Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis

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Cardiovascular pharmacology

Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis Q1

Eman M. Mantawy a, Wesam M. El-Bakly b, Ahmed Esmat a, Amira M. Badr a, Ebtehal El-Demerdash a,n a

Q2

b

Department of Pharmacology & Toxicology, Faculty of Pharmacy, Ain Shams University, Abasia, Cairo, Egypt Department of Pharmacology & Therapeutics, Faculty of Medicine, Ain Shams University, Cairo, Egypt

art ic l e i nf o

a b s t r a c t

Article history: Received 21 October 2013 Received in revised form 25 January 2014 Accepted 29 January 2014

Doxorubicin (DOX) is one of the most effective chemotherapeutic drugs; however, its incidence of cardiotoxicity compromises its therapeutic index. Chrysin, a natural flavone, possesses multiple biological activities, such as antioxidant, anti-inflammatory and anti-cancer. The present study was designed to investigate whether chrysin could protect against DOX-induced acute cardiotoxicity; and if so, unravel the molecular mechanisms of this protective effect. Chrysin was administered to male albino rats once daily for 12 consecutive days at doses of 25 and 50 mg/kg orally. DOX (15 mg/kg; i.p.) was administered on day 12. Chrysin pretreatment significantly protected against DOX-induced myocardial damage which was characterized by conduction abnormalities, increased serum creatine kinase isoenzyme-MB (CK-MB), and lactate dehydrogenase (LDH) and myofibrillar disarrangement. As indicators of oxidative stress, DOX caused significant glutathione depletion, lipid peroxidation and reduction in activities of antioxidant enzymes; catalase (CAT) and superoxide dismutase (SOD). Chrysin pretreatment significantly attenuated DOX-induced oxidative injury. Additionally, DOX provoked inflammatory responses by increasing the expressions of nuclear factor kappa-B (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the levels of tumor necrosis factor-alpha (TNF-α) and nitric oxide while chrysin pretreatment significantly inhibited these inflammatory responses. Furthermore, DOX induced apoptotic tissue damage by increasing Bax and cytochrome c expressions and caspase-3 activity while decreasing the expression of Bcl-2. Chrysin pretreatment significantly ameliorated these apoptotic actions of DOX. Collectively, these findings indicate that chrysin possesses a potent protective effect against DOX-induced acute cardiotoxicity via suppressing oxidative stress, inflammation and apoptotic tissue damage. & 2014 Published by Elsevier B.V.

Keywords: Cardiotoxicity Doxorubicin Chrysin Oxidative stress Inflammation Apoptosis

1. Introduction Doxorubicin (DOX), an anthracycline antibiotic, has long been one of the most effective chemotherapeutic agents for treatment of a variety of human neoplasms, including leukemias, lymphomas, and solid tumors (Sant et al., 2009). However, its clinical utility is markedly hampered by high incidence of a dose-dependent cardiotoxicity; irreversible degenerative cardiomyopathy and congestive heart failure (Smith et al., 2010). With the increasing use of this anthracycline antibiotic, an acute cardiotoxicity has been recognized as a severe complication of DOX chemotherapy (Hayek et al., 2005). n Corresponding author. Tel.: þ 202 2878567, mobile: þ 202 01001925375; fax: þ202 2876271. E-mail addresses: [email protected], [email protected] (E. El-Demerdash).

The pathogenesis of DOX-induced cardiotoxicity is not entirely clear, but a solid body of evidence indicates that oxidative stress, inflammation and apoptosis are involved (Minotti et al., 2004). Nonetheless, oxidative stress remains the cornerstone. DOX has been shown to induce generation of reactive oxygen species which are involved in the interplay of a number of processes, including redox cycling of the quinone moiety of DOX, disturbance of iron metabolism and DOX metabolites in the heart (Chen et al., 2007). Reactive oxygen species ultimately lead to oxidative damage of cellular and mitochondrial membranes and cellular macromolecules (Goormaghtigh et al., 1990). Moreover, there is growing evidence that DOX also elicits inflammatory effects in the vasculature and the myocardium by increasing the expression of nuclear factor kappa-B (NF-κB), a key regulator of genes that are involved in the immune responses and inflammatory responses (Hou et al., 2005). Subsequently, DOX induces the production of several proinflammatory mediators such

http://dx.doi.org/10.1016/j.ejphar.2014.01.065 0014-2999 & 2014 Published by Elsevier B.V.

