Ironing out the role of ferroptosis in immunity.

Ferroptosis is a type of regulated cell death that drives the pathophysiology of many diseases. Oxidative stress is detectable in many types of regulated cell death, but only ferroptosis involves lipid peroxidation and iron dependency. Ferroptosis originates and propagates from several organelles, including the mitochondria, endoplasmic reticulum, Golgi, and lysosomes. Recent data have revealed that immune cells can both induce and undergo ferroptosis. A mechanistic understanding of how ferroptosis regulates immunity is critical to understanding how ferroptosis controls immune responses and how this is dysregulated in disease. Translationally, more work is needed to produce ferroptosis-modulating immunotherapeutics. This review focuses on the role of ferroptosis in immune-related diseases, including infection, autoimmune diseases, and cancer. We discuss how ferroptosis is regulated in immunity, how this regulation contributes to disease pathogenesis, and how targeting ferroptosis may lead to novel therapies.

Selenium Utilization by GPX4 Is Required to Prevent Hydroperoxide-Induced Ferroptosis.

Selenoproteins are rare proteins among all kingdoms of life containing the 21st amino acid, selenocysteine. Selenocysteine resembles cysteine, differing only by the substitution of selenium for sulfur. Yet the actual advantage of selenolate- versus thiolate-based catalysis has remained enigmatic, as most of the known selenoproteins also exist as cysteine-containing homologs. Here, we demonstrate that selenolate-based catalysis of the essential mammalian selenoprotein GPX4 is unexpectedly dispensable for normal embryogenesis. Yet the survival of a specific type of interneurons emerges to exclusively depend on selenocysteine-containing GPX4, thereby preventing fatal epileptic seizures. Mechanistically, selenocysteine utilization by GPX4 confers exquisite resistance to irreversible overoxidation as cells expressing a cysteine variant are highly sensitive toward peroxide-induced ferroptosis. Remarkably, concomitant deletion of all selenoproteins in Gpx4cys/cys cells revealed that selenoproteins are dispensable for cell viability provided partial GPX4 activity is retained. Conclusively, 200 years after its discovery, a specific and indispensable role for selenium is provided.

ALOX5-mediated ferroptosis acts as a distinct cell death pathway upon oxidative stress in Huntington's disease.

Although it is well established that Huntington's disease (HD) is mainly caused by polyglutamine-expanded mutant huntingtin (mHTT), the molecular mechanism of mHTT-mediated actions is not fully understood. Here, we showed that expression of the N-terminal fragment containing the expanded polyglutamine (HTTQ94) of mHTT is able to promote both the ACSL4-dependent and the ACSL4-independent ferroptosis. Surprisingly, inactivation of the ACSL4-dependent ferroptosis fails to show any effect on the life span of Huntington's disease mice. Moreover, by using RNAi-mediated screening, we identified ALOX5 as a major factor required for the ACSL4-independent ferroptosis induced by HTTQ94. Although ALOX5 is not required for the ferroptotic responses triggered by common ferroptosis inducers such as erastin, loss of ALOX5 expression abolishes HTTQ94-mediated ferroptosis upon reactive oxygen species (ROS)-induced stress. Interestingly, ALOX5 is also required for HTTQ94-mediated ferroptosis in neuronal cells upon high levels of glutamate. Mechanistically, HTTQ94 activates ALOX5-mediated ferroptosis by stabilizing FLAP, an essential cofactor of ALOX5-mediated lipoxygenase activity. Notably, inactivation of the Alox5 gene abrogates the ferroptosis activity in the striatal neurons from the HD mice; more importantly, loss of ALOX5 significantly ameliorates the pathological phenotypes and extends the life spans of these HD mice. Taken together, these results demonstrate that ALOX5 is critical for mHTT-mediated ferroptosis and suggest that ALOX5 is a potential new target for Huntington's disease.

ACSL4 upregulates IFI44 and IFI44L expression and promotes the proliferation and invasiveness of head and neck squamous cell carcinoma cells.

