Lipid storage and lipophagy regulates ferroptosis

The synthesis, storage, and degradation of lipids are highly regulated processes. Impaired lipid metabolism is implicated in inflammation and cell death. Although ferroptosis is a recently described form of regulated cell death driven by lipid peroxidation, the impact of lipid droplets on ferroptosis remains unidentified. Here, we demonstrate that lipophagy, the autophagic degradation of intracellular lipid droplets, promotes RSL3-induced ferroptotic cell death in hepatocytes. Lipid droplet accumulation is increased at the early stage but decreased at the late stage of ferroptosis in mouse or human hepatocytes. Importantly, either genetically enhancing TPD52-dependent lipid storage or blocking ATG5-and RAB7A-dependent lipid degradation prevents RSL3-induced lipid peroxidation and subsequent ferroptosis in vitro and in vivo. These studies support an antioxidant role for lipid droplets in cell death and suggest novel strategies for the inhibition of ferroptosis by targeting the lipophagy pathway.     

Lipid peroxidation and the subsequent cell death transmitting from ferroptotic cells to neighboring cells

Ferroptosis regulated cell death due to the iron-dependent accumulation of lipid peroxide. Ferroptosis is known to constitute the pathology of ischemic diseases, neurodegenerative diseases, and steatohepatitis and also works as a suppressing mechanism against cancer. However, how ferroptotic cells affect surrounding cells remains elusive. We herein report the transfer phenomenon of lipid peroxidation and cell death from ferroptotic cells to nearby cells that are not exposed to ferroptotic inducers (FINs). While primary mouse embryonic fibroblasts (MEFs) and NIH3T3 cells contained senescence-associated β-galactosidase (SA-β-gal)-positive cells, they were decreased upon induction of ferroptosis with FINs. The SA-β-gal decrease was inhibited by ferroptotic inhibitors and knockdown of Atg7, pointing to the involvement of lipid peroxidation and activated autophagosome formation during ferroptosis. A transfer of cell culture medium of cells treated with FINs, type 1 or 2, caused the reduction in SA-β-gal-positive cells in recipient cells that had not been exposed to FINs. Real-time imaging of Kusabira Orange-marked reporter MEFs cocultured with ferroptotic cells showed the generation of lipid peroxide and deaths of the reporter cells. These results indicate that lipid peroxidation and its aftereffects propagate from ferroptotic cells to surrounding cells, even when the surrounding cells are not exposed to FINs. Ferroptotic cells are not merely dying cells but also work as signal transmitters inducing a chain of further ferroptosis.Subject terms: Cell death, Autophagy     

Ceruloplasmin suppresses ferroptosis by regulating iron homeostasis in hepatocellular carcinoma cells

Ferroptosis is a regulated form of cell death characterized by the iron-dependent accumulation of lipid hydroperoxides. Ceruloplasmin (CP) is a glycoprotein that plays an essential role in iron homeostasis. However, whether CP regulates ferroptosis has not been reported. Here, we show that CP suppresses ferroptosis by regulating iron homeostasis in hepatocellular carcinoma (HCC) cells. Depletion of CP promoted erastin- and RSL3-induced ferroptotic cell death and resulted in the accumulation of intracellular ferrous iron (Fe²⁺) and lipid reactive oxygen species (ROS). Moreover, overexpression of CP suppressed erastin- and RSL3-induced ferroptosis in HCC cells. In addition, a novel frameshift mutation (c.1192-1196del, p.leu398serfs) of CP gene newly identified in patients with iron accumulation and neurodegenerative diseases lost its ability to regulate iron homeostasis and thus failed to participate in the regulation of ferroptosis. Collectively, these data suggest that CP plays an indispensable role in ferroptosis by regulating iron metabolism and indicate a potential therapeutic approach for hepatocellular carcinoma.     

