Epigenetic Mechanisms Regulating Ferroptosis in Ischemic Stroke: From Pathogenesis to Therapeutic Targets

Abstract

Ischemic stroke (IS) remains a leading cause of death and disability worldwide; however, effective neuroprotective therapies are lacking. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a key contributor to neuronal injury following cerebral ischemia. Epigenetic mechanisms, including DNA methylation, histone modification, and microRNA (miRNA) regulation, critically modulate ferroptosis-related gene expression. This review systematically examines how epigenetic modifications regulate ferroptosis in IS by influencing iron homeostasis, antioxidant defense systems, and lipid metabolism. We highlighted key regulatory axes, such as DNMT-mediated GPX4 and PINK1 methylation, HDAC-regulated iron uptake and antioxidant defense, ubiquitination-dependent SLC7A11 and ACSL4 regulation, and miRNA/ferroptosis-related targets. Furthermore, we discuss the therapeutic potential of targeting these epigenetic-ferroptosis interactions for IS treatment. Understanding these mechanistic links may facilitate the development of novel epigenetic-based strategies to inhibit ferroptosis and improve the outcomes of patients with IS.

Graphical Abstract

Epigenetic Regulation and Ferroptosis in IS: Disrupted iron metabolism in the brain under ischemic conditions, coupled with oxidative stress and imbalanced lipid peroxidation, collectively exacerbates ferroptosis. Epigenetic mechanisms are also involved in this process. Targeting of these epigenetic mechanisms offers a viable avenue for mitigating ferroptosis following IS. LOOH: lipid hydroperoxide; ROS: reactive oxygen species.

Introduction

Ischemic stroke (IS) results from the abrupt cessation of blood supply to the brain, resulting in neuronal death and subsequent neurological deficits. It is a major source of morbidity and mortality worldwide, with an estimated 12 million new cases occurring annually, with an increasing incidence due to aging populations and associated risk factors such as hypertension, diabetes, and smoking (An et al. 2025). Neurological deficits severely affect patients’ quality of life and impose a significant burden on healthcare systems globally (Feigin et al. 2025). Oxidative stress, excitotoxicity, inflammation, and mitochondrial dysfunction are the typical pathological features of IS (Zhuang et al. 2025). The current treatment strategies primarily focus on rapidly restoring blood flow through thrombolytic therapy or surgical intervention. However, these methods are time-sensitive, and reperfusion injury exacerbates neuronal damage and adversely affects clinical outcomes (Ho and Powers 2025). Despite the urgent need for neuroprotective interventions during IS, the underlying causes of neuronal death remain poorly understood, hindering the development and implementation of effective therapeutic approaches. Iron accumulation in the brain has been proven to have a critical role in both the initiation and progression of ischemic damage. Ferroptosis is a unique type of cell death that differs greatly from apoptosis, necrosis, and autophagy (Cheng et al. 2024). It is primarily triggered by excessive intracellular iron buildup and lipid peroxidation reactions (Ma et al. 2024). Several typical pathological alterations associated with IS, including glutathione depletion, iron overload, mitochondrial dysfunction, and elevated reactive oxygen species (ROS) levels, are involved in the core processes of ferroptosis (Zhang et al. 2025a). This also offers an alternative explanation for IS onset and development. Consequently, iron deposition and ferroptosis may represent viable therapeutic targets for IS treatment. Epigenetic modifications are genetic mechanisms that do not alter DNA sequences but can influence gene expression through chemical modifications or structural changes. Research indicates that epigenetic modifications, particularly DNA methylation, histone modifications, and abnormalities in miRNA expression, are also implicated in IS (Shehjar et al. 2025; Arruri et al. 2025). In particular, emerging reports have begun to illuminate the epigenetic regulation of iron metabolism and ferroptosis (Jing et al. 2025). Despite growing evidence implicating ferroptosis and epigenetic dysregulation in IS pathogenesis, the mechanistic interplay between these processes remains poorly understood. Specifically, how epigenetic modifications regulate ferroptosis-related gene expression in the context of cerebral ischemia has not been systematically examined. This review aims to (1) systematically summarize the epigenetic mechanisms regulating ferroptosis in IS, (2) identify key regulatory axes linking epigenetic modifications to ferroptosis pathways, and (3) discuss the therapeutic potential of targeting these mechanisms for IS treatment. By elucidating these mechanistic links, we aimed to provide a foundation for developing novel epigenetic-based strategies to inhibit ferroptosis and improve outcomes in patients with IS.

The Role of Iron Metabolism in IS

Iron is an essential metal in the brain that plays a crucial role in multiple neuronal functions, including myelin formation, neurotransmitter synthesis, and mitochondrial respiration. It also serves as a cofactor for numerous enzymes in the nervous system, including iron-sulfur clusters in the mitochondria, heme-containing proteins, such as hemoglobin or neuroglobin, and oxygen-containing diiron enzymes, such as ribonucleotide reductase (Alrouji et al. 2024). Intracellular iron homeostasis is precisely regulated by proteins such as transferrin receptor 1 (TFR1), divalent metal transporter 1 (DMT1), ferritin, and ferroportin (FPN), which collectively coordinate iron absorption, storage, and efflux. However, this finely tuned regulatory system is disrupted by cerebral ischemia, leading to pathological iron accumulation (Lei et al. 2025). Specifically, ischemia causes endothelial cell damage, disrupts the blood–brain barrier (BBB), and increases permeability, thereby permitting a substantial influx of iron into brain tissue. This leads to the local disruption of iron metabolism and intracellular iron overload (Wang et al. 2024b). In contrast, anaerobic glycolysis following ischemia significantly lowers the pH of local brain tissue. This acidic environment causes iron to dissociate from transferrin (TF). Concurrently, the upregulation of ferritin, TFR1, and DMT1 promotes iron accumulation, whereas the downregulation of ferroportin 1 (FPN1) impedes cellular iron efflux (Gu et al. 2025; Wu et al. 2024a). Increased intracellular concentrations of Fe²⁺ interact with hydrogen peroxide (H₂O₂) through the Fenton reaction, producing highly reactive oxygen species, including hydroxyl radicals (Fig. 1). These reactive species can intensify oxidative stress and induce damage to lipids, proteins, and nucleic acids (Ratan 2020). Experimental models have demonstrated that iron accumulation occurs in the ischemic penumbra and core regions following stroke, exacerbating infarct size and neurological deficits (Yan et al. 2020). In patients with IS, elevated iron levels within ischemic regions are associated with worse prognosis (Uchida et al. 2024). Iron chelators, such as deferoxamine (DFO), minimize oxidative damage and improve functional prognosis. Notably, both DFO and the iron chelator deferiprone have demonstrated neuroprotective effects in preclinical models and early clinical trials (Millán et al. 2021; Ayton et al. 2025). This finding provides compelling evidence for the possibility of targeting iron metabolism during IS treatment. As research advances, increasing evidence suggests that iron accumulation plays a critical role in the etiology of IS (Zheng et al. 2025). Importantly, iron overload and lipid peroxidation induced by disrupted iron metabolism directly contribute to ferroptosis, which has emerged as a critical mechanism underlying neuronal injury in IS (Lei et al. 2025).

Fig. 1.

Schematic of iron metabolism in IS. Under physiological conditions, TF tightly binds to Fe³⁺. TFR1 exhibits high affinity for TF. TF (carrying two Fe³⁺ ions) binds to TFR1 to form a complex, which enters cells via clathrin-mediated endocytosis to form endocytic vesicles (endosomes). The acidic environment (decreased pH) following cerebral ischemia causes iron ions (Fe³⁺) to dissociate from TF. The dissociated Fe³⁺ must be reduced to Fe²⁺ (e.g., via reductases such as STEAP3) and is then primarily transported out of the endosome into the cytoplasmic solute via the DMT1. FPN1 is responsible for transferring intracellular Fe²⁺ to the extracellular space. Ischemic injury triggers inflammation, leading to elevated hepcidin levels, which bind to and degrade FPN1. Oxidative stress can directly damage the FPN1 protein or its regulatory pathways. Increased intracellular concentrations of Fe²⁺ interact with H₂O₂ through the Fenton reaction, producing highly reactive oxygen species, including HO•. When both iron uptake (TFR1, DMT1) and impaired iron efflux (FPN1) lead to a dramatic net accumulation of intracellular iron, creating a severe iron overload environment. This is a key driver of iron-dependent oxidative stress (Fenton reaction) and lipid peroxidation. TF, transferrin; HO•, hydroxyl radical; H2O2, hydrogen peroxide; TFR1, transferrin receptor 1; DMT1, divalent metal ion transporter 1; FPN1, ferroportin 1; pH, potential of Hydrogen; STEAP3, six-transmembrane epithelial antigen of the prostate 3; ROS, reactive Oxygen Species

The Role of Ferroptosis in IS

Ferroptosis is an iron-dependent programmed cell death characterized by elevated oxidative stress and compromised antioxidant defenses. This process often results in structural changes in mitochondria, including cristae loss and membrane disruption (Mishima et al. 2025). Over the past decade, research has clarified the pathogenic role of ferroptosis in a range of diseases, such as cancer, cardiovascular conditions, metabolic disorders, kidney injury, and neurological disorders. Increasing evidence suggests that it is also implicated in ischemic events (Zhang et al. 2025b; Walravens et al. 2024).

