Exosomal let-7a-5p derived from human umbilical cord mesenchymal stem cells alleviates coxsackievirus B3-induced cardiomyocyte ferroptosis via the SMAD2/ZFP36 signal axis. / 人脐带间å 质干细胞来源的外泌体let-7a-5p通过SMAD2/ZFP36ä¿¡å·è½´å‡è½»æŸ¯è¨å¥‡ç— æ¯’B3诱导的心肌细胞铁死亡.

Viral myocarditis (VMC) is one of the most common acquired heart diseases in children and teenagers. However, its pathogenesis is still unclear, and effective treatments are lacking. This study aimed to investigate the regulatory pathway by which exosomes alleviate ferroptosis in cardiomyocytes (CMCs) induced by coxsackievirus B3 (CVB3). CVB3 was utilized for inducing the VMC mouse model and cellular model. Cardiac echocardiography, left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) were implemented to assess the cardiac function. In CVB3-induced VMC mice, cardiac insufficiency was observed, as well as the altered levels of ferroptosis-related indicators (glutathione peroxidase 4 (GPX4), glutathione (GSH), and malondialdehyde (MDA)). However, exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exo) could restore the changes caused by CVB3 stimulation. Let-7a-5p was enriched in hucMSCs-exo, and the inhibitory effect of hucMSCs-exolet-7a-5p mimic on CVB3-induced ferroptosis was higher than that of hucMSCs-exomimic NC (NC: negative control). Mothers against decapentaplegic homolog 2 (SMAD2) increased in the VMC group, while the expression of zinc-finger protein 36 (ZFP36) decreased. Let-7a-5p was confirmed to interact with SMAD2 messenger RNA (mRNA), and the SMAD2 protein interacted directly with the ZFP36 protein. Silencing SMAD2 and overexpressing ZFP36 inhibited the expression of ferroptosis-related indicators. Meanwhile, the levels of GPX4, solute carrier family 7, member 11 (SLC7A11), and GSH were lower in the SMAD2 overexpression plasmid (oe-SMAD2)+let-7a-5p mimic group than in the oe-NC+let-7a-5p mimic group, while those of MDA, reactive oxygen species (ROS), and Fe2+ increased. In conclusion, these data showed that ferroptosis could be regulated by mediating SMAD2 expression. Exo-let-7a-5p derived from hucMSCs could mediate SMAD2 to promote the expression of ZFP36, which further inhibited the ferroptosis of CMCs to alleviate CVB3-induced VMC.

PLAG1 interacts with GPX4 to conquer vulnerability to sorafenib induced ferroptosis through a PVT1/miR-195-5p axis-dependent manner in hepatocellular carcinoma.

BACKGROUND: Sorafenib is a standard first-line treatment for advanced hepatocellular carcinoma (HCC), yet its effectiveness is often constrained. Emerging studies reveal that sorafenib triggers ferroptosis, an iron-dependent regulated cell death (RCD) mechanism characterized by lipid peroxidation. Our findings isolate the principal target responsible for ferroptosis in HCC cells and outline an approach to potentially augment sorafenib's therapeutic impact on HCC. METHODS: We investigated the gene expression alterations following sgRNA-mediated knockdown induced by erastin and sorafenib in HCC cells using CRISPR screening-based bioinformatics analysis. Gene set enrichment analysis (GSEA) and the "GDCRNATools" package facilitated the correlation studies. We employed tissue microarrays and cDNA microarrays for validation. Ubiquitination assay, Chromatin immunoprecipitation (ChIP) assay, RNA immunoprecipitation (RIP) assay, and dual-luciferase reporter assay were utilized to delineate the specific mechanisms underlying ferroptosis in HCC cells. RESULTS: Our study has revealed that pleiomorphic adenoma gene 1 (PLAG1), a gene implicated in pleomorphic adenoma, confers resistance to ferroptosis in HCC cells treated with sorafenib. Sorafenib leads to the opposite trend of protein and mRNA levels of PLAG1, which is not caused by affecting the stability or ubiquitination of PLAG1 protein, but by the regulation of PLAG1 at the transcriptional level by its upstream competitive endogenous long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1). Data from 139 HCC patients showed a significant positive correlation between PLAG1 and GPX4 levels in tumor samples, and PLAG1 is instrumental in redox homeostasis by driving the expression of glutathione peroxidase 4 (GPX4), the enzyme that reduces lipid peroxides (LPOs), which further leads to ferroptosis inhibition. CONCLUSIONS: Ferroptosis is a promising target for cancer therapy, especially for patients resistant to standard chemotherapy or immunotherapy. Our findings indicate that PLAG1 holds therapeutic promise and may enhance the efficacy of sorafenib in treating HCC.

