ARE pathway

Chemico-Biological Interactions 305 (2019) 54–65

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Chitosan oligosaccharides prevent doxorubicin-induced oxidative stress and cardiac apoptosis through activating p38 and JNK MAPK mediated Nrf2/ ARE pathway

T

Yongtian Zhanga, Khalil Ali Ahmada,b, Farhan Ullah Khanb,c, Simin Yana, Awais Ullah Ihsanc, Qilong Dinga,* a b c

Experimental and Teaching Center of Medical Basis for Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China Shanghai Jiao Tong University, School of Pharmacy, 800 Dongchuan Road, Shanghai, 200240, China Department of Clinical Pharmacy, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu Province, 211198, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Doxorubicin Chitosan oligosaccharides Oxidative stress Apoptosis Nrf2/ARE

Doxorubicin (DOX) is one of the most effective chemotherapeutic drugs; however, the incidence of cardiotoxicity compromises its therapeutic index. Oxidative stress and apoptosis are believed to be involved in DOX-induced cardiotoxicity. Chitosan oligosaccharides (COS), the enzymatic hydrolysates of chitosan, have been reported to possess diverse biological activities including antioxidant and anti-apoptotic properties. The objective of the present study was to investigate the potential role of COS against DOX-induced cardiotoxicity, and the effects of COS on apoptosis and oxidative stress in rats and H9C2 cells. Furthermore, we also shed light on the involved pathways during the whole process. For this purpose, first, we demonstrated that COS exhibited a significant protective effect on cardiac tissue by not only inducing a decrease in body and heart growth but also ameliorated oxidative damage and ECG alterations in DOX-treated rats. Second, we found that COS reversed the decrease of cell viability induced by DOX, reduced the intracellular reactive oxygen species (ROS), increased the mitochondrial membrane potential (MMP) and Bcl-2/Bax ratio. COS treatment also results in reduced caspase-3 and caspase-9 expressions, and an increase in the phosphorylation of MAPKs (mitogen-activated protein kinases) in DOX-exposed H9C2 cells. Additionally, cellular homeostasis was re-established via stabilization of MAPK mediated nuclear factor erythroid 2-related factor 2/antioxidant-response element (Nrf2/ARE) signaling and transcription of downstream cytoprotective genes. In summary, these findings suggest that COS could be a potential candidate for the prevention and treatment of DOX-induced cardiotoxicity.

1. Introduction Doxorubicin (DOX) is an effective and potent anthracycline antibiotic widely used to treat a broad scope of cancers including leukemia, lymphoma, and solid tumors [1,2]. However, the toxic side effects of DOX to healthy tissues, specifically to the heart, has limited DOX in the clinical therapy because of its ability to induce a dose-dependent cardiotoxicity [3,4]. Up to date, the exact mechanisms underlying of DOX-induced cardiotoxicity have not been fully explained but are likely to be involved with multiple pathways. Particularly, increased oxidative stress, overproduction of reactive oxygen species (ROS) and apoptosis have been proposed as main mechanisms of DOX-induced cardiotoxicity [5,6].

Also, accumulated evidences have indicated that DOX-induced mitochondrial dysfunction is a key trigger of DOX-induced cardiotoxicity [7,8]. Mitochondria are among the key intracellular sites for the production of ROS intermediates [9,10]. DOX has a high affinity for cardiolipin which is included in the processes and reactions of mitochondrial biogenesis [11]. DOX binds to cardiolipin after which enters the mitochondria and leads to the respiratory chain inhibition and ultimately leads to cardiomyocyte apoptosis [12–14]. Furthermore, mitochondrial membrane potential rapid depolarization; stimulation of mitochondrial DNA destruction [15], impaired expression of sundry vital cardiac proteins [16]; damage of mitochondrial bioenergetics [17] have been linked also to DOX-induced cardiotoxicity mechanisms. Since DOX is capable of dictating intracellular ROS production, it

* Corresponding author. Department of Pharmacology, Experimental and Teaching Center of Medical Basis for Pharmacy, China Pharmaceutical University, Longmian Avenue, 639, Nanjing, Jiangsu, 211198, China. E-mail address: [email protected] (Q. Ding).

https://doi.org/10.1016/j.cbi.2019.03.027 Received 19 January 2019; Received in revised form 14 March 2019; Accepted 26 March 2019 Available online 28 March 2019 0009-2797/ © 2019 Published by Elsevier B.V.

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Abbreviations (ARE) (Bcl-2) (CAT) (COS) (CK) (Cyt-c) (DOX) (ECG) (ERKs) (GR)

(GSH) (GSSG) (HO-1) (JNK) (LDH) (MAPK) (MDA) (MMP) (Nrf-2) (ROS) (SOD)

Antioxidant response element B-cell lymphoma-2 Catalase Chitosan Oligosaccharides Creatine kinase Cytochrome-C Doxorubicin Electrocardiography Extracellular signal-regulated protein kinases Glutathione reductase

Reduced glutathione Oxidized glutathione Heme oxygenase-1 c-Jun N-terminal kinases Lactate dehydrogenase Mitogen activated protein kinases Malondialdehyde Mitochondrial membrane potential Nuclear factor erythroid 2-related factor-2 Reactive oxygen species Superoxide dismutase

GIBCO (Grand Island, NY, USA). Primary antibodies against β-actin, Nrf2, Cyt-c, Bcl-2, Bax, Caspase-3, Caspase-9, ERK1/2, p- ERK1/2, JNK, p- JNK, p38, p-p38 and goat anti-mouse, or anti-rabbit IgG-HRP secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end-labeling (TUNEL) cell death detection kit was obtained from Beyotime Institute of Biotechnology (Jiangsu, China).

