The role of ACSL4 in stroke: mechanisms and potential therapeutic target

Abstract

Stroke, as a neurological disorder with a poor overall prognosis, has long plagued the patients. Current stroke therapy lacks effective treatments. Ferroptosis has emerged as a prominent subject of discourse across various maladies in recent years. As an emerging therapeutic target, notwithstanding its initial identification in tumor cells associated with brain diseases, it has lately been recognized as a pivotal factor in the pathological progression of stroke. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is a potential target and biomarker of catalytic unsaturated fatty acids mediating ferroptosis in stroke. Specifically, the upregulation of ACSL4 leads to heightened accumulation of lipid peroxidation products and reactive oxygen species (ROS), thereby exacerbating the progression of ferroptosis in neuronal cells. ACSL4 is present in various tissues and involved in multiple pathways of ferroptosis. At present, the pharmacological mechanisms of targeting ACSL4 to inhibit ferroptosis have been found in many drugs, but the molecular mechanisms of targeting ACSL4 are still in the exploratory stage. This paper introduces the physiopathological mechanism of ACSL4 and the current status of the research involved in ferroptosis crosstalk and epigenetics, and summarizes the application status of ACSL4 in modern pharmacology research, and discusses the potential application value of ACSL4 in the field of stroke.

Graphical abstract

Introduction

Globally, stroke remains the second-leading cause of death and has been one of the paramount health concerns worldwide [1]. According to the 2019 Global Burden of Diseases (GBD) survey, ischemic stroke accounted for 62.4% of all incident strokes, while cerebral hemorrhage accounted for 27.9%, and subarachnoid hemorrhage (SAH) comprised 9.7% [2]. With the rapid growth of the elderly population and the escalating disease trajectory among younger cohorts, the incidence and fatality rates of stroke have risen sharply in recent years. This trend has worsened the socioeconomic burden, especially in low-income countries [3–5]. Given the stringent time constraints associated with thrombolysis in ischemic stroke and the dearth of efficacious treatments for hemorrhagic stroke, there exists a compelling imperative to explore formidable neuroprotective agents for stroke [6–8]. This requires a deeper understanding of the pathological mechanism of stroke.

At present, oxidative stress, inflammatory reaction, excitotoxicity, and apoptosis are recognized as pivotal contributors to the pathogenesis of cardiovascular and cerebrovascular diseases, including cerebral stroke [9]. As an emerging pathological finding, ferroptosis has attracted significant attention within the realm of stroke research. Since normal brain metabolism necessitates iron, any perturbation in iron homeostasis will affect normal brain function [10]. Ferroptosis, a recently unearthed form of programmed cell death, is characterized by lipid peroxidation and glutathione depletion, embodying a distinctive mode of oxidative stress-induced cell demise [11]. Although the specific mechanism of ferroptosis remains elusive, it is widely known for its involvement in the development of cardiovascular and cerebrovascular diseases and cancer [12, 13]. Researchers have been focusing on preventing and treating stroke by inhibiting iron accumulation or ferroptosis in recent years [14].

Acyl-CoA synthetase long-chain family member 4 (ACSL4) is involved in many routines and abnormal physiological processes and is also an essential enzyme in ferroptosis lipid peroxidation [15]. According to the results of the bibliometric analysis of the ferroptosis frontier articles, ACSL4 is one of the most frequently used terms [16]. This result shows that ACSL4, as a sensitive ferroptosis monitor, may provide new therapeutic ideas for ferroptosis-related diseases. Lipid peroxidation is a direct factor in triggering ferroptosis. Polyunsaturated fatty acids (PUFAs) are the main target of peroxidation [17]. ACSL4 is a key enzyme that catalyzes the metabolic pathways of PUFAs, including arachidonic acid (AA). It primarily regulates the metabolism of steroids and AA, thereby influencing the occurrence of lipid peroxidation [18]. A key step in ACSL4-mediated ferroptosis is the incorporation of PUFAs into phospholipids. Since brain tissue is rich in PUFAs, particularly AA, it is evident that targeting ACSL4 to inhibit ferroptosis is an effective approach to alleviate brain injuries, including strokes [19]. Therefore, the specific inhibitors targeting ACSL4 represent the primary pharmacological strategy for suppressing lipid peroxidation to combat ferroptosis. Developing drugs that can efficiently and safely inhibit ACSL4 will offer new support for stroke treatment. Currently, researchers have found evidence of targeting ACSL4 to inhibit ferroptosis in ferroptosis inhibitors, existing clinical treatments, and traditional herbal components [20–22].

The occurrence of ferroptosis exacerbates brain damage following a stroke. Mitigating ferroptosis and lipid peroxidation after a stroke holds significant clinical value for its treatment [23]. Thus, the pivotal role of ACSL4 in lipid metabolism and ferroptosis makes it a potential target for stroke therapy. Additionally, ACSL4 can trigger neuroinflammation and ferroptosis, suggesting that targeting ACSL4 may alleviate post-stroke inflammatory damage and cell death [24]. Pharmacological and genetic approaches have thus emerged as therapeutic strategies for targeting ACSL4 inhibition. Currently, drug development targeting ACSL4 remains in its early stages. The functions of ACSL4 within cells are complex and diverse, and its interactions with other molecular pathways and regulatory mechanisms are not yet fully understood. This complexity presents significant challenges for the clinical application of ACSL4-targeted therapies. Gene therapy offers high precision in regulating ACSL4 gene expression, and researchers have identified some epigenetic regulatory targets of ACSL4. However, gene therapies related to ACSL4 have yet to be explored in the field of stroke treatment. Despite the research challenges in achieving clinical translation, targeting ACSL4 remains a highly promising therapeutic strategy for stroke treatment.

This paper endeavors to consolidate the physiological role of ACSL4 and its participation in the pathological progress of ferroptosis, drawing upon extant literature. Additionally, it seeks to elucidate the extant pharmacological investigations concerning ACSL4 in the realm of stroke, with the aim of steering future clinical pharmacotherapeutic interventions and prognostic research endeavors in the field of stroke management.

Physiological function of ACSL4

The mammalian Acsl family, the enzyme encoded by a multigene family, is significant in lipid metabolism in vivo and is composed of five members (Acsl1, Acsl3, ACSL4, Acsl5, and Acsl6), which have different tissue distribution and substrate specificities. ACSL activates fatty acid (FA) for intracellular metabolism [25]. Fatty acid metabolism involves esterification and activation. Acsl enzymes are essential in humans because long-chain FA are ubiquitous in the diet and are preferentially converted to acyl-CoA by these enzymes [26]. The isoforms of ACSL activate different long-chain FA into different metabolic pathways [27].

Kang et al. identified different Acsl types from previous findings in 1997 and named ACSL4 [28]. The human ACSL 4 gene is located on chromosome Xq22.3-q23, the length is approximately 90 kb [29]. ACSL4 is present in peroxisomes, mitochondria, and endoplasmic reticulum and is widespread in ovary testis, seminal vesicles, brain tissues, and many other tissues [30]. ACSL4 consists of luciferase-like regions 1 and 2, a linker connecting the two luciferase-like regions, an NH₂ terminus, and a COOH terminus. The absence of 50 amino acids corresponding to NH₂ may be the reason ACSL4 exhibits different fatty acid specificities. The FA specificity of ACSLs is influenced by multiple factors, such as the physicochemical properties of FA, acyl-CoA binding proteins, intracellular proteins, and FA-binding proteins [28]. Furthermore, a study suggests that FA recognition requires a FA characteristic motif composed of a set of 25 amino acids. Altering specific amino acid residues can modify ACSL4’s preference for long-chain polyunsaturated fatty acids [31]. Function of the ACSL4 includes the transport of cholesterol from the endoplasmic reticulum to mitochondria, intracellular lipid storage, and regulation of AA and its metabolites [32].

