Ferroptosis inhibitors: past, present and future

Ferroptosis is a non-apoptotic mode of programmed cell death characterized by iron dependence and lipid peroxidation. Since the ferroptosis was proposed, researchers have revealed the mechanisms of its formation and continue to explore effective inhibitors of ferroptosis in disease. Recent studies have shown a correlation between ferroptosis and the pathological mechanisms of neurodegenerative diseases, as well as diseases involving tissue or organ damage. Acting on ferroptosis-related targets may provide new strategies for the treatment of ferroptosis-mediated diseases. This article specifically describes the metabolic pathways of ferroptosis and summarizes the reported mechanisms of action of natural and synthetic small molecule inhibitors of ferroptosis and their efficacy in disease. The paper also describes ferroptosis treatments such as gene therapy, cell therapy, and nanotechnology, and summarises the challenges encountered in the clinical translation of ferroptosis inhibitors. Finally, the relationship between ferroptosis and other modes of cell death is discussed, hopefully paving the way for future drug design and discovery.

With the proposal of ferroptosis, ferroptosis has been found to mediate the pathogenesis of many diseases.In recent years, it has been found that ferroptosis inhibitors also play an important role in neurodegenerative diseases (e.g., stroke (Millán et al., 2021), Alzheimer's disease (AD) (Jakaria et al., 2021), spinal cord injury (SCI) (Yu et al., 2023)), Acute kidney injury (AKI) (Guerrero-Mauvecin et al., 2023).Intervening in ferroptosis may therefore lead to new therapeutic strategies for the disease.Several drugs have been reported to play an inhibitory role in iron-death-mediated diseases.This review systematically describes the metabolic pathways of ferroptosis and summarizes the mechanisms of action of natural and synthetic small-molecule inhibitors of ferroptosis as well as their applications in disease.Non-traditional therapeutic approaches such as gene therapy, cell therapy, drug combinations and nano-delivery in ferroptosis-mediated diseases are also presented.In addition, the challenges encountered in the preclinical experimental stage and clinical translation of ferroptosis inhibitors are summarised.Finally, the relationship between ferroptosis and other modes of cell death is discussed in the hope of providing new ideas for future drug design and development and clinical translational applications.

Small molecule ferroptosis inhibitors 2.1 Inhibition of ferroptosis via the iron metabolism pathway
Iron is one of the important trace elements in the human body and is a key causative factor in ROS accumulation and ferroptosis.Under normal conditions, Fe 3+ is found in serum transferrin (TF), The membrane protein Transferrin Receptor1 (TFR1), Six-Transmembrane Epithelial Antigen of Prostate 3 (STEAP3) enzyme, Divalent Metal Transporter 1 (DMT1), Nuclear Receptor Co-Activator 4 (NRC4), and the serum transferrin (TF) enzyme, and in the serum transferrin (TF) and TFR1 enzymes.Epithelial Antigen of Prostate 3 (STEAP3) enzyme, Divalent Metal Transporter 1 (DMT1), and Nuclear receptor coactivator 4 (NCOA4) (Gryzik et al., 2021), Membrane iron transport protein 1 (Ferroportin1, FPN1), Ferritin, Promimin 2, Ceruloplasmin (CP), etc. are involved in the transport to cells and participate in the reaction in vivo (Scarpellini et al., 2023;Zhang et al., 2024a).However, the excess of Fe 2+ in the organism under certain pathological conditions, on the one hand, induces the accumulation of ROS in the body through the Fenton reaction; on the other hand, iron participates in the catalytic process of metabolic enzymes, such as LOX, as a cofactor of various phospholipid peroxidases, which in turn accelerates lipid peroxidation and induces ferroptosis (Du and Guo, 2022;Sun et al., 2023) (e.g., Figure 1).Therefore, regulating Fe 2+ levels in the body could potentially inhibit ferroptosis and aid in disease treatment.

Iron chelators
Deferoxamine (DFO) has been approved by the Food and Drug Administration (FDA) for subcutaneous injection to mitigate elastin-induced ferroptosis in an in vitro ferroptosis model (Abdul et al., 2021).DFO inhibits ferroptosis by chelating free intracellular Fe 3+ , down-regulating ROS, and up-regulating intracellular levels of GPX4, the ferritin heavy chain (FTH1), and Cystine/glutamic acid reverse transporter (System Xc-) (Zhang et al., 2020;Zeng et al., 2021).Moreover, DFO has been shown to inhibit ferroptosis in SCI (Yao et al., 2019), but whether it directly protects neurons from ferroptosis is unclear.At the same time, DFO also plays a role in ischemic stroke (IS), which significantly reduces the area of cerebral infarction (Millán et al., 2021), and plays a certain neuroprotective role against the damage of neurological function after cerebral ischemia (Jones et al., 2022).However, a short DFO half-life was found during treatment.At the same time, two oral drugs, Deferiprone (DFP) and Deferasirox (DFX), have been developed to address the short half-life of DFO, but side effects such as granulocyte deficiency (Lecornec et al., 2022)and renal failure (Kattamis, 2019)are still present during treatment.To address the limitations of DFO, DFP, and DFX such as low oral activity, low efficacy, and side effects, Chen et al. introduced a sacrificial site for glucuronidation and designed and synthesized a novel oral iron chelator, CN128, and found that CN128 was more effective and efficacious orally (Chen et al., 2020), and it has been used in clinical trials in β-thalassemia patients after regular blood transfusions.
Deferric Amine Compounds DFA1 bind iron through two molecules of oxygen in the phenolic hydroxyl group and one molecule of nitrogen in the amine.Research has shown that DFA1 efficiently chelates iron in vivo and ex vivo, outperforming DFO.It has also been observed to alleviate iron overload-induced ferroptosis and reduce cytotoxicity in a mouse model of iron overload when administered orally and intravenously (Feng et al., 2022).Furthermore, DFA1 shows promise as a lead compound due to its high oral (Feng et al., 2022).
DFP was shown to have nephroprotective effects in a glycerolinduced AKI mouse model.Zhang et al. synthesized 25 novel iron chelator cinnamamide-hydroxypyridone derivatives by combining the iron chelating properties of DFP with the free radical scavenging capabilities of phenolic acids.Their assessment included assays for ABTS radical scavenging, Fe 3+ affinity, oxygen radical absorbance capacity (ORAC), and the inhibition of Erastin-induced ferroptosis in HT22 cells.It was shown that compound 9c has both chelating iron ions and antioxidant properties.Compound 9c exhibited the strongest inhibition of ferroptosis, almost 10 times more potent than DFP (EC 50 = 14.89 μM).Additionally, the researchers observed that compound 9c significantly alleviated cisplatin (CP)-induced AKI in the HEK293T cell model (Rayatpour et al., 2022).
In addition, studies have shown that natural compounds also can chelate iron.The flavonoid baicalein significantly reverses elastin-induced downregulation of iron accumulation, GSH, and GPX4 in cells (Xie et al., 2016).Experimental screening yielded Hinokitiol, a natural molecule carrying an α-hydroxy ketone skeleton with strong iron chelating properties, which can activate nuclear factor red factor 2-related factor 2 (Nrf2), providing a basis for further neuroprotection (Sridharan and Sivaramakrishnan, 2018).Tannins (TA), which complex with iron without binding to endogenous iron-containing molecules, are also an effective measure for the treatment of diseases associated with iron overload (Phiwchai et al., 2018).BMS536924 a dual inhibitor of insulin-like growth and insulin receptor protects against the induction of iron-dead cells and can act as an iron chelator to block ferroptosis (Kuganesan et al., 2021).Thymus β4 is an endogenous iron chelator that regulates ferroptosis by affecting free iron ions and ROS (Lachowicz et al., 2022).Ciclopirox (CPX) (Eberhard et al., 2009;Lin J. et al., 2021;Lu et al., 2022) has been approved by the FDA for antifungal therapy, and in recent years it has been found that intraperitoneal injection can significantly inhibit the growth of non-small cell lung cancer (NSCLC).Therefore, the use of iron chelation therapy in clinical practice still holds great promise.

Non-iron chelators
In addition to iron chelators, several compounds have been found to inhibit ferroptosis by modulating iron metabolism and are used in disease treatment.
Yang et al. screened a non-classical inhibitor of ferroptosis, YL-939, from chemical libraries.They found that YL-939 binds to its target inhibitor 2PHB2 and promotes ferritin expression to reduce iron levels to inhibit ferroptosis and alleviate ferroptosis-mediated liver injury (Yang et al., 2022).Compound YL-939 provides a new intervention strategy for diseases associated with ferroptosis.
Phenotypic screening and structural modification identification of benzimidazole derivatives and discovery of a novel ferroptosis inhibitor compound 9a by Fang et al.They found that compound 9a inhibited ferroptosis mainly by stabilizing intracellular Fe 2+ .In the middle cerebral artery occlusion (MCAO) model, compound 9a was found to significantly alleviate neurological impairment after IS.In addition, compound 9a can bind to NCOA4 and break the NCOA4-FTH1 interaction (Fang et al., 2021).
A National Cancer Institute screen found that the polycyclic aromatic compound NSC306711 blocked iron uptake by the Tf-TfR pathway and inhibited ferroptosis.At the same, unlike classical lattice protein-mediated endocytosis of Tf receptors, this drug is also known as ferritin due to its different previous endocytosis pathway that induces internalization and degradation of unoccupied Tf receptors (Horonchik and Wessling-Resnick, 2008).
The complex Chinese herb Naotai formula extract (NTE) has been reported to modulate FPN-1, downregulate TFR1 and DMT1 levels, and reduce ROS and MDA accumulation (Yang T. et al., 2021).NTE was found to upregulate rat Recombinant Solute Carrier Family 7, Member 11 (SLC7A11), GPX4, and GSH levels in the MCAO model (Lan et al., 2020;Qiu et al., 2022).Meanwhile, the flavonoid compound Carthamin yellow (CY) has been shown in ex vivo and in vivo experiments to be useful in myocardial ischemiareperfusion (MIRI) injury and to reduce ROS.In recent years, Guo et al. found downregulation of Fe 2+ , ROS, reverse transcription of Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4), Fe 2+ , TFR1, Glutathione (GSH), SOD, and MDA levels in the brain and improvement of infarct size in the rat brain after administration of the drug in MCAO models (Guo et al., 2021;Chen G. et al., 2022).In addition, the natural flavonoid Farrerol (FA) may alleviate ferroptosis by inhibiting iron accumulation and lipid peroxidation (Wu et al., 2022).

