Glutathione Peroxidase 4 Levels Research Articles

Acupuncture alleviates CUMS-induced depression-like behaviors of rats by regulating oxidative stress, neuroinflammation and ferroptosis

BackgroundThe treatment of depression with acupuncture has been documented. The mechanism behind acupuncture's curative and preventative effects is still unknown. MethodsThe current study examined the effects of acupuncture on depression-like behaviors in a rat model of chronic unpredictable mild stress (CUMS), while also exploring its potential mechanisms. A total of six groups of rats were randomly assigned: control, CUMS, acupuncture, fluoxetine, acupoint catgut embedding and sham acupoint catgut embedding. Fluoxetine (2.1 mg/kg) and acupoint catgut embedding were used for comparative research to acupuncture. The modelling evaluation is measured by body weight and behavior tests. Western blotting and reverse transcription-polymerase chain reaction were used to detect the proteins and mRNA expression of Silent information regulator 1 (Sirt1)/ nuclear factor-erythroid 2-related factor 2 (Nrf2)/ heme oxygenase-1 (HO-1)/ Glutathione peroxidase 4 (GPX4) pathway in the hippocampus. The expression of oxidative stress (OS)-related proteins and inflammatory cytokines in the serum was detected with ELISA. Immunofluorescence showed microglia and astrocytes activity in the hippocampus. ResultsAcupuncture and fluoxetine could alleviate CUMS-induced depression-like behaviors. Acupuncture was also found to effectively reverse the levels of MDA, SOD, GSH, GSH-PX and T-AOC, IL-1β, IL-6 and TNF-α in the serum of CUMS-induced rats. Rats with CUMS showed decreased levels of Sirt1, Nrf2, HO-1 and GPX4 in the hippocampus, while acupuncture treatment could partly reverse the diminished effects. In addition, acupuncture treatment significantly reduced the activation of hippocampal microglia and astrocytes in CUMS-induced rats. ConclusionThe study's findings indicate that acupuncture has the potential to mitigate depression-like behaviors in rats induced with CUMS by mitigating OS and reducing neuroinflammation.

Ex vivo investigation of betaine and boric acid function as preprotective agents on rat synaptosomes to be treated with Aβ (1-42).

Recent evidence suggests that ferroptosis, an iron-dependent cell death process, may be involved in Alzheimer's disease (AD) pathology. The study evaluated the therapeutic potential of betaine and boric acid (BA) pretreatment administered to rats for 21 days in AD. Then, the rats were sacrificed, and morphological and biochemical analyses were performed in brain tissues. Next, an ex vivo AD model was created by applying amyloid-β (Aβ1-42) to synaptosomes isolated from the brain tissues. Synaptosomes were analyzed with micrograph images, and protein and mRNA levels of ferroptotic markers were determined. Betaine and BA pretreatments did not cause any morphological and biochemical differences in the brain tissue. However, Aβ (1-42) administration in synaptosomes increased the levels of acyl-CoA synthetase long chain family member-4 (ACSL4), transferrin receptor-1 protein (TfR1), malondialdehyde (MDA), and 8-hydroxydeoxyguanosine (8-OHdG) and decreased the levels glutathione peroxidase-4 (GPx4) and glutathione (GSH). Moreover, ACSL4, GPx4, and TfR1 mRNA and protein levels were similar to the ELISA results. In contrast, betaine and BA pretreatments decreased the levels of ACSL4, TfR1, MDA, and 8-OHdG in synaptosomes incubated with Aβ1-42, while promoting increased levels of GPx4 and GSH. In addition, betaine and BA pretreatments completely reversed ACSL4, GPx4, and TfR1 mRNA and protein levels. Therefore, betaine and BA pretreatments may contribute to the prevention of neurodegenerative damage by supporting antiferroptotic activities.

Icariin inhibits osteoblast ferroptosis via Nrf2/HO-1 signaling and enhances healing of osteoporotic fractures

The incidence of osteoporotic fractures is increasing every year because of population aging around the world. The reduced osteoblast activity in osteoporotic fracture has been associated with ferroptosis. A recent study showed that the antioxidant icariin (ICA) reduced iron deposition in the bone marrow of osteoporotic mice, although the underlying regulatory mechanisms were not explored. The objective of present study was to assess the therapeutic effects of ICA in a rat osteoporotic fracture model, with particular focus on its impact on ferroptosis. Primary rat osteoblasts were exposed to the ferroptosis inducer erastin, and then treated with ICA or the ferroptosis inhibitor ferrostatin-1 (Fer-1) as the positive control group. The levels of Nrf2 signaling factors and osteogenesis-related factors were examined by RT-PCR and western blotting. An osteoporotic fracture model was established in rats, and the effect of ICA on bone formation was evaluated by X-ray, Micro CT analysis, histological examination and Safranin O staining. Furthermore, the levels of GPX4, Bax, Nrf2 and Runx2 proteins at the fracture site were examined by immunohistochemistry. ICA significantly reduced ROS levels in the erastin-treated osteoblasts, and downregulated glutathione peroxidase 4 (GPX4) and cystine glutamate antiporter (SLC7A11). Moreover, ICA also upregulated Nrf2, NQO-1, HO-1, Runx2, ALP, OPG and OCN in these cells, which was reversed by inhibitors of the Nrf2 signaling pathway and Nrf2 silencing. X-ray and Micro CT analysis showed that ICA increased the trabecular bone and promoted callus formation in the osteoporotic fracture model, and also enhanced the transition from fibrous to osseous callus. Furthermore, ICA upregulated GPX4, Nrf2 and Runx2 at the fracture site, and significantly reduced the expression of the apoptotic genes of Bax. Taken together, our findings indicate that ICA promotes osteoporotic fracture healing by inhibiting osteoblast ferroptosis via activation of the antioxidant Nrf2/HO-1 signaling pathway.

