Deletion of GPX8 Enhances Irradiation-Induced Ferroptosis Through m6A Hypomethylation-Mediated Upregulation of ACSL4 in Oral Cancer.

Background and Aims Ferroptosis is a regulated form of necrosis which is dependent on cellular iron and is characterized by the accumulation of lipid peroxides and failure of cellular antioxidant defences. We have previously described in vivo that ferroptosis is the primary cause of folic acid-induced acute kidney injury (FA-AKI) and that necroinflammation secondary to ferroptosis may further worsen kidney injury, since ferroptosis inhibition improved kidney function and decreased tubular cell death and oxidative stress. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is involved in the incorporation of polyunsaturated fatty acid (PUFA) into membranes, while 15-Lipoxygenase (Alox15) catalyzes the regio- and enantioselective peroxidation of membrane-esterified PUFAs, forming the ultimate peroxidized species that induce ferroptosis. In the present work, we aim to explore the potential of both proteins as therapeutic targets in AKI, as well as the molecular characterization of kidney ferroptosis in cultured tubular cells. Method Animal model: Female 12- to 14-week-old C57BL/6J wild type mice received a single intraperitoneal (i.p) injection of folic acid or vehicle and were sacrificed 48 hours later. Troglitazone was intravenously administered for in vivo ACSL4 inhibition, and Compound 1 was i.p administered for in vivo ALOX15 inhibition. In vitro characterization of ferroptosis: HK2 human tubular kidney cells were incubated with arachidonic acid (AA) prior to sublethal RSL3 administration for sensitization to ferroptosis. We used Ferrostatin-1 (Fer-1) and Liproxstatin-1 (Lpx-1) as inhibitors of ferroptosis. The thiazolidinedione family members Rosiglitazone, Pioglitazone and Troglitazone were used as pharmacological inhibitors of ACSL4. We also performed transcriptional silencing of ACSL4 using a specific siRNA. Compound 1 and PD1646 were administered for in vitro Alox15 inhibition. Cellular lipid peroxidation was analyzed by flow cytometry with Bodipy 581/591 staining. To characterize the lipid signature of ferroptotic cells, supernatants were collected and 15-HETEs levels were measured by ELISA. Oxido-lipidomic analysis of ferroptotic cells was performed by LC-MS. Real Time PCR and Western Blot of whole kidneys and cultured cells were performed for the detection and quantification of ACSL4 and ALOX15. Cell death was assessed by measurement of cell viability (MTT assay) and cytotoxicity (LDH assay). Tissue cell death was assessed by TUNEL. Results Kidney transcriptomics identified Acsl4 as the most upregulated member of Acsl family during FA-AKI. This was validated at mRNA and protein levels. Supplementation with AA, the preferred ACSL4 substrate, sensitized HK2 tubular cells to ferroptosis under sublethal RSL3 conditions, and this was prevented with specific ferroptosis inhibitors Fer-1 and Lpx-1. Pharmacological inhibition of ACSL4 with Troglitazone and a specific siRNA protected from cell death and lipid peroxidation induced by co-stimulation of AA and RSL3 in HK2 cells. Likewise, ALOX15 proteins levels were also increased in FA-AKI at 48 hours. PD1646 and Compound 1, both used to inhibit ALOX15, protected from ferroptotic cell death and lipid peroxidation in HK2 cells. Lipidomic analysis on HK2 tubular cells stimulated with AA+RSL3 uncovered an increased content of key peroxidized lipid species involved in ferroptosis execution, which was alleviated by targeting ACSL4 or ALOX15. Additionally, renal function of FA-AKI in mice was improved by ACSL4 pharmacological inhibition with Troglitazone and with ALOX15 inhibition with Compound 1. Conclusion Our preliminary results suggest therapeutic potential of ACSL4 and ALOX15 as ferroptotic targes during AKI. High PUFA content sensitizes human tubular HK2 cells to cell death and lipid peroxidation in the presence of sublethal ferroptotic triggers, and this was prevented by targeting ACSL4 or ALOX15 both in vitro and in vivo in FA-AKI. Closely related lipid peroxide species were decreased by ACSL4 or ALOX15 inhibitors.

