Ferroptosis: the potential key roles in idiopathic pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by recurrent injury to alveolar epithelial cells, epithelial-mesenchymal transition, and fibroblast activation, which leads to excessive deposition of extracellular matrix (ECM) proteins. However, effective preventative and therapeutic interventions are currently lacking. Ferroptosis, a unique form of iron-dependent lipid peroxidation-induced cell death, exhibits distinct morphological, physiological, and biochemical features compared to traditional programmed cell death. Recent studies have revealed a close relationship between iron homeostasis and the pathogenesis of pulmonary interstitial fibrosis. Ferroptosis exacerbates tissue damage and plays a crucial role in regulating tissue repair and the pathological processes involved. It leads to recurrent epithelial injury, where dysregulated epithelial cells undergo epithelial-mesenchymal transition via multiple signaling pathways, resulting in the excessive release of cytokines and growth factors. This dysregulated environment promotes the activation of pulmonary fibroblasts, ultimately culminating in pulmonary fibrosis. This review summarizes the latest advancements in ferroptosis research and its role in the pathogenesis and treatment of IPF, highlighting the significant potential of targeting ferroptosis for IPF management. Importantly, despite the rapid developments in this emerging research field, ferroptosis studies continue to face several challenges and issues. This review also aims to propose solutions to these challenges and discusses key concepts and pressing questions for the future exploration of ferroptosis.

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease with an unknown cause, and it typically results in an average survival of only 3 to 5 years after diagnosis [1]. This disease features epithelial injury and abnormal activation of lung fibroblasts, resulting in excessive deposition of extracellular matrix (ECM) proteins, irreversible fibrosis, and destruction of lung architecture. Patients with IPF often present with progressive dyspnea, and lung function continues to deteriorate. Without treatment, IPF can progress to respiratory failure and ultimately lead to death [2]. IPF primarily affects middle-aged and elderly males, with epidemiological surveys indicating that over 3 million people worldwide have the disease. The incidence rate ranges from 0.09 to 1.30 per 10,000 individuals, showing a consistent upward trend [3]. Currently, the two recommended medications for IPF are pirfenidone and nintedanib, however, their efficacy in slowing disease progression or improving patients’ quality of life is limited [4]. Lung transplantation remains the only surgical option for advanced IPF, but it is often associated with complications that result in a high mortality rate. Thus, there is a pressing need to continually explore the pathogenesis of the disease and seek new therapeutic approaches.

The main pathogenic mechanisms associated with IPF include epithelial-to-mesenchymal transition (EMT) and fibroblasts-to-myofibroblasts transition (FMT), which occur following recurrent epithelial injury and abnormal activation of fibroblasts [5, 6]. Recurrent injury to alveolar epithelial cells is triggered by various factors, including environmental exposures, oxidative stress, and aging. Many factors cause cellular oxidative stress, and lipid peroxidation is a key factor that occurs through a process called ferroptosis. Ferroptosis is a unique form of cell death characterized by iron-dependent lipid peroxidation reactions. Although pulmonary fibrosis is primarily associated with EMT, fibroblast activation, and ECM accumulation, cell death, including ferroptosis, also plays a significant role in modulating tissue repair and the pathological processes involved. In some instances, cell death may assist in clearing damaged cells; however, excessive or aberrant cell death may exacerbate tissue injury. Particularly in the context of pulmonary fibrosis, ferroptosis may promote the fibrotic process by releasing pro-fibrotic factors or by enhancing inflammatory responses [7, 8].

Ferroptosis and the accumulation of iron cause repeated damage to epithelial cells and activate fibroblasts [9]. This recurrent damage results in metabolic dysfunction within cells, increased apoptosis, and abnormalities in the activation and repair processes of epithelial cells [10, 11]. Dysregulated epithelial cells undergo epithelial-mesenchymal transition (EMT) via multiple signaling pathways, which leads to the excessive release of cytokines and growth factors. The apoptosis of epithelial cells and the EMT process play critical roles in the formation of fibrosis. This dysregulated environment promotes the differentiation of pulmonary fibroblasts into myofibroblasts, ultimately resulting in fibrotic proliferation and excessive accumulation of ECM proteins in distal lung parenchyma, alongside progressive scarring of the lung tissue [12, 13]. Due to this fibrotic transformation, gas exchange becomes increasingly compromised, ultimately leading to significant and irreversible loss of lung function. Research shows that abnormal collagen produced by reactive oxygen species (ROS) and the activation of fibroblasts, particularly myofibroblasts, are closely linked to damage in alveolar epithelial cells, significantly contributing to the onset and progression of IPF. Furthermore, myofibroblasts exhibit an anti-apoptotic phenotype [14, 15].

Iron metabolism and regulation

Iron is primarily ingested through dietary sources. Ferrous iron can easily be oxidized to ferric iron, which primarily exists in the form of ferric ions (Fe³⁺). The absorption efficiency of ferric ions is relatively low, and their solubility in the gastrointestinal tract is limited [16]. The body’s iron balance is intricately linked to processes of iron absorption, transport, storage, recycling, and loss.

The sources of iron include dietary iron absorbed through the duodenum and upper jejunum of the small intestine, as well as iron recycled from aging red blood cells. When iron is absorbed in the small intestine, it is converted to ferrous iron by cytochrome b. This ferrous iron then enters the cells through the divalent metal transporter 1 (DMT1) [17, 18]. Once inside the cell, iron can either be utilized immediately, stored as ferritin, or exported into the bloodstream through ferroportin (FPN). In the bloodstream, iron mainly exists as transferrin-bound iron (transferrin-iron complex), which helps transport and distribute iron throughout the body. This transferrin-iron complex effectively prevents free iron from catalyzing harmful oxidative reactions. Additionally, the transferrin-iron complex is recognized by specific receptors on the cell surface, primarily transferrin receptor 1 (TFR1, also known as TFRC), allowing tissue cells to uptake iron through receptor-mediated endocytosis [19]. Iron retrieved from aged red blood cells is primarily cleared enzymatically by macrophages, wherein enzymes release iron from hemoglobin, a process mediated by heme oxygenase-1 (HO-1) [20, 21].

