Ferroptosis in liver biology and diseases

Abstract

Ferroptosis is a form of non-apoptotic cell death driven by iron-dependent lipid peroxidation (LPO). Ferroptosis is closely associated with hepatic metabolic pathways, particularly those involving iron, fatty acids, amino acids, and redox changes. This review aims to provide a comprehensive overview of the current molecular pathways and regulatory mechanisms underlying iron homeostasis and intracellular compartmentation in liver biology, emphasizing the roles of ferroptosis in several liver diseases, such as ischemia-reperfusion injury, drug-induced liver injury, viral hepatitis, metabolic dysfunction–associated steatotic liver disease, alcohol-associated liver disease, fibrosis, and hepatocellular carcinoma. We have also discussed the challenges of interpreting ferroptosis-related data, proposed a framework for defining ferroptosis, and highlighted future research directions as well as the therapeutic potential of targeting ferroptosis in the treatment of various liver diseases.

INTRODUCTION

Ferroptosis is a non-apoptotic cell death driven by intracellular iron, uncontrolled lipid peroxidation (LPO), and redox changes, and is governed by molecular networks involving diverse molecules and organelles, such as mitochondria, endolysosomes, and autolysosomes.^1,2 Since the term was introduced in 2012 by the Stockwell lab to describe cell death in erastin-treated RAS-mutated lung cancer cells, ³ ferroptosis has emerged as a crucial mechanism in numerous physiological and pathological contexts, leading to significant therapeutic advances across a wide range of diseases, including liver diseases.^1,4 The liver is a highly dynamic metabolic organ, but it is also vulnerable due to its constant exposure to challenges from both internal and external sources. These challenges include alcohol consumption, drugs and xenobiotics, metabolic disturbances with Western diets, viral infections, and ischemia-reperfusion injuries. Such stressors can result in varying degrees of metabolic and redox changes, as well as organelle dysfunction, thereby impacting liver function. Various forms of cell death can contribute to liver damage and liver diseases, as elegantly reviewed elsewhere.^5–7 Of note, iron and LPO have long been causally associated with lethal injury to hepatocytes from various causes.^8–12 Here, we focus on recent advances in the basic mechanisms of ferroptosis, signaling pathways, intracellular iron compartmentation and homeostasis in hepatocytes, and their role in various liver diseases, including ischemia-reperfusion injury, drug-induced liver injury (DILI), metabolic dysfunction–associated steatotic liver disease (MASLD), alcohol-associated liver disease (ALD), viral hepatitis, fibrosis, and hepatocellular carcinoma (HCC). This review aims to provide a comprehensive understanding of iron homeostasis in liver biology and ferroptosis in liver diseases, with implications for developing new drugs that target ferroptosis pathways to prevent and treat liver conditions.

BASIC MECHANISMS OF FERROPTOSIS

Ferroptosis is governed by opposing pro-oxidant and antioxidant pathways (Figure 1). The pro-oxidant pathway drives iron-dependent LPO of membrane polyunsaturated fatty acids (PUFAs). Conversely, the antioxidant network has expanded from a glutathione peroxidase 4 (GPX4)-centric axis to a multi-organelle, multi-metabolic ecosystem, with hundreds of regulators, pharmacological inducers [ferroptosis inducers (FINs)], and inhibitors, as summarized in ferroptosis databases such as FerrDb V2. ¹³ The key molecular players and their mechanisms in ferroptosis are summarized in Table 1, whereas commonly used tools and biomarkers for ferroptosis are listed in Table 2.

FIGURE 1.

Basic mechanisms of ferroptosis: antioxidant defense networks and pro-oxidant pathways. Ferroptosis sensitivity is governed by the dynamic interplay between protective antioxidant systems and LPO-driving pathways. Antioxidant defense networks (left panel): The classical System xc⁻/GSH/GPX4 axis provides core protection by importing cystine for glutathione (GSH) synthesis, thereby enabling GPX4 to reduce toxic lipid hydroperoxides to non-toxic alcohols. Multiple GPX4-independent defense networks operate across different subcellular compartments. At the plasma membrane, FSP1 acts as a radical-trapping antioxidant via the reduction of CoQ10, while DHODH independently reduces CoQ10 to mitigate LPO within mitochondria. In addition, the GCH1-synthesized BH4 pathway serves as a parallel radical-trapping system. MBOAT1/2 enzymes confer membrane resistance by incorporating monounsaturated fatty acids (MUFAs), thereby displacing oxidizable targets. Finally, the master transcription factor NRF2 coordinates the global upregulation of these cytoprotective networks. Pro-oxidant pathways (right panel): Ferroptosis is driven by the iron-dependent peroxidation of polyunsaturated fatty acids (PUFAs). LPCAT3 and ACSL4 mediate the critical incorporation of PUFAs into membrane phospholipids (PUFA-PL). Intracellular accumulation of free ferrous iron (Fe²⁺) catalyzes LPO directly through the generation of ROS via the Fenton reaction and by serving as an essential cofactor for ALOX enzymes. ALOXs subsequently drive the robust oxidation of bis-allylic PUFAs. The labile iron pool can be further expanded through ferritinophagy, an autophagy-dependent process where NCOA4 mediates the lysosomal degradation of ferritin, releasing stored iron to fuel the ferroptotic cascade. Abbreviations: ACSL4, acyl-CoA synthetase long-chain family member 4; ALOX, arachidonate lipoxygenase; DHODH, dihydroorotate dehydrogenase; FSP1, ferroptosis suppressor protein 1; GCH1, GTP cyclohydrolase 1; GPX4, glutathione peroxidase 4; GSH, glutathione; LPCAT3, lysophosphatidylcholine acyltransferase 3; LPO, lipid peroxidation; MBOAT1/2, membrane-bound O-acyltransferase 1/2; MUFAs, monounsaturated fatty acids; NRF2, nuclear factor erythroid 2–related factor 2; PL, phospholipid; PUFAs, polyunsaturated fatty acids; ROS, reactive oxygen species; System xc⁻, cystine/glutamate antiporter system.

TABLE 1.

Key molecular players, regulators, and pathways in ferroptosis

Abbreviation	Full name	Biological function	Role in ferroptosis
Antioxidant defenses (suppressors)
GPX4	Glutathione peroxidase 4	Selenoprotein antioxidant enzyme	Primary canonical suppressor: Reduces toxic lipid hydroperoxides (LPO-OOH) to non-toxic lipid alcohols (LPO-OH) using GSH 14
System xc⁻	Cystine/glutamate antiporter	Heterodimeric membrane amino acid transporter	Upstream gatekeeper: Imports extracellular cystine in exchange for intracellular glutamate (1:1 ratio) to fuel GSH synthesis 15
FSP1	Ferroptosis suppressor protein 1	Myristoylated flavoprotein at the plasma membrane	Major non-canonical suppressor: Reduces CoQ10 to CoQ10H2 and mediates vitamin K cycling to trap lipid radicals independently of GPX4 16
DHODH	Dihydroorotate dehydrogenase	Mitochondrial inner membrane enzyme	Mitochondrial suppressor: Reduces CoQ10 to CoQ10H2 during pyrimidine synthesis to protect mitochondrial membranes from LPO 17
GCH1	GTP cyclohydrolase 1	Rate-limiting enzyme in BH4 biosynthesis	Parallel suppressor: Produces BH4, a potent radical-trapping antioxidant, and promotes lipid remodeling to counter LPO 18
MBOAT1/2	Membrane-bound O-acyltransferase 1/2	Membrane lipid-remodeling enzymes	Lipid-remodeling suppressor: Esterifies monounsaturated fatty acids (MUFAs) into phospholipids, displacing oxidizable PUFAs 19
NRF2	Nuclear factor erythroid 2–related factor 2	Master transcription factor for antioxidant defense	Master transcriptional suppressor: Orchestrates transcriptional expression of genes controlling GSH synthesis, iron export, and NADPH production 20
Pro-oxidant and lipid metabolism (drivers)
ACSL4	Acyl-CoA synthetase long-chain family member 4	Lipid metabolic enzyme	Core driver and sensitivity determinant: Ligates long-chain polyunsaturated fatty acids (PUFAs, eg, AA, AdA) with CoA for membrane integration 21
LPCAT3	Lysophosphatidylcholine acyltransferase 3	Phospholipid remodeling enzyme	Core driver: Esterifies PUFA-CoAs into membrane phospholipids (PUFA-PLs), providing substrates for LPO 21
ALOXs	Lipoxygenases (eg, ALOX12, ALOX15, ALOX5)	Non-heme iron-containing dioxygenases	Enzymatic driver: Directly catalyzes the stereo-specific oxygenation of free and esterified PUFAs into lipid hydroperoxides 22
POR	Cytochrome P450 oxidoreductase	Membrane-bound flavoprotein	Enzymatic driver: Transfers electrons to drive iron-dependent enzymatic peroxidation of PUFA-PLs 23
Iron homeostasis regulators
TfR1 (TFRC)	Transferrin receptor 1	Plasma membrane glycoprotein	Pro-ferroptotic importer: Imports ferric iron (Fe3+)-bound transferrin via receptor-mediated endocytosis 24
Ferritin (FTH1/FTL)	Ferritin heavy/light chain	Cytosolic iron storage protein complex	Ferroptosis suppressor: Sequestrates cytosolic free iron into a redox-inactive Fe3+ core, limiting Fenton reactions 25
NCOA4	Nuclear receptor coactivator 4	Selective autophagic cargo receptor	Pro-ferroptotic driver: Delivers ferritin to lysosomes for degradation (ferritinophagy), releasing free ferrous iron (Fe2+) into the labile iron pool25,26
FPN1 (SLC40A1)	Ferroportin 1	Transmembrane iron exporter	Ferroptosis suppressor: Exports intracellular ferrous iron (Fe2+) out of the cell, reducing iron overload27,28
ZIP14 (SLC39A14)	Solute carrier family 39 member 14	Divalent metal transporter	Pro-ferroptotic importer: Mediates non–transferrin–bound iron (NTBI) uptake into hepatocytes under iron-overload conditions29,30
Membrane repair and execution
ESCRT-III (CHMP4B, CHMP2A/B)	Endosomal sorting complexes required for transport III	Membrane remodeling protein machinery	Physical membrane repairer: Triggered by localized Ca2+ influx to repair LPO-induced membrane pores via ectocytosis and shedding31,32