Reprogramming of cellular energy metabolism, including deregulated lipid metabolism, is a hallmark of head and neck squamous cell carcinoma (HNSCC). However, the underlying molecular mechanisms remain unclear. Long-chain acyl-CoA synthetase 4 (ACSL4), which catalyzes fatty acids to form fatty acyl-CoAs, is critical for synthesizing phospholipids or triglycerides. Despite the differing roles of ACSL4 in cancers, our data showed that ACSL4 was highly expressed in HNSCC tissues, positively correlating with poor survival rates in patients. Knockdown of ACSL4 in HNSCC cells led to reduced cell proliferation and invasiveness. RNA sequencing analyses identified interferon-induced protein 44 (IFI44) and interferon-induced protein 44-like (IFI44L), encoded by two interferon-stimulated genes, as potential effectors of ACSL4. Silencing IFI44 or IFI44L expression in HNSCC cells decreased cell proliferation and invasiveness. Manipulating ACSL4 expression or activity modulated the expression levels of JAK1, tyrosine kinase 2 (TYK2), signal transducer and activator of transcription 1 (STAT1), interferon α (IFNα), IFNß, and interferon regulatory factor 1 (IRF1), which regulate IFI44 and IFI44L expression. Knockdown of IRF1 reduced the expression of JAK1, TYK2, IFNα, IFNß, IFI44, or IFI44L and diminished cell proliferation and invasiveness. Our results suggest that ACSL4 upregulates interferon signaling, enhancing IFI44 and IFI44L expression and promoting HNSCC cell proliferation and invasiveness. Thus, ACSL4 could serve as a novel therapeutic target for HNSCC.

Chondroprotective effects of bone marrow mesenchymal stem cell-derived exosomes in osteoarthritis.

Chondrocyte ferroptosis constitutes a major cause of the development of osteoarthritis (OA). Bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) have a protective role against ferroptosis in various diseases. Hence, we aimed to determine whether BMSC-Exos alleviated chondrocyte ferroptosis and its effect on OA, and to dissect out the possible mechanisms. An OA rat chondrocyte model was established by interleukin-1ß (IL-1ß) exposure, and treated with BMSC-Exos/ferroptosis inhibitor Ferrostatin-1. Cell viability/ferroptosis-related index levels [reactive oxygen species (ROS)/malondialdehyde (MDA)/glutathione (GSH)]/cell death/ACSL4 mRNA and protein levels and METTL3 levels were assessed by MTT/kits/immunohistochemical method and TUNEL staining/RT-qPCR and Western blot. METTL3/ACSL4 were overexpressed in rat chondrocytes to evaluate their role in BMSC-Exo-produced repression on chondrocyte ferroptosis. Bioinformatics website predicted the presence of m6A modification sites on ACSL4 mRNA, with the m6A level enriched on it assessed by MeRIP/RT-qPCR. ACSL4 mRNA stability was detected by actinomycin D assay. A surgical destabilized medial meniscus rat OA model was also established, followed by injection with BMSC-Exos to verify their function. IL-1ß stimulation in rat chondrocytes inhibited cell viability, elevated Fe2+/ROS/MDA levels, declined GSH levels and increased TUNEL positive cell number and ACSL4 level, which were neutralized by BMSC-Exos. BMSC-Exos limited chondrocyte ferroptosis by down-regulating METTL3, with the effect abrogated by METTL3 overexpression. METTL3 regulated the m6A modification of ACSL4 mRNA, increasing ACSL4 mRNA stability and ACSL4 expression. BMSC-Exos reduced chondrocyte ferroptosis and prevented OA progression via disruption of the METTL3-m6A-ACSL4 axis. BMSC-Exos might exert a chondroprotective effect by attenuating chondrocyte ferroptosis and alleviate OA progression.

MitoQ protects against carbon tetrachloride-induced hepatocyte ferroptosis and acute liver injury by suppressing mtROS-mediated ACSL4 upregulation.