If the demand for iron within the cell is not immediate, iron can be stored in ferritin. This capacity of ferritin to store iron helps buffer fluctuations in iron availability, ensuring a stable supply for critical physiological processes. Hepcidin is a 25-amino-acid peptide hormone produced by the liver. It plays a crucial role in regulating iron homeostasis by modulating the levels of ferritin and iron transport proteins. Its expression is influenced by factors such as hypoxia, anemia, and inflammation [22, 23]. When iron stores are sufficient or excessive, hepcidin levels increase, resulting in the internalization and degradation of iron transport proteins, effectively decreasing iron absorption and blocking iron export. Conversely, when iron levels are low, hepcidin production diminishes, promoting intestinal iron absorption and mobilizing stored iron [24].

Iron metabolism heavily relies on the interactions of iron response elements (IREs) and iron regulatory proteins (IRPs), collectively known as IRE-IRPs, to maintain cellular homeostasis. IREs are specific RNA sequences located in the untranslated regions of target mRNAs. These mRNAs encode proteins involved in iron transport, storage, and utilization, such as DMT1, TFR1, FPN, ferritin heavy chain (FtH), and ferritin light chain (FtL) [25, 26]. IRPs, particularly IRP1 and IRP2, are crucial regulatory proteins that bind to IREs to modulate the expression of these mRNAs, thereby influencing iron homeostasis [27]. In conditions of cellular iron deficiency, IRPs bind with high affinity to IREs, forming inhibitory complexes in the 5'UTR of mRNA for FPN, FtH, and FtL to repress translation, while preventing degradation in the 3'UTR of TFR1. Conversely, in cases of iron overload, the affinity of IRPs for IREs decreases [28]. This dynamic regulatory mechanism aids in preventing ferroptosis, and Erastin can induce ferroptosis by lowering the affinity of IRE-IRPs.

In summary, iron metabolism is a tightly regulated process that maintains a balance between iron availability and its potential toxicity. Through a complex network of absorption, transport, storage, and recycling, the body maintains effective iron homeostasis. Disruptions in any of these processes can lead to ferroptosis, and understanding how tissues manage iron can indicate their susceptibility to ferroptosis (Fig. 1).

Fig. 1

Iron metabolism, regulation, and ferroptosis. The regulatory mechanisms of iron contribute to the correction of iron metabolism disorders. Any disruption in the processes of iron absorption, transport, storage, or regulation can potentially lead to ferroptosis

Ferroptosis

Ferroptosis is a non-apoptotic cell death mechanism characterized by iron-dependent lipid peroxidation. This review will focus on the latest research regarding ferroptosis, encompassing three main aspects: the three essential elements of ferroptosis, the pathways that defend against ferroptosis, and the key genes involved in regulating ferroptosis. Additionally, we will explore the relationship between these three aspects and pulmonary fibrosis.

Three fundamental elements of ferroptosis

Ferroptosis is a form of regulated cell death characterized by the accumulation of lipid peroxides in the presence of reactive oxygen species and iron (Fe), leading to oxidative damage to polyunsaturated fatty acids (PUFAs) in cell membranes. This process ultimately results in membrane injury and cellular death [29]. Therefore, reactive oxygen species, iron, and polyunsaturated fatty acids are the three essential components of ferroptosis.

Abnormal iron metabolism

Abnormal iron metabolism is essential for the pathogenic accumulation of lipid peroxides, and the genes and proteins involved in this process are key regulators of ferroptosis. Firstly, the metabolic enzymes involved in lipid peroxidation and the numerous enzymes responsible for ROS generation require iron as a catalyst [30]. Secondly, excess intracellular Fe²⁺ can generate highly reactive hydroxyl (HO·) radicals through the Fenton reaction, leading to the continuous accumulation of intracellular ROS and resulting in ferroptosis [31, 32]. Additionally, ferritin autophagy has been confirmed as a mechanism of iron metabolism; autophagic degradation of ferritin releases Fe²⁺, ultimately contributing to ferroptosis [31, 33, 34]. Genes involved in iron metabolism can also induce ferroptosis; for instance, BAY 11–7085 (BAY) induces ferroptosis by upregulating heme oxygenase-1 (HO-1), which mediates heme degradation and releases free iron, thereby promoting ferroptosis [35].

In the pathological context of IPF, various cell types exhibit heterogeneous alterations in iron metabolism. For instance, abnormal accumulation of iron ions has been detected in alveolar type II epithelial cells. This accumulation is thought to be closely related to ferroptosis and may accelerate the fibrotic process by exacerbating oxidative stress [36]. Furthermore, abnormalities in iron metabolism within pulmonary fibroblasts may enhance their proliferation and activation potential, thereby promoting excessive deposition of ECM. Additionally, increased iron storage levels in macrophages are believed to correlate with macrophage polarization, which further influences airway inflammation and the progression of fibrosis [37]. Collectively, these observations underscore the significance of iron homeostasis regulation in the pathogenesis of IPF.

In the study of IPF, the metabolism of iron and its influence on disease progression are increasingly gaining attention. Although current research on iron levels in patients with various stages of IPF is still limited, existing literature is beginning to unveil the potential significance of this area. Studies have shown that iron levels in IPF patients may exhibit significant fluctuations across different disease stages, and these fluctuations are closely associated with disease progression and pathophysiological changes [38]. As an essential trace element, iron plays a critical role in various physiological processes; however, excessive accumulation can lead to oxidative stress and exacerbate disease conditions. In the late stages of IPF, significant deposition of iron in lung tissues has been observed, which may be closely related to increased iron-mediated reactive oxygen species generation and heightened apoptosis [39]. This phenomenon of iron deposition not only reflects alterations in the pulmonary microenvironment but also indicates the involvement of iron in the processes of inflammation and fibrosis. Moreover, the relationship between iron metabolism and the formation and progression of pulmonary fibrosis may be even more complex. The use of iron chelators in animal models suggests a potential effect in inhibiting pulmonary fibrosis, indicating that the modulation of iron levels or metabolic pathways could represent a novel therapeutic strategy for IPF in the future. These findings highlight the dual role of iron in the progression of IPF: on one hand, it serves as an essential nutrient, while on the other hand, excessive accumulation may lead to damage to lung tissues.

