The animal protocols performed in the present study were approved by the Ethics Committee of People's Hospital Affiliated to Nanchang University (Nanchang, China; approval no. 2019-037). After the experiments, animals were anesthetized by 5% isoflurane, followed by decapitation.

H9c2 myocardial cells were purchased from BeNa Culture Collection (Beijing Beina Chunglian Institute of Biotechnology; cat. no. BNCC295057) and were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in an incubator with 5% CO₂ at 37°C. The effects of SP on the viability of H9c2 cells were determined using the Cell Counting Kit-8 (CCK-8) assay (Beyotime Institute of Biotechnology). The protective effects of SP on doxorubicin-induced cell injury were evaluated as follows: After cells had adhered to the substrate, culture medium was discarded and the cells were washed with 1X phosphate buffered saline (PBS). Doxorubicin (2 µM; cat. no. D107159; Shanghai Aladdin Biochemical Technology Co., Ltd.; model group) or 2 µM doxorubicin + 1 µg/ml exogenous SP (cat. no. HY-P0201A; MedChemExpress) medium (SP group) were then added to paired wells; after 24 h at 37°C, flow cytometry and western blotting were performed. The cells in the control group did not receive any treatment.

After H9c2 cells had completely adhered to the well, the supernatant was discarded and different concentrations of SP (0, 0.1, 0.5, 1, 5 and 10 µg/ml) suspended in fresh culture medium were added to the cells. The cells were cultured in an incubator at 37°C for 24 h and cell viability was assessed to screen for safe concentrations of SP. Briefly, following treatment, 10 µl CCK-8 solution was added to each well and incubated at 37°C for 1.5 h. Optical density was measured at a 450 nm using a microplate reader to calculate cell viability at each SP concentration and 1 µg/ml SP was selected to investigate its effect on doxorubicin-induced cardiomyocyte injury.

Following treatment for 24 h, cells were collected, washed with PBS and centrifuged at 897 × g for 3 min at 4°C. Annexin V-fluorescein isothiocyanate (3 µl) and propidium iodide (5 µl) were added to the cells according to the instructions of the assay kit (cat. no. C1062S; Beyotime Institute of Biotechnology). After gentle mixing, the cells were incubated at room temperature for 10 min in the dark and apoptosis was measured by flow cytometry (NovoCyte^® 2060R; ACEA Biosciences, Inc.) and analyzed using FlowJo 7.6 (FlowJo, LLC), The percentage of early + late apoptotic cells were counted.

A total of 18 male Sprague Dawley rats (age, 2 months; weight, 200±20 g) were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. [license no. SCXK (Xiang) 2019-0004]. The animals were housed in a specific pathogen-free condition that was automatically maintained at a temperature of 23±2°C, a relative humidity of 45–65%, and with a controlled 12 h light/dark cycle and free to access to food and water. The animals were divided into three groups (n=6/group): i) Control group; ii) model group; and iii) SP treatment group. The heart-failure rat model was prepared as previously described (23). Briefly, doxorubicin was injected intraperitoneally once every 3 days (3 mg/kg) with a cumulative total of 15 mg/kg. The injections were completed within 2 weeks to establish the rat myocardial injury model. SP was injected via the caudal vein at a dose of 6.7 µg/kg once every 4 days as previously described (24). The rats in the control and model groups were injected with the same amount of saline. An electrocardiogram (ECG) was used to monitor heart function after treatment and heart rate was automatically recorded. Thereafter, the rats were decapitated following anesthesia (5% isoflurane). Myocardial tissue was collected and fixed in 4% paraformaldehyde at 4°C overnight for determination of pathological changes.

Fixed tissue was washed with running water for several hours, serially dehydrated in 70, 80 and 90% ethanol, a mixture of ethanol and xylene for 15 min, and then xylene for 30 min. The tissues were subsequently immersed in a mixture of xylene and paraffin for 15 min, and then in paraffin for 50–60 min. The paraffin-embedded tissues were then sectioned (10 µm). After warming, dewaxing and rehydrating, the sections were stained with hematoxylin (3%) and eosin (3%) for 5 min at room temperature. The sections were observed under a light microscope (BX53, Olympus Corporation).

After dewaxing (xylene treatment for 10 min) and rehydrating in a gradient ethanol series at room temperature, 50 µg/ml proteinase K was added and the sections were incubated at 37°C for 30 min. The tissues were then washed with PBS three times (5 min/wash). The PBS was removed and TUNEL solution (5 µl/ml; Beyotime Institute of Biotechnology) was added to each slide and incubated at 45°C for 2 h in the dark, followed by DAPI (5 µg/ml) staining at room temperature for 5 min. The liquid on the slide was dried using absorbent paper and the slide was sealed and observed under a fluorescence microscope.

Myocardial tissues were placed in 2.5% glutaraldehyde at 4°C for 4 h followed by fixation with 1% osmium tetroxide for 1.5 h. and washed three times with pre-cooled PBS. After dehydration with a gradient ethanol series and acetone, the tissues were incubated in epoxy resin overnight at room temperature prior to sectioning (2 nm slices). After that, the slices were stained with 2% uranyl acetate and 0.5% lead citrate for 5 min at room temperature. Autophagic ultrastructure was observed by transmission electron microscopy (HT7700; Hitachi High-Technologies Corporation; magnification, ×8,000).

