FTH1 Expression Research Articles

Autophagy mediated FTH1 degradation activates gasdermin E dependent pyroptosis contributing to diquat induced kidney injury

Acute kidney injury (AKI) induced by diquat (DQ) progresses rapidly, leading to high mortality, and there is no specific antidote for this chemical. Our limited knowledge of the pathogenic toxicological mechanisms of DQ has hindered the development of treatments against DQ poisoning. Pyroptosis is a form of programmed cell death and was recently identified as a novel molecular mechanism of drug-induced AKI. To explore the role of pyroptosis in HK-2 cells exposed to DQ, the plasma membrane damage of the cells was detected by LDH release assay. Western blot was performed to detect the cleavage of GSDME. Proteomics analysis was performed to explore the mechanism of DQ induced nephrotoxicity. FerroOrange probe was used to measure the intracellular Fe2+ levels. Herein, we show that DQ induces pyroptosis in HK-2 cells. Mechanistically, DQ induces the accumulation of mitochondrial ROS and initiates the cleavage of gasdermin E (GSDME) in an intrinsic mitochondrial pathway. Knockout of GSDME attenuated DQ-induced cell death. Further analysis revealed that loss of FTH1 induces Fe2+ accumulation, contributing to DQ-induced pyroptosis. Knockdown LC3B could help restore the expression of FTH1 and improve cell viability. Moreover, we found DFO, an iron chelator, could reduce cellular Fe2+ levels and inhibit pyroptosis. Collectively, these findings suggest an unrecognized mechanism for GSDME-dependent pyroptosis in DQ-induced AKI.

Ferroptosis is involved in focal segmental glomerulosclerosis in rats

To explore whether ferroptosis is involved in focal segmental glomerulosclerosis (FSGS) and its mechanism. The FSGS rat model was constructed by single nephrectomy combined with fractional tail vein injection of doxorubicin. 24-hour urine protein, serum biochemistry, HE, PAS and Masson pathological staining were measured to assess renal injury. Glomerular and morphological changes of ferroptosis were observed by transmission electron microscopy. Iron content in renal tissue was assessed by Prussian blue staining and iron detection. GSH/GSSG kit was used to detect the content and proportion of reduced/oxidized glutathione. Lipid peroxidation related proteins including MDA expression was assessed by colorimetry. The iron metabolism biomarkers such as hepcidin, ferroportin and TFR, ferroptosis biomarkers such as GPX4, ACSL4, and ferritinophagy biomarkers such as LC3II/LC3I, NCOA4, and FTH1 were detected by Western blot. Significant urinary protein, hyperlipidemia, azotemia, increased serum creatinine and hypoproteinemia were observed in FSGS rats. Histology and electron microscopy showed segmental sclerosis of glomeruli, compensatory enlargement of some glomeruli, occlusion of capillary lumen, balloon adhesion, increased mesangial matrix, atrophy of some tubules, and renal interstitial fibrosis in renal tissue of FSGS rats. The morphology of glomerular foot processes disappeared; the foot processes were extensively fused and some foot processes detached. Mitochondria became smaller, membrane density increased, and mitochondrial cristae decreased or disappeared. In addition, iron deposition was observed in renal tissue of FSGS rats. Compared with the control group, the levels of GSH, GSH/GSSG, GPX4, and ferroportin were reduced and the expression of GSSG, MDA, ACSL4, hepcidin, and TFR was increased in the renal tissue of FSGS rats; meanwhile, the expression of LC3II/LC3I and NCOA4 was increased and the expression of FTH1 was decreased. Ferroptosis is involved in the pathological progression of FSGS, which is probably associated with activation of ferritinophagy. This represents a potential therapeutic target for FSGS.

Sunitinib induces cardiotoxicity through modulating oxidative stress and Nrf2-dependent ferroptosis in vitro and in vivo

SUN, a multi-targeted tyrosine kinase inhibitor, exerts cardiotoxicity which hinders its clinical use. It is necessary to elucidate molecular mechanism of SUN-induced cardiotoxicity. To elucidate molecular mechanism of SUN-induced cardiotoxicity and whether it is related to Nrf2-dependent ferroptosis, in vitro model with H9c2 cells derived from rat heart tissue and in vivo model (C57BL/6J male mouse) were used in the present study. In vivo model was established by oral treatment of SUN at dose of 10, 20, 40 mg/kg for 14 days. Body weight, ECG, plasma enzyme activities, histology staining were performed to evaluate heart function. Western-blot was performed to analyze the level of ferroptosis-related proteins. In vitro results indicated that SUN markedly induced ferroptosis embodied as collapsed MMP, accumulated iron and elevated ROS. In vivo results showed that SUN significantly impaired cardiac function. Abnormal electrocardiogram, increased serum CK and lactate LDH levels were significantly observed in SUN groups. Histology staining showed that SUN caused structural injuries and fibrosis deposition. Moreover, SUN increased the level of MDA and Fe2+ content, decreased the level of GSH. Both in vitro and in vivo experiments indicated that SUN reduced the expression of Nrf2, HO-1, NQO1, GPX4 and FTH1, enhanced the TfR expression. This study suggested that oxidative stress and Nrf2-dependent ferroptosis played a vital role in SUN-induced cardiotoxicity.