ACSL4 plays a pivotal role in lipid metabolism processes. ACSL4 links long-chain FA with Coenzyme A to form fatty acyl-coenzyme A (FA-CoA), which enters the lipid synthesis pathway, producing phospholipids (PL), triacylglycerol (TG), and cholesterol esters (CE). ACSL4 also participates in the process of FA β-oxidation, facilitating the catabolism of FA. The β-oxidation reaction is a lipid metabolic pathway, and ACSL4’s ability to bind free FA with CoA enables these FA to enter the mitochondria and participate in β-oxidation [33]. As a member of the ACSL family, ACSL4 can catalyze a variety of PUFAs preferentially and is regarded as a critical isoenzyme for PUFAs metabolism [34, 35]. PUFAs can modulate inflammation and immune responses [36]. Among the numerous PUFAs, ACSL4 exhibits a predilection for recognizing AA and eicosapentaenoic acid (EPA) as substrates [37]. The synthesis of AA-CoA by ACSL4 is the first step in incorporating arachidonic acid (AA) into PL. In other words, ACSL 4 combines free AA and Coenzyme A (CoA) in the endoplasmic reticulum, oxidizing them to form the derivative AA-CoA, which subsequently undergoes esterification into membrane phosphatidylethanolamine (PE) by lysophosphatidylcholine acyltransferase 3 (LPCAT3) [38]. This process allows long-chain PUFAs into the cellular lipid membrane, increasing the membrane fluidity [39, 40]. Moreover, AA incorporation into PL undergoes lipid peroxidation, producing toxic products such as 4-hydroxynonenals (4-HNE) and malondialdehydes (MDAs) [41]. ACSL4 ablation can lower oxygen consumption in adipocytes of high-fat diet (HFD) mice and prevent the harmful effects of elevated 4-HNE levels [32]. Lipid droplets are highly dynamic organelles composed of triglycerides (TG) and cholesterol esters, which buffer and store excess lipids. ACSL4-catalyzed FA-CoA participates in TG synthesis. LPCAT3 is a determinant of TG secretion. Research suggests that hepatic ACSL4 and LPCAT3 regulate plasma TG metabolism through the activation of PPARδ [42]. A study has shown that in high-fat diet (HFD) model, elevated expression of ACSL4 leads to increased TG levels in the liver, while inhibiting ACSL4 improves hepatic steatosis and fibrosis [43]. Another characteristic of ACSL4 is its role in regulating steroidogenesis. Acsl4 is a key enzyme involved in the regulation of steroid production through the release of AA, which is induced by steroidogenic hormones. Intracellular free AA are translocated into the mitochondrial compartment via the ACSL4/DBI/TSPO/ACOT2 pathway, subsequently enhancing steroidogenesis by regulating the steroidogenic acute regulatory protein (StAR) [44]. Moreover, ACSL4 deficiency can reduce the storage of intracellular CE, thereby diminishing steroidogenesis [45].

The ability of ACSL4 to regulate neural development and function by fatty acid metabolism is the result of ACSL4 being a gene involved in non-specific X-linked low intelligence. Diminished expression of ACSL4 may precipitate premature apoptosis of neuronal cells and disrupt brain development [46]. Beyond its involvement in lipid metabolism, ACSL4 serves as a crucial mediator of signal transduction and other cellular processes. ACSL4 regulation of cell growth, differentiation, and death provides a new perspective on preventing and treating cell death-related diseases [47]. Thus far, ACSL4 has been implicated in the pathological processes of various diseases, including neurodegeneration, brain injury, and cancer. (Fig. 1).

Fig. 1.

The function of ACSL4. ACSL4 is involved in pathological progression of disease by regulating fatty acid metabolism and neuronal development as well as growth, differentiation, and death of cells

Ferroptosis

The initial recognition of ferroptosis stems from screening studies for small molecule compounds that selectively induce gene death in cancer cells with RAS mutations [48]. Ferroptosis, conceptualized by Dr. Brent R. Stockwell in 2012, distinguishes itself from apoptosis, necrosis, and autophagy in terms of morphology, biochemistry, and autophagy [49]. Morphologically, ferroptosis exhibits necrotic-like alterations characterized by compromised plasma membrane integrity, swelling of cytoplasmic components and organelles, and moderate chromatin condensation. Concurrently, mitochondrial morphology undergoes discernible changes, marked by diminished mitochondrial size, increased membrane density, and reduced cristae [50]. Ferroptosis, a regulated mode of cell death, is propelled by phospholipid peroxidation. Key features include iron accumulation, lipid peroxidation, and altered gene expression [40, 51]. The essence of ferroptosis is the depletion of glutathione (GSH), glutathione peroxidase (GPX 4) activity decreases, and lipid peroxides cannot participate in the gpx 4-catalyzed reduction reactions. As a result, excess Fe2+ accumulates inside the cell, leading to a significant production of ROS through Fenton reactions. This, in turn, intensifies oxidative damage [52–54].

Lipid peroxidation is one feature of ferroptosis. Lipid peroxidation of mitochondrial polyunsaturated membranes is an important source of ROS [55, 56]. In addition to the Fenton reaction, lipid peroxides from lipid autoxidation and oxidized PUFAs esterified by lipoxygenases (Loxs) contribute significantly to ROS accumulation. ACSL4 and lysophosphatidylcholine acyltransferase 3 (LPCAT3) also play important roles in this process [57]. The peroxidation reaction, driven by ROS and PUFAs on the cell membrane, destabilizes the lipid bilayer. This destabilization leads to membrane disintegration, ultimately promoting ferroptosis [58]. Consequently, iron-dependent lipid peroxidation emerges as a critical facet of ferroptosis. Inhibiting lipid peroxidation to impede the progression of ferroptosis represents a novel pharmacological strategy for disease diagnosis and treatment.

Redox system is an important way to defence ferroptosis. Currently, four antioxidant pathways have been elucidated in relation to ferroptosis: The system Xc-GPX4, NAD(P) H/ferroptosis suppressor protein 1 (FSP1)/coenzyme Q10 (CoQ10), GTP cyclohydrolase 1 (GCH1)/tetrahydropterin (BH4)/dihydrofolic acid reductase (DHFR), and the dihydroorotate dehydrogenase (DHODH)-CoQ10 [59]. Among them, the SLC7A11-GSH-GPX 4 axis is the main pathway against ferroptosis. GSH can mediate the reduction of phospholipid hydrogen peroxide (PL-OOH) to (PL-OH) through GPX 4, thereby mitigating the accumulation of lipid peroxides and inhibiting ferroptosis [60]. The NAD(P)H/FSP1/CoQ10 system is an antioxidant system independent of the GPX 4 pathway, and FSP 1 is a NAD (P) H-dependent oxidoreductase that defends ferroptosis by reduced ubiquinone in the absence of GPX 4 [61, 62]. Furthermore, the DHODH-CoQH 2 system is another newly discovered ferroptosis defense mechanism localized within mitochondria and independent of the glutathione peroxidase 4 (GPX4) pathway. This pathway counteracts the effects of lipid peroxidation by reducing CoQ10 in the inner mitochondrial membrane [63]. GCH 1 inhibits ferroptosis through its metabolites dihydrobiopterin (BH2) and BH4 [64, 65]. BH4, as a lipophilic radical trapping antioxidant (RTA), prevents ferroptosis by inhibiting lipid peroxidation through the action of DHFR [66].

As a pivotal regulatory mechanism in stroke, ferroptosis impacts cell survival and engages in crosstalk with other pathological processes [67] (Fig. 2).

Fig. 2.

The mechanism of ferroptosis. The Xc-system composed of SLC7A11 and SLC3A2 ingests cystine, which is further converted to cysteine, and GSH is oxidized to oxidized glutathione (GSSG) in response to GPX 4. GSH mediates the reduction of phospholipid hydrogen peroxide (PL-OOH) to (PL-OH) through GPX 4, which alleviates the accumulation of lipid peroxides and inhibits ferroptosis. Inactivation of GPX 4 enzymes (e.g., RSL 3) or GPX4 will cause accumulation of lipid peroxides, further leading to an increase in ROS. Fe3 + enters the cell through the ferroportin receptor (TFR 1) and subsequently transforms into Fe2 + in the endosome. Then, Fe2 + accumulation produces a fenton response, leading to ROS accumulation and triggering ferroptosis. Additionally, Fe2 + can elevate levels of lipid ROS by catalyzing the conversion of phospholipids (PUFA-PL) to PLOO·. In addition, ACSL4/LPCAT3/ 15-LOX-dependent enzymatic reaction is another way to produce lipid peroxides. Moreover, ACSL4 can induce ferroptosis by mediating GPX 4 inhibition. Alternatively, FSP1-CoQ10, GCH1-BH4, and DHODH-CoQ10 pathways inhibit ferroptosis independent of the system Xc-GPX4. Abbreviations: GSH glutathione; GSSG oxidized glutathione; GPX4 glutathione peroxidase 4; RSL3 RAS-selective lethal 3; TFR1 transferrin receptor 1; ACSL4 acyl-CoA synthetase long-chain family member 4; LPCAT3 lysophosphatidylcholine acyltransferase 3; 15-LOX acid-15-lipoxygenase; PL phospholipid; PL-OOH phospholipid hydroperoxides; PL-OH phospholipid hydroxides; CoQ10 coenzyme Q10; FSP1 ferroptosis suppressor protein 1; CoQ10H2 ubiquinol; BH2 dihydrobiopterin; BH4 tetrahydrobiopterin; DHODH the dihydroorotate dehydrogenase; DHFR dihydrofolic acid reductase; ROSI Rosiglitazone; NBP Dl-3-n-Butylphthalide; As-IV Astragaloside IV; BCP β-caryophyllene; DHM Dihydromyricetin

