No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Ferroptosis in Central Nervous System Hypoxia–Ischemia
Abstract
Millions of people suffer from acute hypoxic–ischemic brain injuries (HIBIs) such as stroke and neonatal HIBI worldwide each year, resulting in significant mortality and lifelong morbidity. Therapeutic options for HIBI remain limited, but many investigators are actively seeking out novel therapies based on our evolving understanding of the pathophysiology. Ferroptotic cell death has recently been demonstrated to play a significant part in the complex cellular injury resulting after HIBI. This chapter will discuss the current literature evaluating the role of ferroptosis-related reactive oxygen species release and lipid peroxidation in the pathophysiology of HIBI. Based on the current mechanistic understanding, several investigators have begun to develop interventions for HIBI that target the ferroptosis pathways. As such, this chapter will also discuss many of the ferroptosis inhibitors that have shown neuroprotective effects in cell culture and animal models of HIBI.
ResearchGate has not been able to resolve any citations for this publication.
Microglia play an important role in maintaining central nervous system homeostasis and are the major immune cells in the brain. In response to internal or external inflammatory stimuli, microglia are activated and release numerous inflammatory factors, thus leading to neuroinflammation. Inflammation and microglia iron accumulation promote each other and jointly promote the progression of neuroinflammation. Inhibiting microglia iron accumulation prevents neuroinflammation. Ferroptosis is an iron-dependent phospholipid peroxidation-driven type of cell death regulation. Cell iron accumulation causes the peroxidation of cell membrane phospholipids and damages the cell membrane. Ultimately, this process leads to cell ferroptosis. Iron accumulation or phospholipid peroxidation in microglia releases a large number of inflammatory factors. Thus, inhibiting microglia ferroptosis may be a new target for the prevention and treatment of neuroinflammation.
Background:
Neuronal death due to over-oxidative stress responses defines the pathology of cerebral ischemic/reperfusion (I/R) insult. Ferroptosis is a form of oxidative cell death that is induced by disruption of the balance between antioxidants and pro-oxidants in cells. However, the potential mechanisms responsible for cerebral I/R-induced ferroptotic neuronal death have not been conclusively determined. UBIAD1, is a newly identified antioxidant enzyme that catalyzes coenzyme Q10 (CoQ10) and vitamin K2 biosynthesis in the Golgi apparatus membrane and mitochondria, respectively. Even though UBIAD1 is a significant mediator of apoptosis in cerebral I/R challenge, its roles in ferroptotic neuronal death remain undefined. Therefore, we investigated whether ferroptotic neuronal death is involved in cerebral I/R injury. Further, we evaluated the functions and possible mechanisms of UBIAD1 in cerebral I/R-induced ferroptotic neuronal death, with a major focus on mitochondrial and Golgi apparatus dysfunctions.
Results:
Ferroptosis occurred in cerebral I/R. Ferroptotic neuronal death promoted cerebral I/R-induced brain tissue injury and neuronal impairment. UBIAD1 was expressed in cerebral tissues and was localized in neurons, astrocytes, and microglia. Under cerebral I/R conditions overexpressed UBIAD1 significantly suppressed lipid peroxidation and ferroptosis. Moreover, upregulated UBIAD1 protected against brain tissue damage and neuronal death by alleviating I/R-mediated lipid peroxidation and ferroptosis. However, UBIAD1 knockdown reversed these changes. Enhanced UBIAD1-mediated ferroptosis elevated the antioxidative capacity by rescuing mitochondrial and Golgi apparatus dysfunction in cerebral I/R-mediated neuronal injury. They improved the morphology and biofunctions of the mitochondria and Golgi apparatus, thereby elevating the levels of SOD, T-AOC and production of CoQ10, endothelial nitric oxide synthase (eNOS)-regulated nitric oxide (NO) generation as well as suppressed MDA generation.
Conclusions:
The neuroprotective agent, UBIAD1, modulates I/R-mediated ferroptosis by restoring mitochondrial and Golgi apparatus dysfunction in damaged brain tissues and neurons, thereby enhancing antioxidative capacities. Moreover, the rescue of impaired mitochondrial and Golgi apparatus as a possible mechanism of regulating ferroptotic neuronal death is a potential treatment strategy for ischemic stroke.
SLC7A11/xCT is an antiporter that mediates the uptake of extracellular cystine in exchange for glutamate. Cystine is reduced to cysteine, which is a rate-limiting precursor in glutathione synthesis; a process that protects cells from oxidative stress and is, therefore, critical to cell growth, proliferation, and metabolism. SLC7A11 is expressed in different tissues and plays diverse functional roles in the pathophysiology of various diseases, including cancer, by regulating the processes of redox homeostasis, metabolic flexibility/nutrient dependency, immune system function, and ferroptosis. SLC7A11 expression is associated with poor prognosis and drug resistance in cancer and, therefore, represents an important therapeutic target. In this review, we discuss the molecular functions of SLC7A11 in normal versus diseased tissues, with a special focus on how it regulates gastrointestinal cancers. Further, we summarize current therapeutic strategies targeting SLC7A11 as well as novel avenues for treatment.
Ischemic stroke is a common cerebrovascular disease and recovering blood flow as early as possible is essential to reduce ischemic damage and maintain neuronal viability, but the reperfusion process usually causes additional damage to the brain tissue in the ischemic area, namely ischemia reperfusion injury. The accumulated studies have revealed that transplantation of exogenous neural stem cells (NSCs) is an ideal choice for the treatment of ischemia reperfusion injury. At present, the source and efficacy of exogenous NSCs after transplantation is still one of the key issues that need to be resolved. In this study, human umbilical cord mesenchymal stem cells (hUC-MSCs) were obtained and induced into NSCs byadding growth factor and neuregulin1β (NRG1β) was introduced during the differentiation process of NSCs. Then, the rat middle cerebral artery occlusion/reperfusion (MCAO/R) models were established, and the therapeutic effects were evaluated among groups treated by NRG1β, NSCs and NSCs pretreated with 10 nM NRG1β (NSCs-10 nM NRG1β) achieved through intra-arterial injection. Our data show that the NSCs-10 nM NRG1β group significantly improves neurobehavioral function and infarct volume after MCAO/R, as well as cerebral cortical neuron injury, ferroptosis-related indexes and mitochondrial injury. Additionally, NSCs-10 nM NRG1β intervention may function through regulating the p53/GPX4/SLC7A11 pathway, and reducing the level of ferroptosis in cells, further enhance the neuroprotective effect on injured cells.
Alzheimer's disease (AD) is the most prevalent form of dementia, with complex pathophysiology that is not fully understood. While β‐amyloid plaque and neurofibrillary tangles define the pathology of the disease, the mechanism of neurodegeneration is uncertain. Ferroptosis is an iron‐mediated programmed cell death mechanism characterised by phospholipid peroxidation that has been observed in clinical AD samples. This review will outline the growing molecular and clinical evidence implicating ferroptosis in the pathogenesis of AD, with implications for disease‐modifying therapies.
image
Kaempferol has been shown to protect cells against cerebral ischemia/reperfusion injury through inhibition of apoptosis. In the present study, we sought to investigate whether ferroptosis is involved in the oxygen-glucose deprivation/reperfusion (OGD/R)-induced neuronal injury and the effects of kaempferol on ferroptosis in OGD/R-treated neurons. Western blot, immunofluorescence, and transmission electron microscopy were used to analyze ferroptosis, whereas cell death was detected using lactate dehydrogenase (LDH) release. We found that OGD/R attenuated SLC7A11 and glutathione peroxidase 4 (GPX4) levels as well as decreased endogenous antioxidants including nicotinamide adenine dinucleotide phosphate (NADPH), glutathione (GSH), and superoxide dismutase (SOD) in neurons. Notably, OGD/R enhanced the accumulation of lipid peroxidation, leading to the induction of ferroptosis in neurons. However, kaempferol activated nuclear factor-E2-related factor 2 (Nrf2)/SLC7A11/GPX4 signaling, augmented antioxidant capacity, and suppressed the accumulation of lipid peroxidation in OGD/R-treated neurons. Furthermore, kaempferol significantly reversed OGD/R-induced ferroptosis. Nevertheless, inhibition of Nrf2 by ML385 blocked the protective effects of kaempferol on antioxidant capacity, lipid peroxidation, and ferroptosis in OGD/R-treated neurons. These results suggest that ferroptosis may be a significant cause of cell death associated with OGD/R. Kaempferol provides protection from OGD/R-induced ferroptosis partly by activating Nrf2/SLC7A11/GPX4 signaling pathway.
