Crocin treatment prevents doxorubicin-induced cardiotoxicity in rats

Life Sciences 157 (2016) 145–151

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Crocin treatment prevents doxorubicin-induced cardiotoxicity in rats Nasser Razmaraii a,d, Hossein Babaei a,b,⁎, Alireza Mohajjel Nayebi b, Gholamreza Assadnassab c, Javad Ashrafi Helan e, Yadollah Azarmi b a

Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 5165665811, Iran School of Pharmacy, Tabriz University of Medical Sciences, Tabriz 5166414766, Iran Department of Clinical Sciences, Tabriz Branch, Islamic Azad University, Tabriz 5157944533, Iran d Student Research Committee, Tabriz University of Medical Sciences, Tabriz 5166614756, Iran e Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz 5166617564, Iran b c

a r t i c l e

i n f o

Article history: Received 1 February 2016 Received in revised form 4 June 2016 Accepted 10 June 2016 Available online 11 June 2016 Keywords: Crocin Cardioprotection Doxorubicin Cardiomyopathy Echocardiography ECG

a b s t r a c t Doxorubicin (DOX)-induced cardiotoxicity is well-known as a serious complication of chemotherapy in patients with cancer. It is unknown whether crocin (CRO), main component of Crocus sativus L. (Saffron), could reduce the severity of DOX-induced cardiotoxicity. Therefore, this study was undertaken to assess the protective impact of CRO on DOX-induced cardiotoxicity in rats. The rats were divided into four groups: control, DOX (2 mg/kg/ 48 h, for 12 days), and CRO groups that receiving DOX as in group 2 and CRO (20 and 40 mg/kg/24 h, for 20 days) starting 4 days prior to first DOX injection and throughout the study. Echocardiographic, electrocardiographic and hemodynamic studies, along with histopathological examination and MTT test were carried out. Our findings demonstrate that DOX resulted in cardiotoxicity manifested by decreased the left ventricular (LV) systolic and diastolic pressures, rate of rise/drop of LV pressure, ejection fraction, fractional shortening and contractility index, as compared to control group. In addition, histopathological analysis of heart confirmed adverse structural changes in myocardial cells following DOX administration. The results also showed that CRO treatment significantly improved DOX-induced heart damage, structural changes in the myocardium and ventricular function. In addition, CRO did not affect the in vitro antitumor activity of DOX. Taken together, our data confirm that CRO is protective against cardiovascular-related disorders produced by DOX, and clinical studies are needed to examine these findings in human. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Doxorubicin (DOX), a secondary metabolite of Streptomyces peucetiusvar. Caesius, is used as a very effective chemotherapeutic drug for the treatment of a variety of human cancers such as solid tumors, leukemia, lymphomas and breast cancer [34]. Unfortunately, in addition to its excellent properties as an antitumor agent, the clinical use of DOX is associated with a number of cardiotoxic effects such as transient cardiac arrhythmia, nonspecific electrocardiographic abnormalities, and cardiomyopathy [5,6]. These cardiotoxic effects can occur immediately or several weeks to months after the treatment of patients with repetitive DOX administration [2]. Several hypotheses have been proposed to explain DOX-induced cardiotoxicity and among them the free radicals hypothesis has major role in explaining cell death by apoptosis or cell necrosis [21,34,48]. Since the heart is vulnerable to free radicals due to its less developed antioxidant defense mechanisms ⁎ Corresponding author at: Drug Applied Research Center, Research and Development Complex, Tabriz University of Medical Sciences, Daneshgah St., Tabriz 51656658, Iran. E-mail addresses: [email protected], [email protected] (H. Babaei).

http://dx.doi.org/10.1016/j.lfs.2016.06.012 0024-3205/© 2016 Elsevier Inc. All rights reserved.

