Ferroptosis: a new mechanism of traditional Chinese medicine for treating ulcerative colitis.

Ulcerative colitis (UC), a subtype of inflammatory bowel disease, manifests with symptoms such as abdominal pain, diarrhea, and mucopurulent, bloody stools. The pathogenesis of UC is not fully understood. At present, the incidence of UC has increased significantly around the world. Conventional therapeutic arsenals are relatively limited, with often poor efficacy and many adverse effects. In contrast, traditional Chinese medicine (TCM) holds promise due to their notable effectiveness, reduced recurrence rates, and minimal side effects. In recent years, significant progress has been made in the basic research on TCM for UC treatment. It has been found that the inhibition of ferroptosis through the intervention of TCM can significantly promote intestinal mucosal healing and reverse UC. The mechanism of action involves multiple targets and pathways. Aim of the review: This review summarizes the experimental studies on the targeted regulation of ferroptosis by TCM and its impact on UC in recent years, aiming to provide theoretical basis for the prevention, treatment, and further drug development for UC. Results: Ferroptosis disrupts antioxidant mechanisms in intestinal epithelial cells, damages the intestinal mucosa, and participates in the pathological process of UC. TCM acts on various pathways such as Nrf2/HO-1 and GSH/GPX4, blocking the pathological progression of ferroptosis in intestinal epithelial cells, inhibiting pathological damage to the intestinal mucosa, and thereby alleviating UC. Conclusion: The diverse array of TCM single herbs, extracts and herbal formulas facilitates selective and innovative research and development of new TCM methods for targeting UC treatment. Although progress has been made in studying TCM compound formulas, single herbs, and extracts, there are still many issues in clinical and basic experimental designs, necessitating further in-depth scientific exploration and research.

Brucella Manipulates Host Cell Ferroptosis to Facilitate Its Intracellular Replication and Egress in RAW264.7 Macrophages.

Brucella virulence relies on its successful intracellular life cycle. Modulating host cell death is a strategy for Brucella to survive and replicate intracellularly. Ferroptosis is a novel regulated cell death characterized by iron-triggered excessive lipid peroxidation, which has been proven to be associated with pathogenic bacteria infection. Thus, we attempted to explore if smooth-type Brucella infection triggers host cell ferroptosis and what role it plays in Brucella infection. We assessed the effects of Brucella infection on the lactate dehydrogenase release and lipid peroxidation levels of RAW264.7 macrophages; subsequently, we determined the effect of Brucella infection on the expressions of ferroptosis defense pathways. Furthermore, we determined the role of host cell ferroptosis in the intracellular replication and egress of Brucella. The results demonstrated that Brucella M5 could induce ferroptosis of macrophages by inhibiting the GPX4-GSH axis at the late stage of infection but mitigated ferroptosis by up-regulating the GCH1-BH4 axis at the early infection stage. Moreover, elevating host cell ferroptosis decreased Brucella intracellular survival and suppressing host cell ferroptosis increased Brucella intracellular replication and egress. Collectively, Brucella may manipulate host cell ferroptosis to facilitate its intracellular replication and egress, extending our knowledge about the underlying mechanism of how Brucella completes its intracellular life cycle.

Interferon-α stimulates DExH-box helicase 58 to prevent hepatocyte ferroptosis.

BACKGROUND: Liver ischemia/reperfusion (I/R) injury is usually caused by hepatic inflow occlusion during liver surgery, and is frequently observed during war wounds and trauma. Hepatocyte ferroptosis plays a critical role in liver I/R injury, however, it remains unclear whether this process is controlled or regulated by members of the DEAD/DExH-box helicase (DDX/DHX) family. METHODS: The expression of DDX/DHX family members during liver I/R injury was screened using transcriptome analysis. Hepatocyte-specific Dhx58 knockout mice were constructed, and a partial liver I/R operation was performed. Single-cell RNA sequencing (scRNA-seq) in the liver post I/R suggested enhanced ferroptosis by Dhx58hep-/-. The mRNAs and proteins associated with DExH-box helicase 58 (DHX58) were screened using RNA immunoprecipitation-sequencing (RIP-seq) and IP-mass spectrometry (IP-MS). RESULTS: Excessive production of reactive oxygen species (ROS) decreased the expression of the IFN-stimulated gene Dhx58 in hepatocytes and promoted hepatic ferroptosis, while treatment using IFN-α increased DHX58 expression and prevented ferroptosis during liver I/R injury. Mechanistically, DHX58 with RNA-binding activity constitutively associates with the mRNA of glutathione peroxidase 4 (GPX4), a central ferroptosis suppressor, and recruits the m6A reader YT521-B homology domain containing 2 (YTHDC2) to promote the translation of Gpx4 mRNA in an m6A-dependent manner, thus enhancing GPX4 protein levels and preventing hepatic ferroptosis. CONCLUSIONS: This study provides mechanistic evidence that IFN-α stimulates DHX58 to promote the translation of m6A-modified Gpx4 mRNA, suggesting the potential clinical application of IFN-α in the prevention of hepatic ferroptosis during liver I/R injury.

