ALKBH5 targets ACSL4 mRNA stability to modulate ferroptosis in hyperbilirubinemia-induced brain damage.

Bilirubin-induced brain damage is a serious clinical consequence of hyperbilirubinemia, yet the underlying molecular mechanisms remain largely unknown. Ferroptosis, an iron-dependent cell death, is characterized by iron overload and lipid peroxidation. Here, we report a novel regulatory mechanism of demethylase AlkB homolog 5 (ALKBH5) in acyl-coenzyme A synthetase long-chain family member 4 (ACSL4)-mediated ferroptosis in hyperbilirubinemia. Hyperdifferential PC12 cells and newborn Sprague-Dawley rats were used to establish in vitro and in vivo hyperbilirubinemia models, respectively. Proteomics, coupled with bioinformatics analysis, first suggested the important role of ferroptosis in hyperbilirubinemia-induced brain damage. In vitro experiments showed that ferroptosis is activated in hyperbilirubinemia, and ferroptosis inhibitors (desferrioxamine and ferrostatin-1) treatment effectively alleviates hyperbilirubinemia-induced oxidative damage. Notably, we observed that the ferroptosis in hyperbilirubinemia was regulated by m6A modification through the downregulation of ALKBH5 expression. MeRIP-seq and RIP-seq showed that ALKBH5 may trigger hyperbilirubinemia ferroptosis by stabilizing ACSL4 mRNA via m6A modification. Further, hyperbilirubinemia-induced oxidative damage was alleviated through ACSL4 genetic knockdown or rosiglitazone-mediated chemical repression but was exacerbated by ACSL4 overexpression. Mechanistically, ALKBH5 promotes ACSL4 mRNA stability and ferroptosis by combining the 669 and 2015 m6A modified sites within 3' UTR of ACSL4 mRNA. Our findings unveil a novel molecular mechanism of ferroptosis and suggest that m6A-dependent ferroptosis could be an underlying clinical target for the therapy of hyperbilirubinemia.

Polydopamine Nanoparticles-Encapsulated Ferroptosis Inhibitor Ferstatin-1 Promotes GPX4 Expression by Down-Regulating NOX4 to Alleviate Myocardial Ischemia-Reperfusion Injury.

OBJECTIVE: Polydopamine nanoparticles (PDA NPs) are a promising topic in the fields of drug delivery, tissue engineering, bioimaging, etc. The present study aims to explore the impact of PDA NPs carrying ferroptosis inhibitor ferstatin-1 (Fer-1) on myocardial ischemia-reperfusion injury (MIRI). METHODS: After establishment of a rat model of MIRI and PDA NPs, the rats were divided into 4 groups: model group, sham operation group, Fer-1 group, and nano+Fer-1 group (n=8). To detect the effect of PDA NPs encapsulating Fer-1 on ferroptosis in MIRI rats, we further set up NOX4 overexpression group (pc-NOX4 group), NOX4 inhibitor group (Fulvene-5 group), nano+Fer-1+pc-NOX4 group, and nano+Fer-1+Fulvene-5 group (n=8). A CCK-8 assay was conducted to assess cell viability and staining to detect cardiomyocyte apoptosis and observe myocardial infraction. RESULTS: PDA NPs loaded with Fer-1 were successfully prepared with good safety and biocompatibility. Administration of PDA NPs carrying Fer-1 notably alleviated myocardial injury and hindered the process of ferroptosis in MIRI rats when inducing downregulation of NOX4 expression. Additionally, overexpression of GPX4 significantly attenuated myocardial injury in MIRI rats. While Fer-1 was shown to inhibit the expression of NOX4, the NOX4 inhibitor Fulvene-5 greatly elevated GPX4 and FTH1 expression in cardiomyocytes, and down-regulated the content of Fe2+, especially in the nanometer+Fer-1+Fulvene-5 group. CONCLUSION: With promising safety and biocompatibility, PDA NPs encapsulated Fer-1 decrease GPX4 and FTH1 expression by inhibiting the level of NOX4 in myocardial cells of MIRI rats, thereby suppressing ferroptosis of cardiomyocytes and alleviating myocardial injury.