Targeting BMAL1 reverses drug resistance of acute myeloid leukemia cells and promotes ferroptosis through HMGB1-GPX4 signaling pathway.

PURPOSE: Acute myeloid leukemia (AML) is a refractory hematologic malignancy that poses a serious threat to human health. Exploring alternative therapeutic strategies capable of inducing alternative modes of cell death, such as ferroptosis, holds great promise as a viable and effective intervention. METHODS: We analyzed online database data and collected clinical samples to verify the expression and function of BMAL1 in AML. We conducted experiments on AML cell proliferation, cell cycle, ferroptosis, and chemotherapy resistance by overexpressing/knocking down BMAL1 and using assays such as MDA detection and BODIPY 581/591 C11 staining. We validated the transcriptional regulation of HMGB1 by BMAL1 through ChIP assay, luciferase assay, RNA level detection, and western blotting. Finally, we confirmed the results of our cell experiments at the animal level. RESULTS: BMAL1 up-regulation is an observed phenomenon in AML patients. Furthermore, there existed a strong correlation between elevated levels of BMAL1 expression and inferior prognosis in individuals with AML. We found that knocking down BMAL1 inhibited AML cell growth by blocking the cell cycle. Conversely, overexpressing BMAL1 promoted AML cell proliferation. Moreover, our research results revealed that BMAL1 inhibited ferroptosis in AML cells through BMAL1-HMGB1-GPX4 pathway. Finally, knocking down BMAL1 can enhance the efficacy of certain first-line cancer therapeutic drugs, including venetoclax, dasatinib, and sorafenib. CONCLUSION: Our research results suggest that BMAL1 plays a crucial regulatory role in AML cell proliferation, drug resistance, and ferroptosis. BMAL1 could be a potential important therapeutic target for AML.

Blue light induced ferroptosis via STAT3/GPX4/SLC7A11/FTH1 in conjunctiva epithelium in vivo and in vitro.

The prevalence of Light-emitting diodes (LEDs) has exposed us to an excessive amount of blue light (BL) which causes various ophthalmic diseases. Previous studies have shown that conjunctiva is vulnerable to BL. In this study, we aimed to investigate the underlying mechanism of BL-induced injury in conjunctiva. We placed C57BL/6 mice and human conjunctival epithelial cell lines (HCECs) under BL (440 nm ± 15 nm, 0.2 mW/cm2) to establish a BL injury model in vivo and in vitro. Immunohistochemistry and MDA assay were used to identify lipid peroxidation (LPO) in vivo. HE staining was applied to detect morphological damage of conjunctival epithelium. DCFH-DA, C11-BODIPY 581/591, Calcein-AM, and FeRhoNox™-1 probes were performed to identify ferroptosis levels in vitro. Real-time qPCR and Western blotting techniques were employed to uncover signaling pathways of blue light-induced ferroptosis. Our findings demonstrated that BL affected tear film instability and induced conjunctival epithelium injury in vivo. Ferrostatin-1 significantly alleviated blue light-induced ferroptosis in vivo and in vitro. BL downregulates the levels of solute carrier family 7 member 11 (SLC7A11), Ferritin heavy chain (FTH1), and glutathione peroxidase (GPX4) by inhibiting the activation and translocation of the Signal transducer and activator of transcription 3 (STAT3) from inducing Fe2+ burst, ROS and LPO accumulation, ultimately resulting in ferroptosis. This study will offer new insight into BL-induced conjunctival injury and LED-induced dry eye.

Pinobanksin ameliorated DSS-induced acute colitis mainly through modulation of SLC7A11/glutathione-mediated intestinal epithelial ferroptosis.