[11], the search for strategies using natural dietary/non-dietary products as cardioprotective agents with antioxidant properties and no toxicity could be a therapeutic strategy to reduce or prevent the risk of developing DOX cardiotoxicity. However, the challenge for the future is to find antioxidant drugs that are cardioprotective without hindering the antitumor activity of DOX. CRO is well known as a unique water soluble carotenoids which is found in the stigmas of Crocus sativus Linne and in the fruits of Gardenia jasminoides Ellis [33]. This natural compound has attracted research attention for its extensive pharmacological actions such as anti-inflammatory, antitumor, anti-hyperlipidemic, free radical scavenging, antioxidant and anti-atherosclerotic effects as well as protective against DNA damage [1]. Several studies have also demonstrated that CRO has various neuroprotective activities in different animal models of brain disorders including cerebral ischemia [45], anxiety [36], depression [23], memory impairment [17], and Alzheimer's disease [28]. This evidence has demonstrated the therapeutic potentials of CRO in the amelioration of different diseases; however, the efficacy of CRO for reducing DOX-induced cardiotoxicity has not yet been evaluated. Given that, the high antioxidant capacity of CRO in vivo and in vitro

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2. Material and methods

electrodes inserted subcutaneously into the left forepaw and hind paws of the rats [41]. They were connected to a bio-amplifier (Bio Amp ML136; ADInstruments; Australia) to record and analyze ECG data using Lab Chart7 software (ADInstruments; Australia). Each recording was continued for at least 5 min.

2.1. Materials

2.6. Left ventricular function analysis

The following materials were used in the experiments: DOX hydrochloride (Exir Nano Sina Company, Iran), crocin (Bu ali Research Institute, Iran), ketamine hydrochloride and xylazine (Alfasan, Netherlands), heparin (Hospira, USA), human breast adenocarcinoma MCF7 cell line (Pasteur Institute of Iran,), MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), RPMI 1640, DPPH (1, 1-diphenyl-2-picrylhydrazyl; Sigma; Germany), fetal calf serum (FCS), DMSO (Dimethyl sulfoxide), penicillin, streptomycin, L-glutamine and sodium pyruvate (Gibco, USA).

Adult male Wistar rats (180–220 g, aged 8–10 weeks) were obtained from Pasteur institute of Iran. Animals were housed in a room with a 12:12-h light/dark cycle (light on at 07:00 a.m.) under controlled temperature (25 ± 2 °C). All animals had ad libitum access to rodent chow and tap water. All experiments were performed according to the protocols approved by the Committee on the Ethics of Animal Experiments of the Tabriz University of Medical Sciences. All efforts were made to minimize animal suffering.

Animals were anesthetized with ketamine (100 mg/kg/i.p.) and xylazine (10 mg/kg/i.p.). In order to prevent blood coagulation, rats received a subcutaneous injection of heparin (2000 U/kg). After 5-10 min of ECG recording [41], the neck of the rat was opened longitudinally and the left carotid artery (LCA) was exposed and released, ligated distally and stay sutures were placed proximal to the LCA. A small opening was then made in the artery with mini-scissors and a 2F micromanometer-tipped pressure transducer catheter (SPR-407; Millar Instruments) was inserted to the LCA for evaluation of arterial blood pressure (BP). A catheter was inserted gently into the LV to collect hemodynamic data for analysis using Lab Chart 7 (ADInstruments) [41]. The heart rate (HR), LV pressure at the ends of both systole and diastole (LVESP, LVEDP), maximum rate of rise of left ventricular pressure (max dP/dt), minimum rate of rise of left ventricular pressure (min dP/dt), end-diastolic pressure (EDP) and contractility index, a major determinant of cardiac output and an important factor in cardiac compensation, were calculated. The R-R interval, which is the interval from the peak of one QRS complex to the peak of the next and the QT interval on the surface electrocardiogram, an indirect measure of time between ventricular depolarization and repolarization, was also measured.

