro could upregulate the level of ferritin and alleviate the free iron content in neurons; indicators related to ferroptosis also improved, and ferroptosis was suppressed after IRI. These results suggest that NCOA4 is a control target of ferritinophagy, thereby regulating ferroptosis in ischemic stroke (Li et al., 2021). One study synthesized a benzimidazole derivative (9a) and confirmed that it bound to NCOA4 in cells to inhibit ferritinophagy (Fang et al., 2021). Furthermore, the infarct volumes 24 h after IRI were reduced in the experimental rats who were administered 9a by intraperitoneal injection.

Amino acid and glutathione metabolism in ferroptosis after ischemic stroke

The Cystine/glutamate antiporter system Xc^– is one of the main components of the cellular ferroptosis regulatory signaling pathway. System Xc^– is an amino acid transporter located on the cell membrane that regulates the exchange of both extracellular cystine and intracellular glutamate. After entering the cell, the cystine is reduced to cysteine, which is further used to make GSH. Free radical scavenging, antioxidation, and control of cellular aging are the main physiological functions of GSH. GSH binds to ferrous iron in the labile iron pool, an iron exchange site in lysosomes, to prevent it from being oxidized, thereby preventing the production of excess ferrous ions that can cause a harmful Fenton reaction (Hider and Kong, 2011). In ferroptosis, depletion of GSH was observed to release iron to produce hydroxyl radicals and then induce lipid peroxidation. Moreover, GSH is used as a cofactor by GSH peroxidase (GPX) to reduce hydrogen peroxide to water (Aki et al., 2015). By attacking the terminal oxygen of lipid peroxide with selenocysteine, GPX4 reduces lipid peroxides into hydroxyeicozatetraenoic acids or hydroxyoctadecadiene acids (Maiorino et al., 1988; Takebe et al., 2002). GPX4 is another key component in the ferroptosis signaling pathway (Seibt et al., 2019).

Lu et al. (2020) examined the roles of the long noncoding RNA plasmacytoma variant 1 (PVT1) and microRNA (miR)-214 in ferroptosis after ischemic stroke. In their study, products of lipid peroxidation and iron were deposited in the brains of IRI-model mice, demonstrating that IRI facilitates ferroptosis in the brain. miR-214 overexpression or PVT1 silencing notably decreased p53 levels and increased SLC7A11 levels compared with the IRI model control group. In this regard, miR-214 might increase SLC7A11 levels by decreasing p53 levels. Conversely, silencing miR-214 or overexpressing PVT1 notably counteracted the effects of Fer-1 on ferroptosis in vitro. Further experiments found that a decrease in miR-214 expression removed the influence on the ferroptosis pathway caused by the silencing of PVT1, demonstrating an interaction between PVT1 and miR-214 in ferroptosis regulation. To find the specific mechanisms of miR-214 in regulating ferroptosis, they utilized bioinformatics database prediction, a luciferase reporter gene assay, and co-immunoprecipitation to show that the 3′ untranslated region of TFR1 was the binding site for miR-214, indicating that miR-214 modulates ferroptosis partly by controlling iron input through TFR1 modulation. A study by Chen et al. (2021c) identified a decline in GPX4 levels after stroke, and the ferroptosis inhibitor liproxstatin-1 reversed this and reduced iron accumulation, which indicated that ferroptosis occurs following cerebral infarction.

Lipid metabolism of ferroptosis after ischemic stroke

Because lipids are important regulators of cell death, lipid signals can play a role in inducing, regulating, or inhibiting apoptotic or non-apoptotic pathways in mammals. A critical element of ferroptosis is lipid oxidative stress and membrane damage caused by lipid stress, particularly caused by polyunsaturated fatty acids (Yang et al., 2016; Stockwell et al., 2017). Lipidomics study has demonstrated that polyunsaturated fatty acids contain phospholipids such as adrenergic acids (ADAs) and arachidonic acids (AAs) that induce ferroptosis (Kagan et al., 2017). According to Doll et al. (2017), acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) drives ferroptosis through the accumulation of oxidized cellular membrane phospholipids. In fact, ACSL4 catalyzes AA or ADA to produce AA or ADA acyl-coenzyme A derivatives, which are then esterified by lysophosphatidyl cholinyltransferase 3 into phosphatidylglycolamine (AA-PE or ADA-PE). Then, 15-LOX oxidizes AA-PE and ADA-PE to produce lipid hydrogen peroxide, which causes ferroptosis (Yuan et al., 2016; Latunde-Dada, 2017).