may serve as a mechanism to regulate redox-sensitive elements to respond to their environment. Nrf2 (nuclear factor-erythroid 2-related factor 2) is a redox-sensitive transcription factor that regulates the ARE (antioxidant-response element), which controls the expression of numerous antioxidants and detoxifying enzymatic systems such as NAD(P) H dehydrogenase quinone-1 (NQO1), heme oxygenase-1 (HO1), glutathione reductase (GR) and Cu/Zn-SOD [18,19]. There are various potential mechanisms by which Nrf2 can be regulated and some of these appear to be more or less active, depending on the physiological circumstances [20]. Cumulative studies have showed that the mitogenactivated protein kinases (MAPKs) are significant mediators of oxidative stress-induced apoptosis, are also involved in the Nrf2/ARE signal transductions [21,22]. Since DOX changes mitochondrial functions in cardiac cells [23], improving and protecting the crucial biological functions of Nrf2/ARE must be a reliable and effective strategy for counteracting DOX-induced cardiotoxicity. Chitosan oligosaccharide (COS), the hydrolyzed product of chitosan, is a mixture of oligomers of β-1,4-linked D-glucosamine residues and is abundant in the exoskeleton of crustaceans and cell walls of fungi and insects [24]. COS has plenty of biological activities such as immunity regulation [25], antitumor [26], antimicrobial [27] and anti-apoptotic activities [28]. Recently, the antioxidant property of COS has attracted growing attention [29] because of its biocompatibility, biodegradability, non-toxicity, and better absorption properties. Previous studies have shown that chitosan prevents oxidative stress-induced amyloid-β formation and cytotoxicity in NT2 neurons via mediating Nrf2 and NFκB pathways [30]. Earlier reports have also shown that COS attenuated oxidative-stress related retinal degeneration in rats [31]. These enormous numbers of studies suggest that COS has a significant role in the prevention of oxidative stress. However, little is known about the effect of COS on DOX-induced cardiotoxicity and the exact mechanisms involved. The present study was designed to examine the protective ability of COS against DOX-induced cardiotoxicity in rats and to illustrate the mechanisms of its protective effects on oxidative stress and cardiac apoptosis on cultured cardiomyoblasts cells (H9c2). Cells are originally derived from embryonic heart rats and are commonly recognized to be an effective model for cardiomyoblasts cells. These cells have been used practically to examine cardiac protection and cellular mechanisms [32–34].

The rats were randomly divided into seven groups, consisting ten rats per group. The first two groups served as control groups. The first normal control group received only normal saline as vehicle, orally for 2-weeks, while the second control group, a toxin control group, received DOX (2.5 mg/kg body weight) intraperitoneally (i.p.) in 6 injections over a 2-week period to yield a cumulative dose of 15 mg/kg body weight [35]. The remaining five experimental groups were treated with five different doses of COS (10, 30, 50, 70 and 100 mg/kg body weight) intragastrically in normal saline, for 2-weeks concomitantly with DOX treatment (with the same dose in the aforementioned group). Fresh serum was used for analysis of CK (creatine kinase) activity using a commercially available assay kit following manufacturer instructions (Cat.A032, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2. Materials and methods

2.4. Experimental design for in vivo treatments

2.1. Chemicals

After acclimatization, rats were divided randomly into four groups each group consisted of 10 rats and received the following treatment; (1) Control group: rats received only normal saline i. p for 2-weeks; (2) COS group: rats received only COS (50 mg/kg body weight) intragastrically in normal saline for two weeks; (3) DOX group: rats received only DOX (2.5 mg/kg body weight) intraperitoneally (i.p.) in six injections over a 2-week period to yield a cumulative dose of 15 mg/kg body weight, as previously mentioned [35]; (4) DOX + COS group: rats received the same dose of COS as the ‘‘COS group’‘, concomitantly with

2.2. Experimental animals Adult male Sprague-Dawley rats (weighing 200 ± 20 g) were used in this study. Rats were purchased from the Qing Longshan Standard Animal Propagation Center (Nanjing, China). Rats were adopted in a temperature and humidity-controlled environment on a 12-hr light/ dark cycle and ad libitum access to food and water. Three to five days were given for rats to acclimatize to the laboratory environments prior to the experiments. The animal protocols were approved by the Animal Care and Welfare Committee of China Pharmaceutical University (Nanjing, China) and followed the regulatory animal care guidelines of the US National Institutes of Health (Bethesda, MD, USA). 2.3. Determination of dose-dependent activity of COS by creatine kinase (CK) assay

Doxorubicin, Chitosan oligosaccharide, Dichlorofluorescein diacetate (DCFH-DA), 3-(4,5-dimethyl– thiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT), penicillin and streptomycin, SP600125, SB203580 were purchased from Sigma-Aldrich (St. Louis, MO, USA). MitoSOX™ Red mitochondrial superoxide indicator was purchased from molecular probes (Eugene, Oregon, USA). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from 55

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containing 150 μl of DMEM with 10% FBS and incubated overnight. After the drug treatment, 20 μl MTT (5 mg/ml) was added to each well, and the cells were cultured for another 4 h at 37 °C. Next, the culture medium with dye was discarded and 100 μl of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystal. The absorbance of each well was recorded at 490 nm using BioTek Eon microplate spectrophotometer (BioTek Instruments Inc., Winooski, Vermont, USA).

a dose of DOX identical to the ‘‘DOX group’‘. 2.5. Harvest of serum and cardiac samples At the end of the experimental period, the rats were weighed and the anesthetized with i. p. injection of thiopentone (35 mg/kg). Then, blood samples were collected from the left ventricle and centrifuged at 3000×g for 10 min to collect the serum and stored at −20 °C for further assessment of the biochemical parameters. Rats were decapitated, the cardiac tissues were separated and washed quickly with ice-cold saline. The cardiac tissues were fixed in 10% phosphate-buffered formalin for histopathologic examination or stored at −80 °C for later analysis.

