acyl-CoA Synthetase Long-chain Family Research Articles

Protosappanin A Protects DOX-Induced Myocardial Injury and Cardiac Dysfunction by Targeting ACSL4/FTH1 Axis-Dependent Ferroptosis.

Doxorubicin (DOX) is an effective anticancer agent, but its clinical utility is constrained by dose-dependent cardiotoxicity, partly due to cardiomyocyte ferroptosis. However, the progress of developing cardioprotective medications to counteract ferroptosis has encountered obstacles. Protosappanin A (PrA), an anti-inflammatory compound derived from hematoxylin, shows potential against DOX-induced cardiomyopathy (DIC). Here, it is reported that PrA alleviates myocardial damage and dysfunction by reducing DOX-induced ferroptosis and maintaining mitochondrial homeostasis. Subsequently, the molecular target of PrA through proteome microarray, molecular docking, and dynamics simulation is identified. Mechanistically,PrA physically binds with ferroptosis-related proteins acyl-CoA synthetase long-chain family member 4 (ACSL4) and ferritin heavy chain 1 (FTH1), ultimately inhibiting ACSL4 phosphorylation and subsequent phospholipid peroxidation, while also preventing FTH1 autophagic degradation and subsequent release of ferrous ions (Fe2+) release. Given the critical role of ferroptosis in the pathogenesis of ischemia-reperfusion (IR) injury, this further investigation posits that PrA can confer a protective effect against IR-induced cardiac damage by inhibiting ferroptosis. Overall, a novel pharmacological inhibitor is unveiled that targets ferroptosis and uncover a dual-regulated mechanism for cardiomyocyte ferroptosis in DIC, highlighting additional therapeutic options for chemodrug-induced cardiotoxicity and ferroptosis-triggered disorders.

Proteomic and lipidomic analysis of the mechanism underlying astragaloside IV in mitigating ferroptosis through hypoxia-inducible factor 1α/heme oxygenase 1 pathway in renal tubular epithelial cells in diabetic kidney disease

Ethnopharmacological relevanceThe limitations of modern medicine in mitigating the pathological process of diabetic kidney disease (DKD) necessitate novel, precise, and effective prevention and treatment methods. Huangqi, the root of Astragalus membranaceus Fisch. ex Bunge has been used in traditional Chinese medicine for various kidney ailments. Astragaloside IV (AS-IV), the primary pharmacologically active compound in A. membranaceus, is involved in lipid metabolism regulation; however, its potential in ameliorating renal damage in DKD remains unexplored. Aim of the studyTo elucidate the specific mechanism by which AS-IV moderates DKD progression. Materials and methodsA murine model of DKD and high glucose-induced HK-2 cells were treated with AS-IV. Furthermore, multiomics analysis, molecular docking, and molecular dynamics simulations were performed to elucidate the mechanism of action of AS-IV in DKD, which was validated using molecular biological methods. ResultsAS-IV regulated glucose and lipid metabolism in DKD, thereby mitigating lipid deposition in the kidneys. Proteomic analysis identified 12 proteins associated with lipid metabolism regulated by AS-IV in the DKD renal tissue. Additionally, lipid metabolomic analysis revealed that AS-IV upregulated and downregulated 4 beneficial and 79 harmful lipid metabolites, respectively. Multiomics analysis further indicated a positive correlation between the top-ranked differential protein heme oxygenase (HMOX)1 and the levels of various harmful lipid metabolites and a negative correlation with the levels of beneficial lipid metabolites. Furthermore, enrichment of both ferroptosis and hypoxia-inducible factor (HIF)-1 signaling pathways during the AS-IV treatment of DKD was observed using proteomic analysis. Validation results showed that AS-IV effectively reduced ferroptosis in DKD-affected renal tubular epithelial cells by inhibiting HIF-1α/HMOX1 pathway activity, upregulating glutathione peroxidase-4 and ferritin heavy chain-1 expression, and downregulating acyl-CoA synthetase long-chain family member-4 and transferrin receptor-1 expression. Our findings demonstrate the potential of AS-IV in mitigating DKD pathology by downregulating the HIF-1α/HMOX1 signaling pathway, thereby averting ferroptosis in renal tubular epithelial cells. ConclusionsAS-IV is a promising treatment strategy for DKD via the inhibition of ferroptosis in renal tubular epithelial cells. The findings of this study may help facilitate the development of novel therapeutic strategies.