Lung ischemia-reperfusion (IR) injury is a common clinical pathology associated with high mortality. Ferroptosis, a novel mode of cell death elicited by iron-dependent phospholipid peroxidation, has been implicated in ischemic events. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is one of the main enzymes in pro-ferroptotic lipid metabolism. In this study, the involvement of ferroptotic death in different durations of reperfusion was evaluated by assessing the iron content, malondialdehyde, and glutathione levels, ferroptosis-related protein expression, and mitochondria morphology. The roles of ferroptosis-specific inhibitor, liproxastin-1 (Lip-1), and ACSL4 modulation in a preventive regimen were assessed in vivo and in vitro. The hallmarks of pulmonary function, such as histological lung injury score, wet/dry ratio, and oxygenation index, were evaluated as well. Results showed that lung IR increased the tissue iron content and lipid peroxidation accumulation, along with key protein (GPX4 and ACSL4) expression alteration during reperfusion. Pretreatment with Lip-1 inhibited ferroptosis and ameliorated lung IR-induced injury in animal and cell models. In addition, administering ACSL4 inhibitor rosiglitazone before ischemia diminished the ferroptotic damage in IR-injured lung tissue, consistent with the protective effect of ACSL4 knockdown on lung epithelial cells subjected to hypoxia/reoxygenation. Thus, this study delineated that IR-induced ferroptotic cell death in lung tissue and ACSL4 were correlated with this process. Inhibition of ferroptosis and ACSL4 mitigated the ferroptotic damage in IR-induced lung injury by reducing lipid peroxidation and increasing the glutathione and GPX4 levels.

Acyl-CoA Synthetase long-chain family member 4 (ACSL4) is a member of acyl-CoA synthetase protein long-chain family, which is associated with amino acid synthesis, lipid synthesis and lipid peroxidation dependent iron death. However, the role of ACSL4 in generalized carcinoma remains unclear. We aim to analyze the expression and prognostic value of ACSL4 in pan-cancer, and further explore the correlation between ACSL4 and immune infiltration. Through ONCOMINE, TIMER (Tumor Immune Estimation Resource), GEPIA (Gene expression Profiling Interactive), UALCAN and HPA, ACSL4 expression patterns of in pan-cancer were analyzed. The prognostic value of ACSL4 was analyzed using PrognoScan and Kaplan-Meier Plotter databases. Furthermore, gene variation and epigenetic modification of ACSL4 were analyzed by cBioPortal and GSCA databases. Meanwhile, GEPIA and TIMER databases applied to evaluate the relationship between ACSL4 expression and immune infiltration. These results indicate that ACSL4 expression is down-regulated and associated with prognosis in most tumors. In general, lower ACSL4 expression shows more beneficial prognosis. The most common genetic alteration of ACSL4 is point mutation. ACSL4 is negatively correlated with DNA methylation levels in most cancers. ACSL4 mutations or hypomethylation are associated with poor prognosis. In addition, ACSL4 is positively correlated with immune infiltration in cancers. ACSL4 and immune infiltration are strongly associated with prognosis in BRCA (Breast invasive carcinoma) and SKCM (Skin Cutaneous Melanoma). ACSL4 mutation caused significant changes of immune infiltration in UCEC (Uterine Corpus Endometrial Carcinoma) and SARC (Sarcoma). ACSL4 may be a promising prognostic biomarker for pan-cancer and is closely associated with immune infiltration in the tumor microenvironment.