Inhibition of ferroptosis in intestinal epithelial cells serves as an attractive target for the development of therapeutic strategies for colitis. Pinobanksin, one of the main flavonoids derived from propolis, possesses significant anti-inflammatory effects and inhibits the cell death of several cell lines. Here, we evaluated whether pinobanksin influenced colitis by modulation of epithelial ferroptosis. Mice treated with 2.5% DSS dissolved in sterile distilled water were established for an acute colitis model. The mitochondrial morphology, colonic iron level, lipid peroxidation products MDA/4-HNE, and lipid reactive oxygen species levels were measured to assess ferroptosis in epithelial cells. RNA-seq and functional analyses were performed to reveal key genes mediating pinobanksin-exerted modulation of ferroptosis. We found that pinobanksin, at different doses, induced significant anti-colitis effects and inhibited the elevated ferroptosis in colonic epithelial cells isolated from DSS-treated mice largely by activating GPX4 (negative regulator of ferroptosis). Furthermore, RNA-seq assays indicated that pinobanksin significantly increased the cystine transporter SLC7A11 in colonic tissues from mice with colitis. Depletion of SLC7A11 largely blocked pinobanksin-induced promotion of cystine uptake/glutathione biosynthesis and suppression of ferroptosis in epithelial cells from mice with colitis or IEC-6 cells pretreated with RSL3. Altogether, pinobanksin alleviated DSS-induced colitis largely by inhibition of ferroptosis in epithelial cells. Activation of SLC7A11 by pinobanksin resulted in the promotion of cystine uptake and enhancement of glutathione biosynthesis. This work will provide novel guidance for the clinical use of pinobanksin to treat colitis through inhibition of epithelial ferroptosis.

LncRNA FRMD6-AS1/miR-491-5p/USP13 pathway attenuated ferroptosis and contributed to liver fibrosis.

Liver fibrosis is an invertible pathophysiologic process featured by excessive accumulation of extracellular matrix (ECM) which injures liver cells and activates hepatic stellate cells (HSCs). Besides, inducing ferroptosis in activated HSCs can alleviate liver fibrosis. LncRNAs modulate ferroptosis in activated HSCs and ECM deposition in liver fibrosis. However, the role of lncRNA FRMD6-AS1 in liver fibrosis is not discovered. In this study, lncRNA FRMD6-AS1 was dramatically up-regulated in activated HSCs. Knockdown of FRMD6-AS1 markedly increased iron ion, ROS and MDA levels, decreased GSH level, SLC7A11 and GPX4 protein expressions in activated HSCs. In addition, HSCs activation markers α-SMA and COL1α1 expressions were up-regulated in activated HSCs; knockdown of FRMD6-AS1 markedly down-regulated α-SMA and COL1α1 expressions in HSCs. Besides, lncRNA FRMD6-AS1 could interact with miR-491-5p, and negatively modulate miR-491-5p expression. USP13 was a target of miR-491-5p, and could be negatively modulated by miR-491-5p. Moreover, FRMD6-AS1 knockdown increased iron ion and ROS levels, decreased SLC7A11 and GPX4 protein expressions, facilitated HSCs viability, and up-regulated α-SMA and COL1α1 expressions via miR-491-5p/USP13 pathway. Finally, FRMD6-AS1 knockdown restored liver tissue structure and abrogated fibrosis in livers in a CCL4 liver fibrosis mouse model. Hence, lncRNA FRMD6-AS1/miR-491-5p/USP13 pathway repressed ferroptosis, promoted ECM deposition and facilitated liver fibrosis in vitro and in vivo models.

FAdV-4-induced ferroptosis affects fat metabolism in LMH cells.

Ferroptosis is a form of controlled cell death that was first described relatively recently and that is dependent on the formation and accumulation of lipid free radicals through an iron-mediated mechanism. A growing body of evidence supports the close relationship between pathogenic infections and ferroptotic cell death, particularly for viral infections. Ferroptosis is also closely tied to the pathogenic development of hepatic steatosis and other forms of liver disease. Fowl adenovirus serotype 4 (FAdV-4) is a hepatotropic aviadenovirus causing hydropericardium syndrome (HPS) that is capable of impacting fat metabolism. However, it remains uncertain as to what role, if any, ferroptotic death plays in the context of FAdV-4 infection. Here, FAdV-4 was found to promote ferroptosis via the p53-SLC7A11-GPX4 axis, while ferrostain-1 was capable of inhibiting this FAdV-4-mediated ferroptotic death through marked reductions in lipid peroxidation. The incidence of FAdV-4-induced fatty liver was also found to be associated with the activation of ferroptotic activity. Together, these results offer novel insights regarding potential approaches to treating HPS.