Targeting FTO induces colorectal cancer ferroptotic cell death by decreasing SLC7A11/GPX4 expression.

Ferroptosis is a newly identified iron-dependent form of death that is becoming increasingly recognized as a promising avenue for cancer therapy. N6-methyladenosine (m6A) is the most abundant reversible methylation modification in mRNA contributing to tumorigenesis. However, the crucial role of m6A modification in regulating ferroptosis during colorectal cancer (CRC) tumorigenesis remains elusive. Herein, we find that m6A modification is increased during ferroptotic cell death and correlates with the decreased m6A demethylase fat mass and obesity-associated protein (FTO) expression. Functionally, we demonstrate that suppressing FTO significantly induces CRC ferroptotic cell death, as well as enhancing CRC cell sensitivity to ferroptosis inducer (Erastin and RSL3) treatment. Mechanistically, high FTO expression increased solute carrier family 7 member 11 (SLC7A11) or glutathione peroxidase 4 (GPX4) expressions in an m6A-YTHDF2 dependent manner, thereby counteracting ferroptotic cell death stress. In addition, we identify Mupirocin as a novel inhibitor of FTO, and Mupirocin induces CRC ferroptosis and inhibits tumor growth. Clinically, the levels of FTO, SLC7A11, and GPX4, are highly correlated expression in CRC tissues. Our findings reveal that FTO protects CRC from ferroptotic cell death in promoting CRC tumorigenesis through triggering SLC7A11/GPX4 expression.

Age-Dependent Changes in Nrf2/Keap1 and Target Antioxidant Protein Expression Correlate to Lipoxidative Adducts, and Are Modulated by Dietary N-3 LCPUFA in the Hippocampus of Mice.

The brain has a high metabolism rate that may generate reactive oxygen and nitrogen species. Consequently, nerve cells require highly efficient antioxidant defenses in order to prevent a condition of deleterious oxidative stress. This is particularly relevant in the hippocampus, a highly complex cerebral area involved in processing superior cognitive functions. Most current evidence points to hippocampal oxidative damage as a causal effect for neurodegenerative disorders, especially Alzheimer's disease. Nuclear factor erythroid-2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/Keap1) is a master key for the transcriptional regulation of antioxidant and detoxifying systems. It is ubiquitously expressed in brain areas, mainly supporting glial cells. In the present study, we have analyzed the relationships between Nrf2 and Keap1 isoforms in hippocampal tissue in response to aging and dietary long-chain polyunsaturated fatty acids (LCPUFA) supplementation. The possible involvement of lipoxidative and nitrosative by-products in the dynamics of the Nrf2/Keap1 complex was examined though determination of protein adducts, namely malondialdehyde (MDA), 4-hydroxynonenal (HNE), and 3-nitro-tyrosine (NTyr) under basal conditions. The results were correlated to the expression of target proteins heme-oxygenase-1 (HO-1) and glutathione peroxidase 4 (GPx4), whose expressions are known to be regulated by Nrf2/Keap1 signaling activation. All variables in this study were obtained simultaneously from the same preparations, allowing multivariate approaches. The results demonstrate a complex modification of the protein expression patterns together with the formation of adducts in response to aging and diet supplementation. Both parameters exhibited a strong interaction. Noticeably, LCPUFA supplementation to aged animals restored the Nrf2/Keap1/target protein patterns to the status observed in young animals, therefore driving a "rejuvenation" of hippocampal antioxidant defense.

