Tectorigenin attenuates myocardial damage by doxorubicin-induced ferroptosis by activating the p62-Keap1-Nrf2/HO-1/GPX4 axis

Abstract

Background

Anticancer agent doxorubicin is essential for cancer treatment but often causes cardiotoxicity. Tectorigenin has shown potential cardioprotective effects, but underlying mechanisms remain unclear. We aimed to investigate whether Tectorigenin attenuates doxorubicin-induced myocardial injury.

Methods

Doxorubicin (DOX) was utilized in C57BL/6J mice and cardiomyocytes H9c2, to establish in vivo and in vitro cardiotoxicity models, then treated with Tectorigenin. The levels of ferroptosis-related factors were measured using specific assay kits. The Liperfluo and DHE stainings were used to detect levels of lipid ROS. Cardiac function in rats was assessed using echocardiography. Immunofluorescence staining was used to detect Nrf2 nuclear translocation. ELISA assay was employed to check serum CK-MB, BNP and Tn-T levels. Cardiac injury and fibrosis were evaluated through HE and Masson stainings. Furthermore, TEM was employed to observe mitochondrial ultrastructure. Western blot and immunofluorescence staining were utilized to detect protein levels.

Results

DOX induced ferroptosis in H9c2 cells concentration-dependently and time-dependently, which was alleviated by Tectorigenin treatment. ML385 or K67 abolished Tectorigenin's inhibition on DOX-induced H9c2 cell ferroptosis. Mechanistically, Tectorigenin promoted the expressions of p62 and p-p62, leading to decreased Keap1 expression. This cascade facilitated Nrf2 nuclear translocation and subsequently elevated HO-1 and GPX4 expressions. Moreover, Tectorigenin treatment improved cardiac function, myocardial injury, fibrosis and mitochondrial function in C57BL/6J mice induced by DOX, as well as ferroptosis.

Conclusion

Our findings reveal that Tectorigenin attenuates DOX-induced ferroptosis and myocardial damage by activating the p62-Keap1-Nrf2/HO-1/GPX4 axis, this may provide a therapeutic strategy for mitigating cardiotoxicity associated with chemotherapeutic agents.

Graphical abstract

1. Introduction

Doxorubicin (DOX), also known as adriamycin, is a widely used anthracycline anticancer drug.¹ It is frequently used to treat many cancers, including leukemia, lymphoma, and sarcomas.2, 3, 4 However, one of the limitations of DOX is its potential for severe and irreversible toxic effects, particularly its cardiotoxicity.⁵ The mechanisms underlying DOX-mediated cardiotoxicity include inflammation, oxidative stress, apoptosis, impairment of mitochondria function, dysregulation of autophagy, and ferroptosis. These processes contribute to the detrimental effects on the heart caused by DOX treatment.^6,7

Ferroptosis is a unique type of iron-induced cell death characterized by the oxidation of lipids and reactive oxygen species (ROS) buildup in cells.⁸ At the center of this intricate regulatory process is glutathione peroxidase 4 (GPX4), a vital player in the control of ferroptosis. When the function of GPX4 is compromised, it ultimately paves the way for the occurrence of ferroptosis.⁹ It is particularly interesting to note that ferroptosis is involved in DOX-mediated cardiotoxicity. DOX has been found to generate cardiotoxicity by reducing nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and glutathione peroxidase 4 (GPX4) levels.^10,11 HO-1 has been shown to be a protein activated in response to stress and the key downstream target of Nrf2. It is essential for lowering oxidative stress and inflammation.¹² The study reported that Nrf2 and HO-1 are both important factors in the synthesis of GPX4.¹³

