Amelioration of nephrotoxicity by targeting ferroptosis: role of NCOA4, IREB2, and SLC7a11 signaling

Nephrotoxicity is a common complication that limits the clinical utility of cisplatin. Ferroptosis is an iron-dependent necrotic cell death program that is mediated by phospholipid peroxidation. The molecular mechanisms that disrupt iron homeostasis and lead to ferroptosis are yet to be elucidated. In this study, we aimed to investigate the involvement of nuclear receptor coactivator 4 (NCOA4), a selective cargo receptor that mediates ferroptosis and autophagic degradation of ferritin in nephrotoxicity. Adult male Sprague-Dawley rats were randomly-assigned to four groups: control group, cisplatin (Cis)-treated group, deferiprone (DEF)-treated group, and Cis+DEF co-treated group. Serum, urine, and kidneys were isolated to perform biochemical, morphometric, and immunohistochemical analysis. Iron accumulation was found to predispose to ferroptotic damage of the renal tubular cells. Treatment with deferiprone highlights the role of ferroptosis in nephrotoxicity. Upregulation of NCOA4 in parallel with low ferritin level in renal tissue seems to participate in iron-induced ferroptosis. This study indicated that ferroptosis may participate in cisplatin-induced tubular cell death and nephrotoxicity through iron-mediated lipid peroxidation. Iron dyshomeostasis could be attributed to NCOA4-mediated ferritin degradation.

Role of ferritinophagy in the pathogenesis of diabetes and its complications:A review of literature / 实用医学杂志

Diabetes mellitus(DM)is a common metabolic disease,and its hyperglycemia can induce many complications and even threaten human health and life.Ferritinophagy,currently a research focus,has been proven to be related to the occurrence and development of DM and its complications.Ferritinophagy,which is cell-selective,is mediated by nuclear receptor coactivator 4(NCOA4),which degrades ferritin in autophagosomes and releases excessive iron ions so that irons are overloaded and ROS are accumulated.This process contributes to the upstream ferroptosis.This article reviews the mechanism of ferritinophagy and its pathogenesis in DM and its complications,and further analyzes the effects of regulated ferritinophagy on DM and its complications,pro-viding new insight into the prevention and treatment of DM and its complications.

Total saponins of Panax japonicus regulates the inhibitory effect of AMPK/mTOR/ULK1 pathway mediated ferritinophagy on ferroptosis of cardiomyocytes in diabetic cardiomyopathy rats / 中华内分泌代谢杂志

Objective:To investigate the effect of total saponins of Panax japonicus(TSPJ) on ferroptosis of myocardial cells in diabetic cardiomyopathy(DCM) rats and underlying mechanism.Methods:Experiment 1: SD rats were divided into control group, DCM group, low-dose TSPJ group, high-dose TSPJ group, and metformin(Met) group, with 10 rats in each group. Experiment 2: SD rats were divided into control group, DCM group, TSPJ group, adenosine monophosphate-activated protein kinase(AMPK) inhibitor Compound C group, and TSPJ+ AMPK agonist AICAR group, with 10 rats in each group. Except for the control group, all rats were intraperitoneally injected with streptozotocin to construct a DCM model. After 8 weeks of corresponding drug intervention, the body weight as well as glucose and lipid metabolism of rats in each experimental group were assessed, and the cardiac function indicators were detected with echocardiography. The levels of serum lactate dehydrogenase(LDH), cardiac troponin I(cTnI) and creatine kinase isoenzyme MB(CK-MB) were detected by ELISA technique. The pathological changes of myocardial tissue were observed using hematoxylin-eosin(HE) staining. The levels of dismutase(SOD), glutathione(GSH), malondialdehyde(MDA), reactive oxygen species(ROS) and Fe 2+ in myocardial tissue were detected. Western blot was used to detect ferroptosis, ferritinophagy, and the AMPK/mammalian target of rapamycin/UNC-51-like kinase 1(mTOR/ULK1) signaling pathway related proteins expression in myocardial tissue. Results:Compared with control group, left ventricular ejection fraction(EF), left ventricular short axis shortening rate(FS), peak blood velocity ratio(E/A) between early and late diastolic periods were significantly decreased in DCM group, left ventricular inner diameter(LVEDd) was increased, and the serum LDH, cTnI, CK-MB were increased, the levels of SOD, GSH were decreased, MDA, ROS, Fe 2+ were increased in myocardial tissue, the expressions of TFR1, NCOA4 LC3-II/LC3-I, Beclin-1, phosphorylated AMPK and phosphorylated ULK1 were increased, the expressions of GPX4, SLC7A11 and phosphorylated mTOR were decreased. Compared with DCM group, the above indicators of rats were significantly improved in each treatment group. Compared with the TSPJ group, the AMPK agonist AICAR reversed the effects of TSPJ on ferroptosis and ferritinophagy mediated by the AMPK/mTOR/ULK1 pathway in DCM rat cardiomyocytes. Conclusion:TSPJ can inhibit ferroptosis in DCM rat cardiomyocytes and improve myocardial injury by regulating AMPK/mTOR/ULK1 mediated ferritinophagy.