Netrin-1 Alleviates Early Brain Injury by Regulating Ferroptosis via the PPARγ/Nrf2/GPX4 Signaling Pathway Following Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a type of stroke with high morbidity and mortality. Netrin-1 (NTN-1) can alleviate early brain injury (EBI) following SAH by enhancing peroxisome proliferator-activated receptor gamma (PPARγ), which is an important transcriptional factor modulating lipid metabolism. Ferroptosis is a newly discovered type of cell death related to lipid metabolism. However, the specific function of ferroptosis in NTN-1-mediated neuroprotection following SAH is still unclear. This study aimed to evaluate the neuroprotective effects and the possible molecular basis of NTN-1 in SAH-induced EBI by modulating neuronal ferroptosis using the filament perforations model of SAH in mice and the hemin-stimulated neuron injury model in HT22 cells. NTN-1 or a vehicle was administered 2 h following SAH. We examined neuronal death, brain water content, neurological score, and mortality. NTN-1 treatment led to elevated survival probability, greater survival of neurons, and increased neurological score, indicating that NTN-1-inhibited ferroptosis ameliorated neuron death in vivo/in vitro in response to SAH. Furthermore, NTN-1 treatment enhanced the expression of PPARγ, nuclear factor erythroid 2-related factor 2 (Nrf2), and glutathione peroxidase 4 (GPX4), which are essential regulators of ferroptosis in EBI after SAH. The findings show that NTN-1 improves neurological outcomes in mice and protects neurons from death caused by neuronal ferroptosis. Furthermore, the mechanism underlying NTN-1 neuroprotection is correlated with the inhibition of ferroptosis, attenuating cell death via the PPARγ/Nrf2/GPX4 pathway and coenzyme Q10-ferroptosis suppressor protein 1 (CoQ10-FSP1) pathway.

Ancient dormant virus remnant ERVW-1 drives ferroptosis via degradation of GPX4 and SLC3A2 in schizophrenia.

Human endogenous retroviruses (HERVs) are remnants of retroviral infections in human germline cells from millions of years ago. Among these, ERVW-1 (also known as HERV-W-ENV, ERVWE1, or ENVW) encodes the envelope protein of the HERV-W family, which contributes to the pathophysiology of schizophrenia. Additionally, neuropathological studies have revealed cell death and disruption of iron homeostasis in the brains of individuals with schizophrenia. Here, our bioinformatics analysis showed that differentially expressed genes in the human prefrontal cortex RNA microarray dataset (GSE53987) were mainly related to ferroptosis and its associated pathways. Clinical data demonstrated significantly lower expression levels of ferroptosis-related genes, particularly Glutathione peroxidase 4 (GPX4) and solute carrier family 3 member 2 (SLC3A2), in schizophrenia patients compared to normal controls. Further in-depth analyses revealed a significant negative correlation between ERVW-1 expression and the levels of GPX4/SLC3A2 in schizophrenia. Studies indicated that ERVW-1 increased iron levels, malondialdehyde (MDA), and transferrin receptor protein 1 (TFR1) expression while decreasing glutathione (GSH) levels and triggering the loss of mitochondrial membrane potential, suggesting that ERVW-1 can induce ferroptosis. Ongoing research has shown that ERVW-1 reduced the expression of GPX4 and SLC3A2 by inhibiting their promoter activities. Moreover, Ferrostatin-1 (Fer-1), the ferroptosis inhibitor, reversed the iron accumulation and mitochondrial membrane potential loss, as well as restored the expressions of ferroptosis markers GSH, MDA, and TFR1 induced by ERVW-1. In conclusion, ERVW-1 could promote ferroptosis by downregulating the expression of GPX4 and SLC3A2, revealing a novel mechanism by which ERVW-1 contributes to neuronal cell death in schizophrenia.

Zuoguiwan Treats Cyclophosphamide-induced Premature Ovarian Failure by Regulating SLC7A11/GPX4 Pathway to Inhibit Ferroptosis / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Zuoguiwan on the ovarian function in the rat model of cyclophosphamide-induced premature ovarian failure （POF） based on the changes of ferroptosis pathway. MethodForty SD rats were randomized into blank， model， and low- and high-dose （2， 8 g·kg-1， respectively） Zuoguiwan groups， with 10 rats in each group. The rats in the other groups except the normal group were intraperitoneally injected with CTX at a dose of 50 mg·kg-1 on the first day and 8 mg·kg-1 from the second day to the fifteenth day for the modeling of POF. After modeling， the rats were administrated with corresponding drugs or normal saline by gavage for four weeks. Hematoxylin-eosin staining was performed to observe the pathological changes in the ovarian tissue. The mitochondria of the ovarian tissue was observed by electron microscopy. The serum levels of follicle-stimulating hormone （FSH）， estradiol （E2）， luteinizing hormone （LH）， anti-Mullerian hormone （AMH）， malondialdehyde （MDA）， catalase （CAT）， superoxide dismutase （SOD）， and iron ion were measured by biochemical methods and enzyme-linked immunosorbent assay. Western blot was employed to determine the protein levels of glutathione peroxidase 4 （GPX4）， solute carrier family 7 member 11 （SLC7A11）， ferritin heavy chain （FTH1）， and acyl-CoA synthetase long chain family member 4 （ACSL4）. ResultCompared with the blank group， the model group showcased significantly increased atretic follicles， atrophied， fragmented， and vacuolated mitochondria， and reduced， loose， and disordered cristae in mitochondria. Compared with the model group， high-dose Zuoguiwan increased mature follicles， the volume of mitochondria in the ovary， alleviated the vacuolation， and improved the number and arrangement of mitochondrial cristae. Compared with the blank group， the modeling elevated the levels of iron， MDA， FSH， and LH， up-regulated the expression of GPX4， SLC7A11， and FTH1 （P<0.05， P<0.01）， decreased the activities of SOD and CAT， lowered the levels of E2 and AMH， and down-regulated the expression of ACSL4 （P<0.05， P<0.01）. Compared with the model group， drug interventions lowered the levels of iron， MDA， FSH， and LH， down-regulated the expression of GPX4， SLC7A11， and FTH1 （P<0.05， P<0.01）， increased the activity of CAT， elevated the levels of E2 and AMH， and up-regulated the expression of ACSL4 （P<0.05， P<0.01）. ConclusionZuoguiwan may inhibit the occurrence of ferroptosis by regulating the SLC7A11/GPX4 axis， thereby improving the ovarian function of POF rats.