Cerebral ischemia triggers a cascade reaction ultimately leading to intracellular iron overload, which induces the Fenton reaction to produce the highly reactive HO•. Concurrently, Fe²⁺ activates lipoxygenases (LOXs), a family of iron-containing enzymes that catalyze the dioxygenation of polyunsaturated fatty acids (PUFAs). This enzymatic oxidation, together with non-enzymatic iron-mediated Fenton reactions, synergistically promotes lipid peroxidation in cell and mitochondrial membranes (Chang et al. 2025). Excessive lipid peroxides in the cell membrane, along with their degradation products 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), disrupt the integrity of the lipid bilayer, increase membrane permeability, and induce ferroptosis (Xu et al. 2023b; Jiang et al. 2025). Ischemia also inhibits the cystine/glutamate antiporter (system Xc⁻), depleting the precursor for reduced glutathione (GSH) synthesis and causing a sharp drop in GSH levels. GSH is the primary reducing substrate for the key antioxidant enzyme glutathione peroxidase 4 (GPX4), and its depletion impairs GPX4's ability to convert lipid hydroperoxide (LOOH) to harmless lipid alcohol (LOH) (Fang et al. 2022; Wei 2024). This represents the collapse of the antioxidant defense system. Thus, lipid peroxidation and malfunction of the antioxidant defense system constitute the core processes in ferroptosis and are major contributors to neuronal injury following IS (Fig. 2).

Fig. 2.

Regulatory network of ferroptosis following IS. Lipid peroxidation triggers: PUFAs catalyzed by LPCAT3 and ACSL4 form PL-PUFA, which is oxidized by LOXs or HO• generated via the Fenton reaction to produce LOOH. LOOH further decomposes into highly reactive LOO•, which proliferates LOOH through chain reactions. Both compounds synergistically induce mitochondrial disintegration via a positive feedback cycle. The accumulation of Fe.²⁺after IS activates LOXs and the Fenton reaction. Collapse of the antioxidant defense system: cells transfer glutamate to the extracellular space via the Xc⁻ system (composed of SLC7A11 and SLC3A2) while simultaneously taking up cystine. This cystine is reduced to cysteine using NADPH as an energy source, which is then used to generate GSH. GSH serves as an essential cofactor for GPX4, reducing toxic LOOH to harmless LOH and thereby blocking the lipid peroxidation chain. During this process, GSSG is regenerated into GSH by GR using NADPH. Ischemia inhibits the Xc⁻ system, depleting GSH and inactivating GPX4, leading to uncontrolled accumulation of LOOH. Central Regulatory Hub (NRF2): the NRF2-KEAP1 protein complex is a key inhibitor of ferroptosis. When oxidative stress is induced, Keap1 dissociates from NRF2 and undergoes degradation, enabling NRF2 nuclear translocation. NRF2 activation increases cellular GSH and NADPH levels, directly upregulating the transcription of GPX4 and SLC7A11. PUFAs, polyunsaturated fatty acids; ACSL4, acyl-CoA synthetase long chain family member 4; LPCAT3, lysophosphatidylcholine acyltransferase 3; PL-PUFA, phospholipid polyunsaturated fatty acid; LOXs, lipoxygenases; LOO•, lipid peroxide radicals; LOOH, lipid hydroperoxide; SLC7A11, solute carrier family 7 member 11; NADPH, nicotinamide adenine dinucleotide phosphate; SLC3A2, solute carrier family 3 member 2; GR, glutathione reductase; NRF2, nuclear factor erythroid 2-related factor 2; GSH, glutathione; GPX4, glutathione peroxidase4; GSSG, oxidized glutathione; HO•, hydroxyl radical; LOH, harmless lipid alcohol; ROS, reactive Oxygen Species; Keap1, Kelch-like epichlorohydrin-related protein-1

In vivo studies have confirmed that lipid peroxidation levels increase in ischemic brain tissues. As a key protein in ferroptosis, GPX4 effectively reduces the levels of lipid hydroperoxides, inhibits ferroptosis, and mitigates IS-induced neurological damage (Li et al. 2023). In contrast, acyl-CoA synthase long-chain family member 4 (ACSL4) is an essential driver of lipid peroxidation following GPX4 inactivation. It specifically esterifies long-chain PUFAs to generate polyunsaturated fatty acyl-coenzyme A (PUFA-CoA), thereby supplying substrates for lipid peroxidation. In rodent models, ACSL4 was downregulated after IS and promoted neuronal death by enhancing lipid peroxidation (Zhuo et al. 2025; Kloska et al. 2020). Nuclear factor erythroid 2-related factor 2 (NRF2) is a key transcription factor that is implicated in ferroptosis and iron homeostasis in the brain. Under basal conditions, NRF2 is sequestered in the cytoplasm by Kelch-like epichlorohydrin-related protein-1 (KEAP1), which facilitates its ubiquitination and proteasomal degradation. Upon oxidative stress, reactive oxygen species modify critical cysteine residues in KEAP1, disrupting the KEAP1-NRF2 interaction. This allows NRF2 to escape degradation, translocate to the nucleus, and induce an increase in cellular glutathione and NADPH levels (Attri and Kuwar 2025). On the other hand, activated NRF2 can directly upregulate the transcription of GPX4 and Solute Carrier Family 7 member 11 (SLC7A11) (Fig. 2). Interestingly, NRF2 also limits free iron and enhances iron storage capacity by promoting the expression of ferritin heavy chain 1 (FTH1), a cellular iron-storage protein (Fan et al. 2024; Yang et al. 2025b).

Additionally, ferroptosis in glial cells, particularly in microglia and astrocytes, affects IS. Under hypoxic conditions during cerebral ischemia, microglia absorb non-transferrin-bound iron via transferrin-independent mechanisms and TfR1. The hypoxia-inducible factor 1 (HIF-1) pathway modulates these iron transporters, leading to increased intracellular iron levels in microglia during ischemia. Excessive iron accumulation in microglia stimulates ROS production (Xu et al. 2023a), which is crucial for promoting lipid peroxidation and initiating ferroptosis. Astrocytes can absorb iron via the TfR1/DMT1 pathway and export iron through Fpn1 while also coupling with ceruloplasmin to inhibit iron-induced lipid peroxidation. Under ischemic conditions, astrocytes exhibit upregulation of TFR1, DMT1, and FPN1, leading to enhanced iron uptake and release (Moro et al. 2025). Oligodendrocytes are responsible for myelin production in the central nervous system, and their normal function requires high levels of iron, particularly during myelin synthesis. Under ischemic conditions, oligodendrocytes increase intracellular iron uptake, thereby enhancing cellular iron retention within the cells. Research indicates that this accumulation of iron leads to oligodendrocyte injury, accelerating demyelination and axonal degeneration (Kim et al. 2021).

A novel hematological diagnostic model was established using machine learning LASSO regression analysis, incorporating seven key genes associated with ferroptosis in IS (Liao et al. 2023). Furthermore, T2-weighted MRI is commonly used to map iron levels in the brain in neurological disorders. During the early clinical stages of cortical ischemia, a reduction in signal strength on T2-weighted and proton density-weighted MR can be observed in the subcortical regions, indicating the presence of iron deposition. In patients with IS, T2-weighted MRI has detected low-signal phenomena in the ipsilateral thalamic or basal ganglia regions, remote from the primary ischemic area, which are associated with poor functional, cognitive, and emotional outcomes as well as localized microstructural and metabolic abnormalities (Guo et al. 2023; Zhan et al. 2025). This information demonstrates prominent diagnostic accuracy and performance in several patients with IS, demonstrating that ferroptosis plays a significant role in IS. Importantly, a growing body of evidence has demonstrated the therapeutic potential of several compounds against IS, which is achieved through the suppression of ferroptosis. For example, hirudin (Liao et al. 2025), quercetin (Peng et al. 2024), Loureirin C (Liu et al. 2023b), deferoxamine (Abdul et al. 2021), and apelin (Luo et al. 2022) have demonstrated the potential to reduce cerebral ischemia–reperfusion injury and relieve neurological impairments in animals by restraining ferroptosis. Similar to OGD (oxygen–glucose deprivation) cell models, baicalein (Li et al. 2022), N-salicyloyl tryptamine derivatives (Wu et al. 2024b), vitexin (Guo and Shi 2023), ferrostatin-1 (Liu et al. 2023a), and kellerin (Mi et al. 2024) also function as ferroptosis inhibitors, enhancing cell viability and reducing ROS production. These findings indicate that targeting the ferroptosis pathway has significant potential for the development of treatment approaches for IS. However, the clinical application of ferroptosis-targeting therapies for IS is limited by several challenges. A primary hurdle is the inefficient transport of drugs across the BBB, which limits the efficacy of many potential drugs. Furthermore, the long-term safety and toxicity profiles of iron chelators and ferroptosis inhibitors remain to be fully elucidated. Clinical trials are critical in determining the appropriate dosing, safety, and effectiveness of these therapies in patients with stroke. Moreover, combination therapies that integrate ferroptosis modulation with other neuroprotective strategies, such as anti-inflammatory agents or agents that promote neurogenesis, may offer synergistic benefits for improving IS outcomes. Although ferroptosis clearly contributes to neuronal injury in IS, the upstream regulatory mechanisms controlling this process remain incompletely understood. Emerging evidence suggests that epigenetic mechanisms play a crucial role in modulating ferroptosis susceptibility (Cercel et al. 2025).

