acyl-CoA Synthetase Long-chain Family Member Research Articles

Soot nanoparticles promote ferroptosis in dopaminergic neurons via alteration of m6A RNA methylation in Parkinson’s disease

Soot nanoparticles (SNPs) are black carbon prevalent in atmospheric environment with significant impacts on public health, leading to neurodegenerative diseases including development of Parkinson’s disease (PD). This study investigated the effects of SNPs exposure on PD symptoms, employing both in vivo and in vitro PD models. In the in vivo experiments, animal behavior assessments showed that SNPs exposure exacerbated motor and cognitive impairments in PD mice. Molecular biology techniques further unveiled that SNPs aggravated degeneration of dopaminergic neurons. In vitro experiments revealed that SNPs exposure intensified ferroptosis of PD cells by increasing reactive oxygen species and iron ion levels, while reducing glutathione levels and mitochondrial membrane potential. Sequencing tests indicated elevated N6-methyladenosine (m6A) alteration of the ferroptosis-related protein, acyl-CoA synthetase long chain family member 4 (ACSL4). This study demonstrates that SNPs may exacerbate the onset and progression of PD by recruiting YTH domain-containing family protein 1 (YTHDF1) protein, enhancing m6A methylation in the ACSL4 5′UTR, amplifying ACSL4 protein expression, and accelerating the ferroptosis process in dopaminergic neurons. These molecular mechanisms underlying SNPs exacerbation of PD development may provide crucial insights for formulating environmental safety regulations and potential therapeutic strategies addressing PD in populations residing in regions with varied air quality.

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.

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.

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.

Preparation of Pueraria lobata Root-Derived Exosome-Like Nanovesicles and Evaluation of Their Effects on Mitigating Alcoholic Intoxication and Promoting Alcohol Metabolism in Mice.

Pueraria lobata (P. lobata), a dual-purpose food and medicine, displays limited efficacy in alcohol detoxification and liver protection, with previous research primarily focused on puerarin in its dried roots. In this study, we investigated the potential effects and mechanisms of fresh P. lobata root-derived exosome-like nanovesicles (P-ELNs) for mitigating alcoholic intoxication, promoting alcohol metabolism effects and protecting the liver in C57BL/6J mice. We isolated P-ELNs from fresh P. lobata root using differential centrifugation and characterized them via transmission electron microscopy, nanoscale particle sizing, ζ potential analysis, and biochemical assays. In Acute Alcoholism (AAI) mice pre-treated with P-ELNs, we evaluated their effects on the timing and duration of the loss of the righting reflex (LORR), liver alcohol metabolism enzymes activity, liver and serum alcohol content, and ferroptosis-related markers. P-ELNs, enriched in proteins, lipids, and small RNAs, exhibited an ideal size (150.7 ± 82.8 nm) and negative surface charge (-31 mV). Pre-treatment with 10 mg/(kg.bw) P-ELNs in both male and female mice significantly prolonged ebriety time, shortened sobriety time, enhanced acetaldehyde dehydrogenase (ALDH) activity while concurrently inhibited alcohol dehydrogenase (ADH) activity, and reduced alcohol content in the liver and serum. Notably, P-ELNs demonstrated more efficacy compared to P-ELNs supernatant fluid (abundant puerarin content), suggesting alternative active components beyond puerarin. Additionally, P-ELNs prevented ferroptosis by inhibiting the reduction of glutathione peroxidase 4 (GPX4) and reduced glutathione (GSH), and suppressing acyl-CoA synthetase long-chain family member 4 (ACSL4) elevation, thereby mitigating pathological liver lipid accumulation. P-ELNs exhibit distinct exosomal characteristics and effectively alleviate alcoholic intoxication, improve alcohol metabolism, suppress ferroptosis, and protect the liver from alcoholic injury. Consequently, P-ELNs hold promise as a therapeutic agent for detoxification, sobriety promotion, and prevention of alcoholic liver injury.

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.