Polyunsaturated fatty acids (PUFAs)

The first critical factor in ferroptosis is the availability of oxidizable PUFAs. The phospholipids composed of PUFAs (PUFA-PLs) undergo peroxidation in the cell membrane, producing lipid hydroperoxides that react with iron, leading to ferroptosis [40]. Research has identified two mechanisms by which lipid hydroperoxides are generated: one involves the peroxidation of PUFA-PLs [41], while the other is directly synthesized by lipoxygenases [42]. The relative importance of these mechanisms in ferroptosis remains unclear and may be related to specific cell types. The synthesis of PUFA-PLs is co-regulated by key enzymes, including Acyl-CoA Synthetase Long Chain 4 (ACSL4) and Lysophosphatidylcholine acyltransferase 3 (LPCAT3). ACSL4 converts free PUFAs into their corresponding PUFA-CoAs, while LPCAT3 integrates PUFA-CoAs into PUFA-PLs [43].

Recent studies have confirmed the downstream mechanisms promoting ferroptosis resulting from membrane lipid peroxidation. It has been observed that lipid peroxidation during ferroptosis leads to increased membrane tension, which activates Piezo1 and TRP channels, resulting in enhanced cation permeability. This process enhances the influx of Na + and Ca2 +, while simultaneously depleting K +, further compromising Na +/K + -ATPase activity, and ultimately culminating in membrane rupture. This research identifies dysregulation of cation permeability as a crucial downstream mechanism that promotes ferroptosis due to membrane lipid peroxidation [44].

Reactive oxygen species (ROS)

Reactive oxygen species (ROS), such as hydrogen peroxide, superoxide anions, and free radicals, are crucial in initiating ferroptosis [45]. The ROS involved in ferroptosis are generated from Fenton and Haber–Weiss reactions, which convert hydrogen peroxide into highly reactive hydroxyl radicals [46]. These radicals then interact with PUFAs in lipid membranes, leading to in the formation of lipid ROS. The accumulation of lipid ROS within the cell is toxic, damaging membrane structures and ultimately causing ferroptosis [47]. Current research has identified four primary pathways for ROS generation: (1) Mitochondria, which are a major source of ROS. Elevated mitochondrial ROS levels contribute to oxidative stress and can trigger ferroptosis by promoting the lipid peroxidation of cellular membranes [48]. Mitochondrial autophagy plays a dual role in ferroptosis. It can inhibit ferroptosis by phosphorylating acetyl-CoA carboxylase alpha (ACC) through AMP-activated protein kinase (AMPK) [49]. However, AMPK can also promote ferroptosis by targeting BECN1 [50]. (2) NADPH Oxidase (NOX). NOXs are transmembrane enzymes dedicated to ROS generation, producing ROS as byproducts of NADPH oxidation [51]. (3) Enzymatic Reactions. Enzymatic reactions involving cytochrome P450, cytochrome P450 oxidoreductase (POR), cytochrome b5 and its reductase, lead to the production of ROS [52]. (4) Fenton Reaction. In the Fenton reaction, ferrous iron (Fe²⁺) interacts with hydrogen peroxide (H₂O₂), resulting in the generation of hydroxyl radicals (HO·), which are among the most reactive ROS [53]. Studies show that increased ROS accumulation is directly linked to the severity of pulmonary fibrosis and decreased lung function, while elevated ROS levels also worsen lung inflammation and contribute to fibrosis development.

Defense pathways against ferroptosis

The human body contains a pro-ferroptosis system that promotes lipid peroxide production and a ferroptosis defense system that detoxifies these peroxides. When the capacity of the pro-ferroptosis system surpasses that of the ferroptosis defense system, there is an excessive accumulation of lipid peroxides in the cell membrane, which ultimately leads to ferroptosis [54, 55]. Therefore, the ferroptosis defense system plays a crucial role in determining whether ferroptosis occurs. Current research has identified several primary pathways involved in ferroptosis defense.

GPX4-GSH defense pathway

The enzyme glutathione peroxidase 4 (GPX4) reduces toxic lipid hydroperoxides (L-OOH) to lipid alcohols (L-OH), thereby repairing lipids and inhibiting ferroptosis. GPX4 is currently recognized as the most crucial enzyme in preventing the formation and accumulation of lipid hydroperoxides [56, 57]. Several isoforms of GPX4 exist in the cytoplasm, nucleus, and mitochondria. Studies show that cytoplasmic GPX4 plays a more significant role in preventing ferroptosis than its nuclear and mitochondrial counterparts [58, 59]. In vivo experiments have definitively demonstrated the critical role of GPX4 in preventing ferroptosis, as the knockout of Gpx4 triggers ferroptosis, leading to acute renal failure in mice. Furthermore, research confirms that Liproxstatin-1 effectively inhibits ferroptosis. Furthermore, Tsubouchi K et al. found that the expression of GPX4 is decreased in IPF, resulting in increased lipid peroxidation and enhanced growth factor-β (TGF-β) signaling, which promotes the differentiation of myofibroblasts and the progression of fibrosis. Targeting GPX4-mediated lipid peroxidation may offer a novel therapeutic strategy for antifibrotic treatment in IPF [60].

