DOX (CAS number: 23214-92-8, lot number: A396641337769) was purchased from APE × BIO (United States). DEX (CAS number: 24584-09-6, lot number: 0448942-18) was purchased from Cayman (United States). Thy (CAS number: 65-71-4, lot number: C14851700) was purchased from Shanghai Macklin Biochemical Co., Ltd.

Tg (cmlc2: EGFP) and wild-type AB zebrafish with EGFP-labeled hearts were provided by the Drug Screening Platform of the Institute of Biology, Shandong Academy of Sciences. Male and female zebrafish were housed separately at 28 °C under standard conditions and a 14 h light/10 h dark cycle. After natural mating, embryos were collected, transferred to zebrafish culture water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, and 0.33 mM MgSO₄) containing 0.02% methylene blue, and placed in a constant-temperature light incubator (SPX-280B-G, Shanghai Genestar Biotechnology Co., Ltd., Shanghai, China) at 28.0 °C ± 0.5 °C. The culture water was changed every 24 h. All experiments were performed in accordance with standard ethical guidelines and approved by the Animal Ethics Committee of the Institute of Biology, Qilu University of Technology, under animal welfare ethics review approval number SWS20220929.

Tg (cmlc2: EGFP) zebrafish that had developed normally at 48 hpf were selected and placed in 24-well plates, with 15 juveniles per well. In the pilot experiment, a blank control group (zebrafish culture water), a DOX model group (60 μM DOX), a DEX-positive control group (60 μM DOX + 10 μM DEX) and Thy-treated group (60 μM DOX + 10, 20 or 40 μM Thy) were used. The wells for each group were set up in triplicate, and the plates were placed in a light incubator for 24 h.

Changes in the size of the pericardial area directly reflect abnormalities in the zebrafish heart. Opening of the S-loop of the heart results in a greater sinus venous–glomus arteriae (SV-BA) distance. Therefore, the pericardial area and SV-BA distance, as measured under a microscope, can serve as direct indicators of zebrafish heart abnormalities (Wu et al., 2023; Li et al., 2021). Twenty-four hours after drug administration, the zebrafish were anesthetized with an anesthetic agent (0.3% tricaine by mass concentration) and then fixed with 4% methylcellulose. After anesthesia, pictures were taken from the side under a fluorescence stereo microscope (Olympus, SZX2-ILLTQ, Tokyo, Japan), and the morphology of the hearts of zebrafish in each group was photographed and recorded. The SV-BA distance and pericardial area were measured using Image-Pro Plus 6.0 software.

The clinical manifestations of DCM are arrhythmia, ventricular dilatation, and systolic dysfunction. Heart rate, stroke volume, short-axis shortening rate, and ejection fraction can reflect heart rhythm, ventricular dilatation, and systolic function (Begay et al., 2016). Twenty-four hours after drug administration, the zebrafish were fixed in 4% methylcellulose. After fixation, a video of the heartbeat was recorded under an inverted fluorescence microscope (Olympus, SZX16, Tokyo, Japan) with zebrafish in a prone position, and images of ventricular end-diastole and end-systole were extracted. The lengths of the major and minor axes at end-diastole and end-systole were measured using Image-Pro Plus 6.0 software, and the stroke volume, SAS rate and ejection fraction were calculated (Song et al., 2020).

Patients with cardiomyopathy often experience heart failure, resulting in cardiac pump dysfunction. In this study, blood flow velocity was measured to evaluate the effect of Thy on the blood flow velocity of zebrafish with DOX-induced heart injury (Eriksson et al., 2013). Twenty-four hours after drug administration, the zebrafish were anesthetized and fixed with 4% methylcellulose. Arterial blood flow velocity was measured using a blood flowmeter. Video of blood flow was analyzed, and the blood flow velocity was calculated using MicroZebraLab BloodFlow 3.4.6 software (ViewPoint, Lyon, France) (Gao et al., 2023).

O-dianisidine binds to hemoglobin and forms a rust red precipitate in cells. In this study, O-dianisidine was used to analyze hemoglobin production in zebrafish. Twenty-four hours after drug administration, the zebrafish were stained with 1 mg/mL O-dianisidine staining solution for 20 min, and 10 zebrafish were randomly selected from each group and observed under a fluorescence stereo microscope (Zeiss,AXIO-V16,Germany). The erythrocytes in the heart region were photographed. Image-Pro Plus 6.0 image processing software was used to analyze the area of erythrocyte staining in the heart quantitatively to evaluate the antimyocardial ischemic action of the studied compounds (Fan et al., 2022).