2.3. Experimental design

2.7. Body weight, heart weight and heart/body weight ratio

All experiments were conducted in a quiet room during the light period (between 8:00 a.m. and 1:00 p.m.). Twenty-four rats were divided into four groups (six animals in each group). Drug solutions were freshly prepared before administration. Group 1 received saline only intraperitoneally (i.p.) and served as control (Ctrl), group 2 received DOX (2 mg/kg/48 h, i.p. for 12 days; DOX was dissolved in saline) [41] and groups 3, 4 (CRO 20 and CRO 40) received DOX as in group 2 and CRO (20 and 40 mg/kg/24 h, for 20 days) starting 4 days prior to first DOX injection and throughout the study.

Initial and final body weight of the rats in all study groups and their heart weights were determined during and at the end of the study (just before doing surgery), respectively and the heart/body weight ratio was calculated and compared with those from the Ctrl group. [41].

has been shown to be the most interesting subject for research in recent years, in this study, we determined whether CRO could have cardioprotective effects in an animal model.

2.2. Subjects

2.4. Echocardiographic study Rats were sedated with ketamine (10–20 mg/kg, i.p.) [14,15,26] and transthoracic echocardiography (ECHO) was performed with a digital color doppler ultrasound system (iVis 60 Expert Vet CHISON Medical Imaging, China) as described previously [41]. Animals were then positioned in a chest closed supine form. The transducer was placed in the left parasternal position. The left ventricular end diastolic dimension (LVEDD) and left ventricular end systolic dimension (LVESD) were measured using M-mode tracing. The percentage of change in LV cavity dimension; fractional shortening (FS) and ejection fraction (EF) were measured as follows: Fractional shortening (%) = [(LVEDD − LVESD) / LVEDD] × 100; Ejection fraction ð%Þ ¼

h


i  LVEDD3 −LVESD3 =LVEDD3 Þ  100 ([7])

2.5. Electrocardiography Forty-eight hours after last administration of DOX or CRO, rats were anesthetized with a combination of xylazine (10 mg/kg/i.p.) and ketamine (100 mg/kg/i.p.) and kept warm with a heating lamp. Electrocardiograms (ECG) were recorded using three stainless steel needle

2.8. Histopathological examination At the end of study, the animals were euthanized and the hearts were excised, weighted, then washed with normal saline and finally fixed in 10% formalin, as previously described [41]. After fixation, the tissues were processed using the standard histological method, embedded in paraffin and the hearts were transversely cut at the middle third that consist of septum, right and left ventricle muscles and stained with hematoxylin and eosin (H&E). The histopathologic slides were examined by a veterinary pathologist and compared under a light microscope. The H&E stained sections were used for the following purposes: 1) morphological analysis of the myocardium, 2) inflammation and tissue damage assessment. Inflammation and tissue damage were determined by counting the number of mononuclear inflammatory cells (Lymphocytes and Macrophages) in H&E stained sections by randomly counting 100 microscopic fields over a total area 1.5 mm2 at 400× magnifications [18]. 2.9. Evaluation of antitumor activity In order to determine the effect of CRO on DOX-inhibited growth and proliferation of the malignant cell line MCF-7 (human breast cancer cells), cell viability was evaluated by MTT assay according to the manufacturer's instructions. Briefly, the cells were distributed (5000 cells/well) in 96-well plates, and maintained in RPMI-1640 medium supplemented with 10% fetal-calf serum (FCS) and antibiotics (Penicillin G 50,000 units/l. Streptomycin 38,850 units/l and Nystatin 9078 units/l), in an incubator at 37 °C with a humidified atmosphere of 10% CO2, and the cells were grown for 24 h. The cells were then exposed to a series of concentrations of free DOX (0.1, 0.5, 1, 5, 10 μg/

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ml) and/or CRO (25, 50 and 100 μg/ml) and incubated for 24 h (the drugs were dissolved in 100 μl of DMSO and then diluted with RPMI 1640). At the end of incubation time, MTT (20 μl with the concentration of 5 mg/ml) was added to each well and the plates incubated for further 6 h. Then, the culture medium was removed, 200 μl of DMSO was added to each well and the plates were shaken for 10 min. Finally, the optical density was measured at 550 nm using a microplate reader (AD 340; Beckman Coulter). All the experiments were performed in triplicate.