Glutathione (GSH) serves as a cofactor for GPX4 and is ultimately synthesized through the actions of cysteine-glutamate ligase and glutathione synthase [61]. Cysteine is generated from cystine, which is transported into cells and converted via enzymatic reactions. Glutamate and cystine enter the cell via system Xc, which includes two proteins: solute carrier family 7 member 11 (SLC7 A11) and solute carrier family 3 member 2 (SLC3 A2) [62]. Additionally, research has highlighted that cysteine can also be acquired through extracellular proteins, which containing cysteine, via cellular endocytosis [63]. Cysteine can also be released by Gamma-glutamyl transferase 1 (GGT1) cleaving the γ-glutamyl bond in glutathione tripeptide or obtained through the action of dipeptidases acting on cysteine dipeptides at the cell membrane [64]. Some studies indicate that sulfur-containing metabolites from cysteine metabolism are also crucial in inhibiting ferroptosis, akin to glutathione [65, 66]. However, further investigations are needed.

Research has found that selenium is present in GPX4 and plays a crucial role in its function of preventing ferroptosis. GPX4, a selenoprotein, relies on selenium utilization to protect against hydrogen peroxide-induced ferroptosis, particularly safeguarding certain intermediate neurons from lethal seizures. Additionally, the dependence of GPX4 on selenocysteine is essential for embryonic development as it significantly enhances cellular resistance to irreversible oxidation [67]. Yao Y and colleagues discovered that selenium is vital for maintaining the survival of follicular helper T (TFH) cells by enhancing the expression of GPX4, which prevents ferroptosis. The selenium-GPX4 axis is critical for the homeostasis of TFH cells and promotes effective immune responses during infections and vaccinations [68].

FSP1-CoQ defense pathway

The ferroptosis suppressor protein 1 (FSP1)-Coenzyme Q (CoQ) defense pathway plays a role equal in importance to that of the GPX4-GSH defense pathway in inhibiting membrane lipid peroxidation. FSP1 is a redox enzyme that operates through its NAD(P)H-binding domain and FAD-binding domain, enabling it to exist in reduced forms, specifically CoQH2 and VKH2, from CoQ and vitamin K (VK), respectively. CoQH2 and VKH2 act as lipophilic radical-trapping antioxidants (RTAs) that terminate lipid peroxidation chain reactions, thereby inhibiting ferroptosis [69]. The N-myristoylation of FSP1 is crucial for its specific localization to the plasma membrane, facilitating its role in ferroptosis suppression. The activation of FSP1’s N-terminus by methionine is inhibited. Instead, the binding of a 14-carbon fatty acid myristate to a glycine residue directs FSP1 to the plasma membrane [70]. The FSP1-CoQ defense pathway works independently of the GPX4-GSH pathway. Studies show that when the GPX4 gene is knocked out in cells, FSP1 moves from the mitochondria to the plasma membrane to exert its anti-ferroptosis effects. Besides CoQ and VK, which act as RTAs to inhibit ferroptosis, reduced forms of vitamin E, vitamin A, and tetrahydrobiopterin (BH4) are also included. Research indicates that different RTAs play predominant roles in inhibiting ferroptosis in various cell types [71, 72]. Sugizaki et al. found that idebenone, a synthetic analogue of coenzyme Q10, prevents bleomycin-induced pulmonary fibrosis and increases levels of ROS in the lungs. It also suppresses TGF-β-induced collagen production and exerts inhibitory effects on the function of lung fibroblasts. Importantly, administration of idebenone after the onset of fibrosis improves pulmonary fibrosis and lung function [73].

DHODH-CoQ defense pathway

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that plays a crucial role in pyrimidine biosynthesis, thereby influencing the formation of DNA and RNA. It catalyzes the conversion of dihydroorotate to orotate while coupling this reaction with the reduction of CoQ. Consequently, DHODH can reduce CoQ to CoQH2 in the mitochondria, which inhibits ferroptosis [74]. The StAR-related lipid transfer protein 7 (STARD7) is involved in transporting CoQ from the mitochondria to the plasma membrane, where it participates in the protection against ferroptosis. Studies have demonstrated that the inhibition of DHODH leads to a decrease in CoQ levels, thereby impairing the mitochondrial respiratory chain and increasing the production of ROS, ultimately triggering iron-dependent cell death [75]. Supplementing DHODH substrates and products can either mitigate or worsen ferroptosis triggered by GPX4 inhibition. Specifically, inactivating DHODH causes significant mitochondrial lipid peroxidation and ferroptosis in cancer cells with low GPX4, likely due to DHODH's role in reducing CoQ to CoQH2 [76]. Additionally, DHODH inhibitors can promote ferroptosis by reducing the expression or activity of FSP1 [77]. This finding indicates that DHODH inhibitors have anti-cancer potential and may also improve therapeutic efficacy by promoting ferroptosis. Furthermore, drug combinations aimed at increasing DHODH expression or activity have been shown to prevent neuronal death in models of neurodegenerative diseases, highlighting the therapeutic potential of targeting this pathway [78].

GCH1-BH4 defense pathway

The GTP cyclohydrolase 1 (GCH1)-tetrahydrobiopterin (BH4) pathway is another significant route for combating ferroptosis. Research has shown that BH4 serves as a potent antioxidant, maintaining cellular redox balance and acting as an important cofactor in antioxidant defense [79]. GCH1 promotes the synthesis of BH4, thereby preventing lipid peroxidation and inhibiting ferroptosis. Kraft et al. further investigated the mechanism and discovered that GCH1 promotes the production of reduced coenzyme Q10 (CoQ10) by synthesizing its precursor, 4-hydroxybenzoate. The synthesis of 4-hydroxybenzoate relies on the BH4-mediated conversion of phenylalanine to tyrosine, which allows this pathway to inhibit ferroptosis by promoting CoQ10 synthesis. Additionally, this study found that the GCH1-BH4 pathway prevents the depletion of phospholipids in the cell membrane by using two polyunsaturated fatty acid acyl tails, which helps protect against cell membrane damage and ferroptosis. This finding suggests that the GCH1-BH4 pathway operates as a unique mechanism for ferroptosis defense, independent of the GPX4-GSH system. Additionally, studies have indicated that BH4 alleviates pulmonary interstitial fibrosis by inhibiting endothelial-to-mesenchymal transition (EnMT) in IPF [80], and it can also regulate the oxidative balance in macrophages [81].