The ferroptosis landscape in acute myeloid leukemia.

Ferroptosis induction through the suppression of glutathione peroxidase 4 (GPX4) and apoptosis-inducing factor mitochondria-associated 2 (AIFM2) has proven to be an effective approach in eliminating chemotherapy-resistant cells of various types. However, a comprehensive understanding of the roles of GPX4 and AIFM2 in acute myeloid leukemia (AML) has not yet been achieved. Using cBioPortal, DepMap, GEPIA, Metascape, and ONCOMINE, we compared the transcriptional expression, survival data, gene mutation, methylation, and network analyses of GPX4- and AIFM2-associated signaling pathways in AML. The results revealed that high expression levels of GPX4 and AIFM2 are associated with an adverse prognosis for AML patients. Overexpression of AIFM2 correlated with elevated mutation frequencies in NPM1 and DNMT3A. GPX4 upregulation modulated the following pathways: GO:0045333, cellular respiration; R-HSA-5389840, mitochondrial translation elongation; GO:0009060, aerobic respiration; R-HSA-9609507, protein localization; and R-HSA-8953854, metabolism of RNA. On the other hand, the overexpression of AIFM2 influenced the following processes: GO:0048704, embryonic skeletal system morphogenesis; GO:0021546, rhombomere development; GO:0009954, proximal/distal pattern formation; and GO:0048732, gland development. This study identifies the high expression of GPX4 and AIFM2 as novel biomarkers predicting a poor prognosis for AML patients. Furthermore, ferroptosis induction may improve the stratified treatment of AML.

Quercetin ameliorates ferroptosis of rat cardiomyocytes viaactivation of the SIRT1/p53/SLC7A11 signalingpathway to alleviate sepsis‑induced cardiomyopathy.

Sepsis‑induced cardiomyopathy (SIC) is a manifestation of multiple organ failure as a result of sepsis and is a serious threat to life. Here, the effect and mechanisms of quercetin (QUE) in SIC were assessed. It was found that patients with SIC expressed lower serum levels of glutathione peroxidase 4 (GPX4) and SIRT1 but higher levels of CK‑MB, cTnI, TNF‑α, and IL‑6 compared with healthy individuals. A dose of 80µM QUE increased the viability and reduced the ferroptosis of H9C2 cells treated with 1.0µg/ml LPS invitro. The administration of QUE decreased the levels of MDA, NADPH, lipid peroxidation and cytoplasmic cytochrome C and upregulated the levels of GSH and TOM 20, thus exerting an anti‑oxidative effect via mediating SIRT1 expression. It also activated the SIRT1/p53/SLC7A11 signaling pathway to reduce cellular Fe2+ and PTGS2 levels, decreased cell apoptosis rate, and upregulated the levels of GPX4 and ferritin to inhibit ferroptosis of H9C2 cells invitro. Injection of QUE into rats activated the SIRT1/p53/SLC7A11 signaling pathway, reduced the levels of CK‑MB, cTnI, inflammatory cell infiltration, MDA, NADPH, cytoplasmic cytochrome C, cellular Fe2+, and PTGS2 but upregulated the levels of GSH, TOM 20, GPX4, and ferritin to alleviate SIC in a dose‑dependent manner invivo. To conclude, QUE exerted an anti‑ferroptotic effect via activation of the SIRT1/p53/SLC7A11 signaling pathway to dampen SIC both invivo and invitro. These findings highlighted a potential therapeutic strategy for the management of SIC.