Hepatic Oxidative Stress and Cell Death Influenced by Dietary Lipid Levels in a Fresh Teleost

Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation, affecting physiological and pathological processes. Fatty liver disease associated with metabolic dysfunction is a common pathological condition in aquaculture. However, the exact role and mechanism of ferroptosis in its pathogenesis and progression remains unclear. In this study, an experiment was conducted using different dietary lipid levels in the feeding of largemouth bass (Micropterus salmoides) for 11 weeks. The results revealed that the growth performance and whole-body protein content significantly increased with the elevation of dietary lipid levels up to 12%. The activities of antioxidant enzymes as well as the content of GSH (glutathione) in the liver initially increased but later declined as the lipid levels increased; the contents of MDA (malondialdehyde) and GSSG (oxidized glutathione) demonstrated an opposite trend. Moreover, elevating lipid levels in the diet significantly increased liver Fe2+ content, as well as the expressions of TF (Transferrin), TFR (Transferrin receptor), ACSL4 (acyl-CoA synthetase long-chain family member 4), LPCAT3 (lysophosphatidylcholine acyltransferase 3), and LOX12 (Lipoxygenase-12), while decreasing the expressions of GPX4 (glutathione peroxidase 4) and SLC7A11 (Solute carrier family 7 member 11). In conclusion, the optimal lipid level is 12.2%, determined by WG-based linear regression. Excess lipid-level diets can up-regulate the ACSL4/LPCAT3/LOX12 axis, induce hepatic oxidative stress and cell death through a ferroptotic-like program, and decrease growth performance.

Overexpression of USP8 inhibits inflammation and ferroptosis in chronic obstructive pulmonary disease by regulating the OTUB1/SLC7A11 signaling pathway.

Chronic obstructive pulmonary disease (COPD) is a familiar disease, and owns high morbidity and mortality, which critically damages the health of patients. Ubiquitin-specific peptidase 8 (USP8) is a pivotal protein to join in the regulation of some diseases. In a previous report, it was determined that USP8 expression is down-regulated in LPS-treated BEAS-2B cells, and USP8 restrains inflammatory response and accelerates cell viability. However, the regulatory roles of USP8 on ferroptosis in COPD are rarely reported, and the associated molecular mechanisms keep vague. To investigate the regulatory functions of USP8 in COPD progression. The lung functions were measured through the Buxco Fine Pointe Series Whole Body Plethysmography (WBP). The Fe level was tested through the Fe assay kit. The protein expressions were assessed through western blot. The levels of tumor necrosis -factor-α, interleukin 6, and interleukin 8 were evaluated through enzyme-linked immunosorbent serologic assay. Cell viability was tested through CCK-8 assay. In this work, it was discovered that overexpression of USP8 improved lung function in COPD mice. In addition, overexpression of USP8 repressed ferroptosis by regulating glutathione peroxidase 4 and acyl-CoA synthetase long-chain family 4 expressions in COPD mice. Overexpression of USP8 suppressed inflammation in COPD mice. Furthermore, overexpression of USP8 suppressed ferroptosis in COPD cell model. At last, it was verified that overexpression of USP8 accelerated ubiquitin aldehyde-binding protein 1 (OTUB1)/solute carrier family 7 member 11 (SLC7A11) pathway. This study manifested that overexpression of USP8 restrained inflammation and ferroptosis in COPD by regulating the OTUB1/SLC7A11 signaling pathway. This discovery hinted that USP8 could be a potential target for COPD treatment.

ACSL3 regulates breast cancer progression via lipid metabolism reprogramming and the YES1/YAP axis.

Mitochondrial fatty acid oxidation is a metabolic pathway whose dysregulation is recognized as a critical factor in various cancers, because it sustains cancer cell survival, proliferation, and metastasis. The acyl-CoA synthetase long-chain (ACSL) family is known to activate long-chain fatty acids, yet the specific role of ACSL3 in breast cancer has not been determined. We assessed the prognostic value of ACSL3 in breast cancer by using data from tumor samples. Gain-of-function and loss-of-function assays were also conducted to determine the roles and downstream regulatory mechanisms of ACSL3 in vitro and in vivo. ACSL3 expression was notably downregulated in breast cancer tissues compared with normal tissues, and this phenotype correlated with improved survival outcomes. Functional experiments revealed that ACSL3 knockdown in breast cancer cells promoted cell proliferation, migration, and epithelial-mesenchymal transition. Mechanistically, ACSL3 was found to inhibit β-oxidation and the formation of associated byproducts, thereby suppressing malignant behavior in breast cancer. Importantly, ACSL3 was found to interact with YES proto-oncogene 1, a member of the Src family of tyrosine kinases, and to suppress its activation through phosphorylation at Tyr419. The decrease in activated YES1 consequently inhibited YAP1 nuclear colocalization and transcriptional complex formation, and the expression of its downstream genes in breast cancer cell nuclei. ACSL3 suppresses breast cancer progression by impeding lipid metabolism reprogramming, and inhibiting malignant behaviors through phospho-YES1 mediated inhibition of YAP1 and its downstream pathways. These findings suggest that ACSL3 may serve as a potential biomarker and target for comprehensive therapeutic strategies for breast cancer.

