The purpose of this study was to clarify the effect of Huotan Jiedu Tongluo Decoction on atherosclerosis(AS) injury in ApoE~(-/-) mice by regulating the ferroptosis pathway. Seventy-five ApoE~(-/-) mice were randomly divided into model group, low-, medium-, and high-dose of Huotan Jiedu Tongluo Decoction groups, and evolocumab group(n=15), and 15 C57BL/6J mice were selected as the blank group. Mice in the blank group were fed with a normal diet, and those in the other groups were fed with a high-fat diet to induce AS. From the 9th week, mice in Huotan Jiedu Tongluo Decoction groups were administrated with Huotan Jiedu Tongluo Decoction at corresponding doses by gavage, and those in the blank group and the model group were given an equal volume of distilled water. Mice in the evolocumab group were treated with evolocumab 18.2 mg·kg~(-1 )by subcutaneous injection every 2 weeks. After 8 weeks of continuous intervention, oil red O staining and hematoxylin-eosin(HE) staining were employed to observe the lipid deposition and plaque formation in the aortic root. Masson staining was used to evaluate the collagen content in the aortic root. The serum levels of total cholesterol(TC), triglycerides(TG), high-density lipoprotein cholesterol(HDL-C), and low-density lipoprotein cholesterol(LDL-C) were determined by biochemical kits. The levels of Fe~(2+), superoxide dismutase(SOD), malondialdehyde(MDA), and glutathione(GSH) in the aorta were measured by colorimetry. The protein and mRNA levels of nuclear factor erythroid 2-related factor 2(Nrf2), glutathione peroxidase 4(GPX4), solute carrier family 7 member 11(SLC7A11), and acyl-CoA synthetase long chain family member 4(ACSL4) in the aorta were detected by Western blot and RT-qPCR, respectively. The expression of Nrf2, GPX4, and SLC7A11 was localized by immunofluorescence. The results showed that low-, medium-, and high-dose Huotan Jiedu Tongluo Decoction reduced the plaque formation of aortic root and increased the collagen content in AS mice. At the same time, Huotan Jiedu Tongluo Decoction improved the lipid metabolism by lowering the levels of TC, LDL-C, and TG and elevating the level of HDL-C in the serum. Huotan Jiedu Tongluo Decoction enhanced the antioxidant capacity by elevating the levels of GSH and SOD and lowering the level of MDA in the aorta and inhibiting the accumulation of Fe~(2+) in the aorta. In addition, Huotan Jiedu Tongluo Decoction up-regulated the protein and mRNA levels of Nrf2, GPX4, and SLC7A11, while down-regulating the protein and mRNA levels of ACSL4. In summary, Huotan Jiedu Tongluo Decoction can effectively alleviate AS lesions in ApoE~(-/-) mice by activating the Nrf2/GPX4 pathway, reducing lipid peroxidation, and inhibiting ferroptosis.
Animals ; Ferroptosis/drug effects* ; Atherosclerosis/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; NF-E2-Related Factor 2/genetics* ; Mice ; Mice, Inbred C57BL ; Apolipoproteins E/metabolism* ; Male ; Phospholipid Hydroperoxide Glutathione Peroxidase/genetics* ; Signal Transduction/drug effects* ; Humans ; Mice, Knockout

Objective To explore the role and mechanism of mammalian target of rapamycin (mTOR) in oxidized low-density lipoprotein (oxLDL)-induced ferroptosis in vascular smooth muscle cells (VSMCs). Methods A model of oxLDL-induced VSMC ferroptosis was established. VSMCs were co-treated with either the mTOR inhibitor rapamycin or the autophagy inducer carbonyl cyanide m-chlorophenylhydrazone (CCCP), followed by detection of autophagy and ferroptosis-related indexes. Quantitative real-time PCR and Western blot were used respectively to analyze the expression of mTOR, glutathione peroxidase 4 (GPX4), sequestosome 1 (p62), and microtubule-associated protein 1 light chain 3 (LC3). Flow cytometry was employed to assess VSMC death. C11 BODIPY fluorescent staining was used to measure cellular lipid peroxidation levels. Colorimetric assays were performed to determine the contents of malondialdehyde (MDA), ferrous ion (Fe²⁺) and glutathione (GSH). Results oxLDL significantly upregulated mTOR expression in VSMCs, while increasing p62 expression and reducing LC3 expression, thereby suppressing VSMC autophagy. Compared with oxLDL treatment alone, rapamycin co-treatment reversed oxLDL-induced VSMC ferroptosis, as characterized by reduced VSMC death, increased GPX4 expression and GSH contents, along with decreased MDA content, Fe²⁺ content and lipid peroxidation levels. Similarly, CCCP co-treatment activated autophagy characterized by reduced p62 expression and elevated LC3 expression, which subsequently alleviated oxLDL-induced ferroptosis, showing reduced VSMC death, increased GPX4 expressions and GSH contents, and decreased MDA content, Fe²⁺ content and lipid peroxidation levels. Moreover, mTOR inhibition by rapamycin significantly reversed the oxLDL-induced upregulation of p62 and downregulation of LC3. Conclusion mTOR may promote oxLDL-induced VSMC ferroptosis by suppressing autophagy.