Epigenetics in IS

In 1942, Waddington introduced the concept of “epigenetics” to describe the mechanisms by which a genotype translates into a phenotype during development (Waddington 2012). Recent advances in epigenomic analysis technologies have garnered significant attention from researchers because of their potential roles in various diseases, including cancer, cardiovascular conditions, metabolic disorders, and neurological diseases (Atzemian et al. 2025; Sherif et al. 2024; Gaba et al. 2025). Maintenance of a healthy nervous system relies heavily on epigenetic mechanisms. Epigenetic markers mediate the effects of genetics and environment on phenotypes, giving them the potential to elucidate the etiology of IS. In this section, we focus on several alterations in epigenetic mechanisms associated with IS, including DNA methylation, histone modifications, and microRNAs (miRNAs) (Fig. 3).

Fig. 3.

Epigenetic modulation after IS. In the context of IS, an imbalance between DNA methyltransferase (DNMT1, DNMT3A) and TET dioxygenase (TET1-3) activities leads to a dynamic remodeling of DNA methylation and hydroxymethylation; at the same time, HMTs catalyze the addition of methyl groups to histone proteins; HATs and HDACs dynamically modulate histone acetylation status; E3 ubiquitin ligases (such as RNF2) and deubiquitinating enzymes (such as USP14/30) regulate the monoubiquitination of histone H2A at lysine 119 and histone H2B at lysine 120. These histone modifications collectively alter chromatin conformation and regulate gene transcription. These epigenetic mechanisms act synergistically to affect ROS, cell death, mitochondrial function, BBB, and neuroinflammation after ischemia. DNMT, DNA methyltransferase; TET, ten-eleven translocation; Me, methylation; Ac, acetylation; HMTs, histone methyltransferases; HDACs, histone deacetylases, HATs, histone acetyltransferases; 3'UTR, 3' untranslated region; Ub, ubiquitination; USP14/30, ubiquitin-specific peptidase 14/30; RNF2, ring finger protein 2; ROS, reactive oxygen species; BBB, blood–brain barrier

DNA Methylation in IS

DNA methylation (i.e., methylation of the fifth carbon atom of cytosine, 5mC) is the most thoroughly studied epigenetic modification and mechanism of stroke (Jiménez-Balado et al. 2024). Cytosine-guanine (CpG) dinucleotide is the major site of DNA methylation, and in mammalian cells, the majority (70%-80%) of CpG sites are methylated. 5mC is mainly distributed in heterochromatin regions and gene bodies (Mattei et al. 2022). Bisulfite sequencing is the pioneering and most commonly used method for measuring the DNA methylation status. Other techniques, including whole-genome bisulfite sequencing, methylation-based DNA immunoprecipitation, methylation-sensitive restriction endonuclease followed by sequencing, and methylation BeadChip, have also been used to study DNA methylation (Saini et al. 2023). These approaches help us understand the role of genetics in health and disease. DNA methylation is involved in pathophysiological events that occur after IS by regulating oxidative stress, excitotoxicity, mitochondrial dysfunction, BBB disruption, cell death, and inflammation (Choi et al. 2022).

DNA Methyltransferases in IS

Preclinical studies have demonstrated that cerebral ischemia is associated with increased overall DNA methylation in the brain, leading to inhibition of gene transcription and expression. This suppression exacerbates brain damage and is linked to increased DNA methyltransferase (DNMT) activity in the brain (Asada et al. 2020). However, inhibiting DNA methylation may improve the resistance to ischemia (Choi et al. 2022). DNA methylation is catalyzed by DNMT, including DNMT1 and DNMT3. DNMT1 maintains the methylation status of the genome and is critical for embryogenesis. It also plays crucial roles in chromatin structure, neuronal survival, and cell cycle regulation. The DNMT3 family comprises DNMT3A, DNMT3B, and DNMT3L. DNMT3A has been implicated in the regulation of mitophagy after cerebral I/R injury and its overexpression leads to increased neuronal apoptosis after OGD/R. DNMT1 was upregulated in the ischemic penumbra tissue 24 h after cerebral I/R injury. Reduced DNMT1 expression and 5-mC levels protect synaptic function in the rat hippocampus (Shi et al. 2023a; Zhu et al. 2024). In addition, DNMT1 has been associated with post-stroke microglial polarization (Tan et al. 2022).

TET Enzymes in IS

DNA demethylation is facilitated by the ten-eleven translocation (TET) protein family. TET proteins act as 5mC hydroxylases, initiating DNA demethylation by adding hydroxyl groups to 5mC, resulting in the formation of 5-hydroxymethylcytosine (5hmC). The TET enzyme family consists of three isoforms: TET1-3, all of which are highly expressed in the brain (Choi et al. 2022). A typical case of IS involves elevated 5hmC levels in the DNA of blood cells from patients with acute ischemic stroke (AIS). Notably, the increase in 5hmC levels after ischemia was dependent on TET3 and TET2 levels. Inhibition of these enzymes not only decreases 5hmC levels but also exacerbates ischemic brain injury (Zhao et al. 2016). This suggests that TET2, TET3, and 5hmC have neuroprotective effects. In vivo and in vitro studies have found that overexpression of TET1 in astrocytes has been found to resists autophagy and apoptosis, triggers promoter hypomethylation, and potentially attenuates brain damage caused by IS (Zhou et al. 2021). Conversely, TET1 deficiency correlates with learning and memory deficits as well as oxidative stress (Ma et al. 2022). In contrast, overexpression of TET1 in primary mouse microglia promotes M1 polarization and exacerbates cerebral ischemia/reperfusion-induced neuroinflammation (Lin et al. 2024). Thus, it is evident that a dynamic balance of TET1 expression is essential for the maintenance of normal physiological functions.

Clinical and Biomarker

An epigenome-wide association study demonstrated significant hypomethylation of zinc finger homeobox 3 and mitogen-activated protein kinase kinase kinase 1 in patients with IS. Altered DNA methylation in these genes has been identified as a risk factor for cardiogenic embolism and atherosclerotic stroke (Cullell et al. 2022). HTRA serine protease 1 (HTRA1) is a predominantly secreted serine protease that degrades substrates for various diseases. Changes in HTRA1 methylation are detectable two years prior to the stroke clinic, suggesting that differential DNA methylation profiles in peripheral blood may be potential biomarkers for stroke risk assessment and preclinical testing (Liu et al. 2022). DNA methylation of genes with pro-oxidant or antioxidant effects (GCLM, GSTP1, and TXNRD1) has also been observed in patients with acute phase stroke (Bushueva et al. 2021). This suggests that DNA methylation contributes to oxidative stress after stroke. However, precise alterations in the methylation of certain key genes in individuals with IS or in cerebral ischemia models have not been thoroughly documented. Additional research is required to elucidate the relationship between DNA methylation changes and IS, as well as their potential role in IS pathogenesis.

Histone Modifications in IS

While DNA methylation provides a relatively stable epigenetic mark, histone modifications offer a more dynamic layer of regulation that can rapidly respond to ischemic insults (Cercel et al. 2025). Histones encapsulate and organize DNA into chromatin. Histones undergo various post-translational changes, including methylation, ubiquitination, acetylation, and phosphorylation. These modifications are catalyzed by specific enzymes and lead to nucleosome repositioning by altering DNA conformation, thereby activating or inhibiting transcription (Liu and Schneider 2025). Histone modifications occur mainly on the lysine residues of histones H3 and H4. These modifications are highly reversible and can dynamically regulate gene expression in response to the cellular environment. Histone post-translational modifications (HPTMs) are significantly involved in the initiation and progression of human diseases, including inflammation, cancer, cardiovascular disease, renal disease, metabolic disorders, and neuropsychiatric disorders (Yao et al. 2024). In recent years, a number of sophisticated techniques have been developed to study epigenetic protein mapping of genomes (Zhang et al. 2023a; Fu et al. 2024), such as ChIP-seq, to study protein-DNA interactions and histone modifications throughout the genome. DNase-seq and ATAC-seq analyses of chromatin accessibility. Faireseq and MNase-seq were used to identify open chromatin areas and to map nucleosome locations, respectively. Furthermore, the landscape of DNA and RNA modifications is now accessible through high-throughput sequencing strategies, including BS-Seq, oxBSSeq, fCAB-Seq, and CAB-Seq. These research approaches have helped us understand the key role of histone modifications in IS. In this section, we provide an overview of different histone modifications linked with IS, specifically histone methylation and acetylation, which have been widely studied in HPTM.

Histone Methylation in IS

As a cornerstone of epigenetic modification along with DNA methylation, histone methylation carries essential epigenetic information. It is extensively involved in crucial physiological and pathological processes by regulating gene expression, chromatin dynamics, and genomic integrity (Park et al. 2024). Nucleosomes are the most basic units of chromatin, and each nucleosome contains an octamer consisting of two molecules each of the core histones H2A, H2B, H3, and H4. Histone methylation usually occurs at the N-terminal arginine or lysine residues of H3 and H4 histones (Gold and Shilatifard 2024). Recent studies have shown that methylation of histone 3 at lysine 4 (H3K4), histone 3 at lysine 36 (H3K9), and histone 3 at lysine 79 (H3K24) participates in IS mouse pathology by regulating genes associated with inflammation, apoptosis, oxidative stress, and neuronal survival (Su et al. 2022). In stroke patients, a significant association was found between circulating tumor necrosis factor-α levels and H3K9ac and H3K4me3, which may alter the prognosis of stroke (Gómez-Uriz et al. 2014). This suggests that alterations in histone H3 methylation levels are important for IS development.

