Cardamonin protects against doxorubicin-induced cardiotoxicity in mice by restraining oxidative stress and inflammation associated with Nrf2 signaling

Biomedicine & Pharmacotherapy 122 (2020) 109547

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Cardamonin protects against doxorubicin-induced cardiotoxicity in mice by restraining oxidative stress and inflammation associated with Nrf2 signaling

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Wang Qia,1, Wang Boliangb,1, Tian Xiaoxia, Fu Guoqianga, Xiao Jianboa, Wang Gangb,* a b

Emergency Department of the Second Affiliated Hospital of Air Force Medical University, Xi'an, 710000, China Department of Critical Care Medicine, The Second Affiliated Hospital of Xi 'an Jiaotong University, Xi'an, 710000, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Doxorubicin (DOX) Cardamonin (CAR) Nrf2 Apoptosis Inflammation

The clinical application of doxorubicin (DOX) for cancer treatment is limited due to its cardiotoxicity. However, the basic pathophysiological molecular mechanisms underlying DOX-induced cardiomyopathy have not yet been completely clarified, and the disease-specific therapeutic strategies are lacking. The aim of the present study was to investigate the potential cardioprotective effect of cardamonin (CAR), a flavone found in Alpinia plant, on DOX-induced cardiotoxicity in a mouse model. At first, in DOX-treated mouse cardiomyocytes, CAR showed significantly cytoprotective effects through elevating nuclear factor erythroid-2 related factor 2 (Nrf2) signaling, and reducing the degradation of Nrf2. This process then improved the anti-oxidant system, as evidenced by the up-regulated expression levels of haem oxygenase-1 (HO1), NAD(P)H:quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase modifier subunit (GCLM), superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT). In contrast, DOX-induced increases in malondialdehyde (MDA) and reactive oxygen species (ROS) were highly inhibited by CAR treatments. Additionally, DOX-induced apoptosis and inflammatory response in cardiomyocytes were diminished by CAR through reducing the Caspase-3 and nuclear factor-κB (NF-κB) signaling pathways, respectively. Then, in the DOX-induced animal model with cardiotoxicity, we confirmed that through improving Nrf2 signaling, CAR markedly suppressed oxidative stress, apoptosis and inflammatory response in hearts of mice, improving cardiac function eventually. Together, our findings demonstrated that CAR activated Nrf2-related cytoprotective system, and protected the heart from oxidative damage, apoptosis and inflammatory injury, suggesting that CAR might be a potential therapeutic strategy in the prevention of DOX-associated myocardiopathy.

1. Introduction The long-term clinical application of a potent anthracycline, doxorubicin (DOX), is limited due to its cumulative dose-dependent cardiotoxicity [1]. DOX has been postulated to induce cardiotoxicity via redox cycling and ROS production [2]. Moreover, disturbances of free iron and calcium are also involved in the cardiotoxic effects [3,4]. Thus, controlling ROS accumulation and sustaining iron and calcium homeostasis can be an interesting approach for the treatment of DOX-triggered cardiotoxicity. However, the ROS hypothesis has been tempered by a series of studies in which treatment with a ROS scavenger failed to prevent cardiac toxicity caused by DOX [5–7]. Therefore, further study is still needed to find effective treatments against DOX-induced cardiotoxicity [8]. DOX-induced cardiotoxicity seems to be a multifactorial process, such as the increased apoptosis, decreased mitochondrial

function and promoted inflammatory response, which are the typical alterations in DOX-induced heart failure [9,10]. It has been believed that the activation of the innate immune system with the ensuing proinflammatory cytokines release are at the basis of the pathogenesis of DOX-triggered cardiotoxicity [11]. Inflammatory cytokines are involved in several cardiac diseases in that they influence heart rate to a negative inotropic effect, contributing to deleterious left ventricular remodeling [12]. Furthermore, cardiomyocyte apoptosis, including caspase activation, is a key event in heart failure progression in general after DOX injury [13,14]. Accumulating studies have indicated that finding and developing promising therapeutic strategies to inhibit ROS production, apoptotic cell death and inflammatory response are effective for the treatment of DOX-related cardiotoxicity. Flavonoids have a wide range of biological activities. The most common known antioxidant activity has a putative role in suppressing

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Corresponding author. E-mail address: [email protected] (W. Gang). 1 The first authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.109547 Received 14 August 2019; Received in revised form 27 September 2019; Accepted 8 October 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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Fig. 1. Cardamonin increased the expression of Nrf2 and elevated Nrf2 nuclear translocation in HL-1 cells. (A) H9C2 and (B) HL-1 cells were treated with the indicated concentrations of CAR (0, 1.5, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 300 μM) for 24 h. Then, all cells were harvested for cell viability measurements using MTT analysis. (C) HL-1 cells were treated with CAR (0, 12.5, 25, 50 and 100 μM) for 24 h, and then western blotting analysis was used to determine Nrf2 expression levels in cells. (D) HL1 cells were treated with 100 μM of CAR for the indicated time. Then, the cells were collected for western blot analysis of Nrf2 in nucleus and cytoplasm. (E) HL-1 cells were treated with 0, 25, 50 and 100 μM of CAR for 24 h. Then, immunofluorescent analysis was used to measure Nrf2 expression levels. (F) Western blot analysis for Keap1 in HL-1 cells treated with the indicated concentrations of CAR (0, 12.5, 25, 50 and 100 μM) for 24 h. (G) Western blot results for Keap1 in HL-1 cells treated with CAR (100 μM) for 0, 2, 4, 8, 12 and 24 h. (H) ARE induction in HL-1 cells incubated with the indicated concentrations of CAR (0, 12.5, 25, 50 and 100 μM) for 24 h. The values shown were the means ± SEM (n = 3 independent observations). *P < 0.05, **P < 0.01 and ***P < 0.001 vs the Con group.

present work identified that CAR might be a potential treatment for DOX-associated cardiomyopathy.

the risk of cardiovascular diseases such as atherosclerosis [15,16]. Cardamonin (CAR) is isolated from different herbs, including Alpinia katsumadai, Ginkgo biloba and Carya cathayensis Sarg [17–19]. As reported, CAR has multiple pharmacological effects, such as antitumor, anti-oxidative actions and anti-inflammation [20–22]. Recently, CAR was reported to attenuate pressure overload-induced cardiac dysfunction by suppressing oxidative stress [23]. CAR could also inhibit COX and iNOS expression through reducing p65/NF-κB nuclear translocation and IκB phosphorylation in macrophage cells [24]. Thus, we hypothesized that CAR might be a promising therapeutic strategy for the treatment of cardiotoxicity induced by DOX. In the study, we explored the protective effects of CAR against DOXtriggered injury in cardiomyocytes and in an experimental animal model with cardiotoxicity. The results indicated that CAR could activate Nrf2 signaling, suppressing oxidative stress, reducing apoptotic cell death and inhibiting pro-inflammatory response, which contributed to the improvement of cardiac damage induced by DOX. Herein, our