USP8-governed GPX4 homeostasis orchestrates ferroptosis and cancer immunotherapy.

Ferroptosis is an iron-dependent type of regulated cell death resulting from extensive lipid peroxidation and plays a critical role in various physiological and pathological processes. However, the regulatory mechanisms for ferroptosis sensitivity remain incompletely understood. Here, we report that homozygous deletion of Usp8 (ubiquitin-specific protease 8) in intestinal epithelial cells (IECs) leads to architectural changes in the colonic epithelium and shortens mouse lifespan accompanied by increased IEC death and signs of lipid peroxidation. However, mice with heterozygous deletion of Usp8 in IECs display normal phenotype and become resistant to azoxymethane/dextran sodium sulfate-induced colorectal tumorigenesis. Mechanistically, USP8 interacts with and deubiquitinates glutathione peroxidase 4 (GPX4), leading to GPX4 stabilization. Thus, USP8 inhibition destabilizes GPX4 and sensitizes cancer cells to ferroptosis in vitro. Notably, USP8 inhibition in combination with ferroptosis inducers retards tumor growth and enhances CD8+ T cell infiltration, which potentiates tumor response to anti-PD-1 immunotherapy in vivo. These findings uncover that USP8 counteracts ferroptosis by stabilizing GPX4 and highlight targeting USP8 as a potential therapeutic strategy to boost ferroptosis for enhancing cancer immunotherapy.

Protective Role of Dioscin against Doxorubicin-Induced Chronic Cardiotoxicity: Insights from Nrf2-GPX4 Axis-Mediated Cardiac Ferroptosis.

Recent evidence suggests that ferroptosis, an iron-facilitated cell death with excessive lipid peroxidation, is a critical mechanism underlying doxorubicin (DOX)-induced cardiotoxicity (DIC). Although dioscin has been reported to improve acute DIC, direct evidence is lacking to clarify the role of dioscin in chronic DIC and its potential mechanism in cardiac ferroptosis. In this study, we used chronic DIC rat models and H9c2 cells to investigate the potential of dioscin to mitigate DIC by inhibiting ferroptosis. Our results suggest that dioscin significantly improves chronic DIC-induced cardiac dysfunction. Meanwhile, it significantly inhibited DOX-induced ferroptosis by reducing Fe2+ and lipid peroxidation accumulation, maintaining mitochondrial integrity, increasing glutathione peroxidase 4 (GPX4) expression, and decreasing acyl-CoA synthetase long-chain family 4 (ACSL4) expression. Through transcriptomic analysis and subsequent validation, we found that the anti-ferroptotic effects of dioscin are achieved by regulating the nuclear factor-erythroid 2-related factor 2 (Nrf2)/GPX4 axis and Nrf2 downstream iron metabolism genes. Dioscin further downregulates nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and upregulates expression of frataxin (FXN) and ATP-binding cassette B8 (ABCB8) to limit mitochondrial Fe2+ and lipid peroxide accumulation. However, Nrf2 inhibition diminishes the anti-ferroptotic effects of dioscin, leading to decreased GPX4 expression and increased lipid peroxidation. This study is a compelling demonstration that dioscin can effectively reduce DIC by inhibiting ferroptosis, which is dependent on the Nrf2/GPX4 pathway modulation.

Ferroptosis in liver cancer: a key role of post-translational modifications.

Ferroptosis is an emerging form of regulated cell death in an oxidative stress- and iron-dependent manner, primarily induced by the over-production of reactive oxygen species (ROS). Manipulation of ferroptosis has been considered a promising therapeutic approach to inhibit liver tumor growth. Nevertheless, the development of resistance to ferroptosis in liver cancer poses a significant challenge in cancer treatment. Post-translational modifications (PTMs) are crucial enzymatic catalytic reactions that covalently regulate protein conformation, stability and cellular activities. Additionally, PTMs play pivotal roles in various biological processes and divergent programmed cell death, including ferroptosis. Importantly, key PTMs regulators involved in ferroptosis have been identified as potential targets for cancer therapy. PTMs function of two proteins, SLC7A11, GPX4 involved in ferroptosis resistance have been extensively investigated in recent years. This review will summarize the roles of PTMs in ferroptosis-related proteins in hepatocellular carcinoma (HCC) treatment.