Background
Ischemic stroke is the main cause of disability worldwide, leading to a serious socioeconomic burden. Ferroptosis is a non-apoptotic form of programmed cell death and is related to various diseases. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is considered a target of ferroptosis, but its specific role in ischemic stroke remains unclear. In this study, we investigate whether the inhibition of ACSL4 promotes the recovery of neurological function in a way that prevents ferroptosis.
Methods
A transient cerebral ischemia model was established for mice by middle cerebral artery occlusion (MCAO); glutathione peroxidase 4 (GPx4), ACSL4 and cyclooxygenase 2 (COX2) were detected by Western blot, and changes to mitochondria were observed by a transmission electron microscope. A kit was used to determine iron levels and lipid peroxide indicators, such as glutathione peroxidase (GPx), reduced glutathione (GSH), total glutathione/oxidized glutathione (GSH/GSSG), lipid peroxidation, reactive oxygen species, superoxide and malonaldehyde. Following MCAO, a ferroptosis inhibitor, liproxstatin-1, was administered intranasally immediately at a concentration of 10 mg/kg. Rosiglitazone was used to inhibit ACSL4 and was administered intravenously 1 h before MCAO at a concentration of 0.4 mg/kg. Brain injury was determined by neurological deficit scores, neuroscore (28-point), corner test and gait analyses, at 24 and 72 h after stroke. Brain infarct volume was determined by 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining at 72 h after stroke.
Results
After MCAO, GPx4 protein expression decreased, ACSL4 and COX2 protein expression increased, GPx activity decreased and iron accumulation. Transmission electron microscopy confirmed that the outer mitochondrial membrane of neurons had ruptured and mitochondrial cristae had decreased or disappeared. Liproxstatin-1 could significantly attenuate the decrease of GPx4 and the increase of COX2 after MCAO, dramatically reducing iron accumulation and decreasing GPx activity, accompanied by a marked reduction in changes in lipid peroxidation indicators. The use of rosiglitazone to inhibit ACSL4 could significantly improve neurological function and reduce the brain infarct volume at 72 h after stroke. Importantly, inhibiting ACSL4 could significantly attenuate the decline of GPx4 after MCAO and markedly attenuate iron accumulation and a decrease in GPx activity. Additionally, changes in lipid peroxidation indicators were also significantly inhibited.
Conclusion
This study indicates that inhibiting ACSL4 can promote the recovery of neurological function after stroke by suppression of ferroptosis.
Therapeutic hypothermia does not improve outcomes in neonatal hypoxia ischemia (HI) complicated by perinatal infection, due to well-described, pre-existing oxidative stress and neuroinflammation that shorten the therapeutic window. For effective neuroprotection post-injury, we must first define and then target CNS metabolomic changes immediately after endotoxin-sensitized HI (LPS-HI). We hypothesized that LPS-HI would acutely deplete reduced glutathione (GSH), indicating overwhelming oxidative stress in spite of hypothermia treatment in neonatal rats. Post-natal day 7 rats were randomized to sham ligation, or severe LPS-HI (0.5 mg/kg 4 h before right carotid artery ligation, 90 min 8% O2), followed by hypothermia alone or with N-acetylcysteine (25 mg/kg) and vitamin D (1,25(OH)2D3, 0.05 μg/kg) (NVD). We quantified in vivo CNS metabolites by serial 7T MR Spectroscopy before, immediately after LPS-HI, and after treatment, along with terminal plasma drug concentrations. GSH was significantly decreased in all LPS-HI rats compared with baseline and sham controls. Two hours of hypothermia alone did not improve GSH and allowed glutamate + glutamine (GLX) to increase. Within 1 h of administration, NVD increased GSH close to baseline and suppressed GLX. The combination of NVD with hypothermia rapidly improved cellular redox status after LPS-HI, potentially inhibiting important secondary injury cascades and allowing more time for hypothermic neuroprotection.
Carthamin yellow (CY), a flavonoid compound extracted from safflower, has been reported to attenuate cardiac ischemia and reperfusion injury. However, whether CY could ameliorate ischemic stroke is not completely understood. In the present study, the preventive effects of CY on experimental ischemic stroke were investigated using middle cerebral artery occlusion (MCAO) model rats. Neurological scores, brain edema, infarct area and microtubule‑associated protein 2 (MAP‑2) immunoreactivity were assessed to evaluate the effects of CY on ischemic brain injury. The involvement of inflammation and ferroptosis were examined to investigate the mechanism underlying the effects of CY. The results demonstrated that 2‑week CY treatment attenuated the neurological deficit score, brain water content and infarct area, and increased MAP‑2 immunoreactivity in the cortex in MCAO model rats. CY administration also deactivated the cortex NF‑κB/NLR family pyrin domain containing 3 inflammasome signaling pathway, and decreased serum TNF‑α, IL‑1β and IL‑6 concentrations. Moreover, CY treatment inhibited Fe2+ and reactive oxygen species accumulation, and reversed acyl‑CoA synthetase long‑chain family member 4, transferrin receptor 1, glutathione peroxidase 4 and ferritin heavy chain 1 protein expression levels in the brain. The levels of glutathione, superoxide dismutase and malondialdehyde in the serum were also reversed by CY treatment. Collectively, the results of the present study demonstrated that CY protected rats against ischemic stroke, which was associated with mitigation of inflammation and ferroptosis.
Ferroptosis is an iron-dependent form of regulated necrosis associated with lipid peroxidation. Despite its key role in the inflammatory outcome of ferroptosis, little is known about the molecular events leading to the disruption of the plasma membrane during this type of cell death. Here we show that a sustained increase in cytosolic Ca ²⁺ is a hallmark of ferroptosis that precedes complete bursting of the cell. We report that plasma membrane damage leading to ferroptosis is associated with membrane nanopores of a few nanometers in radius and that ferroptosis, but not lipid peroxidation, can be delayed by osmoprotectants. Importantly, Ca ²⁺ fluxes during ferroptosis induce the activation of the ESCRT-III-dependent membrane repair machinery, which counterbalances the kinetics of cell death and modulates the immunological signature of ferroptosis. Our findings with ferroptosis provide a unifying concept that sustained increase of cytosolic Ca ²⁺ prior to plasma membrane rupture is a common feature of regulated types of necrosis and position ESCRT-III activation as a general protective mechanism in these lytic cell death pathways.
The blood–brain barrier (BBB) is a dynamic interface responsible for maintaining the central nervous system homeostasis. Its unique characteristics allow protecting the brain from unwanted compounds, but its impairment is involved in a vast number of pathological conditions. Disruption of the BBB and increase in its permeability are key in the development of several neurological diseases and have been extensively studied in stroke. Ischemic stroke is the most prevalent type of stroke and is characterized by a myriad of pathological events triggered by an arterial occlusion that can eventually lead to fatal outcomes such as hemorrhagic transformation (HT). BBB permeability seems to follow a multiphasic pattern throughout the different stroke stages that have been associated with distinct biological substrates. In the hyperacute stage, sudden hypoxia damages the BBB, leading to cytotoxic edema and increased permeability; in the acute stage, the neuroinflammatory response aggravates the BBB injury, leading to higher permeability and a consequent risk of HT that can be motivated by reperfusion therapy; in the subacute stage (1–3 weeks), repair mechanisms take place, especially neoangiogenesis. Immature vessels show leaky BBB, but this permeability has been associated with improved clinical recovery. In the chronic stage (>6 weeks), an increase of BBB restoration factors leads the barrier to start decreasing its permeability. Nonetheless, permeability will persist to some degree several weeks after injury. Understanding the mechanisms behind BBB dysregulation and HT pathophysiology could potentially help guide acute stroke care decisions and the development of new therapeutic targets; however, effective translation into clinical practice is still lacking. In this review, we will address the different pathological and physiological repair mechanisms involved in BBB permeability through the different stages of ischemic stroke and their role in the development of HT and stroke recovery.