Please cite this article as: Mantawy, E.M., et al., Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.01.065i

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as tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-2) and nitric oxide (Abd El-Aziz et al., 2012). Besides, accumulating evidence indicates that cellular apoptosis or programmed cell death plays a critical role in the pathogenesis of DOX-induced cardiotoxicity (Reeve et al., 2007). As oxidative stress evoked by DOX triggers the intrinsic mitochondria-dependent apoptotic pathway in cardiomyocytes (Kluza et al., 2004). This phenomenon results in mitochondrial dysfunction and myofibrillar degeneration (Arola et al., 2000). As DOX continues to be a mainstay in chemotherapy, so the search for a safe and effective remedy to reverse DOX-induced cardiotoxicity remains a critical issue in both cardiology and oncology. Nowadays, much of attention has been given to the usage of phytochemicals as a protective strategy against DOX-induced cardiotoxicity (Xiao et al., 2012). Flavonoids are natural polyphenolic phytochemicals that are beneficial in preventing and treating many diseases such as cancer, cardiovascular diseases, neurodegenerative diseases as well as diabetes (Khan et al., 2012a). Chrysin (5,7-dihydroxyflavone) belongs to this category which is found in bee propolis, honey and various plants (Barbaric et al., 2011). It has multiple biological properties such as antioxidant, anti-inflammatory, antiapoptotic and anti-cancer (Sultana et al., 2012). Recently, chrysin has been proven to protect against DOXinduced hepatotoxicity and nephrotoxicity (Rashid et al., 2012). Accordingly, this study aimed to investigate the potential protective effect of chrysin against DOX-induced cardiotoxicity in rats and to elucidate the underlying molecular mechanisms in terms of oxidative stress, inflammatory and apoptotic mediators.

2. Materials and methods 2.1. Drugs and chemicals DOX was purchased as Adriablastine (50 mg doxorubicin hydrochloride, Pharmacia & Upjohn, Milan, Italy). Chrysin, reduced glutathione (GSH), Ellman's reagent [3,3'-dithiobis(6-nitrobenzoic acid)], bovine serum albumin, dimethylsulfoxide (DMSO) and thiobarbituric acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). N-butanol, dipotassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4) and trichloroacetic acid were purchased from El-Nasr Chemical Co. (Egypt). All other chemicals were of the highest purity grade commercially available. 2.2. Animals The study was conducted according to ethical guidelines (Ain Shams University, Egypt). Male albino rats (150–250 g) were obtained from Nile Co. for Pharmaceutical and Chemical Industries, Egypt. Rats were housed in an air-conditioned atmosphere, at a temperature of 25 1C with alternatively 12 h light and dark cycles and allowed free access to food and water. Animals were acclimated for 2 weeks before experimentation. They were kept on a standard diet and water ad libitum. Standard diet pellets (El-Nasr, Abu Zaabal, Egypt) contained not less than 20% protein, 5% fiber, 3.5% fat, 6.5% ash and a vitamin mixture. 2.3. Experimental design Rats were randomly assigned to five groups (ten animals per group) and treated as follows; the first group (control group) received 2.5 ml/kg of mixture of DMSO and corn oil (1:9) which was used as a vehicle for chrysin through oral gavage once daily for 12 consecutive days,. The second group received mixture of DMSO and corn oil (1:9) through oral gavage once daily for