The Nrf2 is a nuclear transcription factor that protects cells by resisting oxidative stress and inflammation.¹⁴ Under non-stress situations, Keap1 sequesters Nrf2 in the cytoplasm and degrades it by ubiquitin-proteasome degradation.¹⁵ p62 is involved in signaling through competitive inhibition of Keap1-Nrf2 interaction.¹⁶ It was shown that phosphorylation of p62 Ser349 (equivalent to murine Ser351) significantly increased the binding affinity of p62 to Keap1. This modification further induced Nrf2 release from Keap1 and promoted Nrf2 accumulation.¹⁷ The accumulating Nrf2 was transported from the cytoplasm to the nucleus, where it stimulated the production of its target genes. In addition, the p62-Keap1 heterodimer recruits LC3 and mediates autophagic degradation of Keap1, further promoting Nrf2 accumulation.¹⁸ Increasing data shows that activation of the p62-Keap1-Nrf2 pathway is critical for cardiac protection. However, whether p62 is involved in ferroptosis by regulating the Keap1-Nrf2/HO-1 axis remains unknown.

Flavonoids are a class of low molecular weight plant metabolites with polyphenolic structures that possess antioxidant, anti-inflammatory, and anti-apoptotic properties.^19,20 According to research, Tectorigenin is a widely recognized natural flavonoid aglycone and a bioactive compound found in numerous plant species,²¹ and it has been shown to have protective effects on the heart, liver, and stomach.22, 23, 24 Studies have shown that flavonoids protect the heart by activating the Nrf2 signaling pathway.²⁵ In addition, the flavonoid licorice chalcone A inhibited the interaction between Nrf2 and Keap1 through phosphorylation at the p62 Ser349 site to inhibit arthritis.²⁶ Tectorigenin has been reported to be involved in ferroptosis inhibition, which relieved sepsis-induced myocardial ferroptosis by blocking Smad3.²² Nevertheless, the underlying mechanism by which Tectorigenin inhibits ferroptosis to improve myocardial function is largely unknown.

Herein, we put forward the hypothesis that Tectorigenin has the potential to mitigate myocardial damage caused by DOX-mediated ferroptosis by activation of the p62-Keap1-Nrf2/HO-1/GPX4 axis. Investigating this mechanism will help to discover more effective methods to improve DOX-induced cardiac toxicity.

2. Materials and methods

2.1. Cell culture

ATCC (VA, USA) provided rat cardiomyocytes (H9c2 cells). Cells were cultured in DMEM (Gibco, MD, USA) containing 10 % FBS (Gibco), 100 U/ml penicillin (Sigma-Aldrich, MO, USA), and 100 mg/mstreptomycin (Sigma-Aldrich) at 37 °C with 5 % CO₂.

2.2. Animal experiments

The Nanhua Hospital affiliated to Nanhua University authorized the experimental protocols, which followed the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. 8-week-old C57BL/6J mice were placed in rooms with constant temperature and humidity, and could freely access water and food. C57BL/6J mice were randomly divided into 3 groups (n = 4): control group, DOX group and DOX + Tectorigenin group. The DOX groups received intraperitoneal injections of DOX (15 mg/kg DOX diluted with 10 % dimethyl sulfoxide at the first day, and 10 mg/kg DOX diluted with 10 % dimethyl sulfoxide for the second administration at the eighth day), whereas the control groups received intragastric administration of equivalent quantities of 10 % dimethyl sulfoxide. DOX + Tectorigenin group administered 75 mg/kg Tectorigenin to DOX-induced animals every other day. The animals were then sacrificed, and blood samples were collected. The serum samples were retrieved after a 10-min centrifugation (3000 r/min, 4 °C), and the cardiac samples were immediately removed. A portion of the heart tissue was fixed, and the remaining samples were kept at 80 °C for subsequent testing.

2.3. Cell Counting Kit-8 (CCK-8)

H9c2 cells were cultured in 96-well culture plates (5 × 10³ cells per well) for 24 h. Cells were then incubated with 10 μl CCK8 reagent (Dojindo, Kyushu, Japan) for 2 h at 37 °C. The OD at 450 nm was then measured using Spectra Max M3 (Molecular Devices, CA, USA).