2.12. Detection of intracellular and mitochondrial reactive oxygen species (ROS) For the detection of intracellular and mitochondrial ROS, DCFH-DA probe and MitoSOX™ Red mitochondrial superoxide indicator were used respectively. Briefly, H9C2 cells were plated on 24-well plates in DMEM containing 10% serum. After drug treatment, cells were washed with phosphate buffer saline (PBS) and then stained with 10 μM DCFHDA and 5 μM MitoSOX Red in serum-free medium for 30 min at 37 °C in the dark. Cells were then washed with PBS for two times and the fluorescence density was measured by Varioskan Flash Multimode Reader (Thermo Scientific, MA, USA).

2.6. Preparation of cardiac tissue homogenates The rat hearts were minced, washed, and homogenized in a Dounce glass homogenizer in 10 mM HEPES-KOH/1 mM EDTA buffer (pH 7.5) containing 250 mM sucrose and supplemented with protease and phosphatase inhibitors. The homogenates were centrifuged at 2000×g for 10 min to discard the myofilaments. The supernatant was collected and used for in vivo experiments. The protein concentration was determined with a BCA kit (Pierce, US) following manufacturer instructions.

2.13. Measurement of mitochondrial membrane potential (MMP) Mitochondrial membrane potential was assessed using the JC-1 fluorescent probe，a mitochondria-specific dual-emission potential sensor. Polarized mitochondria accumulate JC-1 within the matrix, where the JC-1 aggregates and fluoresces red. In depolarized mitochondria, JC-1 does not aggregate, remains in the monomer form, and fluoresces green. Staining was performed using 2.5 mg/ml JC-1 at 37 °C for 15 min. After staining, cells were rinsed three times with phosphate buffer saline (PBS) and the fluorescence density was measured by Varioskan Flash Multimode Reader (Thermo Scientific, MA, USA).

2.7. Serum and myocardial tissue biochemical assays The heart activities of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and oxidized glutathione (GSSG), plasma levels of creatine kinase (CK), lactate dehydrogenase (LDH), total cholesterol and HDL cholesterol levels were measured by using a commercially available assay kit purchased from Nanjing Jiangcheng Bioengineering Institute (Nanjing, China), following the manufacturer instructions.

2.14. Cell death assay 2.8. Electrocardiography (ECG) Electrocardiography (ECG) analysis was done one day after completion of drug treatment duration in all groups. Briefly, rats were anesthetized with thiopentone (35 mg/kg; i. p) and followed by placing the needle electrodes under the skin for the limb lead at position II. ECG recordings was recorded using a Biopac ECG recorder (Bioscience, Washington, USA). The QRS and S-T segments were analyzed.

The TUNEL method was performed to label 3′-end of fragmented DNA of the apoptotic H9C2 cells. The cells were fixed with 4% paraform PBS, rinsed with PBS, then permeabilized by 0.1% Triton X-100 for FITC end-labeling fragmented DNA of the apoptotic H9C2 cells using TUNEL cell apoptosis detection kit. The fluorescence density was measured by Varioskan Flash Multimode Reader (Thermo Scientific, MA, USA).

2.9. Histopathological examination of the heart tissue

2.15. Western blot analysis

Paraformaldehyde-fixed heart tissue samples were embedded in paraffin wax were serial sections were at 7-μm thickness. The sections were stained with hematoxylin and eosin for the assessment of histopathological changes. A histopathologist who was blinded to the treatment groups examined the sections under a light microscope.

After treatment, the cells were washed three times with ice-cold PBS. Proteins were solubilized and extracted with 200 μl lysis buffer (Tris 50 mM, pH 7.4, NaCl 150 mM, Triton X-100 1%, EDTA 1 mM, and PMSF 2 mM). The lysates were used to estimate their protein content with BCA method. Equal amount of protein (30 μg) from each sample was subjected to electrophoresis on a SDS-polyacrylamide gel, transferred onto PVDF membrane (Millipore, USA) in pre-cooled transfer buffer and blocked with 4% bovine serum albumin in TBST (1 M TrisHCl, 0.15 M NaCl, 0.1% Tween, PH 7.4) for 1 h at room temperature. The membranes were then incubated with the specific primary antibodies against Nrf2 (1:1000), Cyt-c (1:1000), Bcl-2 (1:1000), Bax (1:1000), Caspase-3 (1:1000), Caspase-9 (1:1000), ERK1/2 (1:1000), pERK1/2 (1:1000) JNK (1:1000), p-JNK (1:1000), p38 (1:1000), p-p38 (1:1000) and β-actin (1:1000) at 4 °C overnight with slight shacking. After washing, the membranes were incubated with the corresponding anti-mouse, or anti-rabbit IgG secondary antibody (1:2000) conjugated to horseradish peroxide for 1 h at room temperature. Then the membranes were washed in TBST for 30 min and exposed to enhanced chemiluminescence reagents. Protein band intensity was measured using ImageJ software (National Institutes of Health, Bethesda, MD).

2.10. Cell culture and treatment Rat cardiac H9C2 cells were obtained from the cell bank of type culture collection the Chinese Academy of Sciences, Shanghai, China. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, 100 U/ml streptomycin. Cells were incubated at 37C with 95% humidity and 5% CO2. The medium was changed every 2–3 days and subcultured once they reached ~90% confluence. 2.11. Cell viability assay The cell viability was determined by MTT assay. Briefly, H9C2 cells were seeded in 96-well plates at a density of 1 × 105/ml, each well 56

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3.2. Inhibition of the cytotoxic effects of DOX on body and heart growth by COS

2.16. RNA extraction and real-time PCR analysis Cells were harvested and total RNA was extracted using the RNeasy Micro kit (Invitrogen, USA). Total RNA was treated with DNase I (Invitrogen, USA) and subsequently reverse transcribed using random hexamers and Superscript II reverse transcriptase enzyme (Invitrogen, USA) according to the manufacturerinstructions. Real-time PCR was done with SYBR Green Real-Time Core Reagents (Applied Biosystems) according to the manufacturer instructions on the ABI Prism 7900 Sequence Detection System (Applied Biosystems, CA, USA). The specific primers were as follows: GAPDH:forward primers:5′-GGCCAAGGTCAT CCATGA-3′, reverse primers:5′-TCAGTGTAGCCCAGGATG-3′, NQO1:forward primers:5′-AACGTCATTCTCTGGCCAATTC-3′, reverse primers:5′-GCCAATGCTGTACACCAGTTGA-3’; HO-1:forward primers: 5′-ATGCCCCACTCTACTTCCCTGA-3′, reverse primers:5′-TGCTGTGTG GCTGGTGTGTAAG-3’; GR: forward primers: 5′-TTGCTGGCCTCTATTC ACTGG-3′, reverse primers:5′-ATTACCTCCGCCCTCTCTTTG-3’; Cu/ ZnSOD: forward primers: 5′-GATTAACTGAAGGCGAGCAT-3′, reverse primers:5′-CCGCCATGTTTCTTAGAGT-3’. The mRNA expression level was normalized against GAPDH expression [36].