Both hemoglobin and hemin cause damage to retinal pigment epithelium through the iron ion accumulation.

Subretinal hemorrhages result in poor vision and visual field defects. During hemorrhage, several potentially toxic substances are released from iron-based hemoglobin and hemin, inducing cellular damage, the detailed mechanisms of which remain unknown. We examined the effects of excess intracellular iron on retinal pigment epithelial (RPE) cells. A Fe2+ probe, SiRhoNox-1 was used to investigate Fe2+ accumulation after treatment with hemoglobin or hemin in the human RPE cell line ARPE-19. We also evaluated the production of reactive oxygen species (ROS) and lipid peroxidation. Furthermore, the protective effect of-an iron chelator, 2,2'-bipyridyl (BP), and ferrostatin-1 (Fer-1) on the cell damage, was evaluated. Fe2+ accumulation increased in the hemoglobin- or hemin-treated groups, as well as intracellular ROS production and lipid peroxidation. In contrast, BP treatment suppressed RPE cell death, ROS production, and lipid peroxidation. Pretreatment with Fer-1 ameliorated cell death in a concentration-dependent manner and suppressed ROS production and lipid peroxidation. Taken together, these findings indicate that hemoglobin and hemin, as well as subretinal hemorrhage, may induce RPE cell damage and visual dysfunction via intracellular iron accumulation.

Rhythm gene PER1 mediates ferroptosis and lipid metabolism through SREBF2/ALOX15 axis in polycystic ovary syndrome.

OBJECTIVE: This work aimed to investigate the role of rhythm gene PER1 in mediating granulosa cell ferroptosis and lipid metabolism of polycystic ovary syndrome (PCOS). METHODS: We injected dehydroepiandrosterone and Ferrostatin-1 (Fer-1) into mice to explore the mechanism of ferroptosis in PCOS. The effect of PER1 on ferroptosis-like changes in granulosa cells was explored by overexpression of PER1 plasmid transfection and Fer-1 treatment. RESULTS: We found that Fer-1 ameliorated the characteristic polycystic ovary morphology, suppressed ferroptosis in the PCOS mice. PER1 and ALOX15 were highly expressed in PCOS, whereas SREBF2 was lowly expressed. Overexpression of PER1 decreased granulosa cell viability and inhibited proliferation. Meanwhile, overexpression of PER1 increased lipid reactive oxygen species, 4-Hydroxynonenal (4-HNE), Malondialdehyde (MDA), total Fe, and Fe2+ levels in granulosa cells and decreased Glutathione (GSH) content. Fer-1, SREBF2 overexpression, or ALOX15 silencing treatment reversed the effects of PER1 overexpression on granulosa cells. PER1 binds to the SREBF2 promoter and represses SREBF2 transcription. SREBF2 binds to the ALOX15 promoter and represses ALOX15 transcription. Correlation analysis of clinical trials showed that PER1 was positively correlated with total cholesterol, low-density lipoprotein cholesterol, luteinizing hormone, testosterone, 4-HNE, MDA, total Fe, Fe2+, and ALOX15. In contrast, PER1 was negatively correlated with SREBF2, high-density lipoprotein cholesterol, follicle-stimulating hormone, progesterone, and GSH. CONCLUSION: This study demonstrates that the rhythm gene PER1 promotes ferroptosis and dysfunctional lipid metabolism in granulosa cells in PCOS by inhibiting SREBF2/ALOX15 signaling.

Ferroptosis inhibitors reduce celastrol toxicity and preserve its insulin sensitizing effects in insulin resistant HepG2 cells.