2.4. Western blot

The proteins were isolated from cells with RIPA (Beyotime, Shanghai, China), and quantified by a BCA kit (Beyotime). Subsequently, total protein (20 μg) was isolated by 10 % SDS-PAGE and transferred to a PVDF membrane (Millipore, MA, USA). Then, the membranes were blocked and incubated with antibodies against p62 (1:10000, 18420-1-AP, Proteintech), p-p62 (Ser349) (1:5000, 29503-1-AP, Proteintech), Keap1 (1:5000, a10503-2-AP, Proteintech), Nrf2 (1:5000, 16396-1-AP, Proteintech), HO-1(1:1000, 10701-1-AP, Proteintech), GPX4 (1:1000, 30388-1-AP, Proteintech) and anti-β-actin (1:1000, 20536-1-AP, Proteintech) overnight at 4 °C. After being rinsed by TBST, the membranes hybridized with the secondary antibody (Beyotime) at room temperature for 1h. The bands were examined by ECL (Beyotime). The grayscale of those protein bands was analyzed by using Image J.

2.5. Immunofluorescence staining

H9c2 cells were fixed in 4 % paraformaldehyde for 15 min and then washed three times. The cell slides were transferred sequentially to 0.5 % TritonX-100 for 15 min and 5 % BSA solutions for 30 min. The solution was then discarded and the cells were immediately incubated with Nrf2 antibody (1:500,16396-1-AP, Proteintech) for 16 h at 4 °C. The sections were washed three times and incubated with fluorescent secondary antibodies at 37 °C for 1 h in the dark. The slides were sealed with mounting medium containing DAPI. Immunofluorescence images were obtained with a laser-scanning confocal microscope. Cardiac sections underwent dual 5-min washes with fresh xylene, followed by sequential ethanol dehydration. After antigen retrieval using citric acid buffer, the slices were incubated overnight at 4 °C with primary antibodies against anti-p62 (1:500, ab56416, Abcam), p-p62 (Ser349) (1:500, 29503-1-AP, Proteintech), Keap1 (1:500, 18420-1-AP, Proteintech), Nrf2 (1:500,16396-1-AP, Proteintech), HO-1(1:200, 10701-1-AP, Proteintech), and GPX4 (1:500, 30388-1-AP, Proteintech). The sections were then washed and treated for 15 min with the appropriate secondary antibody (1:500, SA00004-1, Proteintech). The sections reacted with DAB solution and stained with hematoxylin.

2.6. Nuclear-cytoplasmic separation

Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime, Shanghai, China) was applied for cytoplasmic and nuclear protein extraction. All operations were strictly carried out according to the instructions.

2.7. Detection of GSH, MDA and Fe²⁺

The levels of ferroptosis indicators (MDA, GSH and Fe²⁺) in the supernatant of H9c2 cells were assessed using commercial kits (Nanjing Jiangcheng Bioengineering Institute). To measure MDA content, the H9c2 cells were sonicated. Then, MDA content was measured according to cell protein concentrations. Total GSH content was determined using a GSH assay kit and evaluated by comparison with a GSH standard curve. To determine the concentration of cellular Fe²⁺, H9c2 cells were seeded in a six-well plate (1 × 10⁵ cells/well). The Fe²⁺ content was measured using an iron assay kit as directed by the manufacturer.

2.8. Detection of lipid ROS

ROS level in cells was detected using Liperfluo (Dojindo). Cells were treated for 30 min with 5 μM Liperfluo at 37 °C. Cells were then washed twice with PBS before replacing the original media in each chamber. The images were captured with a Nikon fluorescence microscope (Tokyo, Japan) from three separate dishes for each treatment. The ROS activities in myocardial tissue were detected by dihydroethidium standing (DHE; Sigma-Aldrich). The process of DHE staining involves incubating cryosections with a solution containing 10 μM DHE at 37 °C for 30 min. After incubation, the samples are observed using confocal microscopy to determine the percentage of the area stained by DHE.