Ferroptosis/drug effects* ; Lipoproteins, LDL/metabolism* ; TOR Serine-Threonine Kinases/physiology* ; Autophagy/drug effects* ; Muscle, Smooth, Vascular/metabolism* ; Animals ; Rats ; Myocytes, Smooth Muscle/cytology* ; Cells, Cultured ; Lipid Peroxidation/drug effects* ; Sequestosome-1 Protein/genetics* ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism* ; Microtubule-Associated Proteins/genetics* ; Sirolimus/pharmacology*

OBJECTIVE: To investigate the effect of piperlongumine(PL) on the proliferation and ferroptosis of human adriamycin-resistant chronic myeloid leukemia K562/ADR cells, and to explore its possible molecular mechanism. METHODS: CCK-8 assay was used to detect the effect of PL on the survival rate of K562/ADR cells and to screen the appropriate drug concentration. After K562/ADR cells were treated with low, medium and high concentrations of PL(2, 4, and 6 μmol/L), EdU proliferation assay and plate colony formation assay were used to detect cell proliferation and colony formation ability. CCK-8 assay was used to detect the effects of different inhibitors (Fer-1, Z-VAD, Nec-1) combined with PL on cell proliferation. The intracellular Fe²⁺, ROS, malondialdehyde(MDA) and glutathine(GSH) contents were respectively detected by iron ion colorimetry, DCFH-DA fluorescent probe, MDA and GSH kits. RT-qPCR and Western blot were respectively used to detect the expression level of GPX4 mRNA and protein in cells. Bioinformatics websites predicted miRNA that could target and regulate GPX4 . RT-qPCR was used to detect the effects of different concentrations of PL on the expression levels of the predicted miRNA. Dual luciferase gene reporter assay was used to verify the targeting relationship between miR-214-3p and GPX4 . After treating cells with PL or PL+miR-214-3p inhibitor, the Fe²⁺, ROS, MDA, GSH centents and GPX4 protein expression levels in cells were detected. RESULTS: PL inhibited K562/ADR cell proliferation in a concentration-dependent manner（r =0.979）. Compared with the blank control group, the survival rate, EdU positive cells rate in low, medium and high concentration PL groups were significantly decreased (P < 0.01). Compared with the PL group alone, the survival rate of cells in the Z-VAD+PL group was increased slightly (P < 0.05). The cell survival rate was significantly increased in medium or high concentration PL+Fer-1 group (P < 0.01). Compared with blank control group, ROS expression level in low concentration PL group was slightly increased (P < 0.05), and GSH content was slightly decreased (P < 0.05). In medium and high concentration PL groups, the contents of Fe²⁺, ROS and MDA were significantly increased (P < 0.01), while the contents of GSH, expression of GPX4 mRNA and protein were significantly decreased(P < 0.01). Bioinformatics prediction and double luciferase reporter gene experiment confirmed the targeting relationship between GPX4 and miR-214-3p. Compared with the blank control group, the expression level of miR-214-3p in cells of medium and high concentration PL groups was significantly increased (P < 0.01). Compared with PL group alone, the intracellular Fe²⁺, ROS and MDA contents in PL+miR-214-3p inhibitor group were all decreased (P < 0.01), while GSH content and GPX4 protein expression levels were significantly increased (P < 0.01). CONCLUSION Medium and high concentrations of PL can inhibit the proliferation of K562/ADR cells by inducing ferroptosis, which is related to the regulation of miR-214-3p pathway.