The ACSL4 inhibitor rosiglitazone could decrease the accumulation of lipid ROS after IRI, validating the role of ACSL4 as a target for inhibiting ferroptosis in ischemic stroke (Chen et al., 2021c). In terms of detailed research on the molecular mechanisms, the research of Cui et al. (2021) showed that hypoxia-inducible factor 1α-mediated downregulation of ACSL4 inhibited ferroptosis in neurons, but not in microglia, in the early stages after ischemic stroke. ACSL4 knockdown protected mice from cerebral ischemia, and neurons in which ACSL4 was silenced were resistant to OGD-induced ferroptosis. Despite ACSL4 silencing, lipid peroxidation was not reduced by ACSL4 silencing in microglia; however, Xu et al. (2022) posited that ferroptosis is comprehensively induced after cerebral IRI and not limited to a single pathway. They proposed for the first time that ferroptosis peaked 24 hours after ischemic stroke. In addition, their clinical data showed a positive association between prostaglandin E2 levels and ferroptosis. Further studies in rats confirmed that prostaglandin E2 supplementation inhibited cerebral IRI-induced ferroptosis and that the use of inhibitors of this pathway could aggravate ferroptosis. Liproxstatin-1 and Fer-1, which have previously been shown to be effective in alleviating cerebral IRI damage, cannot salvage neuronal damage caused by permanent MCAO. Therefore, Tuo et al. (2022b) suggested that ferroptosis occurs in the reperfusion stage. Their further study confirmed that thrombin could induce ferroptosis independent of iron accumulation after cerebral IRI by influencing lipid metabolism, which is related to ACSL4 (Tuo et al., 2022b). Polymerase I and transcript release factor affects lipid peroxidation and ferroptosis in cerebral IRI by regulating the expression of phospholipase A2 group IVA (PLA2G4A), another important protein in lipid metabolism (Jin et al., 2022). As a result of cerebral IRI, levels of UbiA prenyltransferase domain containing 1—a newly discovered antioxidant enzyme—were inhibited both in vivo and in vitro. Lipid peroxidation and ferroptosis were inhibited during cerebral IRI when the UbiA prenyltransferase domain containing 1 level was upregulated. This may be related to the regulation of catalyzed coenzyme Q10 expression in the Golgi apparatus membrane and the regulation of vitamin K2 synthesis, thus affecting mitochondrial function (Huang et al., 2022b).

Other pathways of ferroptosis after ischemic stroke

Two studies (Chen et al., 2021b; Fan et al., 2022) based on the same three human peripheral blood ischemic stroke-related microarray Gene Expression Omnibus datasets aimed to understand the importance of ferroptosis in ischemic stroke from a bioinformatics perspective. Three biomarkers associated with ferroptosis—namely PTGS2, MAP1LC3B, and TLR4—were proposed as potential diagnostic biomarkers for ischemic stroke, and there are several possible therapeutic compounds that are directed at these three genes that might be effective in treating ischemic stroke (Chen et al., 2021b). Fan et al. (2022) screened cyclin dependent kinase inhibitor 1A/AP-1 transcription factor subunit as a reliable and promising diagnostic biomarker for identifying patients with ischemic stroke. The C9orf106/C9orf139-miR-22-3p-CDKN1A and GAS5-miR-139-5p/miR-429-JUN axes are the potential pathways regulating ferroptosis during ischemic stroke.

A study focusing on ferroptosis and cerebral vascularization in a rat model of ischemic stroke with diabetes (Abdul et al., 2021) showed that, as a result of deferoxamine treatment, diabetic rats had less vascular drop-out, improved neurovascular integrity, better sensorimotor results, and were protected from further deterioration of their cognitive deficits. In addition, the initial immunohistochemistry of iron responsive element binding protein 2 and citrate synthase—both markers of ferroptosis—showed strong vascular localization. As a result, microvascular endothelial cells showed an increase in the iron-mediated lipid peroxidation in the brain of both the control and diabetes groups. However, markers of ferroptosis were significantly higher in the diabetes group, and deferoxamine improved cell survival in this group. Another study examined the similar role of ferroptosis in vitro (Chen et al., 2021a). Increased expression of Meg3 (a long non-coding RNA) and stronger ferroptosis indicators was seen in rat brain microvascular endothelial cells were treated after OGD combined with hyperglycemia. Ferroptosis indicators, such as the iron concentration, the generation of lipid ROS, and the products of lipid peroxidation, could be reversed by Meg3 knockdown. Additionally, p53 was confirmed as a downstream target of Meg3, and p53 interacted with the GPX4 promoter to inhibit its transcription after OGD combined with hyperglycemia. An overview of the mechanisms of ferroptosis induced by ischemic stroke or OGD/reperfusion (OGD/R) is shown in Figure 1.

Figure 1.

Overview of the mechanisms of ferroptosis induced by ischemic stroke or oxygen glucose deprivation/reperfusion.

