The emerging role of ferroptosis in intestinal disease

Ferroptosis is a newly recognised type of regulated cell death (RCD) characterised by iron-dependent accumulation of lipid peroxidation. It is significantly distinct from other RCDs at the morphological, biochemical, and genetic levels. Recent reports have implicated ferroptosis in multiple diseases, including neurological disorders, kidney injury, liver diseases, and cancer. Ferroptotic cell death has also been associated with dysfunction of the intestinal epithelium, which contributes to several intestinal diseases. Research on ferroptosis may provide a new understanding of intestinal disease pathogenesis that benefits clinical treatment. In this review, we provide an overview of ferroptosis and its underlying mechanisms, then describe its emerging role in intestinal diseases, including intestinal ischaemia/reperfusion (I/R) injury, inflammatory bowel disease (IBD), and colorectal cancer (CRC).


Introduction
Ferroptosis is a form of regulated cell death (RCD) that was first proposed by Dixon and colleagues in 2012 1 . It is morphologically, biochemically, and genetically different from other kinds of RCD, such as apoptosis, necroptosis, and autophagy 1,2 . Iron metabolism and the lipid peroxidation pathway are central mediators of the ferroptotic process 3,4 (Fig. 1). Excessive iron regulates ferroptosis by producing lethal reactive oxygen species (ROS) via the Fenton reaction, while reduced glutathione (GSH) depletion and/or glutathione peroxidase 4 (GPX4) inhibition trigger ferroptosis through the accumulation of intracellular lipid ROS and overwhelming lipid peroxidation 1,4,5 . In addition, ROS attack the polyunsaturated fatty acids (PUFAs) of lipid membranes, producing massive lipid peroxides and leading to membrane damage and cell death 4,6 . Specific small-molecule compounds, such as erastin and RAS-selective lethal 3 (RSL3) can induce ferroptosis, while ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1), and iron chelators deferoxamine (DFO) inhibit it 7,8 . Accumulating evidence suggests that ferroptosis participates in multiple diseases, including neurological disorders, ischaemia/reperfusion (I/R) injury, kidney failure, cardiac disease, and cancer 1,4,[9][10][11] . Recent studies have also implicated ferroptosis in intestinal diseases, including intestinal I/R injury, inflammatory bowel disease (IBD), and colorectal cancer (CRC) [12][13][14][15][16] (Fig. 2 and Table 1). Ferroptosis has been reported in ulcerative colitis (UC) in both humans and mice; moreover, blocking the ferroptotic process alleviated dextran sulphate sodium (DSS)-induced colitis 12,13 . Furthermore, ferroptosis can limit the migration, invasion, and proliferation of CRC. Indeed, RSL3 drives ferroptotic cell death by promoting cellular ROS accumulation and increasing iron load 17 . Another report indicated that in CRC, resibufogenin inhibited cell growth and tumorigenesis by inducing ferroptosis through GPX4 inactivation 18 . Taken together, ferroptosis appears to play a key role in the pathophysiological processes and may provide new ideas and means for the treatment of intestinal diseases. This review presents a comprehensive description of ferroptosis and its emerging role in multiple intestinal diseases.
Ferroptosis: an iron-dependent type of regulated cell death with clinical significance

Definition and measurement
Since 2003, Stockwell and colleagues have successively identified novel compounds, including erastin and RSL3, that activate new, nonapoptotic cell death in particular cancer cells 19,20 . Inhibitors specific to known RCDs did not inhibit this chemically induced cell death; however, antioxidants and iron chelators could block and reverse the process 21 . The definition of ferroptosis was proposed in 2012: nonapoptotic, iron-dependent cell death characterised by the accumulation of lipid peroxidation products and the depletion of membrane PUFAs 1 . Ferroptosis was added to the RCD family by the Nomenclature Committee on Cell Death (NCCD) in 2018 22 . Morphologically, ferroptosis manifests as small mitochondria with concentrated membrane density, decreased or vanishing mitochondrial cristae, and outer mitochondrial membrane rupture 4,23 . The biochemical properties of ferroptosis are iron accumulation, lethal ROS production, and overwhelming lipid peroxidation 4,10 . Multiple molecules, including GPX4, p53, solute carrier family 7 member 11 (SLC7A11), acyl-CoA synthetase long-chain family member 4 (ACSL4), NADPH oxidase (NOX), and nuclear factor E2-related factor 2 (NRF2) positively or negatively regulate ferroptosis 1,[24][25][26] .