Epigallocatechin gallate modulates ferroptosis through downregulation of tsRNA-13502 in non-small cell lung cancer

Ferroptosis, an iron-dependent regulated cell death mechanism, holds significant promise as a therapeutic strategy in oncology. In the current study, we explored the regulatory effects of epigallocatechin gallate (EGCG), a prominent polyphenol in green tea, on ferroptosis and its potential therapeutic implications for non-small cell lung cancer (NSCLC). Treatment of NSCLC cell lines with varying concentrations of EGCG resulted in a notable suppression of cell proliferation, as evidenced by a reduction in Ki67 immunofluorescence staining. Western blot analyses demonstrated that EGCG treatment led to a decrease in the expression of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) while increasing the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4). These molecular changes were accompanied by an increase in intracellular iron, malondialdehyde (MDA), and reactive oxygen species (ROS), alongside ultrastructural alterations characteristic of ferroptosis. Through small RNA sequencing and RT-qPCR, transfer RNA-derived small RNA 13502 (tsRNA-13502) was identified as a significant target of EGCG action, with its expression being upregulated in NSCLC tissues compared to adjacent non-tumorous tissues. EGCG was found to modulate the ferroptosis pathway by downregulating tsRNA-13502 and altering the expression of key ferroptosis regulators (GPX4/SLC7A11 and ACSL4), thereby promoting the accumulation of iron, MDA, and ROS, and ultimately inducing ferroptosis in NSCLC cells. This study elucidates EGCG’s multifaceted mechanisms of action, underscoring the modulation of ferroptosis as a viable therapeutic approach for enhancing NSCLC treatment outcomes.

Effects of ferroptosis-related gene HSPB1 on acute myeloid leukemia.

The purpose of this study was to investigate the effects and potential mechanisms of ferroptosis-related gene heat shock protein beta-1 (HSPB1) on acute myeloid leukemia (AML). The RNA-seq and clinical data of AML samples were obtained from the Genomic Data Commons database, and the FerrDb database was used to screen the marker, drive and suppressor of ferroptosis. Besides, DESeq2 was applied for differential expression analysis on AML samples and screening for differentially expressed genes (DEGs). The screened DEGs were subjected to the intersection analysis with ferroptosis-related genes to identify the ferroptosis-related DEGs. Next, the functional pathways of ferroptosis-related DEGs were further be discussed by Gene Ontology as well as Kyoto Encyclopedia of Genes and Genomes enrichment analysis of DEGs. Additionally, lasso regression analysis was employed to determine the differential genes related to prognosis in patients with AML and the survival analysis was performed. Subsequently, quantitative real-time polymerase chain reaction and western blot assay were applied to detect the mRNA and protein expression levels of HSPB1 in normal/AML bone marrow tissues and human normal (HS-5)/AML (HL-60) bone marrow cells, respectively. Furthermore, HSPB1 was knocked down to assess the expression changes of glutathione peroxidase 4 and acyl-CoA synthetase long-chain family member 4. Ultimately, the viability and oxidative stress levels of HL-60 were analyzed by Cell Counting Kit-8 and biochemical detection. A total of 4986 DEGs were identified in AML samples, with 3324 up-regulated and 1662 down-regulated. The enrichment analysis illustrated that ferroptosis-related DEGs were significantly enriched in response to metal irons, oxidative stress, and other pathways. After lasso regression analysis, 17 feature genes related to the prognosis of patients with AML were obtained, with HSPB1 exhibiting a significant correlation. The reliability of our models was verified by Cox regression analysis and survival analysis of the hazard model. Furthermore, the outcomes of quantitative real-time polymerase chain reaction and western blot showed that mRNA and protein expression levels of HSPB1 were significantly increased in the AML Group and HL-60 cells. The knockdown of HSPB1 in HL-60 cells reduced the protein level of glutathione peroxidase 4, increased the protein level of acyl-CoA synthetase long-chain family member 4, decreased the cell viability, and aggravated oxidative stress. Ferroptosis-related gene HSPB1 is highly expressed in patients with AML. In addition, HSPB1 may be involved in the occurrence and development of AML by regulating oxidative stress and ferroptosis-related pathways. This study provides new clues for further understanding of AML molecular mechanisms. Also, HSPB1 is expected to be a potential therapeutic target for AML in the future.