2. Materials and methods 2.1. Animals and treatments The male C57BL/6 J mice (8 weeks old, 20–22 g) were purchased from the experimental animal center of Xi’an Jiao Tong University (Shaanxi, China) and fed in a temperature-controlled room (at 20 ± 5 °C) on a 12/12 h dark/light cycle with free access to water and food. All animal procedures were performed in accordance with the institutional guidelines for the care and use of laboratory animals and approved by the ethics committee of the Second Affiliated Hospital of Air Force Medical University (Shaanxi). After 1 weeks of acclimatization, mice were randomly divided into 5 groups: (i) the Control group; (ii) DOX group; (iii) DOX + CAR (20 mg/kg); (iv) DOX + CAR (40 mg/ 2

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Fig. 2. Cardamonin promoted the Nrf2-ARE regulated cytoprotective genes in HL-1 cells dependent on Nrf2. (A) RT-qPCR and (B) western blot analysis were used to determine Nrf2, HO1, NQO1 and GCLM expression levels in HL-1 cells treated with the indicated concentrations of CAR (0, 25, 50 and 100 μM) for 24 h. (C) HL-1 cells were transfected with Nrf2 siRNA (siNrf2) for 24 h. Then, transfection efficacy was determined by western blot analysis. HL-1 cells were transfected with siNrf2 for 24 h, and then were exposed to 100 μM of CAR for another 24 h. Next, all cells were harvested for (D) RT-qPCR and (E) western blot analysis of Nrf2, HO1, NQO1 and GCLM. The values shown were the means ± SEM (n = 3 independent observations). *P < 0.05, and **P < 0.01 ***P < 0.001 vs the Con group.

measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocols. Proteins were then subjected to 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The membranes were then incubated with 5% nonfat dry milk in TBST for 1.5 h at room temperature and subsequently incubated with primary antibodies (Supplementary table S1) overnight at 4 °C. Then, the membranes were incubated with HRP-conjugated secondary antibodies (Beyotime Institute). Blots were then visualized using enhanced chemiluminescence (ECL, Thermo Fisher Scientific) substrate and exposed to X-ray film. Relative protein expression was normalized to GAPDH.

kg); and (v) DOX + CAR (80 mg/kg). Mice were received intraperitoneal injections of DOX (5 mg/kg/week) for 4 weeks. The CAR treatment group (DOX + CAR) was pretreated with CAR (20, 40 or 80 mg/kg/day) by gavage, and mice were then intraperitoneal injected with DOX. The concentrations of DOX and CAR used in our study were referred to previous studies [25–27]. DOX was purchased from APExBIO (USA). CAR was purchased from MedChemExpress (USA). Mice in the control group were intraperitoneally injected with equal volumes of 0.9% saline. Finally, the echocardiography (ECG) of mice was calculated before the mice were sacrificed. 2.2. Cells and culture

2.5. Immunofluorescent staining The rat myocardium-derived cardiomyoblast H9C2 and mouse cardiomyocyte HL-1 were purchased from the Chinese Academy of Sciences Shanghai Institute for Cell Resources (Shanghai, China) and cultured in 5% CO2 at 37 °C. The designed siRNA against mouse Nrf2 and control scrambled siRNA were purchased from GenScript (GenScript, China). Cells were then transfected with 50 nM siRNA against Nrf2 or scrambled duplex with Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.

The immunofluorescence analysis was performed as indicated previously [28]. Briefly, the cultured cells and cardiac sections were fixed with 4% paraformaldehyde, incubated in 3% normal donkey serum (Solarbio, Beijing, China) supplemented with 0.3% Triton X-100 at 37 °C for 1 h, and incubated with primary antibodies overnight at 4 °C. Then, the slides were incubated with corresponding secondary antibodies (Solarbio). Next, the samples were stained with DAPI (Solarbio) and observed using a fluorescent microscope. The primary antibodies used in the study were listed as follows: anti-Nrf2 (1:150, Invitrogen, USA) and anti-NF-κB (1:150, Abcam, USA).

2.3. Cell viability analysis Cell viability after treatments was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Beyotime Institute, Shanghai, China) according to the manufacturer’s instructions. The absorbance at 490 nm was measured with a microtiter plate reader.

2.6. Real time-quantitative PCR (RT-qPCR) assays Total RNA was extracted from harvested cells or cardiac tissues using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. RNA was ten quantified and reverse-transcribed into complementary DNAs using specific stem-loop reverse transcription primers [29]. First-strand mRNAs were synthesized with a HiScript II Q RT SuperMix for qPCR (Vazyme Biotech, USA), and the RT-qPCR was conducted using a ChamQTM Universal SYBR qPCR

2.4. Western blot assays The cardiac tissue and cell samples were harvested and lysed using RIPA solution (Beyotime Institute). Then, the protein concentration was 3

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Fig. 3. Cardamonin improved the anti-oxidative capacity in HL-1 cells. (A) Western blot analysis for SOD1, SOD2, GPx1 and CAT in HL1 cells incubated with CAR (0, 25, 50 and 100 μM) for 24 h. *P < 0.05, **P < 0.01 and ***P < 0.001 vs the Con group. (B-H) HL-1 cells were treated with DOX (5 μM) for 24 h with or without CAR (50 and 100 μM). Then, all cells were harvested for further analysis. (B) SOD, (C) GSH-px and (D) CAT activities were measured in cells. Calculation of (E) GSH/ GSSG ratio, (F) MDA levels and (G) ATP levels in cells. (H) DCF-DA analysis was used to determine ROS production in cells. +P < 0.05 and ++P < 0.01 vs the Con group; *P < 0.05 and **P < 0.01 vs the DOX group. The values shown were the means ± SEM (n = 3 independent observations).

2.7. Calculation of biochemical parameters

Master Mix (Vazyme Biotech) with a Bio-Rad CFX96TM R-T PCR instrument (Bio-Rad) [30]. GAPDH was served as a loading control. PCR primers used in the present study were synthesized by Generay Biotech (Shanghai, China) and the sequences were shown in Supplementary table S2.

The Caspase-3 activity in heart tissue was measured using a Caspase-3 colorimetric assay kit (Clontech, USA) according to the manufacturer’s instructions. Creatine kinase (CK), lactate 4

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Fig. 4. Cardamonin alleviated cell death in DOX-incubated HL-1 cells. (A) HL-1 cells were treated with different concentrations of DOX (0, 1.25, 2.5, 5 and 10 μM) for 24 h in the absence or presence of CAR (0, 25, 50 and 100 μM). Then, the cell viability was measured by MTT analysis. (B-D) HL-1 cells were exposed to DOX (5 μM) for 24 h with or without CAR (25, 50 and 100 μM) for another 24 h. Then, the following studies were performed. (B) Calculation of LDH in cells. (C) TUNEL staining for cells. Quantification of apoptosis was displayed. (D) Western blot analysis was used to determine Bcl-2, Bax and cleaved Caspase-3 expression levels in cells. The values shown were the means ± SEM (n = 3 independent observations). +P < 0.05, ++P < 0.01 and +++ P < 0.001 vs the Con group; *P < 0.05, **P < 0.01 and ***P < 0.001 vs the DOX group.

2.10. Immunohistochemistry staining

dehydrogenase (LDH) contents, malondialdehyde (MDA) levels, CAT activity, superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) and glutathione (GSH) concentration were measured using commercial kits (Nanjing Jiancheng Institute of Biotechnology, Nanjing, China) following the manufacturer's instructions. The ratio of GSH/GSSG and troponin T (Tn-T) were measured using commercially available kit according to the manufacturer’s protocols (GSH and GSSG Assay Kit, Beyotime, Nanjing, China). The levels of inflammation factors, such as interleukin 6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α) and IL-18 were measured using ELISA kits according to the manufacturer’s recommendations.