Mechanisms and mediators of ferroptosis GSH/GPX4-lipid peroxidation pathway
Ferroptosis is triggered by excessive lipid peroxidation arising from iron-dependent ROS accumulation. As it can occur when GSH-dependent lipid peroxide repair systems are compromised, maintaining ROS and lipid peroxides at physiological concentrations is a critical factor in minimizing susceptibility 2,35 . Lipophilic antioxidants (e.g. Fer-1, Lip-1) have been defined as specific ferroptosis suppressors that inhibit ROS accumulation caused during lipid oxidation. GSH is a thiol-containing tripeptide that plays an essential role in intracellular antioxidant defences. Its depletion causes increased oxidative stress, macromolecular damage, and subsequent cell death 36 . GPX4 is a member of the glutathione peroxidase family capable of reducing cytotoxic lipid hydroperoxides (L-OOH) to non-toxic lipid alcohols (L-OH) or catalysing free hydrogen peroxide into water to prevent the formation and accumulation of lethal lipid ROS at the expense of GSH 37,38 .
Accumulating evidence has implicated GPX4 as a master regulator of ferroptosis; its inhibition by pharmacological or genetic methods can trigger ferroptotic cell death through the accumulation of lipid peroxides 5,39,40 . Indeed, RSL3 has been shown to induce ferroptosis by directly inhibiting GPX4 activity through covalent binding with the selenocysteine active site of GPX4 5,41 . GPX4 can also be inactivated by indirect methods, such as cellular GSH depletion. The biosynthesis of GSH requires the participation of glutamate, cysteine, and glycine in a twostep reaction catalysed by glutamate-cysteine ligase (GCL) and glutathione synthetase (GSS) 42,43 . Thus, GSH depletion can result either from direct inhibition of GSH synthesis (e.g. by the known GCL inhibitor buthionine sulphoximine (BSO) 6 ) or from cysteine/glutamate unavailability. Cysteine, the rate-limiting substrate for GSH biosynthesis, is produced from dipeptide cystine imported by the cell surface cystine/glutamate antiporter system X c − , or from methionine via the transsulphuration  MiRNAs (let-7c, let-7e, miR-150-5p) CRC patient samples Inhibition Downregulated miRNAs including let-7c, let-7e and miR-150-5p modulated the TP53 gene targeting the process of ferroptosis in CRC. 107 Erastin/Artesunate HCT116 cells; Orthotopic xenografts mice Induction The p53-independent PUMA axis is involved in ferroptosis in human colon cancer HCT116 cells. 108 Sorafenib HCT116/CX-1/LS174T cells; Orthotopic xenografts mice Induction Ferroptosis-inducing agents, such as sorafenib enhanced TRAIL-induced apoptosis through upregulation of DR5. 109 pathway 44,45 . Inhibiting system X c − can reduce GSH levels and GPX4 activity, contributing to ferroptotic cell death. Erastin and other molecules (e.g. sulphasalazine, sorafenib) are inhibitors of system X c − and thus induce ferroptosis 1,3,4 . Studies have shown that regulating the expression of SLC7A11, the functional subunit of system X c − , affects system X c − activity and ferroptosis sensitivity in cancer cells 24,26,44 . Furthermore, cysteinyl-tRNA synthetase (CARS) has been found to participate in the transsulphuration pathway; its knockdown causes upregulation of this pathway and erastin-induced ferroptosis resistance 2 .