Preeclampsia (PE), characterized by hypertension and proteinuria, involves placental dysfunction linked to oxidative stress and ferroptosis. The role of estrogen receptor α (ERα) in hypoxia-induced trophoblast ferroptosis remains unclear. A PE rat model was established using L-NAME injection. Placental tissues were analyzed for protein expression via Western blot. In vitro, HTR-8/SVneo cells were cultured under hypoxic conditions to simulate PE-related hypoxia. ERα was knocked down using siRNAs, and acyl-CoA synthetase long-chain family member 4 (ACSL4) was overexpressed via plasmid transfection. Cell viability, apoptosis, migration, oxidative stress parameters, and ferroptosis-related indicators were assessed using CCK-8, flow cytometry, Transwell assays, and specific biochemical kits. In addition, primary syncytiotrophoblast (STB) cells were isolated from placentas of healthy and PE rats for validation experiments. The transcriptional regulation of ACSL4 by ERα was examined using dual luciferase reporter assays. In PE rat placentas, ERα and ACSL4 were significantly upregulated, while GPX4 and SLC7A11 were downregulated. In addition, the lipid peroxidation products 4-HNE and MDA were significantly increased in PE placental tissues. Hypoxia induced ferroptosis in HTR-8/SVneo cells, characterized by increased Fe2 + levels, elevated 4-HNE and MDA levels, lipid peroxidation, and activation of the ERα/ACSL4 axis. Knockdown of ERα attenuated hypoxia-induced cell injury, oxidative stress, and ferroptosis, whereas ACSL4 overexpression reversed these protective effects. Furthermore, experiments in primary STB cells isolated from PE rats showed that ERα knockdown similarly alleviated ferroptosis-associated injury and suppressed ACSL4 expression. Mechanistically, ERα bound directly to the ACSL4 promoter and transcriptionally activated its expression. The ERα-ACSL4 axis plays a critical role in hypoxia-induced ferroptosis in placental trophoblasts. ERα transcriptionally activates ACSL4, promoting lipid peroxidation and ferroptosis, thereby contributing to placental dysfunction in PE. These findings provide a novel theoretical basis for understanding PE pathogenesis and highlight potential therapeutic targets for intervention.

ZFP36L1 Enhances Microglial Ferroptosis in Ischemic Stroke by Reducing FTO-Mediated N6-Methyladenosine Demethylation of ACSL1 mRNA.

ACSL4 promotes ferroptosis and M1 macrophage polarization to regulate the tumorigenesis of nasopharyngeal carcinoma

Paeonol inhibits ACSL4 to protect chondrocytes from ferroptosis and ameliorates osteoarthritis progression

e21594 Background: Skin cutaneous melanoma (SKCM) has a high incidence and mortality. Immune checkpoint inhibitors (ICIs) are promising but show heterogeneous efficacy in the SKCM treatment. T cell-promoted tumor ferroptosis is a vital anti-tumor mechanism of ICIs. Acyl-CoA synthetase long chain family member 4 (ACSL4) can sensitize ferroptosis by facilitating lipid peroxidation. So we proposed that ACSL4 is a positive predictor for ICIs efficacy and correlated with tumor-infiltrating immune cells (TIICs) in SKCM. Methods: The responses of SKCM patients to ICIs were evaluated from Tumor Immune Dysfunction and Exclusion (TIDE) using the gene expression data of The Cancer Genome Atlas (TCGA)-SKCM downloaded from UCSC Xena browser and datasets within TIDE. The correlation between ACLS4 expression and survival was obtained from the Online consensus Survival webserver for Skin Cutaneous Melanoma (OSskcm) and TIDE databases. The relationships between ACSL4 and TIICs were evaluated using the database of Tumor Immune Estimation Resource 2.0 (TIMER2.0). Results: ACSL4 expression was positively correlated with the predicted responder of ICIs in TCGA dataset (R = 0.12, p = 0.0093) and therapy outcomes of ICIs in Gide2019_PD1+CTLA4 (Progression-free survival(PFS), contimuous z = -2.39, p = 0.0169) and Lauss2017_ACT (PFS, contimuous z = -2.08, p = 0.0371; overall survival(OS), contimuous z = -2.96, p = 0.00309). Favorable OS was observed in the patients with high ACSL4 expression in the TCGA (HR = 0.6567, 95% CI = 0.5015̃0.8599, p = 0.0022) and GSE19234 (HR = 0.4135, 95% CI = 0.1748̃0.9784, p = 0.0445) from OSskcm and the GSE8401 (contimuous z = -2,24, p = 0.025) and GSE54467 (contimuous z = -2.26, p = 0.0239) from TIDE database. TIICs including CD8+ T cells, CD4+ T cells (memory, Th2), B cells, neutrophils, monocytes, M1 macrophages, and cancer-associated fibroblasts (CAFs) were positively associated with the expression level of ACSL4. Conclusions: High ACSL4 expression maybe indicates a good response to ICIs and long survival in SKCM. The increased T cells within the tumor microenvironment correlated with high ACSL4 expression possibly implied the synergism effects of ferroptosis and ICIs, deserving further investigation. Keywords: ACSL4, ICIs, TIICs, SKCM.