The cardiac tissues were fixed in 10% formalin and embedded in paraffin. Then, the heart sections (5-μm-thickness) were subjected to hematoxylineosin (H&E). Masson’s trichrome and Sirius red staining were used for fibrosis analysis [32,33]. After deparaffinization and rehydration, the cardiac sections were covered with 3% H2O2 for 10 min. Then, the tissue preparations were incubated with the primary antibodies (anti-p-IκBα, 1:100, Invitrogen; anti-p-NF-κB, 1:100, Abcam, USA) overnight at 4 °C before incubating with the secondary antibodies (KeyGen Biotech) for 1 h at room temperature, followed by incubation with DAB (KeyGen Biotech). Representative images of the stained sections were obtained with a light microscope.

2.8. ROS production measurements The ROS was measured with 2’,7’-dichlorofluorescein-diacetate (DCFHDA, Beyotime Institute) staining according to the manufacturer’s instructions, and observed using a microscope.

2.11. Measurement of echocardiography (ECG) in mice To determine the cardiac function, the mice were anesthetized with 1% isoflurane in O2 gas and then placed on a heated imaging platform. ECG calculation was then taken with a Vevo 2100 high-resolution imaging system (Visual Sonics Inc., Canada). The parameters of left ventricular (LV) ejection fraction (EF) and the LV fractional shortening (FS) were analyzed.

2.9. TUNEL staining TUNEL staining was measured with the In Situ Cell Death Detection Kit (Roche Ltd., Basel, Switzerland) as described previously [31]. In brief, after preparing the mouse cardiac sections and HL-1 cardiomyocytes, TUNEL staining was conducted according to the manufacturer’s protocols. Five fields in each sample were randomly chosen for apoptotic cell quantification under a light fluorescence microscope in a blinded manner.

2.12. Calculation for ATP ATP levels in cells were measured using ATP Assay Kit (Colorimetric/Fluorometric) (Abcam, catalog #ab83355) according to the manufacturer’s instructions. 5

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Fig. 5. Cardamonin inhibited inflammatory response in DOX-treated HL-1 cells. (A-D) HL-1 cells were treated with DOX (5 μM) for 24 h in the presence or absence of CAR (25, 50 and 100 μM), followed by further analysis. (A) TNF-α, IL-1β, IL-18 and IL-6 levels in cells were measured by ELISA analysis. (B) Protein levels of p-IKKα, p-IκBα and p-NF-κB in cells were determined by western blot analysis. (C) Immunofluorescent analysis was performed to determine NF-κB expression levels in cells. (D) Nuclear and cytoplasm NF-κB expression levels in cells were evaluated by western blotting assays. The values shown were the means ± SEM (n = 3 independent observations). ++P < 0.01 and +++P < 0.001 vs the Con group; *P < 0.05, **P < 0.01 and ***P < 0.001 vs the DOX group.

2.13. Antioxidant response element (ARE) activity assay

3. Results

HL-1 cells were transfected with the Cignal Reporter plasmids for ARE (Qiagen, USA) and treated with CAR (0, 12.5, 25, 50 and 100 μM) for 24 h. Wells were washed using PBS, and 100 μL of passive lysis buffer was added to each well. After 15 min of rocking in passive lysis buffer, plates were stored at −80 °C until ready to read. The dual luciferase kit (Promega, USA) was used to calculate the activity using a luminometer. Each sample was treated in duplicate for each plasmid. The data were expressed as fold induction over control.

3.1. Cardamonin increased the expression of Nrf2 and elevated Nrf2 nuclear translocation in HL-1 cells To explore the effects of CAR on DOX-induced cardiotoxicity, its influence on the cell viability of cells was measured. As shown in Fig. 1A and B, the cell viabilities of H9C2 and HL-1 treated with different concentrations of CAR (0, 1.5, 3.125, 6.25, 12.5, 25, 50 and 100 μM) for 24 h were not remarkably changed, suggesting that CAR under the tested doses showed no significant cytotoxicity to cells. However, CAR at relatively higher concentrations (200 and 300 μM) significantly reduced the cell viability in cardiomyocytes. Then, 12.5 to 100 μM of CAR were selected for in vitro analysis using mouse cardiomyocytes HL1. Then, western blot analysis suggested that CAR markedly increased Nrf2 expression in HL-1 cells in a dose-dependent manner (Fig. 1C). We also found that CAR up-regulated Nrf2 nuclear translocation from cytoplasm by western blot and immunofluorescent analysis (Fig. 1D and E). Kelch-like ECH-associated protein 1 (Keap1) is the predominant repressor protein of Nrf2, and is critical for Nrf2degradation [34,35].

2.14. Statistical analysis All values were presented as the mean ± SEM. Differences were compared by ANOVA followed by Bonferroni correction for post hoc ttest, where appropriate. All statistical tests were conducted with the GraphPad Prism software (GraphPad Software Inc., San Diego, USA). Differences with p < 0.05 were considered statistically significant.

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Fig. 6. Cardamonin treatments alleviated DOX-induced cardiotoxicity in mice. (A) Body weight was measured. (B) The ratio of heart weight to body weight was determined. (C) LVEF% and LVFS% were measured to measure the cardiac function by ECG. (D) LDH, (E) CKMB and (F) Tn-T levels in serum of mice were measured. (G) H&E staining of cardiac sections. (H) AMP and BNP mRNA levels in hearts were measured by RT-qPCR analysis. (I) Masson trichrome staining and sirius red staining were used to determine collagen levels in cardiac sections. (J) Quantification of collagen levels in hearts. (K) TGF-β1 and α-SMA mRNA levels in hearts were assessed using RTqPCR analysis. The values shown were the means ± SEM (n = 8 independent observations). +P < 0.05 and ++P < 0.01 vs the Con group; *P < 0.05 and **P < 0.01 vs the DOX group (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 7. Cardamonin treatments inhibited apoptosis in DOX-challenged mice. (A) TUNEL staining was used to determine apoptosis in heart samples. (B) Caspase-3 activity in hearts was determined. (C) Western blotting analysis was used measure Bcl-2, Bax and cleaved Caspase-3 expression levels in hearts. The values shown were the means ± SEM (n = 8 + P < 0.05, independent observations). ++ P < 0.01 and +++P < 0.001 vs the Con group; *P < 0.05 and **P < 0.01 vs the DOX group.