Inhibition of ferroptosis via the lipid metabolism pathway
Lipid peroxidation is central to triggering ferroptosis.It was shown that Polyunsaturated fatty acids (PUFA) of arachidonic acid (AA)/adrenaline (AdA) were acylated and esterified to AdA-Phosphatidyl Ethanolamine (AA-PE) and AA-PE with the participation of ACSL4, coenzyme A, and lysophosphatidylcholine acyltransferase-3 (LPCAT3).Ultimately, PE-PUFA leads to lipid peroxidation and induces ferroptosis through both non-enzymatic and enzymatic reactions (Naowarojna et al., 2023;Pope and Dixon, 2023).The nonenzymatic reaction that requires the participation of ROS generated by Fe 2+ mediated Fenton reaction.In contrast, enzymatic reactions may be more complex.The enzymatic reaction generally catalyzes the oxidation of PUFAs with the involvement of LOX to produce PE-PUFA-OOH and its derivatives and reactive aldehydes, including Malondialdehyde (MDA) and 4-hydroxynonenal (4HNE), which in turn induce ferroptosis, but these aldehydes, in turn, lead to impaired nucleic acid and protein functions (Pope and Dixon, 2023).Cytochrome P450 oxidoreductase (POR) also promotes lipid peroxidation and induces ferroptosis with the involvement of two cofactors, Flavin mononucleotide (FMN) and Flavin adenine dinucleotide (FAD) (Koppula et al., 2021).In addition, iron can also catalyze the metabolic activity of two enzymes, LOX and POR (Zou et al., 2020) (e.g., Figure 2).

Free radical trapping antioxidants
Free radical trapping antioxidants (RTAs) inhibit ferroptosis and protect hydrocarbon systems by trapping lipid peroxyl radicals.
Fer-1 and Liproxstatin-1 (Lip-1) (Zilka et al., 2017), both obtained by high-throughput screening of small molecule libraries, rapidly transfer H atoms from their arylamine portions to lipid radicals to inhibit lipid peroxidation and prevent free radical chain reactions in membrane PUFA lipids (Bayır et al., 2020;Scarpellini et al., 2023).Current researchers have identified quite a few other exogenous RTAs and many endogenous RTAs that play a role in iron-deathmediated diseases (e.g., Table 1).

Endogenous free radical trapping antioxidants
Vitamin E and the trace mineral selenium (Se) form a complementary antioxidant system (Saito, 2021).Vitamin E inhibits lipid peroxide production by reducing Fe 3+ in LOX-15 (Tavakol and Seifalian, 2022), but it acts less potently than Fer-1 and Lip-1.Furthermore, it was found that neurological damage caused by vitamin E deficiency in COVID-19 patients was strongly associated with ferroptosis.(Tavakol and Seifalian, 2022).

Exogenous free radical trapping antioxidants
Fer-1, the first synthetic inhibitor of ferroptosis, stabilises free radicals, reduces ROS, and has been strongly implicated in a variety of diseases (Skouta et al., 2014;Wang et al., 2023), including acute lung injury (ALI) (Liu et al., 2020).In structure-activity relationship (SAR) analysis (Saito, 2021), it was found that N-cyclohexyl acts as a lipophilic anchor playing an important role in maintaining Fer-1 activity (Scarpellini et al., 2023).Moreover, both amine groups and lipophilic anchors are essential for maintaining Fer-1 activity.Studies have reported that Fer-1 inhibits ferroptosis both in vivo and in vitro with significant in vitro inhibition.Structural analysis of Fer-1 by researchers yielded the structurally stable compound SRS11-92, but it is less active and less stable in plasma metabolism (Linkermann et al., 2014).Further researchers used elastininduced HT-1080 cells as a model of ferroptosis, using SRS11-92 as a starting point for optimization.They first introduced a sulfonamide instead of the unstable ester and introduced a benzyl ring 2 on the NH to obtain the sulfonamide analogue compound 38 (UAMC-2418).Compound 38 has high stability but low solubility.Thus, the researchers introduced solubilityenhancing groups into compound 38 to obtain compound 39 (UAMC-3203).Compound 39 showed stronger solubility, stability, and pharmacokinetic values than 38, was protective against multiple organ damage in mice showed no toxicity, and was overall superior to Fer-1 (Scarpellini et al., 2023).Meanwhile, the nanomaterial poly(2-oxazoline)-Fer-1 significantly improved the potency of Fer-1 (Morrow et al., 2022), providing a new idea for the treatment of ferroptosis.
Lip-1 is a newly discovered ferroptosis inhibitor that contains a spiroquinoxaline amine scaffold, functioning similarly to Fer-1 without affecting other cell death pathways.It is both active and soluble, and was introduced around the same time as the Fer-1 derivative UAMC-3203.Various research studies have highlighted the significance of Lip-1 in treating different neurological disorders and cancers.A recent study revealed that a Lip-1 analog, Lip-2, successfully prevented ferroptosis induced by serum in human proximal tubular epithelial cells from patients with lupus nephritis (Class IV), while also exhibiting improved pharmacokinetics (Alli et al., 2023).
To obtain more potent inhibitors, Yang et al. screened the phenothiazine derivatives isoprozine as well as phenothiazine from the Selleck (USA) library of biologically active compounds and they further explored the structure-activity of phenothiazine derivatives (Shah et al., 2017).By substituting different functional groups, they found that compound 51 with methylcytosine had better activity and exerted its antioxidant capacity mainly by trapping free radicals, thus protecting against elastin-induced cellular ferroptosis.It is worth noting that the compound has good pharmacokinetics as well as good BBB penetration, which is essential for the treatment of CNS diseases.Compound 51 was further evaluated in the MCAO model, which showed a significant reduction in lesion volume (Yang et al., 2021).However, it has a high hERG activity, so they further optimized the structure based on the antioxidant and ferroptosis inhibition properties of compounds with phenothiazine scaffolds to obtain 2-vinyl-10H-phenothiazine derivatives.Compound 7J was found to have the best ferroptosis inhibitory activity by SAR study, 7J showed good ROS scavenging ability and could alleviate DOX-induced cardiotoxicity with a good pharmacokinetic profile and no significant toxicity in vitro and ex vivo (You et al., 2022).
Recent studies have reported that mitochondria-targeted nitrogen oxide RTA has an effective inhibitory effect on ferroptosis.Nitrogen oxides catalyze the cross-disproportionation of alkyl peroxyl and hydroperoxyl radicals, allowing them to form two substances in unsaturated hydrocarbons as uniquely effective RTAs.Ability of Targeted Nitrogen Oxides XJB-5-131 and JP4-039 to prevent the Occurrence of Cytosolic ferroptosis in HT-1080 Cells as Olefinic Peptide Isoforms (Krainz et al., 2016).Optimisation of JP4-039 by Manwika Charaschanya et al. found compound (S)-6c to be the most potent inhibitor of ferroptosis in HT-1080 cells (approximately 30 times more active than JP4-039) (Charaschanya et al., 2022a;Charaschanya et al., 2022b).
In recent years, copper complex diacetylbis (N(4)-methylthiosemicarbazonato) copper(II) (CuATSM) (Kuo et al., 2019) has been an efficient RTA.The study reports that CuATSM reduces the area of murine cerebral infarction and oxidative stress and exerts antioxidant and neuroprotective effects in acute IS in a transient MCAO (tMCAO) model (Shi et al., 2021).CuATSM can be used in combination with EDA to upregulate this effect (Shi et al., 2021).In addition, CuATSM also exerts a neuroprotective effect in ALS, but the pathological mechanism is currently unknown (Kuo et al., 2019;Yang et al., 2023).