Mechanism on Banxia Xiexintang Inducing Ferroptosis in Gastric Cancer Cells Based on Nrf2/GPX4 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Banxia Xiexintang （BXT） on the proliferation of human gastric cancer HGC-27， MKN-45， and AGS cells and its mechanism. MethodCell counting kit-8 （CCK-8） was used to detect the effects of different concentrations of BXT-containing serum （5%， 10%， and 20%） on the proliferation of HGC-27， MKN-45， and AGS cells. A mitochondrial membrane potential probe （TMRE） was used to detect the expression of mitochondrial membrane potential in cells. A kit was used to detect iron ion （Fe2+） content， lipid peroxide （LPO）， and superoxide dismutase （SOD） activity. Western blot was used to detect the protein expression levels of glycogen synthase3β （GSK3β）， phosphorylated GSK3β （p-GSK3β）， nuclear factor E2 related factor 2 （Nrf2）， and glutathione peroxidase 4 （GPX4）. The real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of member 11 of the cystine/glutamic acid reverse transporter solute vector family 7 （SLC7A11）， member 2 of the heavy chain solute vector family 3 （SLC3A2）， transferrin receptor 3 （TFRC）， and tumor protein （TP）53. ResultCCK-8 results showed that BXT and capecitabine could significantly reduce the survival rate of three kinds of gastric cancer cells after treatment with drug-containing serum for 24 h （P<0.01）. After 48 h of intervention with drug-containing serum， the survival rate of three kinds of gastric cancer cells was significantly decreased in both the capecitabine group and the BXT group compared with the blank group. The BXT group was dose-dependent， with 20% BXT having the most significant effect （P<0.01）. In terms of biochemical indicators of ferroptosis， compared with the blank group， BXT and capecitabine significantly decreased the expression of mitochondrial membrane potential （P<0.01） and SOD activity （P<0.01） and significantly increased the contents of LPO and Fe2+ （P<0.01）， so as to improve the sensitivity of gastric cancer cells to ferroptosis. In terms of the Nrf2/GPX4 pathway， compared with the blank group， the BXT group could reduce the protein expressions of p-GSK3β， Nrf2， and GPX4 （P<0.01） in gastric cancer cells and increase mRNA expressions of SLC7A11 and SLC3A2 （P<0.05）. It could also increase the protein expression of GSK3β （P<0.01） and mRNA expression of TP53 and TFRC （P<0.05， P<0.01） in gastric cancer cells. Inhibition of the Nrf2/GPX4 pathway induces ferroptosis in gastric cancer cells. Compared with the capecitabine group， the 20% BXT group showed a more obvious effect. ConclusionBanxia Xiexintang can induce ferroptosis in gastric cancer cells HGC-27， MKN-45， and AGS by inhibiting the Nrf2/GPX4 pathway.

