Ferroptosis in Neonatal Hypoxic-Ischemic Encephalopathy: Mechanisms and the Therapeutic Potential of Vitamin D/VDR Signaling

Abstract

As a major neonatal brain disorder, hypoxic-ischemic encephalopathy(HIE) presents with elevated risks of long-term disability and neonatal death. Ferroptosis is a distinct mode of regulated cell death marked by excess intracellular iron, oxidative lipid injury, and suppressed GPX4 activity, and has gained attention as a pivotal mechanism in the development of HIE. Signaling pathways such as Nrf2, TLR4/NF-κB, and endoplasmic reticulum stress(ERS) play critical roles.Vitamin D (VD) and its receptor (VDR), beyond their classical roles in calcium-phosphate homeostasis, as neuroprotective modulators of ferroptosis. VD/VDR signaling promotes antioxidant defenses (e.g., via the Nrf2/HO-1 pathway), restores GPX4 activity, regulates iron and lipid metabolism, and mitigates neuroinflammation.These insights provide a rationale for exploring VD/VDR-based interventions as adjunctive strategies to therapeutic hypothermia, which could potentially be explored to improve neurodevelopmental outcomes in affected neonates.

Graphical Abstract

In neonatal HIE, ferroptosis involves iron overload, lipid peroxidation, GPX4 suppression, and activation of TLR4/NF-κB and ER stress, alongside Nrf2 inhibition. VD/VDR signaling activates Nrf2/HO-1, restores GPX4, regulates iron/lipid metabolism, and reduces neuroinflammation—attenuating ferroptosis and promoting neuroprotection.

Introduction

Hypoxic-ischemic encephalopathy (HIE) in newborns is a pathophysiological disorder triggered by perinatal oxygen deprivation, resulting in brain injury due to insufficient blood and oxygen supply. This injury induces characteristic neuropathological changes and a wide spectrum of neurological dysfunctions (Rodríguez et al. 2020). The incidence of HIE is estimated at 1 to 3 per 1,000 live births in high-income countries (Acun et al. 2022; Cornet et al. 2023), while in low- and middle-income countries, the incidence ranges from approximately 4 to 26–31 cases per 1,000 live births (Yang et al. 2024; Peeples et al. 2025; Hope for HIE 2023). Approximately 10–15% of affected neonates succumb within the first weeks of life, and among survivors, 25–30% develop long-term sequelae such as intellectual disability, epilepsy, cerebral palsy, and learning difficulties (Yang et al. 2024; Hope for HIE 2023). Such consequences greatly reduce life quality and impose heavy demands on both families and the medical system.

Currently, therapeutic hypothermia (TH) is the only widely accepted neuroprotective treatment for moderate to severe HIE. This approach involves controlled cooling of the neonate’s core temperature to 33.5–34.5 °C for 72 h. It is grounded in robust evidence from large randomized controlled trials and meta-analyses, which have shown that TH significantly improves neurodevelopmental outcomes when initiated within 6 h of birth (Laptook et al. 2024). Although the efficacy of TH stems from the attenuation of multiple secondary injury pathways, including reduced cerebral metabolic rate, suppression of excitotoxicity and neuroinflammation, and inhibition of apoptosis (Prakash et al. 2023; Laptook et al. 2024). However, TH is only effective within a narrow therapeutic window (initiation within 6 h of birth), significantly limiting its global applicability, especially in resource-limited settings (Prakash et al. 2023). Critically, even with timely treatment, a substantial proportion of infants (approximately 40–50%) still suffer from death or severe disability (Acun et al. 2022; Cornet et al. 2023). This underscores the urgent need for adjunct neuroprotective strategies that can extend the therapeutic window and synergize with therapeutic hypothermia.

The pathophysiology of HIE is a multifactorial process evolving through distinct phases (Babbo et al. 2024; Yang et al. 2024). The primary phase, during the initial insult, is marked by immediate energy failure, leading to neuronal depolarization and glutamate-mediated excitotoxicity. Following reperfusion, a more protracted secondary phase unfolds over hours to days, driven by mitochondrial dysfunction, rampant oxidative stress from reactive oxygen species (ROS) production, and a potent neuroinflammatory response involving microglial activation and cytokine release. These interconnected pathways —excitotoxicity, oxidative stress, and neuroinflammation—converge to trigger various forms of regulated cell death, including apoptosis and, as increasingly recognized, ferroptosis (Greco et al. 2020; Ranjan and Gulati 2023; Korf et al. 2023). This intricate cascade underscores the challenge of neuroprotection and highlights the need for therapies targeting multiple injury mechanisms. Recently, ferroptosis—an iron- and lipid peroxide–dependent form of regulated cell death—has been increasingly implicated in the neuronal damage observed in HIE. Furthermore, in the search for effective interventions against the multifaceted pathology of HIE, vitamin D (VD) and its receptor (VDR) have garnered significant interest. Unlike single-target therapeutic approaches, the VD/VDR axis represents a compelling pleiotropic regulator, capable of simultaneously modulating key ferroptotic pathways—including antioxidant defense (e.g., via Nrf2/HO-1), iron and lipid homeostasis, and neuroinflammation. This review aims to synthesize current evidence on the role of ferroptosis in HIE and critically evaluate the potential of VD/VDR signaling as a multifaceted strategy to mitigate this cell death pathway.