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Table 2 Electrocardiogram parameters. Group Parameter

Ctrl

DOX

CRO 20

CRO 40

HR (BPM) RRI (S) QA (μV) QTI (S)

221.9 ± 10.9 0.274 ± 0.014 1.171 ± 3.18 0.072 ± 0.005

186.5 ± 11.1† 0.328 ± 0.020† 14.07 ± 5.760† 0.076 ± 0.004

224.6 ± 13.37⁎ 0.27 ± 0.018⁎ 7.25 ± 1.03 0.083 ± 0.004

240.37 ± 12.05⁎⁎ 0.25 ± 0.012⁎⁎ 4.74 ± 1.99 0.073 ± 0.004

Ctrl = control, DOX = doxorubicin, CRO 20 = crocin (20 mg/kg) + DOX (12 mg/kg), CRO 40 = crocin (40 mg/kg) + DOX (12 mg/kg), RRI = RR interval, the interval from the peak of one QRS complex to the peak of the next on an electrocardiogram, HR = heart rate, S =

2.10. Statistical analysis All data were analyzed using SPSS software (Version 13.0). Student's t-test or one-way analyses of variance (ANOVA), followed by a Tukey's HSD post hoc test were used to analyze the statistical significance of the differences between groups. All data are expressed as the mean ± standard error of the mean (SEM), a pb0.05 was considered statistically significant.

second, BPM = beats per minute. QA: Q wave amplitude, QTI: QT interval, the QT interval is a measure of the time between the start of the Q wave and the end of the T wave on an electrocardiogram. The values are expressed as mean ± SEM (n = 6). † p b 0.05 vs. Ctrl group. ⁎ p b 0.05 vs. DOX group. ⁎⁎ p b 0.01 vs. DOX group.

3. Results

the systolic pressure (p b 0.05 and p b 0.01), diastolic pressure (p b 0.05) and mean pressure (p b 0.05 and p b 0.01), as compared to the DOX group.

3.1. Echocardiographic analysis To assess the effect of the CRO and DOX on LV remodeling and function, a series of echocardiography studies were performed. As illustrated in Table 1, data analyses indicated that DOX treatment significantly decreased the FS (p b 0.01) and EF (p b 0.01), as compared with the Ctrl group. Moreover, the results showed that CRO 20 mg/kg and CRO 40 mg/kg treatments at both doses significantly increased the FS (p b 0.05 and p b 0.01) and EF (p b 0.05 and p b 0.01) in comparison with the DOX group. 3.2. Electrocardiographic recordings Table 2 summarizes significant alterations in the ECG recordings. ECG pattern in the Ctrl was normal. Statistical analysis revealed that DOX exposure significantly altered the HR, RR interval, QA and PA parameters (p b 0.05), as compared to the Ctrl group. In addition, the data indicated that CRO 20 mg/kg and CRO 40 mg/kg treatments at both doses significantly improved the ECG pattern, in term of HR and RR interval (p b 0.05 and p b 0.01), as compared to the DOX group. 3.3. Blood pressure measuring Table 3 indicates that DOX administration consistently and significantly decreased the systolic pressure (p b 0.001), diastolic pressure (p b 0.05) and mean pressure (p b 0.01) in comparison with the Ctrl group. Moreover, the data analysis revealed that CRO 20 mg/kg and CRO 40 mg/kg in both doses reversed the effects of DOX exposure on

Table 1 Echocardiographic analyses of left ventricular fractional shortening and ejection fraction in rat heart. Group

Ctrl

DOX

CRO 20

CRO 40

6.60 ± 0.05 4.40 ± 0.07 33.31 ± 1.24 70.19 ± 1.71

6.30 ± 0.12 4.97 ± 0.217 21.16 ± 2.88†† 50.03 ± 5.10††

6.3 ± 0.07 4.42 ± 0.10 29.94 ± 1.02⁎ 65.50 ± 1.50⁎

6.55 ± 0.07 4.45 ± 0.11 31.98 ± 2.11⁎⁎ 68.07 ± 3.01⁎⁎

Parameter LVDD (mm) LVDS (mm) FS (%) EF (%)

