A combined nanotherapeutic approach targeting farnesoid X receptor, ferroptosis, and fibrosis for nonalcoholic steatohepatitis treatment.

Obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist with favorable effects on fatty and glucose metabolism, has been considered the leading candidate drug for nonalcoholic steatohepatitis (NASH) treatment. However, its limited effectiveness in resolving liver fibrosis and lipotoxicity-induced cell death remains a major drawback. Ferroptosis, a newly recognized form of cell death characterized by uncontrolled lipid peroxidation, is involved in the progression of NASH. Nitric oxide (NO) is a versatile biological molecule that can degrade extracellular matrix. In this study, we developed a PEGylated thiolated hollow mesoporous silica nanoparticles (MSN) loaded with OCA, as well as a ferroptosis inhibitor liproxsatin-1 and a NO donor S-nitrosothiol (ONL@MSN). Biochemical analyses, histology, multiplexed flow cytometry, bulk-tissue RNA sequencing, and fecal 16S ribosomal RNA sequencing were utilized to evaluate the effects of the combined nanoparticle (ONL@MSN) in a mouse NASH model. Compared with the OCA-loaded nanoparticles (O@MSN), ONL@MSN not only protected against hepatic steatosis but also greatly ameliorated fibrosis and ferroptosis. ONL@MSN also displayed enhanced therapeutic actions on the maintenance of intrahepatic macrophages/monocytes homeostasis, inhibition of immune response/lipid peroxidation, and correction of microbiota dysbiosis. These findings present a promising synergistic nanotherapeutic strategy for the treatment of NASH by simultaneously targeting FXR, ferroptosis, and fibrosis.

Alpha-synuclein interaction with mitochondria is the final mechanism of ferroptotic death induced by erastin in SH-SY5Y cells.

Ferroptosis has been characterized as a form of iron-dependent regulated cell death accompanied by an accumulation of reactive oxygen species and lipid oxidation products along with typical morphological alterations in mitochondria. Ferroptosis is activated by diverse triggers and inhibited by ferrostatin-1 and liproxstatin-1, apart from iron chelators and several antioxidants, and the process is implicated in multiple pathological conditions. There are, however, certain ambiguities about ferroptosis, especially regarding the final executioner of cell death subsequent to the accumulation of ROS. This study uses a typical inducer of ferroptosis such as erastin on SH-SY5Y cells, and shows clearly that ferroptotic death of cells is accompanied by the loss of mitochondrial membrane potential and intracellular ATP content along with an accumulation of oxidative stress markers. All these are prevented by ferrostatin-1 and liproxstatin-1. Additionally, cyclosporine A prevents mitochondrial alterations and cell death induced by erastin implying the crucial role of mitochondrial permeability transition pore (mPTP) activation in ferroptotic death. Furthermore, an accumulation of α-synuclein occurs during erastin induced ferroptosis which can be inhibited by ferrostatin-1 and liproxstatin-1. When the knock-down of α-synuclein expression is performed by specific siRNA treatment of SH-SY5Y cells, the mitochondrial impairment and ferroptotic death of the cells induced by erastin are markedly prevented. Thus, α-synuclein through the involvement of mPTP appears to be the key executioner protein of ferroptosis induced by erastin, but it needs to be verified if it is a generalized mechanism of ferroptosis by using other inducers and cell lines.

Baicalein triggers ferroptosis in colorectal cancer cells via blocking the JAK2/STAT3/GPX4 axis.

