Glutathione Peroxidase Protein Research Articles

GPX4 Promoter Hypermethylation Induced by Ischemia/Reperfusion Injury Regulates Hepatocytic Ferroptosis.

Glutathione peroxidase 4 (GPX4) is a key factor in ferroptosis, which is involved in ischemia-reperfusion injury. However, little is known about its role in hepatic ischemia-reperfusion injury (HIRI). This study aimed to investigate the role of GPX4 methylation in ferroptosis during HIRI. For the in vitro experiments, an oxygen and glucose deprivation cell model was established. For the in vivo experiments, an ischemia-reperfusion model was created by subjecting mice to simulated HIRI. Ferroptosis occurrence, GPX4 promoter methylation, and global methylation levels were then assessed. Ferroptosis was observed in oxygen and glucose deprivation, characterized by a significant decrease in cellular viability (P < 0.05), an increase in lipid peroxidation (P < 0.01), iron overload (P < 0.05), and down-regulation of GPX4 (P < 0.05). This ferroptosis was exacerbated by GPX4 knockdown (P < 0.01) and mitigated by exogenous glutathione (P < 0.01). Similarly, ferroptosis was evident in mice subjected to HIRI, with a down-regulation of GPX4 mRNA and protein expression (all P < 0.01), and an upregulation of acyl-CoA synthetase long-chain family member 4 mRNA and protein (all P < 0.01), as well as prostaglandin-endoperoxide synthase 2 mRNA and protein expression (all P < 0.05). Methylation levels increased, evidenced by upregulation of DNA methylation transferase expression (P < 0.05) and down-regulation of Ten-eleven translocation family demethylases (P < 0.01), along with an upregulation of GPX4 promoter methylation. Ferroptosis may be the primary mode of cell death in hepatocytes following ischemia-reperfusion injury. The methylation of the GPX4 promoter and elevated levels of global hepatic methylation are involved in the regulation of ferroptosis.

Fibronectin type III domain containing protein 5/irisin alleviated sepsis-induced acute kidney injury by abating ferroptosis through the adenosine 5'-monophosphate-activated protein kinase/nuclear factor erythroid-2-related factor 2 signaling pathway

Objective: Ferroptosis has been described in association with acute kidney injury (AKI)-induced sepsis. Fibronectin type III domain containing protein 5 (FNDC5)/irisin plays a crucial role in renal protection. The objective of this study was to investigate whether FNDC5/irisin is involved in AKI-induced sepsis by modulating ferroptosis, and the molecular mechanisms that may be involved. Material and Methods: A sepsis-induced AKI model was built in vivo and in vitro through lipopolysaccharide (LPS) intervention. FNDC5, adenosine 5'-monophosphate-activated protein kinase (AMPK), phospho-AMPK (p-AMPK), nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), glutathione peroxidase 4 (GPX4), and acyl-CoA synthetase long-chain family member 4 (ACSL4) concentrations in cells and mouse kidney tissues were appraised by Western blot. Pro-inflammatory cytokines concentrations in cell supernatants and mouse kidney tissues were appraised by enzyme-linked immunosorbent assay. Fe2+ concentration in cells and mouse kidney tissue was appraised by kit. The apoptosis rate of cells and mouse kidney tissue was measured by flow cytometry. Automatic biochemical analyzer was to test serum creatinine (SCr) and blood urea nitrogen (BUN). The kidney tissue sections from each groups were observed by hematoxylin and eosin staining. Results: LPS abated FNDC5 concentration in human kidney-2 cells and mouse kidney tissue (P &lt; 0.001). Overexpression of FNDC5 can abated proinflammatory cytokines concentrations in cells and mouse kidney tissue (P &lt; 0.01). Meanwhile, overexpression of FNDC5 can boost GPX4 protein concentration, abate ACSL4 protein, and abate Fe2+ concentration in cells and mouse kidney tissues (P &lt; 0.05). In addition, the overexpression of FNDC5 can reduce the rate of apoptosis (P &lt; 0.01). In vivo experiments showed that FNDC5 overexpression reduced serum BUN and SCr concentrations and alleviated pathological damage in the mouse renal tissues (P &lt; 0.05) and exhibited a certain renal protective effect. FNDC5 overexpression can boost p-AMPK/AMPK, Nrf2, and HO-1 protein concentrations (P &lt; 0.01). Conclusion: FNDC5/irisin improves sepsis-induced acute renal injury by abating ferroptosis through the AMPK/Nrf2 signaling pathway.