Created using Microsoft PowerPoint 2019. ACSL4: Acyl-coenzyme A synthetase long-chain family member 4; APP: amyloid precursor protein; CoQ10: coenzyme Q10; FPN: ferroportin; GPX4: glutathione peroxidase 4; HIF-1α: hypoxia inducible factor 1 subunit α; HTF: holotransferrin; IRI: ischemia/reperfusion injury; NCOA4: nuclear receptor coactivator 4; OGD/R: oxygen glucose deprivation/reperfusion; PLA2G4A: phospholipase A2, group IVA; PTRF: polymerase I and transcript release factor; SLC7A11: solute carrier family 7, member 11; TFR1: transferrin receptor 1.

Endoplasmic reticulum stress signaling pathway in ischemic stroke

The initiation of the UPR is associated with type-I transmembrane proteins, such as protein kinase RNA-like ER kinase (PERK), inositol requirement protein 1 (IRE1), and activated transcription factor-6 (ATF6). During cellular homeostasis, the major molecular ER chaperone glucose-regulated protein 78 (GRP78) is bound to all of these transmembrane proteins by its peptide-binding domains, keeping them in an inactive state. When GRP78 binds to misfolded proteins, it dissociates itself from these transmembrane proteins, causing oligomerization, autophosphorylation, and enzyme activation, indicating initiation of the UPR (Bertolotti et al., 2000). Other reports showed that, in addition to restoring correct protein conformation and maintaining homeostasis, GRP78 protected cell survival during ischemic stroke and helped neurons resist stress (Lee, 2001; Rao et al., 2004). The expression of oxygen regulated protein 150 (a chaperone that resides in the ER) increased during hypoxia to prevent cell death caused by ischemia. The biosynthesis and biological functions of GRP78 also depend on oxygen regulated protein 150 (Takizawa et al., 2007). The cycloxygenase-2 inhibitor parecoxib enhances the expression of GRP78 and oxygen regulated protein 150 after focal cerebral ischemia and prevents IRI damage to the brain (Ye et al., 2013). GRP78 mRNA was induced as early as 2 hours after cerebral ischemia in rats and persisted for 48 hours (Higashi et al., 1994). The protein appeared in the ischemic cortex as early as 3.5 hours after reperfusion, and immunoreactivity in degenerated neurons peaked at 24–48 hours after ischemia (Ito et al., 2001). A selective inducer of GRP78 significantly induced GRP78 expression in vivo and in vitro and reduced tunicamycin-induced cell death in SH-SY5Y cells, cell death in gerbils with forebrain ischemia, and apoptosis in the hippocampal CA1 subregion (Oida et al., 2008). The cellular and subcellular localization of GRP78 showed that the expression of GRP78 was almost completely restricted to neurons in healthy rats, but after transient focal cerebral ischemia, activated microglia/macrophages expressed GRP78 and, in the peri-infarct region, it was primarily expressed in reactive astrocytes and neurons (Jin et al., 2018).

PERK is activated to dimerizes by autophosphorylation, leading to phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which prevents protein accumulation in the ER by inhibiting protein translation (Marciniak et al., 2006). However, phosphorylated eIF2α increases activating transcription factor 4 (ATF4) translation, which promotes the expression of various adaptive genes, including ER chaperone genes and the key transcription factor C/EBP-homologous protein (CHOP). CHOP translocates to the nucleus and is associated with ER stress-mediated apoptosis (Rzymski et al., 2010). Apoptosis induced by ER stress has been highlighted as another important process in the pathophysiology of cerebral ischemia. The PERK/eIF2α pathway is associated with the inhibition of protein synthesis after transient cerebral ischemia (Kumar et al., 2001; Owen et al., 2005). After knocking out PERK in forebrain neurons, the cerebral ischemia-induced inhibition of protein synthesis was reported as attenuated, and stroke outcomes were worse (Wang et al., 2020). In another study, neuronal apoptosis, cerebral infarction, and neurological dysfunction that increase after knockdown of Hairy and enhancer of split 1 after MCAO were significantly rescued by selective PERK inhibitor treatment, which can be explained by the promoted activation of the PERK/eIF2α/ATF4/CHOP pathway and subsequently elevated ER stress (Li et al., 2020c).