Colorectal cancer (CRC) is a prevalent form of gastrointestinal malignancy with challenges in chemotherapy resistance and side effects. Effective and low toxic drugs for CRC treatment are urgently needed. Ferroptosis is a novel mode of cell death, which has garnered attention for its therapeutic potential against cancer. Baicalein (5, 6, 7-trihydroxyflavone) is the primary flavone extracted from the dried roots of Scutellaria baicalensis that exhibits anticancer effects against several malignancies including CRC. In this study, we investigated whether baicalein induced ferroptosis in CRC cells. We showed that baicalein (1-64 µM) dose-dependently inhibited the viability of human CRC lines HCT116 and DLD1. Co-treatment with the ferroptosis inhibitor liproxstatin-1 (1 µM) significantly mitigated baicalein-induced CRC cell death, whereas autophagy inhibitor chloroquine (25 µM), necroptosis inhibitor necrostatin-1 (10 µM), or pan-caspase inhibitor Z-VAD-FMK (10 µM) did not rescue baicalein-induced CRC cell death. RNA-seq analysis confirmed that the inhibitory effect of baicalein on CRC cells is associated with ferroptosis induction. We revealed that baicalein (7.5-30 µM) dose-dependently decreased the expression levels of GPX4, key regulator of ferroptosis, in HCT116 and DLD1 cells by blocking janus kinase 2 (JAK2)/STAT3 signaling pathway via direct interaction with JAK2, ultimately leading to ferroptosis in CRC cells. In a CRC xenograft mouse model, administration of baicalein (10, 20 mg/kg, i.g., every two days for two weeks) dose-dependently inhibited the tumor growth with significant ferroptosis induced by inhibiting the JAK2/STAT3/GPX4 axis in tumor tissue. This study demonstrates that ferroptosis contributes to baicalein-induced anti-CRC activity through blockade of the JAK2/STAT3/GPX4 signaling pathway, which provides evidence for the therapeutic application of baicalein against CRC.

Liproxstatin-1 Alleviated Ischemia/Reperfusion-Induced Acute Kidney Injury via Inhibiting Ferroptosis.

Ferroptosis, as a novel regulable cell death, is characterized by iron overload, glutathione depletion, and an accumulation of lipid peroxides. Recently, it has been discovered that ferroptosis is involved in ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) and plays a crucial role in renal tubular cell death. In this study, we tried to investigate the effect and mechanism of liproxstatin-1 (Lip-1) in I/R-induced AKI and seek the key regulator of ferroptosis in I/R-induced AKI. Mice were administrated with clamping bilateral renal pedicles for 30 min. We found that early growth response 1 (EGR1) might be a key regulator of ferroptosis, and Lip-1 could suppress ferroptosis via EGR1. Meanwhile, Lip-1 could reduce macrophage recruitment and the release of inflammatory cytokines. These findings indicated that Lip-1 alleviated I/R-induced AKI via regulating EGR1, and it might pave the theoretical basis of a new therapeutic strategy for I/R-induced AKI.

Liproxstatin-1 alleviates cartilage degradation by inhibiting chondrocyte ferroptosis in the temporomandibular joint.

BGROUND INFORMATION: Ferroptosis contributes to temporomandibular joint osteoarthritis (TMJOA) lesion development and is still poorly understood. RESULTS: In this study, we used different TMJOA animal models to examine whether ferroptosis was related to disease onset in TMJOA induced by monosodium iodoacetate (MIA), IL-1ß, occlusion disorder (OD), and unilateral anterior crossbite (UAC). Immunohistochemical staining and Western blot analysis were used to detect ferroptosis- and cartilage degradation-related protein expression. Our results revealed reduced levels of the ferroptosis-related protein GPX4 in the cartilage layer, but the levels of ACSL4 and P53 were increased in the condyle. Injection of the ferroptosis inhibitor liproxstatin-1 (Lip-1) effectively decreased ACSL4, P53 and TRF expression. In vitro, IL-1ß reduced cartilage extracellular matrix expression in mandibular condylar chondrocytes (MCCs). Lip-1 maintained the morphology and function of mitochondria and ameliorated the exacerbation of lipid peroxidation and reactive oxygen species (ROS) production induced by IL-1ß. CONCLUSION: These results suggest that chondrocyte ferroptosis plays an important role in the development and progression of TMJOA. SIGNIFICANCE: Inhibiting condylar chondrocyte ferroptosis could be a promising therapeutic strategy for TMJOA.