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Fig. 8. Cardamonin decreased oxidative stress in hearts of DOX-treated mice. (A) SOD, (B) GSH-px, (C) ratio of GSH to GSSG and (D) MDA levels in hearts were measured. (E) DCF-DA and Nrf2 staining for cardiac samples. (F) Nrf2, HO1, NQO1 and GCLM mRNA levels in hearts were measured by RT-qPCR analysis. (G,H) Nrf2, HO1, NQO1 and GCLM protein expression levels in hearts were determined by western blotting analysis. The values shown were the means ± SEM (n = 8 independent observations). +P < 0.05 and ++P < 0.01 vs the Con group; *P < 0.05 and **P < 0.01 vs the DOX group.

by induced by a wide range of oxidative agents, including DOX [39]. In this regard, RT-qPCR and western blotting analysis suggested that Nrf2, HO1, NQO1 and GCLM mRNA and protein expression levels were markedly induced by CAR in a dose-dependent manner (Fig. 2A and B). Then, Nrf2 expression was knocked down in HL-1 cells to further investigate its role in DOX-induced cardiac damage (Fig. 2C). Nrf2 knockdown clearly decreased the mRNA and protein expression levels of Nrf2 and its targeting genes HO-1, NQO1 and GCLM when compared to the Con group in the absence of any treatments. Also, RT-qPCR and western blot analysis confirmed the significantly up-regulated expression of Nrf2, HO-1, NQO1 and GCLM in CA R-T reated cells compared with the Con group; however, in comparison to the CAR group, Nrf2 silence markedly abrogated the CAR-induced up-regulation of Nrf2, HO-1, NQO1 and GCLM in HL-1 cells (Fig. 2D and E). Collectively, these findings indicated that CAR could activate Nrf2 signaling through a Nrf2-dependent molecular mechanism in cardiomyocytes.

Inhibiting Keap1 activity could hinder the Nrf2 degradation through ubiquitinproteasome system, leading to the accumulation of newly synthesized Nrf2 and its translocation to the nucleus [36]. Then, Keap1 expression levels were measured using western blot analysis. As shown in Fig. 1F and G, we found that in contrast to Nrf2 expression change, CAR treatment markedly reduced the expression of Keap1 in a doseand time-dependent manner. We then found that CAR exhibited a concentration-dependent pattern for ARE inductivity (Fig. 4H). Therefore, results above showed that CAR could not only induce the expression of Nrf2, but also repressed Nrf2 degradation mainly through reducing Keap1 expression levels, which significantly promoted the activation of Nrf2-ARE signaling. 3.2. Cardamonin promoted the Nrf2-ARE regulated cytoprotective genes in HL-1 cells dependent on Nrf2 Through binding to the enhancer sequence in the gene promoter regulatory region that is termed the ARE, Nrf2 modulates the expression of a large number of genes encoding antioxidant proteins, detoxifying enzymes, metabolic alteration enzymes as well as stress response proteins, most of which play critical roles in the cellular defence system, particularly in oxidative stress regulation [37,38]. HO-1, NQO1 and GCLM are Nrf2 cytoprotective targeting genes, which could be induced

3.3. Cardamonin improved the anti-oxidative capacity in HL-1 cells We then explored the effects of CAR on key antioxidant enzymes, including SOD, CAT and GPx1 [40,41]. As shown in Fig. 3A, we found that CAR dose-dependently increased the expression levels of SOD1, SOD2, GPx1 and CAT in HL-1 cells. As shown in Fig. 3B-D, DOX 8

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Fig. 9. Cardamonin treatments attenuated inflammatory response in the hearts of DOXtreated mice. (A) Immunohistochemistry staining of F4/80 in heart samples of mice. (B) TNF-α, IL-1β and IL-18 mRNA levels in hearts of mice were determined using RT-qPCR analysis. (C,D) The protein expression levels of pIKKα, p-IκBα and p-NF-κB in hearts were measured by western blot analysis. (E,F) Immunohistochemistry staining was used to calculate p-IκBα and p-NF-κB expression levels in cardiac sections. The values shown were the means ± SEM (n = 8 independent observations). ++P < 0.01 and +++P < 0.001 vs the Con group; *P < 0.05 and **P < 0.01 vs the DOX group.

Fig. 10. Schematic diagram of the protective effects of cardamonin on cardiotoxicity induced by DOX.

while being decreased by CAR in a dose-dependent manner (Fig. 3H). These data suggested that CAR showed anti-oxidant effects in DOXstimulated cardiomyocytes.

treatment could slightly improve the activity of SOD, GSH-px and CAT in HL-1 cells. However, treatment with CAR markedly improved the activities of these antioxidant enzymes. GSH is a critical endogenous antioxidant protein, which could rescue cellular homeostasis through inhibiting damages to important cellular components caused by ROS [42]. DOX reduced the ratio of GSH/GSSG, indicating the immune response and stress conditions, in HL-1 cells, which was restored by CAR (Fig. 3E). The levels of MDA, a biomarker of oxidative stress, increased by DOX were markedly decreased in HL-1 cells treated with CAR (Fig. 3F). At the molecular level, ATP is primarily generated by the mitochondria and assists in the cardiac contractile function [43]. Then, we found that CAR treatment markedly restored ATP contents in DOXincubated HL-1 cells (Fig. 3G). Subsequently, DCF-DA staining suggested that DOX significantly induced ROS production in HL-1 cells,

3.4. Cardamonin alleviated cell death in DOX-incubated HL-1 cells In this regard, MTT analysis demonstrated that DOX dose-dependently reduced the cell survival rate in HL-1 cells, which were markedly rescued by CAR also in a dose-dependent manner (Fig. 4A). LDH, a marker for cardiac injury [44], was enhanced by DOX; however, CAR treatment greatly reduced LDH levels in HL-1 cells stimulated by DOX (Fig. 4B). TUNEL staining indicated the role of CAR in reducing the number of TUNEL-positive cells triggered by DOX, illustrating the suppression of apoptotic cell death (Fig. 4C). Then, the expression 9

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challenged mice (Fig. 7C). These findings indicated that CAR could suppress apoptosis to alleviate DOX-induced cardiac injury.

levels of anti-apoptotic signal Bcl-2 [45] were significantly reduced by DOX, whereas the expression levels pro-apoptotic molecules Bax and cleaved Caspase-3 [46] were highly enhanced. Notably, these effects were reversed by CAR (Fig. 4D). Taken together, these findings demonstrated that CAR-alleviated injury in cardiomyocytes was associated with the suppression of apoptosis.

3.8. Cardamonin decreased oxidative stress in hearts of DOX-treated mice In this part, we found that SOD and GSH-px activities in hearts were slightly up-regulated by DOX, which were further promoted by CAR administration (Fig. 8A and B). The ratio of GSH to GSSG decreased by DOX was markedly rescued by CAR in a dose-dependent manner (Fig. 8C). Then, MDA levels induced by DOX were significantly reduced by CAR, along with decreased ROS production by DCF-DA staining. Meanwhile, Nrf2 stimulated by DOX was further elevated by CAR, showing anti-oxidant effects (Fig. 8D and E). Subsequently, RT-qPCR and western blotting analysis illustrated that Nrf2, HO1, NQO1 and GCLM expression levels were slightly increased by DOX, which were notably further potentiated by CAR treatments in a dose-dependent manner (Fig. 8F-H). Therefore, we confirmed that CAR could prevent oxidative stress to alleviate DOX-induced cardiac injury.

3.5. Cardamonin inhibited inflammatory response in DOX-treated HL-1 cells Inflammatory response has been reported to be involved in DOXinduced cardiotoxicity [47]. DOX exposure markedly enhanced the levels of TNF-α, IL-1β, IL-18 and IL-6 in HL-1 cells. However, these increased expression levels of pro-inflammatory cytokines were highly down-regulated by CAR in a dose-dependent manner (Fig. 5A). NF-κB (p65) signaling pathway is critical in inducing the release of pro-inflammatory factors [48]. Subsequently, western blot analysis indicated that DOX incubation significantly promoted the expression of phosphorylated IKKα, IκBα and NF-κB in HL-1 cells, which were, however, markedly reduced by CAR treatments in a dose-dependent manner (Fig. 5B). Also, NF-κB nuclear translocation induced by DOX was also clearly blunted by CAR, accompanied with increased expression of cytoplastic NF-κB by immunofluorescent staining and western blotting analysis (Fig. 5C and D). Together, results in this part elucidated that CAR could impede DOX-triggered inflammation in cardiomyocytes, demonstrating its role in suppressing DOX-related cardiac injury.