Zhenwutang Ameliorates Diabetic Kidney Disease in Mice with Spleen-kidney Yang Deficiency via Nrf2/HO-1/GPX4 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect and mechanism of Zhenwutang on renal oxidative damage in the mouse model of diabetic kidney disease with the syndrome of spleen-kidney Yang deficiency via the nuclear factor erythroid 2-related factor-2 （Nrf2）/heme oxygenase-1 （HO-1）/glutathione peroxidase 4 （GPX4） signaling pathway. MethodTwenty-five 7-week-old SPF-grade male db/m mice and 95 7-week-old SPF-grade male db/db mice were adaptively fed for a week. A blank group was set with the db/m mice without treatment， and the other mice were administrated with Rhei Radix et Rhizoma decoction and hydrocortisone for the modeling of diabetic kidney disease with the syndrome of spleen-kidney Yang deficiency. The modeled mice were randomized into the model， irbesartan （25 mg·kg-1）， and high-， medium-， low-dose （33.8， 16.9， 8.45 g·kg-1） Zhenwutang groups （n=15） and administrated with corresponding drugs for 8 weeks. The survival status of mice was observed， and the traditional Chinese medicine （TCM） syndrome score was recorded. The indicators related to spleen-kidney Yang deficiency， fasting blood glucose （FBG）， and renal function indicators were determined. Hematoxylin-eosin staining was employed to observe the histopathological changes of the renal tissue in each group. Biochemical kits were used to determine the oxidative stress-related indicators in the renal tissue. Real-time polymerase chain reaction and Western blotting were employed to determine the mRNA and protein levels， respectively， of Nrf2， HO-1， glutamate-cysteine ligase catalytic subunit （GCLC）， and GPX4 in the renal tissue of mice in each group. ResultCompared with the blank group， the modeling increased the TCM syndrome score （P<0.05）， elevated the estradiol （E2） and FBG levels （P<0.05）， lowered the testosterone （T）， triiodothyronine （T3）， and tetraiodothyronine （T4） levels （P<0.05）， and weakened the renal function （P<0.05）. In addition， the modeling led to glomerular hypertrophy and glomerular mesangial and basal thickening， decreased the catalase （CAT） activity， total antioxidant capacity （T-AOC）， and glutathione （GSH） content （P<0.05）， increased the malondialdehyde （MDA） content （P<0.05）， and down-regulated the mRNA and protein levels of Nrf2， HO-1， GCLC， and GPX4 in the renal tissue （P<0.05）. Compared with the model group， high and medium doses of Zhenwutang decreased the TCM syndrome score and E2 content （P<0.05）， increased the T， T3， and T4 content （P<0.05）， improved the renal function （P<0.05）， alleviated the pathological changes in the renal tissue， increased CAT， T-AOC， and GSH （P<0.05）， reduced MDA （P<0.05）， and up-regulated the mRNA and protein levels of Nrf2， HO-1， GCLC， and GPX4 in the renal tissue （P<0.05）. ConclusionZhenwutang can improve the general state and renal function and reduce the oxidative damage and pathological changes in the renal tissue of db/db mice with spleen-kidney Yang deficiency by regulating the Nrf2/HO-1/GPX4 signaling pathway.

LncRNA-XIST Promotes Lung Adenocarcinoma Growth and Inhibits Ferroptosis by Regulating GPX4.

This study aimed to explore the regulatory effects and molecular mechanisms of long non-coding RNA X-inactive-specific transcript (LncRNA-XIST) in lung adenocarcinoma. si-XIST or glutathione peroxidase 4 (GPX4) plasmids were transfected in PC-9 cells to suppress LncRNA-XIST expression or over-express GPX4, respectively. The mRNA expression levels of LncRNA-XIST and GPX4 in lung adenocarcinoma tissues or cells were assessed using RT-qPCR. CCK-8 assay was performed to examine cell activity, and corresponding biochemical kits were used to measure the levels of Fe2+, reactive oxygen species (ROS), malondialdehyde (MDA) in cells. Western blot is used to examine relative protein expression of FANCD2, SLC7A11, and GPX4 in lung adenocarcinoma cells. The mRNA and protein expression levels of LncRNA-XIST in clinical tissues and cells of lung adenocarcinoma were significantly higher than those in adjacent tissues and normal cells. Functional analysis showed that knockdown of LncRNA-XIST notably weakened the viability of lung adenocarcinoma cells and promoted ferroptosis (manifested by significantly up-regulated levels of ROS, MDA, and Fe2+ and down-regulated the expression of SLC7A11 and FANCD2, P < 0.05). Further mechanism analysis revealed that knockdown of LncRNA-XIST markedly inhibited the expression of GPX4 in lung adenocarcinoma cells and that GPX4 was significantly over-expressed in clinical tissues and cells of lung adenocarcinoma. Notably, the expression of GPX4 was positively correlated with that of LncRNA-XIST. Over-expression of GPX4 remarkably promoted cell proliferation and inhibited ferroptosis in lung adenocarcinoma. Besides, the GPX4 over-expression reversed the LncRNA-XIST knockdown-induced ferroptosis and decrease in lung adenocarcinoma cell viability. LncRNA-XIST increases the activity of lung adenocarcinoma cells and inhibits ferroptosis by up-regulating GPX4. Knocking down LncRNA-XIST may be an effective treatment for lung adenocarcinoma.