OBJECTIVE: Research has shown that celastrol can effectively treat a variety of diseases, yet when passing a certain dosage threshold, celastrol becomes toxic, causing complications such as liver and kidney damage and erythrocytopenia, among others. With this dichotomy in mind, it is extremely important to find ways to preserve celastrol's efficacy while reducing or preventing its toxicity. METHODS: In this study, insulin-resistant HepG2 (IR-HepG2) cells were prepared using palmitic acid and used for in vitro experiments. IR-HepG2 cells were treated with celastrol alone or in combination with N-acetylcysteine (NAC) or ferrostatin-1 (Fer-1) for 12, 24 or 48 h, at a range of doses. Cell counting kit-8 assay, Western blotting, quantitative reverse transcription-polymerase chain reaction, glucose consumption assessment, and flow cytometry were performed to measure celastrol's cytotoxicity and whether the cell death was linked to ferroptosis. RESULTS: Celastrol treatment increased lipid oxidation and decreased expression of anti-ferroptosis proteins in IR-HepG2 cells. Celastrol downregulated glutathione peroxidase 4 (GPX4) mRNA. Molecular docking models predicted that solute carrier family 7 member 11 (SLC7A11) and GPX4 were covalently bound by celastrol. Importantly, we found for the first time that the application of ferroptosis inhibitors (especially NAC) was able to reduce celastrol's toxicity while preserving its ability to improve insulin sensitivity in IR-HepG2 cells. CONCLUSION: One potential mechanism of celastrol's cytotoxicity is the induction of ferroptosis, which can be alleviated by treatment with ferroptosis inhibitors. These findings provide a new strategy to block celastrol's toxicity while preserving its therapeutic effects. Please cite this article as: Liu JJ, Zhang X, Qi MM, Chi YB, Cai BL, Peng B, Zhang DH. Ferroptosis inhibitors reduce celastrol toxicity and preserve its insulin sensitizing effects in insulin resistant HepG2 cells. J Integr Med. 2024; 22(3): 286-294.

Ferroptosis contributes to airway epithelial E-cadherin disruption in a mixed granulocytic asthma mouse model.

Aberrant expression of airway epithelial E-cadherin is a key feature of asthma, yet the underlying mechanisms are largely unknown. Ferroptosis is a novel form of regulated cell death involved in asthma pathogenesis. This study was aimed to evaluate the role of ferroptosis and to investigate whether ferroptosis mediates E-cadherin disruption in mixed granulocyte asthma (MGA). Two murine models of MGA were established using toluene diisocyanate (TDI) or ovalbumin with Complete Freund's Adjuvant (OVA/CFA). Specific antagonists of ferroptosis, including Liproxstatin-1 (Lip-1) and Ferrostatin-1 (Fer-1) were given to the mice. The allergen-exposed mice displayed markedly shrunk mitochondria in the airway epithelia, with decreased volume and denser staining accompanied by down-regulated GPX4 as well as up-regulated FTH1 and malondialdehyde, which are markers of ferroptosis. Decreased pulmonary expression of E-cadherin was also observed, with profound loss of membrane E-cadherin in the airway epithelia, as well as increased secretion of sE-cadherin. Treatment with Lip-1 not only showed potent protective effects against the allergen-induced airway hyperresponsiveness and inflammatory responses, but also rescued airway epithelial E-cadherin expression and inhibited the release of sE-cadherin. Taken together, our data demonstrated that ferroptosis mediates airway epithelial E-cadherin dysfunction in MGA.

Liproxstatin-1 attenuates acute hypertriglyceridemic pancreatitis through inhibiting ferroptosis in rats.

Ferroptosis is closely associated with inflammatory diseases, including acute pancreatitis (AP); however, the involvement of ferroptosis in hypertriglyceridemic pancreatitis (HTGP) remains unclear. In the present study, we aimed to explore the relationship between lipid metabolism and ferroptosis in HTGP and the alleviating effect of liproxstatin-1 (Lip-1) in vivo. This study represents the first exploration of lipid metabolism and endoplasmic reticulum stress (ERS) in HTGP, targeting ferroptosis as a key factor in HTGP. Hypertriglyceridemia (HTG) was induced under high-fat diet conditions. Cerulein was then injected to establish AP and HTGP models. Lip-1, a specific ferroptosis inhibitor, was administered before the induction of AP and HTGP in rats, respectively. Serum triglyceride, amylase, inflammatory factors, pathological and ultrastructural structures, lipid peroxidation, and iron overload indicators related to ferroptosis were tested. Moreover, the interaction between ferroptosis and ERS was assessed. We found HTG can exacerbate the development of AP, with an increased inflammatory response and intensified ferroptosis process. Lip-1 treatment can attenuate pancreatic injury by inhibiting ferroptosis through lipid metabolism and further resisting activations of ERS-related proteins. Totally, our results proved lipid metabolism can promote ferroptosis in HTGP by regulating ACSL4/LPCAT3 protein levels. Additionally, ERS may participate in ferroptosis via the Bip/p-EIF2α/CHOP pathway, followed by the alleviating effect of Lip-1 in the rat model.