Changes of left ventricular fractional shortening in rats treated with DOX and/or CRO. Ctrl = control, DOX = doxorubicin, CRO 20 = crocin (20 mg/kg) + DOX (12 mg/kg), CRO 40 = crocin (40 mg/kg) + DOX (12 mg/kg), LVSD = left ventricular systolic dimension, LVDD = left ventricular diastolic dimension, FS = fractional shortening, EF = ejection fraction, mm = millimeter, the values are expressed as mean ± SEM (n = 6). †† p b 0.01 vs. Ctrl group. ⁎ p b 0.05 vs. DOX group. ⁎⁎ p b 0.01 vs. DOX group.

3.4. Left ventricular function analysis As shown in Table 3, DOX administration significantly decreased the max pressure (p b 0.001), contractility index (p b 0.05) and the max dP/ dt (p b 0.05), and increased the EDP (p b 0.001), min pressure (p b 0.01) and, the min dP/dt (p b 0.05), as compared to the Ctrl group. In addition, CRO 20 mg/kg and CRO 40 mg/kg in both doses significantly reversed the effects of DOX on max pressure (p b 0.001), contractility index (p b 0.01), the max dP/dt (p b 0.01 and p b 0.001), and the min dP/dt (p b 0.01 and p b 0.001) as well as CRO only at the dose of 40 mg/kg decreased the EDP (p b 0.05) and min pressure (p b 0.05) relative to the DOX group. 3.5. Body weight development and heart/body weight ratio As it can be seen in Table 4, the results indicated that DOX exposure significantly led to decreased body weight (p b 0.001), heart weight (p b 0.001) and heart/body weight ratio (p b 0.001) relative to the Ctrl group. In addition, the data analysis revealed that CRO 20 mg/kg and CRO 40 mg/kg in both doses, especially at higher dose, significantly increased body weight (p b 0.001), heart weight (p b 0.01 and p b 0.001) and heart/body weight ratio only at the dose of 40 mg/kg; (p b 0.01) relative to the DOX group. 3.6. Histopathological results of heart tissue The histopathological changes in the rats' myocardium treated with DOX alone or in combination with CRO are illustrated in Fig. 1. The morphological evidence showed no pathological change in the Ctrl group. The results indicated that DOX treatment caused significant alterations such as cytoplasmic vacuolization, interstitial edema, hyaline degeneration, and Zenker's necrosis, as compared to the Ctrl group. Moreover, DOX appeared to have significant adverse effects on rat cardiac tissue, i.e. focal to extensive hemorrhages, accumulation of acute inflammatory cells, injured vascular structures, necrotic changes in the nuclei of cardiomyocytes and mild cardiac fibrosis. On the other side, co-administration of CRO 20 and CRO 40 treated groups with DOX dramatically attenuated the myocardial damage especially at higher dose, as compared to the DOX group. Hence, our findings showed that CRO treatment slightly resulted in pathological changes in cardiomyocytes. It therefore seems reasonable to speculate that CRO could lead to cell preservation and decreased necrosis, cytoplasmic vacuolization and maintained a normal morphology and structure for the cardiac muscle. As shown in Fig. 2, the numbers of mononuclear inflammatory cells

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Table 3 Arterial and left ventricular function parameters in study groups.

Left Ventricle

Artery

Parameter

Group

Ctrl

DOX

CRO 40

85.76 3.42*

88.72 6.73**

Systolic pressure

88.06 1.85

Diastolic pressure

68.25 2.28

53.32 4.66

67.99 3.11*

68.91 4.73*

Mean pressure (mmHg)

78.27 1.51

62.38 3.39

77.79 3.12*

78.59 5.72**

Max Pressure (mmHg)

86.92 1.98

23.17 2.24

62.26 5.03***

78.62 4.92***

Min Pressure (mmHg)

1.03 0.86

5.01 0.95

4.00 2.95

0.95 1.87*

EDP (mmHg)