2.9. Echocardiography

To evaluate cardiac function in mice, transthoracic echocardiography was performed using a VEVO-1100 ultrasound system (VisualSonics). The procedure involved lightly anesthetizing the mice with 2 % isoflurane inhalation while maintaining their heart rates at 450 to 550 beats per minute. The parasternal long-axis view, as well as the short-axis view at the papillary muscle level, were captured using 2-D Guided M-mode images. Ejection fraction (EF), fractional shortening (FS), left ventricular thickness, and internal diameters were calculated by averaging measurements taken over a minimum of five consecutive cardiac cycles per sample. The researchers who collected the data and took the measurements were not aware of the previous treatment assignments. Electrocardiograms were captured and measured prior to the ultrasound operation utilizing a biopotential amplifier (PowerLab AD Instruments) and Labchart software.

2.10. Measurement of serum CK-MB, BNP and Tn-T levels

Blood samples were collected and centrifuged for 10 min at 3000 rpm to obtain serum. Serum CK-MB, BNP and Tn-T were measured by commercially available kits from Huili Biotech (Changchun, China) according to the manufacturer's instructions.

2.11. Hematoxylin-eosin staining (HE staining) and Masson's trichrome staining

Cardiac tissues were fixed in 10 % formalin for 24 h. The paraffin section (5 μm in thickness) was prepared. The sections were then stained with H&E dye or Masson's trichrome staining reagents following the standard steps. The photographs were captured with an Olympus light microscope (Tokyo, Japan).

2.12. Transmission electron microscopy (TEM)

Mice were anesthetized and given PBS before being given 4 % paraformaldehyde. Cardiac tissues (1 mm³) were immediately dissected and preserved in 2.5 % glutaraldehyde overnight at 4 °C. Tissues were implanted and sliced at a thickness of 60–80 nm along the coronal plane. TEM (JEM-1400 PLUS, Japan) was used to examine the mitochondrial morphology. The extent of mitochondrial damage was quantified using the Flameng score.²⁷

2.13. Statistical analysis

All experimental data were analyzed using GraphPad Prism7 statistical software (CA, USA) and presented as the mean ± SD. Data between two groups were compared by student's t-test and Mann-Whitney U test, and data among multiple groups were analyzed by one-way ANOVA with the Kruskal-Wallis test. P < 0.05 was considered as a statistically significant difference. All experiments were repeated at least 3 times.

3. Results

3.1. Tectorigenin upregulated DOX-induced H9c2 cell viability

Firstly, we investigated the effects of DOX on H9c2 cell viability. H9c2 cells were exposed to different concentrations of DOX (0, 0.25, 0.5, 1, 2, 4 μM) for 24 h, the results revealed a concentration-dependent inhibition of cell viability by DOX. Notably, when the DOX concentration reached 0.5 μM, the cell viability dropped to approximately 50 %. Therefore, we selected this concentration for further experiments (Fig. 1A). Subsequently, we treated cells with 0.5 μM DOX for different times (0, 12, 24, 36, 48h), as the incubation time increased, cell viability was increasingly inhibited. And there was a significant downregulation in cell viability after being treated with DOX for 24h (Fig. 1B). Surprisingly, when H9c2 cells were incubated with different concentrations of Tectorigenin, cell viability remained unchanged, indicating that Tectorigenin has a non-toxic effect on H9c2 cells (Fig. 1C). Next, we investigated the effect of Tectorigenin on DOX-induced H9c2 cells, H9c2 cells were treated with 0.5 μM DOX and incubated with different concentrations of Tectorigenin (0, 5, 10, 20 μM) for 24 h. The results revealed that Tectorigenin had a positive impact on DOX-treated H9c2 cells activity at the concentration of 20 μM (Fig. 1D). 0.5 μM DOX and 20 μM Tectorigenin co-incubated H9c2 cells for a continuous time, which revealed a significant increase in H9c2 cell viability after 12 h of treatment with Tectorigenin (Fig. 1E). Altogether, Tectorigenin could upregulate the cell viability in DOX-induced H9c2.

Fig. 1.

