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. 2026 Aug 4;125(1):137. doi: 10.1007/s00436-026-08719-w

Mechanisms of ferroptosis in Echinococcus granulosus cyst fluid-induced hepatic injury and the protective role of Nrf2 activation

Jiaqi Chen 1,#, Jiangtao Cao 2,#, Jiuming Yan 1, Haiwen Li 1, Xinyi Du 1, Cheng xiao Ke 1, Hailong Lv 3,✉, Yufeng Jiang 1,✉
PMCID: PMC13719821  PMID: 42957908

Abstract

Cystic echinococcosis (CE) is a globally distributed parasitic zoonosis caused by infection with Echinococcus granulosus. This study investigated the ferroptosis mechanism in hepatocyte injury induced by Echinococcus granulosus cyst fluid (EgCF) and its relationship with the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway using an in vitro model. We employed the Cell Counting Kit-8(CCK-8) assay, Calcein AM/PI staining, and flow cytometry to assess cell viability, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP). Additionally, we measured lipid peroxides, malondialdehyde (MDA), glutathione (GSH), and ferrous iron (Fe²⁺) levels. Western blot analysis examined the expression of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11(SLC7A11), prostaglandin-endoperoxide synthase 2 (Ptgs2), ferritin heavy chain (FTH1), Nrf2, and heme oxygenase-1 (HO-1). Results indicated that EgCF inhibited hepatocyte proliferation and promoted hepatocyte apoptosis while inducing ferroptosis. Treatment with ferroptosis inhibitors Deferoxamine (DFO) or N-acetylcysteine (NAC) significantly attenuated EgCF-induced cytotoxicity. Nrf2 activation markedly mitigated EgCF-induced hepatocyte injury. This study elucidates the role of EgCF-induced ferroptosis and Nrf2 signaling in CE-associated liver injury, providing novel theoretical insights and new targets for disease prevention and treatment strategies.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s00436-026-08719-w.

Keywords: Echinococcus granulosus, Echinococcus granulosus cyst fluid, Ferroptosis, Deferoxamine, Nrf2

Introduction

Echinococcosis is a globally prevalent zoonotic disease classified by the World Health Organization as one of the neglected tropical diseases(Agudelo Higuita et al. 2016). The condition manifests primarily as two forms cystic echinococcosis (CE) and alveolar echinococcosis—which arise from infections by Echinococcus granulosus and Echinococcus multilocularis, respectively(Wen et al. 2019). Human infection as an intermediate host typically occurs through ingestion of Echinococcus granulosus eggs released by canids. These eggs enter the body via the gastrointestinal tract, spread through the bloodstream to organs particularly the liver and lungs, and form hydatid cysts at these sites(Budke et al. 2006). As cysts enlarge, they not only compress surrounding organs and tissues, leading to cellular and tissue atrophy, but also induce local ischemia, necrosis, and immune responses (Casulli et al. 2019; Jiménez et al. 2020; Wen et al. 2019).

Present therapeutic approaches for CE mainly consist of surgical removal and pharmacological treatment. Surgical excision is considered an effective curative approach for CE, particularly for larger cysts or those with complications. However, due to surgical risks, patient physical condition, and the complexity of cyst location, surgical treatment is not the optimal choice for all patients (Chen et al. 2017). Additionally, while medications such as albendazole and metoclopramide are widely used clinically, their efficacy is limited by factors including drug resistance and treatment duration (Nasseri-Moghaddam et al. 2011; Wan et al. 2022; Wang et al. 2022a). External trauma, such as blunt force injury or surgical procedures, may cause cyst rupture and fluid leakage, triggering acute hypersensitivity reactions that can be life-threatening in severe cases (Ye et al. 2016). Echinococcus granulosus cyst fluid (EgCF) is a complex biological mixture containing parasite secretions, shed surface antigens, and host-derived proteins(Ahn et al. 2015; Yang et al. 2021). EgCF has been reported to suppress miR-19 expression and activate the TGF-β/Smad pathway, promoting hepatic stellate cell proliferation and increasing collagen (COL1A1/COL3A1) deposition, which led to pericystic fibrosis formation around the echinococcal cysts (Ye et al. 2016). Furthermore, researchers have discovered that excretory-secretory products (ESPs) from the Echinococcus granulosus protoscoleces (PSCs) inhibited hepatocyte proliferation and induced apoptosis (Luo et al. 2024). ESPs secreted by other parasites can resist host immune attacks while also exerting toxic effects on the host(Motran et al. 2018; Zheng et al. 2013).

