DOX was obtained from Selleck. DMF was purchased from Selleck, which was dissolved in 0.8% Carboxymethyl cellulose (CMC) for in vivo tests and 0.1% dimethylsulfoxide (DMSO) for in vitro experiments. The nuclear and cytoplasmic protein extraction kits were purchased from KEYGEN Biotech. Co., Ltd. (Nanjing, China). Bicinchoninic acid (BCA) protein assay kit and cell lysis buffer kit were obtained from Beyotime Institute of Biotechnology (Jiangsu, China).

NRCMs were isolated from the ventricles of 1- to 3-day-old neonatal Sprague–Dawley (SD) rats as previously described (Liu et al., 2014). Briefly, neonatal rat hearts were minced into 1-mm³ pieces and were digested with 0.125% trypsin and 0.1% collagenase type I. Tow hours differential attachment culture was performed to separate cardiac fibroblasts from cardiomyocytes. Then NRCMs were cultured in medium with 5-BrdU. Through this method, the purity of cardiomyocytes could reach more than 90%. After incubation for 24 h, 90% of cardiomyocytes exhibited spontaneous pulsing, which indicated good viability.

Male-SD rats weighing 230–250 g (8-weeks old) were obtained from the Shanghai Jihui Laboratory Animal Care Co., Ltd (Shanghai, China) and maintained under SPF conditions in a controlled environment of 20–22 °C, with a 12/12 h light/dark cycle and 50–70% humidity, and food and water provided ad libitum. Rats were randomly divided into five groups: control groups, DOX-treated groups, solvent control groups, and DMF-treated groups. DMF was dissolved in 0.8% CMC and administered to rats by oral gavage, with CMC as solvent control. Based on preliminary data and previous studies, the rats were treated with DMF at a total daily dose of 40 mg/kg/d and 80 mg/kg/d twice a day (Li et al., 2018; Motterlini et al., 2019). The rats in DOX-treated groups were intraperitoneally injected with DOX (15 mg/kg diluted with 0.9% saline). The rats in solvent control groups and DMF-treated groups were pretreated with 0.8% CMC or DMF a week prior to DOX treatment (15 mg/kg diluted with 0.9% saline, intraperitoneally) and maintained until the end of the experiment. Six days after DOX treatment, all rats were sacrificed. The serum samples were obtained from blood by centrifugation (3000 r/min, 4°C) for 10 min, and the heart tissues were removed for further testing. All animal experiments were approved by the Institutional Review and Ethics Board of Shanghai Xinhua Hospital, Shanghai Jiao Tong University School of Medicine.

Rats were anesthetized with 1% isoflurane inhalation and placed on a heated pad to maintain 37°C body temperature. The ejection fraction (EF) and fractional shortening (FS) were measured from M-mode images of echocardiography (Vivid 7; GE Medical, Milwaukee, WI, United States) with a 15 MHz transducer.

Cell counting kit 8 (CCK8) assay was performed according to the manufacturer’s instructions. Briefly, after the cells were exposed to various treatments, CCK-8 (10 µl) was added to each well of the 96-well plate, and the plate was incubated for 4 h at 37°C. Cell viability was calculated by absorbance measurements at 450 nm using a Synergy H1 Multi-Mode Reader (BioTek, Winooski, VT, United States).

Cells were seeded in the 24-well plates and were incubated with different interventions. At the end of the incubation period, cells were fixed, washed with PBS three times, and stained with Hoechst 33258 staining solution (Beyotime, Shang Hai, China) for 5 min at room temperature and observed by fluorescence microscope (OLYMPUS, Tokyo, Japan). Fragmented or condensed nuclei were considered apoptotic cells.

At the end of different interventions, DCFH-DA (10 μM) was added in the well for 20 min induction at 37°C after removing the medium. The samples were observed using fluorescence microscopy (Olympus, Tokyo, Japan).

NRCMs were stained with antibodies against α-actinin (A5044,1:100) and Nrf2 (ab137500,1:100), heart sections were stained with antibody against Nrf2 (ab137500,1:100) in a humidified box at 4°C overnight and followed by incubation with fluorescein-labeled secondary antibody for 1 h at 37°C. The cell nuclei were stained with DAPI for 5 min. All images were captured with a fluorescence microscope or scanner by Caseviewer software (3D Histech).

The CK-MB, LDH, ALT, creatinine levels in serum were detected using the commercial kits (Changchun Huili Biotech Co., Ltd.) according to the instructions. In addition, the heart tissues were placed in cold saline (1: 10, w/v) and then homogenized with a homogenizer machine. Next, the supernatant was obtained through centrifuging at 3000 r/min to detect the MDA, SOD, GSH, and GSH- Px levels in heart tissues according to the instructions (Jiancheng Biotech Co. Ltd, China).