Cell death can be executed through different subroutines. Since the description of ferroptosis as an iron-dependent form of non-apoptotic cell death in 2012, there has been mounting interest in the process and function of ferroptosis. Ferroptosis can occur through two major pathways, the extrinsic or transporter-dependent pathway and the intrinsic or enzyme-regulated pathway. Ferroptosis is caused by a redox imbalance between the production of oxidants and antioxidants, which is driven by the abnormal expression and activity of multiple redox-active enzymes that produce or detoxify free radicals and lipid oxidation products. Accordingly, ferroptosis is precisely regulated at multiple levels, including epigenetic, transcriptional, posttranscriptional and posttranslational layers. The transcription factor NFE2L2 plays a central role in upregulating anti-ferroptotic defense, whereas selective autophagy may promote ferroptotic death. Here, we review current knowledge on the integrated molecular machinery of ferroptosis and describe how dysregulated ferroptosis is involved in cancer, neurodegeneration, tissue injury, inflammation, and infection.
Many new types of regulated cell death have been recently implicated in human health and disease. These regulated cell deaths have different morphological, genetic, biochemical, and functional hallmarks. Ferroptosis was originally described as a carcinogenic RAS-dependent non-apoptotic cell death, and is now defined as a type of regulated necrosis characterized by iron accumulation, lipid peroxidation, and the release of damage-associated molecular patterns (DAMPs). Multiple oxidative and antioxidant systems, acting together autophagy machinery, shape the process of lipid peroxidation during ferroptosis. In particular, the production of reactive oxygen species (ROS) that depends on the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) and the mitochondrial respiratory chain promotes lipid peroxidation by lipoxygenase (ALOX) or cytochrome P450 reductase (POR). In contrast, the glutathione (GSH), coenzyme Q10 (CoQ10), and tetrahydrobiopterin (BH4) system limits oxidative damage during ferroptosis. These antioxidant processes are further transcriptionally regulated by nuclear factor, erythroid 2-like 2 (NFE2L2/NRF2), whereas membrane repair during ferroptotic damage requires the activation of endosomal sorting complexes required for transport (ESCRT)-III. A further understanding of the process and function of ferroptosis may provide precise treatment strategies for disease.
Traumatic brain injury (TBI) is a structural and physiological disruption of brain function caused by external forces. It is a major cause of death and disability for patients worldwide. TBI includes both primary and secondary impairments. Iron overload and ferroptosis highly involved in the pathophysiological process of secondary brain injury. Ferroptosis is a form of regulatory cell death, as increased iron accumulation in the brain leads to lipid peroxidation, reactive oxygen species (ROS) production, mitochondrial dysfunction and neuroinflammatory responses, resulting in cellular and neuronal damage. For this reason, eliminating factors like iron deposition and inhibiting lipid peroxidation may be a promising therapy. Iron chelators can be used to eliminate excess iron and to alleviate some of the clinical manifestations of TBI. In this review we will focus on the mechanisms of iron and ferroptosis involving the manifestations of TBI, broaden our understanding of the use of iron chelators for TBI. Through this review, we were able to better find novel clinical therapeutic directions for further TBI study.
Acute central nervous system (CNS) injuries, such as stroke, traumatic brain injury (TBI), and spinal cord injury (SCI) present a grave health care challenge worldwide due to high morbidity and mortality, as well as limited clinical therapeutic strategies. Established literature has shown that oxidative stress (OS), inflammation, excitotoxicity, and apoptosis play important roles in the pathophysiological processes of acute CNS injuries. Recently, there have been many studies on the topic of ferroptosis, a form of regulated cell death characterized by the accumulation of iron-dependent lipid peroxidation. Some studies have revealed an emerging connection between acute CNS injuries and ferroptosis. Ferroptosis, induced by the abnormal metabolism of lipids, glutathione (GSH), and iron, can accelerate acute CNS injuries. However, pharmaceutical agents, such as iron chelators, ferrostatin-1 (Fer-1), and liproxstatin-1 (Lip-1), can inhibit ferroptosis and may have neuroprotective effects after acute CNS injuries. However, the specific mechanisms underlying this connection has not yet been clearly elucidated. In this paper, we discuss the general mechanisms of ferroptosis and its role in stroke, TBI, and SCI. We also summarize ferroptosis-related drugs and highlight the potential therapeutic strategies in treating various acute CNS injuries. Additionally, this paper suggests a testable hypothesis that ferroptosis may be a novel direction for further research of acute CNS injuries by providing corresponding evidence.
Melatonin (Mel) has neuroprotective effects; however, its roles in hypoxic-ischemic brain damage (HIBD) and the underlying mechanisms remain unknown. We aimed to explore its roles and mechanisms in a HIBD rat model. We found that exogenous Mel treatment ameliorated HIBD-induced pathological changes, inhibited neuronal ferroptosis, and promoted hippocampal neuronal survival. Moreover, Mel improved the learning and memory abilities of the HIBD rats. Further, we found that glutathione peroxidase 4 (Gpx4) inhibition with RSL3, Akt inhibition with LY29400, and nuclear factor erythroid-2-related factor 2 (Nrf2) inhibition with ML385 abolished the Mel protective effects in HIBD. Our findings indicate that exogenous Mel treatment has a protective effect on HIBD via the Akt/Nrf2/Gpx4 pathway.
Ferroptosis is a novel regulated cell death pattern discovered when studying the mechanism of erastin-killing RAS mutant tumor cells in 2012. It is an iron-dependent programmed cell death pathway mainly caused by an increased redox imbalance but with distinct biological and morphology characteristics when compared to other known cell death patterns. Ferroptosis is associated with various diseases including acute kidney injury, cancer, and cardiovascular, neurodegenerative, and hepatic diseases. Moreover, activation or inhibition of ferroptosis using a variety of ferroptosis initiators and inhibitors can modulate disease progression in animal models. In this review, we provide a comprehensive analysis of the characteristics of ferroptosis, its initiators and inhibitors, and the potential role of its main metabolic pathways in the treatment and prevention of various diseased states. We end the review with the current knowledge gaps in this area to provide direction for future research on ferroptosis.
Iron dyshomeostasis is implicated in Alzheimer’s disease (AD) alongside β-amyloid and tau pathologies. Despite the recent discovery of ferroptosis, an iron-dependent form cell death, hitherto, in vivo evidence of ferroptosis in AD is lacking. The present study uniquely adopts an integrated multi-disciplinary approach, combining protein (Western blot) and elemental analysis (total reflection X-ray fluorescence) with metabolomics (¹H nuclear magnetic resonance spectroscopy) to identify iron dyshomeostasis and ferroptosis, and possible novel interactions with metabolic dysfunction in age-matched male cognitively normal (CN) and AD post-mortem brain tissue (n = 7/group). Statistical analysis was used to compute differences between CN and AD, and to examine associations between proteins, elements and/or metabolites. Iron dyshomeostasis with elevated levels of ferritin, in the absence of increased elemental iron, was observed in AD. Moreover, AD was characterised by enhanced expression of the light-chain subunit of the cystine/glutamate transporter (xCT) and lipid peroxidation, reminiscent of ferroptosis, alongside an augmented excitatory glutamate to inhibitory GABA ratio. Protein, element and metabolite associations also greatly differed between CN and AD suggesting widespread metabolic dysregulation in AD. We demonstrate iron dyshomeostasis, upregulated xCT (impaired glutathione metabolism) and lipid peroxidation in AD, suggesting anti-ferroptotic therapies may be efficacious in AD.