Ferroptosis represents a unique form of regulated cell death, characterized by morphological, biochemical, and genetic profiles that differ markedly from apoptosis, necrosis, and autophagy. Its core mechanism involves iron metabolism disorders and excessive accumulation of lipid peroxides (Dixon et al. 2012). As a key enzyme in maintaining redox balance and inhibiting lipid peroxidation, glutathione peroxidase 4 (GPX4) plays a crucial role in regulating ferroptosis (Ingold et al. 2018; Yang et al. 2014). Glutathione (GSH) serves as an essential cofactor for GPX4, GPX4 inactivation or GSH depletion leads to lipid peroxides accumulation, ultimately inducing ferroptosis (Yang et al. 2014). As liver dysfunction worsens, excessive reactive oxygen species (ROS) are generated, depleting the antioxidant GSH to counteract their toxicity (Dixon and Olzmann 2024; Wang et al. 2024). Iron overload is considered a hallmark feature of ferroptosis, wherein excess free iron promotes ROS generation through Fenton reactions, leading to lipid peroxidation and cellular damage (Dixon and Stockwell 2014). Aslam H et al. evaluated naturally infected buffalo livers with CE, revealing elevated lipid peroxide levels, increased malondialdehyde (MDA) and protein carbonyl content, and significantly reduced GSH levels (Aslam et al. 2023).Furthermore, studies have demonstrated that PSCs induce ferroptosis in mouse hepatocytes both in vivo and in vitro (Zhai et al. 2025). These findings suggest that EgCF may induce ferroptosis in hepatocytes via similar mechanisms. However, whether EgCF-induced hepatocyte death directly triggers ferroptosis remains unresolved.

Nuclear factor erythroid 2-related factor 2 (Nrf2) functions as a pivotal transcriptional modulator of the antioxidant defense network, thereby maintaining the redox stability of cells (Dodson et al. 2019). It has been demonstrated that the signaling regulatory mechanism between Nrf2 and ROS plays a crucial role in modulating PSCs activity (Luo et al. 2023). Beyond its well-established function in redox homeostasis, Nrf2 has been implicated in the control of ferroptosis (Xiang et al. 2024). Activation of Nrf2 protects cells against ferroptotic damage by enhancing the expression of antioxidant enzymes and iron‑sequestering proteins (Dodson et al. 2019). Conversely, inhibition or genetic ablation of Nrf2 increases intracellular reactive oxygen species (ROS) accumulation and lipid peroxidation, thereby sensitizing cells to ferroptosis (Panieri et al. 2020). For instance, wogonin reduced cellular GSH levels by inhibiting the Nrf2-mediated GPX4 pathway, leading to enhanced ROS accumulation and lipid peroxidation in pancreatic cancer cells (Liu et al. 2023). Conversely, in acute renal injury models, Nrf2 activation prevented lipid peroxidation and ferroptosis during acute kidney injury(Hu et al. 2022). Evidence has shown that the Nrf2–lipid peroxidation–ferroptosis axis is implicated in the molecular mechanisms of neurodegenerative diseases(Dodson et al. 2019). Further studies indicated that numerous Nrf2 activators alleviate cisplatin-induced nephrotoxicity by reducing ROS production (Fan et al. 2020). Given the interconnectedness of Nrf2, ROS, and ferroptosis, we hypothesize that activating Nrf2 may suppress ferroptosis and thereby mitigate EgCF-induced hepatocyte injury.

This study reveals ferroptosis involvement in EgCF-induced hepatocyte injury and demonstrates that activating the Nrf2 signaling pathway can suppress ferroptosis, thereby effectively protecting hepatocytes from damage. Modulating the Nrf2 signaling pathway holds promise as a novel theoretical basis and potential therapeutic target for clinically preventing and treating liver injury caused by Echinococcus granulosus infection.