Deubiquitinase OTUD5 as a Novel Protector against 4-HNE-Triggered Ferroptosis in Myocardial Ischemia/Reperfusion Injury.

Despite the development of advanced technologies for interventional coronary reperfusion after myocardial infarction, a substantial number of patients experience high mortality due to myocardial ischemia-reperfusion (MI/R) injury. An in-depth understanding of the mechanisms underlying MI/R injury can provide crucial strategies for mitigating myocardial damage and improving patient survival. Here, it is discovered that the 4-hydroxy-2-nonenal (4-HNE) accumulates during MI/R, accompanied by high rates of myocardial ferroptosis. The loss-of-function of aldehyde dehydrogenase 2 (ALDH2), which dissipates 4-HNE, aggravates myocardial ferroptosis, whereas the activation of ALDH2 mitigates ferroptosis. Mechanistically, 4-HNE targets glutathione peroxidase 4 (GPX4) for K48-linked polyubiquitin-related degradation, which 4-HNE-GPX4 axis commits to myocyte ferroptosis and forms a positive feedback circuit. 4-HNE blocks the interaction between GPX4 and ovarian tumor (OTU) deubiquitinase 5 (OTUD5) by directly carbonylating their cysteine residues at C93 of GPX4 and C247 of OTUD5, identifying OTUD5 as the novel deubiquitinase for GPX4. Consequently, the elevation of OTUD5 deubiquitinates and stabilizes GPX4 to reverse 4-HNE-induced ferroptosis and alleviate MI/R injury. The data unravel the mechanism of 4-HNE in GPX4-dependent ferroptosis and identify OTUD5 as a novel therapeutic target for the treatment of MI/R injury.

The ferroptosis activity is associated with neurological recovery following chronic compressive spinal cord injury.

Chronic compressive spinal cord injury in compressive cervical myelopathy conditions can lead to rapid neurological deterioration in the early phase, followed by partial self-recovery, and ultimately an equilibrium state of neurological dysfunction. Ferroptosis is a crucial pathological process in many neurodegenerative diseases; however, its role in chronic compressive spinal cord injury remains unclear. In this study, we established a chronic compressive spinal cord injury rat model, which displayed its most severe behavioral and electrophysiological dysfunction at 4 weeks and partial recovery at 8 weeks after compression. Bulk RNA sequencing data identified enriched functional pathways, including ferroptosis, presynapse, and postsynaptic membrane activity at both 4 and 8 weeks following chronic compressive spinal cord injury. Transmission electron microscopy and malondialdehyde quantification assay confirmed that ferroptosis activity peaked at 4 weeks and was attenuated at 8 weeks after chronic compression. Ferroptosis activity was negatively correlated with behavioral score. Immunofluorescence, quantitative polymerase chain reaction, and western blotting showed that expression of the anti-ferroptosis molecules, glutathione peroxidase 4 (GPX4) and MAF BZIP transcription factor G (MafG), in neurons was suppressed at 4 weeks and upregulated at 8 weeks following spinal cord compression. There was a positive correlation between the expression of these two molecules, suggesting that they may work together to contribute to functional recovery following chronic compressive spinal cord injury. In conclusion, our study determined the genome-wide expression profile and ferroptosis activity of a consistently compressed spinal cord at different time points. The results showed that anti-ferroptosis genes, specifically GPX4 and MafG, may be involved in spontaneous neurological recovery at 8 weeks of chronic compressive spinal cord injury. These findings contribute to a better understanding of the mechanisms underlying chronic compressive spinal cord injury and may help identify new therapeutic targets for compressive cervical myelopathy.

Apolipoprotein E is required for brain iron homeostasis in mice.

BACKGROUND: Apolipoprotein E deficiency (ApoE-/-) increases progressively iron in the liver, spleen and aortic tissues with age in mice. However, it is unknown whether ApoE affects brain iron. METHODS: We investigated iron contents, expression of transferrin receptor 1 (TfR1), ferroportin 1 (Fpn1), iron regulatory proteins (IRPs), aconitase, hepcidin, Aß42, MAP2, reactive oxygen species (ROS), cytokines and glutathione peroxidase 4 (Gpx4) in the brain of ApoE-/- mice. RESULTS: We demonstrated that ApoE-/- induced a significant increase in iron, TfR1 and IRPs and a reduction in Fpn1, aconitase and hepcidin in the hippocampus and basal ganglia. We also showed that replenishment of ApoE absent partly reversed the iron-related phenotype in ApoE-/- mice at 24-months old. In addition, ApoE-/- induced a significant increase in Aß42, MDA, 8-isoprostane, IL-1ß, IL-6, and TNFα and a reduction in MAP2 and Gpx4 in hippocampus, basal ganglia and/or cortex of mice at 24-months old. CONCLUSIONS: Our findings implied that ApoE is required for brain iron homeostasis and ApoE-/--induced increase in brain iron is due to the increased IRP/TfR1-mediated cell-iron uptake as well as the reduced IRP/Fpn1 associated cell-iron export and suggested that ApoE-/- induced neuronal injury resulted mainly from the increased iron and subsequently ROS, inflammation and ferroptosis.