During this experiment, all animals appeared to be alive and no mortality was encountered in either of the groups. Among the four animal groups, the DOX group gained less body weight and heart weight compared with those in the control group at the end of the experiment. However, treatment with COS increased the body and heart weight compared with DOX-treated rats, demonstrating the growth-impeding effect of DOX and the counteracting action of COS against DOX (Table 1). Conversely, the relative heart weight index (heart weight to body weight ratio) was similar among all the four groups at the end of experiment (Table 1). 3.3. COS attenuated DOX-induced oxidative stress cardiotoxicity in rats Results of the present study showed that DOX-induced severe biochemical changes and oxidative damage in the heart tissues. Myocardial damage in the DOX group was assessed by measuring total cholesterol level and HDL cholesterol, LDH and CK activity. DOX exposure significantly increased total cholesterol and LDH levels and CK activity, but it decreased HDL cholesterol level. However, treatment with COS maintained these levels near to the baseline as shown in Table 2. The formation of ROS produced by DOX, is considered the ratelimiting step in lipid peroxidation. The biochemical determination of malondialdehyde (MDA) indicated lipid peroxide formation. We observed that DOX significantly increased the MDA level in heart tissue compared with the normal group. However, pretreatment with COS significantly reduced this level (Table 3). Increased oxidative stress decreased intracellular GSH and increased the formation of GSSG; thereby leading to imbalance in the GSH/GSSG redox couple in the present study. DOX treatment significantly reduced the cardiac GSH level and the GSH/GSSG ratio (Table 3). Treatment with COS, however, maintained the levels near to the baseline as shown in Table 3. Antioxidant enzyme activities (SOD, CAT) reflect the level of oxidative stress of the tissue examined. SOD and CAT activities in the rat hearts of all groups are illustrated in Table 3. Compared with the control group, DOX-exposed hearts possessed significantly less anti-oxidant enzyme activity, whereas COS treatment effectively prevented DOX-induced reduction of antioxidant enzyme activity. The influence of treatment with COS and/or DOX on rat ECG parameters is shown in Table 4. S-T segment prolongation as well as QRS complex prolongation in DOX-treated rats were statistically

2.17. Isolation of cytosolic and mitochondrial proteins Mitochondria were isolated to test the cytochrome c release as described previously [37]. Mitochondria were isolated from H9c2 cells using a mitochondrial isolation kit for cultured cells (Mitoscience) according to the manufacturer's protocol. 2.18. Transient transfection and ARE-Luciferase assay H9C2 cells were plated in 6 well plates to be ~90% confluent and transfected with ARE-luciferase plasmid pGL6-ARE and the Renilla luciferase pRL-TK vector, used as an internal control, at a concentration of 1 mg/ml using Lipofectamine LTX & PLUS Reagent (Invirogen, CA, USA) according to the manufacturer instructions. After transfection for 24 h, cells were pretreated with or without COS (100 μg/ml) for 24 h before DOX (1 μM) induction for 12 h, and the cell lysates were prepared with passive lysis buffer. Dual-Luciferase reporter assay system (Promega, WI, USA) was employed to evaluate the luciferase activity, using a Varioskan Flash Multimode Reader (Thermo Scientific, MA, USA) according to the manufacturerinstructions. Relative firefly luciferase activity was normalized to Renilla luciferase activity. 2.19. Statistical analyses GraphPad Prism software data analysis program version 5 was used for statistical analysis (GraphPad Software, Inc.). Data were presented as the mean ± standard deviation (S.D). Statistical comparisons were performed using one-way ANOVA. A p-value of (p < 0.05) was considered significant. 3. Results 3.1. Dose-dependent study of COS by CK assay Creatine kinase (CK) was assessed to determine the optimum dose necessary for COS to protect the rat heart against DOX-induced cardiotoxicity. The results suggested that DOX intoxication (2.5 mg/kg body weight, i. p. on alternate days) increased the serum CK activity, but it was prevented by COS treatment up-to a dose of 50 mg/kg body weight in normal saline, intragastrically, 1 day before each dose of DOX as shown in Fig. 1. However, a higher dose of COS did not provide additional benefit to reduce serum CK activity. Therefore, COS dose of 50 mg/kg body weight was selected for subsequent in vivo experiments.

Fig. 1. Dose-dependent effect of COS on CK activity. Rats received different doses of COS (10–100 mg/kg) intragastrically on alternate days followed by DOX (2.5 mg/kg) exposure to detect CK activity after four weeks. Each column represents mean ± SD, n = 10. “a” indicates the significant difference between the control and DOX-treated groups and “b” indicates the significant difference between COS + DOX treated and DOX-treated groups. (Pa < 0.05, Pb < 0.05). 57

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Table 1 Effect of DOX, COS, and their combination on the rat heart and body weight. Group Body weight (g) Heart weight (g) Heart/body weight ratio (mg/g)

Control

COS

283.6 ± 9.94 1.035 ± 0.032 3.65 ± 0.33

DOX

279.4 ± 10.77 1.018 ± 0.030 3.64 ± 0.28

COS + DOX a

253.6 ± 10.15 0.883 ± 0.050a 3.48 ± 0.49

268.5 ± 10.49a,b 0.977 ± 0.032a,b 3.64 ± 0.30

Values are expressed as mean ± SD for 10 animals in each group. a values differ significantly from control (Pa < 0.05). b values differ significantly from DOX (Pb < 0.05).

mitochondrial ROS production.

significant compared to the control group (p < 0.01). In COS-pretreated animals, the S-T segment duration and the QRS complex duration were significantly improved compared to DOX-treated rats. DOX-induced myocardial lesions observed during light microscopic examination were very conspicuous compared to the control. Loss of striation, small vacuolization, edema of myocardial tissue and cells, as well as an increment in inflammatory cells were found in DOX-treated group (Fig. 2). However, DOX-induced histopathological changes were ameliorated partly in the COS + DOX group as shown in Fig. 2.