For AO staining, 24 h after drug administration, prepared AO stock solution was added to each well containing zebrafish to reach a final concentration of 5 μg/mL, followed by incubation in an incubator at 28 °C for 30 min. The zebrafish were washed 3 times with phosphate buffered saline (PBS) and observed under a fluorescence stereo microscope to count the number of apoptotic cells in the heart.

For TUNEL staining, 24 h after drug administration, zebrafish were fixed in 4% paraformaldehyde and placed in a refrigerator at 4 °C overnight, after which 0.3% Triton X-100 and sodium citrate antigen retrieval solution (1X) were added at a ratio of 1:300 and subsequent incubation in a refrigerator at 4 °C for 10 min. Tdt enzyme and fluorescence labeling solution were mixed together at a ratio of 1:10. Fluorescence labeling mixture (50 μL) was added to each group of zebrafish, followed by incubation in the dark for 1.5 h in a 37 °C incubator. The zebrafish were observed under a fluorescence stereo microscope (Zeiss, AXIO-V16, Germany) to count the number of apoptotic cells in the heart.

In patients with cardiomyopathy, the ventricular wall gradually becomes thinner, and myocardial cells are disordered. In this study, H&E staining was used to examine whether the heart tissue of zebrafish exhibited pathological changes. Twenty-four hours after drug administration, 10 zebrafish were randomly selected from blank control group, DOX modeling group, and drug-treated group (60 μM DOX + 20 μM Thy) and fixed with 4% paraformaldehyde fixative solution. The zebrafish were subsequently dehydrated with an ethanol gradient and then cleared with xylene. The zebrafish were then embedded in paraffin and sectioned. The sections were deparaffinized in xylene, absolute ethanol, and alcohol in water successively and then stained with hematoxylin and eosin. After dehydration and mounting, the sections were observed under a fluorescence stereo microscope (Olympus, SZX2-ILLTQ, Tokyo, Japan), and heart tissue was photographed and analyzed.

Twenty-four hours after drug administration, 10 zebrafish were randomly selected from blank control group, DOX model group (60 Μm DOX), and drug-treated groups (60 μM DOX + 10, 20 or 40 μM Thy) and were fixed with 4% paraformaldehyde. The next day, the zebrafish were removed from the fixative solution and dehydrated in propylene glycol. Freshly prepared 0.3% Oil red O solution was added to the zebrafish, followed by incubation for 6 h in the dark. Excess staining solution was removed by washing with propylene glycol and PBST. Lipid accumulation in the hearts of zebrafish was observed under a fluorescence stereo microscope (Olympus, SZX2-ILLTQ, Tokyo, Japan) and photographed (Xu et al., 2023).

Twenty-four hours after treatment, 30 zebrafish from blank control group, DOX modeling group and drug-treated group (60 μM DOX + 20 μM Thy) were randomly selected and washed three times with enzyme-free water. The zebrafish were homogenized, and RNA was extracted using a FastPure Cell/Tissue Total RNA isolation kit V2 (Vazyme, Nanjing, China). cDNA was obtained via reverse transcription, and RT‒PCR was performed using ChamQ Universal SYBR qPCR master mix. The RT‒PCR amplification conditions were as follows: predenaturation at 95 °C for 30 s; denaturation at 95 °C for 10 s and annealing at 60 °C for 10 s, for a total of 40 cycles; and 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s, for 1 cycle. Relative quantitative analysis was performed using β-actin as an internal reference gene. The PCR primers for the internal reference gene β-actin and the target genes were synthesized and purified by BioSune Biotechnology Co., Ltd. (Shanghai, China) and passed quality inspection. Primers for all genes are listed in Supplementary Table S1.

The experimental data are expressed as means ± SD, and significant differences between groups were determined using t tests. p < 0.05 indicated a significant difference, and p < 0.01 indicated a highly significant difference. Statistical analyses were performed using GraphPad Prism 8.0 and Image-Pro Plus 6.0 software.

At 24 h, compared with those in the blank control group, pericardial edema and the SV-BA distance were significantly greater in the DOX model group. Compared with the DOX model group, the DEX-positive control group and the Thy treatment group (60 μM DOX + 10, 20, 40 μM Thy) presented reduced pericardial edema (Figure 1A), decreased SV-BA (Figure 1B), and decreased pericardial area (Figure 1C). When the concentration of Thy was 20 μM, the reduction in DOX-induced pericardial edema and the increase in the SV-BA distance were the most significant.

As shown in Figure 1D, after 24 h of drug administration, the ventricular volume in the DOX model group increased. Compared with those in the blank control group, the heart rate, stroke volume, ejection fraction, and SAS rate of the zebrafish in the DOX model group were significantly lower. Compared with the DOX model group, when the Thy concentration was 10 μM or 20 μM, the heart rate, stroke volume, ejection fraction, and SAS rate of the zebrafish significantly increased. With 40 μM Thy, the heart rate, ejection fraction and SAS rate of the zebrafish increased, but the stroke volume did not change (Figures 1E–H).