Ferroptosis has been shown to be involved in carbon tetrachloride (CCl4)-induced acute liver injury (ALI). The mitochondrion-targeted antioxidant MitoQ can eliminate the production of mitochondrial reactive oxygen species (mtROS). This study investigated the role of MitoQ in CCl4-induced hepatocytic ferroptosis and ALI. MDA and 4HNE were elevated in CCl4-induced mice. In vitro, CCl4 exposure elevated the levels of oxidized lipids in HepG2 cells. Alterations in the mitochondrial ultrastructure of hepatocytes were observed in the livers of CCl4-evoked mice. Ferrostatin-1 (Fer-1) attenuated CCl4-induced hepatic lipid peroxidation, mitochondrial ultrastructure alterations and ALI. Mechanistically, acyl-CoA synthetase long-chain family member 4 (ACSL4) was upregulated in CCl4-exposed human hepatocytes and mouse livers. The ACSL4 inhibitor rosiglitazone alleviated CCl4-induced hepatic lipid peroxidation and ALI. ACSL4 knockdown inhibited oxidized lipids in CCl4-exposed human hepatocytes. Moreover, CCl4 exposure decreased the mitochondrial membrane potential and OXPHOS subunit levels and increased the mtROS level in HepG2 cells. Correspondingly, MitoQ pretreatment inhibited the upregulation of ACSL4 in CCl4-evoked mouse livers and HepG2 cells. MitoQ attenuated lipid peroxidation in vivo and in vitro after CCl4 exposure. Finally, MitoQ pretreatment alleviated CCl4-induced hepatocytic ferroptosis and ALI. These findings suggest that MitoQ protects against hepatocyte ferroptosis in CCl4-induced ALI via the mtROS-ACSL4 pathway.

Traumatic acid inhibits ACSL4 associated lipid accumulation in adipocytes to attenuate high-fat diet-induced obesity.

Obesity is a major health concern that lacks effective intervention strategies. Traumatic acid (TA) is a potent wound-healing agent in plants, considered an antioxidant food ingredient. This study demonstrated that TA treatment significantly reduced lipid accumulation in human adipocytes and prevented high-fat diet induced obesity in zebrafish. Transcriptome sequencing revealed TA-activated fatty acid (FA) degradation and FA metabolism signaling pathways. Moreover, western blotting and quantitative polymerase chain reaction showed that TA inhibited the expression of long-chain acyl-CoA synthetase-4 (ACSL4). Overexpression of ACSL4 resulted in the reversal of TA beneficiary effects, indicating that the attenuated lipid accumulation of TA was regulated by ACSL4 expression. Limited proteolysis-mass spectrometry and microscale thermophoresis were then used to confirm hexokinase 2 (HK2) as a direct molecular target of TA. Thus, we demonstrated the molecular basis of TA in regulating lipid accumulation and gave the first evidence that TA may function through the HK2-ACSL4 axis.

ACSL4 accelerates osteosarcoma progression via modulating TGF-ß/Smad2 signaling pathway.

Osteosarcoma (OS) is a malignant bone sarcoma arising from mesenchymal stem cells. The biological role of Acyl-CoA synthetase long-chain family member 4 (ACSL4), recently identified as an oncogene in numerous tumor types, remains largely unclear in OS. In this study, we investigated the expression of ACSL4 in OS tissues using immunohistochemistry staining (IHC) staining of a human tissue microarray and in OS cells by qPCR assay. Our findings revealed a significant up-regulation of ACSL4 in both OS tissues and cells. To further understand its biological effects, we conducted a series of loss-of-function experiments using ACSL4-depleted MNNG/HOS and U-2OS cell lines, focusing on OS cell proliferation, migration, and apoptosis in vitro. Our results demonstrated that ACSL4 knockdown remarkably suppressed OS cell proliferation, arrested cells in the G2 phase, induced cell apoptosis, and inhibited cell migration. Additionally, a subcutaneous xenograft mice model was established to validate the in vivo impact of ACSL4, revealing ACSL4 silencing impaired tumor growth in the OS xenograft mice. Additionally, we discovered that ACSL4 could regulate the phosphorylation level of Smad2 through cooperative interactions, and treatment with a TGF-ß inhibitor weakened the promoting effects of ACSL4 overexpression. In short, ACSL4 regulated OS progression by modulating TGF-ß/Smad2 signaling pathway. These findings underscore ACSL4 as a promising therapeutic target for OS patients and contribute novel insights into the pathogenesis of OS.

Manganese drives ferroptosis of cancer cells via YAP/TAZ phase separation activated ACSL4 in OSCC.