Ferroptosis: mechanisms and implications for cancer development and therapy response.

As cancer cells develop resistance to apoptosis, non-apoptotic cell death modalities, such as ferroptosis, have emerged as promising strategies to combat therapy-resistant cancers. Cells that develop resistance to conventional therapies or metastatic cancer cells have been shown to have increased sensitivity to ferroptosis. Therefore, targeting the regulatory elements of ferroptosis in cancer could offer novel therapeutic opportunities. In this review, we first provide an overview of the known ferroptosis regulatory networks and discuss recent findings on how they contribute to cancer plasticity. We then expand into the critical role of selenium metabolism in regulating ferroptosis. Finally, we highlight specific cases where induction of ferroptosis could be used to sensitize cancer cells to this form of cell death.

Novel perspective in transplantation therapy of mesenchymal stem cells: targeting the ferroptosis pathway. / 间å 质干细胞移植治疗的新视角：靶向铁死亡通路.

Ex vivo culture-amplified mesenchymal stem cells (MSCs) have been studied because of their capacity for healing tissue injury. MSC transplantation is a valid approach for promoting the repair of damaged tissues and replacement of lost cells or to safeguard surviving cells, but currently the efficiency of MSC transplantation is constrained by the extensive loss of MSCs during the short post-transplantation period. Hence, strategies to increase the efficacy of MSC treatment are urgently needed. Iron overload, reactive oxygen species deposition, and decreased antioxidant capacity suppress the proliferation and regeneration of MSCs, thereby hastening cell death. Notably, oxidative stress (OS) and deficient antioxidant defense induced by iron overload can result in ferroptosis. Ferroptosis may inhibit cell survival after MSC transplantation, thereby reducing clinical efficacy. In this review, we explore the role of ferroptosis in MSC performance. Given that little research has focused on ferroptosis in transplanted MSCs, further study is urgently needed to enhance the in vivo implantation, function, and duration of MSCs.

Novel perspective in transplantation therapy of mesenchymal stem cells: targeting the ferroptosis pathway / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Ex vivo culture-amplified mesenchymal stem cells (MSCs) have been studied because of their capacity for healing tissue injury. MSC transplantation is a valid approach for promoting the repair of damaged tissues and replacement of lost cells or to safeguard surviving cells, but currently the efficiency of MSC transplantation is constrained by the extensive loss of MSCs during the short post-transplantation period. Hence, strategies to increase the efficacy of MSC treatment are urgently needed. Iron overload, reactive oxygen species deposition, and decreased antioxidant capacity suppress the proliferation and regeneration of MSCs, thereby hastening cell death. Notably, oxidative stress (OS) and deficient antioxidant defense induced by iron overload can result in ferroptosis. Ferroptosis may inhibit cell survival after MSC transplantation, thereby reducing clinical efficacy. In this review, we explore the role of ferroptosis in MSC performance. Given that little research has focused on ferroptosis in transplanted MSCs, further study is urgently needed to enhance the in vivo implantation, function, and duration of MSCs.

The effects of electroacupuncture on the cortical SLC7A11 and GPX4 expressions in the rat with post stroke-induced limb spasticity / 神经解剖学杂志