Heart tissues were fixed in 10% formalin and embedded in paraffin, and then the sections were stained with hematoxylin-eosin (H&E) solution. Finally, images of the stained sections were obtained by Olympus microscope or scanner (3D Histech). The images were graded by the degree of myocardial necrosis and inflammatory cell infiltration according to the following standards: grade 0, normal; grade 1, lesion not exceeding 25%; grade 2, lesion between 25–50%; grade 3, lesion between 50–75%; grade 4, lesion exceeding 75% (Kanda et al., 2004). Six sections of one heart were graded by an experienced pathologist, who was blinded to the study design. The mean score of the six sections was recorded as the final cardiac pathology score.

The total protein samples from the cells and heart tissues were homogenized using RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime, Shang Hai, China). The protein concentrations of the samples were determined using a BCA Protein Assay Kit. After determining the contents, the proteins were separated by SDS-PAGE (8–12.5%) and then transferred to PVDF membranes (Millipore, Massachusetts, United States). After being blocked with 5% skim milk for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. The following antibodies were used. Hmox1 (10701-1-AP, 1:1000), Bax (50599-2-Ig, 1:2000), Bcl-2 (12789-1-AP, 1:1000), lamin B1 (12987-1-AP, 1:1000) were purchased from proteintech. Antibody against cleaved caspase-3 (9661, 1:1000) was from CST. Antibody against Nrf2 (ab137500, 1:1000) was purchased from Abcam. Then the bands were incubated with secondary antibody for 1 h at room temperature. The protein bands on the membranes were detected using an enhanced chemiluminescence system (WBKLS0500; Millipore, Darmstadt, Germany). Intensity values of the relative protein levels were normalized to β-actin (in vitro) or α-tubulin (in vivo).

Total RNA was extracted with RNAiso Plus (Takara, Kusatsu, Japan) from NRCMs and heart tissues. cDNA was synthesized using Evo M-MLV RT Kit (AG, Hunan, China). Quantitative real-time polymerase chain reaction (RT-PCR) was performed with Hieff^® qPCR SYBR Green Master Mix (Yeasen, Shang Hai, China) on a QuantStudio 3 Real-Time PCR System (Applied Biosisytems, Waltham, MA, United States). The sequences of primers are shown in Supplementary Table S1.

siRNAs were synthesized by Ribobio (Guangzhou, China). We transfected Nrf2-siRNA or negative control siRNA using Rfect siRNA/miRNA Transfection Reagent (Baidai biotechnology, Changzhou, China) according to the manufacturer’s instructions when cells reached 40–50%. The transfection efficiency was evaluated by Western blot. The used siRNA sequences are as follows: SiRNA1: CAAACAGAATGGACCTAAA;SiRNA2:GCAAGAAGCCAGATACAAA;SiRNA3:GGATGAAGAGACCGGAGAA.

The data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed with GraphPad Prism 5.0 software (San Diego, CA, United States). It was performed with one-way analysis of variance (ANOVA) followed by Tukey’s posthoc test when comparing multiple groups, whereas differences within two groups were evaluated by Student’s t-test. Survival analysis was performed using the Kaplan–Meier method. Statistical significance was defined as p < 0.05.

Firstly, NRCMs purity was identified by immunofluorescence for α-actinin, a cardiomyocyte-specific marker (Figure 1B). Then the concentration-dependently cytotoxicity of DMF was evaluated by CCK8 assay. Compared with the control group, the viabilities of NRCMs were significantly reduced over 40 μM DMF for 24 h. As a result, we conducted concentrations of 10 and 20 μM DMF in this research (Figure 1C). NRCMs were pre-treated with DMF for 4 h, then treated with 5 μM DOX for 48 h. Compared to the control group, DOX treatment caused a significant decrease in cell viability and impaired cell morphology. However, DMF could concentration-dependently improve the viability of NRCMs and cell morphology damage compared with the DOX group (Figures 1D,E). Furthermore, the solvent control of 0.1% DMSO showed no effects on the cells.