4.33 0.80

17.16 1.22

16.28 5.09

8.01 3.38*

3366.85 883.1

533.92 79.0

1645.47 220.9**

2426.28 251.2***

72.86 11.3

18.42 2.9

42.03 7.2**

55.70 6.1**

-2865.71 889.4

-541.31 58.3

-1422.76 174.7**

-2001.97 228.0***

Max dP/dt (mmHg/s)

Contractility Index (1/s)

Min dP/dt (mmHg/s)

71.74 1.84

CRO 20

Ctrl = control, DOX = doxorubicin, CRO 20 = crocin (20 mg/kg) + DOX (12 mg/kg), CRO 40 = crocin (40 mg/kg) + DOX (12 mg/kg) and EDP = end diastolic pressure. The values are expressed as mean ± SEM (n = 6). The values are expressed as mean± SEM (n = 6).†p b 0.05, ††p b 0.01 and †††p b 0.001 vs. Ctrl group, ⁎p b 0.05, ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001 vs. DOX group.

was: 80 ± 4.4, 12.70 ± 1.10 and 3.55 ± 0.30 in the DOX, CRO 20 and CRO 40 treated groups. These findings indicated that CRO treatment in both doses significantly decreased (p b 0.001) the number of inflammatory cells induced by DOX.

3.7. Cytotoxicity To evaluate the antitumor activities of CRO alone or in combination with DOX, cell line MCF-7 was used as tumor cells in a MTT assay. As it can be seen in Fig. 3, the data analysis revealed that DOX dose-dependently increased the cell toxicity. In addition, the results indicated that there was no significant change following CRO treatments in combination with DOX on cell toxicity in vitro, while CRO alone at dose 100 μg/ml showed some toxicity in MTT assay.

Table 4 Body weight, heart weight development and heart weight/body weight ratio. Group

IBW (gr)

FBW (gr)

HW (gr)

HW/BW

Ctrl DOX CRO 20 CRO 40

201.33 ± 1.02 210.00 ± 0.89 212.33 ± 0.76 211.33 ± 0.61

221.16 ± 1.70 181.17 ± 1.61††† 222.50 ± 1.34⁎⁎⁎ 225.83 ± 0.79⁎⁎⁎

0.90 ± 0.02 0.54 ± 0.02††† 0.69 ± 0.01⁎⁎ 0.74 ± 0.01⁎⁎⁎

0.004 ± 0.0001 0.003 ± 0.0001††† 0.003 ± 0.00004 0.003 ± 0.00002⁎

IBW = initial body weight; FBW = final body weight (just before surgery); HW = heart weight; BW = body weight. Ctrl = control, DOX = doxorubicin, CRO 20 = crocin (20 mg/kg) + DOX (12 mg/kg), CRO 40 = crocin (40 mg/kg) + DOX (12 mg/kg). The values are expressed as mean ± SEM (n = 6). †††p b 0.001 vs. Ctrl group, ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001 vs. DOX group.

4. Discussion There has not been any specific treatment for DOX-induced cardiotoxicity, in recent years; many researchers have tried to evaluate the effects of different compounds in various animal models to find natural drugs to reduce this cardiotoxicity. It has generally been accepted that DOX-induced cardiotoxicity is associated with decreased body and heart weights [25,27,49]. Our results confirmed the fact that DOX results in reduced both body and heart weights. In the present study, we demonstrated that CRO treatment in combination with DOX in rats increased body and heart weights as compared to the DOX group. In line with previous evidence [34,37,41], our results clearly indicate that DOX resulted in myocardial injury as indicated by the increase in the left ventricular dysfunction, disturbances in the ECG patterns, blood-pressure, Left ventricular function parameters and histopathological analysis. As it can be seen in the results section, CRO treatment in rats significantly reversed the effects of DOX on the measured parameters. There is an increasing evidence showing that CRO is effective for the treatment of cardiovascular-related disorders such as atherosclerosis, hyperlipidemia, hypertension and myocardial injury [1]. For instance, Du et al. demonstrated that intravenous administration of CRO decreased the areas of myocardial injury [12]. It has also been shown that CRO treatment is preventative against myocardial infarction and cardiotoxicity induced by isoproterenol which was supported by enzymatical, histopathological and ultrastructural findings [19]. CRO treatment significantly improved left ventricular dysfunction induced by isoproterenol in rats [19]. Moreover, the protective role of CRO on antioxidant capacity in comparison with vitamin E has been observed, suggesting that CRO may be beneficial for preventing and treating the