ACSL4 in ferroptosis

ACSL4 has been unequivocally established as a pivotal regulator of cellular ferroptosis. ACSL4, a critical biomarker and driving factor of ferroptosis, resists ferroptosis by changing the lipid structure. For resistance to lipid peroxidation, ACSL4 knockout cells showed the same ability to resist ferroptosis. Unlike GPX 4, ACSL4 positively regulates ferroptosis. Furthermore, re-expression of ACSL4 causes GPX 4-inactivated cells to undergo lipid peroxidation and ferroptosis [68]. The PL containing PUFAs induces cell ferroptosis by bursting the cell membranes. ACSL4 is a dependent regulation of PL [69]. Ferroptosis leads to the degradation of most membrane PL, consequently upregulating various oxygenic PL species. Apart from the Fenton reaction, the ACSL4/LPCAT3/acid-15-lipoxygenase (15-LOX)-dependent enzymatic pathway represents an alternative route for generating lipid peroxides [70]. Hence, ACSL4 promotes cell demise by augmenting lipid peroxidation. Notably, the sensitivity of ferroptosis can be modulated by supplementing arachidonic acid (AA) or other PUFAs, while inhibition of ACSL4 and LPCAT3 activity offers a potential strategy for inhibiting ferroptosis.

Acyl-Coenzyme A synthase catalyzes the formation of PUFA-CoA and subsequently esterifies PUFAs into PL. Recent studies have indicated that PKCβII induces ferroptosis by amplifying lipid peroxides through phosphorylation and activation of ACSL4 [71]. Golgi stress triggers the production of reactive oxygen species (ROS) to promote ferroptosis, and ACSL4 knockout can mitigate this process, shielding cells from the threat of ferroptosis [72]. RAS-selective lethal 3 (RSL3) can react with nucleophilic amino acid residues on GPX 4, resulting in GPX4 inactivation and subsequent induction of ferroptosis [73]. Additionally, ACSL4 is implicated as the primary contributor to the heightened sensitivity to ferroptosis mediated by RSL3 [74]. However, the mechanism underlying ACSL4-RSL3-mediated plasma membrane lipid peroxidation in ferroptosis remains uncertain [47]. Hypoxia-inducible factor-1α (HIF1-α), a transcriptionally active nuclear protein, has recently emerged as a regulator of stroke through various mechanisms [75]. Moreover, HIF-1α negatively modulates ACSL4 expression by binding to the ACSL4 promoter, thereby inhibiting ferroptosis. This discovery holds promise as a novel therapeutic avenue for stroke [76]. ACSL4, GPX4, and cystine/glutamate transporter-solute carrier family 7 member 11 (SLC7A11) are all implicated as drivers of ferroptosis induction, with ACSL4 being an essential component for GPX4-induced ferroptosis. Furthermore, ACSL4 induces ferroptosis by mediating GPX4 inhibition and exhibits greater resistance compared to SLC7A11 inhibition. Nonetheless, ACSL4 is not the sole pathway for ferroptosis induction. A study noted the critical role of arachidonate 12-lipoxygenase (ALOX12) in P53-mediated ferroptosis, presenting an alternative pathway for ACSL4-independent ferroptosis. This pathway may offer a promising therapeutic approach for ferroptosis in cancer cells [77, 78].

Stroke initiates a cascade of detrimental events that disrupt cellular homeostasis and cause cell demise through various pathways, leading to significant damage to neurons, endothelial cells, and glial cells. Notably, glial cell activation exerts influence on neuronal ferroptosis by modulating iron homeostasis, lipid metabolism, and oxidative stress. Previous studies have indicated that iron transporters and related proteins involved in iron metabolism are expressed in neuroglial cells, especially in microglia [79].

The hallmark of ferroptosis is iron-dependent lipid peroxidation, and the critical role of Acsl4 in lipid peroxidation and AA metabolism positions it as an important biomarker for ferroptosis. In recent years, numerous studies have indicated that inhibiting ACSL4 exhibits significant anti-ferroptotic effects. Targeted therapies against ACSL4 hold broad implications for various diseases, particularly ischemia–reperfusion (I/R) injury, central nervous system disorders, and cancer.

Ischemia reperfusion (I/R): I/R causes organ damage through intense inflammatory and oxidative stress responses. Following I/R, ferroptosis and upregulation of ACSL4 can be observed in various organs, including the intestines [80], liver, and kidneys [81]. Therefore, targeting ACSL4 inhibition can, on one hand, reduce cell death and inflammatory responses caused by I/R, and on the other hand, alleviate organ damage induced by ferroptosis [82].

Central Nervous System Diseases: Ferroptosis has been shown to be associated with neuronal damage, abnormal brain discharges, and progressive death of motor neurons [27]. Therefore, inhibiting ACSL4 can protect neural cells by counteracting ferroptosis-induced neurodegeneration and neuroinflammation, offering therapeutic potential for conditions such as stroke, spinal cord injury, epilepsy, and Parkinson’s disease [83–86]. Additionally, targeting ACSL4 to regulate the ferroptosis pathway may help reduce the accumulation of harmful byproducts like lipid peroxides and reactive aldehydes, thereby slowing neurodegeneration [87].

Cancer: ACSL4 is abnormally expressed in various cancers and is involved in lipid metabolism, ferroptosis, and immune responses through multiple pathways [69].This highlights the importance of targeting ACSL4 for cancer therapy. Additionally, inhibiting ACSL4 can help mitigate the disruption of redox balance caused by cancer cell metabolism. However, the role of ACSL4 in cancer is dual sided, depending on the cancer type and tissue environment. On one hand, inhibiting ACSL4 suppresses cancer cell growth, while on the other hand, high ACSL4 expression may contribute to cancer treatment [30]. Thus, determining whether inhibiting ACSL4 will ultimately benefit cancer treatment remains a significant challenge for researchers.

In conclusion, ACSL4’s critical role in ferroptosis positions it as a key player in various diseases. Therefore, targeting ACSL4 through pharmacological or genetic inhibition may offer a novel and specific strategy for treating ferroptosis-related conditions. Moreover, the development of targeted drug delivery systems is essential for enhancing therapeutic efficacy and minimizing systemic side effects. Studies have already shown that nanoparticle-based ACSL4-targeted drug delivery can effectively treat I/R injuries [81]. At the same time, opportunities and challenges coexist. FA are at the crossroads of anabolic and catabolic pathways. As a critical target influencing lipid metabolism, while leveraging ACSL4 in anti-ferroptosis strategies, consideration must also be given to its effects on non-ferroptosis pathways. Highly selective and specific ACSL4-targeted therapies may offer promising solutions for efficient diagnosis and treatment of diseases. Moreover, despite the demonstrated advantages of targeting ACSL4 in ferroptosis-related diseases, clinical translation has not yet been achieved, possibly due to off-target effects [88]. ACSL4 has garnered significant attention for its role in cancer prognosis. However, whether it can serve as a biomarker for the onset and prognosis of neurological diseases remains unclear. Therefore, a comprehensive understanding of ACSL4’s metabolic pathways and identifying the precise role of ferroptosis in specific diseases are crucial for treating a wide range of ferroptosis-related conditions.

Ferroptosis crosstalk

The pathological progression of disease frequently manifests complexity. Interactions often arise between ferroptosis and other pathological pathways involving cell demise or metabolic disturbances, due to shared molecular pathways. ACSL4, esteemed as a pivotal biomarker of ferroptosis, maintains close association with diverse forms of cell demise, encompassing autophagy, cuproptosis, apoptosis, and pyroptosis. This suggests hints at ACSL4’s significance as a key participant within the shared molecular pathways facilitating crosstalk between ferroptosis and other necroptotic pathways. Thus, as a signal transduction node of ferroptosis and other cell apoptosis pathways, ACSL4 may be involved in inducing the intersection of multicellular death pathways. Investigating the interplay between ferroptosis and other apoptotic pathways could yield combined therapeutic strategies, potentially identifying novel molecular targets to enhance neural recovery in stroke survivors.

