A total of 60 male Sprague–Dawley rats were obtained from the Guangdong Institute for Product Quality Supervision and Inspection (license no. SYXK [Yue] 2023-0181). The animals were 6–8 weeks old and weighed 180–220 g; they were housed in groups at the institutional animal experimental centre under controlled conditions (temperature 23–27 °C, relative humidity 50%–60%, and a 12-h light/12-h dark cycle). All rats were allowed a one-week acclimatisation period before the start of the experiment. Throughout the study, animals had free access to standard chow and water.

Doxorubicin was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd.; bisoprolol from Hangzhou Minsheng Pharmaceutical Co., Ltd.; and sacubitril/valsartan from Novartis (China) Biomedical Research Co., Ltd. Haematoxylin–eosin staining reagents were obtained from Beyotime Biotechnology (Shanghai, China). Commercial ELISA kits for CK-MB, SOD, BNP, cTnI, and MDA were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. The catalog numbers and specifications were as follows: CK-MB ELISA kit (Cat# ML037723), SOD ELISA kit (Cat# ML077379), BNP ELISA kit (Cat# ML059422), cTnI ELISA kit (Cat# 059111), MDA ELISA kit (Cat# ML077384). Primary antibodies against nuclear factor erythroid 2-related factor 2 (Nrf2), haem oxygenase-1 (HO-1), Kelch-like ECH-associated protein 1 (Keap1), and GAPDH were obtained from Abcam (Cambridge, UK). The following antibodies were used: anti-Nrf2 (ab137550, rabbit monoclonal, 1:1000, validated for rat), anti-HO-1 (ab68477, rabbit monoclonal, 1:1000, validated for rat), anti-Keap1 (ab227828, rabbit monoclonal, 1:1000, validated for rat), and anti-GAPDH (ab181602, rabbit monoclonal, 1:5000, validated for rat). HRP-conjugated secondary antibody (ab205718, goat anti-rabbit IgG, 1:5000) was also purchased from Abcam.

Cardiac function was assessed using a VisualSonics Vevo 2100 high-resolution small-animal ultrasound system (FUJIFILM VisualSonics, Canada). Flow cytometric analyses were performed with an Attune™ NxT flow cytometer (Thermo Fisher Scientific, USA).

Sixty rats were randomly assigned to five groups (n = 12 per group): normal control (Control), doxorubicin (DOX), bisoprolol (Bisoprolol), sacubitril/valsartan (Valsartan), and combination therapy (Combine). Rats in the Bisoprolol, Valsartan, and combination groups received daily oral gavage of the respective drugs for five consecutive weeks, starting 1 week prior to the first doxorubicin administration and continuing throughout the entire experimental period. Specifically, the Bisoprolol group received bisoprolol (1.0 mg/kg), the Valsartan group received sacubitril/valsartan (30 mg/kg), and the combination group received both sacubitril/valsartan (30 mg/kg) and bisoprolol (1.0 mg/kg) once daily by oral gavage. Rats in the Control and DOX groups received an equivalent volume of 0.9% normal saline by oral gavage by oral gavage daily during the same period.

This pretreatment period was designed to ensure that steady-state plasma concentrations of the cardioprotective agents were achieved before the first doxorubicin dose, consistent with standard prophylactic protocols for preventing chemotherapy-induced cardiotoxicity.

One hour after gavage, rats in all groups except the Control group were administered doxorubicin intraperitoneally at a dose of 2.5 mg/kg once weekly for 5 weeks (cumulative dose 15 mg/kg). Rats in the Control group received an equivalent volume of 0.9% normal saline by intraperitoneal injection, and general health status was monitored throughout the experimental period.

Twenty-four hours after the final gavage, rats were fasted for 6 h before echocardiographic assessment. Anaesthesia was induced by intraperitoneal injection of 2% sodium pentobarbital (3 mL/kg), after which the chest hair was removed and the animals were placed supine with limbs secured to the examination platform to optimise intercostal access. Cardiac function was evaluated using a high-resolution small-animal echocardiography system.

Two-dimensional guided M-mode images were obtained from the parasternal long-axis view at the level of the papillary muscles. Left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were measured according to the American Society of Echocardiography guidelines. Left ventricular ejection fraction (LVEF) was calculated using the Teichholz formula: LVEF (%) = [(LVEDD³ - LVESD³)/LVEDD³] × 100. Left ventricular fractional shortening (LVFS) was calculated as: LVFS (%) = [(LVEDD - LVESD)/LVEDD] × 100. All measurements were averaged over three consecutive cardiac cycles.