12 consecutive days and a single intraperitoneal injection of DOX (15 mg/kg) on the 12th day. The third and fourth groups were pretreated with an oral dose of chrysin 25 mg/kg and 50 mg/kg respectively for 12 consecutive days followed by a single intraperitoneal injection of DOX (15 mg/kg) on the 12th day after 1 h of the last treatment of chrysin in both groups. Chrysin doses were determined based on previous studies reporting its antioxidant and antiapoptotic properties while protecting against cisplatininduced nephrotoxicity and jejunum and colon toxicities (Khan et al., 2012a, 2012b; Sultana et al., 2012). The last group received chrysin only at an oral dose of 50 mg/kg for 12 consecutive days. Forty-eight hours after DOX injection, rats were anesthetized with ketamine (75 mg/kg; i.p.) and subjected to ECG recording. Afterthat, blood samples were collected from the retro-orbital plexus and allowed to clot. Serum was separated by centrifugation at 3000g for 10 min and used for biochemical analyses. Rats were sacrificed and heart tissues were dissected out and washed with ice-cold saline. The body and heart weights were measured. Hearts were homogenized in saline then the homogenate was used for assessment of different biochemical parameters. In addition, heart specimens from different groups were fixed in 10% buffered formalin for histopathological and immunohistochemical examination. 2.4. Electrocardiography (ECG) ECG was recorded at the beginning of the experiment to ensure the normal ECG pattern of the rats. At the end of the experiment, ECG was recorded in ketamine anesthetized rats 48 h after DOX injection using Bioscience ECG recorder (Bioscience, Washington, USA). Anesthetized rats were placed in the supine position on a board and needle electrodes were inserted beneath the skin for the limb lead at position II (right forelimb to left hind limb). Every recording lasted for at least 5 min. ECG recording speed was 50 mm/s and the voltage was 1 mV/cm. Noise was minimized by a digital filter. Analysis of ECG waves was done to calculate heart rate (beats/min), QRS duration (ms), QT interval (ms), which was corrected for heart rate using the Bazett formula [QTc¼QT/(square root of RR interval)], and PR interval (ms). For each parameter, measurements were done at three non-consecutive, randomly chosen points in every 5 min recording. The results are reported as mean of the three randomly selected segments. 2.5. Assessment of cardiotoxicity indices Creatine kinase isoenzyme-MB (CK-MB) and lactate dehydrogenase (LDH) activities were determined according to standard methods using available commercial kits (Spectrum diagnostics, Cairo, Egypt). Heart index was calculated according to the formula: (heart weight/body weight)  100. 2.6. Assessment of oxidative stress markers To determine GSH, 0.5 ml homogenate was added to a tube with 0.5 ml of 10% trichloroacetic acid. The tubes were shaken gently and intermittently for 15 min, followed by centrifugation at 1000g for 10 min. An aliquot of the resulting supernatant (0.2 ml) was added to a tube containing 1.7 ml phosphate buffer and 0.1 ml Ellman's reagent then the absorbance was read at 412 nm within 5 min (Ellman, 1959). The results were expressed as μM of GSH/g of wet tissue. Lipid peroxidation was determined by estimating the level of thiobarbituric acid reactive substances measured as malondialdehyde (MDA), according to the method of Mihara and Uchiyama (1978). Briefly, the reaction mixture (0.5 ml homogenate þ2.5 ml 20% trichloroacetic acid þ1.0 ml 0.6% thiobarbituric acid) was heated for 20 min in a boiling water bath followed by

Please cite this article as: Mantawy, E.M., et al., Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.01.065i

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cooling and addition of 4 ml n-butanol with shaking. The alcohol layer was separated by centrifugation at 2000g for 10 min and absorbance was measured at 535 nm. The results were expressed as nmol of MDA/g of wet tissue using 1,1,3,3-tetraethoxypropane as standard.

2.11. Histopathological examination

2.7. Assessment of antioxidant enzyme activities

2.12. Immunohistochemical detection of NF-κB(p65), iNOS, COX-2, Bax, Bcl2 and cytochrome c

Assessment of superoxide dismutase (SOD) and catalase (CAT) activities in heart homogenate was done using kits provided by Biodiagnostics, Giza, Egypt. The assay of SOD activity employs the use of xanthine and xanthine oxidase to generate superoxide radicals which react with 2-(4-iodophenyl)-3-(4-nitropheno)-5phenyltetrazolium-chloride to form a red formazan dye. Superoxide dismutase activity was measured by the degree of inhibition of this reaction (Flohe and Otting, 1984). CAT activity was estimated colorimetrically, where each unit of CAT decomposes 1 μM of hydrogen peroxide (H2O2) per min at 25 1C and pH 7.0 according to the method of Aebi (1984). CAT reacts with a known quantity of H2O2. The reaction is stopped after exactly 1 min with CAT inhibitor. In the presence of peroxidase, remaining H2O2 reacts with 3,5-dichloro-2-hydroxybenzene sulfonic acid and 4aminophenazone to form a chromophore with a color intensity inversely proportional to the amount of CAT in the original sample. Enzyme activities were expressed as unit/mg protein.