Unlocking the anticancer activity of gambogic acid: a shift towards ferroptosis via a GSH/Trx dual antioxidant system.

Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in ferroptosis by regulating the cellular antioxidant response and maintaining redox balance. However, compounds that induce ferroptosis through dual antioxidant pathways based on Nrf2 have not been fully explored. In our study, we investigated the impact of Gambogic acid (GA) on MCF-7 cells and HepG2 cells in vitro. The cytotoxicity, colony formation assay and cell cycle assay demonstrated potent tumor-killing ability of GA, while its effect was rescued by ferroptosis inhibitors. Furthermore, RNA sequencing revealed the enrichment of ferroptosis pathway mediated by GA. In terms of ferroptosis indicators detection, evidences for GA were provided including reactive oxygen species (ROS) accumulation, alteration in mitochondrial membrane potential (MMP), disappearance of mitochondrial cristae, lipid peroxidation induction, malondialdehyde (MDA) accumulation promotion, iron ion accumulation as well as glutathione (GSH)/thioredoxin (Trx) depletion. Notably, Ferrostatin-1 (Fer-1) and Liproxstatin-1 (Lip-1) successfully rescued GA-induced MDA accumulation. In terms of mechanism, Nrf2 was found to play a pivotal role in GA-induced ferroptosis by inducing protein alterations through the iron metabolism pathway and GSH/Trx dual antioxidant pathway. Furthermore, GA exerted good antitumor activity in vivo through GSH/Trx dual antioxidant pathway, and Fer-1 significantly attenuated its efficacy. In conclusion, our findings first provided new evidence for GA as an inducer of ferroptosis, and Nrf2-mediated GSH/Trx dual antioxidant system played an important role in GA-induced ferroptosis.

Design, synthesis, and evaluation of formylpiperazine analogs of Ferrostatin-1 as novel improved ferroptosis inhibitors.

In this study, a series of new formylpiperazine-derived ferroptosis inhibitors were designed and synthesized based on the structure of a known ferroptosis inhibitor, ferrostatin-1 (Fer-1). The anti-ferroptosis activity of these synthetic compounds in human umbilical vein endothelial cells (HUVECs) induced by Erastin was evaluated. It was found that some of the new compounds, especially compound 26, showed potent anti-ferroptosis activity, as evidenced by its ability to restore cell viability, reduce iron accumulation, scavenge reactive oxygen species, maintain mitochondrial membrane potential, increase GSH levels, decrease LPO and MDA content, and upregulate GPX4 expression. Moreover, compound 26 exhibited superior microsomal stability than Fer-1. The present results suggest that compound 26 is a promising lead compound for the development of new ferroptosis inhibitors for the treatment of vascular diseases.

Identification and validation of the biomarkers related to ferroptosis in calcium oxalate nephrolithiasis.