Angiotensin IV ameliorates doxorubicin-induced cardiotoxicity by increasing glutathione peroxidase 4 and alleviating ferroptosis

BackgroundDoxorubicin (DOX)-induced cardiotoxicity is an important cause of poor prognosis in cancer patients treated with DOX. Angiotensin IV (Ang IV) has multiple protective effects against cardiovascular diseases, including diabetic cardiomyopathy and myocardial infarction, but its role in DOX-induced cardiotoxicity is currently unclear. In this study, we investigated the effects of Ang IV on DOX-induced cardiotoxicity. MethodsThe viability of primary cardiomyocytes was measured by Cell Counting Kit-8 assays and Hoechst 33342/propidium iodide staining in vitro. ELISAs (serum cTnT and CK-MB) and echocardiography were performed to assess myocardial injury and cardiac function in vivo. Phalloidin staining, haematoxylin and eosin staining and wheat germ agglutinin staining were conducted to detect cardiomyocyte atrophy. We also performed C11 BODIPY staining, measured the levels of Ptgs2 and malondialdehyde and detected the concentrations of ferrous ions, glutathione and oxidized glutathione to indicate ferroptosis. ResultsAng IV not only attenuated DOX-induced atrophy and cardiomyocyte injury in vitro but also alleviated myocardial injury and improved cardiac function in DOX-treated mice in vivo. Moreover, Ang IV reversed DOX-induced downregulation of glutathione peroxidase 4 (GPX4) and inhibited ferroptosis both in vitro and in vivo. Knockdown of GPX4 by siRNA abolished the cardioprotective effects of Ang IV. Furthermore, Ang IV increased GPX4 levels and ameliorated ferroptosis in RAS-selective lethal 3-treated primary cardiomyocytes. ConclusionsAng IV ameliorates DOX-induced cardiotoxicity by upregulating GPX4 and inhibiting ferroptosis. Ang IV may be a promising candidate to protect against DOX-induced cardiotoxicity in the future.

PUFAs add fuel to Crohn’s disease-associated AIEC-induced enteritis by exacerbating intestinal epithelial lipid peroxidation

ABSTRACT Polyunsaturated fatty acids (PUFAs) have been shown to exacerbate Crohn’s disease (CD) by promoting lipid peroxidation (LPO) of intestinal epithelial cells (IECs). Dysbiosis of the gut microbiota may play a crucial role in this process. CD patients often exhibit an increased abundance of Escherichia coli (E. coli) in the gut, and the colonization of adherent-invasive E. coli (AIEC) is implicated in the initiation of intestinal inflammation in CD. However, the impact of AIEC on LPO remains unclear. In this study, we observed that AIEC colonization in the terminal ileum of CD patients was associated with decreased levels of glutathione peroxidase 4 (GPX4) and ferritin heavy chain (FTH) in the intestinal epithelium, along with elevated levels of 4-Hydroxynonenal (4-HNE). In vitro experiments demonstrated that AIEC infection reduced the levels of GPX4 and FTH, increased LPO, and induced ferroptosis in IECs. Furthermore, arachidonic acid (AA) and docosahexaenoic acid (DHA) supplementation in AIEC-infected IECs significantly aggravated LPO and ferroptosis. However, overexpression of GPX4 rescued AIEC-induced LPO and ferroptosis in IECs. Our results further confirmed that AIEC with AA supplementation, associated with excessive LPO and cell death in IECs, worsened colitis in the DSS mouse model and induced enteritis in the antibiotic cocktail pre-treatment mouse model in vivo. Moreover, treatment with ferrostatin-1, a ferroptosis inhibitor, alleviated AIEC with AA supplementation-induced enteritis in mice, accompanied by reduced LPO and cell death in IECs. Our findings suggest that AIEC, in combination with PUFA supplementation, can induce and exacerbate intestinal inflammation, primarily through increased LPO and ferroptosis in IECs.

Shenqi Dihuang Decoction inhibits high-glucose induced ferroptosis of renal tubular epithelial cells via Nrf2/HO-1/GPX4 pathway

This study aims to explore the effects of Shenqi Dihuang Decoction on high-glucose induced ferroptosis and the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/glutathione peroxidase 4(GPX4) axis in human renal tubular epithelial cells(HK-2) and to clarify the underlying mechanism. The cell injury model was established by exposing HK-2 to high glucose, and the Shenqi Dihuang Decoction-medicated serum was prepared. The optimal concentration and intervention time of Shenqi Dihuang Decoction were determined. HK-2 were divided into normal, high glucose, and low-, medium-, and high-dose Shenqi Dihuang Decoction groups. After interventions, the cell proliferation rate in each group was determined and the cell morphology and mitochondrial ultrastructure were observed. Then, the levels of intracellular reactive oxygen species(ROS), ferrous ion(Fe~(2+)), glutathione(GSH), and malondialdehyde(MDA) and the protein levels of Nrf2, HO-1, GPX4, and xCT were measured. The optimal concentration and intervention time of Shenqi Dihuang Decoction-medicated serum were determined to be 10% and 24 h, respectively. Compared with the high glucose group, high-dose Shenqi Dihuang Decoction promoted the proliferation of HK-2. The cells in the low-, medium-, and high-dose Shenqi Dihuang Decoction groups presented tight arrangement, an increased cell count, improved morphology from a spindle-fiber shape to a cobblestone shape, and improved morphology and structure of mitochondrial membrane and cristae, compared with those in the high glucose group. Meanwhile, all the doses of Shenqi Dihuang Decoction inhibited ROS elevation to mitigate the peroxidation damage, lowered the Fe~(2+) and MDA levels and elevated the GSH level to inhibit lipid peroxidation, and activated the antioxidant pathway to upregulate the protein levels of Nrf2, HO-1, xCT, and GPX4. In conclusion, Shenqi Dihuang Decoction-medicated serum can inhibit high-glucose induced ferroptosis of HK-2 in vitro, which involves the antioxidant effect and the activation of the Nrf2/HO-1/GPX4 pathway.