ACSL4 in autophagy

Autophagy is a cellular mechanism wherein cells undergo self-degradation to eliminate damaged proteins or waste materials. Excessive autophagy may induce cell ferroptosis by influencing the generation of ROS and intracellular iron balance [89, 90]. Ferritin autophagy caused by ferroptosis results in the degradation of ferritin via the NCOA4 pathway, releasing iron and subsequently promoting ferroptosis [91, 92]. A previous study has revealed that human ACSL4 localizes to nascent autophagosomes and contributes to the regulation of autophagy [93].

The AMPK/mTOR pathway consistently participates in the upstream inhibition of autophagy formation, with AMPK demonstrating the ability to upregulate the expression of ACSL4, thereby inducing ferroptosis. Research has indicated that miR-130b-3p modulates the AMPK/mTOR autophagy pathway to suppress ferroptosis [94]. Sequestosome 1 recombinant protein stimulates the expression of ACSL4 via the AGER receptor, facilitating the production of PUFAs and mediating autophagy and ferroptosis [95]. In general, chaperone-mediated autophagy (CMA) degrades ACSL4 in lysosomes through recognition of the autophagic receptor HSC70, while glial maturation factor-β (GMFB) can induce lysosomal dysfunction to promote ferroptosis [96].

ACSL4 in cuproptosis

Cuproptosis, a novel form of cell demise delineated by Peter Tsvetkov et al. in 2022, arises from dysregulated copper metabolism. They indicate that copper accumulation leads to a loss of protein lipoylation function and blocks the tricarboxylic acid (TCA) cycle in the mitochondria. FDX1 serves as an upstream regulator of protein lipoylation and a pivotal modulator of cuproptosis [97]. Furthermore, copper accumulation instigates robust (ROS) generation, precipitating cellular demise [98]. Co-molecular pathways allow for a crosstalk between cuproptosis and ferroptosis. Previous studies noted that in addition to inducing ROS, the copper chelator elesclomol promotes SLC7A11 degradation, stimulating the oxidative stress response and ferroptosis in cancer cells [97, 99]. Another study indicated that copper ions directly bind to GPX 4 to induce the autophagic degradation of GPX 4 to promote ferroptosis in cancer cells [100]. Moreover, heightened ROS and ACSL4 levels in response to metal co-exposure (copper, iron, and zinc) stimulate lipid peroxidation, instigating ferroptosis in neuronal cells [101].

ACSL4 in apoptosis

Apoptosis is a mode of programmed cell death, which is an active cell death process occurring by the regulation of the corresponding genes to maintain internal environmental homeostasis. Apoptosis is a complex cascade reaction process mediated by various proteins, enzymes, receptors, and signaling pathways, including the caspase family, Blc-2 family, and tumor necrosis factor (TNF) receptor superfamily. The endogenous apoptosis pathway is induced by sensing the changes in the mitochondrial inner membrane caused by endogenous cell stress. Meanwhile, the exogenous pathway is activated by exogenous signaling [102, 103].

A study indicated that the inhibitory effect of the ferroptosis inhibitor LPT 1 on hepatocyte apoptosis suggests a potential link between ferroptosis and apoptosis [104]. Lipid peroxidation contributes to apoptosis by activating the apoptosis signaling pathway through its products [105]. In addition, ACSL4 has a bidirectional effect on apoptosis regulation. It can induce apoptosis by directly modulating the corresponding signaling pathways, while also promoting fatty acid oxidation to inhibit apoptosis [106, 107].

ACSL4 in pyroptosis

Pyroptosis is a pro-inflammatory programmed death dependent on Caspase-1. A study about the Co-occurrence of pyroptosis and ferroptosis provides opportunities for cancer treatment [108]. Moreover, ACSL4 significantly correlates with pyroptosis, apoptosis, and autophagy, but its internal mechanism remains vague [109].

Epigenetics regulation of ACSL4 in stroke

Epigenetics refers to a heritable and reversible regulatory pattern of gene expression influenced by environmental and disease factors, without altering the underlying nucleotide sequence. As a complex polygenic disease, stroke is often influenced by various lifestyle-related metabolic disorders. Studies have suggested an epigenetic involvement in the pathological mechanisms contributing to increased stroke risk. Epigenetics play a role in the pathological mechanisms that enhance stroke risk [110]. Furthermore, the differential expression of RNA at various stages of stroke appears to exert a positive impact on disease progression, diagnosis, and treatment [111]. ACSL4 serves as a critical factor in triggering ferroptosis-induced stroke. Understanding the epigenetic mechanisms may aid in identifying sensitive biomarkers and determining therapeutic approaches for ACSL4-induced stroke.

RNA methylation

RNA methylation has emerged as a significant epigenetic modification in recent years, encompassing over 60% of all RNA modifications, with N6-methyladenosine (m6A) being the most prevalent modification at the RNA level in higher organisms. The function of m6A is determined by the “writers,” “erasers,” and “reader” [112]. Studies have indicated an association between ferroptosis and m6A methylation modifications. Under conditions of cerebral ischemic hypoxia, levels of m6A modification were found to increase. This was accompanied by the downregulation of the Fat mass and obesity-associated protein (Fto), resulting in elevated m6A expression after the stroke. In addition, activating transcription factor (ATF) 3 is an important regulator of induced iron death after stroke [113, 114]. A recent study indicated the involvement of Fto in regulating the m6A levels of ACSL4. The interaction between Atf 3 and Fto results in the upregulation of Fto levels, thereby downregulating the m6A levels of ACSL4, inhibiting ferroptosis, and alleviating neuronal cell damage in ischemic stroke. Furthermore, m6A methylated binding protein Ythdf3 regulates ACSL4 levels by binding to the m6 A of ACSL4 [115].

Non-coding RNA regulation

Long non-coding RNA (lncRNA) exerts multifaceted regulation over gene expression. Studies have demonstrated that lncRNA can competitively bind RNA-binding proteins (RBP) to impede the binding of RBPs to downstream targets [116]. Upstream frameshift 1 (UPF1) stands as a pivotal regulatory component of RNA decay, with its activity modulated by lncRNA under specific circumstances [117]. Zheng et al. elucidated that UPF1 directly interacts with ACSL4 to facilitate the degradation of ACSL4 mRNA. Additionally, lncRNA HOX transcript antisense RNA (HOTAIR) upregulates ACSL4 expression levels by competitively binding to UPF1, thereby promoting the development of cerebral hemorrhage [118]. After brain injury, lncRNA H19 participates in the apoptosis and inflammatory processes of neuronal cells by interacting with distinct miRNAs [119, 120]. A previous study showed that H19 inhibits cerebral microvascular endothelial cells ferroptosis by regulating the miR-106b-5p/ACSL4 axis and may be a novel target for the treatment of intracerebral hemorrhage [121]. miRNAs efficiently regulate post-transcriptional gene expression. Various miRNA can downregulate ACSL 4 by binding to their 3′-UTR (untranslated region). Meanwhile, the differential expression of miRNA is involved in the process of brain injury, neuroprotection, and brain repair after stroke, and it is an important modulator of brain injury and repair mechanisms. Carme Gubern et al. observed that overexpression of miR-347 following middle cerebral artery occlusion induces neuronal apoptosis and indirectly modulates the upregulation of ACSL4 levels [107]. Circular RNAs serve as miRNA sponges, regulating cellular processes. Research indicates that miR-3098-3p inhibits the mRNA expression levels of ACSL4, while Circ-Carm 1 acts as a sponge for miR-3098-3p, positively regulating ACSL4 levels in the model of acute cerebral infarction [122].