Ultrasound-guided femoral nerve block reduced the incidence of postoperative delirium after total knee arthroplasty: A double-blind, randomized study

Background: Postoperative delirium (POD) is an acute brain dysfunction that mainly occurs in elderly patients after surgery. Postoperative pain is an important factor in the occurrence of POD, and effective pain management can reduce the risk of POD. Our study aims is to investigate the effect of ultrasound-guided femoral nerve block (FNB) on postoperative pain and the occurrence of POD after knee arthroplasty, and whether its mechanism is related to oxidative stress, inflammatory factors. Methods: Patients aged 65 to 80 years who were scheduled to undergo knee arthroplasty under general anesthesia from 2020 to 2023 were randomly divided into 2 groups: the control group (NC group) and the FNB analgesia group (FNB group). After anesthesia induction, the FNB group received ultrasound-guided FNB, whereas the NC group received an equal volume of normal saline. Visual analog scale (VAS), incidence of POD. the postoperative Quality of Recovery-15-Chinese version (QoR-15C) was been evaluated. Venous blood samples were collected from patients to detect the level of malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and C-reactive protein (CRP). Results: The VAS scores in the FNB group were significantly lower than those in the NC group at 24h. The summation of perioperative remifentanil usage and the number of remedial analgesia in the FNB group were significantly lower than those in the NC group. The incidence rate of POD in the FNB group is lower than the NC group. The occurrence of POD was markedly lower in the FNB group compared to the NC group. Compared with the NC group, the levels of MDA, GSH-Px, and CRP in the FNB group were lower on the first and third days after surgery. The QoR-15C score in the FNB group was higher on the 5th day after surgery. Conclusion: Ultrasound-guided FNB can reduce the incidence of POD after knee replacement surgery, and its mechanism may be related to analgesia improvement and reduction of postoperative inflammatory reaction.

Lipid composition differentiates ferroptosis sensitivity between in vitro and in vivo systems.

Ferroptosis is a regulated non-apoptotic cell death process characterized by iron-dependent lipid peroxidation. This process has recently emerged as a promising approach for cancer therapy. Peroxidation of polyunsaturated fatty acid-containing phospholipids (PUFA-PLs) is necessary for the execution of ferroptosis. Ferroptosis is normally suppressed by glutathione peroxidase 4 (GPX4), which reduces lipid hydroperoxides to lipid alcohols. Some evidence indicates that GPX4 may be a useful target for drug development, yet factors that govern GPX4 inhibitor sensitivity in vivo are poorly understood. We find that pharmacological and genetic loss of GPX4 function was sufficient to induce ferroptosis in multiple adherent ("2D") cancer cell cultures. However, reducing GPX4 protein levels did not affect tumor xenograft growth when these cells were implanted in mice. Furthermore, sensitivity to GPX4 inhibition was markedly reduced when cells were cultured as spheroids ("3D"). Mechanistically, growth in 3D versus 2D conditions reduced the abundance of PUFA-PLs. 3D culture conditions upregulated the monounsaturated fatty acid (MUFA) biosynthetic gene stearoyl-CoA desaturase (SCD). SCD-derived MUFAs appear to protect against ferroptosis in 3D conditions by displacing PUFAs from phospholipids. Various structurally related long chain MUFAs can inhibit ferroptosis through this PUFA-displacement mechanism. These findings suggest that growth-condition-dependent lipidome remodeling is an important mechanism governing GPX4 inhibitor effects. This resistance mechanism may specifically limit GPX4 inhibitor effectiveness in vivo .

Design, synthesis, and in vitro and in vivo biological evaluation of dihydroartemisinin derivatives as potent anti-cancer agents with ferroptosis-inducing and apoptosis-activating properties

Natural products play a pivotal role in drug development, including their direct use as pharmaceuticals and their structural modification, yielding molecules with enhanced therapeutic potential. The discovery of bioactive molecules, lead compounds, and novel drugs is intrinsically linked to the structural optimization of natural products. In this study, forty-one derivatives of dihydroartemisinin (DHA) were synthesized by incorporating fragments with anti-tumour activity via molecular hybridization, and assessed for their anti-proliferative activity against human cancer cell lines (A549, Bel-7402, HCT-116, and SW620) and normal human liver cells (LO2). Most derivatives exhibited superior anti-proliferative activity compared to DHA. Notably, compound A3, featuring a 4-Cl phenyl carbamate moiety, demonstrated significant anti-proliferative activity against HCT-116 cells with an IC50 of 0.31 μM, making it 16-fold more potent than DHA (IC50 = 5.10 μM). The anti-proliferative mechanism did not involve cytotoxicity (SI = 54.13), indicating its superior safety profile compared to DHA (SI = 1.65).Further mechanistic studies revealed that compound A3 inhibits HCT-116 cell proliferation by modulating the expression of PI3K/AKT/mTOR and STAT3 proteins. STAT3 downregulation represses the expression of the critical ferroptosis protein glutathione peroxidase 4 (GPX4), aggravating the accumulation of reactive oxygen species (ROS) and depletion of glutathione (GSH). This redox imbalance triggers and accelerates ferroptosis. Additionally, A3 also induces apoptosis by damaging mitochondria and influencing MAPK signaling. Compound A3 arrested cells in the G2/M phase by regulating p53 expression. In an HCT-116 xenograft mouse model, compound A3 exhibited significant anti-cancer efficacy, with a tumor growth inhibition rate of 58.7 %. Therefore, compound A3 thus has the potential to serve as a lead compound for the development of new anti-tumor drugs.