N6022 attenuates cerebral ischemia/reperfusion injury-induced microglia ferroptosis by promoting Nrf2 nuclear translocation and inhibiting the GSNOR/GSTP1 axis.

Stroke poses a significant risk of mortality, particularly among the elderly population. The pathophysiological process of ischemic stroke is complex, and it is crucial to elucidate its molecular mechanisms and explore potential protective drugs. Ferroptosis, a newly recognized form of programmed cell death distinct from necrosis, apoptosis, and autophagy, is closely associated with the pathophysiology of ischemic stroke. N6022, a selective inhibitor of S-nitrosoglutathione reductase (GSNOR), is a "first-in-class" drug for asthma with potential therapeutic applications. However, it remains unclear whether N6022 exerts protective effects in ischemic stroke, and the precise mechanisms of its action are unknown. This study aimed to investigate whether N6022 mitigates cerebral ischemia/reperfusion (I/R) injury by reducing ferroptosis and to elucidate the underlying mechanisms. Accordingly, we established an oxygen-glucose deprivation/reperfusion (OGD/R) cell model and a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model to mimic cerebral I/R injury. Our data, both in vitro and in vivo, demonstrated that N6022 effectively protected against I/R-induced brain damage and neurological deficits in mice, as well as OGD/R-induced BV2 cell damage. Mechanistically, N6022 promoted Nrf2 nuclear translocation, enhancing intracellular antioxidant capacity of SLC7A11-GPX4 system. Furthermore, N6022 interfered with the interaction of GSNOR with GSTP1, thereby boosting the antioxidant capacity of GSTP1 and attenuating ferroptosis. These findings provide novel insights, showing that N6022 attenuates microglial ferroptosis induced by cerebral I/R injury through the promotion of Nrf2 nuclear translocation and inhibition of the GSNOR/GSTP1 axis.

GPX4 degradation contributes to fluoride-induced neuronal ferroptosis and cognitive impairment via mtROS-chaperone-mediated autophagy.

Ferroptosis is a newly recognized type of programmed cell death that is implicated in the pathophysiological process of neurological disorders. Our previous studies have revealed that exposure to high concentrations of fluoride for long periods of time induces hippocampal neural injury and cognitive deficits. However, whether ferroptosis is involved in fluoride-induced neuronal death and the underlying mechanism remain unknown. In this study, the results indicated that exposure to high fluoride triggered ferroptosis in SH-SY5Y cells and in the hippocampus of mice. Fluoride exposure accelerated the lysosomal degradation of GPX4 and led to neuronal ferroptosis, while GPX4 overexpression protected SH-SY5Y cells against fluoride-induced neurotoxicity. Intriguingly, the enhanced chaperone-mediated autophagy (CMA) induced by fluoride stimulation was responsible for GPX4 degradation because the inhibition of CMA activity by LAMP2A knockdown effectively prevented fluoride-induced GPX4 loss. Furthermore, mitochondrial ROS (mtROS) accumulation caused by fluoride contributed to CMA activation-mediated GPX4 degradation and subsequent neuronal ferroptosis. Notably, the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) or the ROS scavenger N-acetyl-L-cysteine (NAC) alleviated fluoride-evoked hippocampal neuronal death and synaptic injury as well as cognitive deficits in mice. The present studies indicates that ferroptosis is a novel mechanism of fluoride-induced neurotoxicity and that chronic fluoride exposure facilitates GPX4 degradation via mtROS chaperone-mediated autophagy, leading to neuronal ferroptosis and cognitive impairment.

Orexin-A mediates glioblastoma proliferation inhibition by increasing ferroptosis triggered by unstable iron pools and GPX4 depletion.

Glioblastoma (GBM) represents a prevalent form of primary malignant tumours in the central nervous system, but the options for effective treatment are extremely limited. Ferroptosis, as the most enriched programmed cell death process in glioma, makes a critical difference in glioma progression. Consequently, inducing ferroptosis has become an appealing strategy for tackling gliomas. Through the utilization of multi-omics sequencing data analysis, flow cytometry, MDA detection and transmission electron microscopy, the impact of orexin-A on ferroptosis in GBM was assessed. In this report, we provide the first evidence that orexin-A exerts inhibitory effects on GBM proliferation via the induction of ferroptosis. This induction is achieved by instigating an unsustainable increase in iron levels and depletion of GPX4. Moreover, the regulation of TFRC, FTH1 and GPX4 expression through the targeting of NFE2L2 appears to be one of the potential mechanisms underlying orexin-A-induced ferroptosis.