ACSL4 ubiquitination

Ubiquitination refers to the process by which ubiquitin molecules categorize and select target protein molecules. This process is facilitated by a series of specific enzymes, including ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3, ultimately leading to the specific modification of these target proteins [123]. Ubiquitination is a critical mechanism for dynamically regulating programmed cell death. RNF146, an E3 ubiquitin protein ligase, plays a pivotal role in this process. A previous study has revealed the beneficial impact of Ring Finger Protein 146 (RNF146) in ischemic stroke, where activating transcription factor 3 (ATF3) activates transcription and regulates RNF146 expression, thereby mediating the ubiquitination and degradation of ACSL4 to inhibit ferroptosis and alleviate neuronal damage caused by ischemic stroke [124]. Additionally, Cytochrome P450 1B1 (CYP1B1), a member of the cytochrome P450 family, participates in the processes of cell death and angiogenesis [125]. Studies have indicated that the metabolite of CYP1B1, 20-HETE, induces the expression of E3 ubiquitin ligase FBXO10 through the activation of PKC signaling transduction, thereby promoting the ubiquitination of ACSL4 [126]. The E3 ubiquitin ligase MDM2 is involved in inhibiting ferroptosis through its ubiquitination of ACSL4. Predictions based on Ubibrowser data suggest that MDM2 is implicated in the regulation of ACSL4. MDM2 modulates ferroptosis by regulating the translation of ACSL4 protein [127]. In addition, it was recently found that the TCM prescription Tongqiao Huoxue Decoction also inhibited ferroptosis by promoting ACSL4 ubiquitination [128].

ACSL4 in stroke

To date, cellular ferroptosis has been found to be associated with various disease processes, including cancer, neurodegeneration, stroke, cardiomyopathy, and heart failure. Rather than existing in isolation, ferroptosis is typically intertwined with disease pathologies involving inflammation, oxidative stress, and autophagy [129]. The inhibition of ferroptosis, emerging as a treatment approach for stroke, has garnered significant attention. Recent studies have underscored the critical role of ferroptosis in the pathophysiology of stroke, highlighting its impact on neurological deficits through damage to the NVU [130, 131]. Furthermore, given that ACSL4 mediates the interplay between ferroptosis and fatty acid metabolism, it represents a focal point of research interest in stroke (Fig. 3).

Fig. 3.

The Timeline Roadmap of ACSL4 in Stroke. ACSL4 has been attracting attention since it was discovered as an important target of ferroptosis in 2015, and the application of ACSL4 in the field of stroke began to become epidemic after 2021

Ischemic stroke

Ischemic stroke, which is caused by local blood supply disorders in brain tissue for various reasons, leads to the necrosis of hypoxic lesions in brain tissue and then induces the clinical corresponding manifestation of neurological function [132, 133]. Present clinical strategies for managing ischemic stroke prioritize prompt reperfusion, encompassing intravenous thrombolysis and mechanical thrombectomy [134, 135]. Nevertheless, the applicability of these treatments is constrained by the time window limitations, rendering them unsuitable for all patients [136].

Ferroptosis is one of the main culprits involved in neuronal damage. Iron regulates the dynamic balance of brain tissue. After ischemic stroke, the destruction of BBB broke the original balance, and a large amount of ferritin and free iron entered the brain parenchyma through BBB [137]. Following ischemic stroke, disruption of BBB disturbs this equilibrium, allowing substantial influx of ferritin and free iron into the brain parenchyma [138]. Iron accumulation works together with ROS to promote lipid peroxidation and induce ferroptosis. In addition, brain iron accumulation in the aging process is also a “helper” to aggravate iron overload and thus expand the area of cerebral infarction [139]. ACSL4 is detectable in various bodily tissues, but only ACSL4 variant 2 appears to be confined to brain tissue, particularly in the hippocampus [140, 141]. The functional differences between the two variants still need to be apparent. Researchers have speculated that this feature may be related to its specific function [28, 142].

Upstream signaling pathways: In the early stages of ischemic stroke, ACSL4 expression is suppressed, with HIF-1α inhibiting its transcription by binding to the ACSL4 promoter region [143]. Activin A, a multifunctional cytokine produced by various immune cells and a member of the TGF-β superfamily, is upregulated following ischemic brain injury, promoting neuronal repair and functionally resisting ischemic damage [144]. Previous studies have found that Activin A exerts neuroprotective effects post-ischemic injury by inhibiting ferroptosis through enhanced Nrf2 expression and downregulation of ACSL4 protein levels [145]. Additionally, Cytochrome P450 1B1 (CYP1B1), a metabolic enzyme involved in arachidonic acid metabolism, is downregulated after brain ischemia [125]. Recent studies have shown that, in stroke models, CYP1B1 catalyzes the conversion of AA to 20-HETE. And CYP1B1 acts as a signaling molecule to activate the known PKC pathway, promoting the expression of FBXO10 and the degradation of ACSL4 [126]. This offers a potential explanation for neuroprotection following brain injury. Six hours after reperfusion, ACSL4 expression increases, with high levels detectable in all tissues. ACSL 4 mediates thrombin neurotoxicity. Thrombin, a serine protease, is one of the major drug targets for ischemic stroke. After acute cerebral I/R, thrombin is upregulated and stimulates ferroptosis signaling by promoting AA mobilization and the esterification of the ferroptosis-related gene ACSL4. ACSL4 knockout can restrain ferroptosis induced by RSL3, thereby protecting nerve cells. Therefore, targeting the thrombin-ACSL4 axis is crucial for therapeutically ameliorating neuronal injury during ischemic stroke [146]. The expression of ACSL4 is also regulated by SP1, which increases ACSL4 transcription by binding to its promoter region [147]. Under ferroptotic stimuli, such as cerebral ischemia, SP1 is upregulated [148]. Although some drugs protect against ischemic brain injury via the SP1/ACSL4 pathway, further research is needed to determine whether SP1 is directly involved in regulating ACSL4’s pathological mechanisms in stroke injury [149]. Additionally, influenced by epigenetic modifications, ACSL4 transcription in cerebral ischemia is also controlled by factors such as miR-484, RNF146, FTO, MDM2, YTHDF3, miR-347, and miR-3098-3p [124, 150].

Downstream targets: Currently, it has been found that ACSL4 participates in the pathological processes of stroke by regulating apoptosis mechanisms primarily through the synthesis and metabolism of lipid metabolites, thereby influencing cell survival and death. ACSL4 induces the esterification of PUFAs into phospholipids, which promotes neuronal death via ferroptosis during stroke damage. Furthermore, ACSL4-mediated ferroptosis exacerbates inflammatory responses in ischemic stroke. Microglia are a major contributor to neuroinflammation in ischemic stroke. Studies have shown that ACSL4 can enhance microglia-mediated inflammation. During ischemic stroke, excessive expression of ACSL4 in microglia leads to elevated mRNA levels of inflammatory cytokines TNFα, IL-6, and IL-1β, thereby promoting neuroinflammation [143]. Oxidative stress is a major factor contributing to brain tissue damage following ischemic stroke. GPX4, an antioxidant enzyme, protects cells from membrane lipid peroxidation and helps maintain redox homeostasis. A study highlighted that ACSL4 overexpression post-CIRI leads to increased lipid peroxidation, which in turn reduces GPX4 expression, inducing ferroptosis in neurons [128]. Furthermore, this study demonstrated that inhibiting ACSL4 could alleviate oxidative stress and ferroptosis caused by CIRI.

Intracerebral hemorrhage

Spontaneous intracerebral hemorrhage (ICH) is mainly caused by minor blood vessel diseases, such as hypertensive arteriopathy and cerebral amyloid angiopathy [151, 152]. Despite constituting a small proportion of all stroke subtypes and exhibiting a low incidence of complications, ICH carries the highest mortality rate among them. Surveys indicate that the one-month mortality rate for ICH can reach as high as 50% [153]. Secondary brain injury induced by ICH is the “executioner” of cell death, and neuronal ferroptosis plays an important role in this process [154]. BBB dysfunction is closely linked to the pathophysiological processes of ICH. After ICH, ruptured blood vessels hemorrhage allow blood to permeate the damaged BBB and infiltrate brain tissue. This process will trigger a cascade of neuronal cell damage, such as ferroptosis, and, in turn, aggravate ICH, forming a vicious cycle [155]. In addition, unlike ischemic brain injury, iron overload in hemorrhagic stroke mainly occurs in the brain. Hemoglobin undergoes cleavage and binds to transferrin (TF) in serum post-ICH, subsequently metabolizing into ferrous/ferric iron in the presence of microglia and macrophages. Ferrous ions instigate the excessive generation of lethal ROS, precipitating ferroptosis, damaging brain tissue, and compromising the integrity of the BBB [156]. Evidence of ferroptosis being involved in the pathological progression of ICH was first found by Li et al. Fer-1 has been identified as a potent inhibitor of ferroptosis following ICH [157].