Myocardial tissue was ground into powder in liquid nitrogen. Proteins were extracted from tissues and H9c2 cells using a protein isolation kit (cat. no. 28-9425-44; Cytiva) and protein concentration was determined by the bicinchoninic acid method. The proteins (25 µg/lane) were denatured and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis for 2 h (12% gel), followed by transfer to a nitrocellulose membrane (300 mA was applied for 80 min) as described previously (25,26). The membranes were blocked in 5% skimmed milk for 2 h at room temperature. Thereafter, the membrane was incubated with primary antibodies at 4°C overnight, and then after washing, the membrane was incubated with a secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG; 1:100; cat. no. ab6721; Abcam) at room temperature for 2 h. Dye solution from an enhanced chemiluminescence kit (cat. no. RPN2133; Cytiva) was added to the membrane and staining was visualized using a gel imaging system (Bio-Rad Laboratories, Inc.). The gray value was analyzed by Quantity One software (version 4.62; Bio-Rad Laboratories, Inc.). The primary antibodies included mouse monoclonal anti-β-actin (1:2,000; cat. no. TA-09; OriGene Technologies, Inc.), mouse anti-B-cell lymphoma 2 (Bcl-2; 1:500; cat. no. ab692; Abcam), rabbit anti-Bcl-2-associated X protein (Bax; 1:500; cat. no. A0207; ABclonal Biotech Co., Ltd.), rabbit anti-Beclin-1 (1:1,000; cat. no. ab62557; Abcam) and rabbit anti-microtubule-associated protein 1A/1B-light chain 3 (LC3-II; 1:500; cat. no. bs-8878R; BIOSS).

All data were expressed as the mean ± standard deviation with six repeats in both the animal and cell culture experiments. Statistical analysis was carried out with GraphPad Prism 7 (GraphPad Software, Inc.) using one-way ANOVA followed by the Bonferroni test. P<0.05 was considered to indicate a statistically significant difference.

H9c2 myocardial cells were treated with different concentrations of SP for 24 h. Viability of H9c2 cells was detected using the CCK-8 method. SP at concentrations ranging between 0.1 and 1.0 µg/ml did not affect H9c2 cell viability (Fig. 1). By contrast, 5 or 10 µg/ml SP significantly reduced cell viability compared with untreated controls.

Using the results of the CCK-8 assay, 1 µg/ml SP was selected to investigate the effect of SP on doxorubicin-induced injury of H9c2 cells. The apoptosis of H9c2 cells treated with doxorubicin was significantly higher compared with that detected in the control H9c2 cells, whereas SP significantly reduced this effect (Fig. 2).

In order to further explore the specific effects of SP on apoptosis and autophagy, the protein expression levels of Bcl-2, Bax, Beclin-1 and LC3 were monitored in H9c2 cells. As shown in Fig. 3, Bcl-2 expression in doxorubicin-treated H9c2 cells was significantly lower compared with that in the control group, whereas the addition of SP to the doxorubicin-treated cells had no effect on Bcl-2 expression. Bax expression was comparable in all three groups, which indicated that Bax was unaffected by doxorubicin and SP.

Compared with in the control H9c2 cells, doxorubicin treatment reduced the expression levels of Beclin-1 and LC3. SP further reduced Beclin-1 expression, but inhibited the doxorubicin-induced decrease in LC3 (Fig. 3). These data suggested that SP might rescue doxorubicin-induced autophagy dysfunction.

Of the 12 rats used in the present study, none died during the experiments. Compared with the control rats, the food intake and body weight of the rats in the heart-failure group were significantly decreased (Fig. 4). Administration of SP to the heart-failure rats was associated with no significant change in body weight for 12 days; however, food intake rose on days 10–12.

Subsequently, a six-lead ECG was used to monitor heart function and representative traces are shown in Fig. 5. Heart rate was also quantified in the three groups. Compared with in the control group, heart rate in the heart-failure group was significantly decreased, whereas SP treatment inhibited this decrease (Fig. 5).

The results of H&E staining of rat myocardial tissue are shown in Fig. 6. Compared with in the control group, the cardiomyocytes in the doxorubicin-induced heart-failure group were arranged in a disordered pattern and were loosely connected. SP treatment ameliorated these pathological changes caused by doxorubicin.

Compared with in the control group, cardiomyocytes in the heart-failure group exhibited a loss of striations, vacuolation with damaged organelle, indicating dysfunction of autophagy. By contrast, SP reduced the loss of striations, as well as vacuolation (Fig. 7).

TUNEL staining was used to determine the level of apoptosis in myocardial tissue. Apoptosis in myocardial tissue from the heart-failure group was significantly higher compared with that in the control group, whereas this effect was inhibited by SP treatment (Fig. 8).

Compared with in the control group, there was a significant decrease in the protein expression levels of Bcl-2, Beclin-1 and LC3, but not Bax, in the heart-failure group (Fig. 9). SP inhibited these changes in LC3, although Beclin-1 expression was further reduced. Additionally, SP increased Bax expression, while it did not affect Bcl-2 expression compared with the model group. These results indicated that SP promoted autophagy, while reducing apoptosis.