Endogenous hydrogen sulfide regulation of myocardial injury induced by isoproterenol

Previous work has shown that the endogenous cystathionine γ-synthase (CSE)/hydrogen sulfide (H₂S) pathway participates in the regulation of cardiac contraction. We hypothesized that the pathway might participate in the pathophysiological regulation of ischemic heart disease. Isoproterenol injection of rat hearts induced a myocardial ischemic injury model, with reduced myocardial and plasma H₂S levels, decreased CSE activity, and upregulated CSE gene expression. Exogenous administration of the H₂S donor NaHS reduced the mortality rate; increased left-ventricular pressure development and left-ventricular-end systolic pressure; and decreased left-ventricular-end diastolic pressure (LVEDP) and subendocardial necrosis, capillary dilatation, leukocytic infiltration, fibroblast swelling, and fibroblastic hyperplasia. As well, production of lipid peroxidation, including myocardial malondialdehyde (MDA), and plasma MDA and conjugated diene, was reduced. Oxidative stress injury is an important mechanism of isoproterenol-induced myocardial injury. In vitro experiments revealed that NaHS might antagonize myocyte MDA production by oxygen-free radicals and that NaHS directly scavenged hydrogen peroxide and superoxide anions. Our results suggest that the endogenous CSE/H₂S pathway contributes to the pathogenesis of isoproterenol-induced myocardial injury. Administration of exogenous H₂S effectively protects myocytes and contractile activity, at least by its direct scavenging of oxygen-free radicals and reducing the accumulation of lipid peroxidations.     

CISD3 inhibition drives cystine-deprivation induced ferroptosis

Ferroptosis, a new form of programmed cell death, not only promotes the pathological process of various human diseases, but also regulates cancer progression. Current perspectives on the underlying mechanisms remain largely unknown. Herein, we report a member of the NEET protein family, CISD3, exerts a regulatory role in cancer progression and ferroptosis both in vivo and in vitro. Pan-cancer analysis from TCGA reveals that expression of CISD3 is generally elevated in various human cancers which are consequently associated with a higher hazard ratio and poorer overall survival. Moreover, knockdown of CISD3 significantly accelerates lipid peroxidation and accentuates free iron accumulation triggered by Xc^– inhibition or cystine-deprivation, thus causing ferroptotic cell death. Conversely, ectopic expression of the shRNA-resistant form of CISD3 (CISD3res) efficiently ameliorates the ferroptotic cell death. Mechanistically, CISD3 depletion presents a metabolic reprogramming toward glutaminolysis, which is required for the fuel of mitochondrial oxidative phosphorylation. Both the inhibitors of glutaminolysis and the ETC process were capable of blocking the lipid peroxidation and ferroptotic cell death in the shCISD3 cells. Besides, genetic and pharmacological activation of mitophagy can rescue the CISD3 knockdown-induced ferroptosis by eliminating the damaged mitochondria. Noteworthily, GPX4 acts downstream of CISD3 mediated ferroptosis, which fails to reverse the homeostasis of mitochondria. Collectively, the present work provides novel insights into the regulatory role of CISD3 in ferroptotic cell death and presents a potential target for advanced antitumor activity through ferroptosis.Subject terms: Oncogenes, Preclinical research     

GSTZ1 sensitizes hepatocellular carcinoma cells to sorafenib-induced ferroptosis via inhibition of NRF2/GPX4 axis

Increasing evidence supports that ferroptosis plays an important role in tumor growth inhibition. Sorafenib, originally identified as an inhibitor of multiple oncogenic kinases, has been shown to induce ferroptosis in hepatocellular carcinoma (HCC). However, some hepatoma cell lines are less sensitive to sorafenib-induced ferroptotic cell death. Glutathione S-transferase zeta 1 (GSTZ1), an enzyme in the catabolism of phenylalanine, suppresses the expression of the master regulator of cellular redox homeostasis nuclear factor erythroid 2-related factor 2 (NRF2). This study aimed to investigate the role and underlying molecular mechanisms of GSTZ1 in sorafenib-induced ferroptosis in HCC. GSTZ1 was significantly downregulated in sorafenib-resistant hepatoma cells. Mechanistically, GSTZ1 depletion enhanced the activation of the NRF2 pathway and increased the glutathione peroxidase 4 (GPX4) level, thereby suppressing sorafenib-induced ferroptosis. The combination of sorafenib and RSL3, a GPX4 inhibitor, significantly inhibited GSTZ1-deficient cell viability and promoted ferroptosis and increased ectopic iron and lipid peroxides. In vivo, the combination of sorafenib and RSL3 had a synergic therapeutic effect on HCC progression in Gstz1^−/− mice. In conclusion, this finding demonstrates that GSTZ1 enhanced sorafenib-induced ferroptosis by inhibiting the NRF2/GPX4 axis in HCC cells. Combination therapy of sorafenib and GPX4 inhibitor RSL3 may be a promising strategy in HCC treatment.Subject terms: Cancer therapeutic resistance, Cancer therapeutic resistance     