OBJECTIVE: Ferroptosis has been defined as a novel form of regulated cell death characterized by iron-dependent lipid peroxidation. Manganese has been used to induce ferroptosis in cancer cells recently. This study aims to investigate whether manganese can induce ferroptosis in oral squamous cell carcinoma (OSCC) and the underlying biological mechanisms. MATERIALS AND METHODS: Cancer cells with or without manganese treatment were analyzed by RNA-sequencing to identify ferroptosis-related genes. Next, the activation of YAP/TAZ/ACSL4-ferroptosis signaling pathway was detected. Bioinformatic analysis and immunofluorescence assay were used to explore the phase separation of YAP/TAZ. Finally, specimens of OSCC patients were applied to analyze the clinical significance of YAP/TAZ/ACSL4. RESULTS: RNA-sequencing analysis showed the ferroptosis-related genes and YAP/TAZ were upregulated after manganese treatment. The results of immunofluorescence, ELISA, western blotting, etc. further confirmed that manganese-induced ferroptosis depends on YAP/TAZ/ACSL4 signaling pathway. Moreover, the activation of ACSL4 was achieved by YAP/TAZ phase separation. The survival analysis in OSCC specimen suggested that the higher level of YAP/TAZ-ACSL4 axis expression indicates longer survival. CONCLUSIONS: Manganese induces YAP/TAZ phase separation and subsequent ACSL4 activation via YAP/TAZ nuclear translocation, which facilitates ferroptosis of OSCC. Then YAP/TAZ-ACSL4 axis can be used as a potential prognostic predictor of OSCC patients.

Arginine vasopressin induces ferroptosis to promote heart failure via activation of the V1aR/CaN/NFATC3 pathway.

Arginine vasopressin (AVP) is a key contributor to heart failure (HF), but the underlying mechanisms remain unclear. In the present study, a mouse model of HF and human cardiomyocyte (HCM) cells treated with dDAVP are generated in vivo and in vitro, respectively. Hematoxylin and eosin (HE) staining is used to evaluate the morphological changes in the myocardial tissues. A colorimetric method is used to measure the iron concentration, Fe 2+ concentration and malondialdehyde (MDA) level. Western blot analysis is used to examine the protein levels of the V1a receptor (V1aR), calcineurin (CaN), nuclear factor of activated T cells isoform C3 (NFATC3), glutathione peroxidase 4 (GPX4) and acyl-CoA synthase long chain family member 4 (ACSL4). Immunoprecipitation (IP) and luciferase reporter assays are performed to determine the interaction between NFATC3 and ACSL4. Both in vivo and in vitro experiments reveal that the V1aR-CaN-NFATC3 signaling pathway and ferroptosis are upregulated in HFs, which are verified by the elevated protein levels of V1aR, CaN, NFATC3 and ACSL4; reduced GPX4 protein level; and enhanced Fe 2+ and MDA levels. We further find that inhibiting NFATC3 by suppressing the V1aR/CaN/NFATC3 pathway via V1aR/CaN inhibitors or sh-NFATC3 not only alleviates HF but also inhibits AVP-induced ferroptosis. Mechanistically, sh-NFATC3 significantly reverses the increase in AVP-induced ACSL4 protein level, Fe 2+ concentration, and MDA level by directly interacting with ACSL4. Our results demonstrate that AVP enhances ACSL4 expression by activating the V1aR/CaN/NFATC3 pathway to induce ferroptosis, thus contributing to HF. This study may lead to the proposal of a novel therapeutic strategy for HF.

[Effect and mechanism of Jiedu Huoxue Decoction in regulating YAP/ACSL4 pathway to inhibit ferroptosis in treatment of acute kidney injury].