Following echocardiographic examination, rats were euthanised by exsanguination under deep anaesthesia induced by an intraperitoneal injection of 2% sodium pentobarbital (100 mg/kg body weight). Following the confirmation of deep anesthesia (absence of pedal reflexes), the thoracic cavity was opened, blood samples were collected from the inferior vena cava, and the heart was rapidly excised to isolate myocardial tissue. Approximately 50 mg of left ventricular myocardium from each rat was fixed in paraformaldehyde, embedded, and sectioned at a thickness of 4 μm. For each animal, three non-consecutive sections were collected and stained with hematoxylin and eosin (H&E) for histopathological evaluation.

After deparaffinisation in xylene and rehydration through graded ethanol, sections were stained with haematoxylin for 10 min, differentiated briefly in acid alcohol, blued, and counterstained with eosin for 2 min. After dehydration and mounting with neutral resin, histopathological changes in myocardial tissue were examined under light microscopy.

For semi-quantitative analysis, myocardial injury was scored based on the following criteria modified from previous studies [Reference]: 0 = normal myocardium with regularly arranged cardiomyocytes and no visible damage; 1 = mild focal myofibrillar loss or cytoplasmic vacuolization involving <25% of the field; 2 = moderate multifocal myofibrillar loss, cytoplasmic vacuolization, or interstitial edema involving 25–50% of the field; 3 = severe confluent myofibrillar disorganization, nuclear pyknosis, karyorrhexis, or inflammatory cell infiltration involving >50% of the field. Five randomly selected fields per section (15 fields per animal) were evaluated, and the average score was calculated for each rat.

Myocardial tissue (30 mg) and blood samples were collected from rats in each group following the procedures described above. Blood samples were allowed to clot at room temperature for 30 min before centrifugation at 3,000 × g for 10 min (radius 12 cm) to obtain the supernatan. All samples were processed within 2 h of collection to ensure optimal analyte stability. The supernatants from myocardial homogenates and serum samples were used for quantitative determination of CK-MB, MDA, cTnI, BNP and SOD. All measurements were performed using commercially available ELISA kits, strictly according to the manufacturers’ instructions. All ELISA assays were completed within 4 weeks of sample collection, and samples were stored at −80 °C during this period.

Serum samples: Blood samples were centrifuged at 3,000 × g for 10 min (radius 12 cm) to obtain serum within 1 h of collection. To detect reactive oxygen species (ROS) levels in circulating extracellular vesicles (EVs) enriched fractions, we adapted a previously published method [17]. Briefly, 50–100 μL of serum was incubated with an equal volume of DCFH-DA working solution (diluted in serum-free PBS, final concentration 10 μmol/L) in the dark at 37 °C for 30 min. After incubation, samples were diluted with PBS and immediately analyzed by flow cytometry using an Attune™ NxT flow cytometer (Thermo Fisher Scientific, USA). The EV-enriched particle population was gated based on characteristic forward and side scatter properties, as previously described [17]. It should be noted that this scatter-based gating approach, while widely used for EV-rich fractions, cannot definitively exclude potential contributions from other submicron particles such as lipoproteins or protein aggregates that may share similar scatter characteristics. DCF fluorescence intensity within this gate was quantified to represent serum ROS levels. A total of 10,000 EV events were acquired per sample, and results were expressed as mean fluorescence intensity.

Myocardial tissue samples: Fresh myocardial tissue was enzymatically dissociated using type II collagenase and trypsin, followed by incubation at 37 °C for 30 min with gentle agitation every 10 min. Digestion was terminated by the addition of serum-containing medium, and the cell suspension was filtered through a 40 μm cell strainer to remove undigested tissue fragments, then centrifuged at 3,000 × g for 10 min (radius 12 cm). The total processing time from tissue collection to cell isolation was approximately 45 min. The resulting cell pellet was resuspended in pre-cooled PBS and incubated with DCFH-DA working solution (final concentration 10 μmol/L) at 37 °C for 45 min, with gentle mixing every 10 min. After incubation, cells were washed twice with PBS, resuspended in 500 μL PBS, and intracellular ROS fluorescence was measured by flow cytometry. The entire procedure from tissue collection to flow cytometry analysis was completed within 90 min. A total of 10,000 live cell events were acquired per sample, and results were expressed as mean fluorescence intensity. The analysis was performed on total live cells gated based on forward/side scatter properties, without discrimination of specific cardiac cell subtypes.

Approximately 30 mg of myocardial tissue from each rat was lysed in ice-cold lysis buffer for 30 min on ice. Lysates were centrifuged at 12,000 × g for 15 min (radius 12 cm), and the supernatant was collected for protein quantification using the BCA assay within 2 h of extraction. Equal amounts of protein (30 μg per lane) were mixed with loading buffer at a 1:4 ratio, denatured at 95 °C for 5 min, and separated by SDS–PAGE using stacking and resolving gels. Electrophoresis was performed at 80 V until proteins entered the resolving gel and then continued at 120 V until clear separation of the molecular weight marker was achieved. Proteins were transferred onto PVDF membranes, which were blocked with 5% non-fat milk for 60 min at room temperature. Membranes were then incubated overnight at 4 °C with primary antibodies against Nrf2 (1:1,000), HO-1 (1:1,000), Keap1 (1:1,000), and GAPDH (1:5,000), followed by incubation with appropriate secondary antibodies (1:5,000) for 60 min at room temperature. Protein bands were visualised using enhanced chemiluminescence, and band intensities were quantified using ImageJ software.