Tectorigenin upregulated DOX-induced H9c2 cell viability. A. H9c2 cells were exposed to various concentrations of DOX (0, 0.25, 0.5, 1, 2, 4 μM) for 24 h, and cell viability was assessed using CCK8 assay. B. Cell viability was determined by CCK8 assay after H9c2 cells were treated with 0.5 μM DOX for different durations (0, 12, 24, 36, 48h). C. H9c2 cells were subjected to various concentrations of Tectorigenin (0, 5, 10, 20 μM) for 24 h, and cell viability was measured by CCK8 assay. D. H9c2 cells were treated with 0.5 μM DOX and various concentrations of Tectorigenin (0, 5, 10, 20 μM) for 24 h, and CCK8 assay was used to detect cell viability. E. H9c2 cells were co-treated with 0.5 μM DOX and 20 μM Tectorigenin for varying time (0, 12, 24, 36, 48h), and cell survival was evaluated by CCK8 assay. The measurement data were presented as mean ± SD. All data were obtained from at least three replicate experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

3.2. Tectorigenin protected cardiomyocytes from DOX-induced ferroptosis through the Nrf2/HO-1/GPX4 axis

To explore the potential mechanism of the protective effect of Tectorigenin on DOX-induced ferroptosis in H9c2 cells, DOX-treated H9c2 cells were subjected to Tectorigenin or ferroptosis inhibitor Fer-1. The results showed that DOX significantly inhibited the expression of Nrf2, HO-1, and GPX4, but a notable reversal occurred upon either Tectorigenin or Fer-1 addition to DOX-treated H9c2 cells, leading to a significant increase in Nrf2, HO-1, and GPX4 expression levels (Fig. 2A). Additionally, DOX treatment led to a marked increase in H9c2 cell mortality, while pretreatment with Tectorigenin or Fer-1 effectively mitigated cell death (Fig. 2B). Furthermore, the DOX treatment led to an increase in lipid ROS and MDA levels, as well as accumulation of Fe²⁺ and a decrease in GSH levels in H9c2 cells, but the elevated ROS, MDA and Fe²⁺ levels were subsequently reversed by Tectorigenin or Fer-1 (Fig. 2C–F). Conversely, Tectorigenin mitigated the downregulation of Nrf2, HO-1, and GPX4 induced by DOX in H9c2 cells. However, the effects of Tectorigenin were reversed by the presence of ML385 (Fig. 2G). Additionally, ML385 further suppressed the viability of H9c2 cells under DOX treatment, while Tectorigenin counteracted DOX-mediated inhibition on cell viability. However, the protective effects of Tectorigenin were nullified in the presence of ML385 (Fig. 2H). As shown in Fig. 2I–L, ML385 promoted DOX-induced ferroptosis in H9c2 cells. Tectorigenin inhibited DOX-induced ferroptosis in H9c2 cells which was reversed by ML385. These findings collectively highlight that Tectorigenin inhibits DOX-induced ferroptosis by regulating Nrf2.

Fig. 2.

Tectorigenin protected cardiomyocytes from DOX-induced ferroptosis through the Nrf2/HO-1/GPX4 axis. H9c2 cells were categorized into four groups: Control, DOX, DOX + Tectorigenin, and DOX + Fer-1. A. Western blot analysis was used to quantify levels of Nrf2, HO-1, and GPX4. B. Cell viability assessment conducted by CCK8 assay. C. Intracellular lipid ROS content measured through Liperfluo staining (scale bar = 100 μm). D-F. Determination of intracellular GSH, MDA, and Fe²⁺ levels using GSH, MDA and Fe²⁺ detection kits. G. Western blot was applied to analyze the levels of Nrf2, HO-1, and GPX4. H. Cell viability assessment was conducted using the CCK8 assay. I. Lipid ROS was checked through Liperfluo staining (scale bar = 100 μm). J-L. Determination of intracellular GSH, MDA and Fe²⁺ levels using GSH, MDA and Fe²⁺ detection kits. The measurement data were presented as mean ± SD. All data were obtained from at least three replicate experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