5-lipoxygenase (5-LOX) is a non-heme iron-containing dioxygenase expressed in immune cells that catalyzes the two initial steps in the biosynthesis of leukotrienes. It is well known that 5-LOX activation in innate immunity cells is related to different iron-associated pro-inflammatory disorders, including cancer, neurodegenerative diseases, and atherosclerosis. However, the molecular and cellular mechanism(s) underlying the interplay between iron and 5-LOX activation are largely unexplored. In this study, we investigated whether iron (in the form of Fe³⁺ and hemin) might modulate 5-LOX influencing its membrane binding, subcellular distribution, and functional activity. We proved by fluorescence resonance energy transfer approach that metal removal from the recombinant human 5-LOX, not only altered the catalytic activity of the enzyme, but also impaired its membrane-binding. To ascertain whether iron can modulate the subcellular distribution of 5-LOX in immune cells, we exposed THP-1 macrophages and human primary macrophages to exogenous iron. Cells exposed to increasing amounts of Fe³⁺ showed a redistribution (ranging from ~45 to 75%) of the cytosolic 5-LOX to the nuclear fraction. Accordingly, confocal microscopy revealed that acute exposure to extracellular Fe³⁺, as well as hemin, caused an overt increase in the nuclear fluorescence of 5-LOX, accompanied by a co-localization with the 5-LOX activating protein (FLAP) both in THP-1 macrophages and human macrophages. The functional relevance of iron overloading was demonstrated by a marked induction of the expression of interleukin-6 in iron-treated macrophages. Importantly, pre-treatment of cells with the iron-chelating agent deferoxamine completely abolished the hemin-dependent translocation of 5-LOX to the nuclear fraction, and significantly reverted its effect on interleukin-6 overexpression. These results suggest that exogenous iron modulates the biological activity of 5-LOX in macrophages by increasing its ability to bind to nuclear membranes, further supporting a role for iron in inflammation-based diseases where its homeostasis is altered and suggesting further evidence of risks related to iron overload.
Ischemic stroke is one of the leading causes of death and long-term disability worldwide; however, effective clinical approaches are still limited. The transcriptional factor Nrf2 is a master regulator in cellular and organismal defense against endogenous and exogenous stressors by coordinating basal and stress-inducible activation of multiple cytoprotective genes. The Nrf2 network not only tightly controls redox homeostasis but also regulates multiple intermediary metabolic processes. Therefore, targeting Nrf2 has emerged as an attractive therapeutic strategy for the prevention and treatment of CNS diseases including stroke. Here, the current understanding of the Nrf2 regulatory network is critically examined to present evidence for the contribution of Nrf2 pathway in rodent ischemic stroke models. This review outlines the literature for Nrf2 studies in preclinical stroke and focuses on the in vivo evidence for the role of Nrf2 in primary and secondary brain injuries. The dynamic change and functional importance of Nrf2 signaling, as well as Nrf2 targeted intervention, are revealed in permanent, transient, and global cerebral ischemia models. In addition, key considerations, pitfalls, and future potentials for Nrf2 studies in preclinical stroke investigation are discussed.
Ferroptosis is a recently identified form of regulated cell death defined by the iron-dependent accumulation of lipid reactive oxygen species. Ferroptosis has been studied in various diseases such as cancer, Parkinson’s disease, and stroke. However, the exact function and mechanism of ferroptosis in ischemia/reperfusion (I/R) injury, especially in the intestine, remains unknown. Considering the unique conditions required for ferroptosis, we hypothesize that ischemia promotes ferroptosis immediately after intestinal reperfusion. In contrast to conventional strategies employed in I/R studies, we focused on the ischemic phase. Here we verified ferroptosis by assessing proferroptotic changes after ischemia along with protein and lipid peroxidation levels during reperfusion. The inhibition of ferroptosis by liproxstatin-1 ameliorated I/R-induced intestinal injury. Acyl-CoA synthetase long-chain family member 4 (ACSL4), which is a key enzyme that regulates lipid composition, has been shown to contribute to the execution of ferroptosis, but its role in I/R needs clarification. In the present study, we used rosiglitazone (ROSI) and siRNA to inhibit ischemia/hypoxia-induced ACSL4 in vivo and in vitro. The results demonstrated that ACSL4 inhibition before reperfusion protected against ferroptosis and cell death. Further investigation revealed that special protein 1 (Sp1) was a crucial transcription factor that increased ACSL4 transcription by binding to the ACSL4 promoter region. Collectively, this study demonstrates that ferroptosis is closely associated with intestinal I/R injury, and that ACSL4 has a critical role in this lethal process. Sp1 is an important factor in promoting ACSL4 expression. These results suggest a unique and effective mechanistic approach for intestinal I/R injury prevention and treatment.
Objectives:
Traumatic brain injury triggers multiple cell death pathways, possibly including ferroptosis-a recently described cell death pathway that results from accumulation of 15-lipoxygenase-mediated lipid oxidation products, specifically oxidized phosphatidylethanolamine containing arachidonic or adrenic acid. This study aimed to investigate whether ferroptosis contributed to the pathogenesis of in vitro and in vivo traumatic brain injury, and whether inhibition of 15-lipoxygenase provided neuroprotection.
Design:
Cell culture study and randomized controlled animal study.
Setting:
University research laboratory.
Subjects:
HT22 neuronal cell line and adult male C57BL/6 mice.
Interventions:
HT22 cells were subjected to pharmacologic induction of ferroptosis or mechanical stretch injury with and without administration of inhibitors of ferroptosis. Mice were subjected to sham or controlled cortical impact injury. Injured mice were randomized to receive vehicle or baicalein (12/15-lipoxygenase inhibitor) at 10-15 minutes postinjury.
Measurements and main results:
Pharmacologic inducers of ferroptosis and mechanical stretch injury resulted in cell death that was rescued by prototypical antiferroptotic agents including baicalein. Liquid chromatography tandem-mass spectrometry revealed the abundance of arachidonic/adrenic-phosphatidylethanolamine compared with other arachidonic/adrenic acid-containing phospholipids in the brain. Controlled cortical impact resulted in accumulation of oxidized phosphatidylethanolamine, increased expression of 15-lipoxygenase and acyl-CoA synthetase long-chain family member 4 (enzyme that generates substrate for the esterification of arachidonic/adrenic acid into phosphatidylethanolamine), and depletion of glutathione in the ipsilateral cortex. Postinjury administration of baicalein attenuated oxidation of arachidonic/adrenic acid-containing-phosphatidylethanolamine, decreased the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling positive cells in the hippocampus, and improved spatial memory acquisition versus vehicle.
Conclusions:
Biomarkers of ferroptotic death were increased after traumatic brain injury. Baicalein decreased ferroptotic phosphatidylethanolamine oxidation and improved outcome after controlled cortical impact, suggesting that 15-lipoxygenase pathway might be a valuable therapeutic target after traumatic brain injury.
Stroke is ranked as the second leading cause of death worldwide with an annual mortality rate of about 5.5 million. Not only does the burden of stroke lie in the high mortality but the high morbidity also results in up to 50% of survivors being chronically disabled. Thus stroke is a disease of immense public health importance with serious economic and social consequences. The public health burden of stroke is set to rise over future decades because of demographic transitions of populations, particularly in developing countries. This paper provides an overview of stroke in the 21st century from a public health perspective.
Aims
Ferroptosis, a new form of iron‐dependent programmed cell death, has been shown to be involved in a range of diseases. However, the role of ferroptosis in traumatic brain injury (TBI) has yet to be elucidated. We aimed to investigate whether ferroptosis is induced after TBI and whether the inhibition of ferroptosis would protect against traumatic brain injury in a controlled cortical impact injury (CCI) mouse model.