Other inhibitors
Histone methyltransferase inhibitors (BRD4770) is comparable to Fer-1 inhibition at optimal concentrations (Chen et al., 2022b;Chen et al., 2022c) and may be useful in the treatment of stenosis.The researchers screened and found that BRD4770 exhibited significant ferroptosis inhibition.They further found that BRD4770 inhibited lipid peroxidation by down-regulating PLO and MDA levels and increasing 4-HNE expression.And BRD4770 upregulated the mRNA levels of ferroptosis regulators SLC7A11, Recombinant Solute Carrier Family 3, Member 2 (SLC3A2), GPX4, and FSP1.Furthermore, it was found that BRD4770 alleviated cognitive impairment and downregulated aortic lipid peroxidation in a mouse model of AD (Chen et al., 2022b).Thus, BRD4770 is expected to be an effective molecular drug for the treatment of AD.
Recently Florencio Porto Freitas et al. identified 7dehydrocholesterol (7-DHC), an endogenous ferroptosis inhibitor, as one of the lipid components susceptible to autotrophication in vivo.They prepared soybean phosphatidylcholine (PC) monolayers loaded with 7-DHC and found that 7-DHC is preferentially oxidised in vitro and that the oxidation of 7-DHC in vitro is critical for the inhibition of (phospho)lipid peroxidation.Subsequently, using the iron/ ascorbate couple as a model for oxidative sources, they found that 7-DHC accumulation protects (phosphoric acid) lipids from autoxidation and subsequent rupture and is different from previous lipid peroxidation, mainly due to its better reactivity to peroxyl radicals.In addition, 7-DHC accumulation significantly reduces metabolic stress in the body (Angeli et al., 2021;Freitas et al., 2024).Thus, 7-DHC regulates ferroptosis in an easily overlooked manner by inhibiting lipid peroxidation and bringing new therapeutic ideas to ferroptosis-mediated diseases.

Inhibition of ferroptosis via antioxidant action
The antioxidant system is central to impeding ferroptosis by stabilizing or extinguishing free radicals and thereby inhibiting lipid peroxidation (Tan et al., 2023) (e.g., Figure 3.).System Xc-is an important antioxidant system involved in the regulation of ferroptosis.System Xc-passes through a heterodimer of the lightchain subunit SLC7A11 and the heavy-chain subunit SLC3A2, and glutamate-cysteine ligase (GCL) and glutathione synthase (GSS) transfer cystine to the cell and synthesise glutathione (GSH).Then, GSH is converted to Oxidized glutathione (GSSG) with the participation of glutathione peroxidation GPX4 to eliminate toxic lipid peroxides (Dar et al., 2024).Moreover, in the presence of GSH GPX4 converts toxic lipid peroxides into non-toxic lipocalciferol, which protects cells from lipid peroxidation and thus inhibits ferroptosis (Dar et al., 2024).
Other antioxidant pathways involved in ferroptosis include GTP Cyclohydrolase-1-Tetrahydrobiopterin (GCH1-BH4), Heat shock factor-Heat Shock Protein Beta-1 (HSF1-HSPB1), the mevalonate (MVA) pathway, Sulfur transfer pathway, the glutaminolysis pathway, and so on.Overexpression of the ferroptosis regulator GCH1 inhibits lipid peroxidation and promotes BH4 synthesis (Xu et al., 2023).BH4 is a potent endogenous RTA and participates in CoQ 10 H 2 formation.The GCH1-BH4 pathway has endogenous antioxidant effects and is independent of the Xc-GSH-GPX4 axis and the NADPH-FSP1-CoQ 10 axis (Akiyama et al., 2023).Transcription factor HSF1 promotes HSPB1 transcription and forms the HSF1-HSPB1 pathway (Sun et al., 2015).HSPB1 is a negative regulator of ferroptosis that upregulates GPX4, SLC7A11, and G6PD and HSPB1 overexpression attenuates ischemic-hypoxic brain damage in neonatal rats (Doshi et al., 2010;Liang et al., 2023).MVA plays a regulatory role in ferroptosis.On the one hand, by regulating selenocysteine tRNA which in turn promotes GPX4 synthesis.And on the other hand, MVA can synthesize CoQ 10 in the presence of acetyl coenzyme A, which in turn participates in the Xc-GSH-GPX4 axis and the NADPH-FSP1-CoQ 10 pathway and is involved in the regulation of ferroptosis (Xing et al., 2023).The Sulfur transfer pathway plays a role in the maintenance of redox homeostasis and oxidative stress, mainly due to the upregulation of intracellular Cys, GSH, and GSSG and the inhibition of ROS as a result of Cys-tRNA synthetase (CARS) deficiency (Floros et al., 2022;Sun et al., 2023).Glutamine catabolism provides sufficient GSH and ATP for ferroptosis and also plays a supporting role in ferroptosis-mediated diseases (Durante, 2019;Shin et al., 2020;Gagliardi et al., 2023;Xiao et al., 2023) (e.g., Figure 3).Several ferroptosis inhibitors have been reported to inhibit ferroptosis by modulating the antioxidant pathway.