3.6. COS suppressed DOX-induced disruption of mitochondrial membrane potential (MMP) Myocardial mitochondria are the target organelles of DOX-induced toxicity in cardiomyocytes. The above results suggested that DOX-induced ROS generation occurs in the mitochondria, and oxidative stress could induce mitochondria depolarization. Thus, we investigated the effect of DOX on H9C2 cells MMP, which is fundamental to cell survival. JC-1, a dual-emission cationic dye, was used to measure the change of the MMP. An increase in red fluorescence (JC-1 aggregate) correlates with polarization of the mitochondrial membrane, whereas an increase in green fluorescence (JC-1 monomer) is indicative of mitochondrial membrane depolarization. As shown in Fig. 4C, DOX (1 μM) treatment for 12 h caused a significant reduction in JC-1 ratio (JC-1 aggregate/monomer), an indicator of a depolarized mitochondrial membrane. However, COS pretreatment (100 μg/ml, 24 h) resulted in an increase in the polarized mitochondria as showed by an increase in red JC-1fluorescence and a decrease in green fluorescence (Fig. 4C), confirming the disruptive effect of DOX and the preservative effect of COS on MMP.

3.4. COS ameliorated DOX-induced decrease in cell viability of H9C2 rat cardiomyocytes We first investigated the dose-response study of DOX by MTT assay. As shown in Fig. 3A, exposure of H9C2 cells with various concentrations (0.1–2 μM) of DOX for 12 h reduced the number of viable H9C2 cells in a concentration-dependent manner, of which 1 μM dose reduced cell viability to 50% compared with untreated H9C2 cells. Thus, we select 1 μM as the subsequent concentration of DOX in in-vitro studies unless otherwise indicated. Cell viability was not influenced by the presence of 1.0–200 μg/ml COS (Fig. 3B). We then evaluate the doseresponse experiments to find out the optimal concentration of COS for the study. It was found that COS suppressed DOX (1 μM)-induced reductions in cell viability in a concentration-dependent manner with maximum inhibition concentration at 100 μg/ml (Fig. 3B).

3.7. COS protected H9c2 cardiomyocytes from DOX-induced cell death ROS production and the associated cellular oxidative stress are known triggers of cell death. Therefore, we hypothesized that COS could decrease DOX-induced cell death. The number of TUNEL-positive H9C2 cells were increased in DOX group (46.7 ± 5.12%, p < 0.05) when compared to the control group and COS pretreated group (8.9 ± 6.84% and 9.5 ± 7.53%) respectively (Fig. 5A).

3.5. COS ameliorated DOX-induced oxidative stress in H9C2 cells DOX has been reported to cause an increase in cellular ROS production [38] leading to oxidative stress when production of ROS oversaturates the capacity of cellular antioxidant defense [39]. Therefore, we assessed the effect of COS on DOX-induced total cellular ROS and mitochondrial ROS levels in H9C2 cells. Total cellular ROS levels were measured via quantification of fluorescent DCFH-DA. As shown in Fig. 4A, H9c2 cells displayed marked total cellular ROS accumulation after 12 h of treatment with 1 μM DOX, whereas cellular ROS accumulation was clearly inhibited in the cells pretreated with COS. Mitochondrial ROS production was measured with MitoSOX Red fluorescence microscope. MitoSOX Red fluorescence in DOX-treated cells increased by 2.8-fold compared with the control, which suggested a significant increase in mitochondrial ROS production by DOX (Fig. 4B). Interestingly, COS pretreatment dramatically inhibited

3.8. COS inhibited DOX-induced apoptotic pathways in H9c2 cells As COS suppress DOX-induced apoptosis, we determined the release of the mitochondrial protein Cytochrome c (Cyto c) into the cytosol and the expression of Bcl-2 and Bax, which are capable of regulating apoptosis. Cyto c was increased in the cytosolic fraction following DOX exposure, and it was reduced by pretreatment with COS as shown in Fig. 5B. The expression of apoptosis-related genes Bcl-2 and Bax were changed by DOX, with Bcl-2 decreased and Bax increased compared to the control and COS groups as shown in Fig. 5C. In COS pretreated group, the protein level of Bcl-2 was significantly increased while Bax

Table 2 Effect of DOX and COS on the serum markers level related to cardiac dysfunction. Group CK (U/L) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDH (U/L)

Control 315.9 73.28 25.18 755.8

± ± ± ±

COS 20.5 6.25 1.32 67.5

305.8 75.93 23.76 756.3

Values are expressed as mean ± SD for 10 animals in each group. a values differ significantly from control (Pa < 0.05). b values differ significantly from DOX (Pb < 0.05). 58

DOX ± ± ± ±

21.2 6.55 0.98 53.0

COS + DOX a

549.1 ± 21.9 179.18 ± 8.37a 12.65 ± 0.87a 914.9 ± 85.1a

405.9 ± 20.5a,b 128.39 ± 7.11a,b 18.23 ± 1.12a,b 833.7 ± 77.8a,b

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Table 3 Effect of DOX and COS on antioxidants and lipid peroxidation. Group MDA (nmol/mg protein) GSH(nmol/mg protein) Redox ratio (GSH/GSSG) SOD (U/mg protein) CAT (U/mg protein)