Lipid peroxidation-induced ferroptosis as a therapeutic target for mitigating neuronal injury and inflammation in sepsis-associated encephalopathy: insights into the hippocampal PEBP-1/15-LOX/GPX4 pathway.

BACKGROUND: Sepsis-associated encephalopathy (SAE) refers to the widespread impairment of brain function caused by noncentral nervous system infection mediated by sepsis. Lipid peroxidation-induced ferroptosis contributes to the occurrence and course of SAE. This study aimed to investigate the relationship between neuronal injury and lipid peroxidation-induced ferroptosis in SAE. METHODS: Baseline data were collected from pediatric patients upon admission, and the expression levels of various markers related to lipid peroxidation and ferroptosis were monitored in the serum and peripheral blood mononuclear cells (PBMCs) of patients with SAE as well as SAE model mice. The hippocampal phosphatidylethanolamine-binding protein (PEBP)-1/15-lysine oxidase (LOX)/ glutathione peroxidase 4 (GPX4) pathway was assessed for its role on the inhibitory effect of ferroptosis in SAE treatment. RESULTS: The results showed elevated levels of S100 calcium-binding protein beta (S-100ß), glial fibrillary acidic protein, and malondialdehyde in the serum of SAE patients, while superoxide dismutase levels were reduced. Furthermore, analysis of PBMCs revealed increased transcription levels of PEBP1, LOX, and long-chain fatty acyl-CoA synthetase family member 4 (ACSL4) in SAE patients, while the transcription levels of GPX4 and cystine/glutamate transporter xCT (SLC7A11) were decreased. In comparison to the control group, the SAE mice exhibited increased expression of S-100ß and neuron-specific enolase (NSE) in the hippocampus, whereas the expression of S-100ß and NSE were reduced in deferoxamine (DFO) mice. Additionally, iron accumulation was observed in the hippocampus of SAE mice, while the iron ion levels were reduced in the DFO mice. Inhibition of ferroptosis alleviated the mitochondrial damage (as assessed by transmission electron microscopy, hippocampal mitochondrial ATP detection, and the JC-1 polymer-to-monomer ratio in the hippocampus) and the oxidative stress response induced by SAE as well as attenuated neuroinflammatory reactions. Further investigations revealed that the mechanism underlying the inhibitory effect of ferroptosis in SAE treatment is associated with the hippocampal PEBP-1/15-LOX/GPX4 pathway. CONCLUSION: These results offer potential therapeutic targets for the management of neuronal injury in SAE and valuable insights into the potential mechanisms of ferroptosis in neurological disorders.

Glutathione peroxidase 4 restrains temporomandibular joint osteoarthritis progression by inhibiting ferroptosis.

There are few effective therapeutic strategies for temporomandibular joint osteoarthritis (TMJOA) due to the unclear pathology and mechanisms. We aimed to confirm the roles of GPX4 and ferroptosis in TMJOA progression. ELISA assay was hired to evaluate concentrations of ferroptosis-related markers. The qRT-PCR assay was hired to assess gene mRNA level. Western blot assay and immunohistochemistry were hired to verify the protein level. CCK-8 assay was hired to detect cell viability. Human fibroblast-like synoviocytes (FLSs) were cultured to confirm the effects of GPX4 and indicated inhibitors, and further verified the effects of GPX4 and ferroptosis inhibitors in TMJOA model rats. Markers of ferroptosis including 8-hidroxy-2-deoxyguanosine (8-OHdG) and iron were notably increased in TMJOA tissues and primary OA-FLSs. However, the activity of the antioxidant system including the glutathione peroxidase activity, glutathione (GSH) contents, and glutathione/oxidized glutathione (GSH/GSSG) ratio was notably inhibited in TMJOA tissues, and the primary OA-FLSs. Furthermore, the glutathione peroxidase 4 (GPX4) expression was down-regulated in TMJOA tissues and primary OA-FLSs. Animal and cell experiments have shown that ferroptosis inhibitors notably inhibited ferroptosis and promoted HLS survival as well as up-regulated GPX4 expression. Also, GPX4 knockdown promoted ferroptosis and GPX4 overexpression inhibited ferroptosis. GPX4 also positively regulated cell survival which was the opposite with ferroptosis. In conclusion, GPX4 and ferroptosis regulated the progression of TMJOA. Targeting ferroptosis might be an effective therapeutic strategy for TMJOA patients in the clinic.