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149

Fig. 1. Effect of CRO (crocin, 20 and 40 mg/kg) on doxorubicin (DOX)-induced histopathological alterations in cardiac tissues (H&E, 400×). A: Cardiac tissues from control group shows normal histological pattern. B: Cardiac tissues from DOX group shows hyaline degeneration and Zenker's necrosis ($), infiltration of acute inflammatory cells (†) and inter cardiomyocytes edema (*). This evidence shows severe pathological changes in the cardiac tissues of DOX-treated rats. C and D: Cardiac tissues from CRO 20 (C) and 40 (D) group's shows hyaline degeneration and Zenker's necrosis ($), infiltration of acute inflammatory cells (†) and inter cardiomyocytes edema (*). This evidence shows mild pathological changes in the cardiac tissues of CRO + DOX-treated rats.

cardiac dysfunction and myocardial infarction in patients with ischemic heart disease [10]. In line with these findings, histopathological analysis of heart performed by Razavi et al. indicated that CRO significantly improved cardiac damage induced by diazinon, including coagulative necrosis of cardiac muscle cells associated with hemorrhage, hypertrophy and infiltration of inflammatory cells, as compared to the control group [40].

Recent pharmacological studies have provided the evidence that CRO can be proposed as a new therapeutic drug, because of its antitumor [13,29], antioxidant and free radical scavenging effects [3,24]. Antioxidants play a key role in preventing free radical-induced injuries through scavenging them [40]. Soeda et al. suggested that CRO may have potential for treating neurodegenerative damage induced by oxidative stress [44]. Although the exact mechanism of CRO as a water

Fig. 2. The numbers of mononuclear inflammatory cells (Lymphocytes and Macrophages) in study groups. DOX = doxorubicin (12 mg/kg) alone or in combination with CRO 20 and 40 (crocin, 20 and 40 mg/kg). Values are expressed as mean ± SEM. (n = 6), †††: p b 0.001 vs. Ctrl group; ***: p b 0.001 vs. DOX group.

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Fig. 3. Cytotoxicities of A) DOX (doxorubicin;0.1-10 μg/ml), B) CRO (crocin, 25, 50 and 100 µg/ml) alone and C) combination of DOX (1, 5 µg/ml) with CRO (crocin, 25, 50 and 100 μg/ml) against MCF-7 cells. The results are mean values ± SEM of three independent experiments performed in triplicate. $: There was no a significant difference between DOX 1 (doxorubicin; 1 μg/ml) alone or in combination with CRO (crocin, 25, 50 and 100 μg/ml) and #: There was no a significant difference between DOX 5 (doxorubicin; 5 μg/ml) alone or in combination with CRO (crocin, 25, 50 and 100 μg/ml), p N 0.05.