Control 1.73 ± 0.16 18.15 ± 0.92 53.71 ± 2.16 78.83 ± 4.82 43.83 ± 3.81

COS

DOX

1.56 ± 0.12 20.81 ± 1.22 55.21 ± 2.85 80.23 ± 5.21 44.84 ± 3.64

COS + DOX a

4.27 ± 0.25 11.87 ± 0.82a 39.61 ± 2.76a 50.34 ± 3.86a 23.83 ± 2.46a

2.52 ± 0.19a,b 15.31 ± 1.13a,b 45.97 ± 2.25a,b 64.18 ± 4.28a,b 35.19 ± 2.61a,b

Values are expressed as mean ± SD, for 10 animals in each group. a values differ significantly from control (Pa < 0.05). b values differ significantly from DOX (Pb < 0.05). Table 4 The influence of COS on ECG parameters of rats treated with DOX. Group QRS (ms) S-T segment (ms)

Control 14.09 ± 1.99 39.26 ± 1.19

COS

DOX

13.84 ± 1.27 38.35 ± 2.22

COS + DOX a

26.19 ± 3.12 54.03 ± 2.43a

17.80 ± 1.81a,b 43.94 ± 1.87 a,b

Values are expressed as mean ± SD, for 10 animals in each group. a values differ significantly from control (Pa < 0.05). b values differ significantly from DOX (Pb < 0.05).

Fig. 2. Effect of COS on histopathological changes in DOX treated cardiac tissue (H&E 200 × ). (CN) Control group; (COS) cardiac tissue treated with COS; (DOX) Cardiac tissue treated with DOX; (COS + DOX) Cardiac tissue treated with COS + DOX. Arrows representing the myocardial lesions.

3.9. Phosphorylation of different MAPKs in DOX-induced cardiotoxicity

level was significantly decreased compared to the DOX group respectively. In addition, we calculated the Bcl-2/Bax ratio and found that it was significantly elevated in the COS pretreated group in comparison with the DOX group. Whereas COS treatment effectively suppressed these DOX-induced pro-apoptotic events. Caspase-3 and caspase-9 are the key ‘‘executer” protease of cell apoptosis, so we also investigated the effect of COS on DOX-induced activation of caspase-3 and caspase-9 in H9C2 cells. Treatment of the H9C2 cells with DOX results in a marked increase in the expressions of caspase-3 and caspase-9 at 12 h and COS inhibited these effects (Fig. 5D).

Similar to caspase-3 activation, the MAPK family, including the extracellular signal-regulated protein kinases (ERKs), p38 kinases, and the c-Jun N-terminal kinases (JNK), is involved in the processes that induce stress-mediated cell death [40]. In view of this evidence, Phosphorylation of ERK1/2, p38, and JNK kinases were examined in H9C2 cells of the four groups (Fig. 6A). Phosphorylation of ERK1/2 was significantly reduced in the DOX group compared with the control group while COS did not prevent this reduction (Fig. 6B). Conversely, phosphorylation of both JNK and p38 was significantly increased in the DOX group compared with the control group (Fig. 6C and D). We also

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Fig. 3. Effect of DOX and COS on cell viability. (A) Cells were incubated with different concentration of DOX (0.1–2.0 μM) for 12 h and followed by MTT assay to evaluate the cell viability. (B) Cells were pretreated with different concentrations of COS (10–200 μg/ml) for 24 h prior to DOX (1.0 μM) exposure for 12 h and followed by MTT assay to evaluate the cell viability. “a” indicates the significant difference between the control and DOXtreated cells. “b” indicates the significant difference between DOX and COS + DOX treated cells. All values are presented as mean ± SD, n = 6; (Pa < 0.05, Pb < 0.05).

Fig. 4. COS inhibits DOX-induced intracellular and mitochondrial ROS formation and MMP. Cells were pretreated with/without 100 μg/ml COS for 24 h and then exposed to 1 μM DOX for 12 h followed by incubation with (A) 10 μM DCFH-DA for 30 min or (B) 5 μM MitoSOX Red for 10 min or (C) 2.5 mg/ml JC-1 for 15 min. Thereafter, the fluorescence density was measured by Varioskan Flash Multimode Reader. “a” indicates the significant difference between the control and DOX-treated cells. “b” indicates the significant difference between DOX and COS + DOX treated cells. All values are presented as mean ± SD, n = 6; (Pa < 0.05, Pb < 0.05).

defending against oxidative stress and survival through a wide variety of cellular pathways. To investigate whether COS treatment alters the Nrf2 nucleus translocation and AER-driven transcriptional activity in H9C2 cells, we incubated the cells with COS 100 μg/ml for 3–12 h. The Nrf2 level in the nuclear fraction of COS-treated cells showed a gradual increase, while they declined concomitantly in the cytoplasm as shown in Fig. 8A. However, Nrf2 was dramatically translocated to the nucleus at 12 h treatment (Fig. 8B). By contrast, DOX-exposed group declined nuclear Nrf2 in time-dependent manner. In addition, H9C2 cells transiently transfected with the ARE-luciferase plasmid were exposed to COS and DOX and alteration in luciferase activity was used as a measurement of ARE-driven transcription activity. The reporter assay showed that COS increased ARE-driven luciferase activity while DOX showed the opposite result (Fig. 8C). The nuclear translocation of Nrf2 subsequently triggered the transcription of a downstream battery of phase II detoxifying enzymes genes. The mRNA level of NQO-1, HO-1, GR and Cu/Zn-SOD is depicted in Fig. 8D. DOX-treated group produced a decrease in these enzymes mRNA levels in a time-dependent manner (Fig. 8D). However, pretreatment with COS inhibited the action to a certain extent. It is known that oxidative stress might confer a relative cooperation between major death signals and cellular defense mechanisms. Therefore, we investigated the effect of MAPK inhibition on the nuclear translocation of Nrf2. We pre-treated cells with 10 μM SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) 1 h before any other