Materials and methods

Reagents and antibodies

Deferoxamine (DFO), tert-Butylhydroquinone (tBHQ), and N-acetylcysteine (NAC) were obtained from MedChemExpress (NJ, USA). Primary antibodies were sourced as follows: anti-SLC7A11 (#98051), anti-Ptgs2 (#12282), anti-FTH1 (#3998), and anti-Nrf2 (#12721) from Cell Signaling Technology (MA, USA); anti-GPX4 (ab125066) from Abcam (Cambridge, UK); Histone H3 (#ET1701-64) and anti-HO-1 (#721854) from Huabio (Hangzhou, China); Alexa Fluor 594 Conjugate secondary antibody(#RGAR004), and α-Tubulin (#66031-1-Ig) from Proteintech (Wuhan, China). Reagents including the BCA protein assay kit, Cell Counting Kit-8(CCK-8), ROS assay kit, RIPA lysis buffer, Phenylmethanesulfonyl fluoride, and nuclear/cytoplasmic protein extraction kit were purchased from Beyotime (Shanghai, China). The MDA assay kit, mitochondrial membrane potential (MMP) assay kit, GSH assay kit, ferrous iron (Fe2+) assay kit, and superoxide dismutase (SOD) assay kit was obtained from Solarbio (Beijing, China); BODIPY™ 581/591 C11 fluorescent probe was purchased from Thermo Fisher Scientific (MA, USA); and FerroOrange probe was from Dojindo Laboratories (Kumamoto, Japan). Calcein-AM/PI dual fluorescence staining kit was purchased from Elabscience (Wuhan, China). Polyvinylidene fluoride (PVDF) membranes were obtained from Merck (MA, USA). DMEM/F12 medium and penicillin-streptomycin were purchasesd from Gibco (NY, USA). Insulin-transferrin-selenium and fetal bovine serum (FBS) were obtained from Pricella (Wuhan, China), Dexamethasone was supplied by Yeasen (Shanghai, China).

Cell culture

AML-12 cells were supplied by Pricella (Wuhan, China). Cells were maintained in DMEM/F12 supplemented with 10% FBS, 40 ng/mL dexamethasone, 1% penicillin-streptomycin, and 1% insulin-transferrin-selenium. The cells were maintained at 37 °C in a humidified incubator containing 5% CO₂.

EgCF preparation

Sheep livers with natural Echinococcus granulosus infection were obtained from a slaughterhouse in Shihezi, Xinjiang, China. The liver surfaces were disinfected by wiping with 75% alcohol for 3 min. EgCF was aseptically aspirated from hydatid cysts using a sterile syringe and transferred to sterile centrifuge tubes. Three independent batches of EgCF (each batch pooled from cysts of different sheep) were used for the three independent experiments. The fluid was centrifuged at 3,000 rpm for 30 min at 4 °C. The supernatant was collected and the pellet was discarded. Protein concentration was determined using a BCA assay kit. The collected EgCF was stored at -80 °C until further use.

Cell viability analysis

To explore the effects of EgCF treatment on cell viability, AML-12 cells were seeded at 5 × 10³ cells per well in a 96-well plate and treated with different concentrations of EgCF (0, 0.5,1, 2, 3, and 4 mg/mL) for 24 h. After replacing the old medium with fresh medium supplemented with CCK-8 reagent, the cells were maintained at for 1 h at 37 °C. The optical density at 450 nm was detected using a microplate reader (PerkinElmer, Massachusetts, USA) to evaluate cell viability.

Calcein-AM/PI staining

After treating cells with 2 mg/mL EgCF for 24 h, add the Calcein-AM/PI working solution and incubated at 37 °C for 20 min. Next, the cells were visualized under a fluorescence microscope (Olympus, Tokyo, Japan).

Apoptosis analysis

After treating cells with 2 mg/mL EgCF for 24 h, add Annexin V-FITC/PI working solution and incubated at 37 °C for 30 min. After collection and resuspension, the cells were analyzed by flow cytometry (Agilent, California, USA).

MMP analysis

The MMP was assessed using the JC-1 fluorescence probe. After seeding AML-12 cells into 12-well plates, the cultures were treated with 2 mg/mL EgCF for 24 h. The harvested cells were prepared in accordance with the instructions provided in the assay kit and stained with JC-1 solution, followed by incubation at 37 °C for 30 min. After PBS washing and resuspension, cells were immediately quantified by flow cytometry (Agilent, California, USA) and visualized using a laser confocal microscope (Nikon A1 + Ti2-E + SIM, Tokyo, Japan).

Intracellular ROS levels analysis

Intracellular ROS were quantified using the oxidation-sensitive fluorescent dye DCFH-DA. After treatment with 2 mg/mL EgCF for 24 h, cells were incubated in serum-free medium supplemented with 5 µM ROS probe at 37 °C for 30 min. Upon completion of PBS washes, cells were immediately analyzed by flow cytometry for quantitative detection and visualized under a fluorescence microscope (Olympus, Tokyo, Japan) to obtain images.

Lipid peroxidation analysis

Cellular lipid peroxidation was determined using the fluorescent indicator BODIPY™ 581/591 C11. After 24 h of 2 mg/mL EgCF exposure, cells were incubated with 2 µM BODIPY™ 581/591 C11 working solution at 37 °C for 30 min, washed three times with PBS and immediately analyzed by flow cytometry (Agilent, California, USA).