3.3. Tectorigenin upregulated Nrf2 by phosphorylating p62 in DOX-induced H9c2 cells

DOX-induced H9c2 cells were treated with Tectorigenin or K67 (a specific inhibitor of the interaction between Keap1 and p-p62). Compared with the DOX group, p62 and p-p62 expressions were increased, and Keap1 expression was decreased in the DOX + Tectorigenin group. After treatment with K67, this phenomenon was inhibited. In addition, Tectorigenin treatment up-regulated the levels of HO-1, GPX4, and nuclear Nrf2 of DOX-induced H9c2 cells, and K67 inhibited the promotion of Tectorigenin on the expression of these proteins. However, the results of Keap1 and cytoplasmic Nrf2 presented opposite trends (Fig. 3A). Immunofluorescence staining also showed that DOX inhibited the nuclear translocation of Nrf2 in H9c2 cells, while Tectorigenin alleviated the inhibitory effect of DOX on Nrf2 nuclear translocation, which was inhibited after treatment with K67 (Fig. 3B). As shown in Fig. 3C, Tectorigenin alleviated the inhibitory effect of DOX on H9c2 cell viability, which was inhibited after treatment with K67. In addition, Tectorigenin treatment up-regulated the level of GSH in DOX-induced cells, but decreased the levels of lipid ROS, MDA and Fe²⁺; and K67 reversed the above results (Fig. 3D–G). The above results indicated that Tectorigenin promoted p62 phosphorylation to increase the binding of p62 to Keap1 and elevate the expression of nuclear Nrf2, thereby alleviating DOX-induced ferroptosis in H9c2 cells.

Fig. 3.

Tectorigenin upregulated Nrf2 by phosphorylating p62 in DOX-induced H9c2 cells. H9c2 cells were utilized and categorized into four groups: Control, DOX, DOX + Tectorigenin, and DOX + Tectorigenin + K67. A. Western blot was employed to assess the expressions of p62, p-p62, Keap1, cytoplasmic Nrf2, nuclear Nrf2, HO-1, and GPX4. B. Immunofluorescence was performed to verify the subcellular localization of Nrf2 (scale bar = 100 μm). C. Cell viability evaluated by CCK8 assay. D. Liperfluo staining was used to examine lipid ROS level (scale bar = 100 μm). E-G. Determination of intracellular GSH, MDA and Fe²⁺ levels using GSH, MDA and Fe²⁺ detection kits. The measurement data were presented as mean ± SD. All data were obtained from at least three replicate experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

3.4. Tectorigenin attenuated DOX-induced myocardial injury in mice by inhibiting ferroptosis

To study the effect of Tectorigenin on DOX-mediated cardiotoxicity in vivo, mice were initially exposed to DOX, which induced cardiotoxicity. Subsequently, the mice were treated with Tectorigenin. The administration of Tectorigenin to mice exhibited significant improvements in cardiac function parameters, including EF and FS, compared to mice treated only with DOX (Fig. 4A). As shown in Fig. 4B, the levels of CK-MB, BNP and Tn-T in serum were increased in DOX group, while decreased after Tectorigenin administration. Additionally, Tectorigenin-treated mice exhibited notable reductions in cardiac injury and fibrosis, suggesting its potential as a protective agent for cardiac tissue (Fig. 4C). Furthermore, Tectorigenin treatment effectively mitigated DOX-induced alterations in mitochondrial structure, including increased vacuolization in mitochondria and rupturing of mitochondrial cristae (Fig. 4D). The Western blot and IF results demonstrated that the administration of Tectorigenin to mice resulted in increased p62 and p-p62 in myocardial tissue compared to mice treated with DOX alone. This increase in p-p62 expression led to a decrease in Keap1 expression and subsequent Nrf2 nuclear accumulation, and further promoted the expression of HO-1 and GPX4 (Fig. 4E). This result was also verified in immunofluorescence staining (Fig. S1). Similarly, Tectorigenin attenuated DOX-induced myocardial ferroptosis by lowering MDA, Fe²⁺ and lipid ROS levels but elevating GSH expression (Fig. 4F–I). Collectively, Tectorigenin protected mouse cardiac tissue by reducing DOX-induced ferroptosis.

Fig. 4.