2.8. Assessment of inflammatory markers Both TNF-α and nitric oxide levels in heart homogenate were assessed. In addition, immunohistochemical detection of iNOS and COX-2 was carried out. Determination of TNF-α was performed using commercial ELISA kit (Assaypro Co., USA) according to the manufacturer's instructions. The quantities of rat TNF-α were expressed as pg/mg protein. Nitric oxide content in heart homogenate was estimated spectrophotometrically as formed nitrite using kit provided by Biodiagnostics, Giza, Egypt according to the method described by Miranda et al. (2001). The assay determines total nitrite and nitrate level based on reduction of any nitrate into nitrite by vanadium trichloride followed by detection of total nitrite. As in acid medium and in the presence of nitrite, the formed nitrous acid diazotises sulfanilamide and the product is coupled with N-(1–naphthyl) ethylenediamine. The resulting azo dye has a bright reddish-purple color which can be measured at 540 nm. Nitric oxide content was expressed as mM/g of wet tissue.

2.9. Assessment of apoptotic markers Caspase-3 activity was assayed using a kit purchased from Sigma Chemical Co., USA and following the protocol provided by the manufacturer. Briefly, the heart tissue extract was incubated with the peptide conjugate-substrate DEVD-p-nitroanilide (pNA) at 37 1C for 90 min. Intensity of the colored product p-nitroaniline was read by an automated microplate reader at 405 nm. Caspase-3 activity was expressed in pmol p-nitroaniline/min/mg protein. Additionally, immunohistochemical detection of Bax, Bcl2 and cytochrome c was done.

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Heart specimens were fixed in 10% formalin and processed for paraffin sections of 4 μm thickness. Sections were stained with Hematoxylin and Eosin (H & E) and examined under a light microscope (Olympus BX-50 Olympus Corporation, Tokyo, Japan).

Paraffin embedded tissue sections of 3 μm thickness were rehydrated first in xylene and then in graded ethanol solutions. The slides were then blocked with 5% bovine serum albumin in tris buffered saline for 2 h. The sections were then immunostained with one of the following primary antibodies; rabbit polyclonal anti iNOS antibody (Thermo Fisher Scientific, Cat No. RB-9242-P), rabbit polyclonal anti COX-2 antibody (Thermo Fisher Scientific, Cat No. RB-9072-R7), rabbit polyclonal anti NF-κB p65 antibody (Thermo Fisher Scientific, Cat No. RB-9034-P), rabbit polyclonal anti Bax antibody (Biorbyt, Cat No. orb4655), rabbit polyclonal anti Bcl-2 antibody (Biorbyt, Cat No. orb100697) or mouse monoclonal anti-cytochrome c antibody (Thermo Fisher Scientific, Cat. No. MS 1192-R7) at a concentration of 1 μg/ml containing 5% bovine serum albumin in tris buffered saline and incubated overnight at 4 1C. After washing the slides with tris buffered saline, the sections were incubated with goat anti-rabbit secondary antibody. Sections were then washed with tris buffered saline and incubated for 5–10 min in a solution of 0.02% diaminobenzidine containing 0.01% H2O2. Counter staining was performed using hematoxylin, and the slides were visualized under a light microscope. Immunohistochemical quantification was carried out using image analysis software (Image J, 1.46a, NIH, USA). 2.13. Statistical analysis Data are presented as mean 7S.D. Multiple comparisons were performed using one-way ANOVA followed by Tukey–Kramer as a post-hoc test. The 0.05 level of probability was used as the criterion for significance. All statistical analyses were performed using Instat version 3 software package. Graphs were sketched using GraphPad Prism (ISIs software, USA) version 5 software.