The System Xc-GSH-GPX4 axis
The heat shock protein 90 (HSP90) plays an important role in protein maturation, stabilisation, and activation (Wickramaratne et al., 2023).A triterpenoid compound 2-amino-5-chloro-N, 3dimethylbenzamide (CDDO) was identified by Wu et al.It inhibits HSP90, which in turn inhibits GPX4 degradation and lipid peroxidation, and downregulates ROS, protecting cells from damage caused byferroptosis (Wu et al., 2019).Furthermore, in 2022 Liu et al. found that a series of disulfide compounds, such as disulfiram (DSF) and fursultiamine, could disrupt the interaction of GPX4 with HSC70, which in turn inhibited GPX4 degradation and protected cells from ferroptosis (Liu et al., 2022).
In 2021 Meike Hedwig Keuters et al. found that the novel ferroptosis inhibitor arylthiazyne derivative small molecule (ADA-409-052) inhibited tert-butyl hydroperoxide (TBHP)induced lipid peroxidation and prevented ferroptosis induced by GSH, as well as GPX4 deficiency.It is rapidly absorbed after oral administration in a mouse model.In addition, administration of the drug in a thrombotic mouse model revealed a significant decrease in the area of cerebral edema, the area of cerebral infarction, and the expression of pro-inflammatory factors in mice (Keuters et al., 2021).Thus, small molecule inhibitors such as ADA-409-052 offer new therapeutic strategies for neurological disorders as well as acute brain injury.
In 2023, Qi et al., 2023 reported that the antidiabetic drug mitoglitazone (MGZ) upregulated GPX4 and reduced lipid peroxidation, thereby significantly alleviating renal injury in mice after I/R, providing a new therapeutic strategy to ameliorate renal I/R injury.In addition, MGZ can also exert a nephroprotective effect by maintaining the normal morphology of mitochondria.
The catecholamine neurotransmitter Dopamine (DA) plays a role in human cognitive functions.It was shown that DA enhances the stability of GPX4 protein (Costa and Schoenbaum, 2022).Increased survival of DA ergic neurons and reversal of ROS, GPX4, and FTH1 levels in SNpc after moxibustion application in a Parkinson's disease (PD) model and improvement of motor deficits in it.And DA agonists reduce the risk of side effects during recovery (Ding et al., 2023).In addition, levodopa is approved for use in patients with early or late stroke and can be used in combination with physiotherapy (Obi et al., 2018).DAmediated ferroptosis has been mentioned in other diseases and is expected to lead to new research directions in ferroptosismediated diseases.
The study reports that Se inhibits ferroptosis and protects neurons by enhancing GPX4 expression.Se acts in the antioxidant pathway mainly by containing selenoproteins (Ramakrishnan et al., 2022;Choi et al., 2023).The study reports that Se inhibits ferroptosis and protects neurons by enhancing GPX4 expression (Alim et al., 2019).Inadequate levels of organismal Se downregulate GPX4 and antioxidant enzymes and upregulate ROS, MDA, and LPO (Xu et al., 2023).Se alleviates cerebral ischemia-induced neurological damage by activating GPX4 in IS (Alim et al., 2019).Iron, MDA and 4-HNE were found to be downregulated and promote the FSP1/ GPX4 pathway after sodium selenite injection in a rat model of SCI, which in turn improved motor function in rats (Chen et al., 2022).In addition, injection of double selenium nanospheres (CLNDSe) in an AD mouse model revealed abnormal microglia protofibrils or tau protein-targeted aggregation outside of Aβ42, and its crossing of the BBB into the brain significantly downregulated ROS, ACSL4, and COX-2, and upregulated FTH1, GPX1, and GPX4, ultimately inhibiting ferroptosis and ameliorating cognitive deficits in APP/PS1 mice (Solovyev et al., 2018).Overall, the trace element Se has been found to play a role in a variety of diseases by mediating ferroptosis.
Recently Li et al., 2019 identified a potent GPX4 metastable site and obtained eight GPX4 metastable activators by structural analysis and computational design (Scarpellini et al., 2023).They inhibit ferroptosis by activating GPX4 enzyme activity (IC 50 > 100 μM).Compound PKUMDL-LC-101 and its analogue PKUMDL-LC-101-D04 most efficiently activated and increased GPX4 activity in intact cells while inhibiting ferroptosis in a cellular model (Scarpellini et al., 2023).These compounds provide an effective strategy for the future development of activators for other protein targets and also lead to new therapeutic strategies for lipid peroxidation-related diseases such as neurodegenerative diseases (Li et al., 2018;Li et al., 2019).
Upregulation of GPX4 levels and downregulation of lipid ROS in neurons after treatment with the thromboxane A2 receptor antagonist Seratrodast and consequent inhibition of neuronal cell ferroptosis in erastin-induced hippocampal HT22 cells.And Seratrodast increased GPX4 expression and shortened seizure duration and prolonged seizure latency in a mouse model of epilepsy (Noack et al., 2017).Thus Seratrodast could play a role in several diseases by inhibiting ferroptosis.
Using APP/PS1 mice as subjects, researchers found that the monoterpene glycoside paeoniflorin (PF) improved cognitive performance and downregulated the levels of Fe 2+ , MAD, and ROS in brain tissue, which reduced oxidative damage and thus alleviated the neurological damage in AD mice (Zhang and Wei, 2020;Zhai et al., 2023).And PF can reverse AKI by inhibiting SLC7A11-mediated ferroptosis (Ma et al., 2023).In 2019, Guan et al. found that the monoterpene oenol carvacrol (CAR) (Abdul Ghani et al., 2023) reduced PLO levels in ischemic gerbil brain tissue and inhibited ferroptosis by up-regulating GPX4, which exerted neuroprotective effects in both in vivo and ex vivo models of IS.
Thus, CAR has the potential to be an effective therapeutic agent for IS.In 2021, Li et al. found that injection of Ginkgolide B (GB), the active ingredient of terpene lactones (Wang et al., 2020), in an animal model of AD reversed the levels of TFR1 and NOCA4 and upregulated the expression of Nrf2 and GPX4 in the brains of SAMP8 mice, which exhibited neuroprotective effects in AD mice (Hébert et al., 2022).
The lanolin-type triterpenoid Pachymic Acid (PA) has a variety of pharmacological properties.Liu et al. established a cellular myocardial infarction (MI) model after administration of 20 μg/ mL and found that the inhibitory effect of oxygen-glucose deprivation/reperfusion (OGD/R) on cell viability was reversed and the levels of MDA, ROS, and Fe 2+ were downregulated, and the expression of GSH, SLC7A11, and GPX4 was increased.Meanwhile, PA inhibits cardiomyocyte ferroptosis in a dosedependent manner and attenuates MIRI injury in mice (Liu et al., 2024).
Flavonoids are an important class of phenolic metabolites in plants with good antioxidant effects.The bioflavonoid Kaempferol (KF) has shown neuroprotective effects in neurological disorders such as IS and AD.Recently, it was found that KF activated the Nrf2/ SLC7A11/GPX4 signalling pathway and enhanced antioxidant capacity, which in turn reversed OGDR-induced ferroptosis (Holland et al., 2020;Kim et al., 2023) and alleviated neuronal cell damage.The natural flavonoid compound Icaritin (ICA) plays a role in a variety of diseases such as IS, AD, depression, and others.It directly binds to Nrf2 and promotes GPX4 transcription, which in turn inhibits ferroptosis after IRI and ameliorates brain damage (Choi et al., 2023).In recent years, ICA compounds significantly enhanced GSH-Ps, SOD activities and alleviated oxidative stress injury in mouse brain tissue after gastric administration of ICA compounds in APP/PS1 double transgenic mice.In addition, ICA was found to alleviate cognitive deficits in AD mice in a dosedependent manner in the Morris water maze (VWM) (Angeloni et al., 2019;Zheng et al., 2023).
Total flavonoids from Aspergillus membranaceus (TFA) play a role in neurodegenerative diseases.Gao et al., 2024 found that TFA prevented SH-SY5Y cell neurotoxicity by increasing GSH and GSH/ GSSG ratios and decreasing ROS, and showed significant neuroprotection in MPTP/MPP-induced in vitro and in vivo PD mouse models.Simultaneous injection of the flavonoid galangin (Gal) after I/R injury in VWM revealed a significant reduction in lipid peroxidation levels and an upregulation of SLC7A11 and GPX4 expression in the gerbil brain, which resulted in the inhibition of ferroptosis and the protection of hippocampal neurons in the gerbil brain after I/R (Guan et al., 2021;Hassanein et al., 2023).At the same time, gerbils showed significant improvement in the area of learning memory.Recently, Yang et al. found that Gal prevented iron overload and lipid peroxidation in a MIRI model and significantly attenuated myocardial fiber damage, reduced cerebral infarct size, and improved cardiac function by targeting the GPX4/Nrf2 signalling pathway in mice after MIRI (Salama and Elshafey, 2021).Thus flavonoids are a promising inhibitor of ferroptosis.
A-lipoic acid (LA) reverses folic acid (FA)-induced AKI.In 2021, Xue et al. found that LA upregulated GSH, GPX4, and downregulated ROS as well as lipid peroxidation, and that supplementation with LA reversed the low expression of SLC7A11 (Li et al., 2021).In addition, LA also inhibits ferroptosis and attenuates fluoride-induced liver injury by modulating the Xc-GSH-GPX4 axis and chelating iron (Cho et al., 2022).
In addition to the above-mentioned small molecule drugs, many herbal medicines have also been found to play a role in herbal medicines have also been found to play a role in ferroptosismediated diseases.For example, Lv et al. established a diabetic nephropathy (DN) mouse model and found that Fe 2+ was downregulated and SLC7A11, GPX4 content, and GSH/GSSG content, and GSH/GSSG were upregulated in the renal tissues of mice after San-Huang-Yi-Shen capsule (SHYS) administration and that it alleviated renal injury (Su et al., 2021;Lv et al., 2023).At the same time, Modified Shoutai Pill, also known as Jianwei Shoutai Pill (JSP), upregulated GSH, GPX4, downregulated MDA levels, and ACSL4 protein expression in the placenta of Recurrent Pregnancy Loss (RPL) mice, and was shown to protect against RSL3-induced lipid metabolism (Zhang et al., 2023;Lai et al., 2024).Futhermore, Angong Niuhuang Wan (AGNHW) has recently been found to exert neuroprotective effects by modulating GPX4-related signaling pathways and inhibiting ferroptosis (Bai et al., 2024).
In 2022, Fan et al., 2023 discovered that the anaesthetic Propofol (Islam et al., 2020) could have a strong antioxidant effect by modulating Nrf2/GPX4 through protein blotting, transmission electron microscopy, and glutathione assays.Furthermore, in a mouse model of CIRI, Propofol was shown to effectively prevent ferroptosis and protect against neuronal damage in the brain after cerebral ischemia/reperfusion.
Metformin (Met), a biguanide derivative commonly used in the first-line treatment of type 2 diabetes, has also shown promise in various other conditions such as cancer, cardiovascular disease, and neurological disorders (Ala and Ala, 2021).Recent research has unveiled Met's potential role in spinal cord injury (SCI) by inhibiting oxidative stress through the Nrf2/ARE pathway (Wang et al., 2020) and promoting neural regeneration post-SCI, offering new therapeutic possibilities.Additionally, studies have indicated that Met upregulates GPX4 (Ma et al., 2021), reduces MDA levels, and provides protection against neurological impairments following cerebral ischemia (Cai Z. et al., 2023).
The novel chlorane diterpenoid analogue Ajudecunoid C (ADC) is more effective in ferroptosis inhibition.It has been shown that ADC mainly acts on the Nrf2-AREs pathway and scavenges free radicals to exert an ferroptosis inhibitory effect, and ADC may interfere with the Keap1-Nrf2 pathway and activate the Nrf2-AREs pathway (Tan et al., 2021).In addition, the diterpenoid Dehydroabietic Acid activates the Keap1/Nrf2-ARE signalling pathway and attenuates non-alcoholic fatty liver disease (NAFLD).Therefore, researchers have proposed that diterpenoids may be effective compounds for inhibiting ferroptosis (Tan et al., 2021).
Biopolyphenolics Proanthocyanidins (PACs) are effective free radical scavengers.PACs have been reported to also have the ability to regulate LOX (Zhou et al., 2020).PACs have been found to inhibit ferroptosis and play a role in a variety of diseases by altering the expression of iron-related factors.For example, PACs upregulate the expression of GSH, GPX4, SLC7A11, Nrf2, and HO-1 and  (Zhou et al., 2020).In addition, a large number of studies have shown that PACs activate the Nrf2-HO-1 pathway and ameliorate CIRI, bringing a new direction for CIRI treatment (Chumboatong et al., 2020).In 2022, Yuan et al. found downregulation of oxidative stress and neuronal death after injection of the phenolic substance geraniin by the middle cerebral artery occlusion-reperfusion (MCAO/R), and OGD/R models, as well as by neurological scoring assays, CCK8 assessment, TUNEL staining, detection of cells by flow cytometry, and Western blotting assessment (Chen et al., 2021).Meanwhile, they found that the infarcted portion of brain tissue upregulated ex vivo and in vivo Nrf2, HO-1 protein expression in a concentration-dependent manner in the tMCAO model (Chumboatong et al., 2020).Thus, the neuroprotective effect of geraniin may be closely related to the Nrf2/HO-1 signalling pathway, but further experimental elucidation is needed in the future.
Flavonoids have been implicated in ferroptosis-related diseases through their interaction with Nrf2.For example, quercetin (QCT), a natural flavonoid, has been shown to alleviate renal injury by activating the Nrf2-HO-1 signaling pathway (Kato et al., 2023).Studies using an AKI-I/R model demonstrated that QCT administration led to increased cell viability, upregulation of ALC7A11, SLC3A2, and GSH expression, as well as a reduction in MDA and lipid ROS content in mice (Cruz-Gregorio and Aranda-Rivera, 2023).QCT also shows promise in the treatment of neurological disorders such as Parkinson's disease (PD) (Jiang et al., 2023).Eriodictyol, another flavonoid, was found to upregulate Nrf2/HO-1 and reduce ROS levels in a renal injury model, providing protection against CP-induced AKI (Badi et al., 2024).Furthermore, Eriodictyol has been shown to inhibit ferroptosis and improve cognitive deficits in APP/PS1 mice through activation of the Nrf2/HO-1 pathway (Li et al., 2022;Zhang et al., 2022).Naringenin, a flavonoid derivative from citrus extract, has demonstrated efficacy in alleviating myocardial injury by modulating the Nrf2/system Xc-/GPX4 axis (Shamsi et al., 2021).It also shows potential in mitigating ferroptosis induced by silver nanoparticles in human bronchial epithelial BEAS-2B cells through the Nrf2/HO-1 axis (Zhang et al., 2022b).Hesperidin and naringin have both been found to inhibit ferroptosis by upregulating the Nrf2 pathway, offering protection to human myeloid cells (Tang et al., 2022;Zhu et al., 2023).
The isoflavone tectorigenin protects against unilateral ureteral obstruction and renal injury in rats by affecting NADPH oxidase 4 (NOX4) expression (Li et al., 2022).Biochanin A, an active isoflavone of Astragalus membranaceus, inhibits ferroptosis by modulating the Nrf2/system Xc-/GPX4 signalling pathway, decreasing TFR1 and ferritin levels, and relieves knee osteoarthritis (KOA) (He et al., 2023).Licorice extract chalcone isoiquiritin apioside inhibits ferroptosis mediated ALI through upregulation of HIF-α and HO-1 proteins (Zhongyin et al., 2022).Puerarin, a natural isoflavone extracted from Pueraria Mirifica, was found to be protective against neuronal cell damage in mice by modulating Nrf2 in the OGD/R model (Li and Liu, 2024).It was also demonstrated to downregulate Fe 2+ , cyclooxygenase 2 (COX2), and upregulate Nrf2 and its downstream related ferritin (including SLC7A11, GPX4, HO-1) expression to reduce lipid peroxidation and inhibit retinal ferroptosis (Song et al., 2024).In addition, Puerarin was found to downregulate p53 and effectively improve neurological impairments in patients with ischemic brain injury in combination with conventional treatment in the clinic (Hou et al., 2024).
The non-flavonoid polyphenolic compound Resveratrol (RES) was found to promote ferroptosis-mediated recovery of motor function in SCI mice by modulating the Nrf2/GPX4 pathway in an SCI model (Ni et al., 2023).Meanwhile, RES protects human bronchial epithelial cells (BEAS-2B) from ferroptosis by acting on the ferroptosis pathway and inhibits diabetic periodontitis-induced ferroptosis in alveolar osteoblasts (Li et al., 2023c), which offers the possibility of disease treatment.Furthermore, Kosuke Kato et al. showed that quercetin and RES inhibit ferroptosis by inhibiting iron-catalyzed hydroxyl radicals and are independent of the Nrf2-ARE pathway (Kato et al., 2023).
The natural molecule Hinokitiol has a stronger iron chelating ability than DFO.Platycodone activates Nrf2, upregulates SLC7A11, GPX4, heme oxygenase-1 (HO-1) and exerts antioxidant effects (Abeydeera et al., 2022;Xi et al., 2022).The study demonstrated that the injection of flatulin in the rat MCAO model significantly inhibited ferroptosis and reduced the size of cerebral infarcts and alleviated the neurological damage after cerebral ischemia in rats, providing new insights into the IS (Jayakumar et al., 2013).Meanwhile, Hinokitiol alleviates neurological aspects of behavioural disorders caused by the neurotoxin 6-hydroxy DA (6-OHDA), providing a new treatment option for PD (Xi et al., 2022).Thus Hinokitiol holds promise for more diseases.
The natural anthraquinone derivative aloe-emodin (AE) attenuates adriamycin (the doxorubicin, DOX)-induced cardiomyocyte toxicity in H9c2 rats (He Y. et al., 2023).The researchers assessed the molecular mechanism of action of Nrf2 by Western blot, luciferase reporter gene assay, and qRT-PCR analysis, and detected the changes of intracellular ROS, and lipids by fluorescence assay.The results showed that AE could activate Nrf2, upregulate the expression of SLC7A11 and GPX4, and exhibit significant antioxidant capacity, thus reducing oxidative stress (He et al., 2023).
The bioactive steroid ester Withaferin A (WFA) modulates endothelial cell apoptosis after traumatic brain injury (Şeker Karatoprak et al., 2022).In recent years, WFA has been experimentally found to have the ability to inhibit ferroptosis and to play a role in many diseases associated with ferroptosis.It has been shown that WFA can activate the Nrf2/HO-1 pathway and reduce oxidative stress to inhibit neuronal cell injury after ICH, thus exerting neuroprotective effects.Meanwhile, the combination of WFA with Fer-1 increased this neuroprotective effect (Zhou et al., 2023), bringing new hope for ICH treatment.
Arbutin (ARB) was found to reduce ROS, and MAD accumulation, upregulate GSH, and exert a mitigating effect on alcoholic fatty liver disease (ALD) and liver injury by modulating the Nrf2/HO-1 pathway and oxidative stress.In addition, ARB can promote m6A methylation of SLC7A11 by inhibiting FTO, which in turn inhibits ferroptosis and exerts a mitigating effect on NAFLD in vitro and in vivo (Jiang et al., 2023).
Dl-3-n-butylphthalide (NBP), an extract of celery, has been widely used in stroke, dementia, and ischemic diseases.In 2022 Ye et al. suggested that injection of NBP inhibited erastin-induced accumulation of iron and ROS, downregulated TFH, and upregulated Nrf2, which in turn inhibited ferroptosis and reversed the damage to DAergic neurons (MES23.5 cells) (Ye et al., 2023).
Tetrahydroxy stilbene glycoside (TSG), an active ingredient of Polygonum tigrinum, enhances memory and locomotor activity in aged rats mainly by restoring mitochondrial function and plays a role in AD (Gao et al., 2023).In an AD rat model, TSG administration modulates the Nrf2-HO-1 pathway and protects against hippocampal neuronal damage in mice.Also TSG enhanced antioxidant capacity by activating GSH/GPX4/ROS and Keap1/Nrf2/ARE signalling pathways (Zhao et al., 2023).Recent studies have shown that BFT, a fat-soluble derivative of vitamin B1 synthesis, can also rescue cognitive deficits in mice in the APP/ PS1 mouse model through activation of the Nrf2/ARE pathway (Pan et al., 2016).