Iron content analysis

Following the manufacturer’s instructions, FerroOrange was used to detect the level of Fe2+. Briefly, cells were exposed to 2 mg/mL EgCF for 24 h, followed by incubation with 1 µM FerroOrange working solution at 37 °C for 30 min, followed by Fe2+ levels assessment via laser confocal microscopy (Nikon A1 + Ti2-E + SIM, Tokyo, Japan). Ferrous ion levels were determined using an Fe2+ assay kit, and absorbance was recorded at 590 nm with a microplate reader (PerkinElmer, Massachusetts, USA).

Assay for GSH, MDA, and SOD

To assess the intracellular redox balance, the levels of SOD, GSH, and MDA were measured according to the instructions provided in the respective kits. The brief procedure is as follows: After completing the experimental procedures, AML-12 cells were harvested and lysed. Absorbance was measured at a wavelength of 450 nm to calculate SOD activity. GSH levels were measured by reading absorbance at 412 nm. To determine the concentration of MDA, a lipid peroxidation product, absorbance was read at 532 nm and 600 nm.

Western blot analysis

After collecting cells, add RIPA lysis buffer containing protease inhibitors and incubate on ice for 30 min to extract total protein. Isolate nuclear proteins using a nuclear/cytoplasmic protein extraction kit. Protein concentrations were measured using a BCA protein assay kit. Equal amounts of protein were subjected to SDS–PAGE, followed by transfer onto PVDF membranes. Membranes were blocked with 5% skim milk powder for 1 h and incubated overnight at 4 °C with primary antibodies against GPX4, SLC7A11, FTH1, Nrf2, Ptgs2, HO-1, Tubulin, and H3 at 4 °C overnight. After washing with TBST, membranes were treated with the respective HRP-conjugated secondary antibodies for 1 h at 37℃. Protein bands were detected using Enhanced Chemiluminescent Reagent (Adamas life, Shanghai, China) and the ChemiDoc™ XRS+ imaging system (BioRad, California, USA). Quantification of protein bands on the developed membrane was performed using ImageJ software.

Immunofluorescence staining

Following EgCF exposure, the cells were fixed in 4% paraformaldehyde and rinsed three times with PBS, and then blocked with 5% donkey serum. They were incubated overnight at 4 °C with rabbit anti-GPX4 or anti-Nrf2 antibodies. The following day, cells were incubated at room temperature with fluorescently labelled secondary antibodies for 1 h, then mounted with a DAPI-containing anti-fluorescence quenching mounting medium. Cells were observed using a fluorescence microscope (Nikon A1 + Ti2-E + SIM, Tokyo, Japan).

Statistical analysis

Results were expressed as the means ± standard deviation (SD) and obtained from 3 independent experiments in the present study. Comparisons between two datasets were analyzed using paired t-tests, and one-way ANOVA was conducted when more than two groups were compared. GraphPad Prism 9 software was employed for statistical analysis, with significance defined as p < 0.05.

Results

EgCF-induced hepatocyte toxicity

To evaluate the effects of EgCF on hepatocytes, we treated AML-12 cells with varying concentrations of EgCF (0, 0.5, 1, 2, 3, and 4 mg/mL). CCK-8 assay revealed significantly reduced hepatocyte viability after 24 h treatment with 2 mg/mL EgCF (Fig. 1A), prompting us to select this concentration for subsequent experiments. Flow cytometry results further demonstrated that EgCF treatment induced apoptosis (Fig. 1B). Additionally, Calcein-AM/PI staining, a commonly used assay for detecting cell viability and death status, revealed a significant increase in cell mortality following EgCF treatment (Fig. 1C), providing further visual evidence of EgCF detrimental effects on cells. MMP (ΔΨm) is commonly used to assess mitochondrial function, with ΔΨm loss indicating mitochondrial dysfunction(Skárka and Ostádal 2002). We used the JC-1 staining method to assess changes in ΔΨm. The results showed that in the control group, JC-1 primarily formed red fluorescent aggregates within the mitochondria, indicating a high ΔΨm. Following treatment with EgCF, green fluorescence—indicating that JC-1 was present in the cytoplasm in its monomeric form—was significantly increased, while red fluorescence was significantly reduced, suggesting a decrease in ΔΨm (Fig. 1D).

Fig. 1.