Downregulated GPX4 in salivary gland epithelial cells contributes to salivary secretion dysfunction in Sjogren's syndrome via lipid ROS/pSTAT4/AQP5 axis.

Sjogren's syndrome (SS) is an autoimmune disease characterized by dysfunction of exocrine glands, such as salivary glands. However, the molecular mechanism of salivary secretion dysfunction in SS is still unclear. Given the significance of glutathione peroxidase 4 (GPX4) in cellular redox homeostasis, we hypothesized that dysregulation of GPX4 may play a pivotal role in the pathogenesis of salivary secretion dysfunction observed in SS. The salivary gland of SS patients and the SS mouse model exhibited reduced expression of the ferroptosis inhibitor GPX4 and the important protein aquaporin 5 (AQP5), which is involved in salivary secretion. GPX4 overexpression upregulated and GPX4 knockdown downregulated AQP5 expression in salivary gland epithelial cells (SGECs) and salivary secretion. Bioinformatics analysis of GSE databases from SS patients' salivary glands revealed STAT4 as a key intermediary regulator between GPX4 and AQP5. A higher level of nuclear pSTAT4 was observed in the salivary gland of the SS mouse model. GPX4 overexpression inhibited and GPX4 knockdown promoted STAT4 phosphorylation and nuclear translocation in SGECs. CHIP assay confirmed the binding of pSTAT4 within the promoter of AQP5 inhibiting AQP5 transcription. GPX4 downregulation accumulates intracellular lipid ROS in SGECs. Lipid ROS inhibitor ferrostatin-1 treatment during in vitro and in vivo studies confirmed that lipid ROS activates STAT4 phosphorylation and nuclear translocation in SGECs. In summary, the downregulated GPX4 in SGECs contributes to salivary secretion dysfunction in SS via the lipid ROS/pSTAT4/AQP5 axis. This study unraveled novel targets to revitalize the salivary secretion function in SS patients.

Vitamin B6 alleviates chronic sleep deprivation-induced hippocampal ferroptosis through CBS/GSH/GPX4 pathway.

Several studies have found that sleep deprivation (SD) can lead to neuronal ferroptosis and affect hippocampal function. However, there are currently no effective interventions. Vitamin B6 is a co-factor for key enzymes in the transsulfuration pathway which is critical for maintaining cell growth in the presence of cysteine deprivation. The results showed that SD inhibited cystine-glutamate antiporter light chain subunit xCT protein expression and caused cysteine deficiency, which reduced the synthesis of the glutathione (GSH) to trigger neuronal ferroptosis. Nissl staining further revealed significant neuronal loss and shrinkage in the CA1 and CA3 regions of the hippocampus in SD mice. Typical ferroptotic indicators characterized by lipid peroxidation and iron accumulation were showed in the hippocampus after sleep deprivation. As expected, vitamin B6 could alleviate hippocampal ferroptosis by upregulating the expression of cystathionine beta-synthase (CBS) in the transsulfuration pathway, thereby replenishing the intracellular deficient GSH and restoring the expression of GPX4. Similar anti-ferroptotic effects of vitamin B6 were demonstrated in HT-22 cells treated with ferroptosis activator erastin. Furthermore, vitamin B6 had no inhibitory effect on erastin-induced ferroptosis in CBS-knockout HT22 cells. Our findings suggested chronic sleep deprivation caused hippocampal ferroptosis by disrupting the cyst(e)ine/GSH/GPX4 axis. Vitamin B6 alleviated sleep deprivation-induced ferroptosis by enhancing CBS expression in the transsulfuration pathway.

GPX4, ferroptosis, and diseases.