3. Results 3.1. ECG ECG tracing showed normal cardiac activity in the control and chrysin only treated rats. Rats in DOX-treated group showed several ECG changes including bradycardia, widening of QRS complex and prolongation of both QTc and PR intervals compared to the control group. Such ECG abnormalities were obviously improved in the intoxicated animals pretreated with the higher dose of chrysin (50 mg/kg) as evidenced by normalization of heart rate, QRS complex and both QTc and PR intervals compared to DOX group. However, pretreatment of intoxicated animals with the lower dose of chrysin (25 mg/kg) could only normalize QRS complex and QTc interval while the heart rate and PR interval remain insignificantly different compared to DOX group (Table 1 and Fig. 1).

2.10. Determination of protein content

3.2. Cardiotoxicity indices

The protein content of cardiac tissue homogenates was determined by the Lowry protein assay using bovine serum albumin as standard (Lowry et al., 1951).

DOX intoxication led to loss of body weight but no statistical significance was observed compared to the control group. However, the absolute and relative heart weight significantly decreased

Please cite this article as: Mantawy, E.M., et al., Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.01.065i

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Table 1

Q4 ECG parameters of rats treated with chrysin and/or doxorubicin (DOX). Treated groups

Heart rate (beat/min)

QRS duration (ms)

QTc interval (ms)

PR interval (ms)

Control DOX (15 mg/kg) DOX þChrysin (25 mg/kg) DOX þChrysin (50 mg/kg) Chrysin (50 mg/kg)

361.0 721.60a 250.0 737.34a 327.3 774.72 340.4 749.41a 358.3728.87a

35.07 8.72a 65.07 10.00a 43.37 8.17a 36.07 8.94a 35.57 9.00a

186.77 20.66a 249.07 39.12a 194.37 23.23a 188.37 16.75a 187.87 29.23a

43.07 9.49a 66.77 10.33a 56.07 8.94 44.07 6.99a 43.37 8.17a

Data are the mean 7 S.D. (n¼ 10). a

Significantly different from the control or DOX group respectively at Po 0.05 using ANOVA followed by Tukey–Kramer as a post-hoc test.

revealed marked myocardial degeneration in the form of myofibrillar loss, cytoplasmic vacuolization, inflammatory cell infiltration, edema and congestion (Fig. 2C, D). Interestingly, pretreatment of intoxicated rats with chrysin at both doses (25 and 50 mg/kg) almost preserved the normal myocardium architecture (Fig. 2E, F). Despite, there were still some inflammatory cells in the cardiac tissues of intoxicated rats treated with the lower dose of chrysin. 3.4. Oxidative stress markers and antioxidant enzymes

Fig. 1. Effect of chrysin pretreatment on DOX-induced alterations in ECG pattern. (A) Control group, (B) chrysin (50 mg/kg) treated group, (C) DOX (15 mg/kg) treated group, (D) chrysin (25 mg/kg) þ DOX (15 mg/kg) treated group, and (E) chrysin (50 mg/kg)þ DOX (15 mg/kg) treated group. ECG tracing of control and chrysin only treated rats shows normal heart rate, QRS complex duration and QTc and PR intervals. DOX-treated group shows bradycardia, widening of QRS complex and prolongation of both QTc and PR intervals. Chrysin supplementation at both doses almost normalized heart rate, QRS complex and both QTc and PR intervals.

DOX-induced redox imbalance in the heart was determined by assessing GSH and MDA levels in addition to CAT and SOD activities. As shown in Table 3, DOX treatment significantly reduced GSH level by 26.5% and increased lipid peroxides level by 43% as compared to the control group. Furthermore, DOX induced a significant decrease in the cardiac antioxidant enzyme activities; CAT and SOD by 36 and 31%, respectively, compared to the control levels. Pretreatment of intoxicated animals with the lower dose of chrysin (25 mg/kg) could partially elevate the levels of both of GSH, CAT and SOD and reduce the MDA levels but no significant difference was observed in all these parameters compared to DOX group. Also, in this group, both GSH depletion and MDA elevation was still significant compared to the control group. Meanwhile, pretreatment of intoxicated animals with the higher dose of chrysin (50 mg/kg) could significantly restore the levels of GSH, MDA, CAT, and SOD to almost that of the control group. However, GSH level in this group was still significantly lower than the control group. Furthermore, animals treated with chrysin alone did not show any significant alterations in both GSH and MDA levels and CAT and SOD activities compared to the control group. 3.5. Inflammatory markers