Histone methyltransferase (HMT) is a key “writing enzyme” in the histone methylation process, responsible for adding methyl groups to lysine or arginine residues of histones to regulate gene expression. The activity and expression of multiple HMTs were identified after IS. For example, mild ischemia induced in a mouse model of internal carotid artery occlusion showed dysregulation of multiple HMTs in the striatum, including G9a, SUV39H1, SUV39H2, EZH2, and SUV420H2. This was accompanied by a significant reduction in H3K9me2 levels from 3 h to 15 days of reperfusion (Chen et al. 2023). Similarly, increased levels of histone methyltransferase SUV39H1 were observed in the penumbra tissue 24 h after Photothrombotic Stroke (PTS), whereas G9a was overexpressed at both 4 and 24 h after PTS. Knockdown of SUV39H1 or the G9a inhibitors A-366 and BIX01294 enhanced brain-derived neurotrophic factor (BDNF) expression, improved the survival of rat cortical neurons after OGD, and reduced the volume of PTS-induced cerebral infarction (Sharifulina et al. 2021). The histone methyltransferase Smyd2 has been shown to methylate histones H3K4 and H3K36. In the middle cerebral artery occlusion (MCAO) model, Smyd2 expression is increased in the peri-infarct region of the mouse cortex and mediates the disruption of the blood–brain barrier (BBB) after stroke through methylation. Knockdown of Smyd2 in mice reduces blood–brain barrier permeability and improves functional recovery (Wang et al. 2022b). Although findings related to the regulation of IS processes by histone methylation remain limited, the current findings point to its potential as a target for novel intervention strategies.

Histone Acetylation in IS

Histones are subjected to dynamic acetylation and deacetylation modifications, mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. These modifications regulate the activation or repression of gene transcription depending on the cellular microenvironment (Palomés-Borrajo et al. 2025). Based on their homology and structural features, HDACs have been classified into four groups: class I (HDAC1-3 and 8), class II (HDAC4-7, 9, and 10), class III (sirtuins, SIRT1-7), and class IV (HDAC11) (Wang and Luo 2025). In vivo experiments showed that stroke enhances HDAC expression in the brain in a spatiotemporal manner. For example, 45 min after MCAO, HDAC1-2 expression was reduced in the ischemic core region but elevated in the subventricular zone and ischemic penumbra neurons in the cortex. Increased expression was also observed in glial cells in the subcortical white matter. In the in vitro experiments, HDAC1-3 expression levels were upregulated in all glial cell nuclei and astrocyte protrusions after 60 min of oxygen–glucose deprivation (OGD) treatment. Among these, HDAC3 showed the most significant upregulation (Ji et al. 2024b; Lisek et al. 2025). A human genome-wide association study linked HDAC9 genetic variations to the risk, severity, and short-term prognosis of large-artery atherosclerotic stroke (Hu et al. 2022; Wang et al. 2019a). These studies suggest that histone acetylation is closely associated with IS. Different HDACs play distinct roles in the IS. In vitro studies have indicated that the activation of HDAC1 and HDAC3 induces microglial phenotypic transformation and exacerbates neuroinflammation, whereas defects promote the development of anti-inflammatory microglia (Lisek et al. 2025). Another preclinical study demonstrated that HDAC1 and HDAC3 have cytotoxic effects, promoting apoptosis and impairing mitochondrial transport, and that the neurotoxicity of HDAC3 is inextricably linked to the interaction of HDAC1 (Yang et al. 2024a). Stroke induces nuclear translocation of HDAC4 in mice peri-infarct cortical neurons. This nuclear translocation worsens stroke prognosis by exacerbating neuronal death after OGD and expanding infarct size and functional deficits in MCAO mice. HDAC4 inhibition enhances neuroprotection (Yuan et al. 2016; Chen et al. 2025). However, it seems to have the opposite effect in patients with IS. HDAC4 was found to be generally reduced in patients with AIS and was negatively correlated with National Institutes of Health Stroke Scale (NIHSS) scores. Elevated HDAC4 levels may inhibit disease severity and progression in AIS by suppressing inflammation and inhibiting the development of atherosclerosis (Wang et al. 2022c). The expression of HDAC5, HDAC6, HDAC8, and HDAC9 is also significantly upregulated in animal models of cerebral ischemia, affecting post-ischemic apoptosis, inflammatory responses, and BBB integrity (Guo et al. 2025b). These studies suggest that therapeutic strategies that target specific HDAC subtypes should be considered. Studies have found that sodium valproate and tetrahydrobenzazepine, two widely used HDAC inhibitors, can preserve or restore the normal acetylation levels of H3 and H4 histones. These effects significantly reduce infarct volume and improve neurobehavioral deficits in MCAO rats (Guo et al. 2021; Markus 2023). A recent study observed that intranasal administration of nanotechnology-targeted Sirt1 significantly reduced cerebral edema in mice with cerebral ischemia (Ryu et al. 2024). A clinical trial also reported that the Sirt1 activator resveratrol prolonged the clinical therapeutic window of r-tPA, reduced neurological deficits induced by matrix metalloproteinases, and improved the prognosis of AIS patients (Chen et al. 2016). In addition, SIRT3, SIRT6, and SIRT7 have been shown to play protective roles in IS (Guo et al. 2025b). This evidence suggests that HDACs are potential targets for IS prevention and treatment as well as for clinical translation. CBP/p300, a core member of the HAT family, is upregulated in the ischemic penumbra of the rat cerebral cortex and has been shown to target and inhibit P53 to exert neuroprotective effects (Guzenko et al. 2024). It also interacts with HDAC3 to maintain oligodendrocyte identity (Zhang et al. 2016). Although alterations in HAT activity may be associated with neuroprotective pathways, studies on HAT in stroke remain scarce, emphasizing areas for future research.

Histone Ubiquitination in IS

Histone ubiquitination is mediated by enzymes that covalently link ubiquitin molecules to specific lysine residues in histones. This modification directly affects key biological functions, such as gene transcription, DNA repair, and the cell cycle, by altering the chromatin structure or recruiting regulatory factors. The most commonly ubiquitinated histones are histone H2A (usually at lysine 119 (H2AK119Ub)) and histone H2B (usually at lysine 120 (H2BK120Ub)). Histone ubiquitination is catalyzed by enzymes called E3 ubiquitin ligases and can be reversed by deubiquitinating enzymes (Lopes et al. 2024). Ubiquitin enrichment coupled with nano-liquid chromatography-mass spectrometry/MS and bioinformatics analysis revealed that cerebral ischemia enhances the ubiquitination of proteins in the postsynaptic density region in mice and modulates the activity of ischemia-associated neurokinins (Dhawka et al. 2024). Another in vivo study found that histone ubiquitination during cerebral ischemia may respond to cellular stress and protein aggregation by regulating crosstalk between the ubiquitin–proteasome system and autophagy (Liu et al. 2020). This finding suggests that ubiquitination plays an active role in countering ischemic stress. Additionally, studies have revealed that specific E3 ubiquitin ligases can modulate the prognosis of ischemic brain injury. Among these, E3 ligase ring finger protein 2 (RNF2) is upregulated in the brain tissue of IS rats and exerts neuroprotective effects by inhibiting apoptosis (Shen et al. 2024). Similarly, ubiquitin-specific peptidase 30 (USP30) effectively prevented ischemia–reperfusion injury by reducing OGDR-induced mitofusin 2 ubiquitination and degradation in SK-N-BE (2) cells, thereby preventing mitochondrial fragmentation (Chen et al. 2021). In contrast, selective inhibition of ubiquitin-specific peptidase 14 (USP14) by IU1 preserves BBB integrity and reduces neuroinflammation in MCAO mice (Hou et al. 2023). These results indicate that dynamic equilibrium between ubiquitination and deubiquitination is crucial for neuronal survival.

MicroRNAs in IS

Beyond chromatin-level modifications, post-transcriptional regulation by miRNAs represents another critical epigenetic mechanism that fine-tunes gene expression following IS. miRNAs are a family of endogenous small non-coding RNA molecules, approximately 22 nucleotides in length, that are highly conserved among closely related species. When partial complementary sequences are present in the 3'-untranslated region (3'-UTR) of target mRNAs, they can downregulate gene products by inhibiting translation or promoting mRNA degradation (Wang et al. 2025a).

Dysregulated miRNAs in IS

Ischemia changes the expression patterns of miRNAs in the brain tissue and blood samples from both humans and rodents. In the peripheral blood of patients with IS, miR-140-5p and miR-7-5p were upregulated, while miR-210-3p was downregulated. These are considered specific biomarkers for IS (Mainali et al. 2025). Similarly, dysregulation of miR-424, miR-210, miR-134, and miR-150 has been observed in animal models of cerebral ischemia, and is associated with BBB disruption, oxidative stress, apoptosis, and neuroinflammation (Li et al. 2025b). Recent studies have also identified miR-155, miR-146b, miR-181c, miR-182, miR-34a, miR-92a, miR-122-5p, miR-451a, and miR-409-3p as positively correlated with infarct volume in patients with AIS (Yang et al. 2024b).