Core genes involved in ferroptosis

In addition to the three essential elements and the four main defense pathways of ferroptosis mentioned above, this study summarizes several other core genes that are widely recognized in the current literature as playing a crucial role in the regulation of ferroptosis.

Nuclear factor erythroid 2-like bZIP transcription factor 2 (NRF2)

NRF2 is a key transcription factor in antioxidant response. Under oxidative stress, NRF2 is released from the Kelch-like ECH-associated protein 1 (KEAP1) and translocates from the cytoplasm to the nucleus. There, it binds to specific DNA sequences in the promoters of target genes, like antioxidant response elements (AREs) and electrophilic response elements (EpREs). This binding activates antioxidant signaling pathways, including GPX4 and FSP1. This process ultimately inhibits ferroptosis [82, 83]. Moreover, NRF2 regulates the expression of TGF-β. The NRF2 activator dimethyl fumarate (DMF) upregulates NRF2 expression in macrophages, thereby reducing the macrophage-mediated transition of pulmonary fibroblasts to myofibroblasts and extracellular matrix deposition, thus alleviating pulmonary interstitial fibrosis. The NRF2 activator sulforaphane (SFN) has antifibrotic effects; LOC344887 is a target gene of NRF2 that suppresses the expression of fibrotic genes through the modulation of downstream PI3 K-Akt signaling pathways, exerting its antifibrotic action [84]. Oxidative stress in macrophages significantly contributes to the pathological processes of IPF. The underlying mechanism is associated with SLC15 A3, whereby macrophages upregulate SLC15 A3 expression in the lungs, which weakens its interaction with the scaffold protein p62 and regulates the activation of phosphorylation, consequently inhibiting the antioxidative role of NRF2 against ferroptosis [85].

Tumour protein p53 (TP53)

TP53 is a key transcription factor in ferroptosis, playing dual roles in its regulation that may vary depending on the cell type. In ovarian cancer, MEX3 A promotes tumor progression by inhibiting ferroptosis through the degradation of the TP53 protein [86]. VKORC1L1 reduces the production of phospholipid peroxides by generating reduced forms of vitamin K. In certain cancer cells, TP53 inhibits VKORC1L1 expression, which promotes ferroptosis and suppresses tumor growth [87]. Acetylation of TP53 significantly reduces SLC7 A11 expression and suppresses system Xc activity. This action blocks the GPX4-GSH defense pathway, thus promoting ferroptosis [88]. Conversely, TP53 can inhibit ferroptosis in specific cell types. In colorectal cancer, TP53 suppresses the activity of dipeptidyl peptidase-4 (DPP4) through a transcription-independent mechanism. DPP4 is closely associated with lipid peroxidation of the plasma membrane, and thus, TP53 inhibits ferroptosis by suppressing DPP4. Numerous studies have shown that senescence of alveolar type 2 (AT2) cells in IPF promotes fibrogenesis through a TP53-dependent mechanism, which significantly contributes to the progression of the disease. This mechanism may be related to TP53-mediated ferroptosis, but further research is needed to confirm this relationship.

Heme oxygenase-1 (HO-1)

HO-1 is a crucial enzyme involved in the catabolism of heme, which has garnered significant attention in recent years due to its role in the regulation of ferroptosis. HO-1 catalyzes the conversion of heme into carbon monoxide (CO), biliverdin, and ferrous iron, exhibiting antioxidant properties that confer protection against ferroptosis. Notably, the release of free iron appears to contradict its protective role against ferroptosis; however, HO-1 mitigates ferroptosis through various pathways. For instance, biliverdin, a product derived from HO-1, along with the subsequent bilirubin, possesses antioxidant properties that can counteract lipid peroxidation [89]. The CO generated by HO-1 enzymatic activity can activate cytoprotective pathways, including the NRF2 signaling pathway. Multiple studies have indicated that the interaction between HO-1 and NRF2 may play a regulatory feedback role in the process of ferroptosis [90,91,92]. Research has shown that HO-1 plays a role in IPF. Ye et al. found that increased HO-1 expression promotes the differentiation of lung myofibroblasts induced by TGF-β1 through the activation of the serine/threonine kinase AKT pathway [93]. NRF2 agonists enhance the expression of HO-1, inhibiting the transition of fibroblasts to myofibroblasts and collagen synthesis. However, while HO-1 levels are significantly elevated in IPF, other studies have indicated a reduction in HO-1 levels in the macrophages of IPF patients [94]. These discrepancies may relate to differing molecular mechanisms governing HO-1 expression in various cell types.

HO-1 is a key regulator of oxidative stress, playing a crucial role in iron metabolism, cellular stress response, and antioxidant reactions. During the progression of IPF, HO-1 may have a dual role, which depends on its expression patterns in different cell types. For instance, studies have shown that reduced expression of HO-1 in pulmonary macrophages decreases their antifibrotic effects, whereas expression of HO-1 in fibroblasts may promote the proliferation and differentiation of these cells via the activation of the AKT signaling pathway, thereby exacerbating the fibrotic process [95]. Additionally, the accumulation of HO-1 metabolites, such as carbon monoxide and biliverdin, in the alveolar microenvironment may further influence the inflammatory and oxidative stress status associated with IPF. Consequently, HO-1 could serve as an important target for research and novel therapeutic strategies in IPF. Future studies should focus on elucidating the specific regulatory mechanisms of HO-1 and its feasibility as a potential therapeutic target.