The NLR family pyrin domain containing 3 inflammasome activation is affected by ATF4 through the parkin-mitophagy axis, which protects the brain from IRI (He et al., 2019). In one study, ER stress activators tunicamycin and thapsigargin improved outcomes in mice after MCAO and OGD/R in cells through the eIF2α and downstream ATF4 pathways (Zhang et al., 2014). In another study, CHOP plays a central role in ischemic injury that leads to neuronal death (Tajiri et al., 2004). CHOP was upregulated for up to 14 days after MCAO and was observed to colocalize with caspase-12 positive cells and TUNEL positive cells after IRI in mice, showing that neuronal apoptosis in the MCAO mouse model is closely related to ER stress (Zhao et al., 2018). These effects can be counteracted by chrysophanol. Hypothermia also reversed the increase in CHOP expression and ER oxidoreductin-α induced by ischemic stroke models compared with animals kept at normal temperatures (Poone et al., 2015). OGD/R could induce the activation of the GRP78/eIF2α-ATF4-CHOP signaling pathway in rat primary cortical neurons, which could be reversed by (-)-CLAU (Wu et al., 2020). Melatonin treatment also decreases the levels of cytochrome c and lysed caspase-3, markers of apoptosis induced by IRI, and the underlying mechanism is related to the attenuation of the PERK-elF2α-CHOP pathway (Lin et al., 2018b).

Inositol-requiring enzyme 1α (IRE1α) is another factor involved in the UPR whose cytoplasmic tail has two enzymatic activities, an endoribonuclease (RNase) domain and a serine/threonine kinase domain (Kim et al., 2008). At low levels of ER stress, IRE1α RNase cleaves a 26-nt intron from the mRNA encoding the homeostasis transcription factor X-box protein 1 (XBP1), a transcription factor generating XBP1. As a means of restoring ER homeostasis, it translocates to the nucleus and induces the transcription of numerous genes in the ER (Hetz et al., 2015). When ER stress is highly activated, IRE1α becomes overactive and undergoes homologous oligomerization, directly cleaving microRNAs that inhibit apoptosis. Multiple proinflammatory proteins have been reported to be activated or upregulated by this, including the pro-oxidant protein thioredoxin-interacting protein (Oakes and Papa, 2015). OGD can induce the continuous activation of the IRE1α pathway, and using proteomics to determine differentially expressed proteins after cerebral ischemia and stroke in rats suggested that the increased expression of calretinin 2 protein may mediate the neuronal apoptosis mechanisms induced by ER stress related to IRE1α (Chen et al., 2015). The mRNA level of spliced XBP1 decreased temporarily and then increased in primary neurons after OGD. Exogenous expression of XBP1 significantly inhibited OGD/R stress-induced cell death (Ibuki et al., 2012). It was reported that after OGD/R, the levels of phospho-IRE1α, XBP1, unspliced isomers of XBP1, and neuronal apoptosis were also increased with the increase of interleukin 1 beta, interleukin 6, and tumor necrosis factor alpha in microglia. Moreover, overexpression of IRE1 aggravated OGD/R-induced apoptosis (Mo et al., 2020).

Both in vivo and in vitro studies have demonstrated that taurine, an inhibitory neurotransmitter, inhibited hypoxia-reperfusion-induced ER stress by inhibiting the increase of phospho-IRE1α (Gharibani et al., 2013). The loss of XBP1 in forebrain neurons was also shown to worsen ischemic stroke outcomes in mice. O-linked-N-acetylglucosaminylation, a post-translational modifier that protects cells from many stress conditions, is activated via XBP1 in the ischemic penumbra of young but not old mice, suggesting that neuronal function may be impaired after ischemia in the elderly due to an inability to activate O-linked-N-acetylglucosaminylation, an ER stress pathway (Jiang et al., 2017). Melatonin administration before ischemia attenuated brain IR injury by inhibiting ER stress not only through the PERK pathway but also through the IRE2 pathway. Interestingly, this mechanism is also associated with autophagy (Feng et al., 2017).

The activation of ATF6 is followed by its transfer to the Golgi apparatus, in which it is cleaved by proteases, leading to the nuclear translocation of the N-terminal leucine-zipper transcription factor and the release of activated forms of ATF6 (ATF6 [N]). ATF6 (N) then transports to the nucleus and regulates gene expression there (Ye et al., 2000). As a result of its association with IRE1, ATF6 controls XBP1 translation and ameliorates ER-related degradation by inducing GRP78 and α-mannosidase-like protein 1 (Yamamoto et al., 2007). In one study, on day 7 after cerebral ischemia, the expression of ATF6 increased in the striatum ipsilateral to the lesion in MCAO rats (Rissanen et al., 2006). The selective activation of ATF6 and downstream genes can protect the heart from IRI damage, and it also protects a wide range of tissues, including the brain (Blackwood et al., 2019). It has also been demonstrated that ATF6 mRNA was significantly increased after whole forebrain IRI in rats, and ATF6 protein is believed to enhance the ER’s neuroprotective function during the first 2 hours after reperfusion. Moreover, ATF6 may also be a target for the neuroprotective effects of statins on the attenuated ER stress response after acute IRI (Urban et al., 2009). In addition, ATF6 has a cholesterol-lowering effect (Urban et al., 2009). Following MCAO in mice, the infarct volume and neuronal death increased in the peri-infarct area after Atf6 deletion, which was associated with decreased activation of astroglia and the formation of glial scars (Yoshikawa et al., 2015). By inducing the expression of the active form of ATF6 in mice, forced activation of the ATF6 UPR branch was shown to improve functional outcomes and reduce infarct volumes after stroke, possibly mediated by inhibition of the mammalian target of rapamycin pathway (Yu et al., 2017). However, studies on the protective effects of taurine in ischemic stroke showed that taurine can inhibit apoptosis and improve outcome by inhibiting the lysis of ATF6, suggesting that ATF6 plays a harmful role in ischemic stroke (Gharibani et al., 2013; Prentice et al., 2017a, b). Because ATF6 is related to the initiation of CHOP, the role of ATF6 in ischemic stroke needs to be further studied.