Upstream signaling pathways: Since the discovery of ferroptosis’s role in ICH, researchers have diligently sought to unravel its mysteries. Recent investigations have unveiled an upregulation of ACSL4 expression in post-hemorrhagic hydrocephalus (HPP) rats [158]. LncRNA H19 was found to be highly expressed in the ICH model and induced ferroptosis in brain microvascular endothelial cells. ACSL4, as a microRNA (miR) target gene-106b-5p, will suppress the reversal effect of miR-106b-5p on ferroptosis and induce the dysfunction of brain microvascular endothelial cells. Thus, the knockdown of H19 can protect microvascular endothelial cells by miR-106b-5p/ACSL4 [121]. Another study harnessed SRY-box transcription factor 10 (SOX 10) to target ACSL4, enhancing the expression of miR-29a-3p. This intervention reduced ROS and divalent iron levels, while augmenting glutathione (GSH) and GPX4 levels, thus safeguarding hippocampal neuronal cells in the ICH model [159–161]. Paeonol (PAN), a natural compound derived from the root bark of Paeonia suffruticosa, has emerged as a potential inhibitor of cell ferroptosis [162]. The inhibition of the progression of ICH by PAN may be associated with its inhibition of the LncRNA HOTAIR/UPF1/ACSL4 axis, as found by Jin et al. [118].

Downstream targets: Following cerebral hemorrhage, the overexpression of ACSL4 regulates the production of ROS, contributing to oxidative stress and ferroptosis. Celastrol can alleviate oxidative stress after stroke and promote neurological recovery by binding to specific sites on ACSL4 [163]. Furthermore, studies indicate that ACSL4 rapidly increases after brain injury and catalyzes the elevation of ferritin levels in brain tissue [164]. Further exploration is needed to determine whether ACSL4 induces an increase in transferrin levels following cerebral hemorrhage.

Subarachnoid hemorrhage

Subarachnoid hemorrhage (SAH) and ICH are both classified as hemorrhagic strokes, thus sharing certain pathological similarities [165]. Early brain injury (EBI) stands as a pivotal factor triggering delayed cerebral ischemia and subsequent neurological dysfunction in SAH patients, profoundly influencing prognosis [166]. Similar to ICH, SAH entails the ingress of numerous ruptured erythrocytes into the subarachnoid space, swiftly elevating iron ion concentrations to catalyze the Fenton reaction, thereby instigating lipid peroxidation and subsequent ferroptosis [167, 168]. The initial observation of ACSL4 levels post-SAH revealed a gradual increase commencing at 12 h, peaking at 24 h post SAH. ACSL4 levels surged within the early window of EBI. However, the molecular pathways regulating ACSL4 levels after SAH remain unclear.

Downstream targets: ACSL4 overexpression contributes to EBI following SAH by exacerbating the expression of inflammatory factors, increasing ROS levels, and elevating brain water content, thereby participating in inflammation, oxidative stress, and brain edema associated with SAH [169]. Another study indicated that liproxstatin-1 treatment of SAH by inhibiting ferroptosis involves the downregulation of ACSL4 and cyclooxygenase-2 (COX-2) [170]. Puerarin intervenes in early neurological damage in SAH through antioxidative stress and ferroptosis and will reduce the ACSL4 levels [171, 172]. In conclusion, early intervention proves efficacious in SAH treatment. Given the lower global incidence of SAH compared to ICH, research progress on SAH and the exploration of ferroptosis mechanisms remain relatively insufficient [173, 174].

ACSL4-A potential therapeutic strategy for stroke

Stroke, a neurological disease characterized by a high degree of disability and mortality, has captured considerable attention within the medical field due to the limited availability of effective treatment options in clinical practice. Recent research have shed light on ferroptosis, an emerging form of cell death, which substantially influences the pathophysiology of stroke. Targeted therapeutic strategies aimed at ACSL4 have emerged as a promising avenue for exploring ferroptosis [175]. We provide an overview of the current methodologies targeting ACSL 4 for stroke, including ferroptosis inhibitors, novel findings in existing clinical treatments, herbal and natural components, and novel inhibitors. A comprehensive grasp of these treatment modalities is pivotal in steering future clinical approaches toward stroke management.

Antioxidant

To search for the post-stroke recovery mechanisms and the application of therapeutic drugs, we found that some inhibitors could suppress ferroptosis and reduce the injury caused by stroke by downregulating ACSL4 expression. Potent antioxidants are also excellent inhibitors of ferroptosis [176]. Notably, Lip-1 and Fer-1, potent ferroptosis inhibitors, act as free radical-scavenging antioxidants, underscoring the pivotal role of lipid auto-oxidation in thwarting ferroptosis [177]. Liproxstatin-1 is a proven specific ferroptosis inhibitor, primarily targeting lipid peroxidation during ferroptosis. Experimental research has found that Lip-1 inhibit ferroptosis via the SLC7A11/GSH/GPX4 axis and the downregulation of ACSL4, potentially serving as an efficacious therapeutic agent for CNS disorders [20]. In addition, recent research has affirmed that Fer-1 ameliorates cerebral ischemia–reperfusion injury through its anti-ferroptotic properties, primarily via the downregulation of ACSL4 gene expression, particularly when combined with hyperbaric oxygen therapy, thereby augmenting its therapeutic efficacy [178]. However, in the context of treating early brain injury (EBI), it has been observed that the anti-ferroptotic mechanisms of Lip-1 and Fer-1 do not involve ACSL4 [169]. Ferroptosis inhibitors have heralded remarkable breakthroughs in stroke therapy, yet drugs associated with these inhibitors have not yet transitioned into clinical application.

Clinical treatment

Although the precise pathological mechanisms underlying stroke remain unclear, certain drugs and therapies have exhibited potential in ameliorating post-stroke damage by targeting ACSL4 and ferroptosis (Table 1).

Table 1.

ACSL4 in clinical treatment

Drugs	Type	Target	Signaling	Mechanism	Type of Stroke
Rosiglitazone (ROSI)	Insulin-sensitizing agent	ACSL4	Inhibition of ACSL4	Inhibition of Ferroptosis Attenuate Lipid Peroxidation After Stroke	Cerebral ischemia reperfusion (I/R) [180]
Sevoflurane	Clinical anesthetic	Specificity protein 1 (SP1)	SP1/ACSL4 axis	Reduced intracellular iron accumulation	I/R [149]
Dl-3-n-Butylphthalide (NBP)	Recombinant tissue plasminogen activator (rtPA)	The Bax/Bcl-2 ratio, ACSL4, GPX4 and SLC7A11	Inhibit Cysteine-X-Cysteine chemokine receptor 4 (CXCR4)	Antiapoptosis; Decreased iron accumulation, lipid peroxidation levels, and ferroptosis	I/R [181]
Electroacupuncture (EA)	TCM therapy	ACSL4	Inhibit the proteins and mRNA expression of ACSL4, TFRC, 15-LOX and COX-2	Relieved the mitochondrial morphological changes and inhibited ROS Production	IS [183, 184]
Moxibustion	TCM therapy	ACSL4, malondialdehyde and SLC40A1	Glutathione (GSH)/GPX4) pathways	Regulate lipid metabolism; Reduce ACSL4 production; Decrease iron deposition; Inhibit the accumulation of reactive oxygen species	I/R [185, 186]
Zhuang Medicine Shuanglu Tongnao Formula	TCM therapy	ACSL4, TFR, FTH1, SLC7A11, SOD, GSH and MDA	SIRT1/Nrf2/GPx4 pathways	Reduced intracellular iron accumulation; Inhibit lipid peroxidation	IS [187]
Tongqiao Huoxue Decoction	TCM therapy	ACSL4	Promotes the ubiquitination-mediated degradation of ACSL4	Improves oxidative stress and inhibits the beginning of ferroptosis in cells	I/R [128]

ACSL4 Acyl-CoA synthetase long-chain family member 4; Bax Bcl-2-associated X protein; Bcl-2 B-cell chronic lymphocytic leukemia/lymphoma-2; GPX4 glutathione peroxidase 4; SLC7A11 Cystine/glutamate reverse transporter; TFRC transferrin receptor; 15-LOX acid-15-lipoxygenase; COX-2 cyclooxygenase-2; ROS reactive oxygen species; TCM traditional Chinese medicine; TFR transferrin receptor; FTH1 ferritin heavy chain 1; SOD superoxide dismutase; SIRT1 Silent information regulator 2 homolog 1; Nrf2 nuclear factor erythroid 2-related factor 2

Rosiglitazone (ROSI), a well-known agonist of peroxisome proliferator-activated receptor-γ, has been used in the clinical treatment of diabetes mellitus and can inhibit the ACSL4. ROSI attenuates ferroptosis by suppressing ACSL4, which prevents the reduction in glutathione peroxidase (GPx) activity and iron accumulation. This, in turn, lowers lipid peroxidation levels and facilitates the recovery of neurological function post-stroke [179, 180]. Sevoflurane is a commonly used clinical anesthetic, can inhibit the specificity protein 1 (SP1) in HT22 cells, and its postconditioning can downregulate the SP1/ACSL4 axis to inhibit ferroptosis, reducing neurological deficits and the infarct area in ischemic stroke [149]. Despite preclinical studies have suggested that repurposed drug candidates ROSI and Sevoflurane could target different pathways to inhibit ferroptosis, their targeted effects on stroke have not yet been clinically applied.