Effect of "Xingnao Kaiqiao" needling on expression of ferroptosis-related factors in rats with cerebral ischemia-reperfusion injury

To observe the effect of "Xingnao Kaiqiao" needling on the expression of ferroptosis-related proteins in neurons of rats with cerebral ischemia-reperfusion injury (CIRI), so as to explore its mechanism underlying improvement of CIRI. Male Wistar rats were randomly divided into sham operation, model, acupuncture and deferoxamine (DFO) groups, with 18 rats in each group. The CIRI model was established by occlusion of the middle cerebral artery. In the acupuncture group, "Xingnao Kaiqiao" needling was applied to "Shuigou" (GV26), "Neiguan" (PC6) and "Sanyinjiao"(SP6) for 20 min with electroacupuncture (2 Hz/15 Hz, 1 mA) at PC6 and SP6, twice daily for continuous 3 days. Rats of the DFO group received intraperitoneal injection of iron chelator DFO (0.1 g/kg, once daily). The severity of neurological impairment (neurological deficit score, 0-5 points, the lower the score, the severer is the neurological impairment) was evaluated by using Zausinger 6-poins scaling method. The cerebral infarct volume was measured after 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, and the histopathological changes of the ischemic brain tissue were observed after H.E. staining. The mitochondrial structure of the hippocampal neurons on the ischemic side of the brain was observed by using transmission electron microscope. The levels of iron deposition rate (%) in the ischemic penumbra of the brain tissue and hippocampus were observed after Prussian blue staining, and the reactive oxygen species (ROS) content of the cerebral ischemic penumbra was assayed using flow cytometry, and the content of glutathione (GSH) content in the ischemic penumbra was detected by using microplate method. The real-time quantitative PCR was used to detect the expression of glutathione peroxidase 4 (GPX4), divalent metal transporter 1 (DMT1), transferrin (TF), transferrin receptor 1 (TFR1), and ferroportin 1 (FPN1) mRNAs in the ischemic penumbra, and the Western blot was used to detect the expression of GPX4, DMT1, TF, TFR1, FPN1, and ferritin (FER) proteins in the ischemic penumbra. Compared with the sham operation group, the neurological deficit score, GSH content, expression of GPX4 and FPN1 mRNAs and proteins were significantly decreased (P<0.01), while the percentage of cerebral infarct volume, iron deposit rates of the cerebral ischemic penumbra and hippocampus, ROS content, and the expression levels of DMT1, TF, and TFR1 mRNAs and proteins and FER protein were considerably increased (P<0.01) in the model group. In comparison with the model group, the decrease of neurological deficit score, GSH content, expression of GPX4 and FPN1 mRNAs and proteins, and the increase of the percentage of cerebral infarct volume, iron deposit rates of the cerebral ischemic penumbra and hippocampus, ROS content, and the expression levels of DMT1, TF, and TFR1 mRNAs and proteins and FER protein were all reversed in both DFO and acupuncture groups (P<0.01, P<0.05). The effects of acupuncture were significantly superior to those of DFO in lowering the levels of cerebral cortical and hippocampal iron deposit rates, ROS content and in elevating the expression of GPX4 mRNA and protein (P<0.01, P<0.05). H.E. staining showed large necrotic cells, disordered arrangement of cells in the cerebral cortex and hippocampus, with hyperchromic nuclei, vacuole-like changes, widening of cellular space, and cell swelling in the model group, which was relatively milder in the cell damage in both acupuncture and DFO groups. In addition, the ultrastructure of cells in the hippocampus showed irregular cellular nuclear morphology, atrophy of some mitochondria in the cytoplasm, partial mitochondrial membrane rupture and edema, and loosening of the ridge structure in the model group, which was milder in the mitochondrial impairment (including reduced number of mitochondria, broken mitochondrial membrane and reduced ridge structure in fewer cells) in the acupuncture group. The "Xingnao Kaiqiao" needling intervention has a neuroprotective effect in CIRI rats, which may be related to its functions in regulating ferroptosis-related targets and iron metabolism in cerebral ischemic penumbra, reducing oxidative stress injury, and suppressing neuronal ferroptosis.

Knockdown of nuclear protein 1 delays pathological pro-gression of osteoarthritis through inhibiting chondrocyte ferroptosis.