Ferroptosis mediates airway epithelial E-cadherin dysfunction in LPS-induced acute lung injury.

BACKGROUND: Loss of E-cadherin in the airway epithelial cells is a critical contributor to the development of ALI/ARDS. Yet the underlying mechanisms are largely unknown. Increasing evidences have revealed the significance of ferroptosis in the pathophysiological process of ALI/ARDS. The aim of this study was to investigate the role of ferroptosis in dysregulation of airway epithelial E-cadherin in ALI/ARDS. METHODS: BALB/c mice were subjected to intratracheal instillation of lipopolysaccharide (LPS) to establish an ALI model. Two inhibitors of ferroptosis, liproxstatin-1 (Lip-1, at the dose of 10 mg/kg and 30 mg/kg) and ferrostatin-1 (Fer-1, at the dose of 1 mg/kg and 5 mg/kg), were respectively given to the mice through intraperitoneal injection after LPS challenge. The expression of ferroptotic markers, full-length E-cadherin and soluble E-cadherin (sE-cadherin) were both detected. RESULTS: LPS exposure dramatically down-regulated pulmonary expression of E-cadherin in mice, with profound loss of membrane E-cadherin in the airway epithelial cells and increased secretion of sE-cadherin in the airway lumen. At the same time, we found that the mitochondrial of airway epithelial cells in LPS-exposed mice exhibited significant morphological alterations that are hallmark features of ferroptosis, with smaller volume and increased membrane density. Other makers of ferroptosis were also detected, including increased cytoplasmic levels of iron and lipid peroxidates (MDA), as well as decreased GPX4 expression. 30 mg/kg of Lip-1 not only showed potent protective effects against the LPS-induced injury, inflammation, edema of the lung in those mice, but also rescued airway epithelial E-cadherin expression and decreased the release of sE-cadherin through inhibiting ferroptosis. While no noticeable changes induced by LPS were observed in mice treated with Lip-1 at 10 mg/kg nor Fer-1 at 1 mg/kg or 5 mg/kg. CONCLUSIONS: Taken together, these data demonstrated that ferroptosis mediates airway epithelial E-cadherin dysfunction in LPS-induced ALI.

Ferroptosis inhibition protects vascular endothelial cells and maintains integrity of the blood-spinal cord barrier after spinal cord injury.

Maintaining the integrity of the blood-spinal cord barrier is critical for the recovery of spinal cord injury. Ferroptosis contributes to the pathogenesis of spinal cord injury. We hypothesized that ferroptosis is involved in disruption of the blood-spinal cord barrier. In this study, we administered the ferroptosis inhibitor liproxstatin-1 intraperitoneally after contusive spinal cord injury in rats. Liproxstatin-1 improved locomotor recovery and somatosensory evoked potential electrophysiological performance after spinal cord injury. Liproxstatin-1 maintained blood-spinal cord barrier integrity by upregulation of the expression of tight junction protein. Liproxstatin-1 inhibited ferroptosis of endothelial cell after spinal cord injury, as shown by the immunofluorescence of an endothelial cell marker (rat endothelium cell antigen-1, RECA-1) and ferroptosis markers Acyl-CoA synthetase long-chain family member 4 and 15-lipoxygenase. Liproxstatin-1 reduced brain endothelial cell ferroptosis in vitro by upregulating glutathione peroxidase 4 and downregulating Acyl-CoA synthetase long-chain family member 4 and 15-lipoxygenase. Furthermore, inflammatory cell recruitment and astrogliosis were mitigated after liproxstatin-1 treatment. In summary, liproxstatin-1 improved spinal cord injury recovery by inhibiting ferroptosis in endothelial cells and maintaining blood-spinal cord barrier integrity.

Activated AMPK mitigates diabetes-related cognitive dysfunction by inhibiting hippocampal ferroptosis.