Methods
After establishing the TBI model in mice, we determined the biochemical and morphological changes associated with ferroptosis, including iron accumulation with Perl's staining, neuronal cell death with Fluoro‐Jade B (FJB) staining, iron metabolism dysfunction with Western blotting, reactive oxygen species (ROS) accumulation with malondialdehyde (MDA) assays, and shrunken mitochondria with transmission electron microscopy. Furthermore, a specific inhibitor of ferroptosis, ferrostatin‐1(fer‐1), was administrated by cerebral ventricular injection after CCI. We used cresyl violet (CV) staining to assess lesion volume, along with the Morris water maze and beam walk test to evaluate long‐term outcomes.
Results
TBI was followed by iron accumulation, dysfunctional iron metabolism, the upregulation of ferroptosis‐related genes, reduced glutathione peroxidase (GPx) activity, and the accumulation of lipid‐reactive oxygen species (ROS). Three days (d) after TBI, transmission electron microscopy (TEM) confirmed that the mitochondria had shrunk a typical characteristic of ferroptosis. Importantly, the administration of Fer‐1 by cerebral ventricular injection significantly reduced iron deposition and neuronal degeneration while attenuating injury lesions and improving long‐term motor and cognitive function.
Conclusion
This study demonstrated an effective method with which to treat TBI by targeting ferroptosis.
Cancer cells often upregulate nutrient transporters to fulfill their increased biosynthetic and bioenergetic needs, and to maintain redox homeostasis. One nutrient transporter frequently overexpressed in human cancers is the cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11; also known as xCT). SLC7A11 promotes cystine uptake and glutathione biosynthesis, resulting in protection from oxidative stress and ferroptotic cell death. Recent studies have unexpectedly revealed that SLC7A11 also plays critical roles in glutamine metabolism and regulates the glucose and glutamine dependency of cancer cells. This review discusses the roles of SLC7A11 in regulating the antioxidant response and nutrient dependency of cancer cells, explores our current understanding of SLC7A11 regulation in cancer metabolism, and highlights key open questions for future studies in this emerging research area. A deeper understanding of SLC7A11 in cancer metabolism may identify new therapeutic opportunities to target this important amino acid transporter for cancer treatment.
Ferroptosis is a form of regulated cell death characterized by the accumulation of lipid hydroperoxides. There has been significant research on the pathways leading to the accumulation of oxidized lipids, but the downstream effects and how lipid peroxides cause cell death during ferroptosis remain a major puzzle. We evaluated key features of ferroptosis in newly developed molecular dynamics models of lipid membranes to investigate the biophysical consequences of lipid peroxidation, and generated hypotheses about how lipid peroxides contribute to cell death during ferroptosis.
Lipoxygenases (LOXs) have been implicated as central players in ferroptosis, a recently characterized cell death modality associated with the accumulation of lipid hydroperoxides: the products of LOX catalysis. To provide insight on their role, human embryonic kidney cells were transfected to overexpress each of the human isoforms associated with disease, 5-LOX, p12-LOX, and 15-LOX-1, which yielded stable cell lines that were demonstrably sensitized to ferroptosis. Interestingly, the cells could be rescued by less than half of a diverse collection of known LOX inhibitors. Furthermore, the cytoprotective compounds were similarly potent in each of the cell lines even though some were clearly isoform-selective LOX inhibitors. The cytoprotective compounds were subsequently demonstrated to be effective radical-trapping antioxidants, which protect lipids from autoxidation, the autocatalytic radical chain reaction that produces lipid hydroperoxides. From these data (and others reported herein), a picture emerges wherein LOX activity may contribute to the cellular pool of lipid hydroperoxides that initiate ferroptosis, but lipid autoxidation drives the cell death process.
Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.
Although traumatic brain injury (TBI) is a common cause of death and disability worldwide, there is currently a lack of effective therapeutic drugs and targets. To reveal the complex pathophysiologic mechanisms of TBI, we performed transcriptome analysis of the mouse cerebral cortex and immunohistochemical analysis of human cerebral tissues. The genes Mt1, Mt2, Il33, and Fth1 were upregulated post-TBI and enriched in pathways associated with the inflammatory response, oxidative phosphorylation, and ferroptosis. As an agonist of MT1/2, melatonin (MLT) confers anti-oxidant, anti-inflammatory, and anti-ferroptosis effects after TBI. However, whether these upregulated genes and their corresponding pathways are involved in the neuroprotective effect of MLT remains unclear. In this study, interventions to inhibit MT1/2, IL-33, and ferroptosis (i.e., ferritin H (Fth)-KO) were applied post-TBI. The results showed that MLT attenuated TBI-induced cerebral edema and neurological outcomes by inhibiting inflammation and ferroptosis. Mechanistically, MLT mainly suppressed inflammatory responses and ferroptosis via the activation of MT2 and IL-33 pathways. Building on the previous finding that Fth deletion increases susceptibility to ferroptosis post-TBI, we demonstrated that Fth depletion remarkably exacerbated the post-TBI inflammatory response, and abolished the anti-inflammatory effects of MLT both in vivo and in vitro. Furthermore, the post-TBI anti-inflammatory effect of MLT, which occurs by promoting the polarization of CD206+ macrophages, was dependent on Fth. Taken together, these results clarified that MLT alleviates inflammation- and ferroptosis-mediated brain edema and neurological deficits by activating the MT2/IL-33/Fth pathway, which provides a novel target and theoretical basis for MLT to treat TBI patients.
Accumulating evidence have indicated that ferroptosis plays a crucial role in cerebral ischemia-reperfusion (I/R) injury which is the most serious treatment complication of ischemic stroke. Baicalein (5,6,7-trihydroxyflavone) is a main bioactive ingredient isolated from a traditional Chinese medicine named Baikal Skullcap, which is the root of Scutellaria baicalensis Georgi. This study investigated the potential role of baicalein in cerebral I/R injury using oxygen-glucose deprivation and reoxygenation (OGD/R) HT22 cells, transient middle cerebral artery occlusion (tMCAO) mice and RSL3-sitmulated HT22 cells. Baicalein improved the viability of OGD/R cells and significantly ameliorated cerebral I/R injury in tMCAO mice. Baicalein decreased the iron levels, lipid peroxidation production and morphology features of ferroptosis of the brain tissues in tMCAO mice, which indicated that baicalein ameliorated cerebral I/R injury by inhibiting ferroptosis in vivo and in vitro. We further confirmed that baicalein had the activity of inhibiting ferroptosis in RSL3-stimulated HT22 cells. Western blot revealed that baicalein inhibited the ferroptosis by regulating on the expression levels of GPX4, ACSL4 and ACSL3 in OGD/R cells, tMCAO mice and RSL3-stimulated HT22 cells. Our findings demonstrated that baicalein reversed the cerebral I/R injury via anti-ferroptosis, which was regulated by GPX4/ACSL4/ACSL3 axis. The results suggested that baicalein has therapeutic potential as a drug for cerebral I/R injury.
Ethnopharmacological relevance
Rehmannioside A is derived from Rehmannia glutinosa Libosch, which is widely used as an important ingredient in diverse traditional Chinese medicines to treat diseases caused by “kidney deficiency” such as cerebral arteriosclerosis, aging-related stroke and dementia in China. Recent studies have proved that Rehmannia glutinosa Libosch and Rehmannioside A can improve memory capability and recover nerve damage.
Aim of the study
To investigate the effect of Rehmannioside A on cognitive impairment after ischemia in rats and SH-SY5Y cells, and further evaluate the anti-oxidative and anti-ferroptosis mechanisms.
Materials and methods
Differentially expressed proteins (DEPs) in patients after cerebral ischemic stroke were revealed by a RayBio protein array. Cognitive impairment model was established by middle cerebral artery occlusion and reperfusion (MCAO) 14 days in rats. Rehmannioside A was administered intraperitoneally injection at dose of 80 mg/kg. The SH-SY5Y cells were exposed to H2O2 for 24 h and treated with Rehmannioside A (80 μM) for 24 h. The neuroprotecion of Rehmannioside A were evaluated by infarct volume (TTC), neurological defects (Garcia score) and learning memory (Morris water maze test) in vivo, and cell viability (CCK-8 or LDH) in vitro. Superoxide dismutase (SOD), malondialdehyde (MDA) and myeloperoxidase (MPO) activity of rats, glutathione (GSH), oxidized glutathione (GSSG) and nicotinamide adenine dinucleotide phosphate (NADPH) of cells were detected by biochemical assay. Intracellular reactive oxygen species (ROS) were measured by DCFH-DA assay. Myeloperoxidase (MPO), PI3 kinase (PI3K), p-PI3K, Akt, p-Akt, heme oxygenase-1 (HO-1), nuclear factor-E2-related factor 2 (Nrf2), SLC7A11, glutathione peroxidase 4 (GPX4) of the cerebral cortex in rats or SH-SY5Y cells were examined by western blotting.