Literature Search Methodology

To ensure a comprehensive and systematic review of the current evidence, a structured literature search was conducted. The primary research question guiding this review was: “What is the mechanistic role of ferroptosis in the pathogenesis of neonatal hypoxic-ischemic encephalopathy (HIE), and what is the therapeutic potential of Vitamin D/VDR signaling in attenuating this process?”

A systematic electronic database search was performed in PubMed, Web of Science, and Scopus for relevant articles published from inception until Aug 2025. The search strategy combined keywords for ‘hypoxic-ischemic encephalopathy,’ ‘ferroptosis,’ and ‘Vitamin D/VDR,’ focusing on original research and review articles published in English. The reference lists of retrieved articles were also manually searched for additional relevant publications.​​

Ferroptosis Processes in HIE

Unlike apoptosis, necrosis, or autophagy, ferroptosis is a regulated cell death modality marked by distinct mitochondrial shrinkage, denser membranes, disrupted cristae, intracellular iron excess, and enhanced lipid peroxidation, culminating in reactive oxygen species(ROS) buildup (Ma et al. 2022; Ji et al. 2023). Recent evidence suggests that ferroptosis contributes to the pathogenesis of several disorders, ranging from tumors and neurodegeneration to heart disease and ischemia-reperfusion injury (Li et al. 2024; Wang et al. 2023; Liu et al. 2023a, b; Hou et al. 2022). Its role in neonatal HIE, however, is a relatively new area of research. The pathogenesis of ferroptosis in HIE involves a complex network of mechanisms, including dysregulated iron homeostasis, disrupted lipid oxidation, reduced glutathione (GSH) levels, loss of glutathione peroxidase 4 (GPX4) enzymatic function, and suppression of the cystine–glutamate exchange system (System Xc⁻).

Disrupted Iron Homeostasis

Within cells, iron predominantly occurs as ferrous (Fe²⁺) and ferric (Fe³⁺) ions, serving as a critical cofactor in various redox enzyme systems essential for cellular metabolic functions. In neonatal HIE, imbalance in iron regulation is regarded as a key initiator of ferroptosis. Dysfunctions in iron absorption, retention, or export may result in excess intracellular iron, enhancing ROS generation through the Fenton reaction and triggering lipid peroxidation that leads to neuronal ferroptotic damage (Chen et al. 2020). Clinical magnetic resonance imaging (MRI) studies in severe HIE cases have revealed marked iron deposition in vulnerable brain regions, including the basal ganglia and periventricular white matter (Dietrich and Bradley 1988). Restoring iron homeostasis with chelators such as deferoxamine and erythropoietin has yielded encouraging neuroprotective outcomes in therapeutic studies (Tan et al. 2023; Song et al. 2016).

Experimental studies using hypoxic-ischemic brain damage (HIBD) models in neonatal rats have further elucidated the molecular underpinnings of iron dysregulation. Notably, upregulation of iron-handling proteins—such as transferrin receptor (TFRC), ferritin heavy chain (FTH), and ferritin light chain (FTL)—has been consistently observed in HIBD brain tissues (Lin et al. 2022). Hu et al. (2022) reported a time-dependent increase in brain iron deposition in 3-day-old neonatal rats following hypoxic-ischemic injury, with peak staining intensity observed on day 3, suggesting that iron overload may serve as a key factor in white matter damage. Similarly, Palmer et al. detected early cytoplasmic iron accumulation in pyknotic neurons as soon as 4h after injury via Perls’ staining, with a peak at 24 h and normalization by day 7 (Palmer et al. 1999).

Later studies also observed increased levels of free iron and hydroxyl radicals in the hippocampus of HIBD models, accompanied by elevated TFRC expression and decreased ferritin levels. Administration of deferoxamine significantly reduced hydroxyl radical levels and neuronal apoptosis, providing additional evidence for the pathogenic role of iron overload in ferroptotic injury (Lu et al. 2015). In line with these findings, Tan et al. (2023) demonstrated pronounced iron accumulation observed in the cortical region of the brain of HIBD rats, along with ultrastructural mitochondrial changes characteristic of ferroptosis. Notably, iron chelation therapy attenuated these ferroptotic features, reinforcing the link between iron dysregulation and ferroptosis, and highlighting iron homeostasis with therapeutic relevance in managing neonatal HIBD.

In summary, the dysregulation of iron homeostasis, characterized by iron overload and deposition in vulnerable brain regions, acts as a critical initiating factor that drives the ferroptotic cascade in neonatal HIE.

Aberrant Lipid Peroxidation and PUFA Metabolism

The high polyunsaturated fatty acids (PUFAs) content in brain tissue makes it particularly sensitive to oxidative damage, resulting in lipid peroxidation (LPO) and the accumulation of toxic byproducts such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). LPO is a hallmark of ferroptosis. Higher concentrations of MDA have been linked to greater degrees of HIE and are recognized as established biomarkers for identifying perinatal asphyxia (El Bana et al. 2016). Elevated levels of MDA and 4-HNE, detected in HIBD models both in vivo and in vitro, can be reduced by ferroptosis-targeted interventions (Zhang et al. 2023a, b; Zheng et al. 2023).