Humans ; Ferroptosis/drug effects* ; MicroRNAs/metabolism* ; Dioxolanes/pharmacology* ; Cell Proliferation/drug effects* ; K562 Cells ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Reactive Oxygen Species ; Doxorubicin/pharmacology* ; Signal Transduction ; Piperidones

OBJECTIVES: To investigate the protective effect of catalpol against triptolide-induced liver injury and explore its mechanism to test the Fuzheng Zhidu theory for detoxification. METHODS: C57BL/6J mice were randomized into blank control group, catalpol group, triptolide group and triptolide+catalpol group. After 13 days of treatment with the agents by gavage, the mice were examined for liver tissue pathology, liver function, hepatocyte subcellular structure, lipid peroxidation, ferrous ion deposition and expressions of ferroptosis-related proteins in the liver. In a liver cell line HL7702, the effect of catalpol or the ferroptosis inhibitor Fer-1 on triptolide-induced cytotoxicity was tested by examining cell functions, Fe²⁺ concentration, lipid peroxidation, ROS level and the ferroptosis-related proteins. RESULTS: In C57BL/6J mice, catalpol significantly alleviated triptolide-induced hepatic injury, lowered the levels of ALT, AST and LDH, and reversed the elevation of Fe²⁺ concentration and MDA level and the reduction of GP_X level. In HL7702 cells, inhibition of ferroptosis by Fer-1 significantly reversed triptolide-induced elevation of ALT, AST and LDH levels. Western blotting and qRT-PCR demonstrated that catalpol reversed abnormalities in expressions of SLC7A11, FTH1 and GPX4 at both the mRNA and protein levels in triptolide-treated HL7702 cells. CONCLUSIONS The combined use of catalpol can reduce the hepatotoxicity of triptolide in mice by inhibiting excessive hepatocyte ferroptosis through the SLC7A11/GPX4 pathway.
Animals ; Phenanthrenes/toxicity* ; Ferroptosis/drug effects* ; Diterpenes/toxicity* ; Epoxy Compounds/toxicity* ; Mice, Inbred C57BL ; Hepatocytes/metabolism* ; Mice ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Iridoid Glucosides/pharmacology* ; Liver/metabolism* ; Chemical and Drug Induced Liver Injury/prevention & control* ; Male ; Amino Acid Transport System y+/metabolism*

OBJECTIVES: To explore the mechanism of Shuangshu Decoction (SSD) for inhibiting growth of gastric cancer xenografts in nude mice. METHODS: Network pharmacology analysis was conducted to identify the common targets of SSD and gastric cancer cell ferroptosis, and bioinformatics analysis and molecular docking were used to validate the core targets. In the cell experiment, AGS cells were treated with SSD-medicated serum, Fer-1 (a ferroptosis inhibitor), or both, and the changes in cell viability, ferroptosis markers (ROS, Fe²⁺ and GSH), expressions of P53, SLC7A11 and GPX4, and mitochondrial morphology were examined. In a nude mouse model bearing gastric cancer xenografts, the effects of gavage with SSD, intraperitoneal injection of Fer-1, or their combination on tumor volume/weight, histopathology, and expressions of P53, SLC7A11 and GPX4 levels were evaluated. RESULTS: The active components in SSD (quercetin and wogonin) showed strong binding affinities to P53. In AGS cells, SSD treatment dose-dependently inhibited cell proliferation, increased ROS and Fe²⁺ levels, upregulated P53 expression, and downregulated the expressions of SLC7A11 and GPX4, but these effects were effectively attenuated by Fer-1 treatment. SSD also induced mitochondrial shrinkage and increased the membrane density, which were alleviated by Fer-1. In the tumor-bearing mouse models, gavage with SSD significantly reduced tumor size and weight, caused tumor cell necrosis, upregulated P53 and downregulated SLC7A11 and GPX4 expression in the tumor tissue, and these effects were obviously mitigated by Fer-1 treatment. CONCLUSIONS SSD inhibits gastric cancer growth in nude mice by inducing cell ferroptosis via the P53/SLC7A11/GPX4 axis.