Treating rats with DOX resulted in about 67% mortality compared with the control group. However, pre-treatment with a 40 mg/kg DMF showed a mortality of 42% and 25% with a dose of 80 mg/kg (Figure 2A). The serum CK-MB and LDH levels in DOX groups increased compared with control groups. However, DMF dose-dependently decreased the serum CK-MB and LDH levels (Figure 2B). The pathology of DOX-induced myocardial injury mainly includes sarcoplasmic reticulum expansion, cardiomyocyte edema, fiber rupture, and massive inflammatory cell infiltration (Ferrans et al., 1997). Consistent with previous studies, DOX caused apparent myocardial tissue disturbance, necrosis, and massive inflammatory cell infiltration, as well as higher cardiac pathology scores, which were dose-dependently alleviated by DMF. The control solvents of 0.8% CMC showed no effects on animals (Figure 2C, Supplementary Figure S1).

Considering heart failure is the most severe side effect after DOX treatment, we evaluated indicators related to heart failure. We found that the body weight was dramatically reduced after DOX treatment and can be reversed with an increasing dose of DMF (Figure 2D). The heart weight/body weight ratio, a simple indicator of heart failure, was increased in the DOX group and can be dose-dependently reversed by DMF (Figure 2E). Furthermore, the echocardiography confirmed the cardiac function improvement in DMF after DOX treatment (Figure 2F).

Given that oxidative stress is crucial in DOX-induced cardiac damage, we tested the oxidative stress levels in vitro and in vivo. The intracellular ROS level in NRCMs in the DOX group was remarkably increased compared with the control group and was decreased by DMF (Figure 3A). Then we measured some indicators representing tissue oxidative stress levels in heart tissue. MDA levels were elevated in DOX groups and were significantly decreased by DMF. DMF restored the levels of SOD, GSH, and GSH-Px, which were downregulated in the DOX group (Figures 3B–E). These results indicated that DMF could alleviate DOX-induced oxidative stress in vitro and vivo.

Apoptosis-related cardiomyocyte death is the leading cause of heart failure after DOX treatment, so we analyzed apoptosis indicators in vitro and vivo. The apoptotic cells were notably increased in the DOX group compared with the control group. However, treatment with DMF reduced the number of apoptotic cells (Figure 4A). Then, we evaluated the expression of apoptosis-related proteins. We found that administration of DOX increased the ratio of Bax/Bcl-2 and dramatically elevated the levels of cleaved caspase-3. In contrast, treatment with DMF notably decreased the Bax/Bcl-2 ratio and the levels of cleaved caspase-3 (Figure 4B). Consistent with results in vitro, DMF significantly reduced the ratio of Bax/Bcl-2 and the levels of cleaved caspase-3 in rats after DOX treatment (Figure 4C).

Nrf2 remains inactive in the cytoplasm under normal physiological conditions and enters the nucleus when activated. The expression levels of nuclear and cytoplasmic Nrf2 in NRCMs and heart tissues were assessed by western blotting. The results indicated that the expression levels of nuclear Nrf2 expression were significantly decreased, and the levels of cytoplasmic Nrf2 were slightly reduced in the DOX group. However, DMF significantly increased the expression levels of nuclear Nrf2 and decreased cytoplasmic Nrf2 levels (Figures 5A,C). These results suggested that DOX inhibited the entry of Nrf2 into the nucleus, which can be reversed by DMF. Then we further evaluated the Nrf2 translocation by immunofluorescence assay. Results in vivo confirmed that DOX significantly inhibited Nrf2 translocation, which can be dose-dependent reversed by DMF (Figures 5B,D). Hmox1 is one of the most powerful proteins in the Nrf2 pathway to defeat oxidative stress. We then tested its expression in vitro and in vivo. Results showed that DOX could significantly inhibit the expression of Hmox1, and DMF can dose-dependent increase it (Figure 5E). And other Nrf2 downstream antioxidant genes (NQO1, GCLC) showed similar changes (Supplementary Figure S2). These results indicated that DMF could activate the Nrf2 pathway by promoting Nrf2 transporting to the nucleus.

To confirm the role of Nrf2 in the protective effects of DMF, the Nrf2-siRNA was conducted. Compared with the NC group, the protein levels of Nrf2 in NRCMs were most downregulated in the siRNA-2 group, which was used for further in vitro experiments (Figure 6A). Transfection with Nrf2-siRNA reversed the cell viability improvements by DMF (Figure 6B). Moreover, the cell morphology damage, ROS level, apoptosis levels, and antioxidant genes expression were reserved after Nrf2 silencing (Figures 6C,D, Supplementary Figure S3). These results suggested that DMF protected against DOX through the Nrf2 pathway.

It was reported that Nrf2 activation could promote tumor cell proliferation and lead to chemotherapy resistance (Singh et al., 2016). So, we tested the effects of DMF on tumor cell viability after DOX treatment. Results showed that DMF exerted no protective effects on SHSY-5Y, RenCa, CT26. WT tumor cell lines after DOX (Figure 7).