Abbreviations: ACSL4, acyl-CoA synthetase long-chain family member 4; AdA, adrenic acid; AA, arachidonic acid; ALOX, lipoxygenase; BH4, tetrahydrobiopterin; CHMP2A/B, charged multivesicular body protein 2A/2B; CHMP4B, charged multivesicular body protein 4B; DHODH, dihydroorotate dehydrogenase; ESCRT-III, endosomal sorting complex required for transport III; FPN1, ferroportin 1; FSP1, ferroptosis suppressor protein 1; FTH1, ferritin heavy chain 1; FTL, ferritin light chain; GCH1, GTP cyclohydrolase 1; GPX4, glutathione peroxidase 4; GSH, glutathione; LPO, lipid peroxidation; LPO-OH, lipid alcohol; LPO-OOH, lipid hydroperoxide; LPCAT3, lysophosphatidylcholine acyltransferase 3; MBOAT1/2, membrane-bound O-acyltransferase 1/2; MUFAs, monounsaturated fatty acids; NCOA4, nuclear receptor coactivator 4; NADPH, nicotinamide adenine dinucleotide phosphate; NRF2, nuclear factor erythroid 2–related factor 2; NTBI, non–transferrin-bound iron; POR, cytochrome P450 oxidoreductase; PUFAs, polyunsaturated fatty acids; PUFA-CoAs, polyunsaturated fatty acid coenzyme A conjugates; PUFA-PLs, polyunsaturated fatty acid phospholipids; ROS, reactive oxygen species.

TABLE 2.

Summary of commonly used experimental tools and biomarkers for ferroptosis research

Category	Specific tool	Mechanism of action	Primary applications
Pharmacological inducers (FINs)
Class I FINs	Erastin, imidazole ketone erastin, sulfasalazine	Inhibits System xc⁻, depleting intracellular cystine and GSH.	In vitro cell death assays and in vivo tumor xenograft models (IKE has a superior PK profile over Erastin)3,33,34
Class II FINs	RSL3, ML162	Directly binds and inactivates GPX4 via covalent interaction with selenocysteine.	Robust in vitro driver of lipid ROS; limited in vivo utility due to poor solubility and rapid clearance35,36
Class III FINs	FIN56	Promotes GPX4 degradation and depletes CoQ10 via squalene synthase activation.	Used to dissect dual antioxidant pathway dysregulation in cell culture models 37
Class IV FINs	FINO2	Directly oxidizes iron and indirectly inactivates GPX4.	Used in vitro to study iron-dependent endoperoxide generation and membrane oxidation 38
Pharmacological inhibitors
Radical-trapping antioxidants	Ferrostatin-1 (Fer-1), Liproxstatin-1 (Lip-1)	Scavenges lipid peroxyl radicals in cellular membranes.	Gold-standard ferroptosis inhibitors in vitro and in vivo; Fer-1 has poor stability in plasma (Lip-1 preferred for in vivo)39,40
Iron chelators	Deferoxamine (DFO), deferiprone (DFP)	Chelates free ferrous (Fe2+) or ferric (Fe3+) iron, blocking Fenton reactions.	DFO is membrane-impermeant; DFP is cell-permeant. Used to prove the iron dependence of observed cell death41,42
Organelle-targeted chelators	Starch-Desferal	Sequester iron specifically within lysosomes.	Crucial for identifying subcellular iron translocation and compartmentation during APAP toxicity and IRI43,44
Genetic approaches
Pro-ferroptotic targets	ACSL4, LPCAT3, NCOA4	Disrupts PUFA incorporation into membranes or blocks ferritinophagy.	Routinely used to demonstrate genetic resistance to ferroptosis in vitro and in conditional knockout mice21,25,26
Anti-ferroptotic targets	GPX4, SLC7A11, FSP1	Abolishes antioxidant defenses or cystine import.	GPX4 KO triggers spontaneous ferroptosis in hepatocytes and tissue models; used to test essentiality14–16
Biomarkers and probes
Lipid ROS probes	C11-BODIPY (581/591), Liperfluo	Fluorescent sensors shift spectrum upon reaction with lipid hydroperoxides.	Real-time flow cytometry or confocal microscopy in live cells; highly recommended over non-specific DCFH-DA45,46
End-product biomarkers	MDA, 4-HNE	Measure lipid degradation products.	Widely used in serum/tissue homogenates; non-specific on their own and must be paired with functional rescue 3
Oxidized phospholipids	PE-AdA-OOH, PE-AA-OOH (LC-MS/MS)	Direct quantification of oxygenated polyunsaturated phosphatidylethanolamines.	Gold-standard definitive chemical biomarker of ferroptosis using mass spectrometry-based lipidomics 47
Iron sensors	Mito-FerroGreen, FerroOrange	Fluorescent probes detecting the chelatable labile iron pool.	Enables subcellular imaging of iron mobilization between the cytosol and mitochondria 2

Abbreviations: ACSL4, acyl-CoA synthetase long-chain family member 4; APAP, acetaminophen; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; DFO, deferoxamine; DFP, deferiprone; FIN, ferroptosis inducer; FINO2, ferroptosis inducer 2; Fer-1, ferrostatin-1; FSP1, ferroptosis suppressor protein 1; GPX4, glutathione peroxidase 4; GSH, glutathione; IKE, imidazole ketone erastin; IRI, ischemia-reperfusion injury; KO, knockout; LC-MS/MS, liquid chromatography–tandem mass spectrometry; Lip-1, liproxstatin-1; LPCAT3, lysophosphatidylcholine acyltransferase 3; LPO, lipid peroxidation; MDA, malondialdehyde; NCOA4, nuclear receptor coactivator 4; PE-AA-OOH, phosphatidylethanolamine-arachidonic acid hydroperoxide; PE-AdA-OOH, phosphatidylethanolamine-adrenic acid hydroperoxide; PK, pharmacokinetic; PUFA, polyunsaturated fatty acid; ROS, reactive oxygen species; SLC7A11, solute carrier family 7 member 11; System xc⁻, cystine/glutamate antiporter system.

Antioxidant defense networks

The classical System xc⁻/GSH/GPX4 antioxidant axis

As a cornerstone defense, the System xc⁻/glutathione (GSH)/GPX4 axis imports cystine for GSH synthesis, enabling GPX4 to reduce toxic lipid hydroperoxides to non-toxic alcohols.^14,15,48,49 Disrupting this axis offers therapeutic potential since tumor-specific GPX4 knockout triggers severe LPO and strongly suppresses tumorigenesis, highlighting malignant cells’ dependency on ferroptosis surveillance.^50–52 Notably, System xc⁻ overexpression rescues GPX4-deficient cells via a GPX4-independent mechanism. ⁵³ Beyond GSH, cysteine serves as a sulfur source for sulfane sulfur species (eg, hydropersulfides) that directly scavenge free radicals and suppress LPO, broadening the antioxidant capacity of the System xc⁻ network. ⁵³

GPX4 activity depends on selenium, featuring an indispensable catalytic selenocysteine (Sec) residue.^54–56 Co-translational Sec incorporation enables efficient lipid hydroperoxide detoxification and prevents irreversible overoxidation; substituting Sec with cysteine drastically impairs enzyme efficiency. ⁵⁵ Consequently, selenium deficiency compromises GPX4 translation and stability, whereas supplementation robustly drives its expression.^54–56 This highlights that, alongside GSH and cysteine availability, selenium metabolism is a key metabolic determinant of ferroptosis sensitivity and offers a vital therapeutic target for ferroptosis-associated diseases.

Non‑canonical antioxidant axis

Parallel non-GPX4 defense networks further protect cells. At the plasma membrane, ferroptosis suppressor protein 1 (FSP1) eliminates radicals via CoQ10 reduction and a vitamin K cycle, ¹⁶ while mitochondrial dihydroorotate dehydrogenase (DHODH) mitigates LPO through CoQ10 reduction independently of cytosolic FSP1 and mitochondrial GPX4. ¹⁷ Moreover, the GTP cyclohydrolase 1‑mediated tetrahydrobiopterin pathway acts as a third radical-trapping system. ¹⁸ Spatially, membrane‑bound O‑acyltransferase 1 and 2 (MBOAT1/2) incorporate monounsaturated fatty acids (MUFAs) into phospholipids to inhibit polyunsaturated fatty acid (PUFA) oxidation, with their regulation by sex hormone receptors explaining tumor susceptibility. ¹⁹ Finally, nuclear factor erythroid 2–related factor 2 (NRF2) acts as a master transcription factor, upregulating iron, GSH, and quinone genes to enhance survival. ²⁰ Current evidence increasingly points to highly specialized spatial compartmentalization of the ferroptotic regulators. GPX4/NRF2 provides global defense, DHODH protects mitochondria, and FSP1/MBOATs guard the plasma membrane. Therefore, future therapies should employ multi-organelle-targeting approaches to specifically target these ferroptotic compartments.