Fig. 1

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EgCF-induced hepatocyte cytotoxicity. (A) Cell viability was assessed using the CCK-8 assay after 24 h treatment with various concentrations of EgCF. (B) Apoptosis was assessed by flow cytometry. (C) Calcein-AM/PI staining measured live/dead cells. (D) JC-1 staining measured MMP, Green fluorescence represents the monomeric form of JC-1, indicating low mitochondrial membrane potential. Means ± SD, n = 3. **p < 0.01, ***p < 0.001, and ns, not significant

EgCF promotes ferroptosis by inducing iron overload and lipid peroxidation

The occurrence of ferroptosis is primarily associated with imbalances in key intracellular factors such as iron, ROS, GSH, and GPX4 (Qiu et al. 2020). Our experimental findings indicated that, in comparison to the control group, treatment with EgCF led to a substantial elevation in cellular ROS and lipid peroxide levels (Fig. 2A and B). MDA, a natural end product of lipid peroxidation, is widely recognized as a marker of oxidative stress. Results indicated that cellular MDA levels significantly increased following EgCF treatment (Fig. 2C). Additionally, we assessed changes in the antioxidant defense system. Following EgCF treatment, GSH and SOD levels both decreased significantly, suggesting EgCF may exacerbate oxidative damage by suppressing the antioxidant system (Fig. 2D, E). Iron homeostasis disruption is a hallmark of ferroptosis. Therefore, we assessed intracellular Fe²⁺content. Results indicated that EgCF treatment markedly increased cellular Fe2+ levels (Fig. 2F). Fluorescence signals detected using the FerroOrange iron probe further corroborated these findings, confirming EgCF-induced iron overload (Fig. 2G). Western blotting analysis of ferroptosis-related protein expression in EgCF-treated cells revealed significant downregulation of GPX4, SLC7A11, and FTH1 compared to controls, while Ptgs2 expression was markedly upregulated (Fig. 2H). These data indicated that EgCF promotes ferroptosis in hepatocytes by inducing iron overload, enhancing ROS production, suppressing antioxidant systems (such as GPX4), and altering ferroptosis-related proteins.

Fig. 2.

Fig. 2

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EgCF induces ferroptosis by promoting lipid peroxidation and disrupting iron homeostasis. (A) Measurement of cellular ROS levels. (B) BODIPY™ 581/591 C11 assay for lipid peroxidation. (C-F) Ferroptosis-related marker assay: expression of MDA, GSH, SOD, and Fe²⁺. (G) The iron content in AML-12 cells was detected by FerroOrange staining. (H) Western blot analysis of GPX4, SLC7A11, Ptgs2, and FTH1 in cells treated with EgCF for 24 h. Means ± SD, n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001

The ferroptosis inhibitor DFO mitigates ferroptosis induced by EgCF in AML-12 cells

To corroborate the involvement of ferroptosis in EgCF-induced cytotoxicity, we employed the ferroptosis-specific inhibitor 10 µM DFO in our intervention experiments. First, CCK-8 assay results demonstrated that DFO treatment significantly reversed the EgCF-induced decline in cell viability, indicating that inhibiting ferroptosis effectively mitigates the cytotoxic effects of EgCF (Fig. 3A). DFO treatment markedly reduced EgCF-induced cellular ROS production and lipid peroxide levels (Fig. 3B, C). ROS accumulation is a primary driver of oxidative damage and cell death. Following DFO treatment, MDA and Fe²⁺ levels were significantly decreased, while GSH levels were markedly increased, indicating that DFO effectively suppresses ferroptosis by restoring the antioxidant system (Fig. 3D-F). We further assessed MMP changes. Results showed that DFO effectively reversed the EgCF-induced decline in MMP, suggesting that inhibiting ferroptosis helps maintain MMP stability, thereby protecting cells from further damage (Fig. 3G). Western blot analysis revealed that DFO treatment significantly upregulated the expression levels of key ferroptosis regulators GPX4, SLC7A11, and FTH1, while downregulating Ptgs2 expression (Fig. 4A). Immunofluorescence results further confirmed that GPX4 expression was significantly reduced following EgCF treatment, and this change was reversed by DFO (Fig. 4B). In summary, the ferroptosis inhibitor DFO significantly antagonizes EgCF-induced cytotoxicity by restoring intracellular homeostasis, reversing oxidative stress, iron metabolism disruption, and mitochondrial damage. Our study further demonstrates the pivotal role of ferroptosis in EgCF-induced cellular injury.

Fig. 3.