Ferroptosis is a specific type of programmed cell death characterized by iron-dependent lipid peroxidation. Understanding the involvement of ferroptosis in calcium oxalate (CaOx) stone formation may reveal potential targets for this condition. The publicly available dataset GSE73680 was used to identify 61 differentially expressed ferroptosis-related genes (DEFERGs) between normal kidney tissues and Randall's plaques (RPs) from patients with nephrolithiasis through employing weighted gene co-expression network analysis (WGCNA). The findings were validated through in vitro and in vivo experiments using CaOx nephrolithiasis rat models induced by 1% ethylene glycol administration and HK-2 cell models treated with 1 mM oxalate. Through WGCNA and the machine learning algorithm, we identified LAMP2 and MDM4 as the hub DEFERGs. Subsequently, nephrolithiasis samples were classified into cluster 1 and cluster 2 based on the expression of the hub DEFERGs. Validation experiments demonstrated decreased expression of LAMP2 and MDM4 in CaOx nephrolithiasis animal models and cells. Treatment with ferrostatin-1 (Fer-1), a ferroptosis inhibitor, partially reversed oxidative stress and lipid peroxidation in CaOx nephrolithiasis models. Moreover, Fer-1 also reversed the expression changes of LAMP2 and MDM4 in CaOx nephrolithiasis models. Our findings suggest that ferroptosis may be involved in the formation of CaOx kidney stones through the regulation of LAMP2 and MDM4.

Investigating the Therapeutic Effects of Ferroptosis on Myocardial Ischemia-Reperfusion Injury Using a Dual-Locking Mitochondrial Targeting Strategy.

Research on ferroptosis in myocardial ischemia/reperfusion injury (MIRI) using mitochondrial viscosity as a nexus holds great promise for MIRI therapy. However, high-precision visualisation of mitochondrial viscosity remains a formidable task owing to the debilitating electrostatic interactions caused by damaged mitochondrial membrane potential. Herein, we propose a dual-locking mitochondria-targeting strategy that incorporates electrostatic forces and probe-protein molecular docking. Even in damaged mitochondria, stable and precise visualisation of mitochondrial viscosity in triggered and medicated MIRI was achieved owing to the sustained driving forces (e.g., pi-cation, pi-alkyl interactions, etc.) between the developed probe, CBS, and the mitochondrial membrane protein. Moreover, complemented by a western blot, we confirmed that ferrostatin-1 exerts its therapeutic effect on MIRI by improving the system xc-/GSH/GPX4 antioxidant system, confirming the therapeutic value of ferroptosis in MIRI. This study presents a novel strategy for developing robust mitochondrial probes, thereby advancing MIRI treatment.

Cyclophosphamide activates ferroptosis-induced dysfunction of Leydig cells via SMAD2 pathway†.

Cyclophosphamide (CP) is a widely used chemotherapeutic drug and immunosuppressant in the clinic, and the hypoandrogenism caused by CP is receiving more attention. Some studies found that ferroptosis is a new mechanism of cell death closely related to chemotherapeutic drugs and plays a key role in regulating reproductive injuries. The purpose of this study is to explore ferroptosis' role in testicular Leydig cell dysfunction and molecular mechanisms relating to it. In this study, the level of ferroptosis in the mouse model of testicular Leydig cell dysfunction induced by CP was significantly increased and further affected testosterone synthesis. The ferroptosis inhibitors ferrostatin-1 (Fer-1) and iron chelator deferoxamine (DFO) can improve injury induced by CP. The results of immunohistochemistry showed that Fer-1 and DFO could improve the structural disorder of seminiferous tubules and the decrease of the number of Leydig cells in testicular tissue induced by CP. Immunofluorescence and western blot confirmed that Fer-1 and DFO could improve the expression of key enzymes in testosterone synthesis. The activation of SMAD family member 2 (Smad2)/cyclin-dependent kinase inhibitor 1A (Cdkn1a) pathway can improve the ferroptosis of Leydig cells induced by CP and protect the function of Leydig cells. By inhibiting the Smad2/Cdkn1a signal pathway, CP can regulate ferroptosis, resulting in testicular Leydig cell dysfunction. In this study, CP-induced hypoandrogenism is explained theoretically and a potential therapeutic strategy is provided.

Ferrostatin-1 inhibits ferroptosis of vascular smooth muscle cells and alleviates abdominal aortic aneurysm formation through activating the SLC7A11/GPX4 axis.