Interferon-α could induce liver steatosis to promote HBsAg loss by increasing triglyceride level

BackgroundThe correlation between metabolic syndrome (MetS) and hepatitis B surface antigen (HBsAg) loss remains to be further elucidated, particularly in patients receiving pegylated interferon-α (PEG-IFN) treatment. Methods758 patients with low HBsAg quantification who had received nucleos(t)ide analog (NUC) therapy for at least one year and subsequently switched to or add on PEG-IFN therapy over an unfixed course were enrolled. 412 patients were obtained with baseline data matched. A total of 206 patients achieved HBsAg loss (cured group) within 48 weeks. Demographic and biochemical data associated with MetS were gathered for analysis. HepG2.2.15 cell line was used in vitro experiments to validate the efficacy of interferon-α (IFN-α). ResultsThe proportion of patients with diabetes or hypertension in the uncured group was significantly higher than in the cured group. The levels of fasting blood glucose (FBG) and glycated albumin remained elevated in the uncured group over the 48 weeks. In contrast, the levels of blood lipids and uric acid remained higher in the cured group within 48 weeks. Triglycerides levels and liver steatosis of all patients increased after PEG-IFN therapy. Baseline elevated uric acid levels and hepatic steatosis may be beneficial for HBsAg loss. IFN-α could induce hepatic steatosis and indirectly promote HBsAg loss by increasing triglyceride level through upregulation of acyl-CoA synthetase long-chain family member 1(ACSL1). ConclusionsIFN-α could induce liver steatosis to promote HBsAg loss by increasing triglyceride level through upregulation of ACSL1. Comorbid diabetes may be detrimental to obtaining HBsAg loss with PEG-IFN therapy in CHB patients.

Tetramethylpyrazine alleviates ferroptosis and promotes functional recovery in spinal cord injury by regulating GPX4/ACSL4

ObjectiveTetramethylpyrazine (TMP) has been demonstrated to alleviate neuronal ferroptosis following spinal cord injury (SCI), thereby promoting neural repair. However, the precise underlying mechanisms remain elusive. MethodsThe SCI model was established using a modified version of Allen's method. TMP (40, 80, 120, and 160 mg/kg) and ras-selective lethal 3 (RSL3) (5 mg/kg) were administered intraperitoneally once daily for 7 days. HE and Nissl staining were employed to examine histomorphology and neurons, respectively. Perls staining was used to identify the distribution of iron. A transmission electron microscope was used to observe the microcosmic morphology of mitochondria. Immunofluorescence staining and Western blot were used to analyze neuronal nuclear protein (NeuN) and glial fibrillary acidic protein (GFAP) surrounding injury sites. Additionally, glutathione peroxidase 4 (GPX4)/NeuN + cells and acyl-CoA synthetase long-chain family member 4 (ACSL4)/NeuN + cells were observed. RT-qPCR was conducted to examine the mRNA expression levels of GPX4 and ACSL4. ELISA were used to quantify the concentrations of GPX4, reactive oxygen species (ROS), L-glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and tissue iron. ResultsTMP had an inhibitory effect on the concentrations of tissue iron, ROS, GSH, MDA, and SOD. TMP improved the microcosmic morphology of mitochondria and increased GPX4 level while decreasing that of ACSL4. TMP reduced lesion sizes, enhanced neuronal survival, and inhibited glial scar formation. However, the effect of TMP can be effectively reversed by RSL3. ConclusionTMP alleviates neuronal ferroptosis by regulating the GPX4/ACSL4 axis, thereby protecting the remaining neurons surrounding injury sites and reducing glial scar formation.

Propofol Mitigates Sepsis-Induced Brain Injury by Inhibiting Ferroptosis Via Activation of the Nrf2/HO-1axis.

Sepsis-associated encephalopathy (SAE) develops in 30-70% of hospitalized patients with sepsis. In intensive care units (ICUs), propofol is often administered to ensure an appropriate level of sedation in mechanically ventilated patients. Ferroptosis is a newly identified mode of cellular death characterized by the peroxidation of membrane lipids and excessive iron. This study was conducted to explore the interplay between propofol, sepsis, and ferroptosis. An acute systemic inflammatory model was constructed via the intraperitoneal administration of lipopolysaccharide (LPS). Nissl and Fluoro-Jade C (FJC) staining were employed to display neuronal damage and degeneration. Western blotting and immunofluorescence (IF) staining of Bax and Bcl-2 were used to confirm the neural apoptosis. QPCR of cytokines and DHE staining were used to indicate neuroinflammation. To validate ferroptosis, we assessed the content of malondialdehyde (MDA), GSH, and tissue iron, accompanied by transcription level of CHAC1, PTGS2 and GPX4. Additionally, we examined the content of acyl-CoA synthetase long-chain family member 4 (ACSL4), xCT (SLC7A11, solute carrier family 7 member 11), and glutathione peroxidase 4 (GPX4). The IF staining of Iba1-labeled microglia and GFAP-marked astrocytes were used to measure the gliosis. Erastin was pre-pretreated to confirm the anti-ferroptotic capability of propofol. ML385 was preconditioned to explore the role of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in propofol-repressed ferroptosis. Propofol dose-dependently inhibited the decrease of Nissl-positive neurons and the increase of FJC-stained neurons in septic hippocampus and cortex. Neural cytokines, oxidative stress, apoptosis and gliosis were reduced by propofol. Propofol repressed the level of MDA, iron, CHAC1, PTGS2, ACLS4 and restored the content of GSH, GPX4, xCT, Nrf2 and HO-1, thus inhibiting sepsis-induced ferroptosis. All protections from propofol could be reversed by eratsin and ML385 pretreatment. Propofol protected against sepsis-induced brain damage, neuroinflammation, neuronal apoptosis and gliosis through the activation of the Nrf2/HO-1 axis to combat ferroptosis.