As described below, disruption of lipid repair systems involving GSH and GPX4 can facilitate the accumulation of (lipid) ROS; however, cysteine/GSH depletion and/or GPX4 suppression alone is not sufficient to cause ferroptosis. ROS react with PUFAs of lipid membranes to cause lipid peroxidation, which is central to the final execution of ferroptosis. Free PUFAs are substrates for the synthetic-lipid signal-transduction medium, but they must be esterified and incorporated into membrane phospholipids (PLs) with the help of the enzymes ACSL4 and lysophosphatidylcholine acyltransferase 3 (LPCAT3). Then lipoxygenases (LOXs) catalyse PUFA-containing PLs to produce pro-ferroptotic lipid peroxidation 23 . Researchers have identified ACSL4 as both a biomarker for, and a critical contributor to, ferroptosis 25,46 . ACSL4 expression is positively correlated with ferroptosis sensitivity; in addition, lipid oxidation upon GPX4 inhibition requires the involvement of ACSL4 25,46 . One group has reported that LPCAT3 deletion protected fibroblasts against ferroptosis, suggesting that LPCAT3 is also an important player in ferroptosis 25 . However, this protective effect was mild compared with the protection provided by ACSL4 deletion, suggesting that ACSL4 plays a more extensive role in ferroptosis; moreover, the functional effect of LPCAT3 possibly depends on cellular subtypes 25,47 . Of the different oxidised PL species, PUFAcontaining phosphatidylethanolamines (PEs), especially arachidonic acid (AA)-and adrenic acid (AdA)-containing PEs, are the most susceptible to peroxidation in ferroptosis 48 . Finally, overwhelming lipid peroxidation likely alters lipid bilayer properties, producing cytotoxic reactive fragments, and leading to irreversible cell death 49 .

Iron metabolism and ferroptosis
Iron is a redox-active element that promotes ROS generation through the Fenton reaction, which leads to non-enzymatic lipid peroxidation 3,50 . Iron also serves as a cofactor for iron-containing enzymes, including LOXs, suggesting a necessary role in enzymatic lipid reactions 50 . Thus, as a significant factor for the production of (lipid) ROS via enzymatic or non-enzymatic ways, iron appears to be an indispensable component in ferroptosis 1,2 . The chelation of intracellular iron by DFO and ciclopirox olamine is sufficient to inhibit erastin-induced cell death, reinforcing the importance of iron in ferroptosis 4 . Normally, extracellular iron forms a complex with circulating transferrin (TF), binds to the specific membrane transferrin receptor protein-1 (TFR1), and is delivered into cells. Excess cellular iron is stored as ferritin, the main intracellular iron storage protein that consists of a ferritin light chain (FTL) polymer and FTH1, or exported by iron exporter ferroportin (FPN) 51,52 . Maintaining cellular iron homoeostasis can prevent oxidative damage, and cell toxicity and death. Either reduced iron storage or increased iron uptake can cause iron overload and trigger ferroptosis 4 . Recent studies have revealed an association between genes involved in iron metabolism and ferroptosis. Erastin-induced ferroptosis can be prevented by silencing TFRC, the gene that encodes TFR1, whereas overexpression of haem oxygenase 1 (HO-1) alters iron homoeostasis and aggravates it 6,53 . Autophagic degradation of ferritin, known as ferritinophagy, modulated by the nuclear receptor coactivator 4 (NCOA4), controls cellular liable iron levels and ROS accumulation, thus regulating ferroptotic cell death in some cell lines 54,55 . The pentaspan membrane protein prominin-2 can drive ferroptosis resistance by promoting the formation of ferritincontaining multivesicular bodies and exosomes, thus exporting iron from the cell 56 . Furthermore, iron response element-binding protein 2 (IREB2) encodes the master regulator of iron metabolism, which results in the expression of TRFC, FTH1, and FTL. Inhibiting IREB2 expression contributes to decreased sensitivity to erastininduced ferroptosis 1 . Other targets, such as heat shock protein beta-1 (HSPB1) and CDGSH iron sulphur domain 1 (CISD1), can also regulate ferroptotic cell death by mediating iron uptake and lipid peroxidation 57,58 . Collectively, these findings indicate the iron dependence of ferroptosis.