Preconditioning Exercise Inhibits Neuron Ferroptosis and Ameliorates Brain Ischemia Damage by Skeletal Muscle-Derived Exosomes via Regulating miR-484/ACSL4 Axis.

Aims: Although there is evidence that patients with stroke who exercise regularly before stroke have a better prognosis than those who do not exercise, the detailed mechanism remains unclear. Moreover, neuronal death plays a central role in neurological dysfunction caused by ischemic stroke. Thus, we investigated whether exercise could reduce stroke-induced neuronal death and its associated mediators in the current study. Results: Ferroptosis was the most dominant form of programmed cell death in neurons. Preconditioning exercise before stroke improved the neurological function and decreased the infarct area in rats with ischemic stroke. Preconditioning exercise attenuated stroke-induced ferroptosis by reducing lipid peroxidation (LPO) production, upregulating glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11), and downregulating acyl-CoA synthetase long-chain family member 4 (ACSL4). High-throughput sequencing and dual luciferase reporter assays revealed that exercise-induced exosomal miR-484 inhibits Acsl4 expression. Moreover, we showed that exercise-induced exosomal miR-484 is mainly derived from skeletal muscle, and the neuroprotective effect of preconditioning exercise is suppressed by inhibiting miR-484 production in skeletal muscle. Innovation: This study suggested that neuronal ferroptosis is the most dominant form of programmed cell death in a hypoxic environment. Moreover, we showed that the ferroptosis pathway is a potential therapeutic target in ischemic stroke and that preconditioning exercise could be an effective antioxidant intervention for cerebral ischemia. Conclusion: Our work revealed that preconditioning exercise before stroke exerts neuroprotective effects against brain ischemia by skeletal muscle-derived exosomal miR-484 via inhibiting ferroptosis.

Identification critical host factors for Japanese encephalitis virus replication via CRISPR screening of human sgRNA library

Japanese encephalitis virus (JEV) is a pathogen with a substantial impact on both livestock and human health. However, the critical host factors in the virus life cycle remain poorly understood. Using a library comprising 123411 small guide RNAs (sgRNAs) targeting 19050 human genes, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based screen to identify essential genes for JEV replication. By employing knockout or knockdown techniques on genes, we identified eleven human genes crucial for JEV replication, such as prolactin releasing hormone receptor (PRLHR), activating signal cointegrator 1 complex subunit 3 (ASCC3), acyl-CoA synthetase long chain family member 3 (ACSL3), and others. Notably, we found that PRLHR knockdown blocked the autophagic flux, thereby inhibiting JEV infection. Taken together, these findings provide effective data for studying important host factors of JEV replication and scientific data for selecting antiviral drug targets.

Brazilin Actuates Ferroptosis in Breast Cancer Cells via p53/SLC7A11/GPX4 Signaling Pathway.

To investigate the mechanism of induction of ferroptosis by brazilin in breast cancer cells. Breast cancer 4T1 cells were divided into 6 groups: control, brazilin 1/2 half maximal inhibitory concentration (IC50), IC50, 2×IC50, erastin (10 µg/mL) and capecitabine (10 µg/mL) groups. The effect of brazilin on the proliferation of 4T1 cells was detected by cell counting kit-8 assay, and the treatment dose of brazilin was screened. The effect of brazilin on the mitochondrial morphology of 4T1 cells, and the mitochondrial damage was evaluated under electron microscopy. The levels of Fe2+, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase 4 (GPX4) were estimated using various detection kits. The invasion and migration abilities of 4T1 cells were detected by scratch assay and transwell assay. The expressions levels of tumor protein p53, solute carrier family 7 member 11 (SLC7A11), GPX4 and acyl-CoA synthetase long-chain family member 4 (ACSL4) proteins were quantified by Western blot assay. Compared to the control group, the 10 (1/2 IC50), 20 (IC50) and 40 (2×IC50) µg/mL brazilin, erastin, and capecitabine groups showed a significant decrease in the cell survival rate, invasion and migration abilities, GSH, SLC7A11 and GPX4 protein expression levels, and mitochondrial volume and ridge (P<0.05), and a significant increase in the mitochondria membrane density, Fe2+, ROS and MDA levels, and p53 and ACSL4 protein expression levels (P<0.05). Brazilin actuated ferroptosis in breast cancer cells, and the underlying mechanism is mainly associated with the p53/SLC7A11/GPX4 signaling pathway.