Jiedu Huoxue Decoction(JDHX), first recorded in the Correction on Errors in Medical Works by WANG Qing-ren, is an effective formula screened out from ancient formulas by the traditional Chinese medicine(TCM) master ZHANG Qi to treat acute kidney injury(AKI) caused by heat, toxicity, stasis, and stagnation. This paper elucidated the therapeutic effect of JDHX on AKI and probed into the potential mechanism from ferroptosis. Thirty-two male C57BL/6 mice were randomized into four groups(n=8): normal, model, and low-and high-dose JDHX. Since the clinical treatment of AKI depends on supportive or alternative therapies and there is no specific drug, this study did not include a positive drug group. The low dose of JDHX corresponded to half of clinically equivalent dose, while the high dose corresponded to the clinically equivalent dose. Mice were administrated with JDHX by gavage daily for 7 consecutive days, while those in the normal group and the model group were administered with the corresponding volume of distilled water. On day 5 of drug administration, mice in other groups except the normal group were injected intraperitoneally with cisplatin solution at a dose of 20 mg·kg~(-1) to induce AKI, and the normal group was injected with saline. All of the mice were sacrificed 72 h after modeling, blood and kidney samples were collected for subsequent analysis. The levels of serum creatine(Scr) and blood urea nitrogen(BUN) were measured by the commercial kits. The expression level of kidney injury molecule 1(KIM-1) in the serum was measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin(HE) staining, periodic acid-Schiff(PAS) staining, and Prussian blue staining were employed to observe the pathological changes, glycogen deposition, and iron deposition, respectively, in the renal tissue. In addition, the levels of glutathione(GSH), superoxide dismutase(SOD), and catalase(CAT) in the renal tissue were examined by biochemical colorimetry. Western blot was performed to determine the protein levels of acyl-CoA synthetase long chain family member 4(ACSL4), lysophosphatidylcholine acyltransferase 3(LPCAT3), and Yes-associated protein(YAP, a key molecule in the Hippo pathway) in the renal tissue. Immunohistochemistry was then employed to detect the location and expression of YAP in the renal tissue. Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR) was performed to measure the mRNA levels of ACSL4 and glutathione peroxidase 4(GPX4). Compared with the normal group, the model group showed elevated serum levels of Scr, BUN, and KIM-1. In the AKI model group, the tubular epithelial cells underwent atrophy and necrotic detachment, disappearance of brush border, and some tubules became protein tubules or experienced vacuole-like degeneration. In addition, this group presented widening of the interstitium or even edema, increased renal tubule injury score, and obvious glycogen and iron deposition in parts of the renal tissue. Moreover, the model group had lower GSH, SOD, and CAT levels, higher ASCL4 and LPCAT3 levels, and lower GPX4 expression and higher YAP expression than the normal group. Compared with the model group, high dose of JDHX effectively protected renal function, lowered the levels of Scr, BUN and KIM-1, alleviated renal pathological injury, reduced glycogen and iron deposition, and elevated the GSH, SOD, and CAT levels in the renal tissue. Furthermore, JDHX down-regulated the protein levels of ACSL4, LPCAT3, and YAP and up-regulated the level of GPX4, compared with the model group. In conclusion, JDHX can protect mice from cisplatin-induced AKI by inhibiting ferroptosis via regulating the YAP/ACSL4 signaling pathway.

FLRT2 suppresses bladder cancer progression through inducing ferroptosis.

Bladder cancer is a common tumour worldwide and exhibits a poor prognosis. Fibronectin leucine rich transmembrane protein 2 (FLRT2) is associated with the regulation of multiple tumours; however, its function in human bladder cancer remain unclear. Herein, we found that FLRT2 level was reduced in human bladder cancer and that higher FLRT2 level predicted lower survival rate. FLRT2 overexpression inhibited, while FLRT2 silence facilitated tumour cell growth, migration and invasion. Mechanistic studies revealed that FLRT2 elevated acyl-CoA synthetase long-chain family member 4 (ACSL4) expression, increased lipid peroxidation and subsequently facilitated ferroptosis of human bladder cancer cells. In summary, we demonstrate that FLRT2 elevates ACSL4 expression to facilitate lipid peroxidation and subsequently triggers ferroptosis, thereby inhibiting the malignant phenotype of human bladder cancer cells. Overall, we identify FLRT2 as a tumour suppressor gene.

Astragaloside IV alleviates neuronal ferroptosis in ischemic stroke by regulating fat mass and obesity-associated-N6-methyladenosine-acyl-CoA synthetase long-chain family member 4 axis.