Ligustilide delays senescence of auditory cortex in mice by inhibiting ferritinophagy / 中国药理学通报

Aim To investigate the mechanism of ligu aged 2 months of the same strain were used as the constilide (LIG) in delaying the senescence of auditory trol (Ctrl) group. Auditory brainstem response test was cortex and treating central presbycusis. Methods used to detect the auditory threshold of mice before and Forty C57BL/6J mice aged 13 months were randomly di after treatment. Levels of serum MDA and activity of vided into ligustilide low-dose(L-LIG) group, ligustil serum SOD were detected to display the level of oxidative ide medium-dose (M-LIG) group, ligustilide high-dose stress. The pathological changes of auditory cortex were (H-LIG) group and aging (Age) group, and 10 mice observed by HE staining. Ferroptosis was observed by

Targeting ferroptosis and ferritinophagy: new targets for cardiovascular diseases / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Cardiovascular diseases (CVDs) are a leading factor driving mortality worldwide. Iron, an essential trace mineral, is important in numerous biological processes, and its role in CVDs has raised broad discussion for decades. Iron-mediated cell death, namely ferroptosis, has attracted much attention due to its critical role in cardiomyocyte damage and CVDs. Furthermore, ferritinophagy is the upstream mechanism that induces ferroptosis, and is closely related to CVDs. This review aims to delineate the processes and mechanisms of ferroptosis and ferritinophagy, and the regulatory pathways and molecular targets involved in ferritinophagy, and to determine their roles in CVDs. Furthermore, we discuss the possibility of targeting ferritinophagy-induced ferroptosis modulators for treating CVDs. Collectively, this review offers some new insights into the pathology of CVDs and identifies possible therapeutic targets.

Progress in mechanism and detection methods of ferrinophagy-mediated ferroptosis / 中国病理生理杂志

Ferroptosis is an iron-dependent form of regulated cell death,which is distinct from apoptosis,ne-crosis,and pyroptosis.Recent studies have found that activators of ferroptosis,such as Erastin,can activate autophagy-re-lated proteins,induce the formation of autophagosomes,and ultimately release ferric ions to mediate ferroptosis.This pro-cess,called ferritinophagy,is initiated by the binding of an autophagic cargo receptor protein,nuclear receptor coactivator 4(NCOA4),to iron-laden ferritin.The transfer of NCOA4-ferritin to the lysosome by ferritinophagy results in the proteoly-sis of ferritin,and,in turn,the release of its iron content and lipid-reactive oxygen species(ROS)accumulation.Ferritin-ophagy has been closely associated with central nervous system disorders,circulatory system diseases,and cancer.Fur-thermore,the regulation mechanism of ferritinophagy is also a hot topic in the study of iron-dependent cell death process.With the in-depth study of ferritinophagy,great progress has been made in the study of key components of ferritinophagy as well as its molecular mechanisms and processes.However,a comprehensive summary of the methods for detecting ferritin-ophagy is still unclear.To further deepen the understanding of ferritinophagy and its detection methods,this review focus-es on the concept,characteristics,methods,and precautions during detection of ferritinophagy.This review provided ex-perimental reference for subsequent researchers and promoting the progress of research related to ferritinophagy.

