Ferroptosis intensifies ischemic injury through mechanisms involving iron overload, lipid peroxidation, and mitochondrial dysfunction (Tuo and Lei, 2024) (Figure 2). During cerebral ischemia, disrupted iron homeostasis leads to the accumulation of labile iron, which catalyzes Fenton reactions and generates hydroxyl radicals (Guo et al., 2023). These radicals initiate lipid peroxidation of PUFAs in neuronal membranes, resulting in the production of cytotoxic aldehydes, such as 4-hydroxynonenal (4-HNE) and MDA, which disrupt membrane integrity and organelle function (Delgado-Martín and Martínez-Ruiz, 2024; Ayala et al., 2014). The inactivation of GPX4, a hallmark of ferroptosis, further depletes antioxidant defenses, permitting the unchecked accumulation of lipid peroxides (Wei, 2024). Mitochondrial dysfunction exacerbates ferroptosis injury (Pedrera, 2025; Tian, 2024). Ischemic stress induces mitochondrial membrane depolarization, ATP depletion, and ROS overproduction, creating a vicious cycle that amplifies lipid peroxidation (Huang, 2023). Additionally, endoplasmic reticulum (ER) stress triggered by ischemia activates the unfolded protein response (UPR), which intersects with ferroptosis via the IRE1α-XBP1 axis (Yang, 2024; Li Y., 2024).

Ferroptosis synergizes with neuroinflammatory pathways to exacerbate ischemic damage (Chen, 2023b). Dying neurons release damage-associated molecular patterns (DAMPs), which activate microglia and astrocytes (Lin M. M., 2022). In glutamatergic neurons, ferroptosis-driven GPX4 dysfunction and mitochondrial collapse amplify excitotoxicity, while astrocytes lose their ability to regulate glutamate or maintain the blood–brain barrier (BBB), exacerbating edema (Jacquemyn et al., 2024; Mahmoud, 2019). Ferroptosis cells secrete pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), along with ROS, leading to disruption of the BBB, recruitment of peripheral immune cells, and propagation of secondary injury (Chen, 2023b). Infiltrating macrophages and activated microglia shift to pro-inflammatory states, further amplifying oxidative stress and releasing DAMPs that trigger NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation (Zhan, 2022). Additionally, ferroptosis disrupts mitochondrial energy metabolism in microglia, impairing their ability to clear apoptotic debris via efferocytosis (Liu Z., 2025). Accumulated cellular debris perpetuates DAMP release, activating the NLRP3 inflammasome and caspase-1-dependent pyroptosis in neighboring cells, thereby expanding the ischemic penumbra (Zhan, 2022). Oligodendrocytes contribute to white matter damage through iron-dependent oxidation of myelin lipids, while endothelial cell ferroptosis disrupts vascular integrity, facilitating leukocyte infiltration (Shen, 2022; Lin X., 2022). Collectively, these cross-amplifying mechanisms drive neuroinflammation, neuronal death, and tissue destruction, ultimately worsening neurological outcomes.

The role of ferroptosis in ischemic stroke has been well-documented in experimental studies. Pharmacological ferroptosis inhibitors have been shown to reduce infarct size and improve neurological outcomes in stroke models by targeting lipid peroxidation and disrupting the vicious cycle of oxidative stress (Du and Guo, 2022). However, a deeper dive into the literature reveals a complex and sometimes contradictory landscape of molecular regulators, which must be critically appraised to guide future research and therapy development.