Ischemic stroke (IS) is a detrimental neurological disease with limited treatment options. Astragaloside IV (As-IV) was a promising bioactive constituent in the treatment of IS. However, the functional mechanism remains unclear. Here, IS cell and mouse models were established by oxygen glucose deprivation/re-oxygenation (OGD/R) and middle cerebral artery occlusion (MCAO). Quantitative reverse transcription PCR (RT-qPCR), Western blotting, or Immunofluorescence staining measured related gene and protein expression of cells or mice brain tissues, and the results revealed altered expression of acyl-CoA synthetase long-chain family member 4 (Acsl4), fat mass and obesity-associated (Fto), and activation transcription factor 3 (Atf3) after treatment with As-IV. Then, increased N6 -methyladenosine (m6 A) levels caused OGD/R or MCAO were reduced by As-IV according to the data from methylated RNA immunoprecipitation (MeRIP)-qPCR and dot blot assays. Moreover, through a series of functional experiments such as observing mitochondrial changes under transmission electron microscopy (TEM), evaluating cell viability by cell counting kit-8 (CCK-8), analyzing infract area of brain tissues by 2,3,5-triphenyltetrazolium chloride (TTC) staining, measuring levels of malondialdehyde (MDA), lactate dehydrogenase (LDH), Fe2+ , solute carrier family 7 member 11 (Slc7a11) and glutathione peroxidase 4 (Gpx4) and concentration of glutathione (GSH), we found that Fto knockdown, Acsl4 overexpression or Atf3 knockdown promoted the viability of OGD/R cells, inhibited cell ferroptosis, reduced infract size, while As-IV treatment or Fto overexpression reversed these changes. In mechanism, the interplays of YTH N6 -methyladenosine RNA-binding protein 3 (Ythdf3)/Acsl4 and Atf3/Fto were analyzed by RNA-pull down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assay. Fto regulated the m6 A levels of Acsl4. Ythdf3 bound to Acsl4, and modulated its levels through m6 A modification. Atf3 bound to Fto and positively regulated its levels. Overall, As-IV promoted the transcription of Fto by upregulating Atf3, resulting in decreased m6 A levels of Acsl4, thus, improving neuronal injury in IS by inhibiting ferroptosis.

MiR-20a-5p alleviates kidney ischemia/reperfusion injury by targeting ACSL4-dependent ferroptosis.

Ischemia/reperfusion injury (IRI) is prone to occur after kidney transplantation, leading to delayed graft function (DGF). MicroRNAs play a crucial role in the pathogenesis of ischemia/reperfusion-induced acute kidney injury, and miR-20a-5p was found to be the most significantly upregulated gene in a DGF patient cohort. However, the roles of microRNAs in transplanted kidneys remain largely unknown. In this study, we found that miR-20a-5p was upregulated in the kidneys of acute kidney injury mice and in patients with DGF. We identified early growth response-1 as a critical upstream target and verified the binding of early growth response-1 to a predicted sequence in the promoter region of the miR-20a-5p gene. Functionally, the miR-20a-5p mimic attenuated IRI and postischemic renal fibrosis, whereas the miR-20a-5p inhibitor delivery aggravated IRI and fibrosis. Importantly, delivery of the miR-20a-5p mimic or inhibitor in the donor kidneys attenuated or aggravated renal loss and structural damage in cold storage transplantation injury. Furthermore, our study identified miR-20a-5p as a negative regulator of acyl-CoA synthetase long-chain family member 4 (ACSL4) by targeting the 3' untranslated region of ACSL4 mRNA, thereby inhibiting ACSL4-dependent ferroptosis. Our results suggest a potential therapeutic application of miR-20a-5p in kidney transplantation through the inhibition of ACSL4-dependent ferroptosis.

Kidney tubular epithelial cell ferroptosis links glomerular injury to tubulointerstitial pathology in lupus nephritis.

Ferroptosis is a druggable, iron-dependent form of cell death that is characterized by lipid peroxidation but has received little attention in lupus nephritis. Kidneys of lupus nephritis patients and mice showed increased lipid peroxidation mainly in the tubular segments and an increase in Acyl-CoA synthetase long-chain family member 4, a pro-ferroptosis enzyme. Nephritic mice had an attenuated expression of SLC7A11, a cystine importer, an impaired glutathione synthesis pathway, and low expression of glutathione peroxidase 4, a ferroptosis inhibitor. Lipidomics of nephritic kidneys confirmed ferroptosis. Using nephrotoxic serum, we induced immune complex glomerulonephritis in congenic mice and demonstrate that impaired iron sequestration within the proximal tubules exacerbates ferroptosis. Lupus nephritis patient serum rendered human proximal tubular cells susceptibility to ferroptosis which was inhibited by Liproxstatin-2, a novel ferroptosis inhibitor. Collectively, our findings identify intra-renal ferroptosis as a pathological feature and contributor to tubular injury in human and murine lupus nephritis.