Clinical and preclinical interest in Type 2 diabetes (T2D)-associated cognitive dysfunction (TDACD) has grown in recent years. However, the precise mechanisms underlying TDACD need to be further elucidated. Ferroptosis was reportedly involved in neurodegenerative diseases and diabetes-related organ injuries; however, its role in TDACD remains elusive. In this study, mice fed with a high-fat-diet combined with streptozotocin (HFD-STZ) were used as a T2D model to assess the role of ferroptosis in cognitive dysfunction. We found that ferroptosis was mainly activated in hippocampal neurons but not in microglia or astrocytes. Accordingly, increased levels of transferrin receptor and decreased levels of ferritin, GPX4, and SLC7A11 were observed in hippocampal neurons. In addition, pre-treatment with liproxstatin-1, a ferroptosis inhibitor, attenuated iron accumulation and oxidative stress response, which resulted in improved cognitive function in the HFD-STZ group. Furthermore, we found that p-AMP-activated protein kinase (AMPK) was decreased in the HFD-STZ group. Pre-treatment with AMPK agonist increased the expression of AMPK and GPX4, but decreased lipocalin 2 (LCN2) in the hippocampus that resulted in improved spatial learning ability in the HFD-STZ group. Taken together, we found that activation of neuronal ferroptosis in the hippocampus contributed to cognitive impairment of HFD-STZ mice. Furthermore, AMPK activation may reduce hippocampal ferroptosis, and consequently improve cognitive performance in diabetic mice.

A novel 3-phenylglutaric acid derivative (84-B10) alleviates cisplatin-induced acute kidney injury by inhibiting mitochondrial oxidative stress-mediated ferroptosis.

Cisplatin is one of the most effective chemotherapy drugs and is widely used for cancer treatment. However, its clinical use is limited by nephrotoxicity. Emerging findings suggested that both ferroptosis and mitochondrial dysfunction mediate cisplatin-induced nephrotoxicity. In the current study, a novel 3-phenylglutaric acid derivative 5-[[2-(4-methoxyphenoxy)-5-(trifluoromethyl)phenyl]amino]-5-oxo-3-phenylpentanoic acid (referred to as 84-B10) was found to play a protective role in cisplatin-induced acute kidney injury with no tumor promoting effects. A genome-wide transcriptome analysis indicated that the protective effect of 84-B10 might be dependent on antagonizing ferroptosis. In accordance, lipid peroxide accumulation and downregulation of key ferroptosis suppressors were reversed using 84-B10 treatment both in vivo and in vitro. In addition, 84-B10 inhibited cisplatin-induced mitochondrial damage and mitochondrial reactive oxygen species (mtROS) production and restored superoxide dismutases (SODs). Furthermore, 84-B10 showed similar therapeutic effects to MnTBAP (a cell-permeable SOD mimetic) in eliminating mtROS, restoring mitochondrial homeostasis, and inhibiting ferroptosis under cisplatin challenge. Comparable effects of 84-B10 and liproxstatin-1 in ameliorating cisplatin-induced ferroptosis were observed. However, liproxstatin-1 failed to prevent mitochondrial dysfunction. These data indicated that mtROS might act upstream of cisplatin-induced tubular ferroptosis. Taken together, the novel 3-phenylglutaric acid derivative 84-B10 showed therapeutic potential against cisplatin-induced nephrotoxicity possibly by restoring mitochondria homeostasis and inhibiting mtROS-induced ferroptosis, which suggests the potential use of 84-B10 in preventing and treating cisplatin-nephrotoxicity.

Intrathecal liproxstatin-1 delivery inhibits ferroptosis and attenuates mechanical and thermal hypersensitivities in rats with complete Freund's adjuvant-induced inflammatory pain.