3.9. Cardamonin treatments attenuated inflammatory response in the hearts of DOX-treated mice F4/80, as a marker of macrophage and inflammatory response, was then investigated. Immunohistochemistry staining demonstrated that DOX led to the significant increase of F4/80 in cardiac sections, while being attenuated by CAR in a dose-dependent manner (Fig. 9A). Then, the mRNA expression levels of TNF-α, IL-1β and IL-18 stimulated by DOX were significantly alleviated by CAR (Fig. 9B). Moreover, the protein expression levels of p-IKKα, p-IκBα and p-NF-κB in hearts were highly induced by DOX, which were, however, decreased by CAR treatments also in a concentration-dependent manner by western blotting and immunohistochemical staining (Fig. 9C-F). Collectively, these data suggested that CAR could improve DOX-elicited cardiotoxicity through blocking inflammatory response.

3.6. Cardamonin treatments alleviated DOX-induced cardiotoxicity in mice Results above have indicated the protective role of CAR in DOXinduced cellular injury. Then, we attempted to further explore the potential effects of CAR on DOX-triggered cardiotoxicity in vivo. As shown in Fig. 6A, CAR does-dependently increased the body weight of mice challenged with DOX. We also found that the ratio of heart weight to body weight was markedly increased by DOX, which was, however, decreased by CAR treatment in a dose-dependent manner (Fig. 6B). Subsequently, the ECG was used to calculate the cardiac function [49]. ECG analysis demonstrated that LVEF% and LVFS% were markedly reduced by DOX, while being rescued by CAR supplementation, indicating the improved cardiac function (Fig. 6C). Subsequently, myocardial injury was further evaluated by analyzing the levels of cardiac damage markers, including LDH, CK-MB and Tn-T [50,51]. As shown in Fig. 6D-F, DOX treatment significantly increased serum LDH, CK-MB and Tn-T levels, and these effects were markedly reduced by CAR in a dose-dependent manner. H&E staining suggested that DOX-induced histological changes were improved by CAR (Fig. 6G). ANP and BNP, as critical markers for cardiac injury, were significantly increased in hearts of DOX-challenged mice; however, these results were clearly reduced by CAR treatment (Fig. 6H). Then, by Masson trichrome and sirius red staining, we found that DOX led to significant collagen accumulation in cardiac sections, while being markedly attenuated by CAR treatments in a dose-dependent manner (Fig. 6I and J). Consistently, the mRNA levels of fibrosis markers TGF-β1 and α-SMA were highly induced by DOX. However, these findings were clearly reversed by CAR (Fig. 6K). Together, these findings elucidated that CAR showed protective effects against DOX-triggered cardiotoxicity.

4. Discussion DOX is a powerful drug for the clinical fight against tumor progression. However, its cardiotoxicity is a major challenge, limiting its clinical use [1–4]. Further understanding of the molecular mechanisms could attenuate the cardiotoxicity and improve the clinical efficacy of DOX. Presently, there is no effective treatment to improve cardiac function induced by DOX. Herein, finding an effective treatment still remains an urgent priority. Increasing studies have indicated that oxidative stress and its constant companion inflammatory response are common features and major factors of cardiotoxicity because of DOX and progression, as well as the related complications [52]. Therefore, meditators that regulate this molecular mechanism might be attractive drug targets. ROS accumulation in DOX-induced cardiac injury is accompanied with a disturbed antioxidant defense system in the hearts [53]. Pharmacological evidences have suggested that reducing ROS production, or improving antioxidants is useful for ameliorating cardiomyopathy associated with DOX [54,55]. Nrf2, known as a cytoprotective transcription factor, has a key role in the basal activity and coordinated induction of targeting gene products, which include antioxidant enzymes (SOD, GPx, CAT, HO1, etc), the critical enzymes responsible for glutathione synthesis (GCLC and GCLM), as well as the major detoxifying enzyme NQO1. These molecules play essential roles in cellular antioxidant defense system [40,41,56,57]. Considering the crucial role of oxidative stress and inflammatory response in the pathogenesis of cardiotoxicity induced by DOX, strategies targeting Nrf2 may hold potential for the treatment of myocardiopathy progression for humans. After being activated, Nrf2 translocates to the nuclear area to bind with ARE, which is located in the promoter region of genes that encode the anti-oxidant enzymes [58]. In addition, Keap1 is the known as a predominant

3.7. Cardamonin treatments inhibited apoptosis in DOX-challenged mice As shown in Fig. 7A and B, higher number of TUNEL-positive cells in cardiac sections from DOX mice was observed, which was decreased by CAR, along with significantly reduced Caspase-3 activity. Consistently, western blotting analysis suggested that Bcl-2 expression levels were greatly reduced by DOX, whereas Bax and cleaved Caspase-3 were enhanced. Obviously, CAR treatments significantly rescued Bcl-2, and inhibited Bax and Caspase-3 cleavage in heart tissues from DOX10

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of CAR resulted in the maintenance of normal body weight following DOX exposure. Treatment with CAR also preserved the normal morphology of the heart tissue. Furthermore, we detected up-regulated levels of serum cardiac injury markers (LDH, Tn-T and CK-MB) in DOXchallenged mice [50,51]. Of note, CAR treatment down-regulated the contents of these cardiac injury biomarkers in serum, further confirming its cardioprotective effects. In summary, as exhibited in Fig. 10, we demonstrated that CAR might function as a potential activator of Nrf2, and could also inhibit Nrf2 degradation, which subsequently improved Nrf2-dependent antioxidative signaling and restrained apoptosis and inflammatory response, protecting heart from DOX-induced cardiotoxicity. Therefore, CAR was promising for overcoming the cardiotoxicity of chemotherapy for cancer therapy.