soluble carotenoid for scavenging of free radicals is not yet clearly known, but it has been accepted that the mechanistic paths could be similar to those of the most known carotenoids [35]. Hence, it seems reasonable to speculate that CRO can modulate intracellular oxidative stress by regulation of body's natural antioxidant enzymes, when CRO absorbed into the blood plasma [38]. On the other side, it was found CRO has cytoprotective effect against hydrogen peroxide-induced endothelial cell injury in a dose dependent manner [46]. A study conducted by Xu et al. showed that CRO treatment results in the inhibition and regression of atherosclerosis through preventing the cell apoptosis [47]. In addition, He et al. indicated that CRO administration leads to significant decreases in the levels of cholesterol, triglyceride, LDL-C and a restriction in the formation of aortic plaque in an animal model of atherosclerosis via reducing endothelial cell apoptosis [20]. The protective effect of CRO against diazinon-induced apoptosis in the vascular system by inhibiting caspase-mediated apoptosis in aortic tissue in rats has recently been demonstrated [39]. Besides the antioxidant effects mentioned in the above, increasing evidence suggests that oxidative stress and production of free radicals are the major apoptotic stimulants in various illnesses such as cardiovascular disease and reactive oxygen species is able to induce apoptosis and DNA damage [8,40], it is believed that antioxidant agents can significantly suppress this process [30]. It was shown that CRO dose-dependently reduces methyl methanesulfonate (MMS)-induced DNA damage [22]. In addition, CRO has been shown to influence the apoptotic pathway through suppression of tumor necrosis factor-induced cell death, modulation of the expression of Baxand Bcl2 family proteins and blockade of the caspase-3 activation induced by cytochrome c, and eventually inhibition of DNA fragmentation [31, 43,47]. These data are supported by the findings showing antioxidant agents are able to prevent the occurrence of apoptosis by regulating gene expression and signal transduction pathways. For instance, Razavi et al. demonstrated that CRO has protective impacts against diazinon-

induced cardiotoxicity through reducing lipid peroxidation, histopathological injuries and apoptosis by decreasing Bax/Bcl-2 ratio and cytochrome c and blockade of caspase-3 activation [40]. Bcl-2, an antiapoptotic protein, and Bax, a pro-apoptotic protein, are involved in the control of the instinct apoptotic pathway through stabilizing mitochondrial membrane and inhibiting the release of cytochrome c [16,42]. Hence, disturbances in the balance between these two proteins can extremely affect the progression of apoptosis. Given that DOX results in increased free radicals and apoptosis via reducing Bcl2 expression and increasing Bax expression, and the fact that in vitro and in vivo studies have shown that CRO inhibits apoptosis through increasing Bcl2/Bax ratio expression, in this study, it is conceivable that CRO may reduce DOX-induced cardiotoxicity and improve the heart function through reducing free radicals and increasing Bcl2/Bax ratio expression. Although there was no significant change in MTT assay following CRO treatment in combination with DOX, we found that CRO alone at dose 100 μg/ml resulted in cell toxicity. In support of these findings, it has repeatedly been reported that CRO has the potential to inhibit growth of different types of tumor cells and numerous previous studies highlight the antitumor properties of CRO [1]. For instance, long-term administration of CRO on colorectal cancer resulted in a potent cytotoxic effect on human and animal adenocarcinoma cells in vitro; further confirmation came from three colorectal (HCT-116, SW-480, and HT-29) and two breast cancer cell lines where these results were reinforced by previous findings and suggested CRO can markedly inhibit growth of cancer cells without influencing normal cells [4,9]. Interestingly, encapsulation of CRO in liposomal form also led to cell toxicity in the HeLa and MCF-7 cells. Overall, this evidence clearly shows that CRO can be useful for the treatment of cancers [32]. Taking together, the outcome of the present study indicate that CRO could inhibit the DOX-induced cardiotoxicity with various mechanisms of action, which should be investigated by further experimental designs.

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In conclusion, the results of the present study showed that CRO clearly repressed the toxic effect of DOX in rat hearts, as measured by the echocardiography, electrocardiography, hemodynamic parameters, heart/ body weight ratio, blood pressure histopathological examination; whereas it had no significant effect on antitumor activity of DOX. Considering that clinical studies in humans are still scarce and future in-depth studies in cancer patients undergoing DOX therapy are needed to translate these findings into an effective clinical therapy and to define underlying mechanisms contributing to the therapeutic properties of CRO. Conflicts of interest The authors report no conflicts of interest in this research. Acknowledgements The authors would like to acknowledge their funding support, provided by the Drug Applied Research Center (DARC) at Tabriz University of Medical Sciences (Grant No. 91.71). This study is part of a PhD project submitted by N Razmaraii at DARC. References [1] S.H. 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