A multifunctional nanoplatform for chemotherapy and nanocatalytic synergistic cancer therapy achieved by amplified lipid peroxidation

Traditional cancer chemotherapy suffers from low efficacy and severe side effects, limiting its use as a first-line treatment. To address this issue, we investigated a novel way to induce lipid peroxidation (LPO), which plays an essential role in ferroptosis and may be useful against cancer cells and tumors. In this study, a pH-responsive synergistic cancer therapy nanoplatform was prepared using CaCO3 co-loaded with oleanolic acid (OA) and lipoxygenase (LOX), resulting in the formation OLCaP NP. This nanoplatform exhibited good drug release properties in an acidic tumor environment owing to the presence of CaCO3. As a result of acidic stimulation at tumor sites, the OLCaP NP released OA and LOX. OA, a chemotherapeutic drug with anticancer activity, is already known to promote the apoptosis of cancer cells, and LOX is a natural enzyme that catalyzes the oxidation of polyunsaturated fatty acids, leading to the accumulation of lipid peroxides and promoting the apoptosis of cancer cells. More importantly, OA upregulated the expression of acyl-coenzyme A synthetase long-chain family member 4 (ACSL4), which promoted enzyme-mediated LPO. Based on our combined chemotherapy and nanocatalytic therapy, the OLCaP NP not only had remarkable antitumor ability but also upregulated ACSL4 expression, allowing further amplification of LPO to inhibit tumor growth. These findings demonstrate the potential of this nanoplatform to enhance the therapeutic efficacy against tumors by inducing oxidative stress and disrupting lipid metabolism, highlighting its clinical potential for improved cancer treatment. Statement of significanceThis study presents a novel nanoplatform that combines oleanolic acid (OA), a chemotherapeutic drug, and lipoxygenase (LOX), which oxidizes polyunsaturated fatty acids to trigger apoptosis, for targeted cancer therapy. Unlike traditional treatments, our nanoplatform exhibits pH-responsive drug release, specifically in acidic tumor environments. This innovation enhances the therapeutic effects of OA and LOX, upregulating acyl-CoA synthetase long-chain family member 4 expression and amplifying lipid peroxidation to promote tumor cell apoptosis. Our findings significantly advance the existing literature by demonstrating a synergistic approach that combines chemotherapy and nanocatalytic therapy. The scientific impact of this work lies in its potential to improve cancer treatment efficacy and specificity, offering a promising strategy for clinical applications and future research in cancer therapy.

#1955 Ferroptotic proteins ACSL4 and ALOX15 as therapeutic targets in AKI

Abstract 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.

PUFA-PLs biosynthesis enzymes contribute to pathogenic development of rice blast fungus Magnaporthe oryzae

ABSTRACT Rice blast is one of the most devastating diseases and a serious threat to global food security. It is caused by the ascomycetous fungus Magnaporthe oryzae. During the pathogenic development of M. oryzae, ferroptotic death of conidial cells is critical for appressorium formation and infection to host rice. In this study, we identified and functionally characterised orthologs of fatty acid desaturase (Fad2) and acyl-CoA synthetase long-chain family (Acsl4) in M. oryzae. Pathogenicity was impaired in the fad2Δ or acsl4Δ mutant and targeted lipidomics analysis demonstrated that Fad2 and Acsl4 were involved in the production of polyunsaturated fatty acids (PUFAs)-containing phospholipids (PUFA-PLs) potentially contributing to ferroptosis. Treatment with FeCl3, an oxidative agent to cause lipid peroxidation, could partially restore fad2Δ pathogenicity. Fad2 was also found to potentially interact with proteins involved in cellular redox homoeostasis. Overall, our results elucidate the role of PUFA-PLs biosynthesis in fungal cell death and fungal pathogenicity, providing a theoretical basis for the development of specific pesticides/drugs targeting ferroptosis caused by lipid peroxidation.

Peroxynitrite-Triggered Carbon Monoxide Donor Improves Ischemic Stroke Outcome by Inhibiting Neuronal Apoptosis and Ferroptosis.