To investigate the effect of nuclear protein 1 (Nupr1) on the pathological progression of osteoarthritis and its relationship with ferroptosis of chondrocytes. Chondrocytes of mouse knee were divided into small interfering RNA (siRNA) control group, small interfering RNA targeting Nupr1 (siNupr1) group, siRNA control+IL-1β group (siRNA control interference for 24 h followed by 10 ng/mL IL-1β) and siNupr1+IL-1β group (siNupr1 interference for 24 h followed by 10 ng/mL IL-1β). The protein and mRNA expressions of Nupr1 were detected by Western blotting and real-time RT-PCR. Cell proliferation viabilities were measured using the cell counting kit-8 method. The levels of ferrous ions were detected by FerroOrange staining. Lipid peroxidation levels were detected by C11-BODIPY-591 fluorescence imaging. The contents of malondialdehyde (MDA) and glutathione (GSH) were detected by enzyme-linked immunosorbent assay. The protein expressions of long-chain acyl-coenzyme A synthetase 4 (ACSL4), tumor protein 53 (P53), glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 gene (SLC7A11) were detected by Western blotting. The osteoarthritis model was constructed by destabilization of the medial meniscus (DMM) surgery in 7-week-old male C57BL/6J mice. The mice were randomly divided into 4 groups with 10 animals in each group: sham surgery (Sham)+adeno-associated virus serotype 5 (AAV5)-Short hairpin RNA (shRNA) control group, Sham+AAV5-shRNA targeting Nupr1 (shNupr1) group, DMM+AAV5-shRNA group, and DMM+AAV5-shNupr1 group. Hematoxylin and eosin staining and Safranin O-Fast Green staining were used to observe the morphological changes in cartilage tissue. The Osteoarthritis Research Society International (OARSI) osteoarthritis cartilage histopathology assessment system was used to evaluate the degree of cartilage degeneration in mice. The mRNA expressions of Nupr1, matrix metallopeptidase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), cyclooxygenase 2 (COX2), and glutathione peroxidase 4 (GPX4) were detected by real-time RT-PCR. In vitro experiments showed that knocking down Nupr1 alleviated the decrease of chondrocyte proliferation activity induced by IL-1β, reduced iron accumulation in mouse chondrocytes, lowered the lipid peroxidation, downregulated ACLS4 and P53 protein expression and upregulated GPX4 and SLC7A11 protein expression (all P<0.01), thereby inhibiting ferroptosis in mouse chondrocytes. Meanwhile, in vivo animal experiments demonstrated that knocking down Nupr1 delayed the degeneration of articular cartilage in osteoarthritis mice, improved the OARSI score, slowed down the degradation of the extracellular matrix in osteoarthritis cartilage, and reduced the expression of the key ferroptosis regulator GPX4 (all P<0.01). Knockdown of Nupr1 can delay the pathological progression of osteoarthritis through inhibiting ferroptosis in mouse chondrocytes.

METTL14-Mediated m6A Modification of TUG1 Represses Ferroptosis in Alzheimer's Disease via Inhibiting GDF15 Ubiquitination.

Alzheimer's disease (AD) is a neurodegenerative disease that remains a serious global health issue. Ferroptosis has been recognized as a vital driver of pathological progression of AD. However, the detailed regulatory mechanisms of ferroptosis during AD progression remain unclear. This study aimed to explore the regulatory role and mechanism of methyltransferase like 14 (METTL14) in ferroptosis in AD models. Serum samples were collected from 18 AD patients and 18 healthy volunteers to evaluate clinical correlation. Scopolamine-treated mice and Aβ1-42-stimulated SH-SY5Y cells were served as the in vivo and in vitro models of AD. Ferroptosis was detected by reactive oxygen species (ROS), Fe2+, total iron levels, and ferroptosis-related proteins glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11). Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. The N6-methyladenosine (m6A) modification was detected by RNA methylation quantification kit and methylated RNA immunoprecipitation sequencing-quantitative real-time polymerase chain reaction (MeRIP-qPCR). Molecular mechanisms were investigated by RNA pull-down, RNA immunoprecipitation (RIP), and co-immunoprecipitation (Co-IP) assays. Cognitive disorder of AD mice was measured by Morris water maze test. METTL14 was down-regulated, while lncRNA taurine upregulated gene 1 (TUG1) was up-regulated in clinical patients and experimental models of AD. Functional experiments demonstrated that METTL14 overexpression or TUG1 silencing effectively attenuated Aβ1-42-induced ferroptosis and neurotoxicity in SH-SY5Y cells. Mechanistically, METTL14-mediated m6A modification reduced the stability of TUG1. Moreover, TUG1 promoted the ubiquitination and degradation of growth differentiation factor 15 (GDF15) by directly interacted with Smad ubiquitin regulatory factor 1 (SMURF1), which consequently inactivated nuclear factor erythroid 2-related factor 2 (NRF2). Rescue experiments indicated that GDF15 depletion reversed sh-TUG1-mediated protection against ferroptosis and neurotoxicity. Finally, Mettl14 overexpression repressed ferroptosis to ameliorate the cognitive disorder via modulating Tug1/Gdf15/Nrf2 pathway in vivo. METTL14 inhibited ferroptosis to ameliorate AD pathological development by m6A modification of TUG1 to activate GDF15/NRF2 axis, providing a novel therapeutic target for AD.