Fig. 3

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The ferroptosis inhibitor DFO antagonizes EgCF-induced cellular damage. (A) Cell viability was assessed by the CCK-8 assay after co-treatment with 10 µM DFO and EgCF. (B) Measurement of cellular ROS levels. (C) BODIPY™ 581/591 C11 assay for lipid peroxidation. (D-F) Ferroptosis-related markers assayed by kits: MDA, Fe²⁺, and GSH levels. (G) MMP measured. Means ± SD, n = 3. **p < 0.01 and ***p < 0.001

Fig. 4.

Fig. 4

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DFO antagonizes EgCF-induced ferroptosis by regulating the expression of key proteins such as GPX4. (A) Western blot analysis of GPX4, SLC7A11, Ptgs2, and FTH1 expression in cells treated with EgCF for 24 h. (B) Immunofluorescence staining for GPX4 expression. Means ± SD, n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001

NAC intervention effectively alleviates EgCF-induced oxidative stress and ferroptosis

Intracellular ROS production is a key driver triggering ferroptosis (Su et al. 2019; Wang et al. 2019). As shown in Fig. 1, EgCF treatment significantly induces cellular ROS generation, indicating that EgCF induces oxidative stress by elevating ROS levels. As a key endogenous antioxidant, GSH plays an essential role in preserving cellular redox equilibrium and defending against oxidative injury(Ursini and Maiorino 2020). To intervene in EgCF-induced oxidative stress, we employed NAC as a GSH precursor and ROS scavenger. NAC is a broad-spectrum antioxidant proven to effectively mitigate cellular damage in numerous oxidative stress-related diseases(Roh et al. 2017). Results indicated that 5 µM NAC treatment significantly restored cell viability (Fig. 5A) and effectively reversed EgCF-induced increases in ROS and lipid peroxide levels (Fig. 5B, C). Furthermore, NAC markedly reduced MDA and Fe²⁺ accumulation while elevating GSH levels (Fig. 5D-F). These findings indicated that NAC significantly mitigates EgCF-induced oxidative damage by enhancing cellular antioxidant capacity, thereby effectively alleviating ferroptosis. To further elucidate the mechanism of NAC action, we analyzed changes in ferroptosis-related proteins. Western blot analysis revealed that NAC treatment significantly upregulated the protein expression levels of SLC7A11, a key component of the cystine/glutamate antiporter system Xc⁻, and the core detoxification enzyme GPX4 (Fig. 5G). These data indicate that NAC supplementation enhances cellular antioxidant capacity, effectively blocks EgCF-induced oxidative stress, and upregulates the expression of downstream ferroptosis-related key molecules, thereby effectively antagonizing ferroptosis.

Fig. 5.

Fig. 5

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ROS inhibition mitigates EgCF-induced cellular damage (A) Cell viability assay after 5 µM NAC treatment. (B) Cellular ROS levels. (C) Lipid peroxidation assessment using BODIPY™ 581/591 C11 probe. (D-F) Ferroptosis-related markers assay: MDA, Fe²⁺, and GSH expression. (G) Western blot analysis of GPX4 and SLC7A11. Means ± SD, n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001

Protective effects of Nrf2 activation against EgCF-induced ferroptosis

The transcription factor Nrf2 plays a crucial role in the regulation of ferroptosis. Our results indicated that following EgCF treatment, Nrf2 undergoes significant nuclear translocation, accompanied by marked upregulation of its downstream gene heme oxygenase-1 (HO-1) (Fig. 6A, B). To investigate the role of Nrf2, we used the Nrf2-specific agonist 10 µM tBHQ to elucidate the interaction between Nrf2 and ferroptosis following EgCF treatment. Compared with the control group, tBHQ treatment upregulated the expression levels of nuclear Nrf2 protein. Compared with the group treated with EgCF alone, the combined treatment with tBHQ and EgCF further upregulated the expression levels of nuclear Nrf2 protein (Fig. 6C). Co-treatment with tBHQ and EgCF significantly upregulated GPX4 and SLC7A11 expression, markedly reduced Ptgs2 protein levels, and enhanced the expression of HO-1 (Fig. 6D). According to the immunofluorescence results, treatment with tBHQ promoted a pronounced translocation of Nrf2 from the cytoplasm to the nuclear (Fig. 6E), confirming that Nrf2 was activated. Following tBHQ treatment, flow cytometry revealed a significant reduction in lipid peroxide levels (Fig. 7A). Moreover, MDA and Fe²⁺ levels decreased while GSH content markedly increased (Fig. 7B-D). Finally, CCK-8 assay demonstrated that tBHQ rescued the EgCF-induced decline in cell viability (Fig. 7E). Collectively, these data indicate that Nrf2 activation effectively counteracts EgCF-induced cellular damage.