Accumulating evidence supports the therapeutic targeting of ACSL4 protein stability to modulate ferroptosis sensitivity. ACSL4 exacerbates ischemic stroke via two parallel mechanisms: triggering neuronal ferroptosis via lipid peroxidation and fueling neuroinflammation, the latter being evidenced by suppressed cytokine production upon its downregulation in microglia (Cui, 2021). Melatonin mitigates ferroptosis and neuronal damage in ischemic stroke models, an effect achieved through the MDM2-mediated ubiquitination and degradation of ACSL4, leading to a reduction in ferroptosis markers like intracellular Fe²⁺ (Yu, 2024; Ji et al., 2024). This finding highlights ACSL4 as a promising target. Tongqiao Huoxue Decoction (TQHX) confers neuroprotection against cerebral ischemia–reperfusion injury by promoting the ubiquitin-dependent degradation of ACSL4 (Ou, 2024). Collectively, these findings indicate that regulating ACSL4 stability is a viable anti-ferroptotic strategy.

A significant challenge in the field is the context-dependent functionality of several key players. In mouse tumor xenograft models, Glutaredoxin 5 (GLRX5) was shown to mediate ferroptosis (Lee, 2020). Interestingly, the regulation of this pathway appears to be context-dependent. A study by Zhou et al. demonstrated that Salvia miltiorrhiza Bge processed with porcine cardiac blood alleviated cerebral ischemia–reperfusion injury by inhibiting GLRX5-mediated ferroptosis (Zhou S., 2024). Conversely, Liu et al. found that exosomes from hypoxic pretreated ADSCs attenuated ultraviolet light-induced skin injury by delivering GLRX5 to inhibit ferroptosis (Liu, 2024). This apparent contradiction underscores that the role of molecules like GLRX5 may not be absolute but is probably shaped by cell type, subcellular localization, and the nature of the insult. It cautions against the oversimplified classification of targets as purely “pro-” or “anti-ferroptotic” and emphasizes the need for cell-type-specific mechanistic studies.

Ferroptosis is not an isolated event but a coordinated pathology across the neurovascular unit. Evidence now shows that ferroptosis occurs simultaneously in multiple cell types. In a rat focal ischemia model, Ginsenoside Rd. (G-Rd) preserved blood–brain barrier integrity by activating the NRG1/ErbB4 signaling pathway to enhance tight junction protein (ZO-1, occludin, claudin-5) expression and reduce endothelial cell loss, and by inhibiting endothelial cell ferroptosis (Hu, 2024). Cottonseed oil (CSO) treatment was shown to protect BBB integrity, reduce iron influx, and inhibit ferroptosis in middle cerebral artery occlusion (MCAO) models (Sun, 2023). Piceatannol protects against cerebral ischemia or reperfusion injury by inhibiting neuronal ferroptosis through the USP14/GPX4 axis (Zhao, 2025), and ACSL4 in microglia fuels neuroinflammation (Cui, 2021). The integration of these findings reveals a devastating positive feedback loop: neuronal ferroptosis releases DAMPs that activate microglia, whose inflammatory response further sensitizes neurons and endothelial cells to ferroptosis. This paradigm shift argues that the most effective therapies will be those that can disrupt this intercellular vicious cycle, either through multi-targeted agents like L-F001 or combination regimens (Peng, 2022).

The identification of fatty acid-binding protein 5 (FABP5) as a specific biomarker of ferroptosis in damaged human neurons marks a significant advance (Peng, 2024). FABP5 is uniquely upregulated and stabilizes at the cell surface during early ferroptosis, where it drives sensitivity by enriching pro-ferroptotic, long-chain polyunsaturated fatty acid phospholipids through a positive-feedback loop. This specificity was confirmed as FABP5 marked damaged neurons in the ischemic penumbra in a mouse model and was significantly elevated in hypoxically damaged human postmortem neurons. These findings establish FABP5 as a robust pathological marker for detecting ferroptosis in human hypoxic/ischemic conditions, offering valuable potential for assessing stroke severity and monitoring therapeutic responses. Furthermore, the discovery of metabolic triggers such as the PPM1K/BCKDHA axis expands the understanding of ferroptosis beyond classic redox pathways, linking branched-chain amino acid metabolism to neuronal vulnerability (Li T., 2023).