Ferroptosis, a type of iron-catalyzed necrosis, is responsible for vascular smooth muscle cell (VSMC) death and serves as a potential therapeutic target for alleviating aortic aneurysm. Here, our study explored the underlying mechanism of ferroptosis affecting VSMC functions and the resultant formation of AAA using its inhibitor Ferrostatin-1 (Fer-1). Microarray-based gene expression profiling was employed to identify differentially expressed genes related to AAA and ferroptosis. An AAA model was established by angiotensin II (Ang II) induction in apolipoprotein E-knockout (ApoE-/- ) mice, followed by injection of Fer-1 and RSL-3 (ferroptosis inducer). Then, the role of Fer-1 and RSL-3 in the ferroptosis of VSMCs and AAA formation was analyzed in Ang II-induced mice. Primary mouse VSMCs were cultured in vitro and treated with Ang II, Fer-1, sh-SLC7A11, or sh-GPX4 to assess the effect of Fer-1 via the SLC7A11/GPX axis. Bioinformatics analysis revealed that GPX4 was involved in the fibrosis formation of AAA, and there was an interaction between SLC7A11 and GPX4. In vitro assays showed that Fer-1 alleviated Ang II-induced ferroptosis of VSMCs and retard the consequent AAA formation. The mechanism was associated with activation of the SLC7A11/GPX4 pathway. Silencing of SLC7A11 or GPX4 could inhibit the ameliorating effect of Fer-1 on the ferroptosis of VSMCs. In vivo animal studies further demonstrated that Fer-1 inhibited Ang II-induced ferroptosis and vessel wall structural abnormalities in AAA mouse through activation of the SLC7A11/GPX4 pathway. Fer-1 may prevent AAA formation through activation of the SLC7A11/GPX4 pathway.

Beyond ferrostatin-1: a comprehensive review of ferroptosis inhibitors.

Ferroptosis is an iron-catalysed form of regulated cell death, which is critically dependent on phospholipid peroxidation of cellular membranes. Ferrostatin 1 was one of the first synthetic radical-trapping antioxidants (RTAs) reported to block ferroptosis and it is widely used as reference compound. Ferroptosis has been linked to multiple diseases and the use of its inhibitors could have therapeutic potential. Although, novel biochemical pathways provide insights for different pharmacological targets, the use of lipophilic RTAs to block ferroptosis remains superior. In this Review, we provide a comprehensive overview of the different classes of ferroptosis inhibitors, focusing on endogenous and synthetic RTAs. A thorough analysis of their chemical, pharmacokinetic, and pharmacological properties and potential for in vivo use is provided.

Ferrostatin-1 post-treatment attenuates acute kidney injury in mice by inhibiting ferritin production and regulating iron uptake-related proteins.

Background: Acute kidney injury (AKI) is a common and serious medical condition with high morbidity and mortality. Recent research has highlighted ferroptosis, a novel form of programmed cell death, as a potential therapeutic target in mitigating renal tubular injury in AKI. Ferrostatin-1, a specific ferroptosis inhibitor, has been demonstrated to prevent renal injury through ferroptosis inhibition. Methods: Utilizing a murine AKI model, we investigated the effects of Ferrostatin-1 by administering it post-injury. Through high-throughput sequencing and pathological analysis, we focused on the critical role of ferroptosis-related pathways in the treatment. Results: Ferrostatin-1 post-conditioning effectively mitigated oxidative damage and reduced iron content associated with AKI. Additionally, critical ferroptosis-related proteins, such as GPX4, SLC7A11, NRF2, and FTH1, exhibited increased expression levels. In vitro, Ferrostatin-1 treatment of HK-2 cells significantly diminished lipid peroxidation and iron accumulation. Furthermore, Ferrostatin-1 was found to downregulate the PI3K signalling pathway. Conclusion: Ferrostatin-1 acted as a potential ferroptosis inhibitor with the capacity to enhance antioxidant defences. This study suggests that Ferrostatin-1 could serve as a promising novel strategy for improving the treatment of AKI and promoting recovery from the condition.

The ferroptosis inducing compounds RSL3 and ML162 are not direct inhibitors of GPX4 but of TXNRD1.