Interactions between ferroptosis and endoplasmic reticulum stress signaling pathway in ischemic stroke

Cycloxygenase-2, which is related to ER stress in cerebral IRI, is also a marker of ferroptosis (Chen et al., 2019a), and it is often used as an evaluation index for ferroptosis after stroke (Hu et al., 2022). The coenzyme Q10 in the Golgi apparatus plays a key role in regulating ferroptosis-related lipid peroxidation (Bersuker et al., 2019; Huang et al., 2022b). The relationships between ER and Golgi structures and their physiological functions may also be a potential site of crosstalk between ER stress and ferroptosis in cerebral IRI. It has been demonstrated that polyunsaturated lipids in ferroptosis are oxidized only in the compartments associated with the ER, and only PE molecules with AA or ADA acyl chains are oxidized (Kagan et al., 2017).

This suggests a potential link between ferroptosis and ER stress, and research has found a correlation between ferroptosis and ER stress. During ferroptosis induced by erastin, in situ imaging showed significant increases in pH and the viscosity of the ER, while Fer-1-treated cells showed little change (Song et al., 2022). Cell ER stress induced by lipopolysaccharides could be down-regulated by Fer-1 (Li et al., 2022a). Mesencephalic astrocyte-derived neurotrophic factor is considered a classic ER stress response protein due to its ability to link to the nucleotide-binding structural domain of ADP-bound GRP78 and assist in the maintenance of protein folding homeostasis (Yan et al., 2019). Recombinant murine mesencephalic astrocyte-derived neurotrophic factor pretreatment inhibited ferroptosis in sepsis-associated lung injury via the ER stress-associated GRP78-PERK-ATF4 signaling pathway (Zeng et al., 2023). It was reported that the import of cysteine by the inhibition of system Xc- induced by sorafenib not only led to ferroptosis but also to the induction of the ER stress response, such as the phosphorylation of eIF2α and the upregulation of ATF4 and cation transport regulator homolog 1 (Dixon et al., 2014). Additionally, ATF4 is considered an important regulator of SLC7A11 expression, suggesting that there may be a feedback pathway between ferroptosis and ER stress. By knocking down ATF4, cells are more susceptible to drug-induced ferroptosis (Chen et al., 2017; Gao et al., 2021). GSH transferase inhibitors such as artesunate (ART), which also induces ferroptosis, activate ATF4-dependent genes including CHOP (Lee et al., 2018). Both ART and erastin could increase the expression of GRP78 in a time-dependent manner and induce ER stress. Moreover, DFO inhibits ART-induced ER stress (Hong et al., 2017). Curiously, both AML12 and RAW264.7 cells are insensitive to iron overload but sensitive to iron deficiency, showing ER stress responses such as increased phosphorylation of PERK, ATF4, and CHOP (Wang et al., 2022). In glioma cells, dihydroartemisinin, which can directly inhibit lipid peroxidation, also promote the expression of GRP78 through the PERK/ATF4 pathway, thereby increasing the expression of GPX4 to play a cytoprotective role in inhibiting ferroptosis; this suggests a link between ferroptosis and ER stress (Chen et al., 2019b).