in the DOX-intoxicated group by 19% and 16% respectively, compared to the control group. On the other hand, pretreatment of intoxicated animals with chrysin at both doses increased both the absolute and relative heart weight but statistical significance was only observed with the higher dose of chrysin compared to DOX group. Chrysin alone did not modify the absolute and relative heart weight (Table 2). In addition, the activities of serum markers indicating myocardial injury; CK-MB and LDH were significantly elevated in the DOX-intoxicated group by 122 and 236% respectively, compared to the control group. Pretreatment of intoxicated animals with chrysin at both doses significantly reduced the activities of CK-MB and LDH compared to DOX group. Chrysin alone did not show any significant changes in all these markers when compared to the control group (Table 2). 3.3. Histopathological examination To further characterize the cardiotoxicity induced by DOX, histopathological examination of heart tissue was done. Hearts from control and chrysin only treated rats showed regular cell distribution and normal myocardium architecture (Fig. 2A, B). Histological examination of hearts from DOX-intoxicated animals

The expression of proinflammatory enzymes iNOS and COX-2 was estimated using immunohistochemical staining. Control group showed minimal immunostaining for both iNOS (Fig. 3A) and COX-2 (Fig. 4A). DOX elevated the expression of these enzymes as shown by the intense brown staining (Figs. 3B and 4B) while pretreatment of intoxicated animals with chrysin at both the lower (Figs. 3C and 4C) and the higher doses (Figs. 3D and 4D) prevented this elevation to a large extent. No change in the enzymes expression was observed in the group treated with chrysin alone (Figs. 3E and 4E). The immunohistochemical staining of both iNOS and COX-2 enzymes was quantified as optical density (OD) of the stained regions using the image analysis software, and the results are represented in Figs. 3F and 4F, respectively. Further NF-κB was assessed by detecting the activated subunit p65 in heart tissues. Control rats showed minimal immunostaining for p65 (Fig. 5A). DOX induced an increase in the p65 level in the cardiac tissues, which was evident from the intense brown staining (Fig. 5B). In contrast, pretreatment of intoxicated animals with chrysin at both the lower (Fig. 5C) and the higher doses (Fig. 5D) markedly decreased the p65 expression. No change in the enzyme expression was observed in the group treated with

Please cite this article as: Mantawy, E.M., et al., Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.01.065i

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5

Table 2 Body and heart weight and cardiotoxicity markers of rats treated with chrysin and/or doxorubicin (DOX). Treated groups Control DOX (15 mg/kg) DOX þChrysin (25 mg/kg) DOX þChrysin (50 mg/kg) Chrysin (50 mg/kg)

Body weight (g) 195.007 23.77 185.367 29.32 187.867 30.30 192.927 30.56 193.337 28.75

Absolute heart weight (g) a

0.74 70.081 0.60 70.109a 0.68 70.109 0.71 70.136a 0.73 70.098a

Heart index (%) a

0.387 0.045 0.327 0.035a 0.367 0.059 0.377 0.055a 0.387 0.019a

CK-MB (IU/L)

LDH (IU/L) a

124.29 7 10.43 275.85 7 64.48a 188.96 7 52.99a 159.38 7 35.96a 125.187 10.5a

190.477 39.25a 641.60 7 145.45a 226.39 7 49.47a 201.88 7 50.18a 191.247 38.97a

Data are the mean 7 S.D. (n¼ 10). CK-MB, creatine phosphokinase isoenzyme-MB; LDH, lactate dehydrogenase. a

Significantly different from the control or DOX group respectively at Po 0.05 using ANOVA followed by Tukey–Kramer as a post-hoc test.

Fig. 2. Effect of chrysin pretreatment on DOX-induced histological alterations of the heart tissue (200  ). Photomicrographs of haematoxylin and eosin stained sections of heart depicting (A) control group, (B) chrysin (50 mg/kg) treated group, (C and D) DOX treated group (15 mg/kg), (E) chrysin (25 mg/kg) þ DOX (15 mg/kg) treated group, and (F) chrysin (50 mg/kg) þDOX (15 mg/kg) treated group. Scale bar, 50 mm. (A) and (B) show normal histoarchitecture of the rat heart. (C) and (D) show DOX-induced myofibrillar loss (dotted arrows), cytoplasmic vacuolization (arrowheads), inflammatory cell infiltration (solid arrows), edema (circles) and congestion (star). (E) and (F) show that chrysin pretreatment at both doses prevented cardiomyocyte damage induced by DOX and ameliorated inflammatory cell infiltration but the higher dose of chrysin (F) showed more protection than the lower one (E) where there was still some inflammatory cell infiltration (solid arrows).

chrysin alone (Fig. 5E). The immunohistochemical staining was quantified as OD of the stained regions using the image analysis software, and the results are represented in Fig. 5F.