Evaluation of the expression of genes associated with iron metabolism in peripheral blood mononuclear cells from Type 2 diabetes mellitus patients

AimsType 2 Diabetes (T2DM) has been linked to ferroptosis. This study aimed to assess expression levels of genes linked with iron metabolism in peripheral blood mononuclear cells (PBMCs) from T2DM patients and to investigate the association of these expression levels with anthropometric and clinical parameters. MethodsGene expression of iron metabolism genes (Ferritin Light Chain, FTL; Ferritin Heavy Chain, FTH1; Transferrin Receptor, TFRC; Divalent Metal Transporter 1, SLC11A2; Ferroportin, SLC40A1) in archival PBMCs was assessed using quantitative real-time PCR assays. Correlations of gene expression with anthropometric/biochemical patient data were evaluated. ResultsThe study included 36 (18 male/18 female) T2DM patients and 45 (28 male/17 female) normoglycemic (NGT) subjects with a mean age of 38.1 ± 6.8 years and 47.6 ± 8.6 years respectively. Relative expression of FTL was significantly lower in T2DM females compared to that in NGT females (P = 0.027). Relative expression of SLC40A1 was significantly lower in the T2DM group (P = 0.043) and in the T2DM females (P = 0.021). Relative expression of SLC11A2 was negatively correlated with systolic blood pressure in T2DM male patients. Relative expression of SLC40A1 was negatively associated with serum phosphorous and positively associated with serum thyroid stimulating hormone in male T2DM patients. ConclusionsOur findings indicate a reduction in the expression of FTL in perimenopausal T2DM females. Also, in male T2DM patients and NGT subjects, biochemical markers are significantly correlated with the expression of FTL, FTH1, SLC11A2, and SLC40A1 in PBMCs.

M7G modification of FTH1 and pri-miR-26a regulates ferroptosis and chemotherapy resistance in osteosarcoma.

Doxorubicin and platinum are widely used in the frontline treatment of osteosarcoma, but resistance to chemotherapy limits its curative effect. Here, we have identified that METTL1 mediated N7-Methyladenosine (m7G) low expressed in osteosarcoma tissues, plays a critical oncogenic role, and enhances osteosarcoma chemosensitivity in osteosarcoma. Mechanistically, AlkAniline-Seq data revealed that Ferritin heavy chain (FTH1), the main component of ferritin, which is crucial for iron homeostasis and the inhibition of lipid peroxidation, is one of the top 10 genes with the most significant change in m7G methylation sites mediated by METTL1 in human osteosarcoma cells. Interestingly, METTL1 significantly increased the expression of FTH1 at the mRNA level but was remarkably suppressed at the protein level. We then identified primary (pri)-miR-26a and pri-miR-98 in the Top 20 m7G-methylated pri-miRNAs with highly conserved species. Further results confirmed that METTL1 enhances cell ferroptosis by targeting FTH1 and primary (pri)-miR-26a, promoting their maturity by enhancing RNA stability dependent on m7G methylation. The increase of mature miR-26a-5p that resulted from METTL1 overexpression could further target FTH1 mRNA and eliminate FTH1 translation efficiency. Moreover, the reduction of FTH1 translation dramatically increases cell ferroptosis and promotes the sensitivity of osteosarcoma cells to chemotherapy drugs. Collectively, our study demonstrates the METTL1/pri-miR-26a/FTH1 axis signaling in osteosarcoma and highlights the functional importance of METTL1 and m7G methylation in the progression and chemotherapy resistance of osteosarcoma, suggesting that reprogramming RNA m7G methylation as a potential and promising strategy for osteosarcoma treatment.

Inhibition of Ferroptosis Ameliorates Photoreceptor Degeneration in Experimental Diabetic Mice

Diabetic retinopathy (DR) is a leading cause of vision impairment in the working-age population worldwide. Various modes of photoreceptor cell death contribute to the development of DR, including apoptosis and autophagy. However, whether ferroptosis is involved in the pathogenesis of photoreceptor degeneration in DR is still unclear. High-glucose (HG)-stimulated 661W cells and diabetic mice models were used for in vitro and in vivo experiments, respectively. The levels of intracellular iron, glutathione (GSH), reactive oxygen species (ROS), lipid peroxidation (MDA), and ferroptosis-related proteins (GPX4, SLC7A11, ACSL4, FTH1, and NCOA4) were quantified to indicate ferroptosis. The effect of ferroptosis inhibition was also assessed. Our data showed the levels of iron, ROS, and MDA were enhanced and GSH concentration was reduced in HG-induced 661W cells and diabetic retinas. The expression of GPX4 and SLC7A11 was downregulated, while the expression of ACSL4, FTH1, and NCOA4 was upregulated in the 661W cells cultured under HG conditions and in the photoreceptor cells in diabetic mice. Furthermore, the administration of the ferroptosis inhibitor ferrostatin-1 (Fer-1) obviously alleviated ferroptosis-related changes in HG-cultured 661W cells and in retinal photoreceptor cells in diabetic mice. Taken together, our findings suggest that ferroptosis is involved in photoreceptor degeneration in the development of the early stages of DR.