Tributyltin-induced visceral adiposity is associated with impaired redox balance in white adipose tissue of male rats

Tributyltin (TBT) is an organotin compound that has several adverse health effects, including the development of obesity. Although obesity is strongly associated with adipose redox imbalance, there is a lack of information on whether TBT promotes a pro-oxidative environment in WAT. Thus, adult male Wistar rats were randomly exposed to either vehicle (ethanol 0.4%) or TBT (1000 ng/kg) for 30 days. Body and fat pad masses, visceral fat morphology, lipid peroxidation, protein carbonylation, redox status markers, and catalase activity were evaluated. TBT promoted increased adiposity and visceral fat, with hypertrophic adipocytes, but did not alter body mass and subcutaneous fat. ROS production and lipid peroxidation were elevated in TBT group, as well as catalase protein expression and activity, although protein oxidation and glutathione peroxidase protein expression remained unchanged. In conclusion, this is the first study to demonstrate that subacute TBT administration leads to visceral adipose redox imbalance, with increased oxidative stress. This enlights the understanding of the metabolic toxic outcomes of continuous exposure to TBT in mammals.

Regulation of the p53/SLC7A11/GPX4 Pathway by Gentamicin Induces Ferroptosis in HEI-OC1 Cells.

Gentamicin is a commonly used aminoglycoside antibiotic, with ototoxicity as a significant side effect. Ferroptosis, an iron-dependent form of cell death, has been implicated in a variety of disorders. Whether ferroptosis impacts gentamicin ototoxicity is not yet known. The current work used an in-vitro model to examine the influence of gentamicin-induced ferroptosis on cochlear hair cell damage and probable molecular biological pathways. House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were treated with different concentrations of gentamicin for 24 hours, with or without ferrostatin-1 pretreatment, to observe gentamicin-induced ferroptosis. The role of p53/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling in gentamicin-induced ferroptosis was explored by pretreating cells with the p53 inhibitor pifithrin-α (PFT-α). We investigated the effect of gentamicin on cells by assessing cell viability. Cellular proteins were isolated and Western blots were performed to detect changes in the expression of p53, SLC7A11, and GPX4. Fluorescence staining was used to assess levels of reactive oxygen species. An enzymatic detection kit was used to detect glutathione, Fe, and malondialdehyde markers. Gentamicin reduced cell viability, glutathione content, and SLC7A11 and GPX4 protein levels, and increased levels of p53 protein, reactive oxygen species, malondialdehyde, and Fe. These effects were largely blocked by pretreatment with ferrostatin-1. Pretreatment with the p53 inhibitor PFT-α prevented the gentamicin-induced reduction in SLC7A11 and GPX4, which alleviated several features of ferroptosis including glutathione depletion, iron overload, and lipid peroxidation build-up. Gentamicin induces ferroptosis in the HEI-OC1 cell line, and the mechanism may be related to the p53/SLC7A11/GPX4 signaling pathway.

Inducible RPE-specific GPX4 knockout causes oxidative stress and retinal degeneration with features of age-related macular degeneration

Age-related macular degeneration (AMD) is one of the leading causes of vision loss in the elderly. This disease involves oxidative stress burden in the retina leading to death of retinal pigment epithelial (RPE) cells and photoreceptors. The retina is susceptible to oxidative stress, in part due to high metabolic activity and high concentration of polyunsaturated fatty acids that undergo lipid peroxidation chain reactions. Antioxidant enzymes exist in the retina to combat this stress, including glutathione peroxidase 4 (GPX4). GPX4 specifically reduces oxidized lipids, protecting against lipid peroxidation-induced oxidative stress, which is noted in dry AMD. We hypothesize that Gpx4 knockout within the RPE will result in an environment of chronic oxidative stress yielding degeneration akin to AMD. C57BL/6J mice with a floxed Gpx4 gene were mated with Rpe65Cre/ER mice. Offspring containing Rpe65Cre ± alleles and either Gpx4 WT or Gpx4 fl/fl alleles were administered tamoxifen to induce Gpx4 knockout in Gpx4 fl/fl mice. At sequential timepoints, retinal phenotypes were assessed via in vivo imaging utilizing confocal scanning laser ophthalmoscopy and optical coherence tomography (OCT), and visual function was probed by electroretinography. Retinas were studied post-mortem by immunohistochemical analyses, electron microscopy, plastic sectioning, and quantitative polymerase chain reaction and Western analyses. The RPE-specific Gpx4 knockout model was validated via Western analysis indicating diminished GPX4 protein only within the RPE and not the neural retina. Following Gpx4 knockout, RPE cells became dysfunctional and died, with significant cell loss occurring 2 weeks post-knockout. Progressive thinning of the photoreceptor layer followed RPE degeneration and was accompanied by loss of visual function. OCT and light microscopy showed hyperreflective foci and enlarged, pigmented cells in and above the RPE layer. Electron microscopy revealed decreased mitochondrial cristae and loss of basal and apical RPE ultrastructure. Finally, there was increased carboxyethylpyrrole staining, indicating oxidation of docosahexaenoic acid, and increased levels of mRNAs encoding oxidative stress-associated genes in the RPE and photoreceptors. Overall, we show that RPE-localized GPX4 is necessary for the health of the RPE and outer retina, and that knockout recapitulates phenotypes of dry AMD.