All data were analysed using SPSS software (version 25.0). Continuous variables that conformed to a normal distribution are presented as mean( x ¯ ±s). Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) t-test for post hoc pairwise comparisons. A two-sided P value <0.05 was considered to indicate statistical significance.

Rats in the Control group exhibited a glossy coat, normal food intake and activity, rapid responses, normal defecation, and good overall vitality. In contrast, rats in the DOX group showed dull fur, markedly reduced food intake and spontaneous activity, sluggish responses, and an overall lethargic appearance. Compared with the DOX group, general condition was noticeably improved in the Bisoprolol, Valsartan, and combination groups. The most pronounced improvement was observed in the combination group.

Compared with the Control group, rats in the DOX group showed significant increases in left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD), accompanied by marked reductions in left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) (all P < 0.05). Relative to the DOX group, LVEDD and LVESD were significantly reduced in the Bisoprolol, Valsartan, and combination groups, whereas LVFS and LVEF were significantly increased (all P < 0.05). Among these treatments, the combination therapy produced the greatest improvement in cardiac function (Figure 1).

HE staining revealed normal myocardial architecture in the Control group, with regularly arranged cardiomyocytes, clear striations, and well-defined nuclei and cytoplasm, without evidence of edema or inflammatory infiltration (Figure 2, Control panel). In contrast, the DOX group exhibited severe myocardial injury characterized by disorganized myofibrils, nuclear pyknosis or fragmentation, extensive cardiomyocyte necrosis, marked interstitial edema, and prominent inflammatory cell infiltration (Figure 2, DOX panel).

Myocardial damage was attenuated to varying degrees in the Bisoprolol, Valsartan, and combination groups compared with the DOX group (Figure 2, Bisoprolol, Valsartan, and Combine panels). Semi-quantitative histological scoring confirmed these observations: the DOX group showed significantly higher myocardial injury scores compared with the Control group (2.85 ± 0.42 vs. 0.32 ± 0.18, P < 0.001). All treatment groups significantly reduced injury scores versus the DOX group (Bisoprolol: 1.68 ± 0.35; Valsartan: 1.54 ± 0.31; Combine: 0.86 ± 0.24; all P < 0.05 vs. DOX), with the combination therapy showing the most substantial histological improvement and the lowest injury score among treatment groups (P < 0.05 vs. monotherapy groups). The most substantial histological improvement was observed in the combination group, with near-normal myocardial architecture and minimal residual damage (Figure 2, Combine panel).

Compared with the Control group, levels of creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), and brain natriuretic peptide (BNP) were significantly elevated in the DOX group (all P < 0.05). In contrast, CK-MB, cTnI, and BNP levels were significantly reduced in the Bisoprolol, Valsartan, and combination groups relative to the DOX group (all P < 0.05). Consistent with the functional and histological findings, the greatest reduction in myocardial enzyme levels was observed in the combination group (Figure 3).

Compared with the Control group, rats in the DOX group exhibited significantly increased reactive oxygen species (ROS) fluorescence intensity in both myocardial tissue and the EV-enriched serum fraction (P < 0.05). In contrast, ROS fluorescence intensity was markedly reduced in the Bisoprolol, Valsartan, and combination groups compared with the DOX group (all P < 0.05), with the combination therapy showing the most pronounced reduction (Figure 4). Myocardial ROS levels represent the average fluorescence of total live cells, as specific cell-type markers were not used in this study.

Relative to the Control group, myocardial and serum malondialdehyde (MDA) levels were significantly elevated in the DOX group, whereas superoxide dismutase (SOD) activity was significantly reduced (P < 0.05). Compared with the DOX group, MDA levels were significantly decreased and SOD activity was significantly increased in the Bisoprolol, Valsartan, and combination groups (all P < 0.05). Consistent with other findings, the most substantial improvement in oxidative stress status was observed in the combination group (Figure 5).

Compared with the Control group, protein expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), haem oxygenase-1 (HO-1), and Kelch-like ECH-associated protein 1 (Keap1) were significantly reduced in the DOX group (P < 0.05). In contrast, expression of Nrf2, HO-1, and Keap1 was significantly upregulated in the Bisoprolol, Valsartan, and combination groups compared with the DOX group (all P < 0.05). The greatest increase in protein expression was again observed in the combination group (Figure 6).