DOX-induced proinflammatory response were also evidenced by the significant increase in both TNF-α and nitric oxide contents in the cardiac tissue by 49 and 65% respectively, compared to the

Please cite this article as: Mantawy, E.M., et al., Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.01.065i

Table 3 Oxidative stress markers of rats treated with chrysin and/or doxorubicin (DOX). Treated groups

GSH (μmol/g tissue)

MDA (nmol/g tissue)

CAT (U/mg protein)

SOD (U/mg protein)

Control DOX (15 mg/kg) DOXþ Chrysin (25 mg/kg) DOXþ Chrysin (50 mg/kg) Chrysin (50 mg/kg)

0.53 7 0.054a 0.39 7 0.026a 0.447 0.029a 0.46 7 0.024a 0.52 7 0.041a

75.49 7 4.27a 108.06 7 12.90a 95.76 7 8.13a 88.29 7 7.55a 78.75 7 10.33a

8.047 1.46a 5.177 1.01a 6.94 7 1.77 7.58 7 1.65a 8.017 1.67a

28.83 7 6.62a 19.777 4.12a 24.347 1.92 27.22 7 5.78a 28.26 7 6.05a

Data are the mean 7 S.D. (n¼ 10). GSH, reduced glutathione; MDA, malondialdehyde; CAT, catalase; SOD, superoxide dismutase. a

Significantly different from the control or DOX group respectively at Po 0.05 using ANOVA followed by Tukey–Kramer as a post-hoc test.

Control

1.5

DOX

*

DOX + Chrysin (25 mg/kg) DOX + Chrysin (50 mg/kg)

iNOS OD

Chrysin (50 mg/kg)

1.0 ,

*#

0.5

,

*# #

#

0.0

0.25

Nitric oxide content (μmol/g tissue)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

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Control

#

0.15 0.10

DOX

*

0.20

#

DOX + Chrysin (25 mg/kg)

#

DOX + Chrysin (50 mg/kg)

#

Chrysin (50 mg/kg)

0.05 0.00

Q3 Fig. 3. Expression of inducible nitric oxide synthase (iNOS) by immunohistochemical staining (200  ) and cardiac nitric oxide level expressed as mM/g of wet tissue.

Photomicrographs of histological sections of heart depicting (A) control group, (B) DOX (15 mg/kg) treated group, (C) chrysin (25 mg/kg) þ DOX (15 mg/kg) treated group, (D) Chrysin (50 mg/kg) þDOX (15 mg/kg) treated group, and (E) chrysin (50 mg/kg) treated group. Scale bar, 50 mm. (F) Quantitative image analysis for immunohistochemical staining expressed as optical densities (OD) across 10 different fields for each rat section. (G) Cardiac nitric oxide level. Values are given as mean 7S.D. for groups of 10 rats for each. *Po 0.05 statistically significant from control group and #P o 0.05 statistically significant from DOX group using one-way analysis of variance (ANOVA) followed by Tukey–Kramer as a post-hoc test. For immunohistochemical analyses, brown color (positive) indicates specific immunostaining of iNOS and light blue color (negative) indicates hematoxylin staining. DOX intoxication caused a significant increase in both iNOS expression as indicated by intense brown staining (arrows) and nitric oxide production compared to the control group. Chrysin pretreatment at both doses significantly reduced both iNOS expression and nitric oxide production compared to DOX group. However there was no significant difference in both iNOS expression and nitric oxide production in chrysin only treated group compared to the control group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Expression of cycloxygenase-2 (COX-2) by immunohistochemical staining (200  ) and cardiac tumor necrosis factor-alpha (TNF-α) level expressed as pg/mg protein. Photomicrographs of histological sections of heart depicting (A) control group, (B) DOX (15 mg/kg) treated group, (C) chrysin (25 mg/kg) þ DOX (15 mg/kg) treated group, (D) chrysin (50 mg/kg)þ DOX (15 mg/kg) treated group, and (E) chrysin (50 mg/kg) treated group. Scale bar, 50 mm. (F) Quantitative image analysis for immunohistochemical staining expressed as optical densities (OD) across 10 different fields for each rat section. Values are given as mean 7 S.D. for groups of 10 rats. *P o 0.05 statistically significant from control group and #P o0.05 statistically significant from DOX group using one-way analysis of variance (ANOVA) followed by Tukey–Kramer as a post-hoc test. For immunohistochemical analyses, brown color (positive) indicates specific immunostaining of COX-2 and light blue color (negative) indicates hematoxylin staining. DOX intoxication caused a significant increase in COX-2 as indicated by intense brown staining (arrows) compared to the control group. Chrysin pretreatment at both doses significantly reduced COX-2 expression compared to DOX group. However there was no significant difference in COX-2 expression in chrysin only treated group compared to the control group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