Cerebral ischemia-reperfusion injury produces excessive reactive oxygen and nitrogen species, including superoxide, nitric oxide, and peroxynitrite (ONOO-). We recently developed a new ONOO--triggered metal-free carbon monoxide donor (PCOD585), exhibiting a notable neuroprotective outcome on the rat middle cerebral artery occlusion model and rendering an exciting intervention opportunity toward ischemia-induced brain injuries. However, its therapeutic mechanism still needs to be addressed. In the pharmacological study, we found PCOD585 inhibited neuronal Bcl2/Bax/caspase-3 apoptosis pathway in the peri-infarcted area of stroke by scavenging ONOO-. ONOO- scavenging further led to decreased Acyl-CoA synthetase long-chain family member 4 and increased glutathione peroxidase 4, to minimize lipoperoxidation. Additionally, the carbon monoxide release upon the ONOO- reaction with PCOD585 further inhibited the neuronal Iron-dependent ferroptosis associated with ischemia-reperfusion. Such a synergistic neuroprotective mechanism of PCOD585 yields as potent a neuroprotective effect as Edaravone. Additionally, PCOD585 penetrates the blood-brain barrier and reduces the degradation of zonula occludens-1 by inhibiting matrix metalloproteinase-9, thereby protecting the integrity of the blood-brain barrier. Our study provides a new perspective for developing multi-functional compounds to treat ischemic stroke.

Oxidative Stress, Lipid Peroxidation and Ferroptosis Are Major Pathophysiological Signatures in the Placental Tissue of Women with Late-Onset Preeclampsia.

Preeclampsia, a serious and potentially life-threatening medical complication occurring during pregnancy, is characterized by hypertension and often accompanied by proteinuria and multiorgan dysfunction. It is classified into two subtypes based on the timing of diagnosis: early-onset (EO-PE) and late-onset preeclampsia (LO-PE). Despite being less severe and exhibiting distinct pathophysiological characteristics, LO-PE is more prevalent than EO-PE, although both conditions have a significant impact on placental health. Previous research indicates that different pathophysiological events within the placenta may contribute to the development of preeclampsia across multiple pathways. In our experimental study, we investigated markers of oxidative stress, ferroptosis, and lipid peroxidation pathways in placental tissue samples obtained from women with LO-PE (n = 68) compared to healthy control pregnant women (HC, n = 43). Through a comprehensive analysis, we observed an upregulation of specific molecules associated with these pathways, including NADPH oxidase 1 (NOX-1), NADPH oxidase 2 (NOX-2), transferrin receptor protein 1 (TFRC), arachidonate 5-lipoxygenase (ALOX-5), acyl-CoA synthetase long-chain family member 4 (ACSL-4), glutathione peroxidase 4 (GPX4) and malondialdehyde (MDA) in women with LO-PE. Furthermore, increased ferric tissue deposition (Fe3+) was observed in placenta samples stained with Perls' Prussian blue. The assessment involved gene and protein expression analyses conducted through RT-qPCR experiments and immunohistochemistry assays. Our findings underscore the heightened activation of inflammatory pathways in LO-PE compared to HC, highlighting the pathological mechanisms underlying this pregnancy disorder.

Enrichment of Bioactive Lipids in Urinary Extracellular Vesicles and Evidence of Apoptosis in Kidneys of Hypertensive Diabetic Cathepsin B Knockout Mice after Streptozotocin Treatment.

Cathepsin B (CtsB) is a ubiquitously expressed cysteine protease that plays important roles in health and disease. Urinary extracellular vesicles (uEVs) are released from cells associated with urinary organs. The antibiotic streptozotocin (STZ) is known to induce pancreatic islet beta cell destruction, diabetic nephropathy, and hypertension. We hypothesized that streptozotocin-induced diabetic kidney disease and hypertension result in the release of bioactive lipids from kidney cells that induce oxidative stress and renal cell death. Lipidomics was performed on uEVs isolated from CtsB knockout mice treated with or without STZ, and their kidneys were used to investigate changes in proteins associated with cell death. Lysophosphatidylethanolamine (LPE) (18:1), lysophosphatidylserine (LPS) (22:6), and lysophosphatidylglycerol (LPG) (22:5) were among the bioactive lipids enriched in uEVs from CtsB knockout mice treated with STZ compared to untreated CtsB mice (n = 3 uEV preparations per group). Anti-oxidant programming was activated in the kidneys of the CtsB knockout mice treated with STZ, as indicated by increased expression of glutathione peroxidase 4 (GPX4) and the cystine/glutamate antiporter SLC7A11 (XCT) (n = 4 mice per group), which was supported by a higher reactivity to 4-hydroxy-2-nonenal (4-HNE), a marker for oxidative stress (n = 3 mice per group). Apoptosis but not ferroptosis was the ongoing form of cell death in these kidneys as cleaved caspase-3 levels were significantly elevated in the STZ-treated CtsB knockout mice (n = 4 mice per group). There were no appreciable differences in the pro-ferroptosis enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) or the inflammatory marker CD93 in the kidneys (n = 3 mice per group), which further supports apoptosis as the prevalent mechanism of pathology. These data suggest that STZ treatment leads to oxidative stress, inducing apoptotic injury in the kidneys during the development of diabetic kidney disease and hypertension.