Other antioxidant pathways
Phenotypic analysis of the HT22 cell model revealed that the diphenylbutene derivative DPT could inhibit ferroptosis (EC 50 = 12.0 mM) and was non-toxic.Researchers have designed and synthesised 14 DPT analogues (Fang et al., 2022) using DPT as a backbone and chemically to enhance the inhibitory activity.By analysis, it was found that 3f, 3m, and 3n exhibited stronger inhibition of ferroptosis.Next, scaffolding analysis was carried out and it was found that R1 of compound 3f was substituted with 3-OCH3 and 4-OH on the A ring, and R1 of compounds 3m and 3n was substituted with 3-OCH3 and 4-OH on both the A and B rings.A comparison of activities revealed that compound 3f was significantly more active than the latter two mentioned above.Also, the stronger ferroptosis inhibition ability and superior biological activity of compound 3f were demonstrated by morphological analysis.Further DPPH assay DPT was found to be different from RTAs such as Fer-1 and DFO but inhibited ferroptosis by up-regulating FSP1 protein levels (Fang et al., 2022).In addition, compound 3f was found to alleviate the impairment of neurological function after cerebral ischemia to a certain extent in a rat MCAO model (Fang et al., 2022), which is expected to be a new prospect for the treatment of neurological disorders.
Ginsenoside Rg1, an active component of ginseng, has been shown to protect the kidney from damage by reducing oxidative stress.Following Rg1 treatment, iron content, FTL, FTH, and MDA levels in renal tissues were significantly reduced, while GPX4, FSP1, and GSH levels were elevated (Guo et al., 2022).Recent studies have demonstrated that knocking down FSP1 eliminates the inhibitory effect of ginsenosides on ferroptosis, and the administration of ginsenosides effectively alleviates Sepsis-induced Acute Kidney Injury (SI-AKI) (Guo et al., 2023).
In vivo experiments have shown that the natural chalcone cardamonin (CAD) inhibits ferroptosis and improves cartilage damage in rats by modulating the p53/SLC7A11/GPX4 signaling pathway.Additionally, CAD has been found to have a similar effect as DFO in osteoarthritis (OA), with CAD being more effective in improving cartilage damage in OA (Gong et al., 2023).

Alternative approaches to targeting ferroptosis
In addition to the small molecule ferroptosis inhibitors traditionally used to inhibit ferroptosis that can play a role in ferroptosis-mediated diseases, gene regulatory approaches, cellular therapies, and nano-targeted delivery can also exhibit ferroptosis inhibition by all along the correlates of ferroptosis (Table 2), and there are some differences in treatment efficiency between them (Table 3).