Pro-oxidant pathways

ACSL4-mediated peroxidation

Ferroptosis sensitivity depends on the dynamic balance between pro-ferroptotic and anti-ferroptotic lipids. Membrane PUFAs incorporated by acyl-CoA synthetase 4 (ACSL4) and peroxisomal ether phospholipids drive ferroptosis. ²¹ Alongside endoplasmic reticulum (ER) diacylglycerol phospholipids, peroxisomal PUFA-ether phospholipids can also promote ferroptosis. ⁵⁷ Conversely, ACSL3 and stearoyl-CoA desaturase 1 (SCD1) incorporate MUFAs, thereby displacing membrane PUFAs and enhancing resistance. ⁵⁸ Targeting ACSL4, the specific inhibitor AS-252424 binds its Gln464 residue, robustly blocking LPO and ferroptosis in acute liver injury models. ⁵⁹ Moreover, cells can sequester excess PUFAs within the core of lipid droplets by promoting triglyceride synthesis, exerting a protective effect. ⁵⁸

ALOX-mediated peroxidation

Excess free ferrous ions (Fe²⁺) drive LPO by both fueling free-radical generation from the Fenton reaction and serving as an essential cofactor for the lipoxygenase (ALOX) family.^22,60,61 ALOX enzymes drive ferroptotic LPO by catalyzing bis-allylic PUFA oxidation. ²² This process depends on the labile iron pool, which phosphorylase kinase G2 controls free iron availability. ²² ALOX12 mediates an ACSL4/GPX4-independent ferroptosis pathway, which is crucial for p53-dependent tumor suppression. ⁶² Photosensitizer-activated ALOX12 generates organelle-specific lipid ROS (eg, in the ER and Golgi), directly triggering ACSL4-independent ferroptosis in cancer cells. ⁶³ In the tumor microenvironment, ALOX5 deficiency mediates evasion of ferroptosis. In bladder cancer, EGR1 transcriptionally downregulates ALOX5, enhancing resistance and malignant progression. ⁶⁴ In summary, ALOXs constitute a core driving force for LPO in ferroptosis. Targeting ALOX networks not only provides novel intervention strategies to overcome drug resistance but also offers highly promising therapeutic targets to alleviate pathological processes.

Autophagy‑dependent ferroptosis

International guidelines define autophagy-dependent ferroptosis (ADF) as a mechanism where selective autophagy amplifies LPO by degrading anti-ferroptotic components or remodeling metabolism. ² It operates via: (1) ferritinophagy, where NCOA4 targets ferritin for lysosomal degradation, releasing Fe²⁺ to expand the labile iron pool;^25,26 (2) lipophagy, where RAB7A drives lipid droplet breakdown to supply free PUFA substrates; ⁶⁵ (3) chaperone-mediated autophagy, where HSPA8/LAMP2 degrade GPX4 via its KFERQ-like motif, dismantling antioxidant defense; ⁶⁶ (4) clockophagy, where SQSTM1-mediated BMAL1 degradation alters FABP3/7 to remodel fatty acid storage. ⁶⁷

ADF presents a biological paradox in which cytoprotective autophagy is hijacked to confer pro-death selectivity, yet the molecular switches driving this transition remain unknown. In the liver, this duality offers a tunable therapeutic window. Hyperactivating ADF bypasses apoptotic resistance to trigger ferroptosis in HCC. Conversely, decoupling specific autophagic receptors for ferritinophagy in acute liver injury prevents ferroptotic damage while preserving the basal autophagic flux necessary for hepatic homeostasis.

LPO, MEMBRANE DAMAGE, AND REPAIR DURING FERROPTOSIS

In terminal ferroptosis, LPO alters membrane biophysics via increased tension, thinning, permeabilization, and membrane rupture but also triggers dynamic cellular membrane repair mechanisms. If the membrane repair mechanisms fail, the spatiotemporally complex LPO across diverse organelle membranes ultimately drives organelle damage and plasma membrane rupture, resulting in mixed necrotic and apoptotic cell death.

Membrane damage induced by LPO and its spatiotemporal origins

LPO follows a strict spatiotemporal order, yet its precise initiation site is debated between two distinct paradigms. One paradigm identifies the ER and ER-mitochondria contact sites (EMCSs) as initial hotspots, where PKCβII amplifies LPO signals before spreading to the plasma membrane.^68,69 Alternatively, lysosomal iron triggers local LPO and lysosomal membrane permeabilization (LMP).^70–72 Fentomycin-1 or endogenous iron leakage augments LMP to drive cell-wide LPO,^70,71 resulting in dual ferroptotic and pyroptotic death. ⁷²

This spatiotemporal discrepancy (ER/EMCS vs. lysosome) likely stems from several complex factors: (1) Inducer heterogeneity: small molecules may ignite lysosomal iron while other treatments trigger ER-associated LPO. ⁷⁰ (2) Microscope resolution: spatiotemporal resolution and organelle-targeting affinity heavily bias these divergent findings. ⁷¹ (3) Cell-type-specific susceptibility: ferroptosis-resistant cancer cells exhibit LPO but require lysosomotropic agents like chloroquine to undergo LMP. ⁷¹ To resolve this discrepancy, future research must leverage unbiased, ultra-fast pan-organelle LPO probes and single-organelle lipidomics to map the dynamic trajectory of ferroptotic signaling.

Membrane repair mediated by ESCRT-III

During terminal ferroptosis, LPO-driven permeabilization, LMP-mediated iron leakage, and Piezo1-mediated Na⁺/Ca²⁺ influx collectively form an irreversible lethal signal. To counter this osmotic and structural collapse, cells deploy dual chemical and physical defenses. Chemically, hydropersulfides act as radical-trapping agents that autocatalytically intercept LPO. ⁵³ If this fails, endosomal sorting complexes required for transport III (ESCRT-III)-mediated physical membrane repair serves as the final, evolutionarily conserved defense against ferroptosis.^{31,32,73–75} LPO-induced pores trigger localized Ca²⁺ influx, recruiting ESCRT-III subunits (CHMP4B, CHMP2A/B) to damaged sites. ⁷³ Powered by VPS4 ATPase, they polymerize to drive outward budding and ectocytosis, shedding peroxides to reseal the membrane.^76,77 This repair delays membrane collapse, enhancing cell survival under oxidative stress by reshaping high-curvature membranes.^74,75,78 Conversely, compromising ESCRT function via Chmp4b knockdown or Vps4a interference sensitizes tumors to ferroptosis, offering a precise anti-tumor strategy. ⁷³

Understanding membrane damage and repair opens up exciting avenues for research in chemical biology and translational medicine. Methodologically, wash-free fluorescent probes (eg, MDG1, calcein, mitoferrofluor) enable sensitive, real-time in situ imaging of chelatable iron in relation to membrane permeability loss during ferroptosis and necrosis without physiological disruption.^79–82 Notably, ferroptosis-mediated membrane damage is a double-edged sword. ⁸³ Treating degenerative conditions like liver failure or neurodegeneration requires enhancing membrane repair to preserve organ function, whereas eradicating refractory liver cancer demands disrupting this repair to accelerate lethal collapse and overcome drug resistance. The mechanisms and molecular events regulating membrane damage and repair were summarized in Figure 2.

FIGURE 2.

Mechanisms of membrane damage and repair during ferroptosis. Membrane repair (left panel): To counter osmotic and structural collapse, cells deploy dual defenses. Chemically, hydropersulfides act as radical-trapping agents to intercept LPO. Physically, when chemical defense fails, LPO-induced pores trigger localized Ca²⁺ influx via channels like Piezo1, which recruits endosomal sorting complexes required for transport III (ESCRT-III) complex proteins (including CHMP4B and CHMP2A/B) to damaged sites. Powered by VPS4 ATPase, these proteins polymerize to drive outward budding and ectocytosis, shedding peroxides to reseal the plasma membrane and delay collapse. Membrane damage (right panel): The spatiotemporal initiation of LPO-mediated membrane damage operates through 2 distinct paradigms. In paradigm 1, the endoplasmic reticulum (ER) and ER-mitochondria contact sites (EMCSs) serve as initial hotspots, where PKCβII amplifies LPO signals before they spread to the plasma membrane. In paradigm 2, lysosomal iron triggers localized LPO and lysosomal membrane permeabilization (LMP). Collectively, LPO-driven plasma membrane permeabilization, LMP-mediated iron leakage, and Piezo1-mediated Na⁺/Ca²⁺ influx form an irreversible lethal signal. Abbreviations: CHMP2A/B, charged multivesicular body protein 2A/2B; CHMP4B, charged multivesicular body protein 4B; EMCSs, endoplasmic reticulum–mitochondria contact sites; ER, endoplasmic reticulum; ESCRT-III, endosomal sorting complex required for transport III; LMP, lysosomal membrane permeabilization; LPO, lipid peroxidation; PKCβII, protein kinase C beta II; VPS4, vacuolar protein sorting-associated protein 4.