Functional Roles of Specific miRNAs in IS Pathophysiology

Preclinical studies have demonstrated that multiple miRNAs regulate key pathological processes following IS, including neuroinflammation, BBB integrity, and neuronal apoptosis. miR-155 is among the most extensively studied inflammatory miRNAs in IS, functioning as a pro-inflammatory regulator that modulates oxidative homeostasis and microglial activation. Its inhibition shifts microglia toward an anti-inflammatory phenotype and represents a potential therapeutic target (Hering and Conover 2025). Similarly, miR-128-3p is significantly downregulated in ischemia-challenged neurons and their EVs, leading to increased microglial activation and neuronal injury, and intravenous injection of miR-128-3p mimics significantly improves neuronal survival and reduces neuroinflammation (Li et al. 2025c). In terms of BBB regulation, miR-199a-5p attenuates BBB disruption following IS by activating the PI3K/Akt signaling pathway, which increases the expression of the tight junction proteins Claudin-5 and VEGF in the ischemic penumbra while reducing inflammatory cytokine expression (Ni et al. 2024). Conversely, miR-34a is elevated after ischemic events and increases BBB permeability through its negative effects on mitochondrial function in cerebrovascular endothelial cells (Payne et al. 2023). With respect to neuronal apoptosis and neuroprotection, the miR-17–92 cluster—particularly miR-19a, miR-18a, and miR-92a—contributes to neurogenesis, axonal growth, and reduction of neuronal apoptosis, offering potential therapeutic applications for enhancing neural regeneration and functional recovery (Braicu et al. 2025). These findings highlight the diverse regulatory roles of miRNAs in IS pathophysiology beyond their involvement in ferroptosis, which will be discussed in subsequent sections.

miRNAs as Biomarkers and Therapeutic Targets

A notable feature of miRNAs is their remarkable stability in human body fluids (plasma, serum, and cerebrospinal fluid), largely due to their association with extracellular vesicles or RNA-binding proteins that protect them from degradation (Burlacu et al. 2022). This stability, combined with their disease-specific expression patterns, makes circulating and exosomal miRNAs promising minimally invasive biomarkers for stroke diagnosis, prognosis, and risk stratification. Plasma microvesicles and microvesicle-derived miR-155 have been identified as a biomarker for diagnosing atherosclerotic stroke and show a positive correlation with NIHSS scores (Zhang et al. 2020a). From a therapeutic perspective, miRNA mimics (to restore downregulated miRNAs) or antagomirs (to inhibit upregulated miRNAs) represent potential strategies for modulating IS outcomes, although their delivery to the ischemic brain remains a significant challenge.

Crosstalk Among Epigenetic Mechanisms in IS

The epigenetic mechanisms described above do not function in isolation but engage in extensive crosstalk that coordinates gene expression programs during IS. DNA methylation recruits methyl-CpG-binding domain proteins, which, in turn, recruit histone deacetylases to establish repressive chromatin. Conversely, certain histone modifications (e.g., H3K36me3) can recruit DNMTs to establish DNA methylation. miRNAs can target mRNAs encoding epigenetic enzymes (DNMTs, HDACs, and HMTs), whereas DNA methylation and histone modifications at miRNA gene promoters regulate miRNA expression. For example, miRNA-mediated suppression of SIRT6 mRNA affects histone acetylation patterns, which in turn influence the expression of ferroptosis-related genes in rodent models (Abdelfattah et al. 2025). This interconnected network allows for coordinated, multilayered control of gene expression in response to ischemic stress. Understanding these interactions is crucial for developing epigenetic therapies for IS. Future research should aim to systematically map these interactions and identify key regulatory nodes that could serve as therapeutic targets.

Epigenetic Regulation of Ferroptosis in IS

Recently, researchers have become interested in exploring how epigenetics regulates ferroptosis during IS (Zhou et al. 2024), which provides a new avenue for investigating the associations between ferroptosis, epigenetics, and IS. In this section, we focus on analyzing the roles of different epigenetic modifications in the ferroptosis process and their contributions to the pathological progression of IS, thereby providing a basis for elucidating potential regulatory mechanisms.

Role of DNA Methylation in Regulating Ferroptosis During IS

DNA methylation is the most common epigenetic modification in mammals and is crucial for ferroptosis (Wang et al. 2023b). Lymphocyte-specific helicase (LSH) is a DNA methylation modifier 5-hmC reader. High levels of LSH are detectable in the mouse brain during embryonic development, and their absence affects the growth and death of neural stem cells (Han et al. 2017). One study indicated that LSH directly modifies DNA methylation at the WD repeat domain 76 to activate lipid metabolism genes, including glucose transporter type 1, fatty acid desaturase 2, and stearoyl-CoA desaturase 1. This inhibits ferroptosis by reducing lipid ROS levels and iron concentrations both in vivo and in vitro (Jiang et al. 2017). In MCAO mouse models and OGD cell models, the DNA demethylase TET2 mediates increased 5hmC abundance following ischemic injury. Inhibition of TET2 activity leads to reduced 5hmC modification accompanied by increased infarct volume and decreased BDNF expression (Ma et al. 2021). Interestingly, TET2 can regulate lipid peroxidation via the Gpx4-GSH antioxidant system, thereby influencing ferroptosis (Zeng et al. 2023). This indicated a link between DNA methylation, ferroptosis, and IS. In fact, abnormal DNA methylation serves as a key regulatory mechanism linking oxidative stress to ferroptosis in cerebral ischemia/reperfusion injury. The core of this process is the homeostasis imbalance of the methyl donor S-adenosylmethionine (SAM) (Xia et al. 2024). On the one hand, the enhanced pentose phosphate pathway drives GSH synthesis to counter oxidative stress. This process consumes large amounts of methionine, leading to reduced levels of its downstream product SAM. As the sole methyl donor for DNA methylation, SAM depletion directly impairs methylation capacity across the genome and at specific gene loci. Altered methylation patterns in key ferroptosis-regulating genes (e.g., GPX4, ACSL4, and SLC7A11) may significantly influence their expression levels, thereby determining neuronal susceptibility to ferroptosis (Narne et al. 2017). On the other hand, oxidized glutathione (GSSG) generated by oxidative stress inhibits the activity of S-adenosylmethionine synthase, further exacerbating SAM depletion and promoting a state of DNA hypomethylation (Mersaoui et al. 2022).

Elevated homocysteine (Hcy) levels significantly contribute to the risk of developing IS. An in vitro study indicated that Hcy can upregulate GPX4 methylation, leading to oxidative stress and ferroptosis (Zhang et al. 2020b). Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme that catalyses folate metabolism. It provides methyl groups for SAM synthesis, thereby maintaining the intracellular DNA methylation homeostasis. MTHFR dysfunction can cause hyperhomocysteinemia (HHcy), whose brain injury mechanisms involve not only protein and lipid peroxidation but also specific activation of the N-methyl-D-aspartate receptor (NMDAR) signaling pathway. Under HHcy conditions, elevated Hcy binds with a high affinity to glutamate sites on neuronal and endothelial cell NMDARs, particularly the NR1 subunit. This activates nitric oxide synthase and induces superoxide anion production, thereby triggering significant oxidative stress (Jara-Prado et al. 2003). Further studies have indicated that the Hcy–NMDAR–oxidative stress pathway differentially regulates DNMT expression, specifically upregulating DNMT1 and DNMT3a while suppressing DNMT3b, thereby inducing mitochondrial toxicity and endothelial dysfunction (Gou et al. 2021). Interestingly, Ferrostatin-1 (Fer-1) reduces Hcy-induced oxidative stress by increasing TET levels and decreasing DNA methylation (Shi et al. 2022). These preclinical findings indicated that Hcy exacerbates ferroptosis following IS by inducing DNA methylation, whereas Fer-1 inhibits this process. This suggests that DNA methylation is involved in the pathogenesis of ferroptosis in IS.

ALOX12 functions as a primary effector enzyme in the ferroptosis pathway by catalyzing PUFA peroxidation. This process initiates the lipid peroxidation cascade, resulting in oxidative membrane damage and culminating in ferroptosis. An epigenome-wide association study conducted on acute-phase blood samples of IS patients revealed that hypomethylation of the ALOX12 gene, a lipoxygenase family member, was associated with its elevated expression and correlated with adverse clinical outcomes (Jiménez-Balado et al. 2024). Similarly, elevated levels of Tumor Protein p53 (TP53) promoter methylation have been observed in the peripheral blood of patients with IS. This methylation is associated with carotid intima-media thickness, the degree of carotid atherosclerosis, and circulating homocysteine levels in the peripheral blood (Wei et al. 2019). In the rat MCAO model, p53 participates in ferroptosis through multiple pathways by downregulating SLC7A11 and upregulating spermidine/spermine N1-acetyltransferase 1 and glutaminase 2 genes (Hou et al. 2025). These findings provide evidence that DNA methylation contributes to IS pathophysiology by affecting ferroptosis. ELAVL1 is an RNA-binding protein that acts as a key post-transcriptional regulator. It plays a critical role in controlling gene expression by modulating the stability and translation of the target mRNAs. It is extensively involved in the regulation of diverse cellular processes, including oxidative stress, autophagy, cell proliferation, differentiation, apoptosis, aging, and immune responses. Research indicates that upregulation of ELAVL1 promotes methylation of the PTEN-induced kinase 1 (PINK1) gene promoter region by stabilizing the mRNA of DNA methyltransferase DNMT3B. As a core regulator of mitochondrial autophagy, PINK1 expression is suppressed by high DNA methylation levels (Deng et al. 2024). This suppression leads to mitochondrial dysfunction, iron buildup, and exacerbated oxidative stress, ultimately inducing ferroptosis in the neurons. Downregulation of ELAVL1 mitigates iron-induced neuronal injury in ischemic/reperfused rats by reducing DNMT3B-dependent PINK1 methylation (Du et al. 2022). Another study indicated that activated transcription factor 4 (ATF4) activates DNMT1, inducing the DNA methylation of GPX4 and reducing its expression. ATF4 knockdown protects against cerebral infarction and sensory dysfunction in rats by promoting DNMT1-mediated DNA methylation of GPX4 (Lu et al. 2025). These findings link DNA methylation and ferroptosis to IS pathogenesis and indicate novel combination therapeutic options for IS.