This study aimed to identify key ferroptosis-related genes associated with intervertebral disc degeneration(IDD) through bioinformatics and machine learning algorithms, predict potential TCM, and conduct experimental validation. IDD datasets(GSE124272 and GSE150408) were obtained from the GEO database, while ferroptosis-related genes were retrieved from the FerrDb and GeneCards databases. Data preprocessing was performed using R. Weighted gene co-expression network analysis(WGCNA) was employed to identify differentially expressed genes(DEGs), which were intersected with ferroptosis-related genes to construct a protein-protein interaction(PPI) network. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were subsequently conducted. Three machine learning algorithms, i.e., LASSO regression, support vector machine-recursive feature elimination(SVM-RFE), and random forest, were applied to screen feature genes and construct prediction models. Potential TCM was predicted through the Coremine Medical database and validated through molecular docking. For experimental validation, mouse nucleus pulposus cells were divided into a normal control group, an erastin(1 μmol·L~(-1))-induced model group, and an icariin(10 μmol·L~(-1)) intervention group. Cell viability, oxidative stress indicators [reactive oxygen species(ROS), lipid peroxides(LPO), reduced glutathione(GSH), oxidized glutathione(GSSG), and malondialdehyde(MDA)], and mitochondrial function were assessed. Immunofluorescence staining was used to detect the levels of aggrecan and type Ⅱ collagen(collagen Ⅱ), and Western blot was performed to evaluate the expression of key proteins, including glutathione peroxidase 4(GPX4), signal transducer and activator of transcription 3(STAT3), and acyl-CoA synthetase long-chain family member 1(ACSL1). The results identified 14 ferroptosis-related DEGs associated with IDD. GO analysis revealed enrichment in biological processes such as interleukin(IL)-8 and IL-17 regulation, radial glial cell differentiation, vascular endothelial growth factor regulation, and inflammatory responses; cellular components related to lysosomes; and molecular functions involving protein kinase binding. KEGG pathway analysis showed significant enrichment in the human cytomegalovirus infection pathway. Machine learning algorithms identified four key genes, including perilipin 4(PLIN4), STAT3, lysine demethylase 6B(KDM6B), and ACSL1, with the constructed diagnostic model achieving an area under the curve(AUC) of 0.890. Potential TCM, including Epimedii Folium, Tripterygii Radix, and Cuscutae Semen, were predicted, among which icariin showed high binding affinity to ACSL1 and STAT3. Experimental validation demonstrated that compared with the normal control group, the model group showed significantly decreased cell viability, elevated levels of ROS, LPO, GSSG, and MDA, and increased expression of STAT3 and ACSL1 proteins, along with impaired mitochondrial membrane potential and activity. In contrast, levels of GSH, aggrecan, collagen Ⅱ, and GPX4 protein expression were markedly reduced. Compared with the model group, icariin intervention significantly improved the oxidative stress status of the cells, reduced levels of ROS, LPO, GSSG, MDA, and expression of STAT3 and ACSL1 proteins, improved mitochondrial membrane potential and activity, and increased levels of GSH, aggrecan, collagen Ⅱ, and GPX4 protein expression. In conclusion, the diagnostic model based on PLIN4, STAT3, KDM6B, and ACSL1 provides a new strategy for the early diagnosis and treatment of IDD. Icariin may influence IDD progression by modulating ferroptosis-related genes and holds potential therapeutic value.