MECHANISM OF IRON HOMEOSTASIS AND REGULATION IN THE LIVER

The liver plays a central role in regulating systemic iron homeostasis via hepatocyte-derived hepcidin, ⁸⁴ which targets the exporter ferroportin,^27,28 the sole known cellular iron exporter, for degradation, thereby controlling intestinal absorption and macrophage iron recycling. ⁸⁵ Hepcidin expression is upregulated in response to elevated circulating and hepatocellular iron levels and downregulated under conditions of systemic iron deficiency, which is mediated through sinusoidal endothelial-hepatocyte crosstalk and the bone morphogenetic protein (BMP)2/6–SMAD1/5/8 axis.^86,87 Disruption of this hepatocyte surface machinery impairs SMAD signaling, thereby driving systemic and hepatic iron overload in humans. ⁸⁸

Cellular iron homeostasis is tightly regulated at the levels of uptake, export, utilization, and storage. Iron acquisition occurs via transferrin receptor 1 (TfR1)-mediated endocytosis of circulating transferrin-bound iron (TBI),^24,89 followed by divalent metal transporter 1 (DMT1)-mediated endosomal export into the cytosol or mitochondria. ⁹⁰ This mechanism is also utilized by most, if not all, cell types. Under iron excess, circulating non–transferrin-bound iron (NTBI) enters hepatocytes via ZIP14 (encoded by Slc39a14 gene), driving iron overload in hereditary hemochromatosis.^29,30,91,92 Distinctly, liver macrophages like Kupffer cells accumulate iron by phagocytosing senescent erythrocytes and injury debris. ⁹³

To counter iron-induced oxidative damage, elevated iron drives ferroportin-mediated export and ferritin-dependent sequestration while suppressing TfR1/DMT1-mediated import. ⁹⁴ Conversely, iron deficiency reverses this pattern and activates NCOA4-mediated ferritinophagy,^25,26 delivering ferritin to lysosomes for degradation and iron release. ⁹⁵ These adaptations rely on post-transcriptional regulation via iron regulatory proteins (IRPs) binding to iron-responsive elements (IREs) within target mRNAs (ferroportin, TfR1, DMT1, ferritin). ⁹⁶

Mitochondria utilize iron to synthesize iron-sulfur clusters (ISCs) and heme,^97,98 cofactors vital for metabolism, respiration, and redox control. ⁹⁹ Disrupted ISC biogenesis drives mitochondrial iron accumulation and cytosolic deficiency, ¹⁰⁰ feedback-regulating cellular iron import and export. Consequently, mitochondria play an important role in iron homeostasis, especially in high-ATP-demanding cell types such as hepatocytes.

The mechanisms described above collectively maintain systemic and cellular iron balance under physiological conditions. However, iron homeostasis is frequently disrupted in liver diseases. Suppressed hepcidin production drives systemic and hepatic iron overload in alcohol-associated liver disease (ALD) and chronic viral hepatitis (B and C).^101–103 Conversely, metabolic dysfunction–associated steatotic liver disease (MASLD) exhibits divergent iron phenotypes: 30%–40% of patients manifest mild-to-moderate iron overload in hepatocytes, macrophages, or both. ¹⁰⁴ Paradoxically, up to one-third of predominantly obese females display hepatic iron deficiency. ¹⁰⁵ The mechanisms underlying the divergent iron phenotypes among different groups of MASLD patients are unclear. Despite advances in quantifying total hepatic iron, its subcellular distribution and partitioning (labile iron, ISC/heme, ferritin) remain poorly characterized. Resolving these dynamics will elucidate both the mechanisms of ferroptosis and organelle dysfunction. Notably, mitochondrial iron overload-driven ROS strongly induces hepatocyte ferroptosis.^106,107

The exact intracellular iron threshold required to trigger ferroptosis remains undefined. Although acute iron shocks drive hepatocyte ferroptosis in vitro by depleting antioxidants, ¹⁰⁸ chronic in vivo accumulation rarely reaches toxic levels due to ferritin upregulation. Therefore, mild-to-moderate hepatic iron increases are insufficient to induce ferroptosis when antioxidant defenses remain intact.

FERROPTOSIS IN LIVER DISEASES

Ferroptosis in hepatic ischemia-reperfusion injury

Historically, hepatic ischemia-reperfusion injury (IRI) was hypothesized to stem from xanthine oxidase-mediated oxidative stress driving LPO and cell death.^109,110 This was challenged by low LPO levels, absent intracellular oxidant evidence,^111,112 and allopurinol’s xanthine oxidase-independent preconditioning effects. ¹¹³ However, severe ischemia does provoke brief xanthine oxidase superoxide formation and prolonged mitochondrial oxidant stress, ¹¹⁴ shifting the focus to extracellular inflammatory ROS. During the first few hours of reperfusion, Kupffer cells dominate vascular ROS generation.^115,116 Subsequently, complement and cytokines recruit neutrophils, driving oxidant stress and tissue injury between 6 and 24 hours.^117–121 Crucially, while moderate oxidant-induced liver injury typically elevates LPO markers (HETEs, F2-isoprostanes) by 1200%–3000%, severe IRI necrosis shows increases of <300%. ¹²² Thus, time-dependent ROS from discrete intracellular and extracellular sources drives hepatic IRI pathophysiology without relevant LPO. ¹²³

The concept of ferroptosis revived debates on hepatic oxidant stress and LPO. ¹²⁴ Many studies infer ischemia-reperfusion ferroptosis from non-specific markers, including depleted GSH, decreased GPX4, modulated ACSL4/Ptgs2, mild LPO, and elevated iron.^125–128 However, the lack of specific biomarkers, rigorous interventions, and quantitative correlations leaves current evidence weak and speculative. Yamada et al ¹²⁵ inferred IRI ferroptosis because deferoxamine, ferrostatin-1, and α-tocopherol attenuated severe liver injury. However, these pharmacological tools lack specificity for ferroptosis; deferoxamine is not merely an iron chelator but also mitigates peroxynitrite cytotoxicity, a key IRI mediator,^129,130 while ferrostatin-1 acts as a lipophilic radical-trapping antioxidant with broad free-radical scavenging properties rather than an exclusive ferroptosis inhibitor. ¹³¹ Thus, cytoprotection by deferoxamine or ferrostatin-1 alone cannot be taken as definitive proof of ferroptosis execution. Furthermore, standard rodent diets provide sufficient vitamin E; short-term α-tocopherol supplementation fails to further improve baseline antioxidant defenses. ¹³² Crucially, a major mismatch exists between the massive injury (>7000 U/L ALT) and the minor (1.4-fold to 2-fold) increase in MDA/LPO, even under iron-rich diets. ¹²⁵ These data conflict with the hypothesis that iron-dependent LPO causes reperfusion injury, challenging the involvement of ferroptosis.

Nonetheless, in a hepatocyte model of IRI, intracellular movement of Fe²⁺ during ischemia from the lysosomal/endosomal compartment to mitochondria is a key factor sensitizing cells to lethal injury after reperfusion. ⁴⁴ Because of ATP depletion during ischemia, the vacuolar proton-pumping ATPase (V-ATPase) becomes inhibited, leading to the collapse of the lysosomal/endosomal pH gradient and consequent release of lysosomal iron stores. ¹³³ Such iron enters the cytosol and is then taken up by mitochondria as Fe²⁺ via the mitochondrial calcium uniporter (MCU). Mitochondrial iron then predisposes to the formation of reactive oxygen species (ROS) and activated hydroperoxides after reperfusion, causing opening of high conductance permeability transition (PT) pores, bioenergetic failure, and cell death. Lysosomally targeted iron chelation with starch-desferal and inhibition of MCU-mediated mitochondrial Fe²⁺ uptake by Ru360 attenuate iron-dependent mitochondrial ROS generation, inner membrane permeabilization, and cell death in IRI. The relatively slow time-dependent movement of iron from lysosomes to mitochondria during ischemia provides a mechanistic basis for why lethal IRI occurs after longer periods of ischemia but not shorter.

Taken together, newer data confirm previous findings that there is clear oxidant stress during reperfusion, also involving the generation of aggressive oxidants such as peroxynitrite in hepatocytes ¹³⁰ and hypochlorous acid in neutrophils ¹²¹ during reperfusion. Consistent throughout the literature, the extent of LPO is moderate, and quantitatively, LPO does not correlate with the severity of reperfusion injury. Therefore, evidence that ferroptosis is central to the pathophysiology of hepatic IRI has not been convincing. However, iron translocation into mitochondria promotes IRI. Consequently, increased LPO may be confined to and directed against mitochondrial membranes. Thus, less total LPO may be sufficient to drive lethal cell injury. Nonetheless, given that reactive oxidants are generated, any condition in which cellular antioxidant mechanisms are significantly impaired poses the risk that the mechanism of cell death can switch to ferroptosis.

Ferroptosis in DILI

DILI is the leading cause of acute liver failure, with oxidative damage and ferroptosis recognized as critical pathophysiological nodes. ⁴ However, ferroptosis’s precise contribution depends on the inducing drug, the underlying subcellular mechanisms, and the detection specificity.