repressor protein of Nrf2. Through acting as an E3 ligase adaptor component, Keap1 could facilitate the polyubiquitination of the Nrf2 protein, resulting in the proteasome-dependent Nrf2 degradation [59]. Suppressing Keap1 activity could inhibit the degradation of Nrf2 [36]. As reported, Nrf2-dependent antioxidant response regulated the protective effect of tanshinone IIA on DOX-induced cardiotoxicity [60]. Moreover, pristimerin could protect against DOX-induced cardiac injury and fibrosis by regulating Nrf2 signaling pathway [61]. In this study, results from both in vivo and in vitro investigations showed that CAR treatment increased the expression levels of Nrf2, as well as the down-streaming signals such as HO1, NQO1 and GCLM in HL-1 and cardiac samples challenged by DOX. Meanwhile, the expression levels of Keap1 were significantly reduced by CAR treatments. These results indicated that CAR was capable of inhibiting Keap1, contributing to an increase of Nrf2 expression and its subsequent translocation to the nucleus, which markedly activated the Nrf2-ARE signaling. Subsequently, our findings demonstrated that Nrf2 activation by CAR markedly up-regulated the antioxidant capacity of HL-1 cells through enhancing the activity of critical antioxidant enzymes such as SOD, CAT and GPx, along with the increase in the ratio of GSH/GSSG. Meanwhile, CAR treatment significantly reduced the ROS production and MDA levels both in vitro and in vivo. These data strongly demonstrated that CAR could protect HL-1 cells and cardiac tissues from the DOX-induced injury through the antioxidative signaling pathway. Because DOX has been shown to mediate its deleterious effects via cardiac apoptosis, as well as the disturbance of cardiac energy metabolism [62,63], the anti-apoptotic effects of CAR on cardiomyocytes and heart tissues were investigated. Recently, increasing studies have indicated that programmed cell death or cellular apoptosis are essential in regulating DOX-induced cardiotoxicity [64]. DOX-evoked oxidative stress leads to an intrinsic mitochondrial-dependent apoptotic pathway in cardiomyocytes [65]. Apoptosis is regulated by a series of certain proteins during signal transduction. Members of the Bcl-2 family are critical meditators for apoptosis [66]. Bcl-2 family includes pro-apoptotic signals including Bax, and anti-apoptotic members such as Bcl-2 [45,46,67]. In the present study, we found that DOX led to significant apoptosis in cardiomyocytes, proved by the remarkable Caspase-3 activity, enhanced expression of Bax, as well as the reduced Bcl-2 expression levels. These results were in agreement with other studies [62,63,68]. Notably, CAR treatment significantly ameliorated DOX-induced apoptosis in cardiomyocytes and cardiac tissues, as evidenced by the decreased activation of Caspase-3 and Bax, along with restored Bcl2 expression levels. All of these contributed to the improvement of cardiotoxicity induced by DOX. Oxidative stress could also result in inflammatory responses through activation of redox sensitive transcription factors such as NF-κB [69]. NF-κB signaling plays an important role in immune and inflammatory response induction. DOX provokes a host of inflammatory reactions through modulating NF-κB and activating the subsequent production of pro-inflammatory cytokines [62,70,71]. The release of these inflammatory cytokines contributes to profound pathological alterations in the form of cardiomyopathy, transmural myocarditis and biventricular fibrosis [72]. In this study, DOX exposure led to a significant activation of NF-κB signaling, evidenced by the increased expression of phosphorylated IKKα, IκBα and NF-κB. NF-κB then modulates the expression levels of a series of pro-inflammatory factors such as COX2, TNF-α, IL-1β, IL-18 and IL-6 [73]. Both in vivo and in vitro results demonstrated that DOX treatment significantly enhanced the expression of TNF-α, IL-1β, IL-18 and IL-6. However, the inflammatory response was clearly blunted by CAR treatments in a dose-dependent manner. Moreover, cardiac fibrosis induced by DOX was also ameliorated by CAR through Masson trichrome staining and sirius red staining. TGF-β1 signaling is of great importance in inducing fibrosis [74,75]. Furthermore, we found that TGF-β1 and α-SMA expression levels evoked by DOX were markedly reduced by CAR, which was participated in the amelioration of DOX-induced cardiac damage. These favorable effects

Declaration of Competing Interest The authors declare there is not any conflict of interest in this work. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109547. References [1] X. Wang, X.L. Wang, H.L. Chen, et al., Ghrelin inhibits doxorubicin cardiotoxicity by inhibiting excessive autophagy through AMPK and p38-MAPK, Biochem. Pharmacol. 88 (3) (2014) 334–350. [2] J. Ma, Y. Wang, D. Zheng, et al., Rac1 signalling mediates doxorubicin-induced cardiotoxicity through both reactive oxygen species-dependent and-independent pathways, Cardiovasc. Res. 97 (1) (2012) 77–87. [3] F. Belmonte, S. Das, P. Sysa-Shah, et al., ErbB2 overexpression upregulates antioxidant enzymes, reduces basal levels of reactive oxygen species, and protects against doxorubicin cardiotoxicity, Am. J. Physiol. Heart Circ. Physiol. 309 (8) (2015) H1271–H1280. [4] Y. Ichikawa, M. Ghanefar, M. Bayeva, et al., Cardiotoxicity of doxorubicin is mediated through mitochondrial iron accumulation, J. Clin. Invest. 124 (2) (2014) 617–630. [5] Q. Dong, L. Chen, Q. Lu, et al., Quercetin attenuates doxorubicin cardiotoxicity by modulating B mi‐1 expression, Br. J. Pharmacol. 171 (19) (2014) 4440–4454. [6] R. Lagoa, C. Gañán, C. López-Sánchez, et al., The decrease of NAD (P) H: quinone oxidoreductase 1 activity and increase of ROS production by NADPH oxidases are early biomarkers in doxorubicin cardiotoxicity, Biomarkers 19 (2) (2014) 142–153. [7] L. Xi, S.G. Zhu, A. Das, et al., Dietary inorganic nitrate alleviates doxorubicin cardiotoxicity: mechanisms and implications, Nitric Oxide 26 (4) (2012) 274–284. [8] M.S. Ewer, S.M. Ewer, Cardiotoxicity of anticancer treatments, Nat. Rev. Cardiol. 12 (9) (2015) 547. [9] E.M. Mantawy, W.M. El-Bakly, A. Esmat, et al., Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis, Eur. J. Pharmacol. 728 (2014) 107–118. [10] Z. Sun, B. Yan, W.Y. Yu, et al., Vitexin attenuates acute doxorubicin cardiotoxicity in rats via the suppression of oxidative stress, inflammation and apoptosis and the activation of FOXO3a, Exp. Ther. Med. 12 (3) (2016) 1879–1884. [11] M. Pecoraro, M. Del Pizzo, S. Marzocco, et al., Inflammatory mediators in a shorttime mouse model of doxorubicin-induced cardiotoxicity, Toxicol. Appl. Pharmacol. 293 (2016) 44–52. [12] R.M. Guo, W.M. Xu, J.C. Lin, et al., Activation of the p38 MAPK/NF-κB pathway contributes to doxorubicin-induced inflammation and cytotoxicity in H9c2 cardiac cells, Mol. Med. Rep. 8 (2) (2013) 603–608. [13] F.S. Carvalho, A. Burgeiro, R. Garcia, et al., Doxorubicin‐induced cardiotoxicity: from bioenergetic failure and cell death to cardiomyopathy, Med. Res. Rev. 34 (1) (2014) 106–135. [14] J. Das, J. Ghosh, P. Manna, et al., Taurine protects rat testes against doxorubicininduced oxidative stress as well as p53, Fas and caspase 12-mediated apoptosis, Amino Acids 42 (5) (2012) 1839–1855. [15] R.M. van Dam, N. Naidoo, R. Landberg, Dietary flavonoids and the development of type 2 diabetes and cardiovascular diseases: review of recent findings, Curr. Opin. Lipidol. 24 (1) (2013) 25–33. [16] G. Siasos, D. Tousoulis, V. Tsigkou, et al., Flavonoids in atherosclerosis: an overview of their mechanisms of action, Curr. Med. Chem. 20 (21) (2013) 2641–2660. [17] L.M. Gonçalves, I.M. Valente, J.A. Rodrigues, An overview on cardamonin, J. Med. Food 17 (6) (2014) 633–640. [18] P.G. Niu, Y.X. Zhang, D.H. Shi, et al., Cardamonin inhibits metastasis of lewis lung carcinoma cells by decreasing mTOR activity, PLoS One 10 (5) (2015) e0127778. [19] Y.J. Shen, X.X. Zhu, X. Yang, et al., Cardamonin inhibits angiotensin II-induced vascular smooth muscle cell proliferation and migration by downregulating p38 MAPK, Akt, and ERK phosphorylation, J. Nat. Med. 68 (3) (2014) 623–629.