Other ferroptosis regulatory pathways
The canonical tumour suppressor p53 probably plays dual roles in mediating ferroptosis in multiple cancers (Fig. 3). It can directly adjust the metabolic versatility of cells by modulating metabolic targets, favouring mitochondrial respiration and resulting in ROS-mediated cell death 59 . Researchers have found that p53 represses SLC7A11 protein expression, resulting in decreased cystine import, decreased GSH production, and enhanced ROS-mediated ferroptosis in some cancer cell lines 24,59 . The acetylation-defective mutant p53 3KR , which lacks the ability to trigger apoptosis, cell-cycle arrest, and senescence, can suppress tumourigenesis by inhibiting SLC7A11 and inducing ferroptosis 24 . On the contrary, other studies have reported an inhibitory effect of p53 on ferroptosis in different contexts. Wild-type p53 stabilisation suppresses ferroptosis in specific cancer cell lines in response to cystine deprivation and system X c − inhibition because of the activation of p53-p21 transcriptional axis 60 . Besides, p53 negatively regulates ferroptosis in CRC cells by inhibiting dipeptidyl-peptidase-4, described in more detail in section 'Ferroptosis and colorectal cancer' below 61 . In addition to p53-mediated ferroptosis, the intracellular metabolic process glutaminolysis is required for the initiation of cystine deprivation-induced ferroptosis 9 . The FSP1-CoQ10-NAD(P)H pathway exists as an independent parallel system that cooperates with GSH/GPX4 to mitigate lipid peroxidation and ferroptosis 62 . Targeting the NRF2-related pathway is also a vital strategy to mediate lipid peroxidation and ferroptosis 63 .

The significance of ferroptosis research in disease
In parallel with more basic research, it has been found that inducing or blocking ferroptosis can affect the onset and development of multiple pathogenic conditions, providing a potential target for therapeutics, especially for diseases tolerant/resistant to conventional drugs. Taking drug-resistant cancer as an example, ineffective induction of cancer cell death is an important problem with many chemotherapy and bio-targeted drugs, closely linked to drug resistance 64 . Persister cells are clones that survive initial cancer treatment and induce drug-resistant states across diverse cancer contexts 64,65 . Interestingly, induction of ferroptosis can kill these drug-tolerant persister cells and decrease the emergence of acquired drug resistance 66,67 . In addition, the epithelial-to-mesenchymal transition (EMT) is one of the mechanisms leading to apoptotic failure and drug resistance in epithelial-derived carcinoma cells 68 . Evidence has indicated that tumour cells in a high-mesenchymal state are characterised by enhanced activity of enzymes related to the promotion of PUFA synthesis, making these cells dependent on the lipid peroxidase pathway involving GPX4. Thus, cancer cells in a mesenchymal state can undergo ferroptosis through pharmacological perturbations to overcome cancer therapy resistance 68 . Targeting ferroptosis is a new perspective for the treatment of kidney injury, non-cancer liver diseases, and intestinal diseases, suggesting the significant potential of ferroptosis research 10,69 .

Ferroptosis and intestinal ischaemia/reperfusion (I/R) injury
Intestinal I/R injury is a common clinical condition with high morbidity and mortality, resulting from sudden reduction of intestinal blood flow and reoxygenation after the restoration of blood supply 70 . It occurs in many clinical conditions, including trauma, haemorrhagic shock, acute mesenteric ischaemia, small intestinal volvulus, and intestinal transplantation 71,72 . Intestinal mucosal barrier dysfunction, as a consequence of epithelial cell death, can allow the translocation of bacteria and associated toxins into the bloodstream, leading to inflammation, systemic sepsis, and organ dysfunction 73,74 . Intestinal I/R injury is associated with multiple types of RCD, including apoptosis, necroptosis, and autophagy, but with the discovery of ferroptosis, new potential mechanisms of RCD have attracted attention [75][76][77] . Indeed, ferroptosis has been identified in I/R injury in other organs both in vivo and in vitro; moreover, ferroptosis inhibitors can alleviate these injuries. Studies have shown that treatment with DFO reduced myocardial infarct size and lactate dehydrogenase levels in an ex vivo heart model of I/R stress 9 , while Lip-1 prevented acute renal failure from renal I/R injury 39 . ROS generation and lipid peroxidation are associated with intestinal I/R injury and are primary contributors to the initiation and execution of ferroptosis 78 . Decreased GSH levels and superoxide dismutase activity, as well as increased MDA levels, were observed in rat intestinal tissues after intestinal I/R 79,80 . Furthermore, DFO administration was beneficial in the prevention of intestinal I/R-induced lipid peroxidation and GPX activity reduction was reversed by DFO treatment 81 . Taken together, lipid peroxidation and iron participate in I/ R-induced intestinal injury, but their contribution to ferroptosis is still enigmatic.