Acyl-CoA synthetase long-chain family member 4 (ACSL4) is a rate-limiting enzyme that facilitates the esterification of PUFAs, thereby promoting lipid peroxidation. ACSL4 is recognized as a key modulator involved in the regulation of ferroptosis and neuroinflammatory processes. During ischemic brain injury, the microRNA miR-347 has been shown to transcriptionally upregulate ACSL4, enhancing LPO formation and exacerbating neuronal ferroptosis (Cui et al. 2021). A recent metabolomic study demonstrated that 2-phosphoglyceric acid exerts neuroprotective effects in both OGD/R-induced neuronal HT-22 cells and neonatal rats subjected to HIBD by modulating the ACSL4, thereby inhibiting ferroptosis (Chen et al. 2024a, b).

Lipoxygenases (LOXs), particularly 12/15-LOX, also function as key mediators of PUFA peroxidation and ferroptotic cell death. In models of brain ischemia in mice, the expression of 12/15-lipoxygenase progressively rises over time in the hippocampus, striatum, and cerebral cortex. Genetic deletion of LOX significantly attenuates ischemic brain injury (Yigitkanli et al. 2017). N-acetylcysteine (NAC), a clinically approved antioxidant, has been shown to inhibit 15-LOX activity, reduce lipid peroxidation, and preserve neuronal function (Karuppagounder et al. 2018). In HIBD studies conducted on both cultured cells and neonatal rats, increased LOX expression and enzymatic activity have been closely associated with injury severity. LOX is believed to promote ferroptosis, and blocking its activity with specific drugs has been reported to significantly protect neurons in HIBD models (Jiang et al. 2024).

Thus, aberrant lipid peroxidation, mediated by enzymes like ACSL4 and LOX, is not merely a hallmark but a critical executor of ferroptotic cell death, making it a central therapeutic target in HIE.

Dysfunction of the System Xc⁻–GSH–GPX4 Axis

In neonatal HIE, the dysfunction of the System Xc⁻–GSH–GPX4 axis represents a pivotal event driving ferroptosis. This cascade is often initiated by excessive extracellular glutamate levels—a well-established feature of excitotoxicity in HIE—and the downregulation of the Solute carrier family 7 member 11 (SLC7A11) subunit, which together impair the uptake of cystine (Wu et al 2019; Chen et al. 2023; Pu et al. 2007). This disruption leads to a critical reduction in the synthesis of GSH, the essential cofactor for GPX4.

As the central executor and essential regulator of ferroptosis, GPX4 operates in a GSH-dependent manner to reduce lipid hydroperoxides, thereby preventing the lethal accumulation of LPOs. Its genetic deletion or oxidative inactivation is sufficient to trigger massive LPO accumulation and ferroptotic cell death (Gong et al. 2019). The pathophysiological relevance of this axis is consistently observed in HIBD models, which demonstrate characteristic reductions in SLC7A11, GSH, and GPX4, accompanied by marked LPO accumulation. Hypoxia significantly reduces both the enzymatic activity and protein levels of GPX4, particularly in mitochondria, an event that is pivotal to the enhanced oxidative stress and mitochondrial dysfunction seen in HIBD (Demarest et al. 2016). Therapeutically, ferroptosis inhibitors such as Liproxstatin-1 have been shown to restore System Xc⁻ activity, reduce neuronal loss, and improve outcomes following hypoxic-ischemic brain injury (Zheng et al. 2023; Zhang et al. 2023a, b). Conversely, restoring GPX4 expression or activity is a cornerstone of anti-ferroptotic therapy, as demonstrated by several antioxidants—including catalpol, ginsenoside Rb1, and safflower yellow—which mitigate ferroptosis and ameliorate neuropathological outcomes in HIBD primarily through the preservation or upregulation of GPX4 (Zhang et al. 2023a, b; Lin et al. 2024; Zhou et al. 2023a, b).

In summary, the System Xc⁻–GSH–GPX4 axis constitutes the primary defense system against ferroptosis, and its dysfunction is a pivotal event in the pathogenesis of neonatal HIE. The critical role of GPX4 makes its upstream regulatory pathways promising therapeutic targets. Notably, the transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) serves as a master positive regulator of GPX4 expression. Pharmacological activation of Nrf2 signaling represents a key strategy to enhance cellular defense against ferroptosis by transcriptionally upregulating GPX4, as will be detailed in the next section.

The Nrf2 Signaling Pathway in Ferroptosis Regulation

Nrf2, a member of the basic leucine zipper transcription factor family, plays a key role in modulating the cellular antioxidant defense system.The NFE2L2 gene, located on chromosome 2q31.2, encodes a 605-amino acid protein. Kelch-like ECH-associated protein 1(Keap1) retains Nrf2 in the cytoplasm under non-stressed conditions, promoting its degradation through the ubiquitin–proteasome pathway. Oxidative stress disrupts this interaction, allowing Nrf2 to enter the nucleus and initiate transcription of antioxidant defense genes via antioxidant Response Element (ARE) binding.