Molecular mechanism of ligustilide attenuating OGD/R injury in PC12 cells by inhibiting ferroptosis / 中国中药杂志

The aim of this study is to explore the mechanism of ligustilide, the main active constituent of essential oils of traditional Chinese medicine Angelicae Sinensis Radix, on alleviating oxygen-glucose deprivation/reperfusion(OGD/R) injury in PC12 cells from the perspective of ferroptosis. OGD/R was induced in vitro, and 12 h after ligustilide addition during reperfusion, cell viability was detected by cell counting kit-8(CCK-8) assay. DCFH-DA staining was used to detect the level of intracellular reactive oxygen species(ROS). Western blot was employed to detect the expression of ferroptosis-related proteins, glutathione peroxidase 4(GPX4), transferrin receptor 1(TFR1), and solute carrier family 7 member 11(SLC7A11), and ferritinophagy-related proteins, nuclear receptor coactivator 4(NCOA4), ferritin heavy chain 1(FTH1), and microtubule-associated protein 1 light chain 3(LC3). The fluorescence intensity of LC3 protein was analyzed by immunofluorescence staining. The content of glutathione(GSH), malondialdehyde(MDA), and Fe was detected by chemiluminescent immunoassay. The effect of ligustilide on ferroptosis was observed by overexpression of NCOA4 gene. The results showed that ligustilide increased the viability of PC12 cells damaged by OGD/R, inhibited the release of ROS, reduced the content of Fe and MDA and the expression of TFR1, NCOA4, and LC3, and improved the content of GSH and the expression of GPX4, SLC7A11, and FTH1 compared with OGD/R group. After overexpression of the key protein NCOA4 in ferritinophagy, the inhibitory effect of ligustilide on ferroptosis was partially reversed, indicating that ligustilide may alleviate OGD/R injury of PC12 cells by blocking ferritinophagy and then inhibiting ferroptosis. The mechanism by which ligustilide reduced OGD/R injury in PC12 cells is that it suppressed the ferroptosis involved in ferritinophagy.

Regulatory Mechanism of NCOA4-mediated Ferritinophagy and its Progress in Colitis and Colitis-associated Cancer / 胃肠病学

Abnormal iron metabolism mediated by ferritinophagy is one of the most important mechanisms in the occurrence of ferroptosis.The regulatory mechanism of ferritinophagy mainly involves the transcription of NCOA4 and its corresponding protein modifications.Ferroptosis plays an important role in the development of colitis and colitis-associated cancer,and target-oriented regulation of ferroptosis can alleviate colonic inflammatory response and induce the tumor cell death.This article mainly reviewed the regulatory mechanism of NCOA4-mediated ferritinophagy and its progress in colitis and colitis-related cancer,which may provide a new point for the investigation on mechanism of colitis and inflammation-cancer transformation.

Relationship between cGAS-STING signaling pathway and ferritinophagy in early stage of cerebral ischemia-reperfusion in mice / 中华麻醉学杂志