Results
Compared with model group, the cognitive impairment and neurological deficits of Rehmannioside A group were significantly improved, and the cerebral infarction was reduced in MCAO rats. Moreover, the cell viability obviously increased and the H2O2-induced toxicity was reduced in Rehmannioside A group. Further research indicated that the expression of p-PI3K, p-Akt, nuclear Nrf2, HO-1 and SLC7A11 in Rehmannioside A group was significantly higher than model group.
Conclusions
Rehmannioside A has neuroprotection effect and improves cognitive impairment after cerebral ischemia by inhibiting ferroptosis and activating PI3K/AKT/Nrf2 and SLC7A11/GPX4 signaling pathway. These findings provide valuable insight into the pathogenesis and therapeutic target of ischemic stroke.
Ferroptosis, an iron-dependent form of non-apoptotic cell death, is reportedly responsible for cerebral ischemia/reperfusion (I/R) injury. Evidence has shown that spermidine/spermine N1-acetyltransferase 1 (SSAT1) activation-induced ferroptosis is associated with upregulation of arachidonate 15-Lipoxygenase (ALOX15). Our previous study has revealed that upregulation of ALOX15 contributes to cerebral I/R injury via inducing microglial activation. The current study aimed to investigate the role of SSAT1/ALOX15 axis in neuronal ferroptosis after I/R. We found that the expression of SSAT1 was upregulated in the cortical penumbra of mice subjected to transient middle cerebral artery occlusion and reperfusion (tMCAO/R). Knockdown of SSAT1 mitigated I/R-induced cerebral infarction and neurological impairments, as well as decreased cortical iron contents, reactive oxygen species (ROS) generation and 4-Hydroxynonenal (4-HNE) level. Further in vitro evidence revealed that knockdown of SSAT1 downregulated the expression of ALOX15 in the primary cortical neurons exposed to tertbutyl-hydroksyperoxide (TBH). In addition, loss of neuronal viability and production of lipid hydroperoxides were inhibited in TBH-treated neurons when SSAT1 was knocked down. Mechanistically, SSAT1 overexpression decreased the expression levels of two key ferroptotic repressors, glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) in TBH-stimulated neurons. Treatment with the ALOX15 inhibitor PD146176 or ferroptosis inhibitor ferrostatin-1 partially reversed SSAT1 upregulation-induced ferroptosis and viability loss in TBH-treated neurons. These results together indicate that the activation of SSAT1/ALOX15 axis may aggravate cerebral I/R injury via triggering neuronal ferroptosis, providing novel insights into cerebral injury associated with lipid peroxidation.
The emergence of ferroptosis as a cell death pathway associated with brain disorders including stroke and neurodegenerative diseases emphasizes the need to develop therapeutics able to target the brain and to protect neurons from ferroptotic death. Selenium plays an essential role in reducing lipid peroxidation generated during ferroptosis through its incorporation into the catalytic site of glutathione peroxidase 4. Here, we compared the anti-ferroptotic activity of several organic and inorganic selenium compounds: methylselenocysteine, selenocystine, selenomethionine, selenocystamine, ebselen, sodium selenite, and sodium selenate. All were effective against erastin- and RSL3-induced ferroptosis in vitro. We characterized the ability of the selenium compounds to release selenium and boost glutathione peroxidase expression and activity. Based on our results, we selected organic selenium compounds of similar characteristics and investigated their effectiveness in protecting against neuronal death in vivo using the cerebral ischemia–reperfusion injury mouse model. We found that pretreatment with methylselenocysteine or selenocystamine provided protection from ischemia–reperfusion neuronal damage in vivo. These data support the use of ferroptosis inhibitors for treatment and select selenium compounds for prevention of neuronal damage in ischemic stroke and other diseases of the brain where ferroptosis is implicated.
Inflammation and cell death play important roles in the pathogenesis of hypoxic-ischemic brain damage (HIBD). Toll-like receptor 4 (TLR4) triggers the activation of the inflammatory pathway. Ferroptosis, a newly identified type of regulated cell death, is implicated in various diseases involving neuronal injury. However, the role of ferroptosis in HIBD has not been elucidated. The objectives of this study were to explore the function and mechanism of TLR4 in neuronal ferroptosis in the context of HIBD. A neonatal rat model of hypoxia-ischemia (HI) and a cell model of oxygen-glucose deprivation (OGD) were employed. TAK-242, a TLR4-specific antagonist, was used to evaluate the effect of TLR4 on neuronal ferroptosis in vivo. A TAK-242 inhibitor and a p38 inhibitor (SB203580) were administered to HT22 hippocampal neurons to explore the association between TLR4 in inflammation and ferroptosis in vitro. The effects of TLR4 on ferroptosis were assessed by the Western blot, real-time PCR, immunofluorescence staining, cell viability and transmission electron microscopy (TEM) assays. HI insult significantly upregulated the TLR4, increased the p53 level, reduced the SLC7A11 and GPX4 levels, and caused mitochondrial damage, thereby inducing neuronal ferroptosis in the hippocampus. Inhibition of TLR4 inhibited the expression of ferroptosis-related proteins, decreased the expression of ferroptosis-related genes and the proinflammatory milieu, attenuated oxidative stress and mitochondrial injury and, finally, ameliorated the activation of hippocampal neuronal ferroptosis following HIBD. Consistent with the results of these in vivo experiments, TLR4 inhibition also attenuated OGD-induced ferroptosis by suppressing oxidative stress and p38MAPK signaling, ultimately increasing neuronal cell viability. Finally, the in vitro and in vivo results demonstrated that TAK-242 exerted neuroprotective and antiferroptotic effects by suppressing TLR4-p38 MAPK signaling. TLR4 activation induced neuronal ferroptosis following both HIBD and OGD. Inhibition of TLR4 attenuated oxidative stress-induced damage, decreased the activation of ferroptosis, and attenuated neuroinflammation following HIBD. In this study, we demonstrated that the inhibition of TLR4-p38 MAPK signaling modulates HIBD- or OGD-induced ferroptosis in neuronal cells and may play a novel role in brain homeostasis.
Rupture of the plasma membrane in different forms of cell death was long thought to be a passive process. The finding that it is an active one, mediated by a specific membrane protein, reveals an unexpected feature shared by dying cells. NINJ1 protein mediates rupture of the plasma membrane in dying cells.
s
Acyl-CoA synthetase long-chain family member 4 (ACSL4) is an important isozyme for polyunsaturated fatty acids (PUFAs) metabolism that dictates ferroptosis sensitivity. The role of ACSL4 in the progression of ischemic stroke is unclear. Here, we found that ACSL4 expression was suppressed in the early phase of ischemic stroke and this suppression was induced by HIF-1α. Knockdown of ACSL4 protected mice against brain ischemia, whereas, forced overexpression of ACSL4 exacerbated ischemic brain injury. ACSL4 promoted neuronal death via enhancing lipid peroxidation, a marker of ferroptosis. Moreover, knockdown of ACSL4 inhibited proinflammatory cytokine production in microglia. These data identify ACSL4 as a novel regulator of neuronal death and neuroinflammation, and interventions of ACSL4 expression may provide a potential therapeutic target in ischemic stroke.