Nrf2, a central controller of redox balance, significantly influences a cells’ vulnerability to ferroptosis. Upon activation, Nrf2 translocates to the nucleus and binds to the ARE, initiating the transcription of a battery of cytoprotective genes. Among these, GPX4 is a key downstream effector.Therefore, Nrf2 activation enhances the cellular capacity to detoxify lipid peroxides and resist ferroptotic damage. In contrast, Nrf2 deficiency heightens cellular sensitivity to ferroptosis inducers (Stockwell et al. 2017).

The Nrf2-GPX4 axis is a critical pathway targeted by many neuroprotective agents.For example, resveratrol has been shown to alleviate HIBD by activating the SIRT1/Nrf2 signaling, which in turn leads to the upregulation of GPX4 expression and a reduction in oxidative damage (Li et al. 2022a, b). Similarly, catalpol exerts its anti-ferroptotic effects primarily by activating the Nrf2 pathway, resulting in the coordinated upregulation of the entire GSH synthesis pathway, including GPX4 and the System Xc- subunit SLC7A11 (Lin et al. 2024). The protective effect of melatonin against HIBD is also mediated through the Akt-Nrf2-GPX4 axis, and importantly, this protection is abolished by the GPX4 inhibitor RSL3,underscoring the indispensable role of GPX4 as the ultimate effector of Nrf2-mediated neuroprotection (Gou et al. 2020; Pang et al. 2021).

Certain Nrf2 inhibitors, including all-trans retinoic acid, trigonelline, and brucein A, can promote ferroptosis by suppressing the expression of antioxidant proteins, further highlighting the importance of this pathway in ferroptosis regulation (Sun et al. 2016).

In summary, the Nrf2 signaling pathway serves as a master regulator of the cellular antioxidant response and a pivotal upstream defense against ferroptosis, primarily through its transcriptional control of GPX4. The evidence that diverse neuroprotective agents converge on the Nrf2-GPX4 axis underscores its fundamental role in mitigating oxidative damage in HIE. This established role of Nrf2 as a key therapeutic target provides a strong rationale for investigating upstream modulators, such as the vitamin D/VDR signaling pathway, which can potently activate this protective cascade.

The TLR4/NF-κB Pathway: A Link between Inflammation and Ferroptosis

Toll-like receptor 4 (TLR4) serves as an important pattern recognition receptor that is widely distributed throughout the central nervous system, especially within neurons and glial cells. It plays a pivotal role in mediating innate immune responses and initiating neuroinflammatory cascades following brain injury (Tang et al. 2019; Zhou et al. 2018). TLR4 may become abnormally activated in response to endogenous damage-associated molecular patterns (DAMPs) released from damaged or dying cells during brain injuries such as hypoxic-ischemic events and trauma. This activation links the initial insult to secondary inflammatory responses and exacerbates neuronal injury.

Increasing evidence supports the role of TLR4 as a promising therapeutic target in neonatal HIBD. Inhibition of TLR4 has been shown to confer neuroprotection by reducing both inflammation and neuronal death (Zhou et al. 2023a, b; Chen et al. 2024a, b). Zhu et al. (2021) reported that HIBD leads to concurrent upregulation of ferroptosis markers and TLR4 expression in neonatal brain tissue. Treatment with TAK-242, a selective TLR4 inhibitor, attenuated oxidative stress–induced neuronal injury, suppressed ferroptosis, and alleviated neuroinflammation. Evidence from these observations points to TLR4 signaling as a pivotal upstream regulator involved in ferroptosis during neonatal HIBD.

Upon TLR4 activation, nuclear factor kappa B (NF-κB) undergoes phosphorylation and translocation into the nucleus, where it modulates processes such as inflammation, apoptosis, and immune regulation. In experimental traumatic brain injury, the TLR4/NF-κB signaling axis has been associated with enhanced neuroinflammatory activity and autophagy dysfunction (Feng et al. 2016). Zhou et al. (2020) demonstrated that asiaticoside ameliorates HIBD in neonates by inhibiting the TLR4/NF-κB/STAT3 pathway, further underscoring the therapeutic relevance of this signaling cascade.

Lipocalin-2 (LCN2), an inflammation-associated protein, has also been implicated in ferroptosis regulation. In glutamate-induced ferroptosis models using HT22 hippocampal neurons, LCN2 expression was markedly upregulated. LCN2 knockdown effectively attenuated glutamate-driven ferroptosis, and subsequent mechanistic studies identified the NF-κB pathway as a key mediator of LCN2-induced ferroptosis in HIBD (Luo et al. 2023).

Collectively, these findings highlight the TLR4/NF-κB signaling axis as a crucial upstream driver of both neuroinflammation and ferroptosis in neonatal brain injury. Therapeutically targeting this pathway may provide dual benefits by mitigating inflammation and preventing ferroptosis-mediated neuronal death in HIE.