Astaxanthin alleviates PM2.5-induced cardiomyocyte injury via inhibiting ferroptosis

BackgroundLong-term exposure of humans to air pollution is associated with an increasing risk of cardiovascular diseases (CVDs). Astaxanthin (AST), a naturally occurring red carotenoid pigment, was proved to have multiple health benefits. However, whether or not AST also exerts a protective effect on fine particulate matter (PM2.5)-induced cardiomyocyte damage and its underlying mechanisms remain unclear.MethodsIn vitro experiments, the H9C2 cells were subjected to pretreatment with varying concentrations of AST, and then cardiomyocyte injury model induced by PM2.5 was established. The cell viability and the ferroptosis-related proteins expression were measured in different groups. In vivo experiments, the rats were pretreated with different concentrations of AST for 21 days. Subsequently, a rat model of myocardial PM2.5 injury was established by intratracheal instillation every other day for 1 week. The effects of AST on myocardial tissue injury caused by PM2.5 indicating by histological, serum, and protein analyses were examined.ResultsAST significantly ameliorated PM2.5-induced myocardial tissue injury, inflammatory cell infiltration, the release of inflammatory factors, and cardiomyocyte H9C2 cell damage. Mechanistically, AST pretreatment increased the expression of SLC7A11, GPX4 and down-regulated the expression of TfR1, FTL and FTH1 in vitro and in vivo.ConclusionsOur study suggest that ferroptosis plays a significant role in the pathogenesis of cardiomyocyte injury induced by PM2.5. AST may serve as a potential therapeutic agent for mitigating cardiomyocyte injury caused by PM2.5 through the inhibition of ferroptosis.

Comprehensive analysis of single-cell and bulk RNA-sequencing data identifies B cell marker genes signature that predicts prognosis and analysis of immune checkpoints expression in head and neck squamous cell carcinoma

Recent studies have shown that B cells and the associated tertiary lymphoid structures (TLS) correlate with the response of patients to immune checkpoint inhibitors (ICIs) and predict overall survival (OS) in cancer patients. We screened 145 B cell marker genes (BCMG) by a comprehensive analysis of single-cell RNA-sequencing (scRNA-seq) data of head and neck squamous cell carcinoma (HNSC) from the Gene Expression Omnibus (GEO) database. The BCMG signature (BCMGS) was established using The Cancer Genome Atlas (TCGA) dataset of HNSC and verified in four independent datasets. The multivariate Cox regression analysis identified the signature as an independent prognostic factor. A prognostic nomogram was constructed with independent prognostic factors using the TCGA dataset. GO and KEGG analysis revealed the underlying signaling pathways related to this signature. Study of immune profiles showed that patients in the low-risk group presented discriminative immune-cell infiltrations. Furthermore, the low-risk group was featured by higher TCR and BCR diversity, which suggested that low-risk patients may be more sensitive to ICIs. Immunohistochemistry was performed, and we found that high expression of FTH1 was significantly correlated with poor OS (P = 0.025). The expression of TIM-3, LAG-3 and PD-1 was positively correlated and associated with better OS in HNSC. However, there was no statistically significant difference between PD-L1, PD-L2, CTLA-4, TIGIT and prognosis. The BCMGS was a promising prognostic biomarker in HNSC, which may help to interpret the responses to immunotherapy and provide a new perspective for future research on the treatment in HNSC.

Integrated analysis of ATAC-seq and RNA-seq unveils the role of ferroptosis in PM2.5-induced asthma exacerbation

BackgroundPM2.5 exposure increases asthma exacerbation risk and worsens airway inflammation and mucus secretion, but the underlying mechanisms, especially the epigenetic modification changes, are not fully understood. MethodsATAC-seq was conducted in Beas-2B cells to explore the differential chromatin accessibilities before and after exposure to PM2.5. RNA-seq was applied to screen the differentially expressed genes (DEGs) as well. The integrated analysis of ATAC-seq and RNA-seq was performed. The key up-regulated genes in the ferroptosis signaling pathway were identified by combined analysis with the FerrDb database and then verified. Meanwhile, to access the role of PM2.5-induced ferroptosis in asthma mice, house dust mites (HDM) were employed to conduct an allergic asthma mice model, and the ferroptosis-specific inhibitor (Ferrostatin-1, Fer-1) was used. The H&E staining, PAS staining, airway hyperresponsiveness, and bronchoalveolar lavage fluid (BALF) cell counting were used to investigate the impact of PM2.5-induced ferroptosis in asthma mice. ResultsA total of 4,921 regions with differential accessibility were identified, encompassing 4,031 unique genes. Among these, 250 regions exhibited increased accessibility while 4,671 regions displayed reduced accessibility. Through the integrated analysis of ATAC-seq and RNA-seq, ferroptosis was determined as the key enriched pathway based on up-regulated DEGs and increased chromatin accessibilities. Furthermore, the decreased cell viability, accelerated lipid peroxide and morphological changes in mitochondria observed upon PM2.5 exposure were rescued by Fer-1, which are indicative of ferroptosis. By overlapping with ferroptosis-related genes from the FerrDb database, FTH1 and FTL were identified as the prominent up-regulated genes with increased chromatin accessibility in ferroptosis pathway. In addition, ChIP-qPCR analysis indicated that histone modification like H3K4me3 and H3K27ac positively regulated FTH1 and FTL expression. Subsequently, in PM2.5-exposed asthmatic mice, inhibition of ferroptosis effectively attenuated airway inflammation and mucus secretion. ConclusionThese findings shed light on the molecular mechanisms underlying PM2.5-induced asthma exacerbation, with epigenetic modifications playing a pivotal role. Furthermore, it suggests the therapeutic potential of targeting ferroptosis as an intervention strategy.