Cerebral ischemia is the most common cause of hippocampal neuronal death and the most prevalent cause of stroke with high mortality rate. Ferroptosis has been suggested to affect the role of hippocampal neurons. This study explores the influence of lentivirus infection-induced ferritin overexpression in hippocampal neuronal injury and death through simulations in August Copenhagen Irish rat models. Twenty-four-hour cerebral ischemia–reperfusion injury was induced in the rats after 90-min middle cerebral artery occlusion (MCAO). Ferritin overexpression was induced through lentivirus infection. The Morris Water Maze (MWM) test and tau hyperphosphorylation test were performed on hippocampal neurons to establish a MCAO model. The effect of ferritin overexpression on hippocampal neuronal death was evaluated using hematoxylin–eosin staining and annexin V/propidium iodide flow cytometry. The MWM test revealed that MCAO modeling decreased the cognitive and locomotor capacity of the rats, whereas ferritin overexpression partially reversed the effect of MCAO. In addition, the hyperphosphorylation of tau caused by MCAO was reduced by ferritin. Pathogenic changes, impaired viability, increased apoptosis, and elevated caspase-9 cleavage in hippocampal neurons were clearly recovered by ferritin. Moreover, robust reactive oxygen species production and glutathione consumption, which was induced by MCAO modeling, were ameliorated by ferritin. Furthermore, two key modulators of ferroptosis, p53 and SLC7A11, were demonstrated to be upregulated by MCAO modeling and downregulated by ferritin. Ferritin reduction is essential for cerebral ischemia-induced hippocampal neuronal ferroptosis mediated via p53 and SLC7A11.
Accumulating evidence demonstrates that ferroptosis may be important in the pathophysiological process of traumatic brain injury (TBI). As a major hormone of the pineal gland, melatonin exerts many beneficial effects on TBI, but there is no information regarding the effects of melatonin on ferroptosis after TBI. As expected, TBI resulted in the time‐course changes of ferroptosis‐related molecules expression and iron accumulation in the ipsilateral cortex. Importantly, we found that treating with melatonin potently rescued TBI induced the changes mentioned above and improved functional deficits versus vehicle. Similar results were obtained with a ferroptosis inhibitor, liproxstatin‐1. Moreover, the protective effect of melatonin is likely dependent on melatonin receptor 1B (MT2). Although ferritin plays a vital role in iron metabolism by storing excess cellular iron, its precise function in the brain, and whether it involves melatonin's neuroprotection remain unexplored. Considering ferritin H (Fth) is expressed predominantly in the neurons and global loss of Fth in mice induces early embryonic lethality, we then generated neuron‐specific Fth conditional knockout (Fth‐KO) mice, which are viable and fertile but have altered iron metabolism. In addition, Fth‐KO mice were more susceptible to ferroptosis after TBI, and the neuroprotection by melatonin was largely abolished in Fth‐KO mice. In vitro siFth experiments further confirmed the results mentioned above. Taken together, these data indicate that melatonin produces cerebroprotection, at least partly by inhibiting neuronal Fth‐mediated ferroptosis following TBI, supporting the notion that melatonin is an excellent ferroptosis inhibitor and its anti‐ferroptosis provides a potential therapeutic target for treating TBI.
Aims
To evaluate the impact of galangin treatment on cerebral ischemia-reperfusion (I/R) injury in gerbils and to identify potential mechanisms of the protective effect of galangin on hippocampal neurons after I/R injury.
Principal methods
A cerebral ischemia model using bilateral common carotid artery ligation in gerbils was established. The Morris water maze (MWM) test was used to evaluate the learning and memory ability of gerbils. The cell viability was evaluated with an MTT assay. The levels of lipid peroxide biomarkers were measured to estimate the injury due to lipid peroxide. The morphology was detected by electron micrography, immunofluorescence and Nissl staining. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to measure the molecular characteristics.
Key findings
In the MWM, gerbils treated with galangin after I/R injury showed significant improvements in learning and memory. In addition, galangin treatment reduced the levels of lipid peroxide in the brains of gerbils that underwent I/R as well as reduced the amount of cell death and increased the expression of SLC7A11 and glutathione peroxidase 4 (GPX4). Furthermore, the expression of the marker of ferroptosis was decreased in galangin-treated gerbils, and the effect of galangin was weakened when SLC7A11 was knocked down. These results show that galangin can inhibit ferroptosis by enhancing the expressions of SLC7A11 and GPX4 as well as reduce neuronal cell death.
Significance
Galangin inhibits ferroptosis through activation of the SLC7A11/GPX4 axis and has a protective effect on hippocampal neurons in gerbils after I/R.
Background:
Abnormally hyperphosphorylated tau is a defining pathological feature of tauopathies, such as Alzheimer's disease (AD), and accumulating evidence suggests a role for iron in mediating tau pathology that may lead to cognitive decline in these conditions. The metal chelator deferiprone (DFP), which has a high affinity for iron, is currently in clinical trials for AD and Parkinson's disease. However, the effect of DFP on tau pathology remains underexplored.
Objective:
We aimed to investigate the impact of chronic DFP treatment on tau pathology using a well-characterized mouse model of tauopathy (rTg(tauP301L)4510).
Methods:
Animals were treated daily with DFP (100 mg/kg) via oral gavage for 16 weeks. After 14 weeks, mice were tested in the Y-maze, open field, Morris water maze, and rotorod. At the end of the study, brain tissue was collected to examine metal levels (using inductively coupled plasma-mass spectrometry) and for western blot analysis of DFP on tau and iron associated pathways.
Results:
DFP significantly reduced anxiety-like behavior, and revealed a trend toward improved cognitive function. This was accompanied by a decrease in brain iron levels and sarkosyl-insoluble tau. Our data also showed downregulation of the tau kinases glycogen synthase kinase 3β and cyclin dependent kinase-5 in DFP treated mice and an increase in the methylation of the catalytic subunit of protein phosphatase 2A.
Conclusion:
These data support the hypothesis that suggests that iron plays a neurotoxic role in tauopathies and may be a potential therapeutic target for this class of disorders.
Inflammation and oxidative stress play a key role in mediating the pathophysiology of hypoxic‐ischemic (HI) brain injury. Nrf2 is a transcriptional factor that contributes to the innate defense of the body against oxidative stress and inflammation. The current study investigated the effect of Nrf2 in neonatal HI brain injury using Nrf2‐/‐ mice. Nrf2‐/‐ and wild‐type Nrf2+/+ mice on a C57BL/6J background at postnatal day 9 underwent unilateral common carotid artery ligation, followed by hypoxia. Brain damage was determined by infarct size measurement. Apoptosis was evaluated by measuring the expression of Bax and Bcl‐2. The levels of inflammatory cytokines and mediators involved in oxidative stress were measured. Nrf2 knockout exacerbated HI injury‐induced brain infarct and cell apoptosis in the brain. Nrf2‐/‐ mice showed increased inflammatory cytokines and MDA, and reduced activities of antioxidant enzymes including CAT, GSH‐Px and SOD. Nrf2‐/‐ mice showed reduced HO‐1 expression after HI injury compared with wildtype mice. This study supported a protective effect of Nrf2 in neonatal HI brain injury.
Over the past five decades, thanatology has come to include the study of how individual cells in our bodies die appropriately and inappropriately in response to physiological and pathological stimuli. Morphological and biochemical criteria have been painstakingly established to create clarity around definitions of distinct types of cell death and mechanisms for their activation. Among these, ferroptosis has emerged as a unique, oxidative stress-induced cell death pathway with implications for diseases as diverse as traumatic brain injury, hemorrhagic stroke, Alzheimer's disease, cancer, renal ischemia, and heat stress in plants. In this review, I highlight some of the formative studies that fostered its recognition in the nervous system and describe how chemical biological tools have been essential in defining events necessary for its execution. Finally, I discuss emerging opportunities for antiferroptotic agents as therapeutic agents in neurological diseases.
Objective:
Our previous research showed that Naotaifang (a compound traditional Chinese herbal medicine) extract (NTE) has clinically beneficial effects on neurological improvement of patients with acute cerebral ischemia. In this study, we investigated whether NTE protected acute brain injury in rats and whether its effects on ferroptosis could be linked to the dysfunction of glutathione peroxidase 4 (GPX4) and iron metabolism.