These findings indicate that DNA methylation can contribute to IS progression by exacerbating ferroptosis, as exemplified by the methylation of genes, such as GPX4, SLC7A11, and PINK1. Furthermore, DNA methylation, such as that observed in the ALOX12 and TP53 genes, may also serve as a link between ferroptosis and IS (Fig. 4). However, further research is needed to completely elucidate the complicated link between the DNA methylation of ferroptosis-related genes and IS development. Understanding the role of dynamic DNA methylation in ferroptosis following IS remains in its infancy. Whether the methylation of gene expression plays a crucial role in regulating ferroptosis after IS warrants further investigation.

Fig. 4.

DNA methylation regulates ferroptosis during IS. DNA methylation at CpG islands modulates the expression of key ferroptosis-related genes. Hcy and ATF4 upregulate DNMT1, which methylates the GPX4 promoter, suppressing its expression and promoting ferroptosis. Fer-1 inhibits Hcy-mediated effects. Methylation of TP53 indirectly regulates SLC7A11 expression. ELAVL1 stabilizes DNMT3b, which methylates PINK1, promoting ferroptosis. ALOX12 methylation enhances PUFA peroxidation, contributing to ferroptosis. Me, methylation; DNMT, DNA methyltransferase; GPX4, glutathione peroxidase 4; PINK1, PTEN-induced kinase 1; ALOX12, arachidonate 12-lipoxygenase; SLC7A11, solute carrier family 7 member 11; PUFA, polyunsaturated fatty acid; Fer-1, ferrostatin-1; Hcy, homocysteine; ATF4, activating transcription factor 4; ELAVL1, embryo lethal abnormal vision-like 1; ALOX12, arachidonate 12-Lipoxygenase 12S Type; TP53, tumor protein p53

Histone Modifications of Ferroptosis in IS

As a fundamental form of epigenetic regulation, histone modifications, including acetylation, methylation, and ubiquitination, can alter the cellular vulnerability to ferroptosis by influencing the expression of genes involved in ferroptosis-related metabolic pathways. Therefore, we investigated the effect of histone modifications on IS through ferroptosis (Fig. 5).

Fig. 5.

Histone modifications involved in ferroptosis during IS. A Histone methylation: EZH2 catalyzes H3K27me3 and G9a/GLP catalyzes H3K9me1/2 on the NRF2 promoter, repressing NRF2 transcription and thereby reducing SLC7A11 expression to promote ferroptosis. LSD1 demethylates H3K4me2 on the SLC7A11 promoter, further suppressing its expression. SETD1B deposits H3K4me3 on FPN1, promoting iron efflux and inhibiting ferroptosis. G9a increases H3K9me3 on TFR1, FPN1, and HMOX1 genes, modulating iron metabolism. B Histone acetylation: HDAC1 deacetylates histones at the hepcidin promoter, inhibiting ferroptosis. HDAC9 promotes ferroptosis by increasing 4-HNE (lipid peroxidation marker) and TFR1 while suppressing GPX4 expression. HDAC2 inhibits p21, thereby promoting ferroptosis. C Histone ubiquitination: H2Bub1 positively regulates SLC7A11 transcription; its levels are modulated by the P53-USP7 axis. H2Aub regulates Hsp27, which suppresses ROS accumulation. RNF146 ubiquitinates and degrades ACSL4, inhibiting ferroptosis, while USP14 stabilizes ACSL4 by removing ubiquitin, promoting ferroptosis. SETD1B, SET domain containing 1B; TFR1, transferrin receptor 1; FPN1, ferroportin 1; HMOX1, heme oxygenase 1; EZH2, enhancer of zeste homologue 2; G9a/GLP, G9a and G9a-like protein; H3K9me1/2/3, mono/di/trimethylation of histone H3 at lysine 9; H3K4me2/3, di/trimethylation of histone H3 at lysine 4; H3K27me3, trimethylation of histone H3 at lysine 27; NRF2, nuclear factor E2-related factor; EZH2, enhancer of zeste homologue 2; SLC7A11, solute carrier family 7 member 11; LSD1, lysine-specific demethylase 1; HDAC, histone deacetylase; 4-HNE, 4-hydroxynonenal; GPX4, glutathione peroxidase 4; ROS, reactive oxygen species; USP7/14, ubiquitin specific protease 7/14; ACSL4, acyl-coa synthetase long-chain family member 4; USP14, ubiquitin-Specific Peptidase 14; P53, Tumor protein p53; P21, cyclin-dependent kinase inhibitor 1A; H2Bub1, histone H2B monoubiquitination 1; H2Aub, histone H2A monoubiquitination; HSP27, heat shock protein 27; RNF146, ring finger protein 146

Role of Histone Methylation in Regulating Ferroptosis During IS

In recent years, an increasing number of studies have suggested that histone methylation/demethylation participates in neuronal apoptosis and ferroptosis (Zhang et al. 2023b). This finding implies that investigating the potential role of histone methylation has significant implications in the treatment of IS. The SLC7A11/GPX4 pathway is essential for sustaining intracellular redox homeostasis and suppressing neuronal ferroptosis. Lysine-specific demethylase 1 (LSD1) has been demonstrated to primarily inhibit transcription via H3K4me2 demethylation. LSD1 inhibitors exert protective effects on mouse ganglion cells by blocking ROS-related oxidative stress, preventing H3K4me2 demethylation, and maintaining SLC7A11 expression (Li et al. 2015). Meanwhile, H3K9 demethylase KDM3B can upregulate SLC7A11 expression by cooperating with transcription factor ATF4 (Wang et al. 2020). Additionally, NRF2 serves as another crucial component of the GPX4 pathway. Typically, NRF2 operates as a transcription factor that regulates GPX4 transcription by binding to Kelch-like ECH-associated protein 1 (KEAP1) in the cytoplasm. NRF2 also limits free iron and enhances iron storage capacity by increasing the production of FTH1, a cellular iron-storage protein. Enhancer of zeste homologue 2 (EZH2), a catalytic subunit of the polycomb repressive complex 2, catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3). Recent evidence indicates that in LPS-treated BV2 cells, EZH2 recruits H3K27me3 to the Nrf2 gene promoter site to inhibit Nrf2 transcription (Cai et al. 2020). Heme oxygenase 1 (HMOX1) is a pivotal component integral to the cellular antioxidant defense machinery. By catalyzing heme degradation to generate biliverdin/bilirubin and carbon monoxide with potent antioxidant capabilities, it effectively neutralizes lipid peroxides, thereby inhibiting ferroptosis (Li et al. 2025d). G9a and its homologous protein, G9a-like protein (GLP), are lysine methyltransferases. In IS, G9a is highly expressed and influences HMOX1 expression by regulating H3K4Me3 levels (Yang et al. 2023). Pharmacological inhibition of G9a/GLP enhances learning and memory in mice, increases the gene expression of BDNF and Nrf2 in the hippocampal region, and reduces ROS (Griñán-Ferré et al. 2019). All of these factors are related to the pathogenesis of IS.

In addition to regulating the core pathways of ferroptosis, histone methylation also influences neuronal fate by modulating genes associated with iron metabolism. TF and its receptor TFR are localized in neuronal mitochondria. They enable the transport of extracellular iron into the mitochondria via a TF/TFR-dependent system, leading to iron buildup within the mitochondria. This accumulation induces excessive ROS production, triggering iron-induced neuronal death. Interestingly, mitochondrial iron overload can be negatively regulated by Fpn1, which expels iron from mitochondria. Studies have indicated that H3K9 trimethylation (H3K9me3) suppresses TfR1 expression, thereby alleviating neuronal ferroptosis (Lan et al. 2023). Histone lysine methyltransferase 1 B (SETD1B) is a histone lysine methyltransferase that regulates gene transcription by catalyzing H3K4me3. Human haploid cytogenetic studies have suggested that SETD1B participates in ferroptosis regulation by influencing key nodes in lipid metabolism pathways (Wang et al. 2023a). Elevated SETD1B expression was observed in MCAO mice and HT22 cells cultured under OGD/R conditions, accompanied by increased levels of ferroptosis markers. Knockdown of SETD1B alleviates ferroptosis in IS by reducing H3K4me3 enrichment in the transferrin receptor promoter, thereby decreasing TFR1 protein levels (Wang et al. 2025c). Similarly, in dopaminergic neurons, upregulation of H3K4me3 also exhibits neuroprotective effects against ferroptosis. This mechanism involves increased Fpn1 expression following H3K4me3 upregulation (Mu et al. 2020).

Based on the current limited literature, we can conclude that H3K4me3/me2 exerts an inhibitory effect on ferroptosis. It protects neurons by enhancing the antioxidant capacity and iron efflux by promoting the transcription of genes such as SLC7A11 and FPN1. Conversely, H3K27me3 promotes ferroptosis by suppressing the transcription of key antioxidant genes like NRF2. H3K9me3/me2/me1 exhibited context-dependent effects: H3K9me3 suppressed TFR1 expression, reduced iron uptake, and alleviated ferroptosis, whereas G9a/GLP-mediated H3K9me1/me2 promoted ferroptosis (Fig. 5a). Although these studies have provided insights into the interactions between histone methylation, ferroptosis, and IS, direct evidence demonstrating how histone methylation influences IS through ferroptosis regulation remains insufficient. Several questions remain to be answered in future studies.