Ferroptosis is defined as cell death triggered by iron-dependent lipid peroxidation that is preventable by antioxidant compounds such as ferrostatin-1. Endogenous suppressors of ferroptosis include FSP-1 and the selenoprotein GPX4, the latter of which directly enzymatically reduces lipid hydroperoxides. Small molecules that trigger ferroptosis include RSL3, ML162, and ML210; these compounds are often used in studies of ferroptosis and are generally considered as GPX4 inhibitors. Here, we found that RSL3 and ML162 completely lack capacity of inhibiting the enzymatic activity of recombinant selenoprotein GPX4. Surprisingly, these compounds were instead found to be efficient inhibitors of another selenoprotein, TXNRD1. Other known inhibitors of TXNRD1, including auranofin, TRi-1 and TRi-2, are also efficient inducers of cell death but that cell death could not be suppressed with ferrostatin-1. Our results collectively suggest that prior studies using RSL3 and ML162 may need to be reevaluated in the context of ferroptosis with regards to additional enzyme targets and mechanisms of action that may be involved.

Inhibition of ferroptosis protects sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is a serious and common complication of sepsis. To study the ferroptosis in the pathogenesis of SAE and demonstrate the protection effect of ferroptosis resistance, cognitive function, neurological deficits, blood-brain barrier integrity and neuroinflammation were detected. SAE model was established by cecal ligation and puncture (CLP) in mice and an in vitro model was created by introducing LPS to HT22 cells. Ferroptosis inducer Fe-citrate (Fe) and ferroptosis inhibitor ferrostatin-1 (Fer-1) was post-treated in the models, respectively. SAE caused ferroptosis, as evidenced by an increase in reactive oxygen species (ROS), iron content and malondialdehyde (MDA) and a decrease in glutathione (GSH) level, as well as changes in the expression of ferroptosis-related proteins as acyl-CoA synthetase long-chain family member 4 (ACSL4), glutathione peroxidase 4 (GPX4), and cystine-glutamate antiporter (SLC7A11), and harmed mitochondrial function. In contrast, inhibiting ferroptosis with Fer-1 attenuated ferroptosis. Meanwhile, Fer-1 attenuated neurologic severity score, learning and memory impairment, Fluoro-Jade C (FJC) staining, and decreased Evans Blue (EB) extravasation, microglia activation and TNF-α and IL-1ß production following SAE. The benefit of Fer-1 was diminished by ferroptosis inducer Fe. In addition, Fer-1 up-regulated the nuclear factor erythroid-2-related factor 2 (Nrf2)/ heme oxygenase-1(HO-1) signaling axis both in vivo and in vitro. In conclusion, our study revealed that Fer-1 might inhibit feroptosis in neurons by triggering the Nrf2/OH-1 pathway, thereby providing a therapeutic solution for SAE.

Derivatives of methoxetamine and major methoxetamine metabolites potently block NMDA receptors.

N-Methyl-D-aspartate receptors (NMDARs) in the brain are influenced by psychoactive drugs such as 2-(2-chlorophenyl)-2-(methylamino)cyclohexan-1-one (ketamine) and its analog 2-(ethylamino)-2-(3-methoxyphenyl)-cyclohexanone (methoxetamine). The recreational methoxetamine use can cause several toxicities and methoxetamine-related deaths have also been reported. Therefore, it has been banned in many countries. Since 2020, methoxetamine derivatives, 2-(ethylamino)-2-(m-tolyl)cyclohexan-1-one (deoxymethoxetamine) and 2-(isopropylamino)-2-(3-methoxyphenyl)cyclohexan-1-one (methoxisopropamine), have been sold online as designer drugs. However, how deoxymethoxetamine and methoxisopropamine act on NMDARs remains unknown. In this study, we first performed in silico docking studies of NMDARs, and deoxymethoxetamine and methoxisopropamine in addition to the major methoxetamine metabolites, 2-amino-2-(3-methoxyphenyl)-cyclohexanone (N-desethyl methoxetamine) and 2-(ethylamino)-2-(3-hydroxyphenyl)-cyclohexanone (O-desmethyl methoxetamine). The docking study suggested each compound interacts with NMDARs. We also determined the half-maximal inhibitory concentration (IC50s) of the methoxetamine-related compounds for NMDARs using NMDAR-expressing cartwheel interneurons of mice and patch-clamp recordings. We found that the IC50s of methoxetamine, deoxymethoxetamine, methoxisopropamine, N-desethyl methoxetamine, and O-desmethyl methoxetamine for NMDARs were 0.524, 0.679, 0.661, 1.649, and 0.227 µM, respectively. These results indicate that the methoxetamine-related compounds act as potent NMDAR blockers. Thus, deoxymethoxetamine and methoxisopropamine, both of which may cause damage by blocking NMDARs, are serious concerns. N-Desethyl methoxetamine and O-desmethyl methoxetamine may cause several adverse effects when methoxetamine is metabolized.

