Engeletin (HY-N0436), Doxorubicin (HY-15142A) were purchased from MedChemExpress (China). The antibodies used included Anti-beta Actin Rabbit pAb (Servicebio, 1:1000 dilution, #GB11001), Bax Rabbit pAb (ABclonal, 1:1000 dilution, #A12009), Bcl2 Mouse Monoclonal Antibody (Proteintech, 1:1000 dilution, #66799-1-Ig), Beclin 1 Antibody (Abmart, 1:1000 dilution, #T55092), LC3B Antibody (Abmart, 1:1000 dilution, #T55992), TNF Alpha antibody (Proteintech, 1:500 dilution, #60291-1-Ig), AMPK alpha 1 Antibody (Abmart, 1:1000 dilution, #T55326), Phospho-AMPK alpha (T172) Antibody (Abmart, 1:1000 dilution, #T55608), BNP Antibody (Abmart, 1:1000 dilution, #PK54192), ANP Antibody (Abmart, 1:1000 dilution, #T57175), HRP-conjugated Goat Anti-Mouse IgG (H + L) (Proteintech, 1:5000 dilution, #SA00001-1) and HRP-conjugated Goat Anti-Rabbit IgG (H + L) (Proteintech, 1:5000 dilution, #SA00001-2).

The animal feeding and experimental procedures were conducted in accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH), and were approved by the Animal Ethics Committee of The Central Hospital of Enshi Tujia and Miao Autonomous Prefecture. Male C57BL/6 mice were purchased from the Experimental Animal Center of China Three Gorges University. Prior to the commencement of the experiments, the mice underwent a one-week acclimatization period. All mice were raised under a 12-h light/dark cycle in a pathogen-free environment, with controlled conditions (temperature: 20 °C ± 2 °C; humidity: 40%–60%), and were provided with ad libitum access to water and food. Upon conclusion of the experiment, the mice were anesthetized using 5% isoflurane and subsequently euthanized via cervical dislocation.

To investigate the effects of ENG in vivo, we established a chronic DIC mouse model by intraperitoneal injection of DOX (5 mg/kg/w for 5 weeks) (Wu et al., 2024) and intervened with intraperitoneal injection of ENG (20 mg/kg/d for 2 weeks) (Fang et al., 2023). Twenty 8-week-old male mice were randomly divided into four groups (five per group): (1) Control group: intraperitoneal injection of an equal volume of saline. (2) ENG group: 20 mg/kg/d of ENG for 2 weeks. (3) DOX group: 5 mg/kg/w of DOX for 5 weeks. (4) DOX + ENG group: 20 mg/kg/d of ENG for 2 weeks, and 5 mg/kg/w of DOX for 5 weeks. At the conclusion of week 6, echocardiographic assessments were conducted, followed by the collection of blood samples and heart tissue from the mice.

To assess the cardiac function of mice, after anesthesia and weighing, the left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were recorded using a Vinno6 echocardiography system (Vinno Technology, Suzhou, Jiangsu, China) operating at 15.0 MHz.

After the mice were executed, peripheral blood was collected in an EDTA tube and centrifuged at 1500 rpm for 15 min at 4 °C. Serum was extracted for the determination of malondialdehyde (MDA) levels using an MDA assay kit (#G4300, Servicebio), following the manufacturer’s instructions.

The isolated mice heart tissues were fixed with 4% paraformaldehyde, followed by dehydrated, embedded, and cut into 4-μm thick sections. Hematoxylin and eosin (HE) staining was performed on the sections to examine cardiac histomorphology. Masson staining was used to assess myocardial fibrosis. Apoptosis was detected using TUNEL staining. Additionally, lipid peroxidation was assessed by staining heart sections with 4-hydroxynonenal (4HNE), and images were captured using fluorescence microscopy (Qu et al., 2022).

Fresh heart tissue was cut into small pieces (1–3 mm³), fixed with 2.5% glutaraldehyde at room temperature in the dark for 2 h, and then stored at 4 °C. Next, dehydration and embedding were performed. Ultrathin sections of tissue were made with a diamond knife and stained with uranyl acetate and lead citrate (Zhuo et al., 2023). Transmission electron microscopy was employed to capture images of the sections.

Cell viability was measured using a cell counting kit-8 (CCK-8, #G4103, Servicebio). H9C2 cells (Wuhan Zishan Biotechnology Co., Ltd.) were seeded at a density of 1 × 10⁴/well in 96-well plates and cultured for 24 h. Next, the cells were treated with different concentrations of ENG. Then, according to the kit instructions, 10 μL of CCK8 reagent was added to each group and incubated for 2 h. Absorbance was subsequently measured at 450 nm using a microplate reader to evaluate cell viability (Lu et al., 2023).