Additionally, nuclear factor erythroid 2-related factor 2 (NRF2) activation, as shown in studies involving Icariside II (ICS II) pretreatment, conferred protection by reducing oxidative stress and promoting long-term recovery (Gao, 2023). NRF2 activation orchestrates a coordinated defense against cerebral ischemia by transcriptionally repressing ferroptosis through the GPX4/SLC7A11 axis and iron metabolism regulation, while concurrently mitigating oxidative stress, neuroinflammation, and apoptosis. This integrated mechanism substantiates the therapeutic potential of NRF2 activators like ICS II in fostering neurological recovery (Deng, 2023). Caffeic acid, administered in a permanent MCAO (pMCAO) rat model, was shown to alleviate cerebral ischemic injury in rats by resisting ferroptosis via NRF2 signaling pathway (Li X. N., 2024).

Other components, such as Rehmannioside A, exhibited significant neuroprotective effects by alleviating cognitive impairments and reducing infarct size and neurological deficits (Fu, 2022). Separately, in an MCAO/R rat model, modulation of the HSP90-GCN2-ATF4 pathway was shown to significantly mitigate ferroptosis and necroptosis, suggesting a dose-dependent neuroprotective effect (Zhou Y., 2024). Meanwhile, another study elucidated a distinct mechanism where USP14 deubiquitinates and stabilizes NCOA4, thereby promoting ferritinophagy, increasing free iron, and triggering ferroptosis. Notably, inhibiting either USP14 or NCOA4 blocked this process and protected neurons, identifying this axis as a promising therapeutic target (Li, 2021). Collectively, these discoveries synergize with established targets such as ACSL4 and FABP5, to map a dynamic interplay of redox imbalance, metabolic dysregulation, and cell-type-specific vulnerability, thereby advancing therapeutic strategies for ferroptosis modulation in stroke. A summary of the experimental evidence is provided in Table 1.

The emerging evidence linking ferroptosis to ischemic stroke is accompanied by a growing number of seemingly contradictory findings. Systematically deconstructing the origins of these divergent results, including model system disparities, cell-type-specific pathway engagement, temporal dynamics, and methodological variations, is essential to refine our mechanistic understanding and guide successful therapeutic development.

The investigation of ferroptosis in ischemic stroke is fundamentally shaped by the experimental context, where a stark disparity exists between reductionist in vitro systems and complex in vivo models, a critical consideration for interpreting the literature and guiding translational efforts. In vitro models, such as oxygen–glucose deprivation (OGD) in homogenous neuronal cultures, are powerful for deconstructing the core ferroptosis pathway (Ni, 2022). In these systems, core pathway inhibitors like Ferrostatin-1 and Liproxstatin-1 demonstrate robust efficacy by directly scavenging lipid radicals (Chen, 2023a; Zhang, 2023), while agents such as Piceatannol validate the GPX4 axis by enhancing its stability, and Deferoxamine (DFO) effectively chelates intracellular iron (Zhang S., 2022; Li, 2008).

However, the therapeutic efficacy observed in vitro frequently encounters significant translational barriers in vivo. A potent compound in a dish may fail to cross the blood–brain barrier, be metabolically inactivated systemically, or elicit unexpected off-target effects within the integrated neurovascular unit, where a drug’s action on one cell type can inadvertently impact another, as exemplified by the context-dependent role of molecules like GLRX5 (Zhou S., 2024; Liu, 2024; Liu H., 2025). This principle of context-dependent efficacy is further highlighted by studies on cellular stress response mechanisms (Wang, 2019). For instance, research comparing the differential neuroprotection offered by HSP70-hom gene single nucleotide polymorphisms demonstrated distinct outcomes between in vitro neuronal hypoxic injury models and in vivo rat middle cerebral artery occlusion models, underscoring how a protective genotype in a controlled cellular environment may not fully recapitulate its effects within the complex pathophysiology of a living organism.