Gene regulation method
MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression and are implicated in various diseases.In a study by Huang et al., in 2022, miR-134-5p was found to be upregulated in bronchopulmonary dysplasia (BPD) in preterm infants, leading to ROS and Fe 2+ accumulation and GPX4 downregulation in these patients.Conversely, inhibiting miR-134-5p reversed these effects and significantly improved BPD (Lan et al., 2023).Another key miRNA, miR-3587, regulates HO-1 and is involved in renal ischemia-reperfusion injury.Tao et al. demonstrated in 2022 that injecting a miR-3587 inhibitor postestablishment of an ex vivo IR model enhanced HO-1 and GPX4 expression and cellular activity, thus safeguarding renal tissues from IR injury (Tao et al., 2021).
Prominin2, a pentameric transmembrane protein, plays a crucial role in mediating iron efflux.It facilitates the formation of multivesicular bodies (MVBs) and exosomes containing iron-rich proteins, allowing the removal of excess intracellular iron via these exosomes, thus preventing cellular ferroptosis.Additionally, Fer-1 has been shown to mitigate the decline in cellular function resulting from prominin2 depletion (Brown et al., 2019).In a study conducted in 2017, Yan et al., 2022 discovered that the small molecule proteinprotein interaction (PPI) inhibitor K-181 could suppress p53 transcription and ameliorate neurological impairments postischemic stroke (IS) by increasing the expression of the p53 repressor Mdmx, consequently inhibiting p53 transcription in the brains of mice following IS.Embryonic lethal-abnormal vision like protein 1 (ELAVL1) is an RNA-binding protein that enhances mRNA stability and regulates translation, thereby influencing gene expression (Du et al., 2022).ELAVL1 is notably upregulated in the I/R model.Research has shown that silencing ELAVL1 leads to an increase in GSH, GPX4, and SLC7A11 expression, while decreasing Fe2+, ROS, and MAD levels in rat brain tissues, ultimately preventing ferroptosis and reducing ischemic brain damage in rats (Du et al., 2022).
In addition, several other genes have been discovered to influence ferroptosis-related diseases by regulating factors associated with ferroptosis.For instance, in 2022, Sean K. Ryan et al. identified the ferroptosis susceptibility gene SEC24B through a comprehensive gene screening process.This gene inhibits ferroptosis and is implicated in neurological disorders by modulating iron-regulated proteins such as transferrin, thus maintaining iron homeostasis within cellular iron pools (Ryan et al., 2023).In a separate study in 2023, Hu et al., 2023 demonstrated that CDGSH iron-sulfur domain 2 (CISD2) was upregulated and activated the Nrf2/HO-1 signaling pathway in both the MCAO mouse model and the OGD/R HT22 cell model, mimicking in vivo and ex vivo conditions of cerebral ischemia and reperfusion.This activation led to increased survival rates of HT22 cells, while also reducing Fe 2+ content and exhibiting antioxidant properties.Furthermore, CISD2 was found to be effective in conditions such as IS-I/R and ICH (Yeh et al., 2022).

Neuregulin1β
With the development of stem cell research, the use of neural stem cells (NSCs) in the treatment of diseases is now of great interest.It has been found that exogenous NSCs can repair damaged tissues and play a role in neurological diseases such as IS (Calabrese et al., 2022).In 2022, Zhai et al. introduced human umbilical cord mesenchymal stem cells (hUC-MSCs) obtained by growth factor induction into NSCs and added NSCs by adding growth factor and neuregulin1β (NRG1β) to obtain NSCs-NRG1β.They found improved neurological function and reduced area of cerebral infarction in the rats injected with NSCs-10 nM NRG1β group in the rat MCAO/R model, and they also found an increase in the levels of GPX4 and SLC7A11 and a downregulation of p53 expression (Zhai et al., 2022).2023, they obtained the same results with NSCs-10 nM NRG1β intervention in oxygen OGD/ R-injured PC12 cells (Zhai et al., 2023).Thus NSCs provide a new direction for the future treatment of neurological diseases.However, many issues including ethical issues, therapeutic efficacy, and safety of exogenous NSC transplantation remain to be resolved.

Neutrophils therapy
To address the problems of antioxidant enzymes in IS therapy, in 2022, Wang et al. designed an albumin-conditioned nanoparticle based on co-encapsulation with antioxidases catalase (CAT) and superoxide dismutase 1 (SOD1) in a "neutrophil piggybacking" strategy.In the MCAO model, Neutrophil therapy delivered SOD1/CAT and Se to the site of brain injury in mice and significantly alleviated the area of cerebral infarction in mice after IS (Wang et al., 2022).In addition, the delivery of Se successfully upregulated GPX4 expression.At the same time, in addition to Neutrophil therapy, macrophages and monocytes also have this delivery capacity (Wang et al., 2022).Therefore, this neutrophil piggybacking strategy holds the promise of facilitating the application of nanomedicines in the central nervous system.

Relationship between nanoparticlesmediated ferroptosis and disease
The nanodelivery system is a sub-particulate carrier drug delivery system that modulates drug release rate and increases biofilm permeability.Numerous studies have reported that targeted ferroptosis nanomedicines can improve the low targeting and low solubility of conventional ferroptosis inhibitors (Żur and Farajpour, 2022).Also, several ferroptosis nanocarrier drugs have been used for ferroptosis-mediated diseases such as AKI, stroke, etc.

Individual use of nanoparticles in ferroptosismediated disease
The injection of DFO in a model of Idiopathic pulmonary fibrosis (IPF) was found to increase survival (from 50% to 90%) and reverse the IPF phenotype in mice.However, low solubility and low targeting during drug delivery remain an insurmountable gap in DFO therapy.To solve this problem, the researchers prepared DFO nanomedicines and used a pulmonary drug-delivery system (PDDS) instead of oral administration or injection, and the biological effect of the drugs was significantly improved (Devkota et al., 2021).
The miR-134-5p inhibitor proposed by the investigators for the treatment of bronchopulmonary dysplasia (BPD) achieves a certain therapeutic effect, but it still suffers from the problems of conventional drugs such as drop targeting and low solubility.To better improve drug utilisation, the investigators further designed and synthesised the targeted ROS-responsive nanocarrier PCC-R8-ROS@miR-134-5p inhibitor (Lan et al., 2023), which more efficiently delivered the miR-134-5p inhibitor to the alveolar epithelial cells, providing a more efficient therapeutic strategy for BPD.However, in the future, improving encapsulation efficiency may remain a major challenge for delivering miRNAs.
AKI, a severe syndrome of renal insufficiency, was the focus of a study by Wang et al., 2021.They developed ultrasmall KCa(H 2 O) 2 [FeIII(CN) 6 ]-H 2 O nanoparticles, known as CaPB nano-enzymes, to function as a multienzymatic mimic.These nano-enzymes were effective in inhibiting ferroptosis by scavenging reactive oxygen/ nitrogen species (RO/NSs) and treating AKI.The study found that CaPB nano-enzymes upregulated GPX4 both in vivo and ex vivo post intravenous administration, leading to kidney protection from oxidative damage and improved drug efficiency and targeting.Furthermore, the potential clinical application of CaPB nanoenzymes in AKI and other RO/NSs-related kidney diseases was highlighted.Xie et al., 2022 also contributed to the field by introducing gallic acid-gallium polyvinyl pyrrolidone nanoparticles (GGP NPs) as iron removers that could reduce intracellular free iron and mitochondrial dysfunction.These nanoparticles were able to downregulate iron-death-related substances like NADPH, GSH, GPX4, and ferritin, effectively suppressing ferroptosis-mediated AKI.Additionally, GGP NPs showed promise in ameliorating renal tubular injury and mitochondrial damage.
ICH remains an important cause of morbidity and mortality worldwide.Wang et al. presented a magnetically targeted nanocarrier loaded with the PPARγ agonist 15d-PGJ2-MNPs, which, when administered intravenously, activated PPARγ receptors on macrophages around haematomas, attenuated brain damage, and improved sensory and motor functions in mice after ICH (Wang et al., 2023).In addition, Yang et al. found that curcumin nanoparticles (Curcumin-NPs, Cur-NPs) could be effective in Cur delivery and provide ideas for the treatment of neurological impairment after ICH (Yang et al., 2021).
IS, a prevalent clinical neurological disorder, constitutes 85% of all strokes globally.Li et al., 2022 demonstrated that poly (lactic acid)-glycolic acid copolymerization-nanoparticles (SOD-PLGA-NPs) possess potent free radical scavenging capabilities, reducing cerebral infarct size in mice post-IS and ameliorating neurological deficits resulting from cerebral ischemia in the MCAO mouse model.Moreover, the nano delivery system enhances drug penetration through the blood-brain barrier.
Acute Liver Injury, a rare and life-threatening condition, was addressed by Shan et al. through the synthesis of ultrasmall poly (acrylic) acid coated Mn3O4 nanoparticles (PAA@Mn3O4-NPs, PMO), which effectively scavenge ROS, inhibit lipid peroxidation and ferroptosis, and mitigate Acute Liver Injury induced by acetaminophen and ischemia/reperfusion in mice.Furthermore, intravenous administration of PMO exhibits superior biocompatibility (Shan et al., 2023).
Myocardial Injury is a common complication of sepsis.Liu et al. designed and synthesised small biocompatible and MRI-visible melanin nanoparticles (MMPP) that could attenuate myocardial Injury.In ex vivo experiments, the investigators found that MMPP scavenges ROS and inhibits ferroptosis-mediated cardiomyocyte injury.In addition, MMPP downregulates oxidative stress induced by inflammatory factors (Alcalá-Alcalá et al., 2023).
Retinal pigment epithelial cells (RPE) are essential for maintaining the normal function and survival of photoreceptor cells.Oxidative stress and ferrous ion accumulation play a role in retinal degenerative diseases.Tang et al. found that a potent ironconjugated nanoscale Prussian blue analogue, KCa [FeIII(CN) 6 ] (CaPB), rescued retinal structure and visual function by preventing RPE degradation and was effective in preventing RPE lesions in mouse models (Tang et al., 2021).