Previous studies have confirmed the relationship between iron-dependent ferroptosis and a peripheral nerve injury-induced neuropathic pain model. However, the role of ferroptosis in inflammatory pain remains inconclusive. Therefore, we aimed to explore whether ferroptosis in the spinal cord and dorsal root ganglion contributes to complete Freund's adjuvant (CFA)-induced painful behaviors in rats. Our results revealed that various biochemical and morphological changes were associated with ferroptosis in the spinal cord and dorsal root ganglion tissues of CFA rats. These changes included iron overload, enhanced lipid peroxidation, disorders of anti-acyl-coenzyme A synthetase long-chain family member 4 and glutathione peroxidase 4 levels, and abnormal morphological changes in mitochondria. Intrathecal treatment of liproxstatin-1 (a ferroptosis inhibitor) reversed these ferroptosis-related changes and alleviated mechanical and thermal hypersensitivities in CFA rats. Our study demonstrated the occurrence of ferroptosis in the spinal cord and dorsal root ganglion tissues in a rodent model of inflammatory pain and indicated that intrathecal administration of ferroptosis inhibitors, such as liproxstatin-1, is a potential therapeutic strategy for treating inflammatory pain.

Liproxstatin­1 induces cell cycle arrest, apoptosis, and caspase­3/GSDME­dependent secondary pyroptosis in K562 cells.

Leukemia is a fatal hematopoietic disorder with a poor prognosis. Drug resistance is inevitable after the long­term use of chemotherapeutic agents. Liproxstatin­1, commonly known as a ferroptosis inhibitor, has never been reported to have anticancer effects. In the present study, the antileukemic role of liproxstatin­1 in K562 leukemia cells was investigated. Liproxstatin­1 inhibited K562 cell proliferation in a dose­ and time­dependent manner. RNA sequencing revealed several pathways that were affected by liproxstatin­1, such as the G1/S transition of the mitotic cell cycle and extrinsic or intrinsic apoptotic signaling pathways. The results of flow cytometry indicated that liproxstatin­1 arrests the cell cycle at the G1 phase, and even at the G2/M phase. p21WAF1/CIP1, a cyclin­dependent kinase inhibitor, was upregulated. It was also determined that liproxstatin­1 induced BAX and TNF­α expression, which was accompanied by cleavage of caspase­3 and PARP. The caspase­3­specific inhibitor z­DEVD­FMK rescued some of the apoptotic cells. Interestingly, K562 cells were characterized by swelling and plasma membrane rupture when treated with a high concentration of liproxstatin­1, which was inconsistent with the typical apoptotic appearance. Thus, it was hypothesized that apoptosis­mediated pyroptosis occurs during liproxstatin­1­induced cell death. The expression of the hallmark of pyroptosis, the cleaved N­terminal GSDME, increased. Additionally, it was observed that endoplasmic reticulum stress and autophagy were involved in liproxstatin­1­induced cell death. Collectively, liproxstatin­1 induced cell cycle arrest, apoptosis, and caspase­3/GSDME­dependent secondary pyroptosis in K562 leukemia cells, which provides new hope for the treatment of leukemia.

A novobiocin derivative, XN4, triggers ferroptosis in gastric cancer cells via the activation of NOX4.

CONTEXT: A novobiocin derivative, XN4, has been shown to promote cell apoptosis in chronic myeloid leukaemia. OBJECTIVE: This study explores the mechanism by which XN4 promotes ferroptosis of gastric cancer (GC) cells. MATERIALS AND METHODS: Human GC SGC-7901 and BGC-823 cells were treated with different XN4 concentrations (0, 0.1, 0.5, 1.0, 5.0, and 10.0 µmol/L) to evaluate effects of XN4. Additionally, cells were pre-treated for 24 h with si-NOX4, for 1 h with the iron chelator deferoxamine mesylate (DFO) or for 1 h with the lipid peroxidation inhibitor liproxstatin-1 before being treated with XN4 to analyse the mechanism of XN4. RESULTS: XN4 increased cell death (IC50 values of XN4 on SGC-7901 and BGC-823 cells: 1.592 ± 0.14 µmol/L and 2.022 ± 0.19 µmol/L) and Fe2+ levels in SGC-7901 and BGC-823 cells. These effects of 2.0 µmol/L XN4 were abolished by 100 µmol/L DFO treatment. XN4 enhanced transferrin and transferrin receptor expression to induce Fe2+ accumulation. XN4 decreased mitochondrial membrane potentials in GC cells, similar to erastin. Additionally, XN4 increased MDA, hydrogen peroxide, and ROS levels, but diminished total glutathione levels. Liproxstatin-1 (200 nmol/L) nullified the effects of XN4 (2.0 µmol/L) on MDA levels and cell death. Moreover, GPX4 levels decreased, but NOX4 and ferroptosis-related protein PTGS2 levels increased in GC cells following XN4 treatment, which was nullified by NOX4 knockdown. DISCUSSION AND CONCLUSIONS: The pro-ferroptotic role of XN4 in GC might enable it to become a promising drug for GC treatment in the future despite the need for extensive research.