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W. Qi, et al.

[20] S. Peng, Y. Hou, J. Yao, et al., Activation of Nrf2-driven antioxidant enzymes by cardamonin confers neuroprotection of PC12 cells against oxidative damage, Food Funct. 8 (3) (2017) 997–1007. [21] Y. Li, Y. Qin, C. Yang, et al., Cardamonin induces ROS-mediated G2/M phase arrest and apoptosis through inhibition of NF-κB pathway in nasopharyngeal carcinoma, Cell Death Dis. 8 (8) (2017) e3024. [22] Y.Y. Li, S.S. Huang, M.M. Lee, et al., Anti-inflammatory activities of cardamonin from Alpinia katsumadai through heme oxygenase-1 induction and inhibition of NF-κB and MAPK signaling pathway in the carrageenan-induced paw edema, Int. Immunopharmacol. 25 (2) (2015) 332–339. [23] W. Li, X. Wu, M. Li, et al., Cardamonin alleviates pressure overload-induced cardiac remodeling and dysfunction through inhibition of oxidative stress, J. Cardiovasc. Pharmacol. 68 (6) (2016) 441–451. [24] D.A. Israf, T.A. Khaizurin, A. Syahida, et al., Cardamonin inhibits COX and iNOS expression via inhibition of p65NF-κB nuclear translocation and Iκ-B phosphorylation in RAW 264.7 macrophage cells, Mol. Immunol. 44 (5) (2007) 673–679. [25] Y.F. Xin, G.L. Zhou, Z.Y. Deng, et al., Protective effect of Lycium barbarum on doxorubicin-induced cardiotoxicity, Phytother Res. 21 (11) (2007) 1020–1024. [26] Z. Wei, J. Yang, Y.F. Xia, et al., Cardamonin protects septic mice from acute lung injury by preventing endothelial barrier dysfunction, J. Biochem. Mol. Toxicol. 26 (7) (2012) 282–290. [27] D. Jia, Y. Tan, H. Liu, et al., Cardamonin reduces chemotherapy-enriched breast cancer stem-like cells in vitro and in vivo, Oncotarget 7 (1) (2016) 771. [28] J. Ohlig, C. Henninger, S. Zander, et al., Rac1-mediated cardiac damage causes diastolic dysfunction in a mouse model of subacute doxorubicin-induced cardiotoxicity, Arch. Toxicol. 92 (1) (2018) 441–453. [29] Y. Wang, T. Lei, J. Yuan, et al., GCN2 deficiency ameliorates doxorubicin-induced cardiotoxicity by decreasing cardiomyocyte apoptosis and myocardial oxidative stress, Redox Biol. 17 (2018) 25–34. [30] Y. Xia, Y.L. Zhang, C. Yu, et al., YAP/TEAD co-activator regulated pluripotency and chemoresistance in ovarian cancer initiated cells, PLoS One 9 (11) (2014) e109575. [31] Z. Zhang, L. Zhu, P. Feng, et al., C1q/tumor necrosis factor-related protein-3-engineered mesenchymal stromal cells attenuate cardiac impairment in mice with myocardial infarction, Cell Death Dis. 10 (7) (2019). [32] S. Zhao, H. Wu, W. Xia, et al., Periostin expression is upregulated and associated with myocardial fibrosis in human failing hearts, J. Cardiol. 63 (5) (2014) 373–378. [33] S.S. Pullamsetti, C. Doebele, A. Fischer, et al., Inhibition of microRNA-17 improves lung and heart function in experimental pulmonary hypertension, Am. J. Respir. Crit. Care Med. 185 (4) (2012) 409–419. [34] K. Itoh, J. Mimura, M. Yamamoto, Discovery of the negative regulator of Nrf2, Keap1: a historical overview, Antioxid. Redox Signal. 13 (11) (2010) 1665–1678. [35] V. Sihvola, A.L. Levonen, Keap1 as the redox sensor of the antioxidant response, Arch. Biochem. Biophys. 617 (2017) 94–100. [36] P. Canning, F.J. Sorrell, A.N. Bullock, Structural basis of Keap1 interactions with Nrf2, Free Radic. Biol. Med. 88 (2015) 101–107. [37] T.W. Kensler, N. Wakabayashi, S. Biswal, Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway, Annu. Rev. Pharmacol. Toxicol. 47 (2007) 89–116. [38] J.A. Johnson, D.A. Johnson, A.D. Kraft, et al., The Nrf2-ARE pathway: an indicator and modulator of oxidative stress in neurodegeneration, Ann. N. Y. Acad. Sci. 1147 (2008) 61. [39] X. Han, S. Gao, Y. Cheng, et al., Protective effect of naringenin-7-O-glucoside against oxidative stress induced by doxorubicin in H9c2 cardiomyocytes, Biosci. Trends 6 (1) (2012) 19–25. [40] B.T. Tung, E. Rodriguez-Bies, H.N. Thanh, et al., Organ and tissue-dependent effect of resveratrol and exercise on antioxidant defenses of old mice, Aging Clin. Exp. Res. 27 (6) (2015) 775–783. [41] S. Hamlaoui, M. Mokni, N. Limam, et al., Resveratrol protects against acute chemotherapy toxicity induced by doxorubicin in rat erythrocyte and plasma, J. Physiol. Pharmacol. 63 (3) (2012) 293. [42] Y. Bai, Q. Chen, Y.P. Sun, et al., Sulforaphane protection against the development of doxorubicin‐induced chronic heart failure is associated with Nrf2 Upregulation, Cardiovasc. Ther. 35 (5) (2017) e12277. [43] T. Doenst, T.D. Nguyen, Abel ED. Cardiac metabolism in heart failure: implications beyond ATP production, Circ. Res. 113 (6) (2013) 709–724. [44] D. Sun, J. Huang, Z. Zhang, et al., Luteolin limits infarct size and improves cardiac function after myocardium ischemia/reperfusion injury in diabetic rats, PLoS One 7 (3) (2012) e33491. [45] C. He, H. Zhu, H. Li, et al., Dissociation of Bcl-2–Beclin1 complex by activated AMPK enhances cardiac autophagy and protects against cardiomyocyte apoptosis in diabetes, Diabetes 62 (4) (2013) 1270–1281. [46] C. Zhai, M. Zhang, Y. Zhang, et al., Glycyrrhizin protects rat heart against ischemiareperfusion injury through blockade of HMGB1-dependent phospho-JNK/Bax pathway, Acta Pharmacol. Sin. 33 (12) (2012) 1477. [47] R.A. Thandavarayan, V.V. Giridharan, S. Arumugam, et al., Schisandrin B prevents doxorubicin induced cardiac dysfunction by modulation of DNA damage, oxidative stress and inflammation through inhibition of MAPK/p53 signaling, PLoS One 10 (3) (2015) e0119214. [48] T. Liu, L. Zhang, D. Joo, et al., NF-κB signaling in inflammation, Signal Transduct. Target. Ther. 2 (2017) 17023.