Reversal of H2O2-induced cell death by knockdown of HOTAIR in HTR-8/SVneo cells by mediation of miR-106b-5p/ACSL4 axis.

Preeclampsia is a serious threat to the health of pregnant women. Injury of trophoblasts could contribute to the progression of preeclampsia, and H2O2 was able to induce apoptosis in trophoblasts. LncRNAs have been reported to be involved in the progression of preeclampsia. Additionally, lncRNA HOTAIR is upregulated in patients with preeclampsia. However, the function of HOTAIR in H2O2-treated trophoblasts remains unclear. To explore the function of HOTAIR in preeclampsia, HTR-8/SVneo cells were stimulated with H2O2. RT-qPCR was performed to measure HOTAIR expression in HTR-8/SVneo cells. The apoptosis of HTR-8/SVneo cells was measured using TUNEL staining. The mitochondrial membrane potential was measured using JC-1 staining. Western blotting was performed to detect the expression of ACSL4, GPX4, and FTH1 in HTR-8/SVneo cells. The level of HOTAIR in HTR-8/SVneo cells was upregulated by H2O2. In addition, H2O2 notably inhibited the proliferation of HTR-8/SVneo cells, whereas knockdown of HOTAIR reversed this phenomenon. The mitochondrial membrane potential in HTR-8/SVneo cells was significantly inhibited by H2O2 and partially abolished by HOTAIR silencing. Moreover, HOTAIR could bind to miR-106b-5p; ACSL4 was identified as the downstream target of miR-106b-5p. Furthermore, HOTAIR knockdown reversed H2O2-induced ferroptosis in HTR-8/SVneo cells by regulating miR-106b-5p/ACSL4. Collectively, the knockdown of HOTAIR reversed H2O2-induced ferroptosis in HTR-8/SVneo cells by mediating miR-106b-5p/ACSL4. Thus, HOTAIR may serve as a new therapeutic target against preeclampsia.

Long non-coding RNA PVT1 regulates atherosclerosis progression via the microRNA-106b-5p/ACSL4 axis.

Long non-coding RNAs (lncRNAs) have been associated with atherosclerosis (AS), but the role of lncRNA PVT1 in this disease is still unknown. However, lncRNA PVT1 was found to be significantly upregulated in the serum of AS patients. In vitro experiments using human vascular endothelial cells (HUVECs) showed that oxidized low-density lipoprotein (ox-LDL) treatment enhanced PVT1 expression and impeded HUVEC proliferation, which could be reversed by PVT1 knockdown or miR-106b-5p mimics. Additionally, knockdown of PVT1 and overexpression of miR-106b-5p inhibited the increase of iron content, MDA level, lipid ROS, ACSL4, and PTGS2 in ox-LDL-induced HUVECs, as well as the decrease of GSH and GPX4. We also found that PVT1 knockdown reduced lipid deposition, atherosclerotic plaque number, and size in ApoE-/- mice. These results suggest that PVT1 plays a crucial role in AS progression by regulating the miR-106b-5p/ACSL4 axis in HUVECs, and may therefore be a potential therapeutic target for AS.

Heat shock protein 27 deficiency promotes ferrous ion absorption and enhances acyl-Coenzyme A synthetase long-chain family member 4 stability to promote glioblastoma cell ferroptosis.