Involvement of hydrogen sulfide and homocysteine transsulfuration pathway in the progression of kidney fibrosis after ureteral obstruction

Hydrogen sulfide (H₂S) produced by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in the transsulfuration pathway of homocysteine plays a number of pathophysiological roles. Hyperhomocysteinemia is involved in kidney fibrosis. However, the role of H₂S in kidney fibrosis remains to be defined. Here, we investigated the role of H₂S and its acting mechanism in unilateral ureteral obstruction (UO)-induced kidney fibrosis in mice. UO decreased expressions of CBS and CSE in the kidney with decrease of H₂S concentration. Treatment with sodium hydrogen sulfide (NaHS, a H₂S producer) during UO reduced UO-induced oxidative stress with preservations of catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) expression, and glutathione level. In addition, NaHS mitigated decreases of CBS and CSE expressions, and H₂S concentration in the kidney. NaHS treatment attenuated UO-induced increases in levels of TGF-β1, activated Smad3, and activated NF-κB. This study provided the first evidence of involvement of the transsulfuration pathway and H₂S in UO-induced kidney fibrosis, suggesting that H₂S and its transsulfuration pathway may be a potential target for development of therapeutics for fibrosis-related diseases.     

NMN recruits GSH to enhance GPX4-mediated ferroptosis defense in UV irradiation induced skin injury

Oxidative stress and lipid peroxidation are major causes of skin injury induced by ultraviolet (UV) irradiation. Ferroptosis is a form of regulated necrosis driven by iron-dependent peroxidation of phospholipids and contributes to kinds of tissue injuries. However, it remains unclear whether the accumulation of lipid peroxides in UV irradiation-induced skin injury could lead to ferroptosis. We generated UV irradiation-induced skin injury mice model to examine the accumulation of the lipid peroxides and iron. Lipid peroxides 4-HNE, the oxidative enzyme COX2, the oxidative DNA damage biomarker 8-OHdG, and the iron level were increased in UV irradiation-induced skin. The accumulation of iron and lipid peroxidation was also observed in UVB-irradiated epidermal keratinocytes without actual ongoing ferroptotic cell death. Ferroptosis was triggered in UV-irradiated keratinocytes stimulated with ferric ammonium citrate (FAC) to mimic the iron overload. Although GPX4 protected UVB-injured keratinocytes against ferroptotic cell death resulted from dysregulation of iron metabolism and the subsequent increase of lipid ROS, keratinocytes enduring constant UVB treatment were markedly sensitized to ferroptosis. Nicotinamide mononucleotide (NMN) which is a direct and potent NAD⁺ precursor supplement, rescued the imbalanced NAD⁺/NADH ratio, recruited the production of GSH and promoted resistance to lipid peroxidation in a GPX4-dependent manner. Taken together, our data suggest that NMN recruits GSH to enhance GPX4-mediated ferroptosis defense in UV irradiation-induced skin injury and inhibits oxidative skin damage. NMN or ferroptosis inhibitor might become promising therapeutic approaches for treating oxidative stress-induced skin diseases or disorders.     

Hydrogen sulfide induces human colon cancer cell proliferation: Role of Akt,ERK and p21

H₂S (hydrogen sulfide), regarded as the third gaseous transmitter, is implicated in ulcerative colitis and colorectal cancers. The present study investigates the effects of H₂S on cell proliferation in human colon cancer HCT 116 cells and SW480 cells. We identified the two key enzymes, CBS and CSE, for H₂S synthesis in HCT 116 cells. An exogenously administered H₂S donor NaHS induced cell proliferation in a concentration‐dependent manner, with optimal proliferative concentration at 200 μmol/l. NaHS administration increased Akt and ERK phosphorylation. Blockade of Akt and ERK activation attenuated NaHS‐induced cell proliferation. Cell‐cycle analysis showed that NaHS treatment for 6 h decreased the proportion of cells in G₀–G₁ phase and increased the proportion of cells in S phase. Protein expressions of Cyclin D1 and PCNA (proliferating cell nuclear antigen) were not altered, but the cyclin‐dependent kinase inhibitor p21^Waf1/Cip1 was inhibited significantly by NaHS treatment. NaHS significantly reduced NO metabolite levels. In conclusion, NaHS induced human colon cancer cell proliferation. This effect might be mediated by the increase of Akt and ERK phosphorylation and the decrease of p21^Waf1/Cip1 expression and NO production. The results suggested a role for H₂S in human colonic cancer development.     