Targeting Ferroptosis as a Novel Approach to Alleviate Aortic Dissection.

A variety of programmed cell death types have been shown to participate in the loss of smooth muscle cells (SMCs) during the development of aortic dissection (AD), but it is still largely unclear whether ferroptosis is involved in the development of AD. In the present study, we found that the expression of key ferroptosis regulatory proteins, solute carrier family 7 member 11 (SLC7A11), ferroptosis suppressor protein 1 (FSP1) and glutathione peroxidase 4 (GPX4) were downregulated in aortas of Stanford type A AD (TAAD) patients, and liproxstatin-1, a specific inhibitor of ferroptosis, obviously abolished the ß-aminopropionitrile (BAPN)-induced development and rupture of AD in mice. Furthermore, the expression of methyltransferase-like 3 (METTL3), a major methyltransferase of RNA m6A, was remarkably upregulated in the aortas of TAAD patients, and the protein levels of METTL3 were negatively correlated with SLC7A11 and FSP1 levels in human aortas. Overexpression of METTL3 in human aortic SMCs (HASMCs) inhibited, while METTL3 knockdown promoted SLC7A11 and FSP1 expression. More importantly, overexpression of METTL3 facilitated imidazole ketone erastin- and cystine deprivation-induced ferroptosis, while knockdown of METTL3 repressed ferroptosis of HASMCs. Overexpression of either SLC7A11 or FSP1 largely abrogated the effect of METTL3 on HASMC ferroptosis. Therefore, we have revealed that ferroptosis is a critical cause of AD in both humans and mice and that METTL3 promotes ferroptosis of HASMCs by inhibiting the expression of SLC7A11 and FSP1. Thus, targeting ferroptosis or m6A RNA methylation is a potential novel strategy for the treatment of AD.

Liproxstatin-1 alleviates LPS/IL-13-induced bronchial epithelial cell injury and neutrophilic asthma in mice by inhibiting ferroptosis.

BACKGROUND AND PURPOSE: Ferroptosis is closely associated with respiratory diseases; however, the relationship between ferroptosis and neutrophilic asthma remains unknown. This study investigated whether Liproxstatin-1 (Lip-1) affects the progression of neutrophilic asthma by inhibiting ferroptosis and inflammatory response, while dissecting the underlying molecular mechanisms. METHODS: The bronchial epithelial cells (16HBE and BEAS-2B) were administered with lipopolysaccharide (LPS) and interleukin-13 (IL-13) to generate a cell injury model. This cell model was employed to examine the effect of Lip-1 on airway epithelial-associated inflammation and ferroptosis as well as the underlying molecular mechanism. Meanwhile, we evaluated the effects of Lip-1 on neutrophilic asthma and ferroptosis by using the ovalbumin (OVA)/LPS-induced mouse model. RESULTS: Lip-1 reversed the altered expression of ferroptotic regulators (glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11) and prostaglandin-endoperoxide synthase 2 (PTGS2)), attenuated lipid reactive oxygen species (lipid ROS) and ameliorated cell viability in HBE and BEAS-2B cells administered with LPS and IL-13. Moreover, Lip-1 treatment led to a marked reduction in the expression of IL-33, TSLP, IL-8, IL-6, and HMGB1 in the HBE and BEAS-2B cells. In the meantime, administration with Lip-1 markedly relieved OVA/LPS-induced neutrophilic asthma, as indicated by significant improvement in lung pathological changes, airway mucus secretion, inflammation, and ferroptosis. CONCLUSION: This study provides data suggesting that Lip-1 alleviates neutrophilic asthma in vivo and in vitro through inhibiting ferroptosis, perhaps providing a new strategy for neutrophilic asthma treatment.