NIR Absorbing Organic Chromophores Combination with NSAIDs for Remodeling of the Inflammatory Microenvironment to Amplify Tumor Ferroptosis-Photothermal Synergistic Therapy.

Photothermal therapy has emerged as a promising approach for cancer treatment, which can cause ferroptosis to enhance immunotherapeutic efficacy. However, excessively generated immunogenicity will induce serious inflammatory response syndrome, resulting in a discounted therapeutic effect. Herein, a kind of NIR absorption small organic chromophore nanoparticles (TTHM NPs) with high photothermal conversion efficiency (68.33%) is developed, which can induce mitochondria dysfunction, generate mitochondrial superoxide, and following ferroptosis. TTHM NPs-based photothermal therapy is combined with Sulfasalazine (SUZ), a kind of nonsteroidal anti-inflammatory drugs, to weaken inflammation and promote ferroptosis through suppressing glutamate/cystine (Glu/Cys) antiporter system Xc- (xCT). Additionally, the combination of SUZ with PTT can induce immunogenic cell death (ICD), followed by promoting the maturation of DCs and the attraction of CD8+ T cell, which will secrete IFN-γ and trigger self-amplified ferroptosis via inhibiting xCT and simulating Acyl-CoA synthetase long-chain family member 4 (ACSL4). Moreover, the in vivo results demonstrate that this combination therapy can suppress the expression of inflammatory factors, enhance dendritic cell activation, facilitate T-cell infiltration, and realize effective thermal elimination of primary tumors and distant tumors. In general, this work provides an excellent example of combined medication and stimulates new thinking about onco-therapy and inflammatory response.

Tongqiao Huoxue Decoction inhibits ferroptosis by facilitating ACSL4 ubiquitination degradation for neuroprotection against cerebral ischemia-reperfusion injury

BackgroundCerebral ischemia-reperfusion injury (CIRI) refers to brain tissue injury caused by the temporary interruption of cerebral blood flow ischemia followed by the restoration of reperfusion, which is the main cause of post-stroke brain injury. A traditional Chinese herbal preparation called Tongqiao Huoxue Decoction (TQHX) has shown promise in reducing CIRI in rats. However, the mechanism of this herbal preparation for CIRI remains unclear. PurposeThis study aimed to evaluate the therapeutic effect of TQHX extract on rats with CIRI and to further explore the underlying mechanisms. MethodsThe active ingredients of TQHX extract were quantified by the high-performance liquid chromatography (HPLC) condition. We conducted thorough investigations to assess the effects of TQHX on CIRI and ferroptosis using oxygen-glucose deprivation/reperfusion (OGD/R)-treated PC12 cells as an in vitro model and transient middle cerebral artery occlusion (tMCAO) animals as an in vivo model. The neurological score assessment was performed to evaluate the neuroprotective effects of TQHX extract on tMCAO rats. Using histologic methods to study the extent of cerebral infarction, blood-brain barrier, and rat brain tissue. We examined the impact of TQHX on ferroptosis-related markers of Fe2+, superoxide dismutase (SOD), reactive oxygen species (ROS), and malondialdehyde (MDA) in the brain tissue. In addition, the expression of key proteins and markers of ferroptosis, as well as key factors associated with Acyl-CoA synthetase long-chain family member 4 (ACSL4) were detected by Western blot and quantitative real-time PCR (RT-qPCR). ResultsTQHX extract could decrease the Longa score and extent of cerebral infarction of tMCAO rats, which exerted the function of neuroprotection. Additionally, TQHX treatment efficiently decreased levels of MDA and ROS while increasing the expression of SOD and ferroptosis-related proteins including ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) at the transcription and translation level. Meanwhile, TQHX provided strong protection against oxidative stress and ferritin accumulation by increasing the ubiquitination and degradation of ACSL4. The injection of OE-ACSL4 reversed the effects of TQHX on neuroprotection and ferroptosis inhibition in PC12 cells. The injection of shACSL4 reversely validate the crucial role of ACSL4 in CIRI rat treatment. ConclusionThis work shows that TQHX promotes the ubiquitination-mediated degradation of ACSL4, which improves oxidative stress and inhibits the beginning of ferroptosis in cells. TQHX provides a possible path for additional research in CIRI therapies, advancing translational investigations.