H9C2 cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin. The cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C. When the cells reached 80% confluency, we performed cell passaging and processing. To investigate the role of ENG in the in vitro model of DIC, we established this model using H9C2 cells treated with 1 µM DOX for 24 h (Qiu et al., 2024), and employed ENG at a concentration of 80 µM for 24 h (Li et al., 2022). as an interventional therapy. H9C2 cells were randomly assigned to four groups: the Control group (treated with an equal volume of solvent for 24 h), the DOX group (exposed to 1 µM DOX for 24 h), the ENG group (administered 80 µM ENG for 24 h), and the DOX + ENG group (pre-treated with 80 µM ENG for 2 h, followed by co-treatment with 1 µM DOX for 24 h). To assess the AMPK pathway, H9C2 cells were pre-treated with the specific AMPK inhibitor Compound C (CC) at a concentration of 10 µM for 18 h (Zhou et al., 2022), subsequently treated with ENG for 2 h and then co-incubated with 1 µM DOX for 24 h (DOX + ENG + CC group).

Dihydroethidium (DHE, #G1904, Servicebio) was employed to measure reactive oxygen species (ROS) in H9C2 cells. In accordance with the manufacturer’s protocol, a 5 μM DHE detection working solution was added to the treated cells and incubated in a dark environment at 37 °C for 30 min within a cell culture incubator (Zhuo et al., 2023). Fluorescence images were subsequently acquired, and fluorescence intensity was evaluated using a fluorescence microscope.

Following the fixation and permeabilization of H9C2 cells cultured in 6-well plates, the apoptosis of cells was detected using an in situ apoptosis detection kit. Images were obtained using an Olympus FV 1000 laser scanning confocal microscope (Olympus, Japan).

Following drug treatment, H9C2 cells cultured in 6-well plates were fixed, permeabilized, and subjected to blocking of non-specific binding according to the protocol provided by the immunofluorescence (IF) staining kit. Subsequently, the cells were incubated overnight at 4 °C with a specific monoclonal antibody against Beclin 1 (Abmart, 1:1000 dilution, #T55092) and TNF-α (Proteintech, 1:500 dilution, #60291-1-Ig). After washing three times with phosphate-buffered saline (PBS), the cells were then incubated for 1 h at room temperature with a red fluorescent-labeled secondary antibody. Finally, DAPI was employed to stain the cell nuclei for fluorescence signal imaging (Lu et al., 2023).

The Annexin V-FITC Apoptosis Detection Kit was employed to quantify the apoptosis rate. Cardiomyocytes subjected to various treatments were harvested, rinsed twice with cold PBS, and subsequently incubated with annexin V-FITC and propidium iodide (PI) for 15 min at ambient temperature in the absence of light. Apoptosis was ultimately assessed using a flow cytometer (Zhong Z. et al., 2023).

The myocardial tissues and H9C2 cells were collected and homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors, followed by a 25-min lysis on ice to extract total proteins. The supernatant was collected after centrifugation at 12,000 rpm for 30 min at 4 °C. Protein concentration was determined using the BCA protein quantification kit (#AR0146, BOSTER, Wuhan, China). Samples were diluted with loading buffer and denatured by heating at 95 °C for 5 min. Equal amounts of protein samples (30 μg/well) were subjected to electrophoretic separation on a 12% sodium dodecyl sulfate-polyacrylamide gel and subsequently transferred onto a 0.45 μm PVDF membrane. The membranes were blocked with 5% skimmed milk at room temperature for 1 hour before being incubated overnight with specific primary antibodies at 4 °C. After washing, the membranes were incubated with secondary antibodies at room temperature for 2 h (Fan D. et al., 2023). Protein bands were then visualized using a highly sensitive ECL chemiluminescence kit (#BMU102-CN, Abbkine, China) in the ChemiDoc XRS + system (Bio-Rad). Finally, quantitative analysis was conducted using ImageJ software (National Institutes of Health, United States).

RNA was extracted from H9C2 cells utilizing the RNA Rapid Extraction Kit (RNO7, Aidlab, China) in accordance with the manufacturer’s instructions. 1 μg of RNA was reverse transcribed into cDNA using the cDNA Reverse Transcription Kit in a 20 μL reaction system. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) was performed using a PCR detection system (7500fast, Applied Biosystems, Foster City, CA, United States) to detect the mRNA expression level (Fan D. et al., 2023).