Design, synthesis, and biological evaluation of RSL3-based GPX4 degraders with hydrophobic tags

Ferroptosis is a new type of programmed cell death characterized by iron-dependent lipid peroxidation, during which glutathione peroxidase 4 (GPX4) plays an essential role and is well-recognized as a promising therapeutic target for cancer treatment. Although some GPX4 degradation molecules have been developed to induce ferroptosis, the discovery of GPX4 degraders with hydrophobic tagging (HyT) as an innovative approach is more challenging. Herein, we designed and synthesized a series of HyT degraders by linking the GPX4 inhibitor RSL3 with a hydrophobic and bulky group of adamantane. Among them, compound R8 is a potent degrader (DC50, 24h = 0.019 μM) which can effectively degrade GPX4 in a dose- and time-dependent manner. Furthermore, compound R8 exhibited superior in vitro antitumor potency against HT1080 and MDA-MB-231 cell lines with IC50 values of 24 nM and 32 nM respectively, which are 4 times more potent than parental compound RSL3. Mechanistic investigation evidenced that R8 consumes GPX4 protein mainly through the ubiquitin proteasome (UPS) and enables to induce the accumulation of LPO, thereby triggering ferroptosis. Our work presented the novel GPX4 degrader of R8 by HyT strategy, and provided a promising pathway of degradation agents for the treatment of ferroptosis relevant diseases.

Radiation-Induced Endothelial Ferroptosis Accelerates Atherosclerosis via the DDHD2-Mediated Nrf2/GPX4 Pathway.

This study sought to explore potential roles of endothelial ferroptosis in radiation-associated atherosclerosis (RAA) and molecular mechanisms behind this phenomenon. Here, an in vivo RAA mouse model was used and treated with ferroptosis inhibitors. We found that the RAA group had a higher plaque burden and a reduction in endothelial cells with increased lipid peroxidation compared to the control group, while ameliorated by liproxstatin-1. In vitro experiments further confirmed that radiation induced the occurrence of ferroptosis in human artery endothelial cells (HAECs). Then, proteomics analysis of HAECs identified domain-containing protein 2 (DDHD2) as a co-differentially expressed protein, which was enriched in the lipid metabolism pathway. In addition, the level of lipid peroxidation was elevated in DDHD2-knockdown HAECs. Mechanistically, a significant decrease in the protein and mRNA expression of glutathione peroxidase 4 (GPX4) was observed in HAECs following DDHD2 knockdown. Co-immunoprecipitation assays indicated a potential interaction between DDHD2 and nuclear factor erythroid 2-related factor 2 (Nrf2). The downregulation of Nrf2 protein was also detected in DDHD2-knockdown HAECs. In conclusion, our findings suggest that radiation-induced endothelial ferroptosis accelerates atherosclerosis, and DDHD2 is a potential regulatory protein in radiation-induced endothelial ferroptosis through the Nrf2/GPX4 pathway.

Ultrasound-responsive Bi2MoO6-MXene heterojunction as ferroptosis inducers for stimulating immunogenic cell death against ovarian cancer

BackgroundOvarian cancer (OC) has the highest fatality rate among all gynecological malignancies, necessitating the exploration of novel, efficient, and low-toxicity therapeutic strategies. Ferroptosis is a type of programmed cell death induced by iron-dependent lipid peroxidation and can potentially activate antitumor immunity. Developing highly effective ferroptosis inducers may improve OC prognosis.ResultsIn this study, we developed an ultrasonically controllable two-dimensional (2D) piezoelectric nanoagonist (Bi2MoO6-MXene) to induce ferroptosis. A Schottky heterojunction between Bi2MoO6 (BMO) and MXene reduced the bandgap width by 0.44 eV, increased the carrier-separation efficiency, and decreased the recombination rate of electron–hole pairs under ultrasound stimulation. Therefore, the reactive oxygen species yield was enhanced. Under spatiotemporal ultrasound excitation, BMO-MXene effectively inhibited OC proliferation by more than 90%, induced lipid peroxidation, decreased mitochondrial-membrane potential, and inactivated the glutathione peroxidase and cystathionine transporter protein system, thereby causing ferroptosis in tumor cells. Ferroptosis in OC cells further activated immunogenic cell death, facilitating dendritic cell maturation and stimulating antitumor immunity.ConclusionWe have succeeded in developing a highly potent ferroptosis inducer (BMO-MXene), capable of inhibiting OC progression through the sonodynamic-ferroptosis-immunogenic cell death pathway.Graphical

Effect and mechanism of resveratrol on ferroptosis mediated by p53/SLC7A11 in oral squamous cell carcinoma