In a study of how tagitinin C induces ferroptosis in colorectal cancer cells, researchers found that PERK was an important mediator of nuclear factor erythroid 2-related factor (Nrf2)/heme oxygenase-1 signaling pathway stimulation induced by Tagitinin C (Wei et al., 2021). Nrf2 can be directly phosphorylated by PERK (Cullinan et al., 2003). It has been shown that acrolein can induce ER stress, while pretreatment with a PERK inhibitor significantly inhibited ER stress in MIN6 cells, and the level of ferroptosis was also decreased, suggesting that acrolein-induced ER stress is closely related to ferroptosis in MIN6 cells. Additional co-immunoprecipitation and chromatin immunoprecipitation experiments demonstrated that CHOP regulated peroxisome proliferator-activated receptor γ (PPARγ). Pretreatment with rosiglitazone, a PPARγ inducer, alleviated ferroptosis in MIN6 cells induced by acrolein. PPARγ acted as an adapter between ER stress and ferroptosis (Zhang et al., 2022). In addition, rosiglitazone is known to reduce the expression of ACSL4, which appears to be responsible for inhibiting ferroptosis. In a study on liver injury, the ER stress pathway IRE1α-XBP1 induced the transcription of G protein alpha subunit 12, thereby inhibiting the expression of miR-15a. mir-15a directly interacted with the 3′ untranslated region of arachidonate 12-lipoxygenase (ALOX12) mRNA, thus inhibiting ALOX12. ALOX12 can directly promote ferroptosis (Tak et al., 2022). In another pathway of ER stress, as part of its role in lipid metabolism, ATF6 increases the transcriptional output of PLA2G4A—an enzyme of cytosolic phospholipase A2 group IV family—directly activating AA metabolism (Zhao et al., 2022).

The link between ferroptosis and ER stress has been demonstrated in IRI models. In a myocardial injury model caused by intermittent hypoxia, ferroptosis occurs along with the activation of ER stress, and the interaction of the two may be related to NADPH oxidase 4 and ROS production (Huang et al., 2022a). Irisin upregulated the expression of GPX4 in renal IRI and also decreased the levels of ER stress-related pathway proteins (GRP78, phospho-IRE1α, p-eIF2α, CHOP), while the GPX4 inhibitor RSL3 reversed the regulation by irisin of ER stress (Zhang et al., 2021). In diabetic myocardial reperfusion injury, ferroptosis was accompanied by activation of the ATF4-CHOP pathway, and ROS produced during ferroptosis can trigger ER stress. Inhibiting ferroptosis in a rat model of diabetic myocardial IRI reduced ER stress and myocardial injury (Li et al., 2020b).

A previous study confirmed that CHOP may bind to forkhead box protein O3a and induce the expression of p53 upregulated modulator of apoptosis in neurons (Ghosh et al., 2012). P53 is one of the inducers of ferroptosis (Jiang et al., 2015). Using melatonin as a treatment for traumatic brain injury, hemin-induced ferroptosis and ER stress were attenuated via circPtpn14/miR-351-5p/5-LOX signaling in bEnd3 cells. A further study showed that hemin-induced ER stress could be alleviated by melatonin and Fer-1 by downregulating CHOP and GRP78, and 5-LOX may be another intermediate station between ER stress and ferroptosis (Wu et al., 2022a). In research on intracerebral hemorrhage, it has been found that treatment with Se increases GPX4 expression as well as ATF4 expression (Alim et al., 2019). The Compound Tongluo Decoction simultaneously inhibits ER stress and ferroptosis caused by cerebral ischemia in rats and promotes angiogenesis in rats with cerebral infarction (Hui et al., 2022). In another study, microglial BV2 cells were simultaneously protected against hypoxia-induced ferroptosis and ER stress after administration of wild bitter melon extract (Lin et al., 2022). Disruption of intracellular calcium homeostasis in BV2 promotes harmful calcium-iron cross-talk and ER stress responses, which both increase ROS production and lead to ferroptosis and excessive inflammatory cytokine release in BV2 cells (Wu et al., 2022b). In mouse hippocampal HT22 cells, pharmacological inhibition of the system Xc^– by erastin caused ER stress through activation of the IRE1 pathway (Hirata et al., 2019). These results indicate that there are many interactions between ferroptosis and ER stress, and more precise research on their regulation and interactions in ischemic stroke is needed. An overview of interactions between ferroptosis and ER stress is given in Figure 2.

Figure 2.

Overview of interaction between ferroptosis and endoplasmic reticulum stress.

Created using Microsoft PowerPoint 2019. AA: Arachidonic acid; ALOX12: arachidonate 12-lipoxygenase; ART: artesunate; ATF: activating transcription factor; CHOP: C/EBP-homologous protein; eIF2α: eukaryotic translation initiation factor 2α; Gα12: G protein subunit alpha 12 GPX4: glutathione peroxidase 4; GRP78: glucose-regulated protein 78; HO-1: heme oxygenase-1; IRE1: inositol requirement protein 1; miR-15a: microRNA -15a; Nrf2: nuclear factor-E2-related factor 2; PERK: protein kinase RNA-like ER kinase; PLA2G4A: phospholipase A2 group IVA; PPARγ: peroxisome proliferator-activated receptor; Se: selenium; XBP1: homeostasis transcription factor X-box protein 1; 5-LOX: 5-lipoxygenase.