Acetaminophen (APAP) overdose, the primary cause of DILI in Western countries, sparks debate over whether hepatocyte death is driven by ferroptosis or another form of cell death. A critical analysis of conflicting literature reveals that discrepancies among studies stem primarily from the non-specificity of pharmacological tools or animal diet. While some LPO accumulation and Ferrostatin-1 protection implicate ferroptosis,^4,134 classical toxicology dictates that NAPQI-mediated GSH depletion and mitochondrial oxidative stress with peroxynitrite (ONOO⁻) bursts trigger lethal oncotic necrosis.^135,136 Importantly, peroxynitrite-mediated protein nitration and opening of the mitochondrial PT pores depend on lysosomal ferrous iron taken up into mitochondria via MCU.^137,138 The critical role of mitochondrial protein nitration over LPO was shown by the exaggeration of APAP-induced liver injury in partial MnSOD (SOD2)-deficient mice ¹³⁹ and by the dramatic protection of mitochondrial-targeted SOD mimetics Mito-TEMPO ¹⁴⁰ and mitoquinone. ¹⁴¹ In addition, a 700% increase in α-tocopherol or γ-tocopherol content of the liver did not protect against APAP overdose. ¹³² Thus, most antioxidants preserve hepatocytes by scavenging mitochondrial ROS to block necrosis, rather than specifically inhibiting ferroptosis. These data demonstrate that under normal circumstances, there is extensive mitochondrial protein nitration but limited LPO after an overdose of APAP, and thus, there is no ferroptosis. However, the formation of mitochondrial ROS and the mobilization of ferrous iron during APAP-induced cell death indicate that, although endogenous defense mechanisms are still intact, the cell is at risk of developing LPO. In fact, if animals are fed a diet high in soybean oil to increase the hepatic content of PUFAs and low in vitamin E, an APAP or allyl alcohol overdose triggers massive LPO-dependent liver injury, ¹⁴² which can be classified as ferroptosis. Similarly, if animals are treated with a moderate dose of ferrous iron together with APAP, this combination triggers extensive LPO and aggravates injury compared with APAP alone. Although this also increases iron-dependent protein nitration, LPO, and thus ferroptosis, it is now the dominant mechanism of cell death. ¹⁴³ This is clinically important as the standard antidote, N-acetylcysteine, is no longer protective beyond the metabolism phase. ¹⁴³ Together, these data suggest that ferroptosis is not involved in the injury mechanism of APAP hepatotoxicity under most conditions; however, modulation of PUFA or iron levels and/or impairment of antioxidant defenses can shift the mode of cell death to ferroptosis.

Recent findings unify necrosis and ferroptosis, showing that ferroptosis drives heterogeneous cell death dominated by lysosomal membrane rupture under GSH depletion. ¹⁴⁴ During APAP hepatotoxicity, the metalloreductase STEAP4 maintains lysosomal iron homeostasis, preserving membrane integrity to limit iron overload-driven necrosis and enable liver regeneration. ¹⁴⁵ The underlying mechanism of APAP hepatotoxicity involves mitochondrial dysfunction from mitochondrial oxidant stress and the onset of mitochondrial permeability transition (MPT). ROS play an essential role, and iron is a critical catalyst for ROS formation. Normally, hepatocytes take up Fe³⁺-transferrin bound to transferrin receptors via receptor-mediated endocytosis. As accumulated in lysosomes, controlled release of iron supports mitochondrial biosynthesis of heme and non-heme ISCs. After APAP overdose, the toxic metabolite, NAPQI, permeabilizes lysosomes, causing excess iron release and mitochondrial uptake of Fe²⁺ by MCU. NAPQI also inhibits mitochondrial respiration to promote ROS formation, including H₂O₂. H₂O₂ reacts with mitochondrial Fe²⁺ to form a highly reactive hydroxyl radical (•OH) through the Fenton reaction, leading to LPO, the formation of toxic aldehydes, MPT onset, and ultimately cell death. Moreover, Fe²⁺ also promotes protein nitration. ¹³⁷ Thus, targeting pathways of mitochondrial iron uptake and consequent iron-dependent mitochondrial ROS formation is a promising strategy to intervene against APAP hepatotoxicity. Collectively, APAP-induced necrosis, LPO, and impaired hepatocyte proliferation are mediated by disrupting lysosomal iron homeostasis, establishing lysosomal iron compartmentalization as a critical factor for liver regeneration in DILI.

Unlike the well-characterized APAP model, the role of ferroptosis in non-APAP DILI remains poorly defined. Emerging evidence reveals drug-specific mechanisms: crizotinib triggers hepatocyte ferroptosis by suppressing the STAT1/NRF2 antioxidant axis, ¹⁴⁶ while ionophoric polyether antibiotics activate TFEB/NCOA4-mediated ferritinophagy to release toxic free iron. ¹⁴⁷ Given the heterogeneity of non-APAP drugs, further studies are required to delineate ferroptosis’s broader toxicological contribution.

Advanced multi-target fluorescent probes offer powerful tools to validate subcellular DILI mechanisms. Specifically, dual-targeted near-infrared probes dynamically monitor mitochondrial viscosity in vivo, ¹⁴⁸ while Fe²⁺-specific sensors track labile iron homeostasis in specific subcellular compartments,^79–82 visualizing organelle collapse during hepatotoxicity. Collectively, these imaging innovations shift DILI theranostics from macroscopic histology toward suborganelle mechanics, iron compartmentalization, and pro-regenerative intervention. Targeting the ferroptotic cascade may offer high translational value across DILI subtypes.

Ferroptosis in viral hepatitis

Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections drive liver cirrhosis and HCC,^149,150 with ferroptosis serving as the central molecular hub linking viral infection, iron overload, and persistent injury.^151,152 These hepatotropic viruses hijack host iron metabolism to expand the hepatocyte labile iron pool,^153,154 simultaneously fueling the viral life cycle and providing substrates for Fenton reaction-driven LPO.

However, a critical evaluation of the literature reveals a paradoxical duality: HBV exerts seemingly opposing roles, acting as a pro-ferroptotic driver during chronic infection but switching to an anti-ferroptotic suppressor following malignant transformation. Discrepancies among studies can be reconciled by considering both cell-type specificity and disease-stage-dependent metabolic rewiring.

HBV infection exploits ferroptosis to remodel the immune microenvironment and facilitate viral evasion. Mechanistically, microenvironmental TGF-β1 downregulates GPX4, while palmitic acid upregulates CD36, synergistically driving lipid accumulation and CD8⁺ T-cell ferroptosis-mediated exhaustion. Consequently, ferroptosis inhibitors (eg, selenium, liproxstatin-1) restore T-cell antiviral immunity. In addition, HBV-positive hepatoma-derived exosomal miR-142-3p downregulates SLC3A2, inducing M1 macrophage ferroptosis and selectively impairing host immune surveillance. ¹⁵⁵ Contrasting its pro-ferroptotic role in chronic infection, HBV exerts anti-ferroptotic effects after malignant transformation into HCC and reverses erastin-induced mitochondrial atrophy and LPO, thereby protecting hepatoma cells. ¹⁵⁶ The viral protein HBx upregulates transcription of sirtuin 1 (SIRT1), which remodels tumor metabolism. ¹⁵⁶ This HBx-SIRT1 axis-driven resistance underlies the clinical refractoriness of HBV-associated HCC to systemic therapies. This bidirectional manipulation requires spatiotemporally precise interventions: deploying ferroptosis inhibitors during chronic hepatitis to restore antiviral immunity and attenuate fibrogenesis, while using targeted FINs in HBV-associated HCC to eliminate chemoresistant cells.

Ferroptosis in ALD and MASLD

ALD

ALD is a leading cause of global mortality from chronic liver diseases. ¹⁵⁷ Mechanistically, oxidative ethanol metabolism induces ROS, organelle stress, Mallory-Denk body formation, which cooperatively promote hepatocyte death and immune cell recruitment.^158–160 Multiple hepatocyte death modes (apoptosis, necroptosis, pyroptosis, ferroptosis) occur in ALD. ¹⁶⁰ This review highlights emerging evidence for hepatic ferroptosis. Treatment of alcohol-fed mice with ferrostatin-1, a lipid ROS scavenger and ferroptosis inhibitor, reduced liver injury, supporting a role for ferroptosis in ALD. ¹⁶¹ Subsequent work showed that ferroptosis occurs in mice fed an ethanol diet for 8 weeks plus a single binge, but not in those fed an ethanol diet for 10 days plus one binge. ¹⁶² This was associated with increased LPO products, elevated ACSL4 expression, increased labile iron content, and reduced GSH levels. Similar findings have been reported by another study in both chronic-plus-binge and acute-plus-binge alcohol-feeding models, along with significant downregulation of xCT and GPX4 in the liver. ¹⁶³ Other studies have further shown that intestinal SIRT1 and brown adipose tissue-derived eNAMPT modulate hepatic iron metabolism, thereby regulating ferroptosis vulnerability in ALD.^164,165

Despite these advances, the mechanisms regulating ferroptosis-associated pathways under alcohol-fed conditions remain undefined, and direct evidence of LPO-dependent hepatic cell necrosis is lacking.