In Vivo Assessment of Ferroptosis and Ferroptotic Stress in Mice.

Ferroptosis is iron-dependent, lipid peroxidation-driven, regulated cell death that is triggered when cellular glutathione peroxidase 4 (GPX4)-mediated cellular defense is insufficient to prevent pathologic accumulation of toxic lipid peroxides. Ferroptosis is implicated in various human pathologies, including neurodegeneration, chemotherapy-resistant cancers, ischemia-reperfusion injury, and acute and chronic kidney diseases. Despite the fact that the ferroptotic process has been rigorously interrogated in multiple preclinical models, the lack of specific and readily available biomarkers to detect ferroptosis in vivo in mouse models makes it challenging to delineate its contribution to key pathologic events in vivo. Critical steps to practically evaluate ferroptosis include, but are not limited to, detecting increased cell death and pathologic accumulation of toxic lipid peroxides and testing augmentation of observed pathologic events by genetic inhibition of the glutathione-GPX4 axis or mitigation of the pathologic process by ferroptosis inhibitors. Here, we describe methods to evaluate these key features of the ferroptotic process in mice in vivo. Specifically, we describe methods to detect toxic lipid peroxides (4-hydroxynonenal) and cell death (based on terminal deoxynucleotidyl transferase dUTP nick end labeling staining) as well as a protocol to pharmacologically inhibit ferroptotic stress using liproxstatin-1. These protocols provide tools for understanding the ferroptotic process in mouse genetic or disease models. © 2022 Wiley Periodicals LLC. Basic Protocol 1: How to use liproxstatin-1 Basic Protocol 2: How to evaluate ferroptosis in mouse kidneys.

Nuclear receptor coactivator 4-mediated ferritinophagy contributes to cerebral ischemia-induced ferroptosis in ischemic stroke.

Ischemic stroke poses a significant health risk due to its high rate of disability and mortality. To address this problem, several therapeutic approaches have been proposed, including interruption targeting programmed cell death (PCD). Ferroptosis is a newly defined PCD characterized by iron-dependent accumulation of lipid peroxidation, and is becoming a promising target for treating numerous diseases. To explore the underlying mechanisms of the initiation and execution of ferroptosis in ischemic stroke, we established stroke models in vivo and in vitro simulating ischemia/reperfusion (I/R) neuronal injury. Different from previous reports on stroke, we tested ferroptosis by measuring the levels of core proteins, such as ACSL4, 15-LOX2, Ferritin and GPX4. In addition, I/R injury induces excessive degradation of ferritin via the autophagy pathway and subsequent increase of free iron in neurons. This phenomenon has recently been termed ferritinophagy and reported to be regulated by nuclear receptor coactivator 4 (NCOA4) in some cell lines. Increased NCOA4 in cytoplasm was detected in our study and then silenced by shRNA to investigate its function. Both in vivo and in vitro, NCOA4 deletion notably abrogated ferritinophagy caused by I/R injury and thus inhibited ferroptosis. Furthermore, we found that NCOA4 was upregulated by ubiquitin specific peptidase 14 (USP14) via a deubiquitination process in damaged neurons, and we found evidence of pharmacological inhibition of USP14 effectively reducing NCOA4 levels to protect neurons from ferritinophagy-mediated ferroptosis. These findings suggest a novel and effective target for treating ischemic stroke.

Targeting Ferroptosis against Ischemia/Reperfusion Cardiac Injury.