Nuclear receptor coactivator 4 (NCOA4)

NCOA4 is an essential adaptor protein required for lysosome-mediated ferritin autophagy. NCOA4 links ferritin, which carries iron, to the lysosomal degradation pathway through ferritin autophagy [96]. NCOA4 selectively degrades ferritin to release iron during this process. Furthermore, NCOA4 enhances intracellular iron levels during redox reactions by facilitating the absorption of iron from ferritin, thereby promoting ferroptosis. Studies have shown that NCOA4 mediates the catabolism of ferritin according to the characteristics of the cellular environment, which regulates the mechanism of ferroptosis more significantly than directly inhibiting GPX4 to induce ferroptosis [97]. Cells lacking NCOA4 exhibit reduced susceptibility to ferroptosis [98], indicating that the role of NCOA4 as a promoter of iron availability is indispensable in the execution of cellular death pathways. Overexpression of NCOA4 plays a critical role in ferroptosis of lung epithelial cells (MLE-12) in IPF [99]. Ferritin autophagy-mediated ferroptosis plays a significant role in inhibiting the differentiation of fibroblasts into myofibroblasts during IPF [38] (Fig. 2).

Fig. 2

Regulatory pathways of ferroptosis. The figure illustrates the regulatory pathways involved in ferroptosis, which can be broadly categorized into three types. The first category encompasses the three essential components of ferroptosis: abnormal iron metabolism, polyunsaturated fatty acids, and reactive oxygen species. The second category includes the four major defense pathways against ferroptosis: the GPX4-GSH defense pathway, the FSP1-CoQ defense pathway, the DHODH-CoQ defense pathway, and the GCH1-BH4 defense pathway. The third category consists of key genes that regulate ferroptosis, namely NFR2, Tp53, HO-1, and NCOA4

IPF and ferroptosis

The prevailing pathogenesis of IPF contains epithelial-mesenchymal transition (EMT) and fibroblast to myofibroblast transition following continued epithelial injury and aberrant fibroblast activation. Ferroptosis plays a crucial role in oxidative stress-induced epithelial cell damage, EMT, and fibroblast activation in the context of IPF.

Ferroptosis and epithelial-mesenchymal transition (EMT)

Ferroptosis induces epithelial cell damage and increased apoptosis through oxidative stress in various fibrotic diseases, leading to abnormal epithelial cell activation and repair processes that contribute to disease onset and progression. Abnormal epithelial cell activation and repair trigger EMT. Recent studies suggest that ferroptosis-mediated oxidative stress can regulate the TGF-β signaling pathway, which plays a crucial role in inducing EMT in pulmonary fibrosis. Specifically, TGF-β binds to its receptor and activates downstream Smad2/3 phosphorylation, leading to the transcription of EMT-related genes such as Snail, Twist, and ZEB1. Iron accumulation and lipid peroxidation further enhance this process by stabilizing HIF-1α and exacerbating ROS production, promoting fibroblast activation and ECM deposition. In TGF-β-induced pulmonary interstitial fibrosis cell models, levels of GSH and the expression of SLC7 A11 are reduced, while ROS levels are elevated. Erastin promotes the de-epithelialization of lung epithelial cells and induces EMT [99]. RAS-selective lethal 3 (RSL3), as an ferroptosis activator, can induce ferroptosis and promote EMT in IPF by directly inhibiting GPX4 [100]. BTB and CNC homology 1 (Bach1) is a heme-binding transcription factor that regulates ferroptosis by modulating the heme and iron-related oxidative stress response. Bach1 suppresses the NRF2 signaling pathway by competitively binding to the EpRE sites in the NRF2 dimer and the promoters of NRF2 target genes, thereby promoting EMT [101]. Research indicates that smoking causes iron deposition in the mitochondria of alveolar epithelial cells. This iron overload results in increased ROS production, damaging lung epithelial cells and inducing EMT. The iron chelator deferoxamine (DFO) can reduce smoking-induced damage, death, and EMT in lung epithelial cells, alleviating pulmonary fibrosis [102]. Iron accumulation enhances the vulnerability of lung epithelial cells to iron-driven oxidative damage, promoting EMT and triggering autophagy [103]. Ferroptosis and apoptosis in epithelial cells are central to IPF progression, contributing to EMT and fibroblast activation. In addition to epithelial cells, studies have identified that stromal fibroblasts, mononuclear macrophages, and mesenchymal stem cells may also undergo apoptosis at different stages of IPF, influencing fibrosis formation and resolution [104]. Moreover, apoptosis is not limited to a single phase of the disease; it may occur at early stages, exacerbating inflammatory responses, as well as at later stages, contributing to fibroblast activation and extracellular matrix remodeling (Fig. 3).

Fig. 3

The regulation of epithelial-mesenchymal transition (EMT) in idiopathic pulmonary fibrosis (IPF) by ferroptosis. TGF-β1 stimulation upregulates the expression of transferrin receptor 1 (TFRC) in human lung fibroblasts, leading to increased intracellular Fe²⁺ levels, which promotes EMT. TGF-β1 also suppresses the GPX4-GSH defense pathway against ferroptosis, while RSL3 can induce both EMT and ferroptosis in IPF by directly inhibiting GPX4. NRF2 translocates to the nucleus, dimerizes with small Maf proteins, and binds to antioxidant response elements (ARE), thereby activating transcription of target genes to maintain cellular homeostasis. Bach1 inhibits the NRF2 signaling pathway by competitively binding to NRF2 dimers, thereby facilitating EMT