MASLD

MASLD is another highly prevalent chronic liver disease. Like ALD, it also encompasses a spectrum of pathological conditions, including steatosis, inflammation, fibrosis, and cirrhosis. Compared with ALD, the features of ferroptosis are better defined in mouse models of MASLD. Tsurusaki et al ¹⁶⁶ are among the first to demonstrate that hepatocytes undergo ferroptosis in choline-deficient, ethionine-supplemented (CDE) diet-induced metabolic dysfunction–associated steatohepatitis (MASH). Hepatocyte necrosis was identified through in situ staining with propidium iodide (PI). They showed that this cell death was not necroptosis, as MLKL knockout did not reduce the number of PI-positive cells. In contrast, treatment with deferiprone, an iron chelator, and trolox, a lipid ROS scavenger, significantly reduced the number of PI-positive cells, suggesting iron-mediated LPO-dependent cell death. Ferroptosis has also been implicated in methionine-choline-deficient (MCD) diet-induced MASH, where administration of lipid ROS scavengers alleviated liver injury, inflammation, and fibrosis. ¹⁶⁷ However, many subsequent studies have primarily reported alterations in canonical ferroptosis-related molecules without directly demonstrating ferroptotic cell death in the liver.

A major limitation of the studies described above is their reliance on dietary models that induce profound oxidative stress, mitochondrial dysfunction, and marked depletion of key antioxidant systems, particularly GSH. Consequently, it remains unclear whether ferroptosis occurs under conditions that more closely recapitulate the natural pathogenesis of human MASLD. Similar to the liver diseases discussed above, the molecular mechanisms underlying hepatic ferroptosis in MASLD remain poorly understood. A recent study suggests a protective role for PPARα in MASH-associated ferroptosis, ¹⁶⁸ but the associated pathways require further investigation. To date, most investigations have focused on the disruption of the GPX4-GSH axis as a central mechanism driving ferroptosis in liver diseases. In contrast, the contribution of the CoQ10 antioxidant pathway and the regeneration of its reduced form, CoQ10H2, has received very little attention in MASLD. This pathway is of particular interest because CoQ10 metabolism becomes dysregulated during the metabolic maladaptation that accompanies MASLD progression. Consistent with this concept, a recent study demonstrated that mitochondrial oxidative stress, a hallmark of advanced MASLD, suppresses CoQ8A-dependent CoQ10 biosynthesis, thereby compromising endogenous CoQ10 antioxidant defenses and increasing hepatocyte susceptibility to ferroptosis. ¹⁰⁸ These findings suggest that impaired CoQ10 homeostasis may represent an underappreciated mechanism contributing to ferroptotic vulnerability in MASLD and warrant further mechanistic investigation.

Ferroptosis in liver fibrosis

Hepatic stellate cells (HSCs), pericytes in the space of Disse, centrally mediate liver wound healing. Though quiescent in healthy tissue, injury activates their transdifferentiation into proliferative, contractile, and migratory myofibroblasts. ¹⁶⁹ Activated HSCs drive scar formation by producing extracellular matrix (ECM) proteins like fibrillar collagens. While adaptive during acute tissue repair, chronic liver damage prompts persistent HSC activation and excessive ECM deposition, replacing functional parenchyma with fibrotic tissue and culminating in cirrhosis. ¹⁷⁰

Ferroptosis crucially regulates HSC survival and fibrogenesis. Driven by active lipid metabolism and iron handling during transdifferentiation, activated HSCs are highly susceptible to ferroptosis. ¹⁷¹ Consistently, canonical triggers, such as cysteine deprivation, System xc⁻ (SLC7A11) blockade, or GPX4 inhibition, effectively induce activated HSC ferroptosis. ¹⁷² These perturbations compromise antioxidant defenses and drive LPO-triggered HSC ferroptosis. Thus, ferroptosis serves as an intrinsic mechanism limiting fibrogenic stellate cells during chronic liver injury.

Among the multiple pathways that regulate HSC ferroptotic susceptibility, p53 signaling promotes cell death via mitochondrial translocation. Here, it enhances SLC25A28 activity, driving redox-active iron accumulation and increasing ferroptotic sensitivity. ¹⁷³ During HSC activation, the Hippo/YAP pathway regulates ferroptosis via the p21-GPX4 axis. ¹⁷⁴ Iron availability and autophagic signaling further dictate sensitivity: HO-1 promotes heme degradation, ¹⁷⁵ whereas ferritinophagy releases labile iron to amplify LPO. ¹⁷⁶ In addition, post-transcriptional and epitranscriptomic networks program gene expression, driven by RNA-binding proteins (ZFP36, ELAVL1/HuR), modulating transcript stability^177,178 and m6A RNA methylation.^179,180 Together, these regulatory networks underscore the complex integration of metabolic, transcriptional, and post-transcriptional signals that determine ferroptosis sensitivity in fibrogenic stellate cells.

Since activated HSCs drive hepatic fibrogenesis, inducing their ferroptosis represents a promising antifibrotic strategy, with multiple pro-ferroptotic agents demonstrating preclinical efficacy. Inhibition of the System xc⁻ with erastin suppresses HSC survival and attenuates fibrosis in rodents. Other agents with ferroptosis-inducing properties, including artesunate, ¹⁸¹ magnesium isoglycyrrhizinate, ¹⁸² berberine, ¹⁸³ and sorafenib, ¹⁸⁴ have also been shown to mitigate experimental liver fibrosis through mechanisms linked to increased ferroptosis in activated HSCs. Many of these compounds appear to act by perturbing GSH metabolism, enhancing intracellular iron availability, or promoting LPO within HSC. Clinically, retrospective analyses of sorafenib-treated advanced fibrosis and HCC liver tissues reveal ferroptotic features in primary human HSCs,^173,177,178 suggesting that pharmacologic induction of ferroptosis can occur in fibrogenic HSC in vivo.

However, exploiting ferroptosis in liver fibrosis is highly context-dependent. Systemic inducers, including cyst(e)inase, erastin and its imidazole ketone derivative, iron dextran, and RSL3, can paradoxically exacerbate injury, HSC activation, and inflammation in carbon tetrachloride-induced or bile duct ligation-induced liver injury and MASH models.^171,185 Because chronic disease sensitizes multiple hepatic cell populations to ferroptosis,^185,186 non-selective induction risks aggravating hepatocyte death and amplifying inflammatory cascades, thereby indirectly fueling fibrogenic HSC activation.

These findings underscore a critical therapeutic challenge: selectively inducing ferroptosis in fibrogenic HSCs while preserving hepatocyte viability. Given their distinct iron and redox profiles,^171,187 defining the molecular determinants of cell-type-specific ferroptosis sensitivity is vital. Deciphering these mechanisms will enable precise therapies that eradicate pathogenic myofibroblasts while protecting parenchymal cells, thereby breaking the self-perpetuating cycle of chronic liver injury.

Ferroptosis in HCC

HCC accounts for 80%–90% of primary liver cancers, fueled by chronic viral infections and escalating MASLD incidence. ¹⁸⁸ Beyond standard advanced therapies [tyrosine kinase inhibitors (TKIs)/immune checkpoint inhibitors (ICIs)], ferroptosis represents a promising, exploitable therapeutic vulnerability.

Metabolic reprogramming and ferroptosis sensitivity

HCC cells evade ferroptosis by rewiring their iron availability, lipid profile, and antioxidant systems. Firstly, HCC cells fine-tune iron homeostasis across the cytosol and mitochondria to prevent the accumulation of redox-active iron. The LIFR–NF–κB–Lipocalin 2 (LCN2) axis limits the intracellular labile iron pool through LCN2-mediated iron sequestration. ¹⁸⁹ Complementing this, the COMMD10-HIF1α-CP loop alters the Cu-Fe balance to decrease available iron for LPO, ¹⁹⁰ while the glycolytic enzyme Enolase 1 (ENO1) non-canonically represses mitoferrin-1 to block mitochondrial iron accumulation and subsequent oxidative damage. ¹⁹¹

Secondly, HCC cells rewire lipid metabolism to evade ferroptosis under therapeutic or metabolic stress. Mechanistically, the chaperone-unconventional prefoldin RPB5 interactor (URI) regulates p53-dependent stearoyl-CoA desaturase 1 (SCD1) expression, thereby reprogramming fatty acid desaturation to render p53-wild-type HCC resistant to TKI-induced ferroptosis. ¹⁹² Similarly, the calcium-binding protein S100P mediates the degradation of acetyl-CoA carboxylase 1 (ACC1) to limit the availability of substrates for LPO, ¹⁹³ while fatty acid-binding protein 5 (FABP5) gates fatty acid transport to protect obesity-associated liver cancer from ferroptosis. ¹⁹⁴

Lastly, HCC cells fortify antioxidant networks to neutralize LPO. Mechanistically, FSP1 eliminates LPO via the CoQ10 pool, ¹⁹⁵ which is sustained alongside GPX4 translation by the mevalonate pathway. ¹⁹⁶ Besides, the ATF4–SLC7A11 axis and the membrane protein aminopeptidase N (ANPEP) promote cystine uptake and GSH biosynthesis, ¹⁹⁷ while the checkpoints phosphoseryl-tRNA kinase (PSTK) and apurinic/apyrimidinic endonuclease 1 (APE1) buffer redox balance to drive chemoresistance.^198,199 In contrast, the mitochondrial enzyme glutaminase 2 (GLS2) promotes ROS production and ferroptosis. ²⁰⁰ HCC cells can strategically couple glutaminolysis to the ALDH18A1-spermine biosynthesis axis to build a parallel, GPX4-independent cell-death-evasion program, which establishes metabolic plasticity under nutrient stress and presents a promising therapeutic vulnerability for genetic or small-molecule intervention. ²⁰¹

Mitochondrial reprogramming promoting ferroptosis sensitivity

Despite increased defenses against ferroptosis, proliferating HCC cells are more vulnerable to FINs than non-proliferating hepatocytes. For example, an effect of erastin is to block tubulin-dependent inhibition of VDAC conductance, leading to increased entry of respiratory substrates, mitochondrial hyperpolarization, and formation of mitochondrial ROS with consequent ferroptotic cell death.^202,203 By contrast, VDAC conductance in non-proliferating hepatocytes is uninhibited due to low cytosolic free tubulin concentrations, and erastin has no toxic effect.