Endothelial cell ferroptosis mediates monocrotaline-induced pulmonary hypertension in rats by modulating NLRP3 inflammasome activation.

Inflammation triggers pulmonary vascular remodelling. Ferroptosis, a nonapoptotic form of cell death that is triggered by iron-dependent lipid peroxidation and contributes to the pathogenesis of several inflammation-related diseases, but its role in pulmonary hypertension (PH) has not been studied. We examined endothelial cell ferroptosis in PH and the potential mechanisms. Pulmonary artery endothelial cells (PAECs) and lung tissues from monocrotaline (MCT)-induced PH rats were analysed for ferroptosis markers, including lipid peroxidation, the labile iron pool (LIP) and the protein expression of glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1) and NADPH oxidase-4 (NOX4). The effects of the ferroptosis inhibitor ferrostatin-1 (Fer-1) on endothelial cell ferroptosis and pulmonary vascular remodelling in MCT-induced rats were studied in vitro and in vivo. Ferroptosis was observed in PAECs from MCT-induced PH rats in vitro and in vivo and was characterized by a decline in cell viability accompanied by increases in the LIP and lipid peroxidation, the downregulation of GPX4 and FTH1 expression and the upregulation of NOX4 expression. High-mobility group box 1 (HMGB1)/Toll-like receptor 4 (TLR4)/NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome signalling was measured by western blotting. These changes were significantly blocked by Fer-1 administration in vitro and in vivo. These results suggest that Fer-1 plays a role in inhibiting ferroptosis-mediated PAEC loss during the progression of PH. The ferroptosis-induced inflammatory response depended on the activation of HMGB1/TLR4 signalling, which activated the NLRP3 inflammasome in vivo. We are the first to suggest that pulmonary artery endothelial ferroptosis triggers inflammatory responses via the HMGB1/TLR4/NLRP3 inflammasome signalling pathway in MCT-induced rats. Treating PH with a ferroptosis inhibitor and exploring new treatments based on ferroptosis regulation might be promising therapeutic strategies for PH.

ISRIB plus bortezomib triggers paraptosis in breast cancer cells via enhanced translation and subsequent proteotoxic stress.

Despite the success of proteasome inhibitors (PIs) in treating hematopoietic malignancies, including multiple myeloma (MM), their clinical efficacy is limited in solid tumors. In this study, we investigated the involvement of the integrated stress response (ISR), a central cellular adaptive program that responds to proteostatic defects by tuning protein synthesis rates, in determining the fates of cells treated with PI, bortezomib (Bz). We found that Bz induces ISR, and this can be reversed by ISRIB, a small molecule that restores eIF2B-mediated translation during ISR, in both Bz-sensitive MM cells and Bz-insensitive breast cancer cells. Interestingly, while ISRIB protected MM cells from Bz-induced apoptosis, it enhanced Bz sensitivity in breast cancer cells by inducing paraptosis, the cell death mode that is accompanied by dilation of the endoplasmic reticulum (ER) and mitochondria. Combined treatment with ISRIB and Bz may shift the fate of Bz-insensitive cancer cells toward paraptosis by inducing translational rescue, leading to irresolvable proteotoxic stress.