ObjectiveResveratrol (Res) is a natural phytoestrogen with antitumor activity. This study sought to investigate the role of Res in ferroptosis in oral squamous cell carcinoma (OSCC).MethodsNormal human oral keratinocyte (HOK)/oral OSCC (CAL-27/SCC-9) cell lines were treated with different doses of Res. Res toxicity was determined by MTT assay, with half maximal inhibitory concentration values of Res on CAL-27 and SCC-9 cells calculated. Cell viability/colony formation efficiency/migration/invasion/cycle were assessed by CCK-8/colony formation assay/transwell assay/flow cytometry. The expression of p53 protein in the nucleus and cytoplasm, glutathione peroxidase 4 (GPX4) expression, and SLC7A11 messenger RNA (mRNA) and protein expression levels were determined by Western blot and RT-qPCR. Fe2+ content, reactive oxygen species (ROS) level, reduced glutathione (GSH), and lactate dehydrogenase (LDH) release were assessed.ResultsMedium- to low-dose Res had no toxic effect on HOK cells, while high-dose Res markedly reduced HOK cell viability. Res significantly suppressed the viability of OSCC cells (CAL-27 and SCC-9). Res inhibited OSCC cell colony formation/migration/invasion, and induced G1 phase arrest. Res caused the translocation of p53 protein to the nucleus, obviously increased Fe2+ content, ROS level and LDH release, decreased GSH content and GPX4 protein expression, and induced ferroptosis. Down-regulation of p53 partially reversed the inhibitory effects of Res on CAL-27 cell malignant behaviors. Res inhibited SLC7A11 transcription by promoting p53 entry into the nucleus. SLC7A11 overexpression negated the the regulatory effects of p53 knockout on the role of Res in OSCC cell malignant behaviors and ferroptosis.ConclusionRes accelerated ferroptosis and inhibited malignant behaviors in OSCC cells by regulating p53/SLC7A11.

DEX Inhibits H/R-induced Cardiomyocyte Ferroptosis by the miR-141-3p/lncRNA TUG1 Axis.

Ferroptosis is emerging as a critical pathway in ischemia/reperfusion (I/R) injury, contributing to compromised cardiac function and predisposing individuals to sepsis and myocardial failure. The study investigates the underlying mechanism of dexmedetomidine (DEX) in hypoxia/reoxygenation (H/R)-induced ferroptosis in cardiomyocytes, aiming to identify novel targets for myocardial I/R injury treatment. H9C2 cells were subjected to H/R and treated with varying concentrations of DEX. Additionally, H9C2 cells were transfected with miR-141-3p inhibitor followed by H/R treatment. Levels of miR-141-3p, long noncoding RNA (lncRNA) taurine upregulated 1 (TUG1), Fe2+, glutathione (GSH), and malondialdehyde were assessed. Reactive oxygen species (ROS) generation was measured via fluorescent labeling. Expression of ferroptosis-related proteins glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long-chain family member 4 (ACSL4) was determined using Western blot. The interaction between miR-141-3p and lncRNA TUG1 was evaluated through RNA pull-down assay and dual-luciferase reporter gene assays. The stability of lncRNA TUG1 was assessed using actinomycin D. DEX ameliorated H/R-induced cardiomyocyte injury and elevated miR-141-3p expression in cardiomyocytes. DEX treatment increased cell viability, Fe2+, and ROS levels while decreasing ACSL4 protein expression. Furthermore, DEX upregulated GSH and GPX4 protein levels. miR-141-3p targeted lncRNA TUG1, reducing its stability and overall expression. Inhibition of miR-141-3p or overexpression of lncRNA TUG1 partially reversed the inhibitory effect of DEX on H/R-induced ferroptosis in cardiomyocytes. DEX mitigated H/R-induced ferroptosis in cardiomyocytes by upregulating miR-141-3p expression and downregulating lncRNA TUG1 expression, unveiling a potential therapeutic strategy for myocardial I/R injury.

Autologous Platelet-Rich Gel Accelerates Diabetic Wound Healing Through Inhibition of Ferritinophagy.