ER Stress as an Upstream Mediator of Ferroptosis in HIE

The endoplasmic reticulum (ER) plays a vital role in sustaining cellular homeostasis by coordinating protein synthesis, folding, modification, quality assurance, and transport.Disruption of ER structure or function by intrinsic or extrinsic stressors leads to the buildup of misfolded or unfolded proteins, triggering endoplasmic reticulum stress (ERS) (Oakes and Papa 2015). While transient ERS can activate adaptive responses, persistent or severe ERS can compromise ER function and result in multiple types of programmed cell death, such as ferroptosis (Zhai et al. 2025; Zhang et al. 2022).

A key mechanistic link between persistent ERS and the induction of ferroptosis is the activation of the transcription factor C/EBP homologous protein (CHOP). CHOP promotes the expression of the enzyme ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1), which degrades glutathione (GSH) and thereby depletes this critical antioxidant, profoundly sensitizing cells to ferroptotic death. This CHOP-CHAC1 axis represents a direct molecular pathway through which ERS disrupts cellular redox homeostasis.

Contemporary studies have emphasized ERS as a key pathological mechanism underlying HIBD (Hu et al. 2023; Wu et al. 2024). Elevated expression of ERS markers has been consistently observed in neonatal HIBD models, and inhibiting ERS pharmacologically has demonstrated potential in promoting neuroprotection. Moreover, ERS has been implicated as a mediator of ferroptosis in diverse pathological contexts beyond the brain, such as sepsis-induced lung injury and cadmium-induced hepatotoxicity (Zeng et al. 2022; He et al. 2022), supporting the broader role of ERS in triggering this cell death pathway. ERS suppression mitigated ferroptosis by lowering ROS, lipid peroxidation, and cellular iron levels (Liu et al. 2023a, b).

In a study by Ji et al. (2024), neonatal rats subjected to HIBD exhibited significant ERS activation in brain tissue. Pharmacological inhibition of ERS with 4-Phenylbutyric acid (4-PBA) alleviated both neuronal injury and ferroptosis-associated cell death. Complementary in vitro experiments further confirmed the mechanistic role of ERS in promoting ferroptosis under hypoxic-ischemic conditions.

These cumulative findings implicate ER stress as a key upstream mediator of ferroptosis in neonatal HIE. Therefore, inhibiting ERS signaling may constitute an innovative therapeutic option to alleviate neuronal injury.

Summary

Collectively, this section delineates a complex yet interconnected network of mechanisms driving ferroptosis in neonatal HIE. It involves the foundational disruptions of iron and lipid metabolism, the failure of the central GPX4-dependent defense system, and is critically modulated by upstream signaling pathways such as Nrf2, TLR4/NF-κB, and ER stress. The convergence of these pathways on ferroptosis underscores its significance as a central pathological process in HIE. This comprehensive mechanistic framework sets the stage for exploring targeted interventions, notably the VD/VDR signaling pathway, which possesses the potential to modulate multiple facets of this network (see Fig. 1 and Table 1)​.

Fig. 1.

The cascade of ferroptotic pathways in neonatal hypoxic-ischemic encephalopathy

Table 1.

Summary of compounds targeting key pathways of ferroptosis in neonatal hypoxic-ischemic encephalopathy

Compound	Primary Target/Mechanism	Experimental Evidence and Key Findings
Iron Metabolism
Deferoxamine	Iron chelation	Reduces iron deposition and hydroxyl radicals, attenuating neuronal damage and ferroptosis in neonatal rat HIBD models (Lu et al. 2015; Tan et al. 2023)
Erythropoietin	Iron homeostasis	Clinical studies show improved neurological outcomes in very preterm infants (Song et al. 2016)
Lipid Peroxidation & GPX4 System
Liproxstatin-1	Ferroptosis inhibitor (radical-trapping)	Reduces lipid peroxidation and neuronal death, improving functional outcomes in neonatal rat HIBD models (Zheng et al. 2023)
N-acetylcysteine (NAC)	15-LOX inhibition; GSH precursor	Reduces lipid peroxidation and protects neuronal function in experimental models (Karuppagounder et al. 2018)
2-Phosphoglyceric acid	ACSL4 modulation	Exerts neuroprotective effects in vitro and in neonatal rat HIBD by regulating the ACSL4/GPX4 axis (Chen et al. 2024a, b)
Catalpol	Nrf2 activation, upregulating GPX4 & SLC7A11	Inhibits ferroptosis and ameliorates brain damage in both cellular and neonatal rat HIBD models (Lin et al. 2024)
Resveratrol	Activation of SIRT1/Nrf2 signaling, upregulating GPX4	Alleviates HIBD by activating the SIRT1/Nrf2 pathway, leading to upregulation of GPX4 expression and a reduction in oxidative damage (Li et al. 2022a, b)
Ginsenoside Rb1	GPX4 upregulation	Mitigates ferroptosis and ameliorates neuropathological outcomes in HIBD primarily through the preservation or upregulation of GPX4 (Zhang et al. 2023a, b)​
Melatonin	Akt-Nrf2 axis activation, upregulating GPX4	Confers neuroprotection in neonatal HIBD models; this effect is abolished by the GPX4 inhibitor RSL3 (Gou et al. 2020)
Carthamin yellow	Inhibition of neuronal ferroptosis	Attenuates brain injury by inhibiting neuronal ferroptosis in the hippocampus of a neonatal rat HIBD model (Zhou et al. 2023a, b)
Upstream Signaling Pathways
TAK-242	TLR4-specific inhibitor	Attenuates oxidative stress-induced neuronal injury, suppresses ferroptosis, and alleviates neuroinflammation in neonatal rat HIBD (Zhu et al. 2021)
Asiaticoside	TLR4/NF-κB pathway inhibition	Ameliorates HIBD in neonatal rats (Zhou et al. 2020)
4-Phenylbutyric acid (4-PBA)	Endoplasmic reticulum stress inhibition	Alleviates neuronal injury and ferroptosis by suppressing ER stress markers, reducing lipid peroxidation, and upregulating GPX4 in a neonatal rat HIBD model (Ji et al. 2024)

