Experimental mice were treated in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals, and study was approved by the Ethics Committee for Animal Research of the Wuhan University (Institutional Animal Care and Use Committee Issue No.20181215). C57BL/6 male mice (6–8 weeks old, 21.5–22.5 g) were obtained from Vital River Experimental Animal Technology Co. Ltd. (Beijing, China). The mice were maintained in a standard laboratory at the Cardiovascular Research Institute of Wuhan University, and were housed in standard humidity/temperature-controlled environment (70% relative humidity, 22°C) in a light-controlled room (12/12 h light/dark cycle) with access to sterile rodent food and water. All the mice were individually caged. The mice were used for the experiment after acclimatization to the housing environment for 2 weeks. The mice were randomly divided into three groups: control (CTRL; n = 10), Dox (n = 15), and Dox + RvD1 (n = 10). The control group received only sterile saline. Mice in the Dox group were treated with Dox (20 mg/kg) once intraperitoneally (i.p.) (Wang et al., 2018; Zhang et al., 2020). Mice in the Dox + RvD1 group were treated with RvD1 (2.5 μg/kg, i.p.) 30 min before Dox administration and every day thereafter for the duration of the experiment. Both Dox and RvD1 were dissolved in 0.9% sterile saline. All mice were observed and weighed daily. The mice were sacrificed after 5 days of Dox treatment. The heart weight of mice was collected for the ratio of heart weight (HW)/body weight (BW) and HW/tibia length (TL). The left cardiac tissues were collected for detailed analyses.

Echocardiography was performed on anesthetized (1.5–2% isoflurane) mice using a Mylab30CV ultrasound (Biosound Esaote, Inc.) equipped with a 10 MHz linear array ultrasound transducer. We defined end-systole and end-diastole as the phases in which the left ventricular (LV) end-diastolic diameter (LVEDd) and LV end-systolic diameter (LVEDs) were obtained. LV ejection fraction (LVEF) and LV fractional shortening (LVFS) were also analyzed via LV M-mode tracing with a sweep speed of 50 mm/s at the midpapillary muscle level.

The activity of lactate dehydrogenase (LDH), the concentrations of myocardial-bound creatine kinase (CK-MB) and cardiac troponinwere I (cTnI) assessed as indexes of cardiomyocyte injury. Both LDH activity, CK-MB levels and cTnI levels in serum were detected using kits (all purchased from Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions and as described in our previous study (Ye et al., 2018).

At the end of the experiment, the cardiac tissues were removed and washed in ice-cold phosphate-buffered saline. The cardiac tissues (30 mg) were added to 300 μL of phosphate-buffered saline, ground into homogenates, and centrifuged at 3,000 rpm at 4°C for 15 min to collect the supernatant. The activities of superoxide dismutase 1 (SOD1) and the level of malondialdehyde (MDA) and glutathione (GSH) were detected by commercially available kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) following the manufacturer’s instructions.

Histological analysis was performed as described in our previous study (Ye et al., 2020a). Cardiac tissues were fixed with 4% paraformaldehyde for 5 days. Then, the tissues were embedded in paraffin and sliced into 4–5 μm sections and mounted onto slides. Cardiomyocyte vacuolization was analyzed by hematoxylin and eosin (H&E) staining using a commercially available kit (Millipore) and then visualized by light microscopy. Sections were also subjected to immunofluorescence staining. the sections were autoclaved for antigen retrieval and then blocked with 10% goat serumfor 10min. Next, the sections were incubatedwith primary antibodies against Phospho-NF-κB p65 (Abcam, Cambridge, United Kingdom) overnight at 4°C. The sections were rinsed with PBS for 20 min before incubating with two different IRDye^® 800CW conjugated secondary antibodies for 60 min and subsequently counterstained with the SlowFade Gold antifade reagent containing DAPI. All the figures were captured with fluorescence microscope, and Image Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, United States) was used for relative quantification.

TUNEL staining of the cardiac tissue was performed as described previously (Ye et al., 2020b). The sections of the cardiac tissue were analyzed using a TUNEL kit (Millipore, United States) following the manufacturer’s instructions. Light microscopy was used to evaluate apoptotic cells, and Image Pro Plus software was used for relative quantification.

We extracted and prepared total LV tissue protein with 1× RIPA buffer as reported previously (Ye et al., 2018). Proteins (50 μg) were separated by electrophoresis through a 10 or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene fluoride membrane (IPFL00010, Millipore, Billerica, MA, United States). Then, the blots were blocked with 5% nonfat powdered milk and incubated overnight at 4°C with the primary antibodies. The primary antibodies used in this study were as follows: phospho-nuclear factor kappa-B p65 (p-P65, 1:500 dilution, Abcam, Cambridge, United Kingdom), nuclear factor kappa-B (NF-kB) p65 (T-P65, 1:1000 dilution, Abcam), NADPH oxidase 2 (Nox-2, 1:200, Santa Cruz, CA, United States), NADPH oxidase 4 (Nox-4, 1:200, Santa Cruz), nuclear factor-erythroid 2-related factor 2 (Nrf-2, 1:500, Gene Technology, Shanghai, China), heme oxygenase-1 (HO-1, 1:500, Gene Technology), glucose-regulated protein 78 (GRP78, 1:500 dilution, Cell Signaling Technology), phospho-protein kinase R-like ER kinase (p-PERK, 1:200, Santa Cruz), caspase 12 (1: 1,000 dilution, Cell Signaling Technology), phospho-eukaryotic initiation factor 2α (p-eIF2α, 1:1,000 dilution, Cell Signaling Technology), activating transcription factor 6α (ATF-6α, 1:200 dilution, Santa Cruz Biotechnology), C/EBP homologous protein (CHOP, 1:1,000 dilution, Cell Signaling Technology), cleaved caspase 3 (c-caspase 3, 1:500, Cell Signaling Technology), B cell lymphoma-2 (Bcl-2, 1:500, Abcam), BCL2-associated X (Bax, 1:500, Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1,000, Cell Signaling Technology). After several washes, the blots were incubated with the secondary antibody (goat anti-rabbit antibody) at a dilution of 1:5,000 to 1:10,000 for 1 h at 25°C in the dark. The membranes were sequentially washed several times in the dark and scanned using the Odyssey Imaging System (LI-COR Biosciences, Lincoln, United States), and the band intensities were measured.