Common Mutation in the HFE Gene Modifies Recovery After Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is characterized by bleeding into the brain parenchyma. During an ICH, iron released from the breakdown of hemoglobin creates a cytotoxic environment in the brain through increased oxidative stress. Interestingly, the loss of iron homeostasis is associated with the pathological process of other neurological diseases. However, we have previously shown that the H63D mutation in the homeostatic iron regulatory (HFE) gene, prevalent in 28% of the White population in the United States, acts as a disease modifier by limiting oxidative stress. The following study aims to examine the effects of the murine homolog, H67D HFE, on ICH. An autologous blood infusion model was utilized to create an ICH in the right striatum of H67D and wild-type mice. The motor recovery of each animal was assessed by rotarod. Neurodegeneration was measured using fluorojade-B and mitochondrial damage was assessed by immunofluorescent numbers of CytC+ (cytochrome C) neurons and CytC+ astrocytes. Finally, the molecular antioxidant response to ICH was quantified by measuring Nrf2 (nuclear factor-erythroid 2 related factor), GPX4 (glutathione peroxidase 4), and FTH1 (H-ferritin) levels in the ICH-affected and nonaffected hemispheres via immunoblotting. At 3 days post-ICH, H67D mice demonstrated enhanced performance on rotarod compared with wild-type animals despite no differences in lesion size. Additionally, H67D mice displayed higher levels of Nrf2, GPX4, and FTH1 in the ICH-affected hemisphere; however, these levels were not different in the contralateral, non-ICH-affected hemisphere. Furthermore, H67D mice showed decreased degenerated neurons, CytC+ Neurons, and CytC+ astrocytes in the perihematomal area. Our data suggest that the H67D mutation induces a robust antioxidant response 3 days following ICH through Nrf2, GPX4, and FTH1 activation. This activation could explain the decrease in degenerated neurons, CytC+ neurons, and CytC+ astrocytes in the perihematomal region, leading to the improved motor recovery. Based on this study, further investigation into the mechanisms of this neuroprotective response and the effects of the H63D HFE mutation in a population of patients with ICH is warranted.

Circular RNA circBLNK promotes osteosarcoma progression and inhibits ferroptosis in osteosarcoma cells by sponging miR‑188‑3p and regulating GPX4 expression.

As a newly identified circular RNA (circRNA), the role of circBLNK in cancer progression has not been probed. The objective of the present study was to functionally dissect the role of circBLNK in osteosarcoma (OS) tumorigenesis and progression. With regards of the experimental procedure, the levels of mRNAs and proteins were assessed using reverse transcription‑quantitative PCR and western blot analysis, respectively. The subcellular location of circBLNK in OS cells was determined by cell cytosolic/nuclear fractionation assay. Cell ferroptosis ability was assessed through MTT assay. Cell proliferative abilities were assessed by clonogenic and Cell Counting Kit‑8 assays, and cell apoptosis was measured using flow cytometry. The relationships among circBLNK, miR‑188‑3p, and glutathione peroxidase 4 (GPX4) were validated by luciferase reporter and RNA pull‑down assays, as well as RNA immunoprecipitation. The stability of circBLNK and linear BLNK was confirmed using RNase R treatment assay. The association between circBLNK expression and overall survival rate was assessed by Kaplan‑Meier plot. The correlation between the expression levels of circBLNK, miR‑188‑3p, and GPX4 in OS tissues was assessed by Pearson's χ2 test. The results revealed that CircBLNK and GPX4 were significantly upregulated in OS tissues, which predicted the poor prognosis. CircBLNK knockdown led to suppressed cell proliferation and enhanced cell apoptosis, an effect that could be reversed by the inhibition of miR‑188‑3p. In an invivo circBLNK deficiency model, tumor growth was observed to be markedly suppressed. Moreover, circBLNK deficiency elevated levels of intracellular free iron (Fe2+), malondialdehyde, lipid reactive oxygen species and mitochondrial superoxide, while diminishing mitochondrial membrane potential in Erastin‑treated OS cells, which were eliminated by overexpressing GPX4. Furthermore, mechanistic investigations revealed that circBLNK sponged miR‑188‑3p to regulate the expression of GPX4, thereby affecting OS progression. In conclusion, the present study delineated a new regulatory axis involving circBLNK/miR‑188‑3p/GPX4 in OS progression, adding to the growing evidence that circRNAs are critical gene regulators in cancer progression.