In renal fibrosis, endoplasmic reticulum stress (ERS) activates iron-related apoptotic EMT through the XBP1-HRD1-NRF2 signaling pathway, offering new insights into mechanisms and potential treatments for EMT progression [105]. SIRT7 inhibits EMT by promoting KLF15/NRF2 signaling, reducing iron upregulation and lipid peroxidation in tubular epithelial cells [106]. In liver fibrosis, ferroptosis plays a role in the pathogenesis of liver fibrosis by promoting hepatocyte EMT. The AGE receptor 1 (AGER1) can reverse hepatocyte EMT by inhibiting ferroptosis, thus suppressing liver fibrosis [107]. In uterine endometrial fibrosis, erastin-induced ferroptosis promotes the EMT of endometrial epithelial cells, contributing to the progression of endometrial fibrosis. Conversely, the application of the ferroptosis inhibitor ferrostatin-1 (Fer-1) significantly inhibits EMT and improves endometrial fibrosis, indicating that ferroptosis promotes endometrial fibrosis by facilitating EMT [108]. These studies highlight the role of ferroptosis in promoting EMT and subsequently tissue fibrosis in various fibrotic diseases. In contrast to fibrotic diseases, ferroptosis inhibits EMT in tumor conditions, where cancer cells develop resistance to ferroptosis. These differences may be related to the pathological mechanisms of tumors, such as genetic mutations, alterations in signaling pathways, and changes in the tumor microenvironment [109, 110].

Ferroptosis and fibroblast activation

The activation of fibroblasts, specifically the FMT, is a prominent pathological feature of IPF. When TGF-β stimulates pulmonary fibroblasts to create an IPF cell model, the expression of TFR1 increases, resulting in higher intracellular Fe²⁺ levels. This process facilitates the conversion of fibroblasts into myofibroblasts, suggesting that ferroptosis is involved in the pathological activation of fibroblasts in IPF. NRF2 dimerizes with Maf proteins in the nucleus and attaches to the antioxidant response element (ARE), thereby activating the transcription that maintains cellular homeostasis. Sestrin2, an inhibitor of ferroptosis, suppresses FMT, alleviates IPF, and activates the NRF2 signaling pathway to reduce oxidative stress. The addition of ferroptosis inducers reversed the alleviation of IPF mediated by sestrin2, indicating that sestrin2 mitigates ferroptosis through the NRF2 signaling pathway, thereby benefitting IPF [111]. During the differentiation of fibroblasts into myofibroblasts in IPF, there is a marked increase in iron ions, ROS, and lipid peroxidation. Further research has shown that erastin promotes FMT by increasing lipid peroxidation and inhibiting the expression of GPX4, whereas the addition of Fer-1 mitigates these processes and alleviates pulmonary fibrosis [112]. Therefore, ferroptosis induces the transformation of fibroblasts into myofibroblasts through oxidative stress, contributing to the formation and progression of IPF (Fig. 4).

Fig. 4

The regulation of fibroblast-myofibroblasts transition (FMT) in idiopathic pulmonary fibrosis (IPF) by ferroptosis. TGF-β1 stimulation upregulates the expression of transferrin receptor 1 (TFRC) in human lung epithelial cells, leading to increased intracellular Fe²⁺ levels, which promotes FMT. Sestrin2 acts as a ferroptosis inhibitor by activating the NRF2 signaling pathway to mitigate oxidative stress, thus inhibiting FMT and alleviating ferroptosis. Downregulation of NRF2 significantly reduces the level of heme oxygenase 1 (HO-1), thereby promoting FMT. The ferroptosis inducer erastin promotes FMT by increasing lipid peroxidation and inhibiting the expression of GPX4

Ferroptosis promotes IPF

An increasing body of research has confirmed that ferroptosis plays a crucial role in the pathophysiology of IPF. In IPF, high levels of lipid peroxidation products significantly accumulate in lung tissue [113], with elevated levels of ROS and malondialdehyde, combined with reduced transcription and translation levels of GPX4, contributing to the occurrence of ferroptosis. Ferroptosis-induced oxidative damage not only disrupts epithelial integrity but also promotes fibroblast activation, leading to excessive ECM deposition. Iron overload and oxidative stress activate fibroblasts by upregulating fibrosis-associated proteins such as fibronectin and alpha-smooth muscle actin (α-SMA). Additionally, lipid peroxidation products, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), have been found to stimulate fibroblast proliferation and collagen type I synthesis, further contributing to the excessive ECM accumulation seen in IPF. In a mouse model of bleomycin-induced IPF, iron levels were found to be upregulated in alveolar type II (ATII) cells and fibrotic lung tissue, alongside abnormal expression of TFRC, divalent metal transporter 1, and ferroportin 1. The N-methyl-d-aspartate receptor (NMDAR) initiates the pulmonary fibrosis process by inducing iron deposition in lung tissue and ferroptosis in ATII cells. Upon NMDAR activation, the expression of nNOS and IRP1 is enhanced, leading to altered expression of iron metabolism-related genes, ultimately causing mitochondrial damage and ferroptosis [114]. Another study indicated that in bleomycin-induced mice, elevated Sp3 promotes the expression of ferroptosis-related proteins and markers of mitochondrial DNA (mtDNA) damage by recruiting HDAC2, thus inactivating the keap1/NRF2 signaling cascade, resulting in ferroptosis [115]. Furthermore, research has shown that autophagy-mediated iron death plays a significant role in IPF, with bisphenol A (BPA) enhancing autophagy-mediated ferroptosis through activation of the AMPK/mTOR signaling pathway, thereby exacerbating the progression of IPF [116]. Alveolar macrophages (AMs) secrete exosomes that transport ficolin B (Fcn B), which in turn activates the cGAS-STING pathway, accelerating autophagy and iron death in lung epithelial cells [117]. Epigenetic mechanisms of iron death also play an essential role in IPF, as the methylation regulator UHRF1 promotes the methylation of CpG sites in the promoters of GPX4 and FSP1, epigenetically repressing their expression and promoting lung fibrosis [118]. Additionally, the histone methyltransferase SET domain bifurcated 1 (SETDB1) promotes ferroptosis by epigenetically regulating Snai1, contributing to the progression of IPF [119].