A recent study has reported that ferroptosis plays a critical role in intestinal I/R injury and may be a lethal process triggered by reperfusion 14 . In this study, the expression levels of pro-ferroptotic factors such as ACSL4 and iron increased, while those of anti-ferroptotic factors (GPX4, FTH1, GSH) decreased in ischaemic murine intestinal tissues; moreover, treatment with Lip-1 ameliorated intestinal injury both in vivo and in vitro 14 .
Moreover, an ischaemia model that incorporated different reperfusion durations to examine features of ferroptosis in situ suggested that this form of cell death occurred in the early phase of reperfusion and was distinct from apoptosis, which appeared at a later phase 14 . Inhibition of ischaemia-induced ACSL4 (a key regulator and indicator of ferroptosis) expression via pharmacological and genetic manipulations protected against lipid peroxidation and ferroptosis, as well as alleviated cell damage and intestinal barrier dysfunction induced by intestinal I/R 14,25 . Li and colleagues have further shown that Sp1, a transcription factor that binds to GC-box motifs in target-gene promoters, mediated ACSL4 expression 14 . In addition to intestinal damage, intestinal I/R can cause acute injury to remote organs, including the lungs and liver. Indeed, ferroptosis was reported to exacerbate intestinal I/Rinduced acute lung injury, whereas blocking this process mitigated lung injury after intestinal I/R in mouse models 14,82 . In summary, ferroptosis is related to I/R-induced intestinal injury, but more studies are needed to discover its underlying mechanisms and regulation.

Ferroptosis and inflammatory bowel disease
Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a chronic disease characterised by constant progression and relapse. Although not fully elucidated, the aetiology of IBD is commonly thought to implicate reciprocal interactions between host genetics, environmental factors, the gut microbiome, and immune responses 83 . A better understanding of IBD pathogenesis will be beneficial for improving its treatment; recent studies have underlined the importance of cell death in intestinal epithelial homoeostasis. Excessive cell death is closely correlated with chronic inflammation in IBD patients 84 , but what is the relationship between ferroptosis and IBD? It has been reported that iron supplementation changes gut microbial homoeostasis and exacerbates intestinal inflammation similarly to CD in a murine model 85 . Another study using a rat model of DSS-induced colitis also indicated that excess iron aggravated intestinal inflammation 86 . Recently, a Japanese team showed that high dietary iron intake increased the risk of UC 87 . Mucosal ROS production is increased in UC in proportion to the disease activity, and iron chelators are known to reduce ROS production and ameliorate colonic symptoms in IBD 88,89 .
Taken together, these findings suggest a possible relationship between IBD and ferroptosis in which excess iron in the intestine produces ROS via the Fenton reaction, which triggers oxidative stress. Lipid peroxidation procedurally appears and ferroptotic cell death is induced. Thereby, the intestinal epithelial cells are destroyed, and damage to the intestinal mucosal barrier results in IBD 90 .