Targeted nanotechnology in combination with other drugs
The current use of targeted nanotechnology has to some extent improved the problems of traditional drug targeting, solubility, and time window.Recent studies have reported that targeted nanotechnology in combination with conventional drugs has improved drug targeting even further.In a rat model of cerebral ischemia-induced neurological impairment, the co-administration of tissue plasminogen activator (tPA) with nanoliposomal Fasudil-Lip showed better neuroprotection, and prolonged therapeutic time window (TTW) and improved drug targeting (Fukuta et al., 2017).Therefore, we can speculate whether ferroptosis inhibitors could also better address issues such as drug targeting by combining them with nanoliposome-encapsulated drugs.

Clinical diseases mediated by ferroptosis
Ferroptosis, a newly discovered form of cell death, has been found to have strong links with pathophysiological processes in neurological disorders, organ damage, and cardiovascular diseases.
AD is the most prevalent type of dementia, characterized by amyloid-β (Aβ) deposition in senile plaques (SPs) and intracellular neurofibrillary tangles (NFTs) formed due to hyperphosphorylation of tau proteins (Aggleton et al., 2016).Clinical research indicates that iron accumulation and oxidative stress are the primary pathological changes in the brains of AD patients.Aβ has been reported to convert intracellular iron to ferrous iron, leading to increased Aβ plaque formation, elevated ferritin expression, and worsening oxidative damage and cognitive impairments (Lane et al., 2023;Singh et al., 2024).Moreover, high levels of iron in the brain trigger the production of free radicals and induce oxidative stress.Studies have shown increased levels of HNE and acrolein, as well as upregulation of lipid peroxidation-related enzymes in the brains of individuals with AD.Additionally, reduced levels of GSH and inactivation of GPX4 have been observed in both animal models of AD and postmortem brain specimens (Yoo et al., 2010).Therefore, targeting ferroptosis inhibition could serve as a promising therapeutic approach for AD.
PD is characterized by the loss of dopaminergic neurons in the Substantia Nigra (SN) (Bae et al., 2021).The pathogenic mechanisms are multifaceted and involve α-synuclein accumulation, lipid peroxidation damage, iron deposition, oxidative stress, and inflammation.Among these, the production of lipid peroxides by α-synuclein within the cell membrane to induce ferroptosis plays a crucial role in the development of PD.Both iron accumulation and oxidative stress have long been linked to the progressive loss of dopaminergic neurons in PD patients, with dopaminergic neuron loss potentially further triggering ferroptosis (Thapa et al., 2022).Studies have shown that following the onset of PD, levels of DMT1 were elevated, leading to increased intracellular iron input, subsequently promoting dopaminergic neuronal death and α-syn accumulation.Additionally, a decrease in GSH levels post-PD activated 12-LOX, resulting in LOOH accumulation in the brain and worsening PD symptoms (Shibu et al., 2021).Furthermore, the use of ferroptosis inhibitors like DFP has shown promise in mitigating PD-related damage (Lin et al., 2022;Ding et al., 2023).Overall, exploring the relationship between ferroptosis and PD pathomechanisms may provide effective treatment options for patients with PD.
IS is associated with various pathological changes including ROS accumulation, ion metabolism disorders, and oxidative stress.Following cerebral ischemia, lipid peroxidation and iron accumulation are common characteristics of ferroptosis.Research has demonstrated that altered blood-brain barrier permeability post cerebral ischemia exacerbates ischemic injury, leading to disruptions in iron metabolism within brain tissues (Yang et al., 2022).This disruption induces the production of ROS through the Fenton reaction, along with an increase in ferritin, TFR1, and DMT1 expression in the brain.The accumulation of iron in the brain triggers ROS production via the Fenton reaction, worsening brain damage (DeGregorio-Rocasolano et al., 2019).Moreover, the brain contains high levels of unsaturated lipids that generate excess ROS, activating Nrf2 and p53, which further exacerbates oxidative stress and brain damage.Studies in rat MCAO models have shown elevated lipid peroxidation levels and reduced GSH levels (Millán et al., 2021).Thus, amelioration of neurologic impairment after IS may be facilitated by inhibiting relevant targets of ferroptosis.
SCI is a severe traumatic neurological disorder characterized by high levels of polyunsaturated fatty acids in the spinal cord, leading to oxidative stress.Studies have shown that rats with SCI exhibit iron overload in the motor cortex, resulting in the accumulation of Reactive Oxygen Species (ROS) and the induction of ferroptosis.Pathological changes such as iron deposition, lipid peroxide accumulation, and downregulation of GPX4 are commonly observed in both SCI patients and animal models (Wei et al., 2021).The activation of neuroglia after SCI results in the secretion of inflammatory factors, contributing to iron overload in the motor cortex (Li et al., 2023).Furthermore, the use of ferroptosis inhibitors like DFO and Fer-1 has been found to enhance motor function recovery following SCI (Li and Jia, 2023).
AKI is a common renal disease in clinical practice, and recent research suggests that ferroptosis may play a crucial role in IRI-AKI.During ischemia, AKI is exacerbated by elevated levels of free iron in the kidney, downregulation of GPX4 and SLC7A11, and depletion of blood GSH, leading to the activation of ferroptosis and subsequent tubular necrosis (Borawski and Malyszko, 2020).Zhao et al., 2020 demonstrated that iron deficiency worsened rhabdomyolysis (RM)-induced AKI.Additionally, ferroptosis inhibitors like Fer-1 and pioglitazone have been shown to reverse AKI damage in a mouse model by targeting ferroptosis, offering promising avenues for AKI treatment (Hosohata et al., 2022).Acute liver injury is another condition with high clinical mortality rates, and recent studies have highlighted the potential of Nrf2 activation in mitigating liver injury (Zhu et al., 2024).Furthermore, research by Yamada et al., 2020 indicated that upregulation of ferroptosis markers (iron ions, lipid peroxide levels) exacerbated liver injury in hepatic IRI models, but treatment with ferroptosis inhibitors like Fer-1 significantly reduced injury severity.
Studies have shown that ferroptosis is associated with MIRI, heart failure, atherosclerosis, myocardial infarction, and other cardiovascular diseases.Iron metabolism and lipid metabolism play important roles in the regulation of cardiovascular disease.Of these, iron ion disorders are the most common.TFR1 expression was found to be upregulated during MIRI (Miyamoto et al., 2022).In contrast, TFR1 inhibitors such as DMT1i are protective against ferroptosis-mediated heart disease.In cardiovascular disease, LOX expression is upregulated, which in turn promotes lipid peroxidation and ferroptosis (Fratta Pasini et al., 2023).Meanwhile, inhibition of ACSL4 improves cardiac function and prevents heart failure (Ito et al., 2021).Furthermore, Upregulation of SLC7A11 and GPX4 Downregulates the Mouse Lipid Peroxidation and Iron Levels in the Endothelium of Mouse Aorta and Attenuates Atherosclerotic Injury (Bai et al., 2020).

Challenges encountered in the clinical translation of ferroptosis inhibitors
There have always been several problems in the development of drugs.In general, new drugs tend to face many difficulties and challenges before they are launched on the market.Since ferroptosis was proposed, the development of ferroptosis inhibitors as well as their clinical application has also become a topic of increasing interest for researchers.Although many small-molecule ferroptosis inhibitors have been developed and continuously optimised, most of them are still difficult to use successfully in the clinic.

Challenges encountered in preclinical trials
Several drugs have been shown to inhibit ferroptosis in animal experiments, but they mostly suffer from low stability, low solubility, low targeting, low safety, toxicity, poor pharmacokinetics, and low activity (Guo et al., 2023), so very few of them have entered clinical studies.For example, Fer-1, the first synthetic inhibitor of ferroptosis, has been shown to play a role in many diseases in animal models (Skouta et al., 2014;Wang et al., 2023), but is still in the experimental stage, making it difficult to cross over to clinical trials.Mainly due to its low stability and low solubility, the researchers designed and synthesised compound 39 based on Fer-1, compound 39 can significantly improve its ferroptosis inhibition potency (Linkermann et al., 2014;Scarpellini et al., 2023).Meanwhile, phenothiazine-based derivatives compound 51, although it has shown neuroprotective effects in IS animal models, still has high hERG inhibitory activity (Yang et al., 2021;You et al., 2022).Se has long been shown to alleviate diseases such as iron-death-mediated stroke by enhancing adaptive transcription, but Ishraq Alim et al. found a parabolic dose curve after Se supplementation in vitro and in vivo experiments, raising concerns about the mode of Se delivery as well as its safety.Moreover, Se-mediated GPX4 regulation and neuronal protection are extremely complex and therefore difficult to successfully translate into the clinic (Alim et al., 2019).Although DFO reversed the survival of IPF mice, it still suffers from low solubility and targeting during delivery (Guo et al., 2023).In addition, the natural substance RES is still in the preclinical experimental stage and is difficult to translate because of its problems such as low water solubility, easy degradation, low activity, and poor pharmacokinetics (Pharmacokinetics, PK) (Chung et al., 2020;Ni et al., 2023).Therefore, there is a need for further improvement of the drug thus better marketing of the drug to clinical studies.