In proliferating cancer cells, a metabolic switch occurs from electrogenic mitochondrial ATP/ADP exchange catalyzed by the adenine nucleotide translocator (ANT) and driven by mitochondrial membrane potential (ΔΨ) to non-electrogenic exchange likely catalyzed by the ATP-Mg/phosphate carrier (AMPC).^204,205 AMPC-catalyzed non-electrogenic exchange leads to a ~50–100-fold decrease in cytosolic ATP/ADP relative to non-transformed post-mitotic cells, which stimulates aerobic glycolysis (Warburg effect). However, without energy utilization required for electrogenic ATP/ADP exchange, ATP/O ratios for oxidative phosphorylation increase by 35% for complete glucose oxidation, an advantage for biomass formation by proliferating cancer cells. Through near-equilibrium relationships, decreased cytosolic ATP/ADP increases cytosolic and mitochondrial NAD(P)H/NAD(P)⁺ ratios and the redox span across complexes I and III of the mitochondrial respiratory chain, resulting in increased mitochondrial ΔΨ and ROS generation. A consequence is increased susceptibility to ferroptosis in the event that anti-ferroptotic defenses become compromised. Overall, non-electrogenic ATP/ADP exchange promotes aerobic glycolysis (Warburg effect) and confers specific growth advantages to cancer cells but at the expense of a relative vulnerability to ferroptosis. ²⁰⁵

Ferroptosis in liver tumor microenvironment

Ferroptosis shapes the immune landscape in HCC, exerting a dual effect on tumor progression by recruiting cytotoxic T cells while also attracting immunosuppressive subtypes that lead to T-cell exhaustion. On one hand, ferroptotic HCC cells can trigger a tumor-suppressive immune response. This is primarily mediated by the release of damage-associated molecular patterns (DAMPs), such as HMGB1, which facilitates CXCL10-related cytotoxic CD8⁺ T cells infiltration. ²⁰⁶ Pharmacological inhibition of FSP1 also induces ferroptotic cell death in HCC, subsequently recruiting dendritic cells, macrophages, and T cells to the microenvironment. ¹⁹⁵ In addition, many ferroptosis suppressors were also reported to amplify tumor-suppressive microenvironments, such as FABP5 and FADS. Targeting FABP5 shifts tumor-associated macrophages (TAMs) toward a pro-inflammatory state while inhibiting FADS restores T-cell recruitment and sensitizes HCC cells to ferroptosis.^194,207

On the other hand, ferroptotic cell death can paradoxically induce a pro-tumorigenic microenvironment. Excessive release of HMGB1 from ferroptotic HCC recruits myeloid-derived suppressor cells (MDSCs), suppressing T-cell-mediated tumor-killing effect. ²⁰⁶ Moreover, ferroptosis activates the macrophage/IL-1β/neutrophil axis to drive vascular remodeling and promote HCC lung metastasis. ²⁰⁸ This immunosuppressive feedback is often reinforced by accumulation of 27-hydroxycholesterol (27HC) in HCC cells, an endogenous oxylcholesterol that prevents ferroptosis and induces CD8⁺ T cells exhaustion. ²⁰⁹

Aldehydes as drivers of oncogenic mutagenesis in the setting of fibrosis

Patients with fibrosis and cirrhosis from underlying ALD and MASLD/MASH have an increased risk for HCC, but the mechanistic basis for this predisposition is poorly understood.^210–212 Mutagenic aldehydes generated by LPO and ethanol metabolism may be the underlying cause. Aldehydes are mutagenic by forming adducts selectively on single-stranded DNA (ssDNA), leading to base substitutions largely at the opened-up ssDNA of transcription bubbles.^213,214 A possible explanation for why HCC develops in the setting of fibrosis/cirrhosis is that as hepatocytes are replaced with fibrotic scar, surviving hepatocytes undergo proliferative stress and express proto-oncogenes involved in normal cell growth and division. Aldehydes then mutate these proto-oncogenes at the ssDNA in their transcription bubbles, leading to oncogenic transformation and HCC in the setting of fibrosis. ²¹⁵ Nonetheless, further work is needed to support this hypothesis and determine how other factors, such as viral infection and exposure to environmental toxicants, may also contribute to cancer development.

THERAPEUTIC APPLICATIONS AND CLINICAL TRANSLATION

The clinical translation of ferroptosis-based interventions for liver diseases follows a dual paradigm: selectively inducing ferroptosis to eliminate malignant cells in HCC while suppressing the cascade to preserve parenchymal viability in non-cancerous injuries. In acute settings like APAP overdose, upregulating the NRF2-GPX4 axis or inhibiting ACSL4 robustly prevents LPO-driven necrosis. Similarly, in chronic disorders such as MASLD/MASH, mitigating iron-dependent LPO is vital to halting hepatocyte death and downstream inflammatory and fibrogenic cascades.

In acute liver injury like DILI and IRI, preclinical models demonstrate that lipophilic radical-trapping antioxidants (eg, Ferrostatin-1, Liproxstatin-1) and iron chelators robustly attenuate hepatocyte loss. However, several pivotal hurdles have prevented these agents from entering clinical trials: (1) Unfavorable druggability: Ferrostatin-1 and Liproxstatin-1 were identified via high-throughput cell screening and lack optimal pharmacokinetic properties.^39,40 They suffer from low aqueous solubility, metabolic instability, and short plasma half-lives in vivo, rendering them unsuited for acute systemic administration without advanced formulation science. (2) Lack of targeted delivery and off-target risks: Systemic iron chelation or non-specific ferroptosis inhibitor delivery risks disrupting physiological iron homeostasis,^41,42 blunting systemic immune defenses, or compromising beneficial physiological ROS signaling necessary for tissue repair.

Moreover, a major barrier in chronic liver diseases is profound patient heterogeneity. Consequently, biomarker-guided patient stratification is necessary. A precision-medicine framework must be established to identify patient subsets with active ferroptosis: (1) Biopsy-based spatial lipidomics: Integrating matrix-assisted laser desorption/ionization (MALDI) spatial lipidomics on liver biopsy specimens allows precise spatial mapping of LPO accumulation within specific liver zones and cell types. (2) Liquid-biopsy lipidomic profiling: Quantification of circulating oxidized polyunsaturated fatty acid-containing phospholipids via LC-MS/MS, alongside serum oxylipins and 4-HNE-protein adducts, can provide non-invasive readouts of active systemic/hepatic LPO.^3,47

Unlike non-cancerous injuries, clinical translation in HCC prioritizes novel FINs and combination therapies. This paradigm was pioneered by sorafenib, an FDA-approved TKI and the first identified FIN acting via System xc⁻ blockade. ¹⁵ Although poor pharmacokinetics limit the clinical translation of classical inducers like erastin and RSL3, ²¹⁶ alternative targets show significant promise. Specifically, APE1 inhibition destabilizes the SLC7A11-GPX4 axis, ¹⁹⁹ and FADS targeting sensitizes tumor cells to LPO while restoring cGAS-STING signaling. ²⁰⁷ Furthermore, statins and the anti-CD36 antibody PLT012 demonstrate therapeutic potential by modulating CoQ10 synthesis and lipid uptake, respectively.^196,217 To overcome ferroptosis resistance, current HCC research emphasizes combination regimens with TKIs or immunotherapies. Specifically, a triple combination (withaferin A/CXCR2 inhibitor/anti-PD-1) disrupts the HMGB1-mediated immunosuppressive microenvironment, ²⁰⁶ while the FSP1 inhibitor viFSP1 synergizes with sorafenib to target both tumor cells and TAMs, sensitizing HCC to anti-PD-L1. ²¹⁸ In addition, pairing TKIs with statins, LCN2-neutralizing antibodies, MT-1G inhibitors, or the SCD1 inhibitor Aramchol markedly enhances preclinical efficacy.^{189,192,196,219} Finally, ferroptosis serves as a potent adjuvant: the DHODH inhibitor leflunomide augments oxaliplatin chemotherapy via SLC7A11 degradation, ²²⁰ and COMMD10-driven iron modulation enhances radiosensitivity, ¹⁹⁰ collectively establishing a precision-medicine framework for advanced liver cancer.

A FRAMEWORK FOR DEFINING FERROPTOSIS IN LIVER DISEASES

Emerging evidence discussed above indicates that ferroptosis may represent an important mechanism underlying both acute and chronic liver injury across diverse disease contexts (Figure 3). However, in most studies, hepatic ferroptosis is inferred primarily from increased iron levels, accumulation of LPO products such as MDA and 4-HNE, and impaired antioxidant defenses, including GSH depletion and reduced GPX4 expression. While these features are indicative of ferroptotic susceptibility, they are insufficient to establish that cells have undergone ferroptotic cell death. Importantly, LPO is not unique to ferroptosis and can also contribute to other forms of cell death, including apoptosis. The precise distinctions between LPO associated with ferroptosis and apoptosis remain unclear, although they may differ in their magnitude, composition, and subcellular localization.