Ancient dormant virus remnant ERVW-1 drives ferroptosis via degradation of GPX4 and SLC3A2 in schizophrenia

Human endogenous retroviruses (HERVs) are remnants of retroviral infections in human germline cells from millions of years ago. Among these, ERVW-1 (also known as HERV-W-ENV, ERVWE1, or ENVW) encodes the envelope protein of the HERV-W family, which contributes to the pathophysiology of schizophrenia. Additionally, neuropathological studies have revealed cell death and disruption of iron homeostasis in the brains of individuals with schizophrenia. Here, our bioinformatics analysis showed that differentially expressed genes in the human prefrontal cortex RNA microarray dataset (GSE53987) were mainly related to ferroptosis and its associated pathways. Clinical data demonstrated significantly lower expression levels of ferroptosis-related genes, particularly Glutathione peroxidase 4 (GPX4) and solute carrier family 3 member 2 (SLC3A2), in schizophrenia patients compared to normal controls. Further in-depth analyses revealed a significant negative correlation between ERVW-1 expression and the levels of GPX4/SLC3A2 in schizophrenia. Studies indicated that ERVW-1 increased iron levels, malondialdehyde (MDA), and transferrin receptor protein 1 (TFR1) expression while decreasing glutathione (GSH) levels and triggering the loss of mitochondrial membrane potential, suggesting that ERVW-1 can induce ferroptosis. Ongoing research has shown that ERVW-1 reduced the expression of GPX4 and SLC3A2 by inhibiting their promoter activities. Moreover, Ferrostatin-1 (Fer-1), the ferroptosis inhibitor, reversed the iron accumulation and mitochondrial membrane potential loss, as well as restored the expressions of ferroptosis markers GSH, MDA, and TFR1 induced by ERVW-1. In conclusion, ERVW-1 could promote ferroptosis by downregulating the expression of GPX4 and SLC3A2, revealing a novel mechanism by which ERVW-1 contributes to neuronal cell death in schizophrenia.

Total triterpenes of Euphorbium alleviates rheumatoid arthritis via Nrf2/HO-1/GPX4 pathway

This study aims to investigate the effect and mechanism of total triterpenes of Euphorbium in treating rheumatoid arthritis(RA). The rat model of RA was established with Freund's complete adjuvant(FCA). Male rats were randomly assigned into control, model, Tripterygium glycosides(7.5 mg·kg~(-1)), and low-, medium-, and high-dose total triterpenes of Euphorbium(32, 64, and 128 mg·kg~(-1), respectively) groups, with 10 rats in each group. In other groups except the control group, 0.2 mL FCA was injected into the right hind toe. Rats in the intervention groups were administrated with corresponding drugs by gavage, and the control group and the model group with the same volume of 0.5% CMC-Na solution once a day. During the treatment period, the swelling degree of the hind paw was measured and the arthritis was scored until day 30. At the end of drug administration, the pathological changes of the joint tissue were observed by hematoxylin-eosin staining. The content of malondialdehyde(MDA), glutathione(GSH), and Fe~(2+) and the activity of superoxide dismutase(SOD) in the joint tissue were measured by biochemical colorimetry. RT-PCR was performed to determine the mRNA levels of nuclear factor erythroid 2-related factor 2(Nrf2), glutathione peroxidase 4(GPX4), and acyl-CoA synthetase long chain family member 4(ACSL4) in the joint tissue. Western blot was employed to determine the protein levels of Nrf2, Kelch-like ECH-associated protein 1(Keap1), heme oxygenase-1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1), SOD2, GPX4, and ACSL4 in the joint tissue. The results showed that the treatment with Tripterygium glycosides(7.5 mg·kg~(-1)) and total triterpenes of Euphorbium(32, 64, and 128 mg·kg~(-1)) alleviated the swelling degree of bilateral hind limbs, decreased the arthritis score, reduced joint tissue lesions and the content of MDA and Fe~(2+) in the joint tissue, and increased GSH content and SOD activity. Furthermore, the interventions up-regulated the protein and mRNA levels of Nrf2 and GPX4, down-regulated the protein and mRNA levels of ACSL4, and up-regulated the protein levels of Keap1, NQO1, HO-1, and SOD2 in the Nrf2/HO-1/GPX4. In summary, the total triterpenes of Euphorbium can treat RA by inhibiting lipid peroxidation and abnormal ferroptosis, which may involve the Nrf2/HO-1/GPX4 signaling pathway.

A STING pathway-activatable contrast agent for MRI-guided tumor immunoferroptosis synergistic therapy