Ischemic heart disease is a leading cause of death worldwide. Primarily, ischemia causes decreased oxygen supply, resulting in damage of the cardiac tissue. Naturally, reoxygenation has been recognized as the treatment of choice to recover blood flow through primary percutaneous coronary intervention. This treatment is the gold standard therapy to restore blood flow, but paradoxically it can also induce tissue injury. A number of different studies in animal models of acute myocardial infarction (AMI) suggest that ischemia-reperfusion injury (IRI) accounts for up to 50% of the final myocardial infarct size. Oxidative stress plays a critical role in the pathological process. Iron is an essential mineral required for a variety of vital biological functions but also has potentially toxic effects. A detrimental process induced by free iron is ferroptosis, a non-apoptotic type of programmed cell death. Accordingly, efforts to prevent ferroptosis in pathological settings have focused on the use of radical trapping antioxidants (RTAs), such as liproxstatin-1 (Lip-1). Hence, it is necessary to develop novel strategies to prevent cardiac IRI, thus improving the clinical outcome in patients with ischemic heart disease. The present review analyses the role of ferroptosis inhibition to prevent heart IRI, with special reference to Lip-1 as a promising drug in this clinicopathological context.

Liproxstatin-1 alleviates bleomycin-induced alveolar epithelial cells injury and mice pulmonary fibrosis via attenuating inflammation, reshaping redox equilibrium, and suppressing ROS/p53/α-SMA pathway.

With undetermined etiology and limited treatment option, idiopathic pulmonary fibrosis (IPF) an age related disease is extremely lethal. Persistent injury of epithelial cells, abnormal activation of fibroblasts/myofibroblasts, and superabundant deposition of extracellular matrix protein pathologically characterize IPF. Redox imbalance is reported to play a vital role in both IPF development and senescence. This study aim to investigate whether and how Liproxstatin-1 (Lip-1), a strong lipid autoxidation inhibitor, regulates bleomycin (BLM) induced pulmonary fibrosis both in vivo and in vitro. It's demonstrated that Lip-1 exerted a potent anti-fibrotic function in BLM-induced mice pulmonary fibrosis via alleviating inflammatory, reshaping redox equilibrium, and ameliorating collagen deposition. Lip-1 reduced the level of reactive oxygen species (ROS) and methane dicarboxylic aldehyde (MDA), promoted the expression of glutathione (GSH), catalase (CAT), and total superoxide dismutase (T-SOD) after BLM treatment. Moreover, in vitro experiments verified that Lip-1 protected A549 cells from BLM-induced injury and fibrosis. Lip-1 seemed to attenuate BLM-induced fibrosis by targeting ROS/p53/α-SMA signaling both in vivo and in vitro. In summary, this study demonstrates that Lip-1 administration performs a protective role in against pulmonary fibrosis and lights up the potential of Lip-1 treatment for patient with IPF in future.

Selective Ferroptosis Inhibitor Liproxstatin-1 Attenuates Neurological Deficits and Neuroinflammation After Subarachnoid Hemorrhage.

Ferroptosis is a form of iron-dependent regulated cell death. Evidence of its existence and the effects of its inhibitors on subarachnoid hemorrhage (SAH) is still lacking. In the present study, we found that liproxstatin-1 protected HT22 cells against hemin-induced injury by protecting mitochondrial functions and ameliorating lipid peroxidation. In in vivo experiments, we demonstrated the presence of characteristic shrunken mitochondria in ipsilateral cortical neurons after SAH. Moreover, liproxstatin-1 attenuated the neurological deficits and brain edema, reduced neuronal cell death, and restored the redox equilibrium after SAH. The inhibition of ferroptosis by liproxstatin-1 was associated with the preservation of glutathione peroxidase 4 and the downregulation of acyl-CoA synthetase long-chain family member 4 as well as cyclooxygenase 2. In addition, liproxstatin-1 decreased the activation of microglia and the release of IL-6, IL-1ß, and TNF-α. These data enhance our understanding of cell death after SAH and shed light on future preclinical studies.