Beyond in vitro or in vivo models, the specific pathophysiological context of the ischemic model itself profoundly influences the observed therapeutic mechanisms and windows. Ischemia–reperfusion (I/R) models, such as transient middle cerebral artery occlusion (tMCAO), are characterized by a robust oxidative burst and a significant exogenous iron load from hemoglobin, creating an environment where interventions like DFO are particularly effective (Hanson, 2009). In contrast, permanent ischemia models lack this iron surge and are defined by sustained metabolic failure, thereby placing greater emphasis on the collapse of endogenous antioxidant systems, making strategies that boost the GSH/GPX4 axis more relevant (Zhang J., 2022). Furthermore, other models like global hypoperfusion highlight white matter injury (Ran, 2020). This model-specificity is a primary reason for the varying reported efficacy of ferroptosis inhibitors across studies.

Compounding this complexity, susceptibility to ferroptosis demonstrates marked heterogeneity across distinct neural cell types. Neurons, with their high metabolic rate and GPX4-dependence, are the primary targets (Yuan, 2021). Astrocytes assume a dual role, supporting neuronal antioxidant defenses while being vulnerable themselves (An, 2025). Microglial ferroptosis amplifies neuroinflammation via ACSL4 and the NLRP3 inflammasome, a mechanism distinct from neuronal death (Zhou, 2023). Endothelial ferroptosis directly disrupts the BBB, initiating a vicious cycle of damage (Zheng, 2022). This cellular heterogeneity explains why studies focusing on different cell types emphasize divergent pathways and underscores the need for cell-type-specific or multi-target therapeutic approaches.

Adding a crucial temporal dimension, the contribution of ferroptosis evolves dynamically post-stroke. The hyperacute phase is characterized by rapid antioxidant failure, creating a narrow window for direct inhibitors (Ginsberg, 2008). However, it remains unclear precisely how the efficacy of a given anti-ferroptotic agent shifts throughout the disease continuum of ischemic stroke. As the injury progresses into the subacute phase, secondary neuroinflammation amplifies and intertwines with ferroptosis, potentially diminishing the efficacy of acute-phase interventions (Bernardo-Castro, 2021). This is exemplified by microglia, which promotes injury not only via acute ferroptosis but also through sustained inflammation, a later-phase response that can render a drug administered only at onset ineffective (Liu Z., 2025). This temporal shift means that studies conducted at different time points capture fundamentally distinct pathological snapshots, favoring treatment strategies that are precisely timed or multi-mechanistic.

Finally, the interpretation of ferroptosis is further complicated by significant methodological heterogeneity. Differences in pharmacological inhibitors, genetic tools (e.g., acute knockdown vs. constitutive knockout), and biomarkers (e.g., non-specific MDA vs. specific lipidomics) mean that studies employing different toolkits are effectively measuring different facets of the process (Chen, 2023a; Cui, 2021; Luo, 2025).

While numerous ferroptosis inhibitors show promise in preclinical studies, their relative efficacy, optimal time windows, and inherent limitations vary considerably, necessitating a critical appraisal. The radical-trapping antioxidants Ferrostatin-1 and Liproxstatin-1 robustly suppress lipid peroxidation and reduce infarct volumes, establishing them as foundational tools in the field (Yang, 2022). However, comparative studies suggest that Liproxstatin-1 may exhibit superior metabolic stability and a longer plasma half-life, potentially translating to a broader therapeutic window than Ferrostatin-1 in vivo (Skouta, 2014; Friedmann Angeli, 2014). This distinction is critical, as the narrow temporal window for effective neuroprotection post-stroke demands inhibitors with rapid and sustained action.

Iron chelators such as DFO act proximally by sequestering the catalytic iron required for the Fenton reaction, making them highly effective in the hyperacute phase dominated by iron overload (Moradi, 2025). This is supported by a clinical trial in ischemic stroke patients, which demonstrated DFO’s safety, tolerability, and its mechanism of action in reducing transferrin iron saturation and diminishing the labile iron pool (Millán et al., 2021; Bruzzese, 2023). However, the therapeutic promise of DFO is constrained by a critically narrow window of efficacy and challenges such as poor BBB penetration and potential systemic toxicity. This profile stands in clear contrast to inhibitors like Liproxstatin-1, which targets the lipid peroxidation cascade downstream of iron and may offer a more flexible intervention timeline.