Cadmium aggravates liver injury by activating ferroptosis and neutrophil extracellular traps formation in Nile tilapia (Oreochromis niloticus).

Cadmium (Cd) is a pervasive environmental contaminant and a significant risk factor for liver injury. The present study was undertaken to evaluate the involvement of ferroptosis and neutrophil extracellular traps (NETs) in Cd-induced liver injury in Nile tilapia (Oreochromis niloticus), and to explore its underlying mechanism. Cd-induced liver injury was associated with increased total iron, malondialdehyde (MDA), and Acyl-CoA synthetase long-chain family member 4 (ACSL4), together with reduced levels of glutathione, glutathione peroxidase-4a (Gpx4a), and solute carrier family 7 member 11 (SLC7A11), which are all hallmarks of ferroptosis. Moreover, liver hyperemia, neutrophil infiltration, increased inflammatory factors and myeloperoxidase, as well as elevated serum DNA content in Cd-stimulated Nile tilapia suggested that a considerable number of neutrophils were recruited to the liver. Furtherly, in vitro experiments demonstrated that Cd induced the formation of NETs, and the possible mechanism was related to the generation of reactive oxygen species and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, along with the P38 and extracellular regulated protein kinase (ERK) signaling pathways. We concluded that ferroptosis and NETs are the critical mechanisms contributing to Cd-induced liver injury in Nile tilapia. These findings will contribute to Cd toxicological studies in aquatic animals.

Epigenetic mechanism of SET7/9-mediated histone methylation modification in high glucose-induced ferroptosis in retinal pigment epithelial cells.

Ferroptosis of the retinal pigment epithelial (RPE) cells leads to retinal neuron injury and even visual loss. Our study aims to investigate the role of the SET domain with lysine methyltransferase 7/9 (SET7/9) in regulating high glucose (HG)-induced ferroptosis in RPE cells. The cell model was established by HG treatment. The levels of SET7/9 and Sirtuin 6 (SIRT6) were inhibited and Runt-related transcription factor 1 (RUNX1) was overexpressed through cell transfection, and then their levels in ARPE-19 cells were detected. Cell viability and apoptosis was detected. The levels of reactive oxygen species, malondialdehyde, glutathione, ferrous ion, glutathione peroxidase 4, and acyl-CoA synthetase long-chain family member 4 were detected. SET7/9 and trimethylation of histone H3 at lysine 4 (H3K4me3) levels in the RUNX1 promoter region and RUNX1 level in the SIRT6 promoter region were measured. The relationship between RUNX1 and SIRT6 was verified. SET7/9 and RUNX1 were highly expressed while SIRT6 was poorly expressed in HG-induced ARPE-19 cells. SET7/9 inhibition increased cell viability and inhibited cell apoptosis and ferroptosis. Mechanistically, SET7/9 increased H3K4me3 on the RUNX1 promoter to promote RUNX1, and RUNX1 repressed SIRT6 expression. Overexpression of RUNX1 or silencing SIRT6 partially reversed the inhibitory effect of SET7/9 silencing on HG-induced ferroptosis. In conclusion, SET7/9 promoted ferroptosis of RPE cells through the SIRT6/RUNX1 pathway.

Surufatinib combined with photodynamic therapy induces ferroptosis to inhibit cholangiocarcinoma in vitro and in tumor models.

