All procedures involving animals were approved by the Ethics Committee for Animal Research of Wuhan University (Wuhan, China). All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and the National Research Council (12). Male Sprague-Dawley (SD) rats, aged 6–8 weeks and weighing 150–180 g, were purchased from the Experimental Animal Center of Wuhan University. All animals were acclimated to the laboratory for 7 days prior to the experiment and were maintained in a light-controlled room (12-h light/dark cycle) at an ambient temperature of 25° with free access to water and standard chow.

Sixty male SD rats were randomly divided into four groups, namely the control group, ethyl pyruvate (EP) group, Adriamycin (ADR) group and ADR + EP group (n=15 per group). Rats in the ADR and ADR + EP groups were treated with ADR (Actavis Italy SpA, Nerviano, Italy) at a dose of 2.5 mg/kg/week via intraperitoneal injection for 6 weeks. By contrast, rats in the control and EP groups were treated with normal saline (via intraperitoneal injection) at the same dose as ADR for 6 weeks. From the eighth week, rats in the EP group and the ADR + EP group received EP (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) via stomach lavaging at a dose of 50 mg/kg/day for 30 days. Upon the completion of 30 days treatment with EP, cardiac function was accessed by echocardiography. Rats were then sacrificed by an overdose of anesthesia (3.5 mg/100 g pentobarbital) for the harvesting of heart tissues.

Echocardiography was performed using a high-resolution ultrasound imaging system equipped with a 7V3 probe with a frequency of 6.0 MHz (Acuson Sequoia; Siemens, Washington, DC, USA). Fractional shortening (FS), left ventricular internal dimension diastolic (LVIDD) and left ventricular internal dimension systole (LVIDS) were recorded from the parasternal long-axis M-mode images using averaged measurements from 3–5 consecutive cardiac cycles. End diastolic volume (EDV) and end systolic volume (ESV) were calculated from bi-dimensional long-axis parasternal views by means of the single-plane area-length method. The ejection fraction (EF) was calculated as follows: EF (%) = (LVEDV - LVESV) / LVEDV × 100.

Myocardial tissues excised by horizontal intercept from the middle part of the whole heart were fixed in 10% buffered formalin for 24 h, and then embedded in paraffin and sliced into 5-µm sections. The sections were stained with picrosirius red (PSR) to identify collagen deposition, and then were visualized using light microscopy. Fibrillar collagen was visualized under the microscope and the left ventricular collagen volume fraction was measured using a quantitative digital image analysis system (Image-Pro Plus 6.0; Media Cybernetics, Inc., Rockville, MD, USA). The cardiomyocyte apoptosis rate was also assessed using a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay.

Briefly, sections (3-µm-thick) from formalin-fixed and paraffin-embedded myocardial tissue were deparaffinized with xylene and dehydrated with ethanol. Slides were rinsed twice with PBS and treated with proteinase K (15l g/ml in 10 mM Tris/HCl; pH 7.4–8.0) for 15 min at 37°. Endogenous peroxidases were blocked with 3% hydrogen peroxide in methanol at room temperature for 10 min. Tissue sections were analyzed with an in situ cell death detection kit (Roche Diagnostics, Indianapolis, IN, USA), in accordance with the manufacturer's instructions. Reactions were visualized with fluorescence microscopy and were measured using a quantitative Image-Pro Plus 6.0 digital image analysis system.

RT-qPCR and western blotting were performed as previously described (13). Briefly, after total RNA was extracted from ventricles using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), first strand cDNA was synthesized using a Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics). qPCR was performed using SYBR Green PCR Master Mix (Roche Diagnostics) to determine the expression levels of genes of interest which were transforming growth factor-β1 (TGFβ-1), collagen type 1 α 1 (Colla1), collagen type 1 α 3 (Colla3), tissue inhibitor of metalloproteinase (TIMP)1, TIMP2, matrix metalloproteinase (MMP)2 and MMP9, and the results were normalized against GAPDH gene expression. PCR cycling conditions were as follows: Predenaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec, 60°C for 1 min and 72°C for 20 sec, and a final extension at 60°C for 5 min. The PCR primers that were used are shown in Table I. PCR reactions were repeated twice.

For western blotting, cardiac tissue was lysed in radioimmunoprecipitation assay buffer (Roche Diagnostics). Protein extracts (30 µg per lane) were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes and probed with primary antibodies overnight at 4°C. The primary antibodies included anti-GAPDH (1:1,000; sc-365062), anti-NADPH oxidase-4 (Nox4; 1:1,000; sc-517188), anti-Bax (1:1,000; sc-23959); anti-Bcl-2 (1:1,000; sc-7382) and anti-caspase-3 (1:1,000; sc-65496; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-NADPH oxidase-2 (Nox2; (1:1,000; ab80508; Abcam, Cambridge, UK). Following washing with TBST three times, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit/mouse secondary antibodies (1:100; G1201; Guge Biotechnology Co., Ltd., Wuhan, China) for 1 h at room temperature. Subsequently, the membranes were treated with ECL reagents (Bio-Rad Laboratories, Inc., Hercules, CA, USA) prior to visualization using a FluorChem E imager (ProteinSimple, San Jose, CA, USA) according to the manufacturer's instructions. The specific protein expression levels were normalized to the levels of GAPDH on the same PVDF membrane.