Redox Modulation and Induction of Ferroptosis as a New Therapeutic Strategy in Hepatocellular Carcinoma

Ferroptosis, a newly discovered form of cell death mediated by reactive oxygen species (ROS) and lipid peroxidation, has recently been shown to have an impact on various cancer types; however, so far there are only few studies about its role in hepatocellular carcinoma (HCC). The delicate equilibrium of ROS in cancer cells has found to be crucial for cell survival, thus increased levels may trigger ferroptosis in HCC.In our study, we investigated the effect of different ROS modulators and ferroptosis inducers on a human HCC cell line and a human hepatoblastoma cell line. We identified a novel synergistic cell death induction by the combination of Auranofin and buthionine sulfoxime (BSO) or by Erastin and BSO at subtoxic concentrations. We found a caspase-independent, redox-regulated cell death, which could be rescued by different inhibitors of ferroptosis. Both cotreatments stimulated lipid peroxidation. All these findings indicated ferroptotic cell death. Both cotreatments affected the canonical ferroptosis pathway through GPX4 downregulation. We also found an accumulation of Nrf2 and HO-1, indicating an additional effect on the non-canonical pathway. Our results implicate that targeting these two main ferroptotic pathways simultaneously can overcome chemotherapy resistance in HCC.     

Oxygenated phosphatidylethanolamine navigates phagocytosis of ferroptotic cells by interacting with TLR2

During cancer therapy, phagocytic clearance of dead cells plays a vital role in immune homeostasis. The nonapoptotic form of cell death, ferroptosis, exhibits extraordinary potential in tumor treatment. However, the phagocytosis mechanism that regulates the engulfment of ferroptotic cells remains unclear. Here, we establish a novel pathway for phagocytic clearance of ferroptotic cells that is different from canonical mechanisms by using diverse ferroptosis models evoked by GPX4 dysfunction/deficiency. We identified the oxidized phospholipid, 1-steaoryl-2-15-HpETE-sn-glycero-3-phosphatidylethanolamine (SAPE-OOH), as a key eat-me signal on the ferroptotic cell surface. Enriching the plasma membrane with SAPE-OOH increased the efficiency of phagocytosis of ferroptotic cells by macrophage, a process that was suppressed by lipoprotein-associated phospholipase A₂. Ligand fishing, lipid blotting, and cellular thermal shift assay screened and identified TLR2 as a membrane receptor that directly recognized SAPE-OOH, which was further confirmed by TLR2 inhibitors and gene silencing studies. A mouse mammary tumor model of ferroptosis verified SAPE-OOH and TLR2 as critical players in the clearance of ferroptotic cells in vivo. Taken together, this work demonstrates that SAPE-OOH on ferroptotic cell surface acts as an eat-me signal and navigates phagocytosis by targeting TLR2 on macrophages.Subject terms: Cancer, Cancer microenvironment     

Hydrogen Sulfide Promotes Adipogenesis in 3T3L1 Cells

The effect of hydrogen sulfide (H₂S) on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H₂S was increased after 3T3L1 differentiation. The expression of the H₂S-synthesising enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2), a key regulator of this process, was increased by GYY4137 (a slow-releasing H₂S donor compound) and sodium hydrosulfide (NaHS, a classical H₂S donor) but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor), DL-propargylglycine (PAG, CSE inhibitor) as well as by CSE small interference RNA (siCSE) and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (β3-agonist) to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H₂S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H₂S modulate adipogenesis and adipocyte maturation.     