IPF progression is strongly influenced by ferroptosis, characterized by excessive lipid peroxidation and oxidative cell death in alveolar epithelial cells. Notably, apoptosis plays a role across multiple disease phases. During the inflammatory stage, epithelial apoptosis exacerbates tissue damage and induces immune activation, whereas fibroblast apoptosis in late-stage fibrosis may impact extracellular matrix remodeling and fibrosis resolution [104]. These findings suggest that the timing and cell-type specificity of apoptosis are critical factors in determining the extent of fibrosis and tissue repair.

Although this article systematically summarizes the potential role of ferroptosis in the pathogenesis of IPF, several notable limitations remain. Firstly, the majority of existing studies are based on animal models or in vitro experiments, which may not fully translate to the human disease context in terms of the significance and regulatory mechanisms of ferroptosis. Additionally, research on the dynamic changes of ferroptosis throughout the different stages of IPF pathology and the underlying molecular mechanisms is still relatively limited. Future efforts should focus on multidimensional dynamic tracking studies, such as assessing the temporal changes of iron metabolism-related biomarkers using clinical biological samples, to gain a more comprehensive understanding of the regulatory role of ferroptosis in the progression of IPF.

Potential therapeutic targets for IPF

Given the critical role of ferroptosis in the pathogenesis of IPF, several potential therapeutic targets within the ferroptosis network have been identified for treating IPF. Ferroptosis is driven by lipid peroxidation, which offers a rationale for therapeutic interventions that involve the use of drugs aimed at inhibiting the peroxidation processes. A representative drug, liproxstatin, a lipophilic radical-trapping antioxidant (RTA) that helps prevent lipid peroxidation. Another intervention strategy involves inhibiting the excessive accumulation of intracellular Fe²⁺ and suppressing the synthesis of lipoxygenases. A prominent representative drug for this approach is the iron chelator DFO. Research shows that ferroptosis inhibitors, such as liproxstatin-1 and DFO, reduce pulmonary interstitial fibrosis by inhibiting ferroptosis. Moreover, DFO formulated as a nanoparticle medication delivered via a pulmonary drug delivery system (PDDS) has shown to be more efficient and safer in inhibiting IPF compared to conventional delivery methods [120].

In addition to drugs that prevent lipid peroxidation, agents targeting core genes involved in ferroptosis (such as NCOA4 and NRF2) have also been explored for their potential to inhibit IPF. Mimotopes enhance NRF2 signaling by blocking the IL-6 pathway, thereby reducing the generation of lipid peroxidation products in lung tissue and improving pulmonary fibrosis. Extracellular vesicles derived from menstrual blood stem cells (MenSCs-exo) deliver miR-let-7 to lung epithelial cells, inhibiting the expression of Sp3 and enhancing NRF2 signaling, which in turn suppresses fibrosis [115]. Additionally, empagliflozin (EMPA), a sodium-glucose co-transporter 2 (SGLT2) inhibitor, has been shown to inhibit IPF by enhancing autophagy and modulating the NRF2/HO-1 signaling pathway [121]. Natural constituents extracted from Aspergillus flavus, such as Fraxetin, also inhibit ferroptosis by forming stable complexes with NCOA4, thereby alleviating IPF.

Furthermore, several herb-derived compounds have been identified to inhibit IPF by targeting ferroptosis. Dihydroquercetin (DHQ), a flavonoid, reduces the accumulation of iron and lipid peroxidation products and attenuates ferritin autophagy, thereby suppressing pulmonary fibrosis. The Qingfei Xieding prescription helps reduce IPF by lowering lipid oxidation and ROS production, which blocks ferroptosis [122]. Dihydroartemisinin (DHA) inhibits IPF through the modulation of ferritin autophagy-mediated ferroptosis. Tuberostemonine also alleviates pulmonary fibrosis by enhancing the SLC7 A11/GPX4 pathway and inhibiting ferroptosis.

Regarding the clinical significance of the research findings on the potential link between ferroptosis mechanisms and IPF, several candidate drugs based on these foundational studies have shown clear efficacy in early clinical trials [99]. Furthermore, there are ongoing studies focused on ferroptosis inhibitors that are nearing clinical trial stages, revealing that these drugs may play a critical role in specific pathophysiological processes associated with lung fibrosis. For instance, research has demonstrated that clioquinol alleviates pulmonary fibrosis by inactivating fibroblasts through iron chelation [123].

Although our study reviews the role of ferroptosis in IPF and systematically outlines the progress made in animal model research, the clinical translation of ferroptosis-related drugs for the treatment of IPF still requires further investigation. While preclinical data on ferroptosis inhibitors such as DFO and Liproxstatin-1 demonstrate certain potential, there is a notable scarcity of clinical validation specifically for IPF, which currently limits their practical application as therapeutic targets. Future research should accelerate clinical trials based on findings from cellular and animal models and delve deeper into the efficacy and safety of these drugs. Therefore, the clinical benefits of these ferroptosis-targeting medications in IPF remain to be thoroughly elucidated, and their pharmacological characteristics need further refinement. Future studies should expand clinical trials to determine the optimal efficacy and safety of these drugs.

Conclusions and perspectives

IPF is a fatal progressive lung disease, with recent studies revealing the pivotal role of ferroptosis in its pathogenesis. This review systematically summarizes iron metabolism processes, recent advancements in ferroptosis research, and its role in the pathogenesis of IPF. Additionally, we discuss therapeutic strategies for treating IPF. Nonetheless, several challenges remain in translating these ferroptosis-targeting drugs into clinical use, necessitating broader and more extensive clinical trials for optimization.

In conclusion, while the role of ferroptosis in the pathological processes of IPF and the application of ferroptosis inhibitors necessitate further clinical research, the challenges faced are undeniable. However, delving deeply into the mechanisms of ferroptosis presents new avenues and prospects for effective interventions in the treatment of IPF.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used AI in order to reorganize and integrate some of the languages. After using this tool, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.