Therapeutic agents targeting ferroptosis after ischemic stroke and its function in endoplasmic reticulum stress signaling

Monoterpene phenol carvacrol reduced the expression of GRP78, ATF-6, PERK, IRE1α, and CHOP genes, corrected the rate of misfolding due to λ-cyhalothrin in the ER, and protected the liver and kidneys from damage (Ileriturk and Kandemir, 2023). Monoterpene phenol carvacrol is also thought to inhibit ferroptosis in cerebral IRI in gerbils, thereby demonstrating neuroprotective effects (Guan et al., 2019). By silencing GPX4, carvacrol’s protective effect was driven by increasing Gpx4 expression in hippocampal neurons. Moreover, the upregulated expression of ferroportin 1 and the downregulated expression of TFR in the brains of gerbils treated with carvacrol showed that carvacrol treatment could reduce iron overload in the hippocampus and cortex. Similarly, galangin, a Chinese herb, was shown to increase GRP78 and CHOP expression and induce the release of calcium stores from the ER (Su et al., 2013); it has been shown to inhibit ferroptosis and improve cognitive impairment and hippocampal neuronal damage in gerbils after ischemic stroke by inhibiting the SLC7A11/GPX4 signaling pathway. When knocking down SLC7A11 with small interfering RNA, the inhibition of ferroptosis by galangin was no longer seen (Guan et al., 2021). Baicalin could inhibit ferroptosis in cerebral IRI by increasing the expression of GPX4 and ACSL3 and inhibiting ACSL4 (Li et al., 2022b). Pretreatment with baicalin before H₂O₂ stimulation could significantly limit the expression of GRP78 and CHOP and inhibit ER stress (Cao et al., 2018).

Nrf2 has received attention as a key regulator of ferroptosis after ischemic stroke. Another ingredient used in traditional Chinese medicine that can treat ferroptosis after stroke is rehmannioside A. In SH-SY5Y cells exposed to H₂O₂ and in a rat model of cerebral ischemia, rehmannioside A could regulate the phosphatidylinositol 3-kinase/Nrf2 signaling pathway and enhance the expression of SLC7A11 and GPX4 proteins, which inhibited ferroptosis (Fu et al., 2022). Moreover, rehmannioside A inhibited ER stress in the hippocampus in a rat model of depression (Yuan and Yuan, 2022). One study confirmed that an important bioflavonoid, kaempferol, which has been detected in many vegetables, fruits, and medicinal plants, inhibited lipid peroxidation accumulation and significantly reversed ferroptosis in OGD/R-treated neurons via the Nrf2/SLC7A11/GPX4 signaling pathway (Yuan et al., 2021). Kaempferol also inhibited high-fat diet-induced ER stress in mouse hepatocytes (Xiang et al., 2021).

β-Caryophyllene (BCP) is a natural dicyclosesquiterpene found in essential oils that have beneficial pharmacological effects on a number of diseases. A significant increase in Nrf2 nuclear translocation and activation of the Nrf2/heme oxygenase-1 pathway was observed following BCP treatment, preventing ferroptosis and improving infarct volume, neurological scores, and pathological features after MCAO (Hu et al., 2022). BCP is also an inhibitor of ER stress (Xu and Yan, 2021). Icariside II, a natural flavonoid derived from the epimedium plant, can directly bind to Nrf2 and promote GPX4 transcription, thereby inhibiting ferroptosis after cerebral IRI (Gao et al., 2023), and all three branches of UPR signaling can be activated by icariside II, leading to apoptosis (Tang et al., 2022). Similarly, the inhibitory effects of propofol on ferroptosis in cerebral IRI also target the Nrf2/GPX4 pathway (Fan et al., 2023). Different concentrations of propofol showed protective roles in hypoxic neurons by regulating the expression of the ER residential chaperone GRP78 (Bu et al., 2020). The neuronal damage caused by the ER stress inducer amylin could also be partially rescued by propofol (Wang et al., 2014).

Se is necessary for the biosynthesis of GPX4. Tuo et al. (2021) analyzed the anti-ferroptotic activity of a number of inorganic and organic Se compounds. In vitro, these compounds all increased GPX4 protein levels and showed anti-erastin-induced ferroptosis properties. In vivo, methylselenocysteine was the most potent compound for preventing the neuronal loss and functional impairments after IRI. High Se concentrations also induced the expression of several key markers such as ATF4 and CHOP in endothelial cells in a time- and dose-dependent manner, leading to endothelial dysfunction through ER stress-mediated cell death mechanisms (Zachariah et al., 2021). The ferroptosis inhibitors rosiglitazone and DFO, which protect against cerebral IRI, also contribute to ER stress. The Iron chelating agent DFO induced apoptosis of human gastric cancer cells by activating the ER stress pathway (Kim et al., 2016), whereas rosiglitazone reduced the expression of GRP78 and CHOP through the PPARγ-dependent signaling pathway, thereby inhibiting ER stress and protecting human neural stem cells (Lin et al., 2018a). Potential compounds that target ferroptosis for the treatment of ischemic stroke and their role in ER stress are summarized in Table 1.