At the conclusion of the treatment, the mice were euthanized, and peripheral blood samples were collected in tubes containing EDTA. The blood samples were centrifuged at 1500 rpm for 15 min at 4 °C to isolate serum, which was subsequently used to measure B-type Natriuretic Peptide (BNP) levels, a recognized biomarker of myocardial injury. BNP levels in mouse serum were quantified using an ELISA kit (#AB-K554102, Abmart), following the manufacturer’s recommended protocol (Zhuo et al., 2023).

Statistical analyses were conducted using GraphPad Prism version 9.5 (GraphPad Software, San Diego, CA, United States). Quantitative data are presented as mean ± standard error of the mean (SEM). A one-way analysis of variance (ANOVA) was employed to assess differences among multiple groups, followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant for differences.

To investigate the potential role of ENG in DIC, we established a mice model of chronic myocardial injury by intraperitoneal injection of DOX (5 mg/kg) once a week for 5 weeks. Prior to DOX treatment, mice were treated with ENG (20 mg/kg) intraperitoneally once a day for 14 days (Figures 1A,B). The mice treated with DOX exhibited significant weight loss and a decrease in heart volume, which was mitigated by ENG treatment. HE and Mosson staining were used to evaluate cardiac tissue structure, and the results showed that myocardial morphology was disordered, and perivascular and interstitial fibrosis was increased in DOX group. However, these manifestations were significantly improved in mice treated with ENG (Figures 1C–E). The serum BNP concentration, as measured by the ELISA kit, demonstrated that the DOX group exhibited a significantly higher BNP level compared to the control group, and ENG treatment markedly ameliorated this elevation (Figure 1F). M-mode echocardiography was employed to evaluate cardiac function, and the findings indicated DOX-treated mice had significant cardiac function reduction. LVEF, LVFS, LVEDd, and LVESd of mice were significantly restored after ENG treatment (Figures 1G–K). No significant differences were observed in the aforementioned results between the control group and the ENG group.

Several studies have demonstrated that DOX can exacerbate cardiomyocyte apoptosis and impede autophagy (Zhang et al., 2023; Fan X. et al., 2023). To investigate the potential of ENG in mitigating these effects, we assessed key markers of apoptosis and autophagy in cardiac tissues derived from DOX and ENG-treated mice. TUNEL staining of myocardial tissue revealed a significant elevation in apoptotic cells among the mice administered DOX, whereas a substantial reduction in apoptotic cells was observed in the mice treated with ENG (Figures 2A,B). Western blot analysis indicated that ENG mitigated the upregulation of Bax and downregulation of Bcl-2 induced by DOX, while also inhibiting the downregulation of beclin-1 and LC3B caused by DOX treatment (Figures 2C–F). No significant differences were detected between the control and ENG groups in these parameters. These findings substantiate that ENG effectively inhibits DOX-induced apoptosis and restores autophagic function in myocardial tissues of mice.

Previous studies have implicated oxidative stress in the development of DIC (Chen et al., 2024). In this study, we evaluated several main indicators of oxidative stress, including MDA levels, 4-HNE levels and DHE staining. Firstly, DHE staining of H9C2 cells showed that The levels of ROS were significantly increased in the DOX group compared to the control and ENG groups, whereas ROS were significantly decreased in the DOX + ENG group compared with the DOX group (Figures 3A,B). We further conducted 4-HNE staining on myocardial tissue from various treatment groups, observing minimal green fluorescence intensity in the control and ENG groups. In contrast, the DOX group exhibited a marked increase in green fluorescence intensity, which was significantly diminished in the DOX + ENG group (Figures 3A–C). Additionally, serum MDA levels were measured across different treatment groups, revealing a significantly higher concentration in the DOX group compared to the other groups, with statistical significance (Figure 3D). The results suggested that ENG alleviated the oxidative stress response induced by DOX.

To assess the protective effect of ENG on mitochondrial function, we conducted a transmission electron microscope examination of myocardial tissue to observe mitochondrial morphology. Electron microscopy revealed mitochondrial damage, including matrix swelling, cristae fragmentation, breakage, and disappearance in the DOX group. Conversely, this phenomenon was reversed in the DOX + ENG group (Figure 3E). There was no obvious mitochondrial damage in the control and ENG groups. The findings indicate that ENG reversed DOX-induced mitochondrial damage in cardiomyocytes.

Prior to conducting experiments on H9C2 cells, cardiomyocytes were exposed to varying concentrations of ENG (0, 5, 10, 20, 40, 80, and 160 µM), and cell viability was assessed by CCK-8 kit (Figure 4A). The results showed that ENG at low concentration had no significant cytotoxic effect on cardiac myocytes. Based on the results of previous studies (Li et al., 2022) and our cell viability assay, ENG concentration of 80 µM was selected to treat cardiomyocytes in the subsequent investigations.