Iron affects the sphere-forming ability of ovarian cancer cells in non-adherent culture conditions.

Introduction: Detachment from the extracellular matrix (ECM) is the first step of the metastatic cascade. It is a regulated process involving interaction between tumor cells and tumor microenvironment (TME). Iron is a key micronutrient within the TME. Here, we explored the role of iron in the ability of ovarian cancer cells to successfully detach from the ECM. Methods: HEY and PEO1 ovarian cancer cells were grown in 3D conditions. To mimic an iron rich TME, culture media were supplemented with 100μM Fe3+. Cell mortality was evaluated by cytofluorimetric assay. The invasive potential of tumor spheroids was performed in Matrigel and documented with images and time-lapses. Iron metabolism was assessed by analyzing the expression of CD71 and FtH1, and by quantifying the intracellular labile iron pool (LIP) through Calcein-AM cytofluorimetric assay. Ferroptosis was assessed by quantifying mitochondrial reactive oxygen species (ROS) and lipid peroxidation through MitoSOX and BODIPY-C11 cytofluorimetric assays, respectively. Ferroptosis markers GPX4 and VDAC2 were measured by Western blot. FtH1 knockdown was performed by using siRNA. Results: To generate spheroids, HEY and PEO1 cells prevent LIP accumulation by upregulating FtH1. 3D HEY moderately increases FtH1, and LIP is only slightly reduced. 3D PEO1upregulate FtH1 and LIP results significantly diminished. HEY tumor spheroids prevent iron import downregulating CD71, while PEO1 cells strongly enhance it. Intracellular ROS drop down during the 2D to 3D transition in both cell lines, but more significantly in PEO1 cells. Upon iron supplementation, PEO1 cells continue to enhance CD71 and FtH1 without accumulating the LIP and ROS and do not undergo ferroptosis. HEY, instead, accumulate LIP, undergo ferroptosis and attenuate their sphere-forming ability and invasiveness. FtH1 knockdown significantly reduces the generation of PEO1 tumor spheroids, although without sensitizing them to ferroptosis. Discussion: Iron metabolism reprogramming is a key event in the tumor spheroid generation of ovarian cancer cells. An iron-rich environment impairs the sphere-forming ability and causes cell death only in ferroptosis sensitive cells. A better understanding of ferroptosis sensitivity could be useful to develop effective treatments to kill ECM-detached ovarian cancer cells.

Long non-coding RNA SNHG18 epigenetic regulates ferroptosis in acute myocardial infarction

Abstract Background Loss of terminally differentiated cardiomyocytes is a key pathogenic factor for the occurrence of acute myocardial infarction (AMI) and the development of lethal heart failure. LncRNAs have been confirmed to be critical in regulating AMI, besides, recently published studies disclosed that ferroptosis is a new form of programmed cell death that contributes to AMI. However, whether LncRNAs are involved in the regulation of ferroptosis and correlated underlying mechanisms in AMI remains unknown. Purpose Our present study is to investigate LncRNA that regulates cardiomyocyte ferroptosis in AMI and uncover the potential mechanisms. Method RNA-sequencing and bioinformatical analyses were conducted to identify the candidate LncRNAs for AMI. Adenovirus for LncRNA knockout and overexpression were constructed to alter the SNHG18 expression of cardiomyocytes in vitro. Cardiomyocyte-specific knockout and overexpression mice were also constructed to alter SNHG18 expression in vivo. Ferroptosis characteristic measurement, including glutathione peroxidase 4 (GPX4), Ferritin heavy chain (FTH-1), ptgs2, MDA, ROS, Fe2+, glutathione, and mitochondrial morphology, was performed to verify the ability of SNHG18 to regulate the ferroptosis of cardiomyocytes. Cardiac morphological, structural, functional, and hemodynamic changes as well as the viability of cardiomyocytes were also measured to assess the effect of SNHG18 on AMI. RNA-pulldown+Mass spectrometry, RNA immunoprecipitation were performed to identify the specific binding factors of SNHG18. Results RNA-sequencing results revealed the expression of SNHG18 was significantly and persistently elevated from 3 to 7 days after AMI. SNHG18 was mainly enriched in the nucleus of cardiomyocytes. Besides, the down-regulation of SNHG18 could specifically alleviate the cell viability loss caused by ferroptosis inducers. Thereafter, gain- and loss-of-function experiments were performed both in vitro and in vivo. Functionally, SNHG18 knockout dramatically enhanced cardiomyocyte vitality, reduced infarct size, improved cardiac performance and alleviated cardiac fibrosis by mitigating cardiomyocyte ferroptosis. However, enforced SNHG18 expression resulted in the opposite effects. Mechanistically, the subunit RBBP4 that forms the PRC2 (Polycomb Repressive Complex 2) complex was a direct target of SNHG18, which was also confirmed by RIP assays. In addition, Chip-PCR analysis also demonstrated that PRC2 could directly bind to the promoter region of the GPX4 and FTH1 gene, thus altering the level of H3K27me3 and inhibiting their transcription. Moreover, subsequent rescue experiments further verified that SNHG18 regulated the expression of GPX4 and FTH1 to ameliorate AMI-induced cardiomyocyte ferroptosis in a PRC2-dependent manner. Conclusion This study demonstrates that SNHG18-PRC2-GPX4/FTH1 pathway is a promising therapeutic target for the treatment of AMI by ameliorating cardiomyocyte ferroptosis.