Knockdown of JMJD3 ameliorates cigarette smoke extract-triggered bronchial epithelial cell injury via ACSL4-dependent ferroptosis

ALKBH5 regulates ACSL4 to sensitize erastin-induced ferroptosis via YTHDF2-dependent m6A modification in hypoxic pulmonary hypertension.

Ferroptosis is a non-apoptotic cell death characterized by iron-dependent lipid peroxidation and is implicated in renal diseases, including acute kidney injury and diabetic nephropathy. In renal proximal tubular cells, the regulation of ferroptosis is particularly critical for maintaining cellular homeostasis. While inducing ferroptosis in normal rat kidney proximal tubular epithelial (NRK-52E) cells, we observed the emergence of resistant subpopulations and established two ferroptosis-resistant clones, designated clone A and clone B, to investigate the underlying mechanisms. Transcript and immunoblot analyses revealed that clone A lacked acyl-CoA synthetase long-chain family member 4 (ACSL4), and this deficiency conferred ferroptosis resistance. Furthermore, although clone B expressed ACSL4, its enzymatic activity was markedly reduced, leading us to hypothesize that clone B harbors a mutation that impairs ACSL4 function. Therefore, we performed sequence analysis and identified a novel T237A missense mutation in ACSL4. Sequence alignment and structural superposition of rat ACSL4 and long-chain fatty acyl-CoA synthetase from Thermus thermophilus suggested that Thr237 in rat ACSL4 cooperates with Glu429 in Mg2+ coordination. Functional assays using ACSL4-deficient cells expressing ACSL4 variants (T237A or E429A) confirmed that both residues are essential for catalytic activity. These findings provide new insights into the structural and functional roles of mammalian ACSL4 and may facilitate the development of ACSL4-targeted therapeutics.

Ferroptosis is a recently identified form of regulated cell death defined by the iron-dependent accumulation of lipid reactive oxygen species. Ferroptosis has been studied in various diseases such as cancer, Parkinson’s disease, and stroke. However, the exact function and mechanism of ferroptosis in ischemia/reperfusion (I/R) injury, especially in the intestine, remains unknown. Considering the unique conditions required for ferroptosis, we hypothesize that ischemia promotes ferroptosis immediately after intestinal reperfusion. In contrast to conventional strategies employed in I/R studies, we focused on the ischemic phase. Here we verified ferroptosis by assessing proferroptotic changes after ischemia along with protein and lipid peroxidation levels during reperfusion. The inhibition of ferroptosis by liproxstatin-1 ameliorated I/R-induced intestinal injury. Acyl-CoA synthetase long-chain family member 4 (ACSL4), which is a key enzyme that regulates lipid composition, has been shown to contribute to the execution of ferroptosis, but its role in I/R needs clarification. In the present study, we used rosiglitazone (ROSI) and siRNA to inhibit ischemia/hypoxia-induced ACSL4 in vivo and in vitro. The results demonstrated that ACSL4 inhibition before reperfusion protected against ferroptosis and cell death. Further investigation revealed that special protein 1 (Sp1) was a crucial transcription factor that increased ACSL4 transcription by binding to the ACSL4 promoter region. Collectively, this study demonstrates that ferroptosis is closely associated with intestinal I/R injury, and that ACSL4 has a critical role in this lethal process. Sp1 is an important factor in promoting ACSL4 expression. These results suggest a unique and effective mechanistic approach for intestinal I/R injury prevention and treatment.