The immunotherapy efficiency of stimulator of interferon genes (STING)-activatable drugs (e.g., 7-ethyl-10-hydroxycamptothecin, SN38) is limited by their non-specificity to tumor cells and the slow excretion of the DNA-containing exosomes from the treated cancer cells. The efficacy of tumor ferroptosis therapy is always limited by the elimination of lipid peroxides (LPO) by the pathways of glutathione peroxidase 4 (GPX4), dihydroorotate dehydrogenase (DHODH) and ferroptosis suppressor protein 1(FSP1). To solve these problems, in this study, we developed a STING pathway-activatable contrast agent (i.e., FeGd-HN@TA-Fe2+-SN38 nanoparticles) for magnetic resonance imaging (MRI)-guided tumor immunoferroptosis synergistic therapy. The remarkable in vivo MRI performance of FeGd-HN@TA-Fe2+-SN38 is attributed to its high accumulation at tumor location, the high relaxivities of FeGd-HN core, and the pH-sensitive TA-Fe2+-SN38 layer. The effectiveness and biosafety of the immunoferroptosis synergistic therapy induced by FeGd-HN@TA-Fe2+-SN38 are demonstrated by the in vivo investigations on the 4T1 tumor-bearing mice. The mechanisms of in vivo immunoferroptosis synergistic therapy by FeGd-HN@TA-Fe2+-SN38 are demonstrated by measurements of in vivo ROS, LPO, GPX4 and SLC7A11 levels, the intratumor matured DCs and CD8+ T cells, the protein expresion of STING and IRF-3, and the secretion of IFN-β and IFN-γ.

Effect of electroacupuncture pretreatment on ferroptosis in neurons of rats with cerebral ischemia-reperfusion injury

To observe the effect of electroacupuncture（EA）preconditioning on ferroptosis in rats with cerebral ischemia-reperfusion injury （CIRI）, so as to explore the neuroprotective mechanism of EA preconditioning. Male SD rats were randomly divided into sham operation, model, EA, inhibitor and inducer groups with 20 rats in each group. The CIRI model was established by modified Zea Longa occlusion of the middle cerebral artery. Before modeling, EA treatment （2 Hz/15 Hz, 1-2 mA） was applied to "Baihui"（GV20）, "Fengfu"（GV16） and "Dazhui"（GV14） for rats of the EA group, 20 min a day for 7 consecutive days. Rats of the inhibitor group were intraperitoneally injected with ferristatin-1（25 mg/kg）at a slow and uniform rate. Rats of the inducer group were intraperitoneally injected with Erastin（100 mg/kg） after 7 days of EA preconditioning, once every 2 h for a total of 4 times. The CIRI models were prepared 2 d later after the above interventions finished by thread-occlusion. The degree of neurological impairment was evaluated by modified Zea Longa score. The percentage of infarct size was calculated by TTC staining. The ultrastructure of neurons in hippocampus was observed by transmission electron microscope. The contents of ferrous ion （Fe2+）, malondialdehyde （MDA） and glutathione （GSH） in cerebral tissue and reactive oxygen species （ROS） in serum were determined by biochemical method. The changes of mitochondrial membrane potential in rats brain tissues were detected by flow cytometry. The mRNA and protein expression levels of glutathione peroxidase 4 （GPX4）, acyl-CoA synthetase long-chain family member 4 （ACSL4）, transferrin receptor （TFRC）, 15-lipoxygenase （15-LOX） and cyclooxygenase-2 （COX-2） in the ischemic hippocampal region of CIRI rats were detected by real-time quantitative PCR and Western blot, respectively. Compared with the sham operation group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression levels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01）, and mitochondria in brain tissue were significantly damaged （P<0.01） in the model group. Compared with the model group, the above indexes were all reversed （P<0.05, P<0.01） in the EA group and inhibitor group. Compared with the EA group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression le-vels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.05, P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01, P<0.05）, and mitochondria in brain tissue were significantly damaged （P<0.05） in the inducer group. EA preconditioning has neuroprotective effect on CIRI rats, which may be related to inhibiting ACSL4/TFRC/15-LOX/COX-2 expression and increasing GSH/GPX4 expression.

Sodium pyruvate exerts protective effects against cigarette smoke extract-induced ferroptosis in alveolar and bronchial epithelial cells through the GPX4/Nrf2 axis