[49] L.M. Semeniuk, A.J. Kryski, D.L. Severson, Echocardiographic assessment of cardiac function in diabetic db/db and transgenic db/db-hGLUT4 mice, Am. J. Physiol. Heart Circ. Physiol. 283 (3) (2002) H976–H982. [50] H. Diao, Z.C. Kang, F. Han, et al., Astilbin protects diabetic rat heart against ischemia-reperfusion injury via blockade of HMGB1-dependent NF-κB signaling pathway, Food Chem. Toxicol. 63 (2014) 104–110. [51] S. Ahmad, B.P. Panda, M. Fahim, et al., Ameliorative effect of beraprost sodium on celecoxib induced cardiotoxicity in rats, Iran J Pharm Res. 17 (1) (2018) 155. [52] Y. Gao, Y. Xu, S. Hua, et al., ALDH2 attenuates Dox-induced cardiotoxicity by inhibiting cardiac apoptosis and oxidative stress, Int. J. Clin. Exp. Med. 8 (5) (2015) 6794. [53] S. Sangomla, M.A. Saifi, A. Khurana, et al., Nanoceria ameliorates doxorubicin induced cardiotoxicity: possible mitigation via reduction of oxidative stress and inflammation, J. Trace Elem. Med. Biol. 47 (2018) 53–62. [54] S. Hajra, A.R. Patra, A. Basu, et al., Prevention of doxorubicin (DOX)-induced genotoxicity and cardiotoxicity: effect of plant derived small molecule indole-3carbinol (I3C) on oxidative stress and inflammation, Biomed. Pharmacother. 101 (2018) 228–243. [55] X. Yu, L. Cui, Z. Zhang, et al., α-Linolenic acid attenuates doxorubicin-induced cardiotoxicity in rats through suppression of oxidative stress and apoptosis, Acta Biochim Biophys Sin 45 (10) (2013) 817–826. [56] S. Hajra, A. Basu, S. Singha Roy, et al., Attenuation of doxorubicin-induced cardiotoxicity and genotoxicity by an indole-based natural compound 3, 3′-diindolylmethane (DIM) through activation of Nrf2/ARE signaling pathways and inhibiting apoptosis, Free Radic. Res. 51 (9–10) (2017) 812–827. [57] Y. Zhou, S. Zhong, S. Hu, et al., Comparison of the effects of Shenfu injection and Shenmai injection on doxorubicin-induced chronic heart failure in rats, Int. J. Clin. Exp. Med. 11 (12) (2018) 13169–13179. [58] S. Li, W. Wang, T. Niu, et al., Nrf2 deficiency exaggerates doxorubicin-induced cardiotoxicity and cardiac dysfunction, Oxid. Med. Cell. Longev. 2014 (2014). [59] D.D. Zhang, Mechanistic studies of the Nrf2-Keap1 signaling pathway, Drug Metab. Rev. 38 (4) (2006) 769–789. [60] Z. Guo, M. Yan, L. Chen, et al., Nrf2-dependent antioxidant response mediated the protective effect of tanshinone IIA on doxorubicin-induced cardiotoxicity, Exp. Ther. Med. 16 (4) (2018) 3333–3344. [61] D.S. El-Agamy, K.M. El-Harbi, S. Khoshhal, et al., Pristimerin protects against doxorubicin-induced cardiotoxicity and fibrosis through modulation of Nrf2 and MAPK/NF-kB signaling pathways, Cancer Manage. Res. 11 (2019) 47. [62] R. Guo, Y. Hua, J. Ren, et al., Cardiomyocyte-specific disruption of Cathepsin K protects against doxorubicin-induced cardiotoxicity, Cell Death Dis. 9 (6) (2018) 692. [63] Y. Ma, L. Yang, J. Ma, et al., Rutin attenuates doxorubicin-induced cardiotoxicity via regulating autophagy and apoptosis, Biochim. Biophys. Acta Mol. Basis Dis. 1863 (8) (2017) 1904–1911. [64] E.S.M. El-Sayed, A.M. Mansour, M.S. Abdul-Hameed, Thymol and carvacrol prevent doxorubicin‐induced cardiotoxicity by abrogation of oxidative stress, inflammation, and apoptosis in rats, J. Biochem. Mol. Toxicol. 30 (1) (2016) 37–44. [65] J.J. Guo, L.L. Ma, H.T. Shi, et al., Alginate oligosaccharide prevents acute doxorubicin cardiotoxicity by suppressing oxidative stress and endoplasmic reticulummediated apoptosis, Mar. Drugs 14 (12) (2016) 231. [66] X. Lv, X. Yu, Y. Wang, et al., Berberine inhibits doxorubicin-triggered cardiomyocyte apoptosis via attenuating mitochondrial dysfunction and increasing Bcl-2 expression, PLoS One 7 (10) (2012) e47351. [67] X. Yu, L. Cui, Z. Zhang, et al., α-Linolenic acid attenuates doxorubicin-induced cardiotoxicity in rats through suppression of oxidative stress and apoptosis, Acta Biochim. Biophys. Sin. 45 (10) (2013) 817–826. [68] N. Razmaraii, H. Babaei, A.M. Nayebi, et al., Crocin treatment prevents doxorubicin-induced cardiotoxicity in rats, Life Sci. 157 (2016) 145–151. [69] W. Rui, L. Guan, F. Zhang, et al., PM2. 5‐induced oxidative stress increases adhesion molecules expression in human endothelial cells through the ERK/AKT/ NF‐κB‐dependent pathway, J. Appl. Toxicol. 36 (1) (2016) 48–59. [70] R.M. Guo, W.M. Xu, J.C. Lin, et al., Activation of the p38 MAPK/NF-κB pathway contributes to doxorubicin-induced inflammation and cytotoxicity in H9c2 cardiac cells, Mol. Med. Rep. 8 (2) (2013) 603–608. [71] F. Benzer, F.M. Kandemir, M. Ozkaraca, et al., Curcumin ameliorates doxorubicininduced cardiotoxicity by abrogation of inflammation, apoptosis, oxidative DNA damage, and protein oxidation in rats, J. Biochem. Mol. Toxicol. 32 (2) (2018) e22030. [72] P. Chitra, G. Saiprasad, R. Manikandan, et al., Berberine attenuates bleomycin induced pulmonary toxicity and fibrosis via suppressing NF-κB dependant TGF-β activation: a biphasic experimental study, Toxicol. Lett. 219 (2) (2013) 178–193. [73] T. Zhu, L. Zhang, S. Ling, et al., Scropolioside B inhibits IL-1β and cytokines expression through NF-κB and inflammasome NLRP3 pathways, Mediators Inflamm. 2014 (2014). [74] Y. Zhang, X.R. Huang, L.H. Wei, et al., miR-29b as a therapeutic agent for angiotensin II-induced cardiac fibrosis by targeting TGF-β/Smad3 signaling, Mol. Ther. 22 (5) (2014) 974–985. [75] M.H. Arafa, N.S. Mohammad, H.H. Atteia, et al., Protective effect of resveratrol against doxorubicin-induced cardiac toxicity and fibrosis in male experimental rats, J. Physiol. Biochem. 70 (3) (2014) 701–711.

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