Ferroptosis has been implicated in both clinical UC patients and in murine experimental colitis, with significant downregulation and upregulation of ferroptosis-associated genes 12,13 . Administration of ferroptosis inhibitors, including Fer-1, Lip-1, and DFO, reduced disease activity scores and ameliorated colon length shortening in DSSinduced murine colitis, suggesting the beneficial effect of inhibiting ferroptosis 12,13,91 . In keeping with ferroptosis mechanisms, GPX4 also plays a vital role in negatively regulating ferroptotic cell death in IBD. Curculigoside (CUR) is a botanical ingredient with anti-oxidant and antiinflammatory properties that protects against ferroptosis in UC through the promotion of GPX4 91 . CUR increased selenium sensitivity and enhance GPX4 expression levels in the IEC-6 rat intestinal epithelial cell line, while Gpx4 silencing inhibited the protective effects of CUR on cell death and oxidative stress indicators in ferroptotic IEC-6 cells 91 . Moreover, another group emphasised the importance of GPX4 in gut homoeostasis by showing impaired GPX4 activity and features of lipid peroxidation in the small intestinal epithelial cells (IECs) of CD patients 92 . PUFAs, especially AA, induced the production of interleukin 6 (IL-6) and chemokine (C-X-C motif) ligand 1 (CXCL1) in IECs treated with Gpx4 siRNA in response to iron availability, lipid peroxidation, and ACSL4, similar to ferroptosis mechanisms 92 . Interestingly, a PUFA-enriched Western diet triggered small intestinal inflammation in mice that lacked one Gpx4 allele in IECs, with histological characteristics resembling CD 92 . The link between PUFA uptake, GPX4 activity, and intestinal inflammation further provides evidence for the pathogenesis of CD. However, although the process observed in the study was similar to ferroptosis, no definite cell death was observed in the murine intestinal inflammation model; the scholars speculate that in this case, one Gpx4 allele might be sufficient to protect against ferroptotic cell death 92 .
NRF2 is a critical mediator of the cellular antioxidant response that controls redox homoeostasis-related gene expression; perturbations of the NRF2-lipid peroxidation-ferroptosis axis have been found in cancers 63 . HO-1, a cytoprotective enzyme related to cellular stress, also participates in ferroptosis and has antiinflammatory effects 93 . Chen et al. found that Fer-1 alleviated DSS-induced colitis via NRF2/HO-1 signalling, indicating that the NRF2 pathway may be an important factor regulating ferroptosis in UC 12 . ER stress can induce the cell-death signalling pathway in the form of apoptosis and autophagy 94,95 , but interestingly, ER stress also is implicated in the development of ferroptosis in diseases, including IBD 13,96 . It has been found that ferroptosis contributed to UC via ER stress-mediated IEC cell death; moreover, phosphorylation of NF-κBp65 inhibited ER stress-mediated IEC ferroptosis to relieve the disease as an upstream regulator 13 . Together, these data show that ferroptosis has a key role in IBD, and that targeting it may be a promising method for understanding the development of IBD and to provide new treatments.

Ferroptosis and colorectal cancer
Colorectal cancer (CRC) is a common malignant tumour with high morbidity and mortality and is one of the most pressing global health issues. According to the GLOBOCAN 2018 estimates of incidence and mortality worldwide report, CRC is the third-most diagnosed cancer and the second-most cause of cancer-related deaths globally 97 . Current treatments for CRC include surgery, radiotherapy, chemotherapy, immune therapy, and biotargeted therapy 98 ; however, despite recent progress in therapeutics, some patients exhibit resistance or intolerance to them via apoptosis evasion and anti-apoptotic enhancement 99,100 . Thus ferroptosis, as a form of RCD independent from apoptosis, may provide a promising strategy for cancer therapy. Since its discovery in 2012, the manipulation of ferroptosis by specific molecules has enabled inhibition of the growth and spread of multiple cancer types, including CRC 1,23 . RSL3-induced ferroptosis in a dose-and time-dependent manner in three CRC cell lines; this treatment increased ROS and cellular labile iron pool (LIP) levels 17 . Evidence has showed that the classic chemotherapy drug cisplatin induces ferroptosis; moreover, the combination of cisplatin and erastin was synergistic, indicating that ferroptosis adds an alternative cell-death pathway triggered by classical therapeutic drugs and antitumour mechanisms in CRC 100 . In addition, targeting ferroptosis can overcome conventional CRC drug resistance from a new perspective. Chen et al. reported that the bioactive compound β-elemene (extracted from the Chinese herb Curcumae Rhizoma) is a ferroptosis inducer; they combined treatment with β-elemene and anti-EGFR (epidermal growth factor receptor) antibody cetuximab to produce anti-tumour effects by triggering ferroptosis in CRC patients with RAS mutations that do not respond to cetuximab 99,101 . Another study showed that vitamin C, an antioxidant that can paradoxically initiate oxidative stress at pharmacological doses, targeted cetuximab-persister cells and restricted the emergence of acquired resistance to EGFR blockade in CRC through the induction of ferroptosis 67 . Altogether, the role of ferroptosis in CRC in inhibiting tumour growth and overcoming resistance to current anticancer drugs is a promising avenue for research.