Beyond these classic inhibitors, compounds like NecroX-7 and Eriodictyol-7-O-glucoside, which possess polar structural features, demonstrate efficacy by scavenging ROS and attenuating iron toxicity, yet their blood–brain barrier permeability and precise pharmacokinetics remain less characterized (Lu, 2024). Similarly, indirect modulators like Kinase B Suppressors (KBs), a novel ferroptosis suppressor, target ferroptosis through remote ischemic postconditioning (RIPostC) mechanisms, highlighting its potential for clinical translation (Huang, 2024).

Targeting lipid peroxidation extends beyond radical scavenging to include enzymatic regulation. A key approach involves inhibiting enzymes like ACSL4, which incorporates polyunsaturated fatty acids into membranes, a critical step for ferroptosis (Gan, 2022). This is exemplified by the small molecule TQHX and rosiglitazone, which promote the degradation or inhibition of ACSL4, thereby reducing lipid peroxidation (Ou, 2024; Li, 2019). This mechanism may confer advantages when upstream radical generation is intractable, though chronic use risks disrupting essential lipid metabolism. Beyond direct enzyme targeting, a complementary strategy involves enhancing the body’s endogenous antioxidant systems. Compounds like Rhein and Reganone achieve this by activating the NRF2 signaling pathway, which upregulates a network of protective genes, including SLC7A11 and GPX4 (Fu, 2022; Liu, 2023). The trade-off lies between the precise action of enzyme inhibitors and the broad, systemic protection offered by NRF2 activators (Xiong, 2023).

Neuroinflammation amplifies ferroptosis via DAMPs and cytokines, creating a vicious cycle of damage (He, 2024). While anti-inflammatory agents like edaravone dexborneol and minocycline can attenuate this inflammatory drive (Zhao, 2007; Zhao, 2024), their standalone efficacy in stroke is often limited. This has spurred the development of intrinsically multi-target agents. L-F001 and dimethyl fumarate (DMF) exemplify this paradigm, simultaneously targeting oxidative stress (including ferroptosis), inflammation (e.g., via NF-κB), and in DMF’s case, even promoting astrocytic health (Peng, 2022; Vanani, 2021) The proven efficacy of DMF in other neuroinflammatory contexts like Alzheimer’s disease reinforces the validity of this pleiotropic strategy (Wang, 2024). The key advantage of these agents is their ability to intervene at multiple nodes of the injury cascade, making them potentially effective across a wider temporal spectrum, especially in the subacute phase where pure ferroptosis inhibitors may fail. Consequently, therapeutic strategies that combine the modulation of pathways like astrocytic glutamate metabolism with antioxidant and anti-inflammatory approaches may offer superior outcomes.

Emerging therapeutic platforms aim to overcome the limitations of small molecules through enhanced targeting and delivery. Nanoparticle-based systems, such as recombinant human ferritin (rHF), represent a significant advance by functioning as scavengers for multiple types of ROS and inhibiting parallel cell death pathways (Qi, 2024). Their primary advantage lies in their potential for engineered BBB penetration and targeted delivery, which could maximize local efficacy while minimizing systemic exposure. Similarly, gene therapy approaches that upregulate GPX4 or silence ACSL4 offer the ultimate in specificity, allowing for the precise and potentially long-lasting modulation of key ferroptosis nodes (Bai, 2024; Wang, 2022). However, both platforms face substantial translational hurdles, including immunogenicity, manufacturing complexity, and the safe control of transgene expression. While small molecule inhibitors of enzymes like LOXs continue to be explored for their precision (Cakir-Aktas, 2023), the field is increasingly recognizing that the future may lie in these sophisticated platforms capable of delivering therapeutic payloads with unprecedented accuracy to the injured brain.