Investigating the Drug Resistance Mechanisms of Refractory Diffuse Large B-Cell Lymphoma Based on Proteomic Research

Diffuse large B-cell lymphoma (DLBCL) is the most common histological subtype of non-Hodgkin's lymphoma, more than half of the patients of which can achieve complete remission or even be cured with multidrug immunochemotherapy regimen(R-CHOP). However, for patients with relapsed or refractory DLBCL, there is no effective treatment, while the unfavorable prognosis is related to the primary and acquired resistance to existing therapies. Proteomics is the study of the presence and activity of all proteins in cells. In exploring the mechanisms of DLBCL progression, proteomic technology can identify and quantify changed proteins in DLBCL, reveal the relationship between changes in protein expression levels and different stages of lymphoma occurrence and development, and thus provide information on variation in related proteins and protein-related signaling molecules. Based on proteomic technology research, this study is expected to discover new molecular mechanisms, further clarify resistance mechanisms, and find new therapeutic targets for DLBCL, providing more powerful weapons for better prognosis. We first collected paired clinical patient samples that met the experimental requirements and selected biopsy specimens from 3 patients who relapsed within 1 year after systemic treatment, including biopsy specimens at initial diagnosis and relapse. Next, paraffin sections and HE-stains were performed on the samples, and target areas were micro dissected using a laser microscope. The dissected samples were collected in specific EP tubes, lysed ultrasonically by adding lysis buffer to each group of samples, and the protein concentration of the supernatant was determined after centrifugation. Equal amounts of protein from each sample were digested with trypsin and the final concentration was controlled. Analysis was then performed using the EASY-nLC 1200 ultra-high performance liquid phase system and the Q Exactive TM HF-X mass spectrometer. Finally, bioinformatic analysis was performed to analyze differential proteins and find relevant drug targets or markers. The number of identified peptides was 36,107, the number of identified proteins was 6,338, and the number of quantifiable proteins was 6,165. The top five differentially upregulated proteins in the volcano plot of differential proteins were HOPX, TSNAX, PTMA, FTH1, and IFI44, respectively; and the top five downregulated differential proteins were KRT9, KRT1, KRT10, KRT2, and PIP, respectively. Through enrichment analysis of differential proteins, we noticed the ferroptosis pathway as a key pathway, and found that FTH1, TF, and GSH were involved in iron death-related processes, while FTH1 was one of the top five upregulated differential proteins. Iron death is an iron-dependent, non-apoptotic form of cell death discovered in recent years, activation of which can lead to tumor cell death and inhibit tumor growth. Increased expression of the iron-binding subunit FTH1 can suppress activation of iron death and reduce iron death in tumor cells. This suggests that by developing targeted drugs that act on increasing FTH1 expression or iron death inducers which are capable of slowing down the process, it may be possible to address the problem of traditional chemo-resistance in DLBCL and provide new directions for overcoming DLBCL resistance.

Carbonic Anhydrase Inhibitors Induce Ferroptosis through Inhibition of AKT/FTH1 Signaling in Ewing Sarcoma Tumor Cells.

Ewing sarcoma (ES) is one of the most frequent types of malignant tumors among children. The active metabolic state of ES cells presents a new potential target for therapeutic interventions. As a primary regulator of cellular homeostasis, carbonic anhydrases (CAs; EC 4.2.1.1) have emerged as promising molecular targets for the development of anticancer drugs. Within the present study, we tested the commercial drug acetazolamide and our previously discovered inhibitors to target the CAII isoform, which was overexpressed and positively correlated with ES patient relapse. We employed molecular biology tests to identify effective inhibitors of CAII that can induce ferroptosis by downregulating FTH1 expression in ES cells. In vitro, we have also demonstrated their ability to reduce cell proliferation, decrease invasion, and induce apoptosis- or autophagy-related cell death. Using Western blotting, we confirmed the induction of cathepsin B in cells treated with CA inhibitors. It was found that the suppression of cathepsin B expression during the treatment reduces the anticancer efficacy of selected CAII inhibitors. These experiments highlighted profound antitumor activity of CAII inhibitors attributive to their remarkable ability to trigger ferroptosis in Ewing sarcoma cells without causing substantial host damage. The obtained results suggest that cytosolic CAII may be a prospective target for ES treatment, and CAII inhibitors can be considered as potential single-agent or combination antitumor agents to be used in the treatment of ES.