Additionally, DL-3-n-butylphthalide (DL-NBP) was approved in China for ischemic stroke treatment in 2002 and has been employed in stroke therapy for two decades. Several clinical studies have shown that DL-NBP ameliorates clinical symptoms and facilitates long-term recovery [21]. DL-NBP reverses the elevation of the ferroptosis marker ACSL4 and the decreasing trend of anti-apoptotic proteins GPX4 and SLC7A11 by mediating Cysteine-X-Cysteine chemokine receptor 4 (CXCR4), thereby reducing iron deposition, lipid peroxidation, and ferroptosis, and inhibiting neural cell damage following cerebral ischemia [181].

Electroacupuncture therapy is widely used in the treatment and rehabilitation of stroke [182]. Research has indicated that electroacupuncture treatment can effectively suppress ferroptosis by downregulating ACSL 4/TFRC/15-LOX/COX-2 expression and the increased GSH/GPX 4 expression [183, 184]. Moxibustion, a traditional Chinese therapy involving the burning of mugwort or other herbs to apply heat to specific acupuncture points, has been shown to suppress ferroptosis by modulating lipid metabolism. It reduces ACSL4 production, decreases iron deposition, and inhibits the accumulation of reactive oxygen species [185, 186]. The Zhuang Medicine Shuanglu Tongnao Formula, derived from Chinese Zhuang medicine, has been shown in a recent study to inhibit ACSL4 and against ferroptosis [187]. Tongqiao Huoxue Decoction, a commonly used traditional Chinese medicine formula for invigorating blood circulation and removing blood stasis, is implicated in the ferroptosis mechanism related to cerebral ischemia reperfusion by promoting the ubiquitination and degradation of ACSL4 [128].

Herbal/natural component

Herbs and their extracts have long been integral in the treatment of stroke. Recent research indicates that many herbal remedies may confer therapeutic benefits by modulating ferroptosis and ACSL4 (Table 2). These findings hold promise for informing traditional Chinese medicine practices and dietary recommendations for individuals affected by stroke.

Table 2.

ACSL4 in herbal/natural component

Drugs	Type	Source	Target	Signaling	Animals and cells	Model	Type of stroke
Baicalein	Natural flavonoid	The roots from Scutellaria baicalensis Georgi	GPX4, ACSL4, and ACSL3	GPX4/ACSL4/ACSL3	C57BL/6 mice and Hippocampal neuronal cells (HT22)	Oxygen–glucose deprivation/reoxygenation (OGD/R) and Transient middle cerebral artery occlusion (tMCAO)	Cerebral ischemia reperfusion (I/R) [188]
Calycosin	Phytoestrogen	The root of Astragali radix	ACSL4	Suppressed the upregulation of ACSL4 induced by tMCAO/R or OGD/R	Sprague‒Dawley (SD) rats and PC12 cells	tMCAO/R and OGD/R	I/R [22]
Paeonol (PAN)	phenolic active constituent	Paeonia	ACSL4	HOTAIR/UPF1/ACSL4	HT22 cells and C57BL/6 mice	neuronal cells treated with hemin and collagenase VII-S induces hemoglobin-mediated oxidative stress in the brain	Intracerebral hemorrhage (ICH) [118]
Astragaloside IV (As-IV)	tetracyclic triterpenoid saponin	Radix Astragali	N6-methyladenosine (m6A)	Atf3/Fto/ACSL4	Mice and IS cell	tMCAO and OGD/R	IS [189]
Caffeic acid	phenolic acid	plant	ACSL4 and TFR1	Nrf2 signaling pathway	SD rats	permanent middle cerebral artery occlusion (pMCAO)	IS [193]
β-caryophyllene (BCP)	bicyclic sesquiterpene co[94]nd	various foods and spices	ACSL4, HO-1 and GPX4	NRF2/HO-1 pathway	SD rats and astrocytes	middle cerebral artery occlusion reperfusion (MCAO/R) and OGD/R	I/R [194]
Dihydromyricetin (DHM)	flavonoid compound	Vine tea (Ampelopsis grossedentata)	GPX4, ACSL4 and PEBP1	SPHK1/mTOR pathway	SD rats and HT22 cells	MCAO/R and OGD/R	I/R [195]
Chrysin	flavonoid compound	honey, propolis, fruits, vegetables, and other natural drugs	SLC7A11, GPX4, ACSL4, TFR1, and PTGS2	Regulates the mRNA expression of key targets of ferroptosis	SD rats	tMCAO	I/R [196]
Puerarin	isoflavone phytoestrogen	Pueraria lobata root	ACSL4, GPX4, and GSH	AMPK/PGC1α/Nrf2 Pathway	SD rats	SAH model	subarachnoid hemorrhage (SAH) [171]
Ginkgolide B	bioactive terpenoid lactone	eaves and root bark of Ginkgo biloba	ACSL4, GPX4, and NCOA4-FTH1	Disrupt the NCOA4-FTH1 interaction	SD rats and PC12 calls	tMCAO and OGD/R	I/R [197]
Melatonin	indolamine	pineal gland	ACSL4	CYP1B1/ACSL4 pathway or MDM2/ACSL4 pathway	C57BL/6 mice and HT22 cells	tMCAO and OGD/R	I/R [126, 127]
Ecdysterone	active compound	Achyranthes bidentata Blume	ACSL4, NCOA4, and FTH1	suppressed the upregulation of ACSL4	SD rats and PC12 calls	tMCAO and OGD/R	Acute ischemic stroke (AIS) [198]
Celastrol	pentacyclic triterpenoid compound	Tripterygium wilfordii Hook F	ACSL4	antioxidant properties and aids in neurological recovery	SD rats and PC12 calls	ICH model and OxyHb	ICH [163]

ACSL4 Acyl-CoA synthetase long-chain family member 4; ACSL3 Acyl-CoA synthetase long-chain family member 3; GPX4 glutathione peroxidase 4; HOTAIR HOX transcript antisense RNA; UPF1 Upstream frameshift 1; Fto fat mass and obesity-associated; and Atf3 activation transcription factor 3; TFR1 transferrin receptor 1; Nrf2 nuclear factor erythroid 2-related factor 2; HO-1 Heme oxygenase-1; PEBP1 Phosphatidylethanolamine-binding protein 1; SPHK1 sphingosine kinase 1; mTOR mammalian target of rapamycin; SLC7A11 Cystine/glutamate reverse transporter; PTGS2 prostaglandin-endoperoxide synthase 2; AMPK AMP-activated protein kinase; PGC1α Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; NCOA4 Nuclear receptor coactivator 4; FTH1 ferritin heavy chain 1