Fig. 6.

Fig. 6

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Activation of Nrf2 by tBHQ alleviates EgCF-induced ferroptosis in hepatocytes (A-D) Western blot analysis of Nrf2, HO-1, GPX4, SLC7A11, and Ptgs2. (E) Immunofluorescence staining for Nrf2 expression. Means ± SD, n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 7.

Fig. 7

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tBHQ alleviates EgCF-induced oxidative stress and restores hepatocyte viability. (A) Lipid peroxidation assessment using BODIPY™ 581/591 C11 probe. (B-D) Ferroptosis-related markers assay: MDA, Fe²⁺, and GSH expression. (E) Cell viability assay after 10 µM tBHQ treatment. Means ± SD, n = 3. * p < 0.05, **p < 0.01, and ***p < 0.001

Discussion

The mechanism by which EgCF acts as a key trigger for allergic reactions following cyst rupture remains incompletely understood. Previous studies have indicated that EgCF promotes pericystic fibrosis in CE by suppressing miR-19 expression (Zhang et al. 2016). Furthermore, EgCF inhibited the type I interferon response by promoting ROS in macrophages(Fu et al. 2024). Our findings align with these studies, demonstrating a significant reduction in cell viability following treatment with various concentrations of EgCF. Specifically, flow cytometry analysis revealed markedly increased apoptosis rates after 2 mg/mL EgCF treatment. Further experiments showed that EgCF treatment also caused a significant decrease in MMP, suggesting impaired mitochondrial function. This observation was consistent with other studies reporting EgCF effects on oxidative damage to cells (Mohammadi et al. 2021).

Ferroptosis is a regulated cell death process driven by iron overload, excessive lipid peroxidation, and disruption of cellular antioxidant systems(Han et al. 2020). Studies revealed that liver damage in diabetic patients is frequently associated with the occurrence of ferroptosis(Wu et al. 2024). In brain cells infected with Toxoplasma gondii, reduced GSH levels and downregulated expression of SLC7A11 and GPX4 proteins were observed. Meanwhile, DFP as an iron chelator, effectively reduced iron ion accumulation and inhibited ferroptosis, indicating that Toxoplasma infection induces ferroptosis in brain cells, leading to brain damage (Wang et al. 2023). Ferroptosis also plays a significant role in Echinococcus multilocularis infection. PSCs caused liver damage in mice by triggering ferroptosis. Studies indicated infection alters cellular metabolism and ROS pathways, thereby inducing ferroptosis (Zhai et al. 2025). However, the mechanism by which EgCF participates in cellular damage via the ferroptosis pathway remains unclear. Our study demonstrated that following EgCF treatment, intracellular ROS, lipid peroxidation, and MDA levels significantly increased, while GSH levels markedly decreased. Ferroptosis occurrence is primarily associated with ROS accumulation and lipid peroxidation production(Xu et al. 2023). The primary cause of increased lipid peroxides is reduced GPX4 expression (Yang et al. 2014). Immunoblotting results showed that EgCF treatment significantly decreased the levels of GPX4 and SLC7A11, which are important regulators of ferroptosis. The ferroptosis inhibitor DFO significantly mitigated EgCF-induced cellular lipid peroxidation and ROS accumulation, reduced Fe²⁺ and MDA levels, and reversed the downregulation of GPX4 and SLC7A11. Moreover, co-treatment with NAC markedly reduced intracellular ROS levels. Mitochondrial dysfunction has been demonstrated to enhance cellular susceptibility to ferroptosis (Gao et al. 2019). Our findings further revealed that EgCF treatment induces a decline in cellular MMP, which DFO effectively mitigates. These results conclusively demonstrate that EgCF contributes to liver injury by inducing ferroptosis and that inhibiting ferroptosis effectively prevents EgCF-induced cellular damage.