Aims: The objective was to examine the efficacy of autologous platelet-rich gel (APG) in treating diabetic wound and investigate the association between APG and ferritinophagy. Methods: A total of 32 patients with diabetic foot (DF) and Wagner grade 1 to 2 were included. Within the APG group, individuals with DF received weekly APG treatment. In the non-APG group, DF patients received daily dressing changes. Flow cytometry quantified the proportion of endothelial progenitor cells (EPCs) in peripheral blood on days 0 and 10. The diabetic rat model was induced using Streptozotocin. Two circular skin wounds were created on the backs of rats. The normal glucose group received daily dressing changes on the wound. In the diabetic group, the left wound underwent daily dressing changes, whereas the right wound was treated with APG once a week. CD34 levels were tested 7 days after the skin damage. The levels of glutathione peroxidase 4 (GPX4), Nuclear Receptor Coactivator 4 (NCOA4), Light chain 3 (LC3), and Masson staining were quantified on 14 days. The wound area and wound healing rate were separately measured at 0 and 14 days after the injury, regardless of DF patients or diabetic rats. Results: The wound healing rate was higher in the APG group than in the non-APG group, regardless of DF patients or diabetic rats. The APG group had a greater ΔEPCs% in DF patients than the non-APG group. Regarding rat experiment, the APG group exhibited lower levels of NCOA4, and LC3 expressions and a shorter wound healing time. However, the APG group showed higher levels of CD34 expression, GPX4 protein, and collagen fibers than the non-APG group. Conclusions: Autologous platelet-rich gel accelerated the wound healing rate in diabetic populations and rats. Autologous platelet-rich gel promoted EPCs counts, collagen fiber volume, and vessel numbers. Autologous platelet-rich gel decreased LC3 and NCOA4 expression, but increased GPX4 protein expression. The possible mechanism was the inhibition of ferritinophagy.

Polycytosine RNA-binding protein 1 regulates osteoblast function via a ferroptosis pathway in type 2 diabetic osteoporosis.

Recently, type 2 diabetic osteoporosis (T2DOP) has become a research hotspot for the complications of diabetes, but the specific mechanism of its occurrence and development remains unknown. Ferroptosis caused by iron overload is con-sidered an important cause of T2DOP. Polycytosine RNA-binding protein 1 (PCBP1), an iron ion chaperone, is considered a protector of ferroptosis. To investigate the existence of ferroptosis and specific role of PCBP1 in the development of type 2 diabetes. A cell counting kit-8 assay was used to detect changes in osteoblast viability under high glucose (HG) and/or ferroptosis inhibitors at different concentrations and times. Transmission electron microscopy was used to examine the morphological changes in the mitochondria of osteoblasts under HG, and western blotting was used to detect the expression levels of PCBP1, ferritin, and the ferroptosis-related protein glutathione peroxidase 4 (GPX4). A lentivirus silenced and overexpressed PCBP1. Western blotting was used to detect the expression levels of the osteoblast functional proteins osteoprotegerin (OPG) and osteocalcin (OCN), whereas flow cytometry was used to detect changes in reactive oxygen species (ROS) levels in each group. Under HG, the viability of osteoblasts was considerably decreased, the number of mitochondria undergoing atrophy was considerably increased, PCBP1 and ferritin expression levels were increased, and GPX4 expression was decreased. Western blotting results demonstrated that infection with lentivirus overexpressing PCBP1, increased the expression levels of ferritin, GPX4, OPG, and OCN, compared with the HG group. Flow cytometry results showed a reduction in ROS, and an opposite result was obtained after silencing PCBP1. PCBP1 may protect osteoblasts and reduce the harm caused by ferroptosis by promoting ferritin expression under a HG environment. Moreover, PCBP1 may be a potential therapeutic target for T2DOP.

The Important Role of GPX1 and NF-κB Signaling Pathway in Human Gastric Cancer: Implications for Cell Proliferation and Invasion.

Glutathione peroxidases (GPXs) are crucial antioxidant enzymes, counteracting reactive oxygen species (ROS). GPX overexpression promotes proliferation and invasion in cancer cells. Glutathione peroxidase-1 (GPX1), the most abundant isoform, contributes to invasion, migration, cisplatin resistance, and proliferation in various cancers. Nuclear factor-kappa B (NF-[Formula: see text]B) participates in cell proliferation, apoptosis, and tumor progression. The inhibition of NF-[Formula: see text]B expression reduces the malignancy of esophageal squamous cell carcinoma. This study aimed to explore the GPX1 and NF-[Formula: see text]B signaling pathways and their correlation with gastric cancer cell proliferation and invasion. Cell culture, complementary DNA microarray analysis, western blotting, reverse transcription-polymerase chain reaction, zymography, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, GPX1 knock-down with short hairpin RNA (shRNA), standard two-chamber invasion assay, chromatin immunoprecipitation assay. Hepatocyte growth factor (HGF) up-regulated GPX1 expression in gastric cancer cells. The NF-[Formula: see text]B inhibitor, pyrrolidine dithiocarbamate down-regulated HGF-induced GPX1 protein levels. Furthermore, NF-[Formula: see text]B and urokinase-type plasminogen activators were down-regulated in GPX1-shRNA-treated cells. Treatment with an Akt pathway inhibitor (LY294002) led to the down-regulation of GPX1 and NF-[Formula: see text]B gastric cancer cells. GPX1 knockdown resulted in decreased HGF-mediated in vitro cell proliferation and invasion. The study identified the putative binding site of the GPX1 promoter containing the NF-[Formula: see text]B binding site, confirmed through chromatin immunoprecipitation. HGF induced GPX1 expression through the NF-[Formula: see text]B and Akt pathways, suggesting a central role in gastric cell proliferation and invasion. Hence, GPX1 emerges as a potential therapeutic target for gastric cancer.

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.