Challenges encountered in clinical studies
After a drug has gone through the preclinical phase of experimentation, the drug needs to be tested in clinical trials (Moujalled et al., 2021;Iasonos and OQuigley, 2021).Several ferroptosis inhibitors have also made it to the clinical translational stage in recent years, but they still present challenges in clinical studies.
Vitamin E was found to significantly reduce cognitive performance in patients with mild to moderate AD in a multicentre, randomised, double-blind, placebo-controlled trial of AD from 2007 to 2012.However, some studies claim that vitamin E supplementation does not reduce the risk of AD and slows down its pathogenesis (Dysken et al., 2014;Kryscio et al., 2017;Ashraf and So, 2020).
In 2013, DFP significantly improved motor function in patients with PD in a 12-month phase II clinical trial of PD, but patients who continued to take DFP showed diminished improvement in motor function as well as reduced Neutrophils and granulocyte deficiencies (Devos et al., 2014;Martin-Bastida et al., 2017).In addition, Mohsen Saleh Elalfy et al. conducted a clinical trial on DFP for the treatment of transfusion-dependent thalassemia children in 2016.They found that the safety and efficacy of the drug was improved when DFP was administered to thalassemia patients, but adverse effects such as diarrhea, vomiting, colic, neutropenia, and elevated liver enzymes were still present (Elalfy et al., 2018).
In 2019, an injection of Edaravone dexborneol in phase II multicentre, randomised, double-blind, multi-dose, activecontrolled clinical trial in patients with IS found that patients experienced a reduction in acute brain injury, but continued to have itching and AKI side effects (Xu et al., 2019).
Copper compound CuII(ATSM) exhibits neuroprotective effects in phase I clinical trials in ALS and PD (Southon et al., 2020).Meanwhile, CuII (ATSM) has completed phase I trials in ALS patients and entered phase II clinical trials, but it has been found to exhibit spinal cord immunocompromise in patients in the clinical trial phase (Liddell et al., 2023).
In recent years, it has been found that ferroptosis-mediated neurological impairment can be significantly alleviated by thrombin inhibition.Meanwhile, the thrombin inhibitor Dabigatran is currently in Phase III clinical trial in IS (NCT03961334) (Tuo et al., 2022).In April 2023, the thrombin inhibitor Argatroban concluded a Phase IV clinical trial in IS (NCT03740958) and was found to significantly reduce adverse prognosis when used in combination with Aspirin (Peng, 2023).
Therefore, there is a need for researchers to further optimise drugs to improve drug utilisation, reduce side effects and treat ferroptosis-mediated diseases.

Ferroptosis in relation to other modes of cell death
Ferroptosis is a type of programmed cell death that is distinguished from apoptosis, necrotic apoptosis, autophagy, pyroptosis and cuproptosis in both morphological changes and biochemical functions.However, a growing body of research suggests that there is a link between ferroptosis and all of the above modes of cell death.

Ferroptosis and apoptosis
Apoptosis, the first identified regulatory cell death modality, is governed by apoptosis-related genes.The initiation of apoptosis is often characterized by morphological alterations such as cell shrinkage, chromatin fragmentation, and membrane blistering and disintegration.Apoptosis encompasses caspase-dependent intrinsic apoptosis, death receptor-mediated extrinsic apoptosis, and receptor-dependent extrinsic apoptosis.Research indicates that Tumor Necrosis Factor-α (TNF-α) plays a crucial role as an inflammatory factor that triggers ferroptosis.Additionally, Kai et al. (2020) reported the induction of chondrocyte apoptosis through the upregulation of TNF-α (Yang et al., 2021).Furthermore, Ma et al., 2022 demonstrated that Fer-1 mitigates noise-induced hearing loss by targeting TFR1-mediated ferroptosis and the p53-AIFM2 apoptosis pathway.

Ferroptosis and necroptosis
Necroptosis is a type of regulatory necrosis that is often caused by a variety of physicochemical stimuli, such as inflammatory factors and ATP depletion and is not dependent on caspases.Necrotic apoptosis is followed by morphological changes such as rupture of cell membranes, translucent cytoplasm, swollen organelles, increased cell volume, and chromatin condensation.Iron overload was found to affect cellular redox homeostasis and activate necrotic apoptosis and ferroptosis.In recent years, the ferroptosis inhibitor Lip-1 was found to be effective in inhibiting necrotic apoptosis during cerebral ischemia-reperfusion.Meanwhile, the necrotic apoptosis inhibitor Necrostatin-1 effectively inhibited the activation of proteins associated with ferroptosis (Yuk et al., 2021).In addition, Nigratine (also known as 6E11) was found to inhibit both necrotic apoptosis and ferroptosis in human bronchial-like organs (Delehouzé et al., 2022).Thus, ferroptosis and apoptosis have complex interactions and are closely related.

Ferroptosis and autophagy
Autophagy is the process of removing and degrading intracellular material through lysosomes, which includes macroautophagy, microautophagy, and chaperone-mediated autophagy.This process plays a vital role in maintaining cellular homeostasis and preventing cell overgrowth.Studies have shown a close relationship between ferroptosis and ferritin autophagy, adipose autophagy, and chaperone-mediated autophagy (Lee et al., 2023).Moreover, ROS accumulation, p53 activation, and Nrf2 signaling pathway activation all induced autophagy and ferroptosis.Autophagy-dependent ferroptosis has been implicated in kidney-related diseases and cardiovascular diseases.Additionally, research by Du et al., 2023 demonstrated that rapamycin injection enhanced autophagy, leading to the downregulation of TFR1, which inhibited ferroptosis and reduced cognitive deficits in a Sepsisassociated encephalopathy (SAE) model in mice.

Ferroptosis and pyroptosis
Pyroptosis, also known as cellular inflammatory necrosis, is programmed cell death induced by activation of inflammatory vesicles and is mainly regulated by cysteinyl asparagine-1, -4, -5, and -11-dependent signaling pathways (Teng et al., 2023).Pyroptosis was accompanied by morphological changes such as cell swelling, membrane rupture, and leakage of cytoplasmic components.It has been shown that high intracellular ROS accumulation promotes pyroptosis and that severe oxidative stress induces upregulation of pyroptosis.In recent years, it has been found that a mixed form of cell death, "PAN apoptosis", consisting of TNF-α and interferon gamma (IFNγ) or Proadrenomedullin N-terminal 20 peptide (PAMP), can activate pyroptosis and apoptosis at the same time.IFNγ can form an important anti-ferroptosis system with SLC3A2 (Ai et al., 2024).

Ferroptosis and cuproptosis
Cuproptosis is defined as the aggregation of proteins, proteotoxic stress, and eventual cell death triggered by copper binding to the lipolytic enzymes of the tricarboxylic acid (TCA) cycle.This form of regulated cell death, dependent on copper, was termed "copper tumor" in 2022, and has been linked to the metabolic pathways of ferroptosis.Similar to ferroptosis, cuproptosis leads to notable alterations in mitochondrial structure, such as shrinkage and membrane rupture.The mitochondrial TCA cycle serves as a crucial nexus between ferroptosis and copper tumors, playing a pivotal role in cuproptosis (Jhelum and David, 2022).Furthermore, GSH has been identified as a key player in both ferroptosis and cuproptosis.GSH binds copper, reducing protein aggregation in cuproptosis.Moreover, iron-sulfur cluster proteins, essential cofactors for maintaining redox balance and iron levels, are produced as auxiliary enzymes in cuproptosis, with their levels significantly decreasing following cuproptosis initiation.Notably, protein aggregates can interfere with the function of iron-sulfur clusters (Prasad Panda and Kesharwani, 2023).

Discussion and conclusion
Ferroptosis inhibitors have displayed some efficacy in ferroptosisrelated diseases, yet face significant challenges in making a substantial impact.These obstacles stem from the limitations of current small molecule drugs, characterized by poor selectivity, targeting, solubility, pharmacokinetic properties, adverse reactions, efficacy, in vivo toxicity, and side effects.On the other hand, some of the ferroptosis mechanisms themselves are unclear, and some of the mechanisms have been proposed but are still unknown (e.g., GCH1-BH4 pathway, HSF1-HSPB1 pathway, sulfur-transfer pathway, MVA pathway, etc.), which has become an important obstacle in the development of small molecule drugs.In addition, the complexity of the pathological mechanisms of some diseases is unclear, and the controversial relationship between ferroptosis and other modes of cell death is also an issue that needs to be addressed for the application of ferroptosis inhibitors to disease.
Overall, this review specifically describes the metabolic pathways of ferroptosis.A comprehensive summary of natural and synthetic small molecule drugs that treat ferroptosis-mediated diseases by acting on targets of ferroptosis and their therapeutic efficacy in disease.Meanwhile, this paper describes the mechanism of action of therapeutic approaches such as gene regulatory approaches, cell therapy, and nanodelivery in ferroptosis-mediated diseases.And it summarizes the problems encountered during the clinical translation of ferroptosis inhibitors and drugs that are currently in clinical translation.Finally, the relationship between ferroptosis and other forms of cell death modalities such as apoptosis is discussed.It is hoped that this will provide new ideas for the development of future ferroptosis inhibitors and provide effective therapeutic options for future ferroptosis-mediated diseases.
expression and cell viability and reduces Fe 2+ , ROS, MAD levels Du et al. (2022) SEC24B Alteration of iron regulatory proteins or transferrin to maintain iron homeostasis Ryan et al. (2023) CDGSH iron-sulfur structural domain 2 (CISD2) Activation of the Nrf2/HO-1 signalling pathway and downregulation of Fe 2+ Hu et al. (2023) Cell therapy Neural stem cells transplanted to obtain NSCs-NRG1β

TABLE 2
Alternative approaches to targeted inhibition of ferroptosis.

TABLE 3
Comparing the advantages and disadvantages of different ferroptosis inhibition methods.