Objective:To evaluate the relationship between the second messenger cyclic GMP-AMP (cGAS)-cyclic GMP-AMP receptor stimulator of interferon genes (STING) signaling pathway and ferritinophagy in the early stage of cerebral ischemia-reperfusion (I/R) in mice.Methods:Twenty-four clean-grade healthy male C57BL/6 mice, aged 6-8 weeks, weighing 21-25 g, were divided into 4 groups ( n=6 each) using a random number table method: sham group, cerebral I/R injury group (CIRI group), cerebral I/R injury + cGAS inhibitor group (CIRI + RU group), and cerebral I/R injury + cGAS inhibitor + overexpressed nuclear receptor coactivator 4 (NCOA4) group (MCAO + RU + LV-NCOA4 group). The model of cerebral I/R injury was developed using the middle cerebral artery occlusion (MCAO) in anesthetized animals.In CIRI+ RU group, cGAS inhibitor 5 mg/kg was intraperitoneally injected at 10 min before reperfusion.In CIRI+ RU+ LV-NCOA4 group, NCOA4-overexpressing lentivirus (1×10 9 TU/ml) 2 μl was injected into the ventricle at 7 days before MCAO, and the other operations were the same as those previously described in CIRI+ RU group.After 6 h of reperfusion, the neurological function deficits were assessed and scored, then the mice were sacrificed, and brains were removed for determination of the cerebral infarct size (by TTC method), MDA content (by TBA method), activity of SOD (by WST-1 method), and expression of cGAS, STING, NCOA4, ferritin, and microtubule-associated protein 1 light chain 3B (LC3B) (by Western blot). Results:Compared with Sham group, the neurological function deficit score and cerebral infarct size were significantly increased, SOD activity was decreased, MDA content was increased, the expression of cGAS, STING, NCOA4 and LC3B was up-regulated, and the expression of ferritin was down-regulated in CIRI group ( P<0.05). Compared with CIRI group, the neurological function deficit score and cerebral infarct size were significantly decreased, SOD activity was increased, MDA content was decreased, the expression of cGAS, STING, NCOA4 and LC3B was down-regulated, and the expression of ferritin was up-regulated in CIRI+ RU group ( P<0.05). Compared with CIRI+ RU group, the neurological function deficit score and cerebral infarct size were significantly increased, SOD activity was decreased, MDA content was increased, the expression of cGAS, STING, NCOA4 and LC3B was up-regulated, and the expression of ferritin was down-regulated in CIRI group ( P<0.05), and no significant change was found in the expression of cGAS and STING in CIRI+ RU+ LV-NCOA4 group ( P>0.05). Conclusions:The cGAS-STING signaling pathway can promote the over-activation of ferritinophagy, enhance oxidative stress, and thus induce early CIRI in mice.

The Research on Ferritinophagy, Ferroptosis and Related Diseases / 中国生物化学与分子生物学报

Iron, an important cofactor for heme, mitochondrial respiratory chain complexes, and various biologically important enzymes, participates in biological processes including oxygen transport, redox reactions, and metabolite synthesis. Ferritin is an iron storage protein that maintains iron homeostasis in the body by sequestering and releasing iron. Ferritinophagy is a selective type of autophagy that mediates ferritin degradation, releasing free iron when increased intracellular iron level is needed. Moderate rates of iron autophagy maintain intracellular iron content homeostasis. Excessive ferritinophagy will release a large amount of free iron, causing lipid peroxidation and cell damage via reactive oxygen species (ROS) produced by the Fenton reaction. Therefore, ferritinophagy plays a vital role in maintaining cellular iron homeostasis. Nuclear receptor co-activator 4 (NCOA4) acts as a key regulator of ferritinophagy by targeting ferritin binding and delivery to lysosomes for degradation, leading to release of free iron. Thus, NCOA4-mediated ferritinophagy is an important contributor to iron metabolism. Recent research reveals that NCOA4 is regulated by factors including iron content, autophagy, lysosomes, and hypoxia. NCOA4-mediated ferritin degradation is related to ferroptosis (an autophagic cell death process) . Ferritinophagy acts as an upstream mechanism driving ferroptosis by regulating cellular iron homeostasis and ROS production, which are closely correlated with the occurrence and development of anemia, neurodegenerative diseases, cancer, ischemia / reperfusion injury, and other diseases. In this study, the functional characteristics of NCOA4-mediated ferritinophagy in ferroptosis and the role of NCOA4 in these diseases were reviewed, which may provide new avenues for the treatment of related diseases.

Role of Cytoglobin in Ferroptosis Regulation / 中国生物化学与分子生物学报

Ferroptosis is a recently reported iron-dependent cell death, which is induced by the broken of cellular redox homeostasis and characterized by high level of lipid peroxide. More and more studies have indicated the importance of ferroptosis during disease development and prevention. As a member of globin family, cytoglobin (CYGB), also known as stellate cell activating protein (STAP), can bind to oxygen for its transportation. Moreover, CYGB contains two cysteins within its amino acid sequence and could form the S-S bond when there has change of cellular redox which will result in downstream signaling alternation. Furthermore, CYGB has nitric oxygen dioxygenase activity to scavenge excessive nitric oxygen and prevent the production of ONOO