BackgroundFerroptosis in alveolar and bronchial epithelial cells is one of the main mechanisms underlying the development of chronic obstructive pulmonary disease (COPD). Sodium pyruvate (NaPyr) is a natural antioxidant in the body, exhibiting anti-inflammatory and antioxidant activities. NaPyr has been used in a Phase II clinical trial as a novel therapy for COPD; however, the mechanism underlying NaPyr-mediated therapeutic benefits in COPD is not well understood.ObjectiveWe aimed to assess the protective effects of NaPyr and elucidate its potential mechanism in cigarette smoke extract (CSE)-induced ferroptosis.To minic the inflammatory response and ferroptosis triggered by cigarette smoke in COPD in an invitro cell based system, we expose a human bronchial epithelial cells to CSE.MethodsTo minic the inflammatory response and ferroptosis triggered by cigarette smoke in COPD in an invitro cell based system, the A549 (human lung carcinoma epithelial cells) and BEAS-2B (bronchial epithelial cells) cell lines were cultured, followed by treatment with CSE. To measure cellular viability and iron levels, we determined the levels of malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS), mitochondrial superoxide (MitoSOX), membrane potential (MMP), and inflammatory factors (tumor necrosis factor [TNF] and interleukin [IL]-8), and examined CSE-induced pulmonary inflammation and ferroptosis. To clarify the molecular mechanisms of NaPyr in COPD therapy, we performed western blotting and real-time PCR (qPCR) to determine the expression of glutathione peroxidase 4 (GPX4), nuclear factor E2-related factor 2 (Nrf2), and cyclooxygenase 2 (COX2).ResultsWe found that NaPyr effectively mitigated CSE-induced apoptosis and improved apoptosis induced by erastin, a ferroptosis inducer. NaPyr significantly decreased iron and MDA levels and increased GSH levels in CSE-induced cells. Furthermore, NaPyr suppressed ferroptosis characteristics, such as decreased levels of ROS, MitoSOX, and MMP. NaPyr significantly increases the expression levels of GPX4 and Nrf2, indicating that activation of the GPX4/Nrf2 axis could inhibit ferroptosis in alveolar and bronchial epithelial cells. More importantly, NaPyr inhibited the secretion of downstream inflammatory factors, including TNF and IL-8, by decreasing COX2 expression levels to suppress CSE-induced inflammation.ConclusionAccordingly, NaPyr could mitigate CSE-induced ferroptosis in alveolar and bronchial epithelial cells by activating the GPX4/Nrf2 axis and decreasing COX2 expression levels. In addition, NaPyr reduced the secretion of inflammatory factors (TNF and IL-8), affording a novel therapeutic candidate for COPD.Graphical

SETD1A-mediated H3K4me3 methylation upregulates lncRNA HOXC-AS3 and the binding of HOXC-AS3 to EP300 and increases EP300 stability to suppress the ferroptosis of NSCLC cells.

Histone methyltransferases are crucial regulators in non-small cell lung cancer (NSCLC) development. This study explored the mechanism of histone methyltransferase SET domain containing 1A (SETD1A)-mediated H3K4me2 methylation in NSCLC cell ferroptosis and provides novel targets for NSCLC treatment. Upon downregulation of SETD1A in NSCLC cell lines, cell proliferation potential, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) activities, iron content, and SETD1A, long noncoding RNA HOXC cluster antisense RNA 3 (lncRNA HOXC-AS3), E1A binding protein p300 (EP300), glutathione peroxidase 4 (GPX4) expressions were determined via cell counting kit-8, ELISA, iron assay kits, RT-qPCR, and western blot. Enrichment levels of SETD1A and H3K4me3 in the HOXC-AS3 promotor were measured via chromatin immunoprecipitation, and the binding of HOXC-AS3 and EP300 was analyzed via RNA immunoprecipitation. Rescue experiments were performed to confirm their roles in NSCLC cell ferroptosis. Xenograft tumor models were established to validate the role of SETD1A in vivo. SETD1A, H3K4me3, HOXC-AS3, and EP300 were highly-expressed in NSCLC cells. Silencing SETD1A inhibited NSCLC cell proliferation, increased MDA and iron levels, and decreased SOD, GSH, and GPX4 levels. SETD1A downregulation reduced H3K4me3 level, HOXC-AS3 expression, the binding of HOXC-AS3 to EP300, and EP300 stability. Overexpression of HOXC-AS3 or EP300 reversed the promotion of silencing SETD1A on NSCLC cell ferroptosis. Silencing SETD1A reduced tumor volume and weight and positive rate of ki67 and increased ferroptosis through the HOXC-AS3/EP300 axis. SETD1A-mediated H3K4me2 methylation promoted HOXC-AS3 expression, binding of HOXC-AS3 to EP300, and EP300 stability, thereby suppressing NSCLC cell ferroptosis.

Dexmedetomidine ameliorates diabetic cardiomyopathy by inhibiting ferroptosis through the Nrf2/GPX4 pathway

ObjectiveDexmedetomidine (DEX) has been shown to have anti-apoptotic effects in diabetes mellitus, but its role in mitigating diabetic cardiomyopathy (DCM) through ferroptosis regulation is unclear.MethodsAn in vitro DCM model was established using H9C2 cells induced with high glucose (HG) and treated with DEX at varying doses and a nuclear factor erythroid 2-realated factor 2 (Nrf2) specific inhibitor ML385. Cell viability was evaluated using the MTT method after treatment with DEX or mannitol (MAN), and the dosage of DEX used in subsequent experimentation was determined. The effects of HG-induced high osmotic pressure were assessed using MAN as a control. Cell apoptosis was evaluated using flow cytometry. Protein levels of Bcl2, Bax, nuclear Nrf2, and glutathione peroxidase 4 (GPX4) were measured using Western blot. Superoxide dismutase (SOD) activity, malondialdehyde (MDA) levels, Fe2+ concentration and reactive oxygen species (ROS) levels were measured using corresponding kits and dichlorodihydrofluorescein diacetate, respectively.ResultsTreatment with DEX or MAN had no effect on H9C2 cell viability. HG induction reduced H9C2 cell viability, increased cell apoptosis, upregulated levels of Bax, Fe2+, MDA, and ROS, and downregulated Bcl2 protein levels, SOD activity, and protein levels of nuclear Nrf2 and GPX4. DEX inhibited HG-induced H9C2 cell apoptosis, promoted Nrf2 nuclear translocation, and activated the Nrf2/GPX4 pathway. Inhibition of Nrf2 partially reversed the protective effects of DEX against HG-evoked H9C2 cell injury.ConclusionOur findings demonstrate that DEX attenuates HG-induced cardiomyocyte injury by inhibiting ferroptosis through the Nrf2/GPX4 pathway, providing potential therapeutic targets for DCM treatment.