Ferroptosis is a recently recognized form of regulated cell death, characterized by iron-dependent accumulation of lipid peroxidation. Ample evidence has depicted that ferroptosis plays an essential role in the cause or consequence of human diseases, including cancer, neurodegenerative disease and acute kidney injury. However, the exact role and underlying mechanism of ferroptosis in fibrotic kidney remain unknown. Acyl-CoA synthetase long-chain family member 4 (ACSL4) has been demonstrated as an essential component in ferroptosis execution by shaping lipid composition. In this study, we aim to discuss the potential role and underlying mechanism of ACSL4-mediated ferroptosis of tubular epithelial cells (TECs) during renal fibrosis. The unbiased gene expression studies showed that ACSL4 expression was tightly associated with decreased renal function and the progression of renal fibrosis. To explore the role of ACSL4 in fibrotic kidney, ACSL4 specific inhibitor rosiglitazone (ROSI) was used to disturb the high expression of ACSL4 in TECs induced by TGF-β, unilateral ureteral obstruction (UUO) and fatty acid (FA)-modeled mice in vivo, and ACSL4 siRNA was used to knockdown ACSL4 in TGF-β-induced HK2 cells in vitro. The results demonstrated that inhibition and knockdown of ACSL4 effectively attenuated the occurrence of ferroptosis in TECs and alleviated the interstitial fibrotic response. In addition, the expression of various profibrotic cytokines all decreased after ROSI-treated in vivo and in vitro. Further investigation showed that inhibition of ACSL4 obviously attenuates the progression of renal fibrosis by reducing the proferroptotic precursors arachidonic acid- and adrenic acid- containing phosphatidylethanolamine (AA-PE and AdA-PE). In conclusion, these results suggest ACSL4 is essential for tubular ferroptotic death during kidney fibrosis development and ACSL4 inhibition is a viable therapeutic approach to preventing fibrotic kidney diseases.

The Nuclear Receptor Coactivator 4 (NCOA4)-mediated ferritinophagy and the Acyl-CoA synthetase long-chain family member 4 (ACSL4)-mediated lipid peroxidation promote ferroptosis. Nuclear factor erythroid-2 related factor-2 (Nrf2), a transcription factor, controls antioxidants, detoxifying enzymes, and ferroptosis. The concerted activity of NCOA4, ACSL4, and Nrf2 in periodontitis associated with or without T2DM needs exploration. Forty-two patients were recruited and categorized into 3 groups: Group I: systemically and periodontally healthy (PH, n = 14); Group II: Stage III/IV Periodontitis (PD, n = 14); Group III: Stage III/IV Periodontitis with uncontrolled Type 2 Diabetes mellitus (T2DM + DM, n = 14). The site-specific periodontal clinical parameters were recorded. Gingival tissue biopsies were evaluated for gene expression of NCOA4, Nrf2, and ACSL4 by real time -PCR. In groups II and III, NCOA4 expression increased by 10.07 ± 9.15 and 9.40 ± 6.29-fold, respectively, whereas ACSL4 expression increased by 3.23 ± 2.53 and 2.03 ± 2.22-fold, respectively, compared to group I (P < 0.05). Nrf2 expression decreased in groups II (0.36 ± 0.39) and III (0.83 ± 0.89) compared to group I, with the least expression in group II. In the total sample analysis, NCOA4 FC and Nrf2 CT showed a significant positive correlation with all clinical parameters and with each other (P < 0.05). There was a significant positive correlation (P < 0.05) between NCOA4 FC and ACSL4 CT (P < 0.05). In group II, NCOA4 CT had significant positive and negative correlations with Nrf2 FC and ACSL4 FC values, respectively (P < 0.05). The diagnostic accuracy of NCOA4 and Nrf2 in differentiating between periodontal health and periodontitis was 89% (AUC = 0.96) and 71% (AUC = 0.60), respectively. Likewise, NCOA4 and Nrf2 demonstrated diagnostic accuracy of 67.8% (AUC = 0.73) and 78.5% (AUC = 0.85) in differentiating between diabetic periodontitis and periodontal health, respectively. Ferroptosis in the progression of periodontitis and diabetes-associated periodontitis may be linked to dysregulated expressions of the NCOA4, ACSL4, and Nrf2 genes. However, additional investigation is required to fully comprehend the findings.