The curative effect of single therapy for advanced cholangiocarcinoma (CCA) is poor, thus investigating combined treatment strategies holds promise for improving prognosis. Surufatinib (SUR) is a novel multikinase inhibitor that has been confirmed to prolong survival of patients with advanced CCA. Photodynamic therapy (PDT) can also ablate advanced CCA and relieve biliary obstruction. In this study, we explored the anti-CCA effect of SUR combined with PDT, and explored the underlying mechanism. We found that SUR could effectively inhibit the abilities of proliferation, migration and metastasis in CCA cells (HUCCT-1, RBE). The ability of SUR to inhibit CCA was also confirmed by the HUCCT-1 cell xenograft model in Balb/c nude mice and CCA patient-derived organoids. SUR combined with PDT can significantly enhance the inhibitory effect on CCA, and can be alleviated by two ferroptosis inhibitors (Ferrostatin-1, Deferoxamine). By detecting the level of reactive oxygen species, lipid peroxides, malondialdehyde and glutathione, we further confirmed that SUR combined with PDT can inhibit CCA cells by inducing ferroptosis. Glutathione peroxidase 4 (GPX4) belongs to the glutathione peroxidase family and is mainly responsible for the metabolism of intracellular hydrogen peroxide. GPX4 inhibits ferroptosis by reducing cytotoxic lipid peroxides (L-OOH) to the corresponding alcohols (L-OH). Acyl-CoA synthetase long-chain family member 4 (ACSL4) is a member of the long-chain fatty acid coenzyme a synthetase family and is mainly involved in the biosynthesis and catabolism of fatty acids. ACSL4 induces ferroptosis by promoting the accumulation of lipid peroxides. Both SUR and PDT can induce ferroptosis by promoting ACSL4 and inhibiting GPX4. The regulation effect is found to be more significant in combined treatment group. In conclusion, SUR combined with PDT exerted an anti-CCA effect by inducing ferroptosis. Combination therapy provides a new idea for the clinical treatment of CCA.

Bcl-xL regulates radiation-induced ferroptosis through chaperone-mediated autophagy of GPX4 in tumor cells

ObjectiveTo investigate the role and the molecular mechanisms of apoptotic signaling in ferroptosis to regulate tumor radiosensitivity. MethodsReactive oxygen species (ROS) and lipid peroxide levels were detected in Mouse embryonic fibroblasts(MEFs) with Bcl-xL or Mcl-1 deficiency induced by erastin. Colony formation, ROS, lipid peroxidation and the transcription/translation levels of PTGS2 were measured in Bcl-xL knockdown tumor cells induced by 5 ​Gy γ-rays or co-treated with ferrostatin-1 (Ferr-1). The protein levels of LPCAT3, ACSL4 and PEBP1 in Bcl-xL knockout MEF cells were evaluated in Bcl-xL knockout MEF cells post-radiation. Moreover, the interaction of heat shock protein 90 (HSP90) with Bcl-xL, GPX4, or LAMP2A was detected by protein mass spectrometry and immunoprecipitation assays. ResultsManipulating Bcl-xL levels facilitated radiation-induced ferroptosis by augmenting the enzymatic oxidation of polyunsaturated fatty acids (PUFAs) and enhancing chaperone-mediated autophagy (CMA) of glutathione peroxidase 4 (GPX4) (MEF cell line: t=4.540, P＜0.01; A549 ​cell line: t=56.16, P＜0.0001; t=4.885, P＜0.01; HCT116 ​cell line: t=14.75, P＜0.01; t=7.363, P＜0.05). Downregulating Bcl-xL expression promoted the activity of acyl-CoA synthetase long-chain family member 4 (ACSL4), thus increasing the enzymatic oxidation of PUFAs (t=4.258, P＜0.01). Moreover, depletion of Bcl-xL expedited the CMA process targeting GPX4 by facilitating the association of GPX4 with heat shock protein 90 (HSP90) and LAMP2A following radiation exposure. Subsequent degradation of GPX4 led to the accumulation of lipid peroxides, ultimately triggering ferroptosis. ConclusionsOur study provides initial insights into the regulatory role of Bcl-xL in ferroptosis and underscores the potential of targeting Bcl-xL as a promising therapeutic strategy for cancer by modulating both apoptotic and ferroptotic pathways.