This schematic illustrates the temporal sequence and interrelated cascade of events that drive ferroptosis following hypoxic-ischemic injury. The process is initiated by upstream signaling events triggered by the primary insult, including ERS and TLR4/NF-κB-mediated neuroinflammation. Concurrently, the Nrf2 antioxidant signaling pathway is suppressed, thereby weakening the cellular defense mechanisms. These upstream events converge to initiate the core execution mechanisms of ferroptosis: (1) Disruption of iron homeostasis, characterized by iron overload and Fenton reaction-driven production of ROS; (2) Aberrant lipid metabolism and peroxidation, mediated by enzymes such as ACSL4 and LOX, leading to the accumulation of LPO; (3) Dysfunction of the System Xc⁻–GSH–GPX4 axis, a central antioxidant defense system against lipid peroxidation. The failure of these systems ultimately culminates in oxidative, iron-dependent neuronal death. The studies corresponding to the numbered labels in the figure are detailed below: ① Lu et al. (2015); Tan et al. (2023), ② Wu et al. (2019); Chen et al. (2023); Pu et al. (2007); Tan et al. (2023); Zheng et al. (2023); Zhang et al. (2023a, b), ③ Gong et al. (2019); Zhang et al. (2023a, b); Lin et al. (2024); Zhou et al. (2023a, b); ④ Zhang et al. (2023a, b); Zheng et al. (2023), ⑤ Cui et al. (2021); Chen et al. (2024a, b), ⑥ Yigitkanli et al. (2017); Karuppagounder et al. (2018); Jiang et al. (2024), ⑦ Li et al. (2022a, b); Lin et al. (2024); Gou et al. (2020); Pang et al. (2021); Sun et al. (2016), ⑧ Zhou et al. (2023a, b); Chen et al. (2024a, b); Zhu et al. (2021); Feng et al. (2016); Zhou et al. (2020); Luo et al. (2023), ⑨ Zhai et al. (2025); Zhang et al. (2022); Hu et al. (2023); Wu et al. (2024); Ji et al. (2024).

VD/VDR: Modulators of Ferroptosis and Neuroprotection

As established in the previous section, ferroptosis represents a compelling therapeutic target in HIE. Given the multifaceted nature of its mechanisms, an ideal intervention would be one capable of simultaneously addressing multiple dysregulated pathways. VD, through its receptor VDR, has emerged as such a pleiotropic modulator. This section will evaluate the clinical and preclinical evidence supporting the neuroprotective role of VD/VDR, and delve into the specific mechanisms by which it intersects with and regulates the ferroptotic processes detailed earlier.​

Vitamin D (VD), a fat-soluble secosteroid, is traditionally known for its role in calcium-phosphate homeostasis and skeletal health.Increasing evidence indicates roles in neurodevelopment, synaptic plasticity, neuronal signaling, and neuroimmune regulation (Navale et al. 2022; Pignolo et al. 2022). These data expand the conventional view of VD and support its relevance as a neuroactive steroid in neonatal HIE.

VD exerts its effects primarily through the intracellular VDR. Upon binding its active metabolite 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], VDR translocates to the nucleus, heterodimerizes with retinoid X receptor (RXR), and binds vitamin D response elements (VDREs) to regulate target-gene transcription. In the context of HIE, several of these VDR-dependent programs intersect antioxidant and iron/lipid-handling pathways implicated in ferroptosis, providing a mechanistic bridge to the sections that follow.