Role of histone Acetylation in Regulating Ferroptosis During IS

In histone acetylation modifications, the dynamic equilibrium between HATs and HDACs determines chromatin accessibility and gene transcriptional activity. Following cerebral ischemia, hypoxic conditions compel cells to depend on anaerobic glycolysis for energy production. With reduced aerobic metabolism, the acetyl-CoA levels decline. Histone acetyltransferases require acetyl-CoA as a cofactor to execute histone acetylation steps (Demyanenko and Sharifulina 2021). This also disrupts the equilibrium between HATs and HDACs, directly regulating susceptibility to ferroptosis. Studies have indicated that cerebral ischemia upregulates HDAC9 protein levels both in vivo and in vitro (Sanguigno et al. 2023). Silencing HDAC9 significantly reduced two ferroptosis markers (iron overload and 4-HNE) in the temporoparietal cortex of mice following stroke. Furthermore, HDAC9 silencing prevents the elevation of HIF-1 and reduction of Sp1, thereby blocking the upregulation of its target gene TfR1 and the downregulation of Gpx4 to mitigate neuronal ferroptosis. Crucially, HDAC9 silencing not only prevents the deacetylation of HIF-1 and Sp1 after OGD/R but also inhibits the deubiquitination of HIF-1 and ubiquitination of Sp1 (Sanguigno et al. 2023). Genome-wide association studies have identified the HDAC9 gene region as a key risk locus for human atherosclerotic stroke and coronary artery disease (Prestel et al. 2019). Therefore, the development of medicines capable of precisely suppressing HDAC9 overexpression or activity may constitute a potential pharmacological method for reducing ferroptosis and brain injury following IS. Iron overload in cells arises from excessive iron in the extracellular environment and the inhibition of cellular iron efflux via hepcidin. Hepcidin, commonly termed as an iron regulator, attaches to transferrin to trigger its internalization and lysosomal degradation, thus directly regulating iron export from cells to plasma (Sandnes and Reikvam 2024). HDAC1 modulates hepcidin transcription in a SMAD4-dependent manner. This regulatory mechanism may contribute to the maintenance of iron homeostasis in neuronal cells (Yin et al. 2018). P21 (cyclin-dependent kinase inhibitor 1A) is a ferroptosis inhibitor regulated independently of P53. It maintains GPX4 protein stability (Zheng et al. 2024). Interestingly, inhibition of HDAC2 leads to increased histone H4K16 acetylation levels in the p21 promoter region, upregulating p21 expression and protecting HT22 cells from oxidative stress damage (Peng et al. 2015). Additionally, a study reported that the HDAC2 inhibitor HDI-1 provides neuroprotection by activating the Nrf2 pathway and reducing glutamate accumulation in human brain microvascular endothelial cells during OGD (Ling et al. 2025). These factors were all related to ferroptosis (Fig. 5b). Recent studies have indicated that ferroptosis stimuli (such as depletion of the antioxidant glutathione) appear to enhance the binding capacity of Sp1 and Sp3 at specific protective gene promoters (e.g., Gpx4 and Mkp1/Mkp3) by increasing their acetylation. Selenium or Class I HDAC inhibitors can potentiate this protective response both in vitro and in vivo, thereby triggering neuroprotective effects (Alim et al. 2019).

Role of Histone Ubiquitination in Regulating Ferroptosis During IS

Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is an epigenetic marker typically associated with transcriptional activation. During erastin-induced ferroptosis, H2Bub1 levels decrease, and its absence leads to SLC7A11 expression inactivation, further inducing ferroptosis (Wang et al. 2019b). Additionally, P53 negatively regulates H2Bub1 levels by promoting nuclear translocation of the deubiquitinating enzyme USP7. In contrast, the ubiquitination of histone H2AK119 (H2Aub) is generally associated with gene silencing. In rodent models, H2Aub participates in regulating glycolysis and mitochondrial function by interacting with heat shock protein 27 (Hsp27). This reduced mitochondrial ROS production and inhibited ferroptosis during reperfusion (Shi et al. 2023b) (Fig. 5c). Histone ubiquitination is primarily mediated by E3 ubiquitin ligases, and ACSL4 is recognized as a ferroptosis promoter associated with ubiquitin modifications. RING finger protein 146 (RNF146) operates as an E3 ligase, and studies have indicated that it promotes DNA repair to prevent neuronal death in the ischemic cerebral cortex. During the early phase of OGD/R treatment, RNF146 directly mediates ACSL4 ubiquitination and degradation. RNF146 overexpression alleviated neuronal injury in MCAO mice by reducing ACSL4-regulated ferroptosis. Furthermore, overexpression of RNF146 can block OGD/R-induced elevation of LDH, MDA, and Fe²⁺ levels (Jin et al. 2023). Ubiquitin-specific proteases (USPs) identify ubiquitinylation signals on specific target proteins and catalyze their deubiquitination. Inhibition of USP14 protects neurons from ferritin autophagy-mediated ferroptosis, thereby attenuating iron-induced neuronal injuryin mice (Li et al. 2021). Furthermore, USP14 enhances ACSL4 protein expression through deubiquitination (Hao and Liu 2025).

These results establish an important link between histone modifications and ferroptosis regulation in IS.

Roles of microRNAs in Regulating Ferroptosis During IS

Among non-coding RNAs, miRNAs represent the most thoroughly investigated class within the field of epigenetics. In recent years, a growing body of research has indicated that miRNAs have emerged as key post-transcriptional regulators of ferroptosis-related genes (Qin et al. 2024; Yang et al. 2025a). Zhang et al. employed bioinformatic methods to identify 3,747 miRNA-mRNA-TF regulatory pairs linked to oxidative stress and ferroptosis in patients with IS, including miR-188-3p-GPX4-ATF2 and miR-4469-CDKN1A-BACH2, both of which were significantly correlated with IS (Zhang et al. 2024b). Consistent with this, Fan et al. found six differentially elevated ferroptosis-related genes in patients with IS from the perspective of immune infiltration. Among these, CDKN1A/JUN has emerged as a key ferroptosis-related gene pair regulated by miR-22-3p, miR-429, and miR-139-5p, and plays a crucial role in the IS immune microenvironment (Fan et al. 2022). This suggests that the miRNA-mediated post-transcriptional regulation of these genes may influence ferroptosis during IS. It has been established that miR-10a, miR-34b/c, miR-27a, and miR-300 are dysregulated in patients with IS and linked to stroke risk and mortality (Ryu et al. 2020). Among these, miR-27a is critical for cell survival and is implicated in the progression of IR injury across multiple organs, including the liver, kidneys, heart, and brain (Salimi et al. 2023). In rodent models, miR-27a upregulation exacerbates cerebral ischemia/reperfusion injury by inducing ferroptosis through targeting SLC7A11 and Nrf2 (Zhu et al. 2023; Zhang et al. 2022). Additionally, researchers have found that both miR-9-5p and miR-23b participate in ferroptosis following IS by targeting the Nrf2 axis (Xin et al. 2021; Zhao et al. 2025b).

Clinical analysis indicated that plasma exosomal miR-30a-5p levels were significantly upregulated within 6 h post-ischemia and were markedly downregulated on days 1–3. This suggests that miRNA-30a-5p could serve as a potential biomarker for diagnosing IS and distinguishing between the hyperacute and subacute phases (Wang et al. 2018). Interestingly, preclinical studies have revealed that the overexpression of miR-30a-5p elevates the levels of ROS and MDA following cerebral ischemia/reperfusion injury. Inhibition of miR-30a-5p upregulates Sirt1 and facilitates Nrf2 nuclear translocation. This enhances Gpx4 and GSH levels, and suppresses ferroptosis in HT-22 cells (Wang et al. 2024a). This demonstrated that miR-30a-5p is a positive regulator of ferroptosis. Another study found that Sirt6 participates in the activation of the Nrf2 signaling pathway through deacetylation, whereas miR-370 accelerates cerebral ischemia–reperfusion injury in the rat brain by targeting and inhibiting Sirt6 (Ruan et al. 2020). These findings suggested that miRNA-mediated suppression of mRNA expression may lead to alterations in histone acetylation patterns, thereby affecting the expression of genes associated with ferroptosis.

Autophagy is an evolutionarily conserved pathway that disrupts damaged organelles and macromolecules through lysosomal degradation. This process, which is governed by autophagy-related genes, is essential for meeting the cellular metabolic demands and enabling organelle turnover. For example, autophagy-related genes 5 and 7 (ATG5, ATG7) contribute to ferroptosis. Interestingly, miR-193b acts as a key regulator of ferroptosis by inhibiting autophagy and reducing iron-related damage by targeting ATG7 (Fan et al. 2023).

These findings support the role of miRNAs as important therapeutic targets for alleviating ferroptosis in IS (Fig. 6).