As described above, GPX4 plays a central role in regulating ferroptosis. Recently, several molecules have been implicated in ferroptosis in CRC through their mediation of GPX4. RSL3 inhibits GPX4 activity by directly binding with GPX4, and overexpression of GPX4 rescued CRC cell death induced by RSL3, suggesting a similar role of GPX4 in RSL3-induced ferroptosis in CRC as in other diseases 17 . Shen et al. found that resibufogenin isolated from Asiatic toad dried skin secretions is a potential anticancer agent in the treatment of CRC because it induced ferroptosis in a GPX4 inactivation-dependent manner 18 . In addition to direct GPX4 inhibition, inhibiting SLC7A11, the functional subunit of system X c − , also induces ferroptotic cell death in CRC. It was reported that the benzopyran derivative 2-imino-6-methoxy-2H-chromene-3-carbothioamide (IMCA) has a wide spectrum of biological activities, including those relevant to cancer therapy 102 . Zhang et al. first discovered the anti-CRC effect of IMCA through ferroptosis induction by downregulating SLC7A11 103 . Interestingly, IMCA affected the downstream components of the AMPK/mTOR/p70S6k pathway, which have been linked to SLC7A11 activity and ferroptosis 103 . The role of SLC7A11 and ferroptosis has also been elucidated in colorectal cancer stem cells (CSCs), which can provide resistance to chemotherapy and form secondary tumours in the progression of CRC 104 . Colorectal CSCs are more sensitive to ferroptosis than parental CRC cells; the knockout of SLC7A11 with CRISPR-Cas9 technology facilitated ferroptotic cell death, suggesting that targeting SLC7A11 may specifically suppress the progression of colorectal CSCs and reduce CRC drug resistance 105 . Another key regulator of ferroptosis in many related diseases is ACSL4 25 . A recent study has determined its crucial regulatory role in the induction of ferroptosis by bromelain (a plant extract derived from pineapple) in KRAS-mutant CRC through signalling pathway and miRNA profiling 106 .
The TP53 gene is known as a tumour suppressor and ferroptosis regulator in multiple cancers. It inhibited ferroptotic CRC cell death by blocking dipeptidyl-peptidase-4 (DPP4) activity, while the loss of p53 increased the anticancer activity of erastin in tumour-bearing mice, very different from the positive regulation of ferroptosis by p53 in other cancers (Fig. 3) 61 . Specifically, the loss of p53 restrains DPP4 nuclear localisation and facilitates the formation of the DPP4-NOX1 complex that promotes lipid peroxidation, resulting in ferroptosis in the HCT116 human CRC cell line 59,61 . While p53 can limit ferroptosis by forming a DPP4-p53 complex in the nucleus, disassembly of this complex restores the erastin-induced ferroptosis sensitivity of CRC 59,61 . Moreover, the fact that TP53 can stimulate SLC7A11 expression in CRC protects CRC cells from ferroptosis 61 . Therefore, regulation of TP53 may be highly desirable as part of CRC therapy. In a human miRNome analysis of miRNA-mRNA interactions and multiple pathways involved in CRC pathogenesis, three downregulated miRNAs, let-7c, let-7e, and miR-150-5p, were found to modulate TP53 in CRC and thus could regulate ferroptosis 107 .