As shown in Figures 2A,B, compared with that in the blank control group, the blood flow velocity in the tail of the zebrafish in the DOX model group was significantly lower. Compared with that in the DOX model group, the blood flow velocity in the DEX-positive control group and the 20 μM Thy treatment group was significantly greater and close to the level observed in the control group.

Twenty-four hours after drug administration, erythrocyte staining of the hearts of the zebrafish in each group was performed; the results are shown in Figure 3A. Compared with that in the blank control group, the area of erythrocyte staining in the DOX-treated zebrafish was significantly lower. Compared with that in the DOX model group, the area of staining area in the zebrafish in the DEX-positive control group and the Thy-treated group was significantly greater; 20 μM Thy had the best effect against DOX-induced myocardial ischemia (Figure 3B).

The AO staining and TUNEL staining results are shown in Figures 4A,B. Compared with those in the hearts of zebrafish in the blank group, there were more fluorescent-labeled apoptotic cells in the hearts of zebrafish in the DOX model group. Compared with those in the DOX model group, the number of fluorescent dots and fluorescence intensity in the heart area was significantly decreased after treatment with DEX and 10, 20 or 40 μM Thy, and the antiapoptotic effect on cardiac cells was most significant when the Thy concentration was 20 μM (Figures 4C,D).

The H&E staining results are shown in Figure 5A. The sizes of the atria and ventricles in zebrafish in the normal control group were normal, and the intercellular gap between cardiomyocytes was normal; cardiomyocytes were tightly arranged (black arrow). In the DOX model group, the atria and ventricles in zebrafish were significantly dilated and enlarged, and the intercellular gap between cardiomyocytes was larger than that observed in the normal control group; cardiomyocytes were disorderedly arranged (indicated by the orange arrow). Compared with those in the DOX model group, the degree of dilatation of the heart in zebrafish was significantly greater after treatment with 20 μM Thy, and cardiomyocytes were arranged more tightly. The degree of vacuolization in the heart tissue is shown in Figure 5B. Compared with the control group of zebrafish, the vacuolization area in the DOX model group was significantly larger. Compared with the DOX model group, the vacuolization area in the zebrafish heart decreased after treatment with 20 μM Thy.

After drug administration, Oil red O staining of the zebrafish in each group was performed to observe the effect of Thy on lipid accumulation in the hearts of zebrafish with DOX-induced cardiomyopathy. As shown in Figure 6, compared with those in zebrafish in the blank control group, the hearts of zebrafish in the DOX model group were darker, as shown by Oil red O staining. Compared with that in the DOX model group, the degree of staining in the hearts of zebrafish in the 10, 20, and 40 μM Thy treatment group was significantly lower.

The mRNA expression levels of genes related to the PPAR signaling pathway were assessed via RT‒qPCR. Compared with that in the blank group, the mRNA expression levels of pparg, apoa1a, acsl5, cpt1, pltp, fabp1b.1, slc27a2a, and lpl were lower. Compared with those in the DOX model group, the mRNA expression levels of pparg, apoa1a, acsl5, cpt1, slc27a2a, and lpl were higher in the 10 μM Thy treatment group; in the 20 μM Thy treatment group, the mRNA expression levels of apoa1a, acsl5, cpt1, pltp, fabp1b.1, and slc27a2a were higher, and the expression of the other genes presented no significant changes; and in the 40 μM Thy treatment group, the mRNA expression levels of pparg, apoa1a, acsl5, cpt1, pltp, fabp1b.1, slc27a2a, and lpl were higher (Figure 7).

The mRNA expression levels of ferroptosis pathway-related genes were assessed via RT‒qPCR. Compared with those in the blank control group, the expression levels of zgc:198419 and zgc:92006 tended to be higher and the mRNA expression levels of tfa and si:ch211-254p-10.2 were lower in the DOX model group. Compared with those in the DOX model group, the mRNA expression levels of si:ch211-254p-10.2 were higher and the mRNA expression levels of zgc:92006 were lower in the 10 μM Thy treatment group; in the 20 μM Thy treatment groups, the mRNA expression levels of zgc:198419 and zgc:92006 were lower, and the mRNA expression levels of si:ch211-254p-10.2 were higher; and in the 40 μM Thy treatment group, the mRNA expression levels of zgc:198419 and zgc:92006 were lower, and the mRNA expression levels of tfa and si:ch211-254p-10.2 were higher (Figure 7).