The activity of SOD in myocardial tissue was detected by the xanthine oxidase technique. This procedure is based upon the inhibition of nitrite (NIT) reduction due to the superoxide anion generated by the combination of xanthine and xanthine oxidase. An SOD assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to assess the SOD activity. One unit of SOD was the amount that caused a 50% inhibition in the rate of NIT reduction. The SOD activity was expressed as U/mg protein in myocardial tissue homogenate.

The content of MDA in myocardial tissue was assayed according to thiobarbituric acid (TBA) method. In this method MDA reacts with TBA under high temperature (90–100°C) and acidic conditions to form TBA reactive substances (TBARS). TBARS were measured using a spectrophotometer at 532 nm. An MDA assay kit (Nanjing Jiancheng Bioengineering Institute) was used to assess the MDA concentration. The content of MDA was expressed in units of nmol/mg protein in myocardial tissue homogenate.

All statistical analyses were performed using SPSS software, version 18.0 (SPSS, Inc., Chicago, IL, USA). The inter-group differences were analyzed by one-way analysis of variance. The data are expressed as the mean + standard deviation. All P-values were two-sided and P<0.05 was considered to indicate a statistically significant difference.

Out of 60 rats, 51 completed the study. The mortality rates of the ADR group and the ADR + EP group were 33.3 and 26.7%, respectively, at the end of the interventions, while no deaths were encountered in other groups.

Echocardiography was performed in each rat to measure relative parameters of cardiac functions. As shown in Fig. 1A, two-dimensional and M-mode short-axis views of the left ventricle were acquired at the level of the papillary muscles in rats. There was no difference in terms of LVIDD and EVD between the four groups (data not shown). Compared with the control group, treatment with EP did not affect FS, EF, LVIDS and ESV (Fig. 1B-E). By contrast, FS and EF in the ADR + EP group were significantly higher than those in the ADR group (Fig. 1B and C). Moreover, LVIDS and ESV in the ADR + EP group were greatly lower than those in the ADR group (Fig. 1D and E), indicating that treatment with EP improved the impaired cardiac functions induced by ADR in rats.

SOD is the major defense against ROS production in cells (14), and MDA is produced by the actions of ROS on the lipids existing in the membranes of the cells (15). Therefore, SOD and MDA can be used to experimentally evaluate oxidative injury. To understand the mechanisms underlying the protective role of EP against ADR-induced cardiotoxicity, MDA levels and SOD activity of myocardial tissues were measured in rats. Treatment with EP alone did not affect the production of MDA and SOD compared with that in the control group (Fig. 2A and B). However, in the ADR group, the SOD activity of the myocardial tissue was significantly lower (Fig. 2A), while the MDA level was significantly higher than those in control group, EP group and ADR + EP group (Fig. 2B).

Another two molecules associated with oxidative stress, namely Nox2 and Nox4, were also investigated in rats using western blot analysis. The protein level of Nox4 in the cardiomyocytes in the ADR group was significantly higher than those in the control group, EP group and ADR + EP group, and was reduced by EP (Fig. 2C and D). By contrast, there was no significant difference in the protein levels of Nox2 among the four groups (Fig. 2C and E).

To assess the effects of ADR and EP on myocardial cell apoptosis, markers of apoptosis were assessed in this study. TUNEL staining showed an increase in cardiomyocyte apoptosis rate in the ADR group, whereas the increased apoptosis rate was reduced by EP treatment in the ADR + EP group (Fig. 3). No apoptosis was observed in the control and EP only groups (Fig. 3). To confirm these findings, western blot analysis was applied to assess the expression levels of the apoptosis-related proteins caspase-3, Bax and Bcl-2. As shown in Fig. 4, the expression levels of casepase-3 and Bax in myocardial tissue were significantly higher in the ADR group than in the control group, EP group and ADR + EP group. By contrast, the expression level of Bcl-2 was significantly lower in the ADR group than in the other three groups (Fig. 4C).

The consequence for ADR-induced chronic damage is myocardial tissue fibrosis. Thus, to explore whether treatment with EP can attenuate ADR-induced myocardial tissue fibrosis, PSR staining and RT-qPCR assays were applied to evaluate the fibrosis in myocardial tissues. PSR staining in left ventricular tissue sections showed that the collagen volume fraction (%) in the ADR group was significantly higher than those in the control group, EP group and ADR + EP group (Fig. 5). Furthermore, the results of RT-qPCR indicated that the mRNA levels of TGFβ-1, Colla1 and Colla3, TIMP1, TIMP2, MMP2 and MMP9 in myocardial tissue in the ADR group were significantly higher than those in the other three groups (Fig. 6A-E). The ratios MMP9/TIMP1 and MMP2/TIMP2 in the ADR group were notably lower than those in the ADR + EP group (Fig. 6F and G), however, they were not significantly different from those in the control group and the EP group (Fig. 6F and G).