Objective:To observe the effects of electroacupuncture on the expression of cortical solute carrier family 7 member 11(SLC7A11),glutathione(GSH)and glutathione peroxidase 4(GPX4)in rats with post stroke spasticity(PSS),and to explore the mechanism of electroacupuncture in the treatment ferrozosis in PSS.Methods:Thirty SD male rats were randomly divided into sham group,model group and electroacupuncture group.A modified Zea-Longa wire bolus+internal capsule injection NMDA method was used to produce a rat model of PSS.In the electroacupunc-ture group,the affected side of Yanglingquan and Quchi were needled once/day for 30 min/time for 7 d.In the sham group and the model group,only fixation without intervention was performed during the same period.Zea-Longa Neuro-logical Function Score was used to detect the neurological function of rats,electrophysiological tracing method was used to detect the muscle tone of rat quadriceps,Western Blot was used to detect the protein expression of rat cortical SLC7A11 and GPX4,Enzyme-linked immunosorbent assay(ELISA)was used to detect the GSH content of rat cortex,and real time RT-PCR was used to detect the mRNA of rat cortical SLC7A11 and GPX4 mRNA expression.Results:Neurological function scores were elevated;quadriceps muscle tone was increased;GSH content was decreased;the protein expression of SLC7A11 and GPX4 was significantly decreased;the expression of SLC7A11 mRNA and GPX4 mRNA was significantly decreased.Electroacupuncture treatment resulted in lower neurological function scores,lower quadriceps muscle tone,increased GSH content in rat cortex,significantly up-regulated protein expression of SLC7A11 and GPX4,and significantly increased expression of SLC7A11 and GPX4 mRNA.Conclusion:Electroacupuncture on"Yanglingquan"and"Quchi"could improve limb spasticity and promote the recovery of neurological function in PSS rats,and its mechanism of action may be related to the inhibition of ferroptosis of cortical cells by electroacupuncture regulating the SLC7A11 and GPX4 expression.

Huaier Polysaccharide Attenuates Doxorubicin-Induced Acute Cardiotoxicity by Regulating Ferroptosis.

We studied the effects of Huaier polysaccharide (HP) in doxorubicin-induced myocardial injury in mice. The content of HP in Trametes robiniophila Murr medicinal fungus determined by the phenol-sulfuric acid method was 85.25%. In the in vitro model, the viability of H9c2 cells was significantly increased after HP treatment compared to the control, while doxorubicin (DOX) decreased this parameter. The inhibitory effect of DOX on cell viability was attenuated after HP treatment. In the in vivo model, the body weight of mice in DOX and DOX+HP groups was significantly decreased compared to the control group. ECG showed significantly elevated ST segment in the DOX group, while in the DOX+HP group, ECG was close to normal. The levels of cardiotoxicity markers cTnI and lactate dehydrogenase in the DOX+HP group were significantly lower than in the DOX group. In the DOX group, the myocardial tissue had obvious structural disorder and interfibrillar vacuoles. In the DOX+HP group, the cardiomyocytes were neatly arranged without interfibrillar vacuoles. The expression of the ferroptosis marker glutathione peroxidase 4 was increased in the DOX+HP group compared to the DOX group. Thus, our study reveals that HP attenuated DOX-induced myocardial injury in mice probably by regulating ferroptosis.

Exosomal thioredoxin-1 from hypoxic human umbilical cord mesenchymal stem cells inhibits ferroptosis in doxorubicin-induced cardiotoxicity via mTORC1 signaling.

Doxorubicin (DOX), a clinical chemotherapeutic drug, is often annoyed by its cardiotoxicity which involves ferroptosis in its pathological progress. Human umbilical cord mesenchymal stem cells (HucMSCs)-derived exosomes (HucMSCs-Exo) are proven effective in treating cardiovascular diseases. This study aimed to compare the therapeutic effects between normoxic HucMSCs-Exo (Exo) and hypoxic HucMSCs-Exo (Hypo-Exo) on DOX-induced ferroptosis and explore the underlying mechanisms. An acute cardiotoxicity model was successfully constructed by administrating two doses intraperitoneal injections of DOX (25 mg/kg in total). Exo and Hypo-Exo were extracted by ultracentrifugation and characterized. Compared with Exo, Hypo-Exo and Ferrostatin-1 (Fer-1) exerted superior effects on inhibiting DOX-induced ferroptosis, as evidenced by decreasing malondialdehyde (MDA), iron content and increasing glutathione (GSH) level as well as ferroptosis-related genes expression including prostaglandin-endoperoxide synthase 2 (Ptgs2) mRNA level and glutathione peroxidase 4 (GPX4) protein level. Based on quantitative proteomics analysis, we found that thioredoxin1 (Trx1) was remarkably upregulated in Hypo-Exo and exhibited anti-ferroptosis activity via activating the mechanistic target of rapamycin complex 1 (mTORC1) in neonatal rat cardiomyocytes (NRCMs). Trx1 knockdown and rapamycin (an mTORC1 inhibitor) partially abolished the protective effects of Hypo-Exo. Furthermore, our data indicated that solute carrier family 7 member 11 (SLC7A11) was critical for GPX4 protein synthesis. In conclusion, Hypo-Exo exhibited a better suppression of ferroptosis in DOX-induced cardiotoxicity. Trx1-mediated mTORC1 activation is critical for the Hypo-Exo anti-ferroptosis process, which involves increased GPX4 protein synthesis and decreased iron overload. This study indicated that Hypo-Exo may present a potential strategy against ferroptosis in DOX-induced cardiotoxicity.