There also are connections between ferroptosis and other types of RCDs in CRC. Hong et al. reported molecular crosstalk between ferroptosis and apoptosis when CRC cells were treated with the ferroptotic agents erastin or artesunate (ART) in combination with the apoptotic agent tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) 108 . The combination of erastin/ART and TRAIL significantly promoted TRAIL-induced apoptosis due to ER stress-induced p53-independent PUMA (p53 upregulated modulator of apoptosis) expression 108 . The group further found that ER stress response-mediated expression of the TRAIL receptor death receptor 5 (DR5) also played a vital role in this combinatorial synergy in a variety of CRC cell lines 109 . These studies are preliminary explorations of the mechanisms that may be shared between ferroptosis and apoptosis, but further research is needed to understand the relationship between these RCDs. Autophagy promotes ferroptotic cell death through the degradation of ferritin (ferritinophagy) in fibroblasts and certain cancer cells, modulated by selective cargo receptor NCOA4 54,55 ; its inhibition suppresses ferritin degradation and inhibits ferroptosis in these cells 54,55 . However, this is not the case for CRC cells; Hasan et al. indicated that ferritinophagy was not required for CRC cell growth 110 . Interestingly, knocking out NCOA4 did not alter ferroptosis in CRC 110 . Differences in cell lines may partially explain these conflicting findings, but another possibility is that CRC cells have an alternative mechanism that compensates for the loss of NCOA4 function in response to ferroptosis induction. Future studies should investigate the compensatory and alternative pathways in CRC that enhance cell survival.
Taken together, ferroptosis plays a significant role in CRC, and its regulation may provide new insights into cancer therapy. In addition to the regulators already mentioned, researchers also have identified novel compounds, for example, iron oxide-hydroxide nanospindles, with the potential to promote ferroptosis to inhibit colon cancer 111 . Researchers also have identified new targets which can regulate ferroptosis in CRC, including the short/branched chain acyl-coenzyme A dehydrogenase and anoctamin 6 112,113 . We believe that fully understanding ferroptosis and its underlying mechanisms in CRC, as well as the connections between ferroptosis and other RCDs, can give us hope to improve the treatment and prognosis of this cancer.

Conclusions and perspectives
Ferroptosis is a newly identified type of RCD that is mediated by the iron-dependent accumulation of lipid ROS and has been implicated in the development of a wide variety of disorders, especially intestinal diseases. Inhibiting ferroptosis can attenuate intestinal injury in non-infectious inflammatory conditions such as intestinal I/R injury and IBD, while inducing ferroptosis with pharmacological activators can inhibit the migration, invasion, and proliferation of colorectal neoplasms, suggesting a dual role for ferroptosis in different intestinal diseases. As shown in Fig. 2, the common ferroptotic mechanisms in intestinal diseases include GPX4 inhibition, system X c − suppression, lipid peroxide accretion, and iron overload, which are consistent with other diseases. Key regulators such as GPX4, SLC7A11, ACSL4, and p53 are also important for mediating ferroptosisassociated intestinal diseases. In addition to known ferroptotic inducers (erastin, RSL3) and inhibitors (Fer-1, Lip-1, DFO), researchers have found more drugs and targets related to ferroptosis in intestinal diseases (Table  1). However, whether there are specific regulators or signalling pathways in these diseases remains unclear. Interestingly, some regulators seem to play different roles in intestinal diseases compared with diseases in other organs, for example, p53 and NCOA4 in CRC. As a result, further research is needed to identify disease-specific ferroptotic mechanisms to develop disease contextdependent therapeutic regimens. Furthermore, studies have found correlations between ferroptosis and other forms of cell death in intestinal diseases. These RCDs may share common pathways and key regulators, which can provide new directions for combining different therapeutic interventions.
Although much progress has been made, research on intestinal ferroptosis is still at an early stage, and its specific role remains to be investigated across the spectrum of intestinal disease, including many not presented in this review. Although we have summarized multiple methods for assaying ferroptosis from multiple aspects, no unanimously agreed-upon criteria directly define its occurrence. It is imperative to identify markers and other approaches to assess ferroptosis in vivo. In this way, ferroptosis biomarkers could be promising to indicate intestinal disease severity. We caution that the relationship between ferroptotic cell death and iron/lipid peroxides remains controversial, so more evidence is required to support the links between ferroptosis and iron, oxidative stress, and lipid peroxidation in disease development and progression. In addition, the signalling pathways and main transcriptional regulators of ferroptosis need to be studied so that we can benefit more from its modulation to protect the intestine against injury and carcinogenesis. Therefore, ferroptosis should be further investigated within the field of intestinal disease as a novel therapeutic target.