GPX4 (Glutathione peroxidase 4) serves as a crucial intracellular regulatory factor, participating in various physiological processes and playing a significant role in maintaining the redox homeostasis within the body. Ferroptosis, a form of iron-dependent non-apoptotic cell death, has gained considerable attention in recent years due to its involvement in multiple pathological processes. GPX4 is closely associated with ferroptosis and functions as the primary inhibitor of this process. Together, GPX4 and ferroptosis contribute to the pathophysiology of several diseases, including sepsis, nervous system diseases, ischemia reperfusion injury, cardiovascular diseases, and cancer. This review comprehensively explores the regulatory roles and impacts of GPX4 and ferroptosis in the development and progression of these diseases, with the aim of providing insights for identifying potential therapeutic strategies in the future.

Peroxiredoxin 6 suppresses ferroptosis in lung endothelial cells.

Peroxiredoxin 6 (Prdx6) repairs peroxidized membranes by reducing oxidized phospholipids, and by replacing oxidized sn-2 fatty acyl groups through hydrolysis/reacylation by its phospholipase A2 (aiPLA2) and lysophosphatidylcholine acyltransferase activities. Prdx6 is highly expressed in the lung, and intact lungs and cells null for Prdx6 or with single-point mutations that inactivate either Prdx6-peroxidase or aiPLA2 activity alone exhibit decreased viability, increased lipid peroxidation, and incomplete repair when exposed to paraquat, hyperoxia, or organic peroxides. Ferroptosis is form of cell death driven by the accumulation of phospholipid hydroperoxides. We studied the role of Prdx6 as a ferroptosis suppressor in the lung. We first compared the expression Prdx6 and glutathione peroxidase 4 (GPx4) and visualized Prdx6 and GPx4 within the lung. Lung Prdx6 mRNA levels were five times higher than GPx4 levels. Both Prdx6 and GPx4 localized to epithelial and endothelial cells. Prdx6 knockout or knockdown sensitized lung endothelial cells to erastin-induced ferroptosis. Cells with genetic inactivation of either aiPLA2 or Prdx6-peroxidase were more sensitive to ferroptosis than WT cells, but less sensitive than KO cells. We then conducted RNA-seq analyses in Prdx6-depleted cells to further explore how the loss of Prdx6 sensitizes lung endothelial cells to ferroptosis. Prdx6 KD upregulated transcriptional signatures associated with selenoamino acid metabolism and mitochondrial function. Accordingly, Prdx6 deficiency blunted mitochondrial function and increased GPx4 abundance whereas GPx4 KD had the opposite effect on Prdx6. Moreover, we detected Prdx6 and GPx4 interactions in intact cells, suggesting that both enzymes cooperate to suppress lipid peroxidation. Notably, Prdx6-depleted cells remained sensitive to erastin-induced ferroptosis despite the compensatory increase in GPx4. These results show that Prdx6 suppresses ferroptosis in lung endothelial cells and that both aiPLA2 and Prdx6-peroxidase contribute to this effect. These results also show that Prdx6 supports mitochondrial function and modulates several coordinated cytoprotective pathways in the pulmonary endothelium.

Butylated hydroxyanisole induces vascular endothelial injury via TFEB-mediated degradation of GPX4 and FTH1.

Butylated hydroxyanisole (BHA) is one of the most commonly used antioxidants and is widely used in food, but whether it causes vascular damage has not been clearly studied. The present study demonstrated for the first time that BHA reduced the viability of human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (BEND3) in a dose- and time-dependent manner. Moreover, BHA inhibited the migration and proliferation of vascular endothelial cells (ECs). Further analysis revealed that in ECs, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the BHA-induced increase in Fe2+ and malonaldehyde (MDA) levels. Acridine orange staining demonstrated that BHA increased lysosomal permeability. At the protein level, BHA increased the expression of transcription factor EB (TFEB) and decreased the expression of glutathione peroxidase (GPX4), solute carrier family 7 member 11 (SLC7A11, xCT), and ferritin heavy chain 1 (FTH1). Moreover, these effects of BHA could be reversed by knocking down TFEB. In vivo experiments confirmed that BHA caused elevated pulse wave velocity (PWV) and reduced acetylcholine-dependent vascular endothelial diastole. In conclusion, BHA degrades GPX4, xCT, and FTH1 through activation of the TFEB-mediated lysosomal pathway and promotes ferroptosis, ultimately leading to vascular endothelial cell injury.