Knockdown of HNRNPM inhibits the progression of glioma through inducing ferroptosis

ABSTRACT Purpose Ferroptosis acts as an important regulator in diverse human tumors, including the glioma. This study aimed to screen potential ferroptosis-related genes involved in the progression of glioma. Materials and Methods Differently expressed genes (DEGs) were screened based on GSE31262 and GSE12657 datasets, and ferroptosis-related genes were separated. Among the important hub genes in the protein-protein interaction networks, HNRNPM was selected as a research target. Following the knockdown of HNRNPM, the viability, migration, and invasion were detected by CCK8, wound healing, and transwell assays, respectively. The role of HNRNPM knockdown was also verified in a xenograft tumor model in mice. Immunohistochemistry detected the expression levels of HNRNPM and Ki67. Moreover, the ferroptosis was evaluated according to the levels of iron, glutathione peroxidase (GSH), and malondialdehyde (MDA), as well as the expression of PTGS2, GPX4, and FTH1. Results Total 41 overlapping DEGs relating with ferroptosis and glioma were screened, among which 4 up-regulated hub genes (HNRNPM, HNRNPA3, RUVBL1, and SNRPPF) were determined. The up-regulation of HNRNPM presented a certain predictive value for glioma. In addition, knockdown of HNRNPM inhibited the viability, migration, and invasion of glioma cells in vitro, and also the tumor growth in mice. Notably, knockdown of HNRNPM enhanced the ferroptosis in glioma cells. Furthermore, HNRNPM was positively associated with SMARCA4 in glioma. Conclusions Knockdown of HNRNPM inhibits the progression of glioma via inducing ferroptosis. HNRNPM is a promising molecular target for the treatment of glioma via inducing ferroptosis. We provided new insights of glioma progression and potential therapeutic guidance.

Curcumin reverses erastin-induced chondrocyte ferroptosis by upregulating Nrf2

Osteoarthritis (OA) is associated with ferroptosis, a newly discovered form of programmed cell death associated with lipid peroxidation. Curcumin, the main monomer component in turmeric rhizomes, possesses antioxidant and anti-ferroptosis properties, but its effect on ferroptosis in chondrocytes of OA is unknown. This study aimed to investigate the protective effect and potential mechanism of curcumin on chondrocytes induced by erastin, a ferroptosis inducer. CCK-8 assays were used to assess cell viability in mouse primary chondrocytes treated with 3.33 μM erastin alone or in combination with different doses of curcumin. Various parameters were detected, including LDH, SOD, GSH-PX, MDA, ROS and Fe2+ contents. The ferroptosis-related proteins, such as SLC7A11, GPX4, TFR1, ACSL4, and FTH1, were examined using immunofluorescence and western blotting. Nrf2 was knocked down using siRNA to explore the molecular mechanism through which curcumin protects chondrocytes from erastin-induced ferroptosis. In a mouse model of knee ferroptosis induced by intracavity injection of 10 μL erastin (5 mg/mL), HE staining, Safranin O-Fast Green staining, and immunohistochemistry were employed to evaluate articular cartilage injury. The results demonstrated that erastin significantly suppressed the expression of SOD, GSH-PX, SLC7A11, GPX4, and FTH1 while upregulating the levels of LDH, MDA, ROS, ACSL4, and TFR1 in chondrocytes. Moreover, erastin-induced chondrocyte ferroptosis, lipid ROS, and Fe2+ production were reversed by curcumin. Additionally, curcumin significantly upregulated the expression level of the Nrf2 gene and protein. Silencing Nrf2 reversed the protective effect of curcumin on erastin-induced chondrocyte ferroptosis. In animal experiments, silencing Nrf2 counteracted the impact and damage of curcumin on erastin-induced ferroptosis of cartilage tissue in vivo, leading to significant inhibition of OA progression. Taken together, these findings suggest that curcumin can inhibit chondrocyte ferroptosis by activating the Nrf2 signaling pathway, providing further insight into the regulatory mechanism of curcumin in OA and supporting its potential therapeutic use in OA treatment.

Multi-omics analysis identifies IgG2b class-switching with ALCAM-CD6 co-stimulation in joint-draining lymph nodes during advanced inflammatory-erosive arthritis.