RNA was extracted from LV tissue using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, United States). The concentration of RNA is measured by Nanodrop 2000 (Thermo Fisher Scientific, United States). The cDNA was synthesized from 2 µg of RNA from each group using oligo (DT) primers and the Transcriptor First Strand cDNA Synthesis Kit (Roche). RT+/RT-control was applied when cDNA was synthesized. Water without RNase and DNase was used to dilute DNA for three times before quantitative analysis. Quantitative analysis was conducted using a LightCycler 480 and SYBR Green Master Mix (Roche) follow the MIQE guidelines. The results were analyzed with the 2^−△△Ct method and normalized against GAPDH gene expression. Details of the primer sequences are presented in Supplementary Table S1.

Data were analyzed using SPSS 24.0 software and are presented as the mean ± standard deviation (SD). Comparisons between groups were made using one-way analysis of variance (ANOVA) followed by Tukey’s test. A p value <0.05 was considered statistically significant.

Compared to control mice, mice treated with Dox showed a decrease in the ratio of HW/BW and HW/TL. However, the administration of RvD1 improved the Dox-induced HW/BW and HW/TL ratios (Figures 1A,B). Compared to the control group, the levels of CK-MB, cTNI and activity of LDH in LV tissue were significantly increased in the Dox group. The administration of RvD1 reversed these trends (Figures 1C–E). In addition, the decreased LVEDd, LVEF, and LVFS in the Dox group were significantly improved by RvD1 treatment (Figures 1F–I). Histological examination revealed increased vacuolar accumulation in the Dox-treated mice, which was significantly improved in the Dox + RvD1 group (Figures 1J,K).

The expression of proinflammatory cytokines including IL-1β and IL-6 in the heart was significantly increased by Dox treatment (Figures 2A,B). Compared to the Dox group, a significant reduction in IL-1β and IL-6 level was observed in the Dox + RvD1 group (Figures 2A,B). In addition, the inhibitory effect of RvD1 on inflammation was further confirmed by western blotting and immunofluorescence examination, which showed that RvD1 reduced NF-kB signaling (Figures 2C–E). These results showed that RvD1 protects against heart injury by inhibiting inflammatory responses.

Compared to the control group, the activity of SOD and the expression level of GSH were reduced in the Dox group. Whereas, MDA level was significantly increased in the Dox group (Figures 3A–C). However, treatment with RvD1 significantly restored SOD activity and GSH level, and reduced MDA level compared to the Dox-treated group (Figures 3A–C). In addition, western blotting results showed that the expression of Nox-2 and -4, which are important generators of reactive oxygen species (ROS), was reduced in the Dox + RvD1 group compared to the Dox-treated group (Figures 3D–F). The expression of Nrf-2 and HO-1, which play protective roles against oxidative stress, was increased in the Dox + RvD1 group compared to the Dox group (Figures 3G,H). These findings indicate that RvD1 treatment protects the cardiac tissue against Dox-induced oxidative stress.

We investigated whether RvD1 attenuated ER stress, thereby reducing cardiotoxicity induced by Dox. The western blot results showed that RvD1 treatment reduced the expression of important markers that are indicative of the severity of ER stress (Figure 4A), including GRP78 and CHOP (Figures 4B,G). In addition, the administration of Dox increased the expression levels of caspase-12, p-PERK, p-eif2α, and ATF6α, which were attenuated by RvD1 treatment (Figures 4C–F). These results indicate that RvD1 treatment attenuated Dox-induced ER stress.

Apoptosis is reported to be involved in Dox-induced cardiotoxicity (Wang et al., 2016a). We evaluated the severity of apoptosis and identified the potential signaling pathways associated with the RvD1 treatment on cardiomyocyte apoptosis. Compared to the control group, the mRNA level of Bax was increased while that of Bcl-2 was decreased in the Dox-treated group (Figures 5A,B). These changes were significantly reversed following treatment with RvD1. Similarly, western blotting results showed that Dox increased the expression levels of c-caspase 3 and Bax, and decreased Bcl-2 expression (Figures 5C–G). These changes were attenuated by RvD1. In addition, TUNEL assay showed that RvD1 treatment significantly reduced the number of Dox-induced apoptotic cells (Figure 5H). These results showed that RvD1 reduces Dox-induced cardiomyocyte apoptosis.