Tectorigenin attenuated DOX-induced myocardial injury in mice by inhibiting ferroptosis. A. The cardiac function in mice was evaluated using echocardiography. B. Levels of CK-MB, BNP and Tn-T in serum were tested by ELISA assay. C. HE staining (scale bar = 50 μm) and Masson staining (scale bar = 100 μm). D. Mitochondrial ultrastructure was detected by TEM (scale bar = 500 nm). E. The levels of p62, p-p62, Keap1, Nrf2, HO-1 and GPX4 in myocardial tissue were detected by WB. F-H. The content of GSH, MDA, and Fe²⁺ in myocardial tissue was measured using GSH, MDA and Fe²⁺ detection kits. I. ROS in myocardial tissue was checked by DHE staining (scale bar = 50 μm). The measurement data were presented as mean ± SD. All data were obtained from at least three replicate experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

4. Discussion

Doxorubicin is a powerful antitumor drug that is widely recognized for its ability to combat various types of cancer. Its effectiveness against a broad range of cancer types has made it a popular choice in the medical field.²⁸ DOX is important in the treatment of breast cancer. It is frequently used as part of neoadjuvant or adjuvant therapy, which aims to shrink tumors before surgery or prevent their recurrence.²⁹ Moreover, DOX is an integral component of many standard chemotherapy protocols for the treatment of hematological malignancies, including leukemia and lymphoma.³⁰ Our study aimed to examine the potential cardioprotective effects of Tectorigenin in the context of DOX-induced myocardial damage and the underlying mechanisms involved. We explored the hypothesis that Tectorigenin could alleviate myocardial injury by repressing ferroptosis through the p62-Keap1-Nrf2/HO-1/GPX4 axis, which has potential implications for developing new cardiac protective therapy strategies.

Ferroptosis is a separate biological mechanism of controlled cell death that is characterized by iron-dependent lipid peroxidation, ROS buildup, and membrane disruption.³¹ It has gained recognition as a crucial contributor to tissue injury in various pathological conditions, including cardiotoxicity.^10,32 Concomitant with the previous study, our study revealed that DOX-induced ferroptosis in cardiomyocytes is characterized by increased Fe²⁺ accumulation, lipid peroxidation, and altered redox balance. Furthermore, our research revealed that DOX dramatically reduced the expression of critical components such as Nrf2, HO-1, and GPX4. Nrf2, a crucial transcription factor central to cellular defense against oxidative stress and the maintenance of redox homeostasis, plays a critical role in regulating gene expression through the antioxidant response element (ARE).³³ When cells encounter heightened levels of ROS or encounter oxidative challenges, Nrf2 activation is triggered, leading to the transcription of genes encoding vital antioxidant enzymes and cytoprotective proteins, including HO-1, GPX and superoxide dismutase (SOD), which collectively contribute to the detoxification of ROS and the mitigation of cellular damage.^34,35 The activation of the Nrf2/HO-1/GPX4 axis inhibited ferroptosis by reducing ROS production in liver cells, nerve cells and cardiomyocytes under different pathological conditions.36, 37, 38 In addition, the activation of the Nrf2/HO-1/GPX4 axis reduced cardiomyocyte injury and inflammation during the progression of diabetic cardiomyopathy.³⁹ All the above evidence suggested that the activation of the Nrf2/HO-1/GPX4 axis helped alleviate myocardial injury and ferroptosis. However, the role of the Nrf2/HO-1/GPX4 axis in Tectorigenin-mediated protective effects on DOX-induced cardiotoxicity and ferroptosis remains unclear. Our results revealed that Tectorigenin attenuated DOX-induced myocardial injury in mice as well as DOX-induced inhibition on H9c2 cell viability. Tectorigenin treatment also prevented DOX-induced inhibition on the Nrf2/HO-1/GPX4 axis. Furthermore, Tectorigenin treatment was associated with reduced myocardial iron overload, decreased levels of lipid ROS and MDA in DOX-treated cardiomyocytes, and increased GSH level. All these results suggested that Tectorigenin mitigated DOX-induced cardiotoxicity by reducing ferroptosis through activating the Nrf2/HO-1/GPX4 axis, which was reported for the first time.