Nrf2 is critically involved in regulating lipid peroxidation and modulates ferroptosis through multiple mechanisms, including iron homeostasis, GPX4 synthesis, and lipid peroxidation (Chang et al. 2018; Shin et al. 2018). Studies have indicated that Nrf2 activation directly regulates ferroptosis by upregulating GPX4 protein levels. This mechanism confers resistance to the classical ferroptosis inducer RSL3, effectively protecting cells from cisplatin-induced damage (Wang et al. 2022b). Nrf2 overexpression significantly enhances cell survival and induces chemotherapy resistance in multiple cancers (Liu et al. 2021; Torrente et al. 2020). This study examined the protein expression levels of Nrf2 and its downstream gene HO-1, revealing significantly increased expression of both following EgCF treatment. HO-1 is a crucial antioxidant enzyme that defends against cellular stress and plays an essential role in maintaining redox homeostasis(Lever et al. 2016). Upregulation of HO-1 is recognized as an antioxidant mechanism protecting cells from ROS attacks. Previous studies have demonstrated increased Nrf2 expression during Echinococcus granulosus infection, suggesting active oxidative stress processes(Luo et al. 2023). Our findings similarly indicated that elevated Nrf2 and HO-1 expression represented a response to EgCF-induced oxidative stress. To investigate the role of Nrf2 in EgCF-induced liver injury, we treated cells with the Nrf2 agonist tBHQ. The results demonstrated a significant improvement in cell viability following tBHQ treatment. Under normal physiological conditions, Nrf2 is located in the cytoplasm. When cells are exposed to oxidative stress, Nrf2 migrates to the cell nucleus, where it binds to the antioxidant response element (ARE) and activates a cascade of downstream target genes (Hou et al. 2016; Raghunath et al. 2018). Immunofluorescence and Western blot analyses both confirmed that EgCF treatment induced Nrf2 translocation from the cytoplasm to the nucleus. Co-treatment with tBHQ significantly enhanced this nuclear translocation. Ferroptosis occurrence is closely associated with the expression of GPX4 and SLC7A11, two downstream target genes regulated by Nrf2 (Wen et al. 2019). Results demonstrate that Nrf2 activation effectively reversed EgCF-induced downregulation of GPX4 and SLC7A11 expression while significantly increasing HO-1 protein levels. Furthermore, Nrf2 activation markedly reduced Fe²⁺, MDA levels, and lipid peroxidation. Collectively, these findings suggest that Nrf2 effectively prevents EgCF-induced cellular damage by regulating ferroptosis, highlighting its protective role against tissue damage under both normal and stress conditions. However, it is worth noting that the activation of Nrf2 signaling is ambivalent: sustained activation of Nrf2 exacerbates heavy metal (lead/cadmium)-induced kidney injury by inhibiting autophagy and promoting apoptosis(Fan et al. 2021; Lian et al. 2023). Therefore, caution must be exercised regarding the potential adverse effects of Nrf2-targeted therapies.

In conclusion, our findings confirm that ferroptosis plays a key role in EgCF-induced hepatocyte damage and highlight the importance of Nrf2 activation in mitigating EgCF-induced liver injury. However, this study focused solely on the effects of EgCF on ferroptosis in AML-12 hepatocytes and did not investigate the effects of EgCF on other non-hepatocyte cells in the liver, nor did it clarify whether ESPs can also induce ferroptosis in hepatocytes. In future experiments, we will conduct relevant studies to address these limitations of the present study.

Conclusions

In this study, we investigated the effects of EgCF on hepatocytes in vitro. Results demonstrated that 2 mg/mL EgCF treatment induced oxidative stress, mitochondrial dysfunction, and ferroptosis in hepatocytes. Concurrently, activation of Nrf2 significantly rescued EgCF-induced ferroptosis. Modulating the ferroptosis process may provide novel therapeutic approaches for liver damage caused by echinococcosis.

Supplementary information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (347.6MB, pptx)

Acknowledgements

We would like to thank the staff of Shihezi slaughterhouse in Xinjiang Province for donating their abandoned sheep liver.

Author contributions

**Jiaqi Chen and Jiangtao Cao:** Conceptualization, Methodology, Investigation, Data curation, Formal analysis, Writing–original draft. **Jiuming Yan and Haiwen Li:** Data curation, Formal analysis. **Xinyi Du and Chengxiao Ke:** Methodology, Data curation. **Hailong Lv:** Project adminstration, Funding acquisition, Supervision, Writing-review & editing; **Yufeng Jiang:** Project adminstration, Supervision, Writing-review & editing.

Funding

This work was financially supported by the National Natural Science Foundation of China (Grant number 81560334 to HLL), the Key research and development program of Sichuan Provincial Science and Technology Department (Grant number 2022YFS0231 to HLL), and Chengdu Medical College Graduate Innovation Fund(Grant number YCX20240113). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

Data availability

Data will be made available on request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jiaqi Chen and Jiangtao Cao contributed equally to this work.

Contributor Information

Hailong Lv, Email: lvhl1999@126.com.

Yufeng Jiang, Email: yufengjiang03@126.com.

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Supplementary Materials

Supplementary Material 1 (347.6MB, pptx)

Data Availability Statement

Data will be made available on request.

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