Ferroptosis/drug effects* ; Animals ; Stomach Neoplasms/metabolism* ; Tumor Suppressor Protein p53/metabolism* ; Mice, Nude ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Drugs, Chinese Herbal/pharmacology* ; Humans ; Amino Acid Transport System y+/metabolism* ; Mice ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Xenograft Model Antitumor Assays

OBJECTIVES: To explore the mechanism of Gandou Fumu Decoction (GDFMD) for improving Wilson's disease (WD) in tx-J mice. METHODS: With 6 syngeneic wild-type mice as the control group, 30 tx-J mice were randomized into WD model group, low-, medium- and high-dose GDFMD treatment groups, and Fer-1 treatment group. Saline (in control and model groups) and GDFMD (3.48, 6.96 or 13.92 g/kg) were administered by gavage, and Fer-1 was injected intraperitoneally once daily for 14 days. Oil red and HE staining were used to observe lipid deposition and pathological conditions in the liver tissue; ALT, AST, albumin, AKP levels were determined to assess liver function of the mice. Western blotting and RT-qPCR were used to detect hepatic protein and mRNA expressions of GPX4, ACSL4, ALOX15, FTH1, FLT, TFR1, FAS, SCD1, and ACOX1, and Fe²⁺, MDA, ROS, SOD, GSH and 4-HNE levels were analyzed to assess oxidative stress. RESULTS: The mouse models of WD showed obvious fatty degeneration in the liver tissue significantly increased serum levels of ALT, AST and AKP, decreased albumin level, increased Fe²⁺, MDA, ROS, 4-HNE levels, decreased SOD and GSH levels (P<0.05), lowered protein expressions of ACOX1, GPX4, FTH1, FLT, FAS, and SCD1, and increased protein contents of TFR1, ACSL4 and ALOX15 in the liver. Treatment with GDFMD and Fer-1 improved liver histopathology and liver function of the mouse models, decreased the levels of Fe²⁺, MDA and ROS, increased SOD and GSH levels, and reversed the changes in hepatic protein expressions. CONCLUSIONS GDFMD improves liver steatosis in mouse models of WD possibly by inhibiting hepatocyte ferroptosis through the GPX4/ACSL4/ALOX15 signaling pathway.
Animals ; Ferroptosis/drug effects* ; Mice ; Signal Transduction/drug effects* ; Drugs, Chinese Herbal/therapeutic use* ; Hepatolenticular Degeneration/drug therapy* ; Hepatocytes/metabolism* ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Fatty Liver/metabolism* ; Arachidonate 15-Lipoxygenase/metabolism* ; Coenzyme A Ligases/metabolism* ; Liver/metabolism* ; Male

OBJECTIVES: To evaluate the therapeutic effect of gastrodin against hypoxic-ischemic brain damage (HIBD) in neonatal mice and explore the role of GPX4/SLC7A11/FTH1 signaling in mediating its effect. METHODS: Twenty-four 9- to 11-day-old C57BL/6J mice were randomized equally into 4 groups for sham operation, HIBD modeling by right common carotid artery ligation and subsequent exposure to hypoxia for 1 h, or gastrodin treatment at 100 or 200 mg/kg before and at 1 and 2 days after modeling. The mice then underwent neurological assessment (Zea-Longa scores), and the cerebral cortical penumbra tissue were collected for HE and Nissl staining, detection of ferroptosis biomarkers and protein expressions of GPX4, SLC7A11, and FTH1 with Western blotting and immunofluorescence co-localization, and observation of mitochondrial ultrastructure with electron microscopy. In cultured HT22 neuronal cells with oxygen-glucose deprivation (OGD) for 2 h, the effects of pretreatments with 0.5 mmol/L gastrodin, 10 μmol/L RSL3 (a GPX4 inhibitor), alone or in combination, were analyzed on expressions of ferroptosis-related proteins, cellular Fe²⁺, ROS, lipid peroxidation, MDA, and GSH levels, mitochondrial membrane potential (JC-1), and cell viability. RESULTS: Gastrodin treatment at the two doses both significantly ameliorated HIBD and neurological deficits of the mice, reduced mitochondrial damage and Fe²⁺, MDA and ROS levels, increased GSH level, and upregulated GPX4, SLC7A11, and FTH1 protein expressions. In HT22 cells, gastrodin pretreatment obviously attenuated OGD-induced ferroptosis and improved cell viability and mitochondrial function. Co-treatment with RSL3 potently abrogated the inhibitory effects of gastrodin on Fe²⁺, ROS, BODIPY-C11, and MDA levels and attenuated its protective effects on GSH level, cell viability, and mitochondrial membrane potential. CONCLUSIONS Gastrodin provides neuroprotective effects in neonatal mice with HIBD by suppressing neuronal ferroptosis via upregulating the GPX4/SLC7A11/FTH1 signaling pathway.