BACKGROUND: Glioblastoma is one of the malignant tumors of the central nervous system with high lethality, high disability and low survival rate. Effective induction of its death is one of the existing challenges. In recent studies, heat shock protein 27 (HSP27) has been shown to be associated with ferroptosis; therefore, targeting HSP27 may be a potential therapeutic approach for GBM. METHODS: Immunohistochemistry and western blot analysis were used to detect the expression of HSP27 in GBM tissues. CCK8, plate clone formation assay, EdU proliferation assay for cell proliferation ability, PI, LDH release assay for cell viability. Reactive oxygen, iron levels, and mitochondrial potential for HSP27 silencing were assayed for ferrotosis in vitro. Western blotting and IP were used to verify the relationship between HSP27 and ACSL4. The effect of knockdown of HSP27 on tumor growth capacity was assessed in an intracranial xenograft model. RESULTS: HSP27 was significantly highly expressed in GBM. In vitro experiments, knockdown of HSP27 significantly induced ferroptosis in GBM cells. IP and western blot demonstrated a sumo-ization link between HSP27 and ACSL4. In vivo experiments, HSP27 deficiency retarded tumor growth rate by promoting ferroptosis. CONCLUSIONS: HSP27 deficiency promotes GBM ferroptosis. Targeting HSP27 may serve as a new direction for GBM treatment.

Calycosin decreases cerebral ischemia/reperfusion injury by suppressing ACSL4-dependent ferroptosis.

Ischemic stroke is the second leading cause of death globally. Calycosin is a typical phytoestrogen that protects against cerebral ischemia/reperfusion (I/R) injury. However, the role of ferroptosis in this effect remains unknown. In the present study, we investigated the ferroptosis mechanism of calycosin against cerebral I/R injury using transient middle cerebral artery occlusion/reperfusion (tMCAO/R)-exposed rats and oxygen-glucose deprivation/reperfusion (OGD/R)-stimulated PC12 cells. We found that calycosin treatment significantly improved neurological deficits, brain edema, blood-brain barrier (BBB) breakdown, infarction volume, and neuronal injuries in rats that underwent tMCAO/R; similar to ferrostatin-1 (a ferroptosis inhibitor), calycosin prevented cell viability loss in PC12 cells exposed to OGD/R stimulation. In addition, calycosin intervention decreased ferroptosis, as assessed by iron accumulation, malondialdehyde (MDA), superoxide dismutase (SOD), ceramide, and reactive oxygen species (ROS) levels, as well as ferroptosis-related protein expression (ACSL4, TfR1, FTH1, and GPX4). Furthermore, overexpression of ACSL4 reversed calycosin-induced beneficial efficacy in OGD/R-stimulated PC12 cells. The molecular docking analysis demonstrated that calycosin binds to ACSL4 by forming stable hydrogen bonds at G465, K690, and D573. Collectively, these findings indicate that calycosin ameliorates cerebral I/R injury by depressing ACSL4-dependent ferroptosis.

ACSL4 serves as a novel prognostic biomarker correlated with immune infiltration in Cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CHOL) is the second most common primary hepatic malignant tumor, following hepatocellular carcinoma (HCC). CHOL is highly aggressive and heterogeneous resulting in poor prognosis. The diagnosis and prognosis of CHOL has not improved in the past decade. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is reported to be associated with tumors, however, its role in CHOL has not been revealed. This study is mainly for exploring the prognostic values and potential function of ACSL4 in CHOL. METHODS: We investigated the expression level and prognostic value of ACSL4 in CHOL based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. TIMER2.0, TISIDB and CIBERSORT databases were utilized to assess the associations between ACSL4 and immune infiltration cells in CHOL. Single-cell sequencing data from GSE138709 was analyzed to study the expression of ACSL4 in different types of cells. ACSL4 co-expressed genes were analyzed by Linkedomics. Additionally, Western Blot, qPCR, EdU assay, CCK8 assay, transwell assay and wound healing assay were performed to further confirm the roles of ACSL4 in the pathogenesis of CHOL. RESULTS: We found that the level of ACSL4 was higher in CHOL and it was correlated with the diagnosis and prognosis of CHOL patients. Then, we observed that the infiltration level of immune cells was related to the level of ACSL4 in CHOL. Moreover, ACSL4 and its co-expressed genes were mainly enriched in metabolism-related pathway and ACSL4 is also a key pro-ferroptosis gene in CHOL. Finally, knockdown of ACSL4 could reverse the tumor-promoting effect of ACSL4 in CHOL. CONCLUSIONS: The current findings demonstrated ACSL4 may as a novel biomarker for CHOL patients, which might regulate immune microenvironment and metabolism resulting in poor prognosis.