Baicalein, the primary bioactive compound extracted from the traditional Chinese medicine Baikal Skullcap (Huang Qin), has been reported to reverse post-ischemic brain injury by inhibiting ferroptosis through the regulation of GPX4, ACSL4, and ACSL3 expression levels [188]. Calycosin is a component extracted from traditional Chinese medicine. Research indicates that it inhibits the expression of ACSL4 by forming stable hydrogen bonds with ACSL4 at G465, K690, and D573, thereby suppressing ACSL4-dependent ferroptosis and ameliorating neural damage in a cerebral ischemia model [22]. UPF 1 and ACSL 4 have been identified as downstream targets of HOTAIR and represent a novel strategy for Paeonol (PAN) to treat ICH [118]. Astragaloside IV (As-IV) is a naturally derived bioactive compound primarily obtained from the Chinese medicinal herb Huangqi (Astragalus membranaceus). It has been studied for its potential applications in various fields, including anti-tumor properties, antioxidative effects, anti-inflammatory responses, and cardiovascular health [189–192]. Moreover, As-IV exhibits promising therapeutic efficacy in the treatment of ischemic stroke, as it upregulates activation transcription factor 3 (Atf3) expression, activates fat mass and obesity-associated (Fto) transcription, resulting in reduced N6-methyladenosine (m6A) modification of ACSL4, thereby inhibiting cellular ferroptosis and improving neurological dysfunction caused by ischemic stroke [115]. Caffeic acid, another natural phenolic compound found in various plants, can reduce the expression of TFR1 and ACSL4 through the Nrf2 signaling pathway, increase glutathione production, and counteract iron-dependent cell death, thus alleviating neuronal damage after cerebral ischemia [193]. β-caryophyllene (BCP), a bicyclic sesquiterpene compound found in various foods and spices, significantly alleviates ROS production and iron accumulation by activating the NRF2/HO-1 pathway, inhibiting ferroptosis, and thereby reducing cellular damage in acute ischemic stroke. Furthermore, immunofluorescence results have indicated that BCP downregulates ACSL 4 expression [194]. Dihydromyricetin (DHM) and Chrysin, both flavonoids, have been found to regulate ACSL 4 in the therapeutic mechanism of cerebral ischemia and reperfusion injury [195, 196]. Puerarin, another isoflavone phytoestrogen, regulates ACSL4 in the treatment of EBI after SAH [171]. A recent study suggests that Ginkgolide B regulates oxidative stress and ferroptosis markers to alleviate cerebral ischemia/reperfusion injury. It prevents ferroptosis by inhibiting autophagy and disrupting the NCOA4-FTH1 interaction [197]. Recently, it was found that melatonin inhibits ferroptosis and protects against cerebral ischemic injury by promoting the ubiquitination of ACSL4 through the CYP1B1/ACSL4 and MDM2/ACSL4 pathways [126, 127]. Celastrol, a recently discovered natural compound, exhibits antioxidant properties during ferroptosis by inhibiting ACSL4 expression and aids in the restoration of neurological function following a stroke [163]. Furthermore, Ecdysterone (EDS) mitigates oxidative damage induced by acute ischemic stroke (AIS) by inhibiting ACSL4-mediated ferroptosis in neurons [198].

Others

In addition to the aforementioned methods, other treatment modalities are essential in stroke management. These approaches encompass various drugs and therapies that influence ACSL4 and ferroptosis, providing additional avenues for understanding stroke pathology and offering new possibilities for future treatments.

Cottonseed Oil (CSO), a plant-derived oil commonly used for dissolving lipid-soluble drugs, has been previously studied for its beneficial effects in mitigating intestinal inflammation and suppressing tumor metastasis [199, 200]. Recent research has revealed that in the context of ischemic stroke treatment, CSO intervention leads to reduced iron accumulation, lipid peroxidation, and mitochondrial damage. Additionally, it upregulates the expression of anti-ferroptosis proteins (GPX4, xCT, HO-1, FTH1), while downregulating the levels of the ferroptosis-associated protein ACSL4. Consequently, this intervention significantly reduces infarct volume and neural damage, while preserving the integrity of the blood–brain barrier (BBB) [201]. Additionally, RNF146 is involved in IS process by regulating ACSL4 and ferroptosis [124].

In summary, numerous studies have identified the potential therapeutic benefits of targeting ACSL4 inhibition to alleviate stroke through ferroptosis inhibitors, new discoveries in existing clinical treatments, herbal and natural compounds. Inhibiting ACSL4 to mitigate ferroptosis-induced neuronal injury and neuroinflammation opens a new avenue for stroke treatment. Thus, ACSL4 inhibition may serve as an effective therapeutic strategy for stroke. Nevertheless, nearly all research is still in the laboratory phase, and future investigations must explore the role of ACSL4 in stroke through clinical trials. Thus, investigating the disease benefits of targeting ACSL4 in clinical settings presents a significant challenge for future researchers. Additionally, the regulatory mechanisms of ACSL4-induced ferroptosis in stroke injury remain uncharacterized. There is an urgent need for a more in-depth exploration of the pathological mechanisms underlying ACSL4-induced ferroptosis in the stroke environment. Serum biomarkers hold substantial translational potential in clinical applications.

Due to the complexity and variability of stroke, the search for highly specific and sensitive biomarkers for stroke prediction remains a significant challenge. Previous studies have identified ACSL4 as a biomarker for predicting prognosis in cholangiocarcinoma [202], hepatocellular carcinoma [203], and breast cancer [204]. Furthermore, a recent study indicated that serum ACSL4 holds predictive value for the recovery of motor function post-stroke [205]. This finding is crucial for decision-making regarding stroke treatment plans and improving prognoses. However, further research is needed to confirm whether ACSL4 can serve as a predictive factor for the risk of stroke occurrence and as a biomarker for stroke prognosis, particularly concerning its predictive efficacy and interpretative aspects.

Conclusion and the future direction

Scientists have been trying to decipher the physiopathological mechanism of stroke and try to find effective neuroprotective agents. However, due to the complexity of the stroke, no excellent stroke “specific drug” has been found clinically. Ferroptosis acts as an emerging key mechanism affecting cell death in influencing the progression of neurological disorder through multiple molecular pathways. As research on ferroptosis deepens, the significance of ACSL 4, a key enzyme, in this process becomes increasingly apparent. Particularly, lipid peroxidation and associated FA mediated by ACSL4 are closely intertwined with ferroptosis. Thus, inhibition of ACSL 4-mediated ferroptosis is a novel therapeutic strategy for stroke. Downregulating the expression level of ACSL4 is meaningful for inhibiting ferroptosis. Not only in the laboratory but also the results of the genetic tests have confirmed the abnormal expression of ACSL 4 in neurological disorders [206]. However, the pathological process of stroke involves complex interplays with other mechanisms such as autophagy, apoptosis, cuproptosis, and pyroptosis. Moreover, the regulation of the ACSL 4 gene by various non-coding RNAs, transcription factors, and post-translational modifications underscores the need to explore specific regulatory pathways of ACSL 4 in stroke using proteins, genes, and biological molecules.

The ACSL 4-mediated ferroptosis is used not only in stroke but also in other neurological disorders and cancer fields, and the mechanism of ferroptosis is mainly studied in the field of cancer [207–209]. Meanwhile, ACSL 4-mediated ferroptosis was also found earlier in the cancer field. Consequently, whether the ferroptosis processes in stroke and tumors are identical warrants further investigation, given the scarcity of evidence on ACSL 4-mediated ferroptosis in stroke. Indeed, evidence on the ACSL 4-mediating ferroptosis in stroke remains scarce. The synergy between basic and clinical research will elevate stroke treatment to a precious level. In order to achieve clinical transformation, it is necessary to fully explore the mechanistic pathways and conduct preclinical experiments to identify effective therapeutic agents and the exact molecular mechanisms. Technologies enabling drug delivery across the blood–brain barrier (BBB) create therapeutic platforms for stroke [210, 211]. Conjugated nanocarriers targeted to decompose the ferroptosis defense axis to overcome BBB, avoid cytotoxicity, and perform brain delivery may be a promising approach for stroke.

Despite demonstrating excellence in vitro and in vivo stroke models, there are some limitations that warrant consideration. While numerous basic studies have elucidated the molecular mechanisms of brain injury post-stroke, these studies have yet to be translated into therapeutic and clinical applications, especially for ferroptosis inhibitors, so there are still many challenges in the clinical application of ACSL4-targeted drugs. Additionally, the role of ACSL4 in the iron-related crosstalk pathway in stroke remains unclear, highlighting the need for further investigation. Moreover, whether the ACSL4-mediated ferroptosis mechanism shares the same pathway in stroke and other diseases necessitates exploration.

Author contributions

BFZ, SZD designed the concept of the manuscript. BFZ and CYQ wrote the first version of this paper. HLJ, FT and SKS generated the figures and tables. FC and ZHM reviewed and revised the manuscript. All authors wrote and critically reviewed the manuscript and approved the final manuscript. BFZ, CYQ and SZD contributed equally to this manuscript.

Funding

This work was supported by the National Key R&D Program of China (No. 2018YFC1706001, 2010CB530506); The Science & Technology Development Fund of Tianjin Education Commission for Higher Education (2022KJ170); Open Project of National Clinical Research Center for Chinese Medicine Acupuncture and Moxibustion (Grant No. NCRCOP2023003).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.