Defective lymphatic drainage and translocation of B-cells in inflamed (Bin) joint-draining lymph node sinuses are pathogenic phenomena in patients with severe rheumatoid arthritis (RA). However, the molecular mechanisms underlying this lymphatic dysfunction remain poorly understood. Herein, we utilized multi-omic spatial and single-cell transcriptomics to evaluate altered cellular composition (including lymphatic endothelial cells, macrophages, B-cells, and T-cells) in the joint-draining lymph node sinuses and their associated phenotypic changes and cell-cell interactions during RA development using the tumor necrosis factor transgenic (TNF-Tg) mouse model. Popliteal lymph nodes (PLNs) from wild-type (n=10) and TNF-Tg male mice with "Early" (5 to 6-months of age; n=6) and "Advanced" (>8-months of age; n=12) arthritis were harvested and processed for spatial transcriptomics. Single-cell RNA sequencing (scRNAseq) was performed in PLNs from the TNF-Tg cohorts (n=6 PLNs pooled/cohort). PLN histopathology and ELISPOT along with ankle histology and micro-CT were evaluated. Histopathology of human lymph nodes and synovia was performed for clinical correlation. Advanced PLN sinuses exhibited an increased Ighg2b/Ighm expression ratio (Early 0.5 ± 0.1 vs Advanced 1.4 ± 0.5 counts/counts; p<0.001) that significantly correlated with reduced talus bone volumes in the afferent ankle (R2 = 0.54, p<0.001). Integration of single-cell and spatial transcriptomics revealed the increased IgG2b+ plasma cells localized in MARCO+ peri-follicular medullary sinuses. A concomitant decreased Fth1 expression (Early 2.5 ± 0.74 vs Advanced 1.0 ± 0.50 counts, p<0.001) within Advanced PLN sinuses was associated with accumulation of iron-laden Prussian blue positive macrophages in lymph nodes and synovium of Advanced TNF-Tg mice, and further validated in RA clinical samples. T-cells were increased 8-fold in Advanced PLNs, and bioinformatic pathway assessment identified the interaction between ALCAM+ macrophages and CD6+ T-cells as a plausible co-stimulatory mechanism to promote IgG2b class-switching. Collectively, these data support a model of flare in chronic TNF-induced arthritis in which loss of lymphatic flow through affected joint-draining lymph nodes facilitates the interaction between effluxing macrophages and T-cells via ALCAM-CD6 co-stimulation, initiating IgG2b class-switching and plasma cell differentiation of the expanded Bin population. Future work is warranted to investigate immunoglobulin clonality and potential autoimmune consequences, as well as the efficacy of anti-CD6 therapy to prevent these pathogenic events.

VDAC1 participates in house dust mite-induced asthmatic airway inflammation in mice by inducing ferroptosis of airway epithelial cells

To investigate the role of voltage-dependent anion-selective channel protein 1 (VDAC1) in house dust mite (HDM)-induced asthmatic airway inflammation and its mechanism for regulating ferroptosis in airway epithelial cells. Human airway epithelial (HBE) cells were exposed to a concentration gradient (200, 400 and 800 U) of HDM alone or in combination with treatment with 10 μmol/L VBIT-4 (a VDAC1 inhibitor) for 24 h, and the expressions of VDAC1 and ferroptosis-associated proteins in the cells were examined. Adult male BALB/c mice were treated with intranasal instillation of VBIT-4, HDM, or both, and the level of airway inflammation and the expressions of ferroptosis-associated proteins were detected with immunohistochemistry. In HBE cells, HDM exposure caused a significant increase of mitochondrial ROS (mtROS) production and obviously decreased the mitochondrial membrane potential. The exposed cells showed obviously increased protein expressions of VDAC1 (P=0.005) and FTH1 (P=0.030) but decreased protein expression of GPX4 (P=0.015) and FTH1 (P=0.037), while the treatment with VBIT-4 repressed the expression of GPX4 (P=0.001) and inhibited the expression of VDAC1. In BALB/c mice, treatment with VBIT-4 significantly improved HDM-induced airway inflammation by reducing the number of inflammatory cells (P=0.029) in the airway and the number of eosinophils in the alveolar lavage fluid. Immunohistochemical staining showed that GPX4 expression in the airway epithelial cells was significantly increased after treatment with VBIT-4. VDAC1 participates in HDM-induced chronic airway inflammation in bronchial asthma by causing ferroptosis of the airway epithelial cells.

SYVN1 attenuates ferroptosis and alleviates spinal cord ischemia–reperfusion injury in rats by regulating the HMGB1/NRF2/HO-1 axis

BackgroundThe ferroptosis of neurons is an important pathological mechanism of spinal cord ischemia reperfusion injury (SCIRI). Previous studies showed that synoviolin 1 (SYVN1) is a good prognostic marker of neurodegenerative diseases, but its mechanism is still unclear. This study aims to explore the role of SYVN1 in the ferroptosis of neurons and to clarify its internal mechanism. MethodsRat primary spinal cord neurons were treated with oxygen-glucose deprivation (OGD) for 1, 4 or 8 h, and then cell viability, ROS and MDA levels, glutathione peroxidase (GSH-Px) activity, and the expression of ferroptosis-related proteins GPX4, FTH1 and PTGS2 were detected. OGD/R-induced neurons were transfected with pcDNA-SYVN1 or si-HMGB1, and then cell functions were detected. Transmission electron microscope (TEM) was used to detect cell ferroptosis. The interplay between SYVN1 and high mobility group box 1 (HMGB1) was confirmed with Co-immunoprecipitation (Co-IP) assay. The stability of HMGB1 was measured by ubiquitination assay. Also, cells were treated with pcDNA-SYVN1 or together with ubiquitination inhibitor MG132, as well as treated with pcDNA-SYVN1 and pcDNA-HMGB1 or together with NRF2 activator dimethyl fumarate (DMF), and then Western blotting was used to detect the expression of HMGB1, nuclear NRF2 and HO-1 proteins. In addition, SD rats were occluded left common carotid artery and aortic arch to establish a SCIRI rat model. And rats were injected intrathecal with adenovirus-mediated SYVN1 overexpression vector (Ad-SYVN1, 2 μL, virus titer 5 × 1013 transduction unit [TU]/mL) to overexpress SYVN1. The motion function of rats was quantified using the Basso Rat Scale (BMS) for Locomotion. The ferroptosis and the number of neurons in the spinal cord tissue of rats were detected. ResultsSYVN1 overexpression inhibited ferroptosis of SCIRI rats and OGD/R-treated primary spinal cord neurons, and down-regulated the expression of HMGB1. In terms of mechanism, the binding of SYVN1 and HMGB1 promoted the ubiquitination and degradation of HMGB1, and negatively regulated the expression of HMGB1. Moreover, under OGD/R conditions, MG132 treatment or HMGB1 overexpression eliminated the inhibitory effect of SYVN1 overexpression on the ferroptosis of neurons and the activation of the NRF2/HO-1 pathway, and DMF treatment abolished the inhibition of HMGB1 overexpression on the NRF2/HO-1 pathway. Finally, in vivo experiments showed that SYVN1 overexpression could alleviate the spinal cord ischemia–reperfusion injury in rats by down-regulating HMGB1 and promoting the activation of the NRF2/HO-1 pathway. ConclusionSYVN1 regulates ferroptosis through the HMGB1/NRF2/HO-1 axis to prevent spinal cord ischemia–reperfusion injury.