泛素化在调控铁死亡中的作用

铁死亡是一种由脂质过氧化驱动的铁依赖性的新的细胞死亡方式,越来越多的证据表明,铁死亡与各种病理状态有关,如神经退行性疾病、糖尿病肾病、癌症等,脂质过氧化驱动的铁死亡可能促进或抑制这些疾病的发生发展,细胞中抗氧化系统通过抑制脂质过氧化在抵抗铁死亡过程中发挥着重要作用。铁死亡的关键通路有以SLC7A11-GPX4为关键分子的氨基酸代谢通路、以铁蛋白或转铁蛋白为主的铁代谢通路,以及脂质代谢通路。铁死亡的发生受到细胞内蛋白质的调节,这些蛋白质会发生各种翻译后修饰,包括泛素化修饰。泛素-蛋白酶体系统（ubiquitin-proteasome system,UPS）是细胞内主要降解系统之一,通过酶促级联反应催化泛素分子标记待降解蛋白,随后由蛋白酶体识别并降解目标蛋白质。UPS根据其降解底物的不同在调节铁死亡的反应中发挥双重作用。UPS通过促进铁死亡关键分子（如SLC7A11、GPX4、GSH）以及抗氧化系统成分（如NRF2）的泛素化降解从而促进铁死亡,也可以通过促进脂质代谢通路中相关分子（如ACSL4、ALOX15）的泛素化降解从而抑制铁死亡。本综述介绍泛素化修饰在调控铁死亡进程中作用的最新研究进展,总结了已发表的关于E3泛素连接酶和去泛素酶调控铁死亡的研究,归纳了泛素连接酶、去泛素酶调控铁死亡的作用靶点,有助于确定人类疾病中新的预后指标,为这些疾病提供潜在的治疗策略。     

Cytoglobin promotes sensitivity to ferroptosis by regulating p53-YAP1 axis in colon cancer cells

Ferroptosis is an iron-dependent mode of non-apoptotic cell death characterized by accumulation of lipid reactive oxygen species (ROS). As a regulator of ROS, cytoglobin (CYGB) plays an important role in oxygen homeostasis and acts as a tumour suppressor. However, the mechanism by which CYGB regulates cell death is largely unknown. Here, we show that CYGB overexpression increased ROS accumulation and disrupted mitochondrial function as determined by the oxygen consumption rate and membrane potential. Importantly, ferroptotic features with accumulated lipid ROS and malondialdehyde were observed in CYGB-overexpressing colorectal cancer cells. Moreover, CYGB significantly increased the sensitivity of cancer cells to RSL3- and erastin-induced ferroptotic cell death. Mechanically, both YAP1 and p53 were significantly increased based on the RNA sequencing. The knock-down of YAP1 alleviated production of lipid ROS and sensitivity to ferroptosis in CYGB overexpressed cells. Furthermore, YAP1 was identified to be inhibited by p53 knock-down. Finally, high expression level of CYGB had the close correlation with key genes YAP1 and ACSL4 in ferroptosis pathway in colon cancer based on analysis from TCGA data. Collectively, our results demonstrated a novel tumour suppressor role of CYGB through p53-YAP1 axis in regulating ferroptosis and suggested a potential therapeutic approach for colon cancer.     

The dual role of the cystathionine γ-lyase/hydrogen sulfide pathway in CVB3-induced myocarditis in mice

The present study found that serum H₂S level, H₂S production rate, CSE mRNA and CSE protein levels were increased in CVB3-induced myocarditis. dl-proparglygylcine (PAG), an irreversible CSE inhibitor, decreased the infected myocardium titers on postinfection day 4, while NaHS, a H₂S donor, alleviated myocardial injury and necrosis, inflammatory cell infiltration and interstitial edema on postinfection day 10. These data reveal that the CSE/H₂S pathway is upregulated in the heart in a murine model of CVB3-induced myocarditis and that inhibition of endogenous H₂S is beneficial to treatment early in the disease while administration of exogenous H₂S is protective to infected myocardium during the later stage.     

Endogenous and exogenous hydrogen sulfide facilitates T-type calcium channel currents in Cav3.2-expressing HEK293 cells

Hydrogen sulfide (H₂S), a gasotransmitter, is formed from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE). We have shown that an H₂S donor, NaHS, causes hyperalgesia in rodents, an effect inhibited by knockdown of Ca_v3.2 T-type Ca²⁺ channels (T-channels), and that NaHS facilitates T-channel-dependent currents (T-currents) in NG108-15 cells that naturally express Ca_v3.2. In the present study, we asked if endogenous and exogenous H₂S participates in regulation of the channel functions in Ca_v3.2-transfected HEK293 (Ca_v3.2-HEK293) cells. dl-Propargylglycine (PPG), a CSE inhibitor, significantly decreased T-currents in Ca_v3.2-HEK293 cells, but not in NG108-15 cells. NaHS at 1.5 mM did not affect T-currents in Ca_v3.2-HEK293 cells, but enhanced T-currents in NG108-15 cells. In the presence of PPG, NaHS at 1.5 mM, but not 0.1–0.3 mM, increased T-currents in Ca_v3.2-HEK293 cells. Similarly, Na₂S, another H₂S donor, at 0.1–0.3 mM significantly increased T-currents in the presence, but not absence, of PPG in Ca_v3.2-HEK293 cells. Expression of CSE was detected at protein and mRNA levels in HEK293 cells. Intraplantar administration of Na₂S, like NaHS, caused mechanical hyperalgesia, an effect blocked by NNC 55-0396, a T-channel inhibitor. The in vivo potency of Na₂S was higher than NaHS. These results suggest that the function of Ca_v3.2 T-channels is tonically enhanced by endogenous H₂S synthesized by CSE in Ca_v3.2-HEK293 cells, and that exogenous H₂S is capable of enhancing Ca_v3.2 function when endogenous H₂S production by CSE is inhibited. In addition, Na₂S is considered a more potent H₂S donor than NaHS in vitro as well as in vivo.     