Clinical Evidence Linking Vitamin D Deficiency to HIE

Preliminary clinical evidence indicates that neonates with hypoxic–ischemic encephalopathy (HIE) frequently have mothers with low vitamin D status. In a prospective case–control study of term infants (HIE n = 31; controls n = 30), maternal 25(OH)D concentrations were significantly lower in the HIE group than in controls (9.8 ± 6.8 vs. 16.4 ± 8.7 ng/mL, p = 0.002). In affected neonates, vitamin D levels were already reduced at 6–14 h of life and remained low on day 5; the proportion with severe deficiency (< 5 ng/mL) was also higher than in controls (p < 0.05). Oxidative stress indices were worse in HIE infants (day-1 SOD and MDA elevated, p = 0.001 and p = 0.028; day-5 SOD and GP elevated, p < 0.05) (Mutlu et al. 2016). Complementing these findings, a multicenter analysis reported that ~ 70% of HIE neonates were vitamin D-insufficient at birth and levels declined further over the first 72 h, regardless of hypothermia treatment (Lowe et al. 2017). MRI-based assessments of brain injury severity in HIE neonates show a negative association with serum vitamin D status. In a retrospective cohort at a level IV NICU (≥ 35 weeks’ gestation; n = 43), 22 infants had 25-hydroxyvitamin D [25(OH)D] measured within the first 48 h of life. Lower 25(OH)D concentrations were significantly associated with more severe brain injury on standardized MRI scoring (p = 0.017). Postnatal vitamin D dosing during the first week was not associated with MRI injury severity, although higher doses trended toward fewer ventilator days (p = 0.062) (McGinn et al. 2020).

The association between low VD status and HIE-related brain injury suggests that early correction of deficiency may offer therapeutic value in mitigating neurological outcomes.

Neuroprotective Effects of Vitamin D in HIE

Accumulating evidence from clinical and preclinical studies underscores the neuroprotective potential of VD in the context of HIE. In a phase-I study of hypothermia-treated HIE neonates (n = 30), co-administration of intravenous NAC (25–40 mg/kg q12h) and calcitriol (0.03–0.05 µg/kg) for 10 days markedly improved oxidative-stress indices. Basal-ganglia GSH rose from 1.61 ± 0.28 mM to 1.93 ± 0.31 mM (p = 0.012), while plasma lipid peroxidation markers declined and correlated inversely with GSH changes (rₛ≈−0.8, p < 0.01). About 75% of infants were vitamin D-deficient at baseline, and supplementation normalized 1,25(OH)₂D levels. On 2-year follow-up, all survivors demonstrated normal neurodevelopmental outcomes without cerebral palsy or cognitive delay (Jenkins et al. 2021).

A key mechanism underlying VD’s antioxidant effect is its role in activating the Nrf2 signaling pathway. This has been demonstrated in models of HIBD, where VD administration was shown to upregulate Nrf2 and its downstream effectors, thereby attenuating oxidative damage and improving outcomes (Cai et al. 2022). The relevance of this VD/VDR-Nrf2 axis is further supported by studies in other neurological contexts, including lead-induced neurotoxicity (Hosseinirad et al. 2021) and Alzheimer’s disease models (Saad et al. 2020), underscoring its role as a fundamental neuroprotective pathway. The anti-ferroptotic effect of VD, particularly through the engagement of the Nrf2/HO-1 axis, positions it as a modulator capable of targeting the core oxidative injury processes in HIE.​

VDR-Mediated Regulation of Ferroptosis in HIE

The expression of VDR in brain regions critical for cognition and motor control underscores its significance in neurological health. Conditional knockout of VDR in microglia and macrophages exacerbates cerebral ischemia, leading to larger infarct volumes and heightened neuroinflammation (Cui et al. 2023), while global VDR-deficient mice exhibit motor and cognitive impairments (Mirarchi et al. 2023).

Evidence increasingly identifies VDR as a direct and versatile regulator of ferroptosis. Beyond facilitating Nrf2 signaling, emerging research highlights a more direct role for the VD/VDR axis. For example, VD was shown to ameliorate cognitive decline and ferroptosis in aged mice via the VDR-Nrf2-HO-1 pathway; significantly, this protective effect was abolished in VDR-knockout mice, establishing VDR’s indispensability (Li et al. 2023). Furthermore, of particular mechanistic importance, and despite being shown in a model of cisplatin-induced acute kidney injury, pharmacologic activation of VDR has been demonstrated to protect against cell death by promoting GPX4 expression. Luciferase reporter assays confirmed GPX4 as a direct transcriptional target of VDR (Hu et al. 2020). This discovery reveals a fundamental and novel pathway through which VDR signaling can directly bolster a key defense against ferroptosis, a mechanism that warrants specific investigation in the context of HIE.​​

This direct regulatory role is further supported by studies in neurodegenerative models. The neuroprotective and anti-ferroptotic effects of the flavonoid eriodictyol in APP/PS1 mice were contingent upon VDR presence, providing additional evidence for VDR’s critical function in mediating anti-ferroptotic interventions (Li et al. 2022a, b).