H9C2 cells were then categorized into different experimental groups for comparative analysis using RT-qPCR, Western blot analysis and TUNEL staining. In the DOX group, the mRNA expression levels of the pro-apoptotic marker Bax were significantly elevated compared to those in the control and ENG group. ENG treatment ameliorated the DOX-induced upregulation of Bax and downregulation of Bcl-2 (Figures 4B,C). Western blot analysis corroborated these findings, showing an upregulation of Bax and a downregulation of Bcl-2 in the DOX group, which were reversed in the DOX + ENG group. No significant differences in expression were observed between the control and ENG groups (Figures 4D,E). In the TUNEL staining of cardiomyocytes, we observed a significantly higher number of apoptotic cells in the DOX group than other groups, and no significant difference was observed between the control and ENG groups (Figures 4F,G). These results indicated that ENG significantly attenuated DOX-induced cardiomyocyte apoptosis in vitro. Western blot analysis of the aforementioned cell groups revealed that the expression levels of ANP and BNP were elevated in the DOX group, and these levels were markedly reduced following ENG treatment. Furthermore, no significant difference in expression was observed between the control and ENG groups, indicating that ENG effectively alleviated DOX-induced functional damage in cardiomyocytes (Figures 4H–J).

To verify the protective effect of ENG in DOX-induced inflammatory responses in cardiomyocytes, we conducted in vitro experiments. The RT-qPCR results revealed a marked upregulation in mRNA expression levels of IL-6 and IL-1β in the DOX group, while the control group and ENG groups exhibited low expression levels and not significantly different between the two groups. Treatment with ENG effectively reduced DOX-induced inflammatory mRNA expression (Figures 5A,B). Furthermore, TNF-α immunofluorescence staining in cardiomyocytes corroborated the RT-qPCR findings (Figures 5I,J), thereby substantiating that ENG alleviates the inflammatory response induced by DOX in cardiomyocytes.

At the same time, we evaluated the protective effect of ENG on cardiomyocyte autophagy function through in vitro experiments. First, we determined the Beclin-1 mRNA expression levels of different groups, and the results suggest that autophagy function was impaired in the DOX group, whereas cardiomyocyte autophagy was restored after ENG treatment, and there was no significant difference in expression between the control and ENG groups (Figure 5C). Secondly, Western blot analysis was employed to detect the autophagy markers Beclin-1 and LC3B. The expression of both Beclin-1 and LC3B was significantly reduced in the DOX group but upregulated in the DOX + ENG group (Figures 5D–F). Finally, immunofluorescence staining for Beclin-1 was utilized to validate cardiomyocyte autophagy among different groups, demonstrating that ENG treatment could rectify the DOX-induced decline in autophagic function (Figures 5G,H).

Previous studies have demonstrated that DOX can cause cardiotoxicity through the AMPK pathway and that ENG may play a role in other diseases through the AMPK pathway (Zhuo et al., 2023; Zhong Z. et al., 2023; Kong L. et al., 2022). In our study, Western blot analysis revealed a significantly reduced expression of phosphorylated AMPK (p-AMPK) in the DOX group compared to other groups. No significant difference in p-AMPK expression was observed between the control group and the ENG group. However, p-AMPK expression was upregulated in the DOX + ENG group, and the p-AMPK/AMPK ratio was notably lower in the DOX group compared to the DOX + ENG group (Figures 6A,B). To further verify the mechanism, when cardiomyocytes treated with both DOX and ENG were exposed to Compound C (CC), a specific inhibitor of AMPK, there was a downregulation of p-AMPK expression suggesting that CC blocked activation of the AMPK signaling pathway by ENG (Figures 6C,D).

Subsequently, Western blot analysis was used to detect the apoptosis and autophagy of cardiomyocytes. ENG ameliorated DOX-induced apoptosis and impaired autophagy, whereas the addition of CC inhibited this protective effect of ENG (Figures 6E–H). Apoptosis in cardiomyocytes across different groups was evaluated using flow cytometry, revealing a significantly higher number of apoptotic cells in the DOX group compared to both the control and ENG groups. Furthermore, the DOX + ENG + CC group exhibited a significantly higher number of apoptotic cells than the DOX + ENG group (Figure 6I). Finally, we detected oxidative stress in each group by DHE staining. Consistent with the previous results, oxidative stress was evident in the DOX group, while it was alleviated in the DOX + ENG group, but the addition of CC significantly increased oxidative stress in cardiomyocytes (Figures 6J,K). These results indicated that CC blocked the effects of ENG in ameliorating apoptosis, autophagy and oxidative stress in cardiomyocytes by inhibiting the AMPK pathway, and verified that ENG alleviated DIC by activating AMPK signaling pathway.