Cellular degradation systems in ferroptosis

In eukaryotic cells, macromolecular homeostasis requires selective degradation of damaged units by the ubiquitin-proteasome system (UPS) and autophagy. Thus, dysfunctional degradation systems contribute to multiple pathological processes. Ferroptosis is a type of iron-dependent oxidative cell death driven by lipid peroxidation. Various antioxidant systems, especially the system xc⁻-glutathione-GPX4 axis, play a significant role in preventing lipid peroxidation-mediated ferroptosis. The endosomal sorting complex required for transport-III (ESCRT-III)–dependent membrane fission machinery counteracts ferroptosis by repairing membrane damage. Moreover, cellular degradation systems play a dual role in regulating the ferroptotic response, depending on the cargo they degrade. The key ferroptosis repressors, such as SLC7A11 and GPX4, are degraded by the UPS. In contrast, the overactivation of selective autophagy, including ferritinophagy, lipophagy, clockophagy and chaperone-mediated autophagy, promotes ferroptotic death by degrading ferritin, lipid droplets, circadian proteins, and GPX4, respectively. Autophagy modulators (e.g., BECN1, STING1/TMEM173, CTSB, HMGB1, PEBP1, MTOR, AMPK, and DUSP1) also determine the ferroptotic response in a context-dependent manner. In this review, we provide an updated overview of the signals and mechanisms of the degradation system regulating ferroptosis, opening new horizons for disease treatment strategies.Subject terms: Cell biology, Molecular biology     

Liproxstatin-1 attenuates unilateral ureteral obstruction-induced renal fibrosis by inhibiting renal tubular epithelial cells ferroptosis

Renal fibrosis is a common pathological process that occurs with diverse etiologies in chronic kidney disease. However, its regulatory mechanisms have not yet been fully elucidated. Ferroptosis is a form of non-apoptotic regulated cell death driven by iron-dependent lipid peroxidation. It is currently unknown whether ferroptosis is initiated during unilateral ureteral obstruction (UUO)-induced renal fibrosis and its role has not been determined. In this study, we demonstrated that ureteral obstruction induced ferroptosis in renal tubular epithelial cells (TECs) in vivo. The ferroptosis inhibitor liproxstatin-1 (Lip-1) reduced iron deposition, cell death, lipid peroxidation, and inhibited the downregulation of GPX4 expression induced by UUO, ultimately inhibiting ferroptosis in TECs. We found that Lip-1 significantly attenuated UUO-induced morphological and pathological changes and collagen deposition of renal fibrosis in mice. In addition, Lip-1 attenuated the expression of profibrotic factors in the UUO model. In vitro, we used RSL3 treatment and knocked down of GPX4 level by RNAi in HK2 cells to induce ferroptosis. Our results indicated HK2 cells secreted various profibrotic factors during ferroptosis. Lip-1 was able to inhibit ferroptosis and thereby inhibit the secretion of the profibrotic factors during the process. Incubation of kidney fibroblasts with culture medium from RSL3-induced HK2 cells promoted fibroblast proliferation and activation, whereas Lip-1 impeded the profibrotic effects. Our study found that Lip-1 may relieve renal fibrosis by inhibiting ferroptosis in TECs. Mechanistically, Lip-1 could reduce the activation of surrounding fibroblasts by inhibiting the paracrine of profibrotic factors in HK2 cells. Lip-1 may potentially be used as a therapeutic approach for the treatment of UUO-induced renal fibrosis.Subject terms: Cell death, RNAi, Urinary tract obstruction