​In summary, VDR mediates neuroprotection in HIE through a dual mechanism: (1) indirectly, by enhancing the Nrf2-mediated antioxidant network that includes GPX4, and (2) directly, by transactivating key ferroptosis-related genes like GPX4 itself. This multifaceted role underscores its potential as a high-value therapeutic target for mitigating ferroptotic damage in neonatal brain injury.​

Summary

The evidence presented here positions the VD/VDR signaling pathway as a master regulator capable of countering ferroptosis on multiple fronts. Its ability to enhance antioxidant defenses via Nrf2, directly upregulate GPX4 transcription, and dampen neuroinflammation illustrates a synergistic mechanism of action. This multi-targeted approach offers a distinct advantage, potentially leading to more robust neuroprotection in the complex pathological environment of HIE.​

Discussion

The evidence summarized in this review highlights ferroptosis as a pivotal driver of neuronal injury in neonatal HIE and underscores the emerging role of VD and its receptor (VDR) in modulating this pathway.Unlike single-target approaches, the VD/VDR axis offers a synergistic strategy by enhancing antioxidant defenses via Nrf2 activation, directly transactivating genes like GPX4, and mitigating neuroinflammation, thereby addressing the multifaceted nature of HIE pathology.These findings suggest that targeting VD/VDR may represent a promising adjunctive approach to therapeutic hypothermia.

However, it is crucial to acknowledge the limitations of the current evidence base.The most significant limitation is that the entirety of the experimental data supporting the therapeutic potential of VD/VDR signaling for ferroptosis in HIE is derived from preclinical studies. While these models are invaluable for elucidating mechanisms, they cannot fully recapitulate the complexity of human neonatal HIE. Furthermore, critical translational challenges must be addressed before clinical application can be considered. These include a complete lack of dosing guidelines, safety profiles, and pharmacokinetic data for high-dose VD or VDR agonists in the vulnerable neonatal HIE population. Importantly, the potential risks of excessive VD supplementation—such as hypercalcemia, nephrocalcinosis, or cardiac arrhythmias—cannot be overlooked and must be rigorously evaluated.Therefore, the direct clinical applicability of these findings remains to be established.

​Given these limitations, the translation of these compelling preclinical findings into clinical practice hinges on addressing critical knowledge gaps. Future research should therefore prioritize the following areas:

1. Elucidating Molecular Interplay: Further investigation is needed to decipher the precise crosstalk between VDR activation and other ferroptosis-related pathways, such as the detailed mechanisms of VDR-mediated GPX4 transcription and its interaction with inflammatory signals like TLR4/NF-κB.

2. Cell-Type-Specific Actions: The distinct roles of VD/VDR signaling in different cell types of the neonatal brain—including neurons, astrocytes, microglia, and oligodendrocytes—remain largely unexplored. Cell-specific knockout studies are essential to understand its cell-autonomous versus non-cell-autonomous effects.

3. Clinical Translation: Rigorously designed randomized controlled trials (RCTs) represent the paramount next step. These trials must define critical parameters including the optimal timing of administration, dosing regimens, safety profiles, and most importantly, the long-term neurodevelopmental outcomes of VD supplementation or VDR agonists as an adjunct to therapeutic hypothermia.

4. Biomarker Exploration: The development of biomarkers for assessing VD status, VDR activity, and ferroptosis in real-time within the clinical setting of HIE could guide personalized therapy and monitor treatment response.

Conclusion and Future Perspectives

In conclusion, ferroptosis represents a key mechanistic target in neonatal HIE. The VD/VDR signaling pathway, by acting on multiple molecular levels, provides a biologically plausible and potentially translatable neuroprotective strategy. Future randomized controlled trials are warranted to confirm its efficacy and establish safe therapeutic parameters for clinical use.

Abbreviations

HIE

Hypoxic-ischemic encephalopathy

HIBD

Hypoxic-ischemic brain damage

TH

Therapeutic hypothermia

VD

Vitamin D

VDR

Vitamin D receptor

ROS

Reactive oxygen species

GSH

Glutathione

GPX4

Glutathione peroxidase 4

System Xc⁻

Cystine–glutamate exchange system

TFRC

Transferrin receptor

FTH

Ferritin heavy chain

FTL

Ferritin light chain

PUFAs

Polyunsaturated fatty acids

LPO

Lipid peroxidation

4-HNE

4-hydroxynonenal

MDA

Malondialdehyde

ACSL4

Acyl-CoA synthetase long-chain family member 4

LOXs

Lipoxygenases

NAC

N-acetylcysteine

SLC7A11

Solute carrier family 7 member 11

Nrf2

Nuclear factor erythroid 2-related factor 2

Keap1

Kelch-like ECH-associated protein 1

ARE

Antioxidant Response Element

TLR4

Toll-like receptor 4

DAMPs

Damage-associated molecular patterns

NF-κB

Nuclear factor kappa B

LCN2

Lipocalin-2

ER

Endoplasmic reticulum

ERS

Endoplasmic reticulum stress

4-PBA

4-Phenylbutyric acid

1,25(OH)₂D₃

1,25-dihydroxyvitamin D₃

RXR

Retinoid X receptor

VDREs

Vitamin D response elements

Author Contributions

CYJ was responsible for developing the research framework, and writing the initial draft. ZW performed manuscript evaluation, revisions, and supervised the research process. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Wu Jieping Medical Foundation Clinical Research Special Funding (Grant No.320.6750.2025-9-15) and the Liuzhou Science and Technology Planning Project (Grant No. 2024SB 0104A002).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Ethical Approval

This article is a literature-based review and does not involve any human participants or animal experiments. Therefore, ethical approval and informed consent were not required.

Consent for Publication

Not applicable.