Fig. 6.

miRNA regulation of ferroptosis in IS. miR-30a-5p and miR-23b directly target Nrf2, while miR-9-5p targets ZBTB20 and miR-370 targets SIRT6, both of which positively regulate Nrf2 expression. Reduced Nrf2 activity decreases SLC7A11 transcription, which is also directly inhibited by miR-27a. Downregulation of SLC7A11 leads to decreased GPX4 expression, which is further suppressed by miR-188-3p, ultimately promoting ferroptosis. miR-22-3p and miR-4469 target CDKN1A, leading to increased ROS accumulation and ferroptosis. miR-139-5p targets JUN, resulting in elevated intracellular Fe²⁺ levels that promote ferroptosis. miR-193b targets ATG7, thereby inhibiting ferroptosis. ZBTB20, zinc finger and BTB domain containing 20; SIRT6, sirtuin6; NRF2, nuclear factor erythroid 2-related factor 2; LC7A11, solute carrier family 7 member 11; GPX4, glutathione peroxidase 4; CDKN1A, cyclin dependent kinase inhibitor 1A; JUN, Jun proto-oncogene; ATG7, autophagy related 7; ROS—Reactive Oxygen Species

Epigenetic Regulators as Therapeutic Targets for Ferroptosis in IS

Ferroptosis plays a significant role in IS pathogenesis, making anti-ferroptosis strategies a highly promising therapeutic approach. Crucially, in preclinical models, the ferroptosis inhibitors Liproxstatin-1 and Ferrostatin-1 have shown remarkable efficacy, markedly reducing brain infarct volume and neurological impairments following ischemia–reperfusion injury in mice (Du et al. 2024; Shi et al. 2024). Epigenetic regulation can correct abnormal gene expression patterns without altering DNA sequences. As reviewed herein, epigenetic modifications involving DNA methylation, histone modifications, and miRNAs significantly ameliorate ferroptosis following an experimental stroke. Therefore, targeting epigenetic regulation is a promising strategy for counteracting ferroptosis following IS. Meanwhile, owing to their abundant supply and accessibility, cell-based therapeutic strategies, particularly those utilizing adipose-derived stem cells (ADSCs), have attracted considerable interest. Various preclinical studies have indicated that ADSCs can mitigate IS damage by promoting angiogenesis and synaptic remodeling, lowering neuronal atrophy, inflammatory cytokine levels, and glial scar formation (Wang et al. 2025b, 2022a). Recent research has revealed that intranasal administration of ADSC-Exos during the acute phase suppresses ferroptosis by sending miR-760-3p to neurons, thereby downregulating ChaC glutathione-specific gamma-glutamylcyclotransferase 1 gene expression and reducing ferroptosis-associated protein expression (Wang et al. 2023c). Furthermore, genetically engineered MSC-derived hybrid vesicles overexpressing SOD2 combined with ROS-responsive resveratrol-loaded liposomes have demonstrated efficacy in alleviating ischemia–reperfusion injury by scavenging ROS and restoring mitochondrial homeostasis (Shen et al. 2025), highlighting the potential of engineered vesicle platforms for anti-ferroptosis therapy. Additionally, various natural compounds have demonstrated significant anti-ferroptosis properties through epigenetic regulatory pathways. Piceatannol, a derivative of the polyphenolic compound resveratrol, prevents GPX4 degradation through USP14-mediated deubiquitination, thereby exerting neuroprotective effects against cerebral ischemia (Zhao et al. 2025a). Baicalin is a natural ferroptosis inhibitor that not only suppresses Fe²⁺ formation and reduces GSH consumption but also diminishes GPX4 degradation (Guo et al. 2025a). Recent studies have revealed that baicalin promotes Forkhead box A2 (FOXA2) deacetylation by upregulating SIRT6 expression, thereby suppressing FOXA2 transcription. This leads to increased SLC7A11 expression, ultimately mitigating I/R injury in IS (Fang et al. 2024). Ginsenoside Rd promotes ROS clearance by upregulating miR-139-5p and activating the Nrf2 pathway (Yao et al. 2022). Melatonin, an endogenous hormone primarily secreted by the pineal gland, is known for its antioxidant, anti-inflammatory, and anti-apoptotic properties. It has been discovered to regulate ACSL4 ubiquitination by promoting mouse double minute 2 expression, thereby influencing ferroptosis (Ji et al. 2024a). As a natural product, mangiferin extract can cross the blood–brain barrier and alleviate oxidative stress while improving mitochondrial function by targeting the NRF2/HO-1 and SIRT1/PGC-1α signaling pathways, demonstrating significant therapeutic potential in IS animal models (Zhang et al. 2024a). Although preclinical animal studies have supported the efficacy of these approaches, their clinical translation is still at a preliminary stage. Currently, the U.S. FDA has approved 13 epigenetic drugs for marketing to treat various cancers, but no such drugs have yet entered clinical stage testing for stroke treatment (Dai et al. 2024). Targeting epigenetic modifications in IS therapy remains a challenge.

Conclusion and Perspectives

Evidence supporting the involvement of epigenetic modifications in IS has accumulated substantially. Our review highlights that DNA methylation of ferroptosis-related genes, including GPX4, ALOX12, PINK1, and SLC7A11, represents a critical epigenetic mechanism that links IS pathogenesis to ferroptotic cell death. Furthermore, dysregulation of specific miRNAs (miR-27a, miR-30a-5p, miR-193b, miR-9-5p, and miR-370) and histone modifications (H3K27me3, H3K9me1/2/3, H3K4me2/3, and histone acetylation/ubiquitination) modulates neuronal susceptibility to ferroptosis following cerebral ischemia.

Among the epigenetic mechanisms, histone modifications have been the most extensively characterized in IS-associated ferroptosis. EZH2-mediated H3K27me3 deposition at the NRF2 promoter suppresses antioxidant defense, whereas G9a/GLP-catalyzed H3K9 methylation regulates iron metabolism genes. HDAC9 upregulation promotes ferroptosis by reducing GPX4 expression, whereas HDAC1 and HDAC2 modulate hepcidin and P21, respectively, to influence iron homeostasis and oxidative stress responses. Additionally, histone ubiquitination through the H2Bub1/SLC7A11 and H2Aub/Hsp27 axes provides another layer of ferroptosis regulation in IS.

Notably, histone lactylation has emerged as a novel epigenetic modification bridging metabolic alterations and ferroptosis in IS (Xiong et al. 2024). Elevated lactate levels following cerebral ischemia promote H3K9 lactylation, which upregulates ACSL4 expression and sensitizes neurons to ferroptosis (Li et al. 2025a). This finding opens new avenues for understanding how metabolic reprogramming during ischemia influences cell fate decisions through epigenetic mechanisms.

Translational Implications

The convergence of epigenetic regulation and ferroptosis in IS presents several promising translational opportunities. First, epigenetic biomarkers, particularly circulating miRNAs (e.g., miR-30a-5p) and DNA methylation signatures, hold potential for early IS diagnosis, patient stratification, and therapeutic response monitoring. Second, existing FDA-approved epigenetic drugs may be repurposed for IS treatment: DNMT inhibitors (e.g., 5-azacytidine and decitabine) could restore GPX4 and SLC7A11 expression(Yan et al. 2023; Dong et al. 2024), while HDAC inhibitors (e.g., valproic acid and vorinostat) have demonstrated neuroprotective effects in preclinical stroke models by modulating ferroptosis-related gene expression (Chen et al. 2024; Luo et al. 2025). Third, miRNA-based therapeutics, including miRNA mimics (for ferroptosis-inhibiting miRNAs like miR-193b) and antagomirs (for ferroptosis-promoting miRNAs like miR-27a), represent a highly specific approach to modulate ferroptosis pathways. Fourth, CRISPR-based epigenetic editing technologies (Bindal et al. 2025), employing dCas9 fused with DNMT3A or TET1 for targeted DNA methylation/demethylation or with p300/HDAC for site-specific histone modifications, offer unprecedented precision for therapeutic intervention at specific ferroptosis-related loci (Gupta et al. 2025).

Future Research Directions

Despite significant progress, several critical questions remain to be addressed. Mechanistically, future studies should: (1) determine whether epigenetic modifications primarily trigger ferroptosis or secondarily exacerbate ischemic injury; (2) characterize the temporal dynamics of epigenetic changes across the hyperacute, acute, and chronic phases of IS; (3) investigate cell type-specific epigenetic regulation of ferroptosis in neurons, astrocytes, microglia, and endothelial cells; and (4) explore the roles of understudied modifications, including histone phosphorylation, SUMOylation, and crotonylation, in IS-associated ferroptosis. Technically, advances in single-cell epigenomics, spatial transcriptomics, and multi-omics integration are essential to dissect the heterogeneous epigenetic landscape of the ischemic penumbra. Clinically, priorities include: (1) validating epigenetic biomarkers in large, diverse patient cohorts; (2) conducting preclinical studies evaluating combination therapies targeting both epigenetic modulators and ferroptosis inhibitors; (3) optimizing drug delivery systems (e.g., nanoparticles and exosomes) for CNS penetration; and (4) designing clinical trials stratified by epigenetic profiles to identify patient subgroups that are most likely to benefit from epigenetic-ferroptosis targeted therapies.

In conclusion, epigenetic regulation of ferroptosis represents a highly promising therapeutic frontier for IS. A growing understanding of how DNA methylation, histone modifications, and non-coding RNAs orchestrate ferroptotic cell death provides a strong foundation for the development of novel diagnostic tools and targeted interventions. Continued interdisciplinary collaboration among epigeneticists, neuroscientists, and clinicians will be essential to translate these mechanistic insights into improved outcomes for patients with IS.

Author Contributions

JLG: Writing – review & editing, Writing – original draft. XYY and JNW: Writing – review & editing, Visualization. ZYC, XLZ, and YW: Writing – review & editing, Visualization. WLL and MHW: Funding acquisition, Conceptualization, Project administration. All authors reviewed the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (82274428, 82474435), Natural Science Foundation of Jiangsu Province (BK20241996), Jiangsu Provincial Administration of Traditional Chinese Medicine (ZT202102), and Key Medical Research Project of the Jiangsu Provincial Health Commission (K2023009).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethical Approval

Not applicable.

Consent for Publication

Not applicable.

Consent to Participate

Not applicable.