Low levels of peroxiredoxins are associated with high iron content and lipid peroxidation in seminal plasma from asthenozoospermic infertile men

Asthenospermia is a common cause of male infertility refers to semen samples with spermatozoa that move slowly or immotile. Recent research has implicated oxidative stress-induced ferroptosis in asthenospermia's pathophysiology. Peroxiredoxins are important players in the antioxidant defense to protect cells against oxidative stress. However, some aspects of the antioxidant response necessary for male fertility are not well understood. This case-control study aimed to elucidate the role of peroxiredoxins in regulating oxidative stress in male fertility via their correlation with ferroptosis. It included 90 fertile and 90 asthenospermic subfertile males from Hilla City, Iraq. Total peroxiredoxin activity, peroxiredoxin-6 level, and peroxiredoxin-4 level were measured alongside the ferroptosis biomarkers glutathione peroxidase-4, malondialdehyde, and the reduced/oxidized protein thiol ratio. Infertile males had significantly higher oxidized thiol and malondialdehyde levels than fertile males (p < 0.05). Total peroxiredoxin activities, peroxiredoxin-6 levels, peroxiredoxin-4 levels, glutathione peroxidase-4 levels, and reduced/oxidized protein thiol ratios were significantly lower in infertile males than in fertile males (p < 0.05). Therefore, peroxiredoxin activity and level correlate with reduced/oxidized protein thiol ratio. They are inversely associated with ferroptosis and directly associated with semen quality. These findings suggest that peroxiredoxins are crucial in preventing ferroptosis and have potential implications for treating asthenospermia.

Mechanism of Ziyuglycoside II-mediated Ferroptosis-related Proteins on the Proliferation and Metastasis of Human Lung Adenocarcinoma Cell Lines

Background Ferroptosis is a novel type of regulated cell death and targeting ferroptosis may be a potential treatment strategy for lung cancer. Ziyuglycoside II (ZYG II) has a significant inhibitory effect on the growth of lung cancer cells. However, the selective anti-tumor effect of the ZYG II against lung cancer has not been systemically studied. Objectives We combined ferrostatin-1 and erastin to explore the potential therapeutic mechanism of the ZYG II for lung adenocarcinoma. Materials and Methods A549 and H1299 cells were randomly divided into the control, ZYG II, ferroptosis inhibitor group (ZYG II+ ferrostatin-1), and erastin group (ZYG II+ erastin). Cell proliferation was detected using the CCK-8 method. Cell migration and invasion were evaluated using the Transwell assay. The protein expression levels of Glutathione Peroxidase 4 (GPX4), Solute Carrier Family 7 Member 11 (SLC7A11), and Transferrin receptor 1 (TFR1) were measured using western blotting. Results Compared with the control group, the cell proliferation, migration, and invasion abilities of the ZYG II group significantly decreased, the protein expression levels of GPX4 and SLC7A11 in the ZYG II group declined significantly, and the expression of TFR1 increased significantly ( p &lt; 0.05). After adding ferrostatin 1 (ZYG II+ Ferrostatin 1), the cell proliferation, migration, and invasion abilities of the inhibited cells were significantly increased, the expression of GPX4 and SLC7A11 increased significantly and the expression of TFR1 decreased significantly ( p &lt; 0.05). However, after adding the erastin (ZYG II+ erastin), the cell viability was further inhibited in A549, the expression levels of GPX4 and SLC7A11 were further inhibited and the expression of TFR1 was further increased ( p &lt; 0.05). Conclusion ZYG II significantly inhibited the survival rate, proliferation, migration, and invasion ability of A549 and H1299 cells, possibly by inducing ferroptosis.

Fluorescent Turn-On Probes for Visualizing GPx4 Levels in Live Cells and Predicting Drug Sensitivity.

Glutathione peroxidase 4 (GPx4) is the membrane peroxidase in mammals that is essential for protecting cells against oxidative damage and critical for ferroptosis. However, no live cell probe is currently available to specifically label GPx4. Herein, we report both inhibitory and noninhibitory fluorescent turn-on probes for specific labeling of GPx4 in live cells. With these probes, the GPx4 expression levels and degradation kinetics in live cells could be visualized, and their real-time responses to the cellular selenium availability were revealed. These probes could also potentially serve as staining reagents to predict the sensitivity of GPx4-related ferroptosis drugs. In view of these features, these GPx4-selective probes will offer opportunities for a deeper understanding of GPx4 function in natural habitats and hold great promise for biomedical applications.