Methods:
We established an acute brain injury model of middle cerebral artery occlusion (MCAO) in rats, in which we could observe the accumulation of iron in neurons, as detected by Perl's staining. Using assay kits, we measured expression levels of ferroptosis biomarkers, such as iron, glutathione (GSH), reactive oxygen species (ROS) and malonaldehyde (MDA); further the expression levels of transferrin receptor 1 (TFR1), divalent metal transporter 1 (DMT1), solute carrier family 7 member 11 (SLC7A11) and GPX4 were determined using immunohistochemical analysis, real-time quantitative polymerase chain reaction and Western blot assays.
Results:
We found that treatment with NTE reduced the expression levels of TFR1 and DMT1, reduced ROS, MDA and iron accumulation and reduced neurobehavioral scores, relative to untreated MCAO rats. Treatment with NTE increased the expression levels of SLC7A11, GPX4 and GSH, and the number of Nissl bodies in the MCAO rats.
Conclusion:
Taken together, our data suggest that acute cerebral ischemia induces neuronal ferroptosis and the effects of treating MCAO rats with NTE involved inhibition of ferroptosis through the TFR1/DMT1 and SCL7A11/GPX4 pathways.
Although confirmed as the primary lipophilic antioxidant molecule endogenously produced by cells, the non-mitochondrial pool of CoQ10's functional role is still well debated. Recently, both Bersuker et al. (2019) and Doll et al. (2019) have identified FSP1 as a novel CoQ10 plasma membrane oxidoreductase, protecting cells from glutathione-independent ferroptosis.
Lipid peroxidation underlies the mechanism of oxidative cell death now known as ferroptosis. This modality, distinct from other forms of cell death, has been intensely researched in recent years owing to its relevance in both degenerative disease and cancer. The demonstration that it can be modulated by small molecules in multiple pathophysiological contexts offers exciting opportunities for novel pharmacological interventions. Herein, we introduce the salient features of lipid peroxidation, how it can be modulated by small molecules and what principal aspects require urgent investigation by researchers in the field. The central role of non-enzymatic reactions in the execution of ferroptosis will be emphasized, as these processes have hitherto not been generally considered ‘druggable’. Moreover, we provide a critical perspective on the biochemical mechanisms that contribute to cell vulnerability to ferroptosis and discuss how they can be exploited in the design of novel therapeutics.
Blood brain barrier (BBB) permeability and oxidative stress have been reported to be important mechanisms for brain damage following ischemic stroke and stanniocalcin-1 (STC-1), a neuroprotective protein, has anti-inflammatory and anti-oxidative stress properties. Herein, we report the effect of STC-1 on BBB permeability and brain oxidative stress after stroke in an animal model. Male Wistar received an intracerebroventricularly injection of human recombinant STC-1 (100 ng/kg) or saline and were subjected to sham procedure or global cerebral ischemia/reperfusion (I/R) model. Six and 24 h after I/R, neurological evaluation was performed; at 24 h brain water content was evaluated in the total brain, and BBB permeability, nitrite/nitrate (N/N) concentration, lipid peroxidation, protein carbonyls formation, superoxide dismutase (SOD) and catalase (CAT) activity were determined in the hippocampus, cortex, prefrontal cortex, striatum and cerebellum. Rats exhibited neurological deficit at 6 and 24 h after I/R and STC-1 reduction at 24 h. After I/R there were an increase of brain water content, BBB permeability in the hippocampus, cortex and pre-frontal cortex and N/N in the hippocampus, and STC-1 decreased this level only in the hippocampus. STC-1 decreased lipid peroxidation in the hippocampus, cortex and prefrontal cortex and protein oxidative damage in the hippocampus and cortex. SOD activity decreased in the hippocampus, cortex and prefrontal cortex after I/R and STC-1 reestablished these levels in the hippocampus and cortex. CAT activity decreased only in the hippocampus and cortex and STC-1 increased the CAT activity in the hippocampus. Our data provide the first experimental demonstration that STC-1 reduced brain dysfunction associated with cerebral I/R in rats, by decreasing BBB permeability and oxidative stress parameters.
Objective:
Cerebral ischemia is the most common type of neuronal injury and is characterized by a reduction in the function and number of hippocampal neurons. Carvacrol has a significant neuroprotective effect in cerebral ischemia. However, the mechanisms by which carvacrol affects cerebral ischemia, especially with respect to the regulation of neuronal damage by iron levels, have never been systematically studied. This study aimed to reveal the mechanisms by which carvacrol protects against hippocampal neuron impairment after ischemic stroke in gerbils.
Materials and methods:
The Morris water maze test was performed to evaluate learning and memory impairments. Iron ion content and oxidative stress index were detected by the kit. MTT assay was performed to assess the cell viability. The morphology and molecular characteristics were detected by electron micrographs and western blot.
Results:
In the present study, we demonstrated the neuroprotective effects of carvacrol in vivo and in vitro. The Morris water maze test showed that the learning and memory abilities of the gerbils treated with carvacrol were significantly improved. Lipid peroxide injury was evaluated by measuring the levels of lipid peroxide biomarkers; the results indicated that carvacrol decreased the level of lipid peroxide in ischemic gerbil brain tissue. Histopathological examinations and western blotting were performed to evaluate injury in neurons, and carvacrol reduced cell death. Moreover, ferroptosis in the hippocampus was evaluated by measuring the levels of proteins involved in this iron-dependent form of regulated cell death. These results indicated that carvacrol reduced cell death and that carvacrol inhibited ferroptosis by increasing the expression of glutathione peroxidase 4(GPx4). This study showed that carvacrol may be a valuable drug for treating cerebral ischemia.
Conclusion:
Carvacrol provides protection for hippocampal neurons against I/R in gerbils by inhibiting ferroptosis through increasing the expression of GPx4.
Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke. An adaptive response to ferroptotic stress is uncovered and leveraged to develop a neuroprotectant that reduces cell death and improves function after hemorrhagic stroke in mice.
Damage-associated molecular pattern molecules (DAMPs) are endogenous danger signals that alert the innate immune system and shape the inflammation response to cell death. However, the release and activity of DAMPs in ferroptosis, a recently identified form of regulated necrosis characterized by iron overload and lipid peroxidation, still remain poorly understood. Here, we demonstrate that HMGB1 is a DAMP released by ferroptotic cells in an autophagy-dependent manner. Both type I and II ferroptosis activators, including erastin, sorafenib, RSL3, and FIN56, induce HMGB1 release in cancer and noncancer cells. In contrast, genetic ablation (using ATG5−/− or ATG7−/− cells) or pharmacologic inhibition (the administration of bafilomycin A1 or chloroquine) of autophagy was found to block ferroptosis activator-induced HMGB1 release. Mechanically, autophagy-mediated HDAC inhibition promotes HMGB1 acetylation, resulting in HMGB1 release in ferroptosis. Moreover, AGER, but not TLR4, is required for HMGB1-mediated inflammation in macrophages in response to ferroptotic cells. These studies suggest that HMGB1 inhibition might have some potential therapeutic effects in ferroptosis-associated human disease.
Emerging evidence has demonstrated that vitamin D plays an important role in many adult neurologic disorders, but is also critical in neuronal development and pruning in the neonatal and pediatric populations. Neonates are at a particularly high risk of vitamin D deficiency, in part due to the high prevalence of maternal deficiency during pregnancy. Several preclinical studies have demonstrated that infants born to vitamin D-deficient mothers are at a high risk of developing neonatal brain injury, and recent clinical studies have shown that neonates with hypoxic-ischemic encephalopathy (HIE) tend to be vitamin D-deficient. There are limited data, however, on whether additional prenatal or postnatal supplementation may alter the prevalence or severity of neonatal HIE. This review examines the current data supporting the neuroprotective role of vitamin D, with a focus on how these findings may be translated to neonates with HIE.