A novel target for attenuating T1DM-induced myocardial ferroptosis

Background: The DMCM-AHEAD trial showed that lipotoxicity is a distinct feature of diabetic heart failure, which initiates and exacerbates cardiac pathogenesis in diabetic patients. Circulating iron levels are increased in diabetic patients. The combined presence of lipids and iron suggests a predominant role of ferroptosis in diabetes-induced heart failure. However, molecular targets for diabetes-induced myocardial ferroptosis remain unclear. In the T1DM heart, matrix metalloproteinase-9 (MMP9), a protease, is upregulated, and targeted deletion of MMP9 is cardioprotective. Hypothesis: Targeted deletion of MMP9 mitigates T1DM-induced myocardial ferroptosis. Methods and results: We performed in vitro studies on the loss and gain-of-function of MMP9 using human cardiomyocytes (AC16 cells). Overexpression of MMP9 downregulates (-1.55-fold p<0.001) GPX4, a key marker of ferroptosis, while inhibition of MMP9 upregulates (+2.03-fold p<0.001) GPX4 (n=6), suggesting a key role of MMP9 in GPX4 regulation and ferroptosis signaling. To determine the specific role of MMP9 in T1DM-induced ferroptosis, we evaluated ferroptosis markers in the heart (LV) tissue of male Insulin2 mutant (Ins2+/-) Akita (T1DM), littermate euglycemic Ins2+/+ WT, Akita/MMP9 knockout (MMP9KO), and MMP9KO mice (n=3-6). The cardiac levels of Acyl-CoA synthetase long-chain family member-4 (ASCL4), which initiates the formation of lipid peroxides, and divalent metal transporter-1 (DMT1), which increases redox-active iron, were upregulated in the Akita while downregulated in the Akita/MMP9KO hearts (Akita vs WT: ASCL4: +1.42-fold p<0.05, DMT1: +1.34-fold p<0.05; Akita/MMP9 vs Akita: ACSL4: -1.46-fold p<0.05, DMT1: -1.03-fold p<0.05). Furthermore, cardiac levels of Ferritin-Light Chain (FLC), which stores iron to suppress ferroptosis, was downregulated in the Akita but upregulated in the Akita/MMP9KO hearts (Akita vs WT: -2.01-fold p<0.01, Akita/MMP9 vs Akita: +1.36-fold p<0.05). These results suggest that upregulation of MMP9 promotes while inhibition of MMP9 mitigates ferroptosis in the T1DM heart. Conclusion: This is the first report to reveal MMP9 as a promising therapeutic target for T1DM-induced myocardial ferroptosis. This work was supported in part by the National Institutes of Health (NIH) Grant R56HL156806 to PKM, and University of Nebraska Medical Center (UNMC) Presidential Graduate Fellowship to FIG. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Anti-tumor mechanism of total saponins of Paridis Rhizoma on inducing ferroptosis of breast cancer MCF-7 cells

This study aims to investigate the mechanism of total saponins of Paridis Rhizoma in inducing the ferroptosis of MCF-7 cells and provide a theoretical basis for the clinical treatment of breast cancer with total saponins of Paridis Rhizoma. The methyl thiazolyl tetrazolium(MTT) assay was employed to examine the effects of different concentrations of total saponins of Paridis Rhizoma on the proliferation of MCF-7 cells. A phase contrast inverted microscope was used to observe the morphological changes of MCF-7 cells. The colony formation assay was employed to test the colony formation of MCF-7 cells. The lactate dehydrogenase(LDH) release test was conducted to determine the cell membrane integrity of MCF-7 cells. The cell scratch assay was employed to examine the migration of MCF-7 cells. After that, the level of reactive oxygen species(ROS) in MCF-7 cells was observed by an inverted fluorescence microscope, and the content of Fe~(2+) in MCF-7 cells was detected by the corresponding kit. Transmission electron microscopy was employed to observe the mitochondrial ultrastructure of MCF-7 cells. Western blot was employed to determine the expression of ferroptosis-related proteins, such as p53, solute carrier family 7 member 11(SLC7A11), glutathione peroxidase 4(GPX4), acyl-CoA synthetase long-chain family member 4(ACSL4), and transferrin receptor protein 1(TFR1) in MCF-7 cells. The results showed that 1.5, 3, 4.5, 6, 7.5, and 9 μg·mL~(-1) total saponins of Paridis Rhizoma significantly inhibited the proliferation of MCF-7 cells, with the IC_(50) of 4.12 μg·mL~(-1). Total saponins of Paridis Rhizoma significantly damaged the morphology of MCF-7 cells, leading to the formation of vacuoles and the gradual shrinkage and detachment of cells. Meanwhile, total saponins of Paridis Rhizoma inhibited the colony formation of MCF-7 cells, destroyed the cell membrane(leading to the release of LDH), and shortened the migration distance of MCF-7 cells. Total saponins of Paridis Rhizoma treatment significantly increased the content of ROS, induced oxidative damage, and led to the accumulation of Fe~(2+) in MCF-7 cells. Furthermore, total saponins of Paridis Rhizoma changed the mitochondrial structure, increased the mitochondrial membrane density, led to the decrease or even disappear of ridges, promoted the expression of p53 protein, down-regulated the expression of SLC7A11 and GPX4, and up-regulated the expression of ACSL4 and TFR1. In summary, total saponins of Paridis Rhizoma can significantly inhibit the proliferation and migration of MCF-7 cells and destroy the cell structure by inducing ferroptosis.