FIGURE 3.

The role and mechanisms of ferroptosis in liver diseases. Hepatic ischemia-reperfusion injury (IRI): Reperfusion induces distinct oxidative stress, characterized by the generation of peroxynitrite in hepatocytes and hypochlorous acid by recruited neutrophils. Although classical features such as glutathione (GSH) depletion, decreased GPX4, and ACSL4 modulation are observed, the quantitative extent of LPO is very moderate and does not correlate quantitatively with injury severity. Drug-induced liver injury (DILI): Acetaminophen (APAP) overdose causes NAPQI-mediated GSH depletion, mitochondrial oxidative stress, and a peroxynitrite burst, which trigger oncotic necrosis. The metalloreductase STEAP4 maintains lysosomal iron compartmentalization and membrane integrity to limit this iron overload-driven cell death. Viral Hepatitis (HBV/HCV): Hepatotropic viruses hijack host iron metabolism to expand the intracellular labile iron pool. In the chronic infection microenvironment, TGF-β1 and palmitic acid synergistically upregulate CD36, thereby driving lipid accumulation and inducing ferroptosis-mediated exhaustion of antiviral CD8⁺ T cells, thereby facilitating viral evasion. ALD and MASLD/MASH: In alcohol-associated liver disease (ALD), alcohol intake leads to a significant oxidative stress which can be suppressed by ferroptosis inhibitor (Fer-1). In metabolic dysfunction–associated steatohepatitis (MASH), hepatocyte ferroptosis is significantly blocked by the iron chelator deferiprone (DFP) or the lipid ROS scavenger Trolox. Furthermore, PPARα acts as a protective shield in MASH-associated ferroptosis. Liver fibrosis: During liver injury, quiescent hepatic stellate cells (HSCs) transdifferentiate into proliferative, contractile myofibroblasts. These activated HSCs are highly susceptible to ferroptosis, which can be effectively induced by cysteine deprivation, System xc⁻ blockade, or GPX4 inhibition. This sensitivity is regulated by p53 signaling and is precisely guided by the Hippo/YAP axis. Consequently, the targeted induction of ferroptosis in HSCs emerges as a highly promising antifibrotic strategy. Hepatocellular carcinoma (HCC): Malignant HCC cells evade ferroptosis through metabolic reprogramming and fortified antioxidant networks. Key evasion mechanisms include the LIFR–NF–κB–LCN2 axis sequestering iron via LCN2 to limit the labile iron pool, COMMD10 and ENO1 inhibiting mitochondrial iron accumulation, and the URI–p53–SCD1 axis altering lipid desaturation. Within the tumor microenvironment (TME), ferroptotic HCC cells release damage-associated molecular patterns (DAMPs) such as HMGB1 to promote the infiltration of cytotoxic CD8⁺ T cells.

Until a definitive and specific biomarker of ferroptosis is identified, rigorous interpretation of hepatic ferroptosis requires a multi-layered experimental framework. First, initial screening may include assessment of hepatic iron content, GPX4 expression, GSH levels, and indices of LPO. However, the absence of overt iron accumulation should not be interpreted as evidence against ferroptosis. Under pathological conditions, alterations in the expression of iron transporters, storage proteins, or intracellular iron chaperones may increase the labile iron pool without appreciably affecting total tissue or cellular iron content.

Second, it is essential to demonstrate that liver injury is associated with necrotic cell death, as ferroptosis is fundamentally a form of regulated necrosis rather than merely a state characterized by altered iron homeostasis, antioxidant capacity, or lipid oxidation. Necrosis can be directly visualized by in situ PI staining. Alternatively, combined TUNEL and cleaved caspase-3 staining may help distinguish necrotic from apoptotic cell death, as TUNEL labeling detects both apoptotic and necrotic cells, whereas cleaved caspase-3 is a specific marker of apoptosis. It is also important to demonstrate that pharmacological inhibition of apoptosis fails to reduce liver injury (eg, ALT and AST levels), thereby excluding apoptosis as a major contributor to the observed hepatic damage.

Third, the pathogenic role of LPO should be functionally validated. Because no universally accepted threshold of LPO has been established to define ferroptotic cell death, it is critical to determine whether inhibition of LPO attenuates liver injury. Accordingly, treatment with lipid ROS scavengers, such as liproxstatin-1, should reduce serum ALT/AST activities and decrease the extent of necrotic cell death. Additional evidence may be provided by lipidomic analyses demonstrating accumulation of oxidized PUFAs and by showing that inhibition of alternative forms of regulated necrosis, such as necroptosis, fails to confer protection. Together, these findings would strengthen the conclusion that necrotic death in the liver is driven by LPO-dependent mechanisms.

Fourth, the iron requirement should be verified experimentally. It is important to demonstrate that iron chelation reduces liver injury and decreases the number of necrotic cells, providing critical evidence that iron is an essential piece of the reactions—iron-catalyzed LPO-dependent ferroptosis.

It is worth noting that, although implementation of this experimental framework is technically feasible in animal models, many of these approaches are difficult to apply to clinical liver specimens. Consequently, definitive identification of ferroptosis in human liver disease remains challenging. This limitation underscores the urgent need for specific, reliable biomarkers to distinguish ferroptosis from other forms of cell death in human tissues. Furthermore, the development of new chemical probes to trace ferroptosis from other types of cell death in vitro and in vivo is important to advance the research on ferroptosis in liver diseases.

FUTURE PERSPECTIVES

Future studies should focus on several key areas to determine whether ferroptosis occurs in different liver diseases, elucidate the underlying molecular mechanisms, identify reliable ferroptosis-associated biomarkers, and develop cell-specific therapeutic strategies.

Definitive evidence of ferroptosis is needed using human etiology-relevant models of liver disease

Although numerous studies have investigated the role of ferroptosis in various liver diseases, definitive evidence that it is a major form of cell death during disease progression remains limited. While a few studies provided convincing evidence supporting a role for ferroptosis, they relied on a severe experimental model that does not accurately recapitulate the etiology and pathophysiology in humans. Therefore, future studies should employ clinically relevant disease models and rigorous experimental approaches, as outlined in the section “A framework for defining ferroptosis in liver diseases,” to conclusively determine whether hepatic cells undergo ferroptosis during the development of the liver diseases discussed above. Such efforts are essential for establishing the pathological significance of ferroptosis in liver injury and disease progression.

The molecular mechanisms governing ferroptosis under different liver injury conditions require further investigation

Despite the growing number of studies examining the contribution of ferroptosis to liver injury, the underlying molecular mechanisms remain poorly understood. Most published studies have primarily focused on changes in canonical ferroptosis-associated factors, including iron accumulation, GSH depletion, and GPX4 downregulation. However, the precise mechanisms responsible for these alterations remain largely undefined. Furthermore, it is not yet clear whether changes in the expression or activity of additional regulatory molecules influence the susceptibility of hepatic cells to ferroptosis under different pathological conditions. Future mechanistic studies are therefore needed to identify the upstream signaling pathways and molecular networks that initiate and regulate ferroptosis in liver disease.

The mechanisms of ferroptosis in other liver cell types need to be defined

In addition to hepatocytes, cholangiocytes, and liver sinusoidal endothelial cells, whose ferroptotic death contributes to liver injury, HSCs and HCC cells often exhibit resistance to cell death, including ferroptosis. It is therefore important to determine whether these cell types possess unique protective mechanisms that prevent ferroptotic cell death. Identifying such mechanisms could reveal novel therapeutic targets for treating liver fibrosis, cirrhosis, and HCC. In addition, understanding whether and how ferroptosis occurs in hepatic immune cell populations, including macrophages and neutrophils, may provide new insights into the regulation and resolution of liver inflammation.

Identification of biomarkers for hepatic ferroptosis will facilitate clinical translation

Although the experimental framework described above may help establish the occurrence of ferroptosis in preclinical models of liver disease, validating ferroptosis in human liver tissues remains challenging. Consequently, the development of sensitive and specific biomarkers of hepatic ferroptosis is a critical research priority. In particular, identifying circulating biomarkers that reflect ferroptotic death of specific hepatic cell populations would enable clinicians to determine whether ferroptosis contributes to liver injury in individual patients or patient subgroups. Such biomarkers could improve disease stratification, facilitate patient selection for ferroptosis-targeted therapies, and enhance clinical decision-making.

Development of hepatic cell-specific ferroptosis-targeting strategies will improve therapeutic precision

Current ferroptosis-targeting approaches, including lipid ROS scavengers and iron chelators, exert systemic effects. While these interventions may protect vulnerable cells from ferroptosis, they may also induce unintended consequences in healthy cells and tissues, such as reductive stress or iron deficiency. This limitation is particularly relevant given the pronounced cellular heterogeneity of the liver. Emerging technologies, including single-cell multiomics, spatial transcriptomics, and high-resolution imaging approaches, offer unprecedented opportunities to characterize ferroptosis in a cell-type-specific manner within the hepatic microenvironment. Ferroptosis gene signatures that are unique or common across different cell types in liver diseases should be established. These advances are expected to facilitate the identification of cell-specific regulators of ferroptosis and support the development of targeted therapeutic strategies that selectively modulate ferroptosis in distinct hepatic cell populations while minimizing off-target effects.