Table 1.

Potential compounds targeting ferroptosis and endoplasmic reticulum stress

Compound	Target of action in compound	Response target in ER stress
Carvacrol	GPX4	ATF-6, PERK, IRE1α, GRP78
Kaempferol	Nrf2/SLC7A11/GPX4 axis	ATF-6, PERK, IRE1α, GRP78
Galangin	SLC7A11/GPX4 axis	GRP78/CHOP
Rehmannioside A	PI3K/Nrf2 and SLC7A11/GPX4 axis	ATF-6, ATF-4, XBP1
DFO	TFR	ATF-6, GRP78, CHOP
BCP	Nrf2/HO-1 axis	ATF-4, GRP78, CHOP
Se	Gpx4	ATF-4, GRP78, CHOP
ICS II	Nrf2/GPX4 axis	PERK, IRE1α, ATF6
Propofol	Nrf2/GPX4 axis	GRP78
Baicalin	GPX4, ACSL3 and ACSL4	GRP78/CHOP
Rosiglitazone	ACSL4	GRP78/CHOP

ACSL3: Acyl-coenzyme A synthetase long-chain family member 3; ACSL4: acyl-coenzyme A synthetase long-chain family member 4; ATF: activated transcription factor; BCP: β-caryophyllene; CHOP: C/EBP-homologous protein; DFO: deferiprone; GPX4: glutathione peroxidase 4; GRP78: glucose-regulated protein 78; HO-1: heme oxygenase-1; ICS II: Icariin II; IRE1α: inositol-requiring enzyme 1α; Nrf2: nuclear factor-E2-related factor 2; PERK: protein kinase RNA-like endoplasmic reticulum kinase; PI3K: phosphatidylinositol 3-kinase; Se: selenium; SLC7A11: recombinant solute carrier family 7, member 11; TFR: transferrin receptor 1; XBP1: X-box protein 1.

Perspectives

Our understanding of the mechanisms of ferroptosis in ischemic stroke have been summarized in this review article. We also reviewed the role of ER stress in ischemic stroke and its possible interactions with ferroptosis. Ferroptosis can cause ER stress through various pathways, followed by apoptosis, which in turn positively regulates ferroptosis, forming a positive feedback loop. However, the entire picture is far more complicated; there is also complex crosstalk between ferroptosis, necroptosis (Zhou et al., 2021), and oxidative stress (Ren et al., 2021). The massive network formed by the interaction of different death patterns in nerve cells after ischemic stroke still requires further research. Taking the complex pathophysiological changes after ischemic stroke as a whole, research should focus on finding targets to break or inhibit the function of this network. The molecular mechanisms of the interactions and regulation of ferroptosis and other cell death patterns also require further exploration.

There are still many unanswered questions. For example, it remains unknown whether all three major ferroptosis signaling pathways are present in ischemic stroke, and if so, if all of them are equally important. This review did not find any studies that focused on this question. Protective benefits have been shown in mice with early hemorrhagic brain injury from the oral administration of (-)-epicatechin (Chang et al., 2014), and dauricine can protect against intracerebral hemorrhage-induced disruptions (Li et al., 2020a) by inhibiting ferroptosis. However, whether the neuroprotective effects of (-)-epicatechin (Shah et al., 2010) and dauricine (Pu et al., 2018) on ischemic stroke also involve inhibition of ferroptosis pathways remains unknown. The relationship between the regulation of ferroptosis and age also requires further study. Research has attested that extracellular signal-regulated kinases 1/2, an important marker of ferroptosis, was increased in heme-treated astrocytes (Regan et al., 2001), and another study showed that, in vivo, GPX4 was localized to the nucleus of oligodendrocytes; the inhibition of GPX4 could induce ferroptosis in oligodendrocytes (Fan et al., 2021). After stroke, it is not clear if ferroptosis is regulated differently in different neuronal types. The relationship between nerve cell interactions and ferroptosis after stroke is also worthy of further investigation. Another open question is whether the cross-talk pathways between ferroptosis and ER stress in ischemic stroke are consistent with existing studies in other diseases. Intermediate interactions between the two in ischemic stroke need further study.

In summary, neuronal ferroptosis and ER stress may occur after ischemic stroke, and inhibiting ferroptosis or ER stress after ischemic stroke may prevent the death of neurons. Moreover, there may be mutual interactions between neuronal ferroptosis and ER stress after ischemic stroke.