Altered Iron-Mediated Metabolic Homeostasis Governs the Efficacy and Toxicity of Tripterygium Glycosides Tablets Against Rheumatoid Arthritis

Rheumatoid arthritis (RA), a globally increasing autoimmune disorder, is associated with increased disability rates due to the disruption of iron metabolism. Tripterygium glycoside tablets (TGTs), a Tripterygium wilfordii Hook. f. (TwHF)-based therapy, exhibit satisfactory clinical efficacy for RA treatment. However, drug-induced liver injury (DILI) remains a critical issue that hinders the clinical application of TGTs, and the molecular mechanisms underlying the efficacy and toxicity of TGTs in RA have not been fully elucidated. To address this problem, we integrated clinical multi-omics data associated with the anti-RA efficacy and DILI of TGTs with the chemical and target profiling of TGTs to perform a systematic network analysis. Subsequently, we identified effective and toxic targets following experimental validation in a collagen-induced arthritis (CIA) mouse model. Significantly different transcriptome–protein–metabolite profiles distinguishing patients with favorable TGTs responses from those with poor outcomes were identified. Intriguingly, the clinical efficacy and DILI of TGTs against RA were associated with metabolic homeostasis between iron and bone and between iron and lipids, respectively. Particularly, the signal transducer and activator of transcription 3 (STAT3)–hepcidin (HAMP)/lipocalin 2 (LCN2)–tartrate-resistant acid phosphatase type 5 (ACP5) and STAT3–HAMP–acyl-CoA synthetase long-chain family member 4 (ACSL4)–lysophosphatidylcholine acyltransferase 3 (LPCAT3) axes were identified as key drivers of the efficacy and toxicity of TGTs. TGTs play dual roles in ameliorating CIA-induced pathology and in inducing hepatic dysfunction, disruption of lipid metabolism, and hepatic lipid peroxidation. Notably, TGTs effectively reversed “iron–bone” disruptions in the inflamed joint tissues of CIA mice by inhibiting the STAT3–HAMP/LCN2–ACP5 axis, subsequently leading to “iron–lipid” disturbances in the liver tissues via modulation of the STAT3–HAMP–ACSL4–LPCAT3 axis. Additional bidirectional validation experiments were conducted using MH7A and AML12 cells to confirm the bidirectional regulatory effects of TGTs on key targets. Collectively, our data highlight the association between iron-mediated metabolic homeostasis and the clinical efficacy and toxicity of TGT in RA therapy, offering guidance for the rational clinical use of TwHF-based therapy with dual therapeutic and toxic potential.

Aristolochic acid induces acute kidney injury through ferroptosis.

Aristolochic acid (AA)-induced acute kidney injury (AKI) presents with progressive decline in renal function and rapid progression to end-stage renal disease. Among the multiple mechanisms identified in AKI, ferroptosis has been shown to be involved in various forms of AKI. But few studies have elucidated the role of ferroptosis in AA-induced AKI. In this study, we investigated the role of ferroptosis in AA-induced acute renal tubular injury in vivo and in vitro. Mice with acute aristolochic acid nephropathy showed increased malondialdehyde levels, aggravated lipid peroxidation, decreased superoxide dismutase activity, and glutathione depletion. The expression of glutathione peroxidase 4 was decreased and the expression of acyl-CoA synthetase long-chain family member 4 was increased. Inhibition of ferroptosis by ferrostatin-1 significantly improved the renal function, reduced histopathological lesions, partially alleviated lipid peroxidation, and restored the antioxidant capacity. In vitro studies also revealed that AA significantly reduced cell viability, induced reactive oxygen species production, increased intracellular iron level and decreased ferroptosis-related protein expression. Inhibition of ferroptosis significantly increased cell viability and attenuated AA-induced renal tubular epithelial cell injury. It is suggested that ferroptosis plays an important role in AA-induced acute tubular injury. And inhibition of ferroptosis may exert renoprotective effects possibly by preventing lipid peroxidation, restoring the antioxidant activity or regulating iron metabolism.

Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Promote the Recovery of Spinal Cord Injury and Inhibit Ferroptosis by Inactivating IL-17 Pathway.

Mesenchymal stem cell (MSC)-derived exosomes are considered as alternative to cell therapy in various diseases. This study aimed to understand the effect of bone marrow MSC-derived exosomes (BMMSC-exos) on spinal cord injury (SCI) and to unveil its regulatory mechanism on ferroptosis. Exosomes were isolated from BMMSCs and the uptake of BMMSCs-exos by PC12 cells was determined using PKH67 staining. The effect of BMMSC-exos on SCI in rats was studied by evaluating pathological changes of spinal cord tissues, inflammatory cytokines, and ferroptosis-related proteins. Transcriptome sequencing was used to discover the differential expressed genes (DEGs) between SCI rats and BMMSC-exos-treated rats followed by functional enrichment analyses. The effect of BMMSC-exos on ferroptosis and interleukin 17 (IL-17) pathway was evaluated in SCI rats and oxygen-glucose deprivation (OGD)-treated PC12 cells. Theresults showed that particles extracted from BMMSCs were exosomes that could be taken up by PC12 cells. BMMSC-exos treatment ameliorated injuries of spinal cord, suppressed the accumulation of Fe2+, malondialdehyde (MDA), and reactive oxygen species (ROS), with the elevated glutathione (GSH). Also, BMMSC-exos downregulated the expression of acyl-CoA synthetase long chain family member 4 (ACSL4) and upregulated glutathione peroxidase 4 (GPX4) and cysteine/glutamate antiporter xCT. A total of 110 DEGs were discovered and they were mainly enriched in IL-17 signaling pathway. Further in vitro and in vivo experiments showed that BMMSC-exos inactivated IL-17 pathway. BMMSC-exos promote the recovery of SCI and inhibit ferroptosis by inhibiting the IL-17 pathway, which provides BMMSC-exos as an alternative to the management of SCI.

Ischemia-inhibited ferric chelate reductase 1 improves ferroptosis-mediated intestinal ischemia injury via Hippo signaling

The precise mechanism of ferroptosis as a regulatory cell death in intestinal ischemia injury induced by vascular intestinal obstruction (Vio) remains to be elucidated. Here, we evaluated iron levels, glutathione peroxidase 4 (GPX4) and Acyl-CoA synthetase long-chain family member 4 (ACSL4) changes after intestinal ischemia injury to validate ferroptosis. As an enzyme for Fe3+ reduction to Fe2+, Ferric Chelate Reductase 1 (FRRS1) is involved in the electron transport chain and the tricarboxylic acid (TCA) cycle in mitochondria. However, whether it is involved in ferroptosis and its role in intestinal ischemia injury need to be clarified. In the present study, FRRS1 was overexpressed in vivo and in vitro. The results showed that overexpression of FRRS1 prevented ischemia-induced iron levels, reactive oxygen species (ROS) production, lipid peroxidation, inflammatory responses, and cell death. Meanwhile, FRRS1 overexpression promoted GPX4 expression and suppressed ACSL4 levels. Further studies revealed that FRRS1 overexpression inhibited the activity of large tumor suppressor 1 (LATS1) / Yes-associated protein (YAP) / transcriptional co-activator with PDZ-binding motif (TAZ), a key component of Hippo signaling. In conclusion, this study demonstrates that FRRS1 is intimately involved in the inhibition of ferroptosis and thus protection of the intestine from intestinal ischemia injury, its downstream mechanism was related to Hippo signaling. These data provide new sight for the prevention and treatment of intestinal ischemia injury.

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.