p62, as a multifunctional protein that is a multifunctional protein, is involved in a variety of cellular activities including autophagy, signal transduction, and cell survival.⁴⁰ It serves as a key mediator in various cellular pathways, including those involved in removing damaged or dysfunctional cellular components through autophagy.⁴¹ The interaction among p62, Keap1 and Nrf2 was widely studied. For example, the p62-Keap1-NRF2 pathway activation in hepatocellular cancer cells protected against ferroptosis.¹⁶ Kong et al. demonstrated the that p62-Keap1-Nrf2 signaling pathway was activated in the acute lung injury mice model.⁴² Our findings demonstrated that in DOX-induced mice treated with Tectorigenin, the elevated level of p62 and phosphorylated p62 and a reduced amount of Keap1 in both cells and myocardial tissues. Together, these changes disrupted the interaction between p62 and Keap1 and subsequently prevented the degradation of Nrf2 and facilitated Nrf2 nuclear translocation. And then driving the upregulation of HO-1 and GPX4.

Our research findings provided compelling evidence for the efficacy of Tectorigenin in improving cardiac function parameters in mice treated with DOX. Specifically, we observed significant improvements in EF and FS in mice treated with Tectorigenin compared to those treated with DOX alone. In contrast, Tectorigenin inhibited DOX-induced upregulation of CK-MB, BNP and Tn-T in vivo. These improvements are particularly noteworthy considering that mitochondrial dysfunction is a key characteristic of cardiomyopathy, often manifested by structural abnormalities such as mitochondrial miniaturization and reduced cristae. Moreover, our findings highlighted the potential of Tectorigenin as a cardioprotective agent. We observed a reduction in cardiac injury and fibrosis in mice treated with Tectorigenin, further emphasizing its ability to safeguard the heart from the detrimental effects of DOX. This cardioprotective property is particularly significant in managing cardiomyopathy and holds promise for potential therapeutic applications.

In conclusion, we demonstrated that Tectorigenin inhibited ferroptosis through the p62-Keap1-Nrf2/HO-1/GPX4 axis and improved the cardiotoxicity induced by DOX. These findings suggested that Tectorigenin might act as a novel adjunctive therapy for cardiomyopathy, offering potential avenues for further investigation and clinical translation. However, several limitations warrant attention. Firstly, while we identified the role of p62 in promoting Keap1 degradation and activating the Nrf2/HO-1/GPX4 pathway, the detailed biochemical mechanism underlying p62 phosphorylation in DOX-treated cardiomyocytes remains unclear. Notably, p62 is a key regulator of autophagy-dependent Keap1 degradation, which suggests a potential interplay between ferroptosis and autophagy in this context. Understanding this interplay could provide critical insights into the dual regulatory roles of p62 in these processes. Specifically, further studies should investigate whether Tectorigenin's effect on ferroptosis is partially mediated through autophagy-dependent mechanisms, as suggested by a recent study.⁴³ Moreover, the extent to which Tectorigenin modulates the balance between ferroptosis and autophagy, and whether this balance contributes to its cardioprotective effects, remains to be elucidated. For example, the specific crosstalk between autophagy and ferroptosis in the presence of oxidative stress and DOX toxicity is an important area that warrants further exploration. Additional studies involving autophagy inhibitors or genetic models targeting autophagy-related genes could help clarify this relationship.

Ethics approval and consent to participate

The Nanhua Hospital affiliated to Nanhua University authorized the experimental protocols, which followed the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (2023-KY-08).

Consent for publication

N/A.

Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

This work was supported by the Hunan Provincial Natural Science Foundation of China (grant number: 2025JJ81073); Hunan Provincial Health High-Level Talent Scienctific Research Project (grant number: R2023068) Guidance Program of Health Commission of Hunan Province (20201905), the Clinical Medical Research Center of Hunan (2020SK4007) and the General guidance project of Hunan Provincial Health Commission (No. D202303016868).

diting.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Fig S1. The levels of p62, p-p62, Keap1, Nrf2, HO-1 and GPX4 in myocardial tissue were detected by immunofluorescence staining (scale bar = 50 μm).