performed a time-dependent study with DOX and found that the effect on phospho-JNK and -p38 occurred within 0–12 h of incubation with DOX (Fig. 6E). JNK and p38 phosphorylation was started at 1 h after DOX treatment and with the maximum at 6 h. After this, JNK and p38 phosphorylation was decreased, however, did not recovered to basal level at 12 h. The results showed that COS inhibited DOX-induced activation of p38 and JNK. 3.10. Effect of JNK and p38 MAPK inhibition on DOX-induced apoptosis We next investigated the effect of MAPK inhibition on DOX-induced apoptosis. We pre-treated H9C2 cells with 10 μM SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) separately for 1 h for two different sets of experiments and then studied the effects of DOX and COS on caspase-3 and cell viability (Fig. 7). Results indicates that caspase-3 expression was significantly decreased by co-treatments with DOX- SP600125 and DOX-SB203580, also results indicates that H9C2 cells viability were significantly increased by these co-treatments (Fig. 7). Surely, these findings show that inhibition of JNK and p38 MAPK prevented DOX to induce apoptosis. 3.11. Involvement of MAPK-mediated Nrf2 and transcriptional regulation of downstream genes As previous studies have shown that Nrf2 plays an important role in 60

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Fig. 5. COS protects DOX-induced cell death. (A) H9c2 cells were pretreated with/without 100 μg/ml COS for 24 h and then exposed to 1 μM DOX for 12 h determined by TUNEL assay. (B, C, D), Mitochondrial proteins, cytosolic proteins, and whole-cell extracts were subjected to SDS-PAGE and immunoblotting with antibodies specific to cytochrome c, Bcl-2, Bax, caspase-3, caspase-9 and β-actin as a control. “a” indicates the significant difference between the control and DOXtreated cells. “b” indicates the significant difference between DOX and COS + DOX treated cells. All values are presented as mean ± SD, n = 3 with 3 replicates; (Pa < 0.05, Pb < 0.05).

long threat. Risk of cardiotoxicity remains a serious problem that hampers the clinical usefulness of this potent drug. In the present study, we used COS, a natural product, in the treatment of rat heart and H9C2 cells exposed to DOX-induced cardiotoxicity. The results obtain from this work demonstrate that COS inhibits DOX-induced cardiotoxicity. Treatment of cancer patients with DOX might be responsible for developing cardiomyopathy, which can lead to heart failure [6]. In the present study, DOX administration increased the serum cardiotoxicity indices (LDH and CK) and caused heart failure [41]. This may be due to the lipid peroxidation (oxidative stress) of cardiac myocyte cell

treatment. As shown in Fig. 8E, the combined treatment with inhibitor and COS reduced the nuclear translocation of Nrf2 and increased the level of Nrf2 in the cytosol. The results suggested that JNK and p38 may be partially responsible for transducing signals involved in Nrf2 translocation in DOX-treated H9C2 cells.

4. Discussion DOX-induced cardiotoxicity is an important public health concern because of its little-known etiology for many years and remains a life61

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Fig. 6. Involvement of MAPK signaling pathway in DOX induction. (A, B, C, D) H9c2 cells were pretreated with/without100 μg/ml COS for 24 h, and then exposed to 1 μM DOX. Protein level expression of phosphorylated ERK1/2, JNK and p38 were detected by immunoblotting assay. (E) Phosphorylated JNK and p38 were quantified for varying periods of time (0–12 h) after H9c2 cells exposed to 1 μM DOX.

membrane [43,44]. In addition, the present work has also been confirmed by the histopathological changes induced by DOX, where the heart tissue showed swollen cardiac muscle fibers, interstitial edema and inflammatory infiltration. However, COS pretreatment reduced DOX-induced histopathological changes to a certain extent (Fig. 2). DOX-induced cardiotoxicity is associated with the accumulation of DOX in the mitochondria and ROS production [45]. Cardiac muscle is particularly susceptible to free-radical injury, because it contains low levels of free-radical detoxifying enzymes/molecules like superoxide dismutase, GSH and catalase [46]. Besides, cardiomyocytes are rich in mitochondria, which is the place of principal ROS generation, and are exposed to relatively high oxygen tension compared to other tissues. Furthermore, DOX also has high affinity for the phospholipid component of mitochondrial membrane in cardiomyocyte leading to accumulation of DOX in the heart tissue [46]. The present study demonstrates that COS pretreatment prevents DOX-induced ROS production in the mitochondria of H9C2 cells (Fig. 4). However, NADPH oxidase is

membranes by DOX leading to leakage of CK from cardiac cytosol into the blood. However, COS pretreatment significantly guarded against DOX-induced oxidative stress and cellular injury as monitored by decreased content of LDH and CK. Meanwhile, we also observed that increased lipid peroxidation products (MDA), depleted GSH and increased GSSG levels and decreased antioxidant enzyme activities in DOX-exposed group were effectively reduced with COS supplementation (Table 3). As it is demonstrated in earlier studies that the severity of ECG changes, parallels the DOX cardiotoxicity in patients [42], so we studied ECG alterations. In the present study, S-T segment prolongation as well as QRS complex prolongation in DOX-treated rats were statistically significant in comparison to control and COS pretreatment animals (Table 4) and these results are in accordance with the work that previously reported [43]. All these ECG changes are related to the prolongation of action potential duration, but it is considered that DOX prominently affect the recovery phase of the transmembrane action potential, influencing preferentially Ca2+ movement across the cellular 62

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Fig. 7. Effect of JNK and p38 MAPK inhibition on DOX-induced apoptosis. H9C2 cells were pre-treated with 10 μM SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) separately for 1 h and then exposed to 1 μM DOX for 12 h to detect the protein level of Caspase-3 (A) (C) and cell viability (B) (D). “a” indicates the significant difference between the control and DOX-treated cells. “b” indicates the significant difference between DOX and COS + DOX treated cells. All values are presented as mean ± SD, n = 6; (Pa < 0.05, Pb < 0.05).