Animals ; Ferroptosis/drug effects* ; Hypoxia-Ischemia, Brain/drug therapy* ; Mice ; Mice, Inbred C57BL ; Signal Transduction/drug effects* ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Glucosides/pharmacology* ; Animals, Newborn ; Benzyl Alcohols/pharmacology* ; Amino Acid Transport System y+/metabolism*

OBJECTIVES: To evaluate the inhibitory effect of oridonin against proliferation of pancreatic cancer cells and the mechanism underlying the synergistic effect of low-intensity pulsed ultrasound (LIPUS). METHODS: PANC-1 cells treated with different concentrations of oridonin were examined for changes in cell proliferation using CCK-8 assay and in MDA, GSH and ATP levels using flow cytometry. The protein expressions of GPX4, Nrf2 and HO-1 in the treated cells were detected with Western blotting. The effect of Fer-1, a ferroptosis inhibitor, on proliferation of oridonin-treated cells were assessed, and the effects of oridonin combined with LIPUS on PIEZO1 protein expression was evalauted using Western blotting. A C57BL/6J mouse model bearing pancreatic cancer cell xenograft was established and treated with oridonin, LIPUS, or both, and the histological changes in the tumor tissues and tumor cell proliferation were examined with HE staining and immunohistochemistry for Ki67; the changes in GPX4 expression in the tumor tissues were detected using Western blotting and immunofluorescence staining. RESULTS: In PANC-1 cells, oridonin treatment significantly inhibited cell proliferation, increased intracellular Fe²⁺, ROS, and MDA levels, and decreased GSH and ATP levels. Oridonin also resulted in lowered GPX4 and increased HO-1 and Nrf2 protein expression levels in the cells. The combined treatment with LIPUS signficiantly enhanced the inhibitory effect of oridonin on PANC-1 cell viability in vitro and on xenograft growth in the mouse models, resulting also in more obvious reduction of the intensity of Ki67 staining and GPX4 protein expression and more pronounced increase of PIEZO1 protein expression in the tumor tissues in the mouse models. CONCLUSIONS LIPUS enhances the effect of oridonin to promote ferroptosis of pancreatic cancer cells by activating PIEZO1 through the Nrf2/HO-1/GPX4 pathway.
Ferroptosis/drug effects* ; Animals ; Pancreatic Neoplasms/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Humans ; Cell Line, Tumor ; Mice ; Heme Oxygenase-1/metabolism* ; Diterpenes, Kaurane/pharmacology* ; Cell Proliferation/drug effects* ; Mice, Inbred C57BL ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Ion Channels/metabolism* ; Ultrasonic Waves ; Signal Transduction

OBJECTIVE: To observe the protective effect of Fisetin on sepsis-associated brain injury and explore its possible mechanism from the perspective of ferroptosis. METHODS: Sprague-Dawley (SD) rats (6-8-week-old male) were randomly divided into three groups: sham operation group (Sham group), colonic ligation and puncture (CLP) induced sepsis model group (CLP group) and Fisetin preprocessing group (CLP+Fisetin group), with 18 rats in each group (12 for observing survival rate and 6 for indicator testing). The CLP+Fisetin group was given Fisetin solution 50 mg×kg^-1×d^-1 by gavage continuously for 5 days before CLP, with dimethyl sulfoxide (DMSO) as the solute, while Sham group and CLP group were given the same dose of DMSO. The model was established at 2 hours after the last gavage. The general condition of each group of rats were observed, and the 10-day mortality were record. The behavioral testing (new object recognition experiment, elevated cross maze experiment) were performed after 7 days of modeling. After 24 hours of modeling, nerve reflex scoring was performed, and then the rats were euthanized and brain tissue was collected. The pathological changes of brain tissue were observed under a microscope by hematoxylin-eosin (HE) staining, the deposition of iron ion in brain tissue was observed by Prussian blue staining. The content of iron in brain tissue was determined by tissue iron kit, and the content of malondialdehyde (MDA) in brain tissue was determined by colorimetry. The expressions of tumor necrosis factor-α (TNF-α), neuron damage marker S100β, nuclear factor E2-related factor 2 (Nrf2), heme oxygenases-1 (HO-1) and glutathione peroxidase 4 (GPX4) were detected by Western blotting. RESULTS: On day 10 post-operation, 12, 3, and 7 animals survived in the