Identification of hepatocellular carcinoma-related subtypes and development of a prognostic model: a study based on ferritinophagy-related genes

BackgroundHepatocellular carcinoma still has a high incidence and mortality rate worldwide, and further research is needed to investigate its occurrence and development mechanisms in depth in order to identify new therapeutic targets. Ferritinophagy is a type of autophagy and a key factor in ferroptosis that could influence tumor onset and progression. Although, the potential role of ferritinophagy-related genes (FRGs) in liver hepatocellular carcinoma (LIHC) is unknown.MethodsSingle-cell RNA sequencing (scRNA-seq) data of LIHC were obtained from the Gene Expression Omnibus (GEO) dataset. In addition, transcriptome and clinical follow-up outcome data of individuals with LIHC were extracted from the The Cancer Genome Atlas (TCGA) dataset. FRGs were collected through the GeneCards database. Differential cell subpopulations were distinguished, and differentially expressed FRGs (DEFRGs) were obtained. Differential expression of FRGs and prognosis were observed according to the TCGA database. An FRG-related risk model was constructed to predict patient prognosis by absolute shrinkage and selection operator (LASSO) and COX regression analyses, and its prognosis predictive power was validated. Ultimately, the association between risk score and tumor microenvironment (TME), immune cell infiltration, immune checkpoints, drug sensitivity, and tumor mutation burden (TMB) was analyzed. We also used quantitative reverse transcription polymerase chain reaction (qRT-PCR) to validate the expression of key genes in normal liver cells and liver cancer cells.ResultsWe ultimately identified 8 cell types, and 7 differentially expressed FRGs genes (ZFP36, NCOA4, FTH1, FTL, TNF, PCBP1, CYB561A3) were found among immune cells, and we found that Monocytes and Macrophages were closely related to FRGs genes. Subsequently, COX regression analysis showed that patients with high expression of FTH1, FTL, and PCBP1 had significantly worse prognosis than those with low expression, and our survival prediction model, constructed based on age, stage, and risk score, showed better prognostic prediction ability. Our risk model based on 3 FRGs genes ultimately revealed significant differences between high-risk and low-risk groups in terms of immune infiltration and immune checkpoint correlation, drug sensitivity, and somatic mutation risk. Finally, we validated the key prognostic genes FTH1, FTL, using qRT-PCR, and found that the expression of FTH1 and FTL was significantly higher in various liver cancer cells than in normal liver cells. At the same time, immunohistochemistry showed that the expression of FTH1, FTL in tumor tissues was significantly higher than that in para-tumor tissues.ConclusionThis study identifies a considerable impact of FRGs on immunity and prognosis in individuals with LIHC. The collective findings of this research provide new ideas for personalized treatment of LIHC and a more targeted therapy approach for individuals with LIHC to improve their prognosis.

Puerarin attenuates valproate-induced features of ASD in male mice via regulating Slc7a11-dependent ferroptosis.

Autism spectrum disorder (ASD) is a complicated, neurodevelopmental disorder characterized by social deficits and stereotyped behaviors. Accumulating evidence suggests that ferroptosis is involved in the development of ASD, but the underlying mechanism remains elusive. Puerarin has an anti-ferroptosis function. Here, we found that the administration of puerarin from P12 to P15 ameliorated the autism-associated behaviors in the VPA-exposed male mouse model of autism by inhibiting ferroptosis in neural stem cells of the hippocampus. We highlight the role of ferroptosis in the hippocampus neurogenesis and confirm that puerarin treatment inhibited iron overload, lipid peroxidation accumulation, andmitochondrial dysfunction, as well asenhanced the expression of ferroptosis inhibitory proteins, including Nrf2, GPX4, Slc7a11, and FTH1 in the hippocampus of VPA mouse model of autism. In addition, we confirmed that inhibition of xCT/Slc7a11-mediated ferroptosis occurring in the hippocampus is closely related to puerarin-exerted therapeutic effects. In conclusion, our study suggests that puerarin targets core symptoms and hippocampal neurogenesis reduction through ferroptosis inhibition, which might be a potential drug for autism intervention.