group, but it did not last after 6 h (Fig. 8A). Surprisingly, DOX-induced mitochondrial ROS generation and accumulating evidence shows that mitochondrial ROS activate downstream protective mechanisms, including the Nrf2/ARE pathway [53,54], suggesting DOX activation of Nrf2. This is probably due to up-regulation of Nrf2 in response to oxidative stress. Further research will determine if DOX induced oxidative stress generation stimulate the activation of Nrf2 as a compensatory mechanism to overcome the DOX-induced damage. However, the ROS production by DOX is likely to exceed this compensation and therefore lead to mitochondrial dysfunction and cell death. It is well known that MAPKs play an important role in cell proliferation, differentiation and death. The ERK1/2, JNK and p38 kinases, are involved in the response to DOX-induced oxidative stress [55]. Therefore, we studied the effects of DOX and COS on MAPK (ERK1/2, JNK, and p38-MAPK) signaling pathways. In the present study, DOX exposure significantly increased the phosphorylation of JNK and p38 rather than the phosphorylation of ERK1/2 for 1–12 h in H9C2 cells and the phosphorylation peaked at 6 h. Moreover, a specific inhibitor of JNK, SP600125, or a specific inhibitor of p38, SB203580, suppressed DOX-induced caspase-3 cleavage and the decrease in cell viability. However, COS prevented DOX-induced changes in JNK and p38 phosphorylation, thereby blocking cleavage of caspase-3 and apoptosis (Fig. 8). MAPK signaling has been implicated in the induction of the Nrf2/ ARE pathway as indicated by many previous reports and this may occur via phosphorylation of Nrf2 or through the nuclear translocation of Nrf2 [56,57]. In the present study, the JNK and p38 MAPK inhibitors SP600125, and SB203580 significantly inhibited the nuclear translocation of Nrf2 after DOX exposure in H9C2 cells. This suggests that JNK and p38 MAPKs are important mediators of the Nrf2 signaling network in DOX-treated H9C2 cells. Furthermore, COS treatment significantly protected the nuclear translocation of Nrf2 induced by DOX treatment

another important source of ROS, its contribution in this study could not be excluded, and further studies are needed to investigate the effect of COS on NADPH oxidase. Mitochondria plays a pivotal role in the ROS-mediated apoptotic process [47]. It has been found that DOX induces mitochondrial dysfunction followed by a rapid efflux of intracellular ROS, which further triggers apoptosis of the cells [48]. In the present study, we observed that DOX induced apoptosis (confirmed by TUNEL method), up-regulated pro-apoptotic Bax and down-regulated anti-apoptotic Bcl-2 proteins, reduced mitochondrial membrane potential, increased cytochrome c release into the cytosol, and the cleavage of caspases-3 and 9 in H9C2 cells. However, COS treatment successfully attenuated all these DOX-mediated pro-apoptotic events (Fig. 5). We also wonder whether COS could attenuate intracellular ROS accumulation and protect DOX-induced mitochondrial dysfunction and cell death through its activation of endogenous antioxidant network. Therefore, we tested its effect on Nrf2, a transcription factor that regulates cellular defense against oxidative damage, and plenty of evidences supported that Nrf2/ARE pathway is involved in DOX-induced cardiotoxicity [49]. Nrf2 is normally bound in the reduced form of Keap1 (Kelch-like ECH-associated protein 1) and is inactive in the cytoplasm. The Keap1 protein is consisted a mass of cysteine residues that are susceptible to ROS and modified by reactive electrophiles. Under oxidative stress conditions, the confirmation of Keap1 is changed, which give rise to the release of Nrf2 and then translocate to the nucleus where it accumulates and binds to AREs to transcriptionally regulate expression of genes associated with antioxidant defense pathways [19,50]. Therefore, the expression status of genes such as NQO-1, HO-1, GR and Cu/Zn-SOD can provide an indication of the intracellular ROS level [51,52]. In the present study, we observed that COS increased Nrf2 nuclear translocation as time goes on 3–12 h (Fig. 8A). Nrf2 was also slightly translocated to nucleus in the first 3–6 h in DOX-treated 63

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Fig. 8. COS activates MAPK-medicated Nrf2/ARE pathway. (A) H9C2 cells were pretreated with/without100 μg/ml COS for 24 h, and then exposed to 1 μM DOX for varying periods of time (0–12 h). Nuclear and cytoplasmic extracts were isolated as described under materials and methods and subjected to SDS-PAGE and immunoblotting with antibodies specific to Nrf2 and β-actin as control. (B) The statistic was representative of bands of control and treatment for 12 h group. (C) After 24 h transfection, cells were pretreated with/without 100 μg/ml COS for 24 h, and then exposed to 1 μM DOX. ARE-luciferase reporter gene assay was performed using a commercial kit. (D) The mRNA level of NQO-1, HO-1, GR and Cu/Zn-SOD was quantified for varying periods of time (0–12 h) after H9c2 cells pretreated with/without 100 μg/ml COS for 24 h, and then exposed to 1 μM DOX. (E) Protein level expression of nuclear and cytosolic Nrf2 in the presence of JNK- and p38specific inhibitors, SP600125 and SB203580, respectively. “a” indicates the significant difference between the control and DOX-treated cells. “b” indicates the significant difference between DOX and COS + DOX treated cells. All values are presented as mean ± SD, n = 6; (Pa < 0.05, Pb < 0.05).

Conflicts of interest

and this effect was fully blocked by JNK, SP600125 and p38 MAPK, SB203580 specific inhibitors (Fig. 8).

All authors declare that there is no conflict of interest. 5. Conclusion Acknowledgment In this present study, we demonstrate that COS provide protection against DOX-induced cardiotoxicity through activating MAPK mediated Nrf2/ARE pathway and attenuating Mitochondrion-dependent cell apoptosis and oxidative stress. Therefore, COS appears to have significant potential role in preventing DOX-induced oxidative damage.

We are grateful to the Department of Physiology (China Pharmaceutical University) for providing H9C2 cells line and technical advice.

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