Sham group, CLP group, and CLP+Fisetin group, respectively. Compared with the Sham group, rats in the CLP group showed significantly decreased nerve reflex score, new object discrimination index and open arm dwell time. HE staining showed arranged disorderly of neuronal cells, cytoplasm deep staining, nuclear condensation, unclear structures, neuron loss, and significant inflammation in the hippocampus in the hippocampus. Prussian blue staining showed iron ion deposition in the brain tissue. The contents of iron and MDA in brain tissue were elevated, and the expressions of TNF-α and S100β were up-regulated, while the expressions of Nrf2, HO-1, and GPX4 were down-regulated. Compared with the CLP group, the CLP+Fisetin group showed significantly increased neurological reflex score (7.33±1.15 vs. 4.67±1.53), improved new object discrimination index (0.44±0.02 vs. 0.32±0.04), and longer open arm dwell time (minutes: 78.33±9.29 vs. 41.15±9.64). Neuronal cells in the hippocampus were more organized, with less cytoplasmic staining, nuclear condensation, reduced neuronal loss, and fewer inflammatory cells. Iron ion deposition was reduced, and the contents of iron ions and MDA in brain tissue were decreased [iron ion (μg/g): 151.27±14.90 vs. 224.69±17.64, MDA (μmol/g): 470.0±44.3 vs. 709.3±65.4]. The expressions of TNF-α and S100β were significantly decreased (TNF-α/GAPDH: 0.651±0.060 vs. 0.896±0.022, S100β/GAPDH: 0.685±0.032 vs. 0.902±0.014), while the expressions of Nrf2, HO-1, and GPX4 were significantly increased (Nrf2/GAPDH: 0.708±0.108 vs. 0.316±0.112, HO-1/GAPDH: 0.694±0.022 vs. 0.538±0.024, GPX4/GAPDH: 0.620±0.170 vs. 0.317±0.039). All differences were statistically significant (all P < 0.05). CONCLUSION Fisetin pretreatment can inhibit ferroptosis and reduce sepsis-associated brain injury by Nrf2/HO-1/GPX4 pathway.
Animals ; Ferroptosis/drug effects* ; Rats, Sprague-Dawley ; NF-E2-Related Factor 2/metabolism* ; Sepsis/complications* ; Male ; Rats ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Neurons/drug effects* ; Signal Transduction ; Brain Injuries/metabolism* ; Flavonols ; Flavonoids/pharmacology* ; Heme Oxygenase-1/metabolism* ; Heme Oxygenase (Decyclizing)

Esculetin, a natural dihydroxy coumarin derived from the Chinese herbal medicine Cortex Fraxini, has demonstrated significant pharmacological activities, including anticancer properties. Ferroptosis, an iron-dependent form of regulated cell death, has garnered considerable attention due to its lethal effect on tumor cells. However, the exact role of ferroptosis in esculetin-mediated anti-hepatocellular carcinoma (HCC) effects remains poorly understood. This study investigated the impact of esculetin on HCC cells both in vitro and in vivo. The findings indicate that esculetin effectively inhibited the growth of HCC cells. Importantly, esculetin promoted the accumulation of intracellular Fe²⁺, leading to an increase in ROS production through the Fenton reaction. This event subsequently induced lipid peroxidation (LPO) and triggered ferroptosis within the HCC cells. The occurrence of ferroptosis was confirmed by the elevation of malondialdehyde (MDA) levels, the depletion of glutathione peroxidase (GSH-Px) activity, and the disruption of mitochondrial morphology. Notably, the inhibitor of ferroptosis, ferrostatin-1 (Fer-1), attenuated the anti-tumor effect of esculetin in HCC cells. Furthermore, the findings revealed that esculetin inhibited the Nrf2-xCT/GPx4 axis signaling in HCC cells. Overexpression of Nrf2 upregulated the expression of downstream SLC7A11 and GPX4, consequently alleviating esculetin-induced ferroptosis. In conclusion, this study suggests that esculetin exerts an anti-HCC effect by inhibiting the activity of the Nrf2-xCT/GPx4 axis, thereby triggering ferroptosis in HCC cells. These findings may contribute to the potential clinical use of esculetin as a candidate for HCC treatment.
Umbelliferones/administration & dosage* ; Ferroptosis/drug effects* ; Carcinoma, Hepatocellular/physiopathology* ; NF-E2-Related Factor 2/genetics* ; Humans ; Liver Neoplasms/physiopathology* ; Phospholipid Hydroperoxide Glutathione Peroxidase/genetics* ; Animals ; Cell Line, Tumor ; Mice ; Amino Acid Transport System y+/genetics* ; Mice, Inbred BALB C ; Male ; Signal Transduction/drug effects* ; Lipid Peroxidation/drug effects* ; Reactive Oxygen Species/metabolism* ; Mice, Nude