control group. On the contrary, pretreatment of intoxicated animals with chrysin at both doses exhibited anti-inflammatory effects by significantly reducing cardiac nitric oxide and TNF-α contents compared to DOX group. Treatment of animals with chrysin alone showed no significant effect on cardiac nitric oxide and TNF-α contents compared to the control group (Figs. 3G and 5G) respectively. 3.6. Apoptotic markers Expression levels of apoptosis-related proteins Bax and Bcl-2 were examined immunohistochemically. Control rats showed minimal immunostaining for the proapoptotic protein, Bax (Fig. 6A)

while intense immunostaning for the antiapoptotic protein, Bcl-2 (Fig. 6F), DOX-induced apoptosis was evidenced by increasing the expression of Bax as evidenced by the intense brown staining compared to the control group (Fig. 6B). Meanwhile, DOX decreased the expression of Bcl-2, as evidenced by the faint brown staining compared to control group (Fig. 6G). On the other hand, pretreatment of intoxicated animals with chrysin at both doses; 25 mg/kg (Fig. 6C) and 50 mg/kg (Fig. 6D) significantly decreased Bax expression compared to DOX group. Besides, chrysin at both doses; 25 mg/ kg (Fig. 6H) and 50 mg/kg (Fig. 6I) also significantly increased Bcl-2 expression compared to DOX group. Moreover, the group treated with chrysin alone showed no significant change in the expression of both Bax and Bcl-2 in comparison to the control group (Fig. 6E

Please cite this article as: Mantawy, E.M., et al., Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.01.065i

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Fig. 5. Expression of nuclear factor kappa B (NF-κB) by immunohistochemical staining (200  ). Photomicrographs of histological sections of heart depicting (A) control group, (B) DOX (15 mg/kg) treated group, (C) chrysin (25 mg/kg) þ DOX (15 mg/kg) treated group, (D) chrysin (50 mg/kg) þDOX (15 mg/kg) treated group, and (E) chrysin (50 mg/kg) treated group. Scale bar, 50 mm. (F) Quantitative image analysis for immunohistochemical staining expressed as optical densities (OD) across 10 different fields for each rat section. (G) Cardiac TNF-α. Values are given as mean7 S.D. for groups of 10 rats for each. *P o0.05 statistically significant from control group and #Po 0.05 statistically significant from DOX group using one-way analysis of variance (ANOVA) followed by Tukey–Kramer as a post-hoc test. For immunohistochemical analyses, brown color (positive) indicates specific immunostaining of NF-κB and light blue color (negative) indicates hematoxylin staining. DOX intoxication significantly increased NF-κB expression as indicated by intense brown staining (arrows) and TNF-α level compared to the control group. Chrysin pretreatment at both doses significantly reduced both NF-κB expression and TNF-α level compared to DOX group. However there was no significant difference in both NF-κB expression and TNF-α level in chrysin only treated group as compared to the control group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

and J). The immunohistochemical staining of Bax and Bcl-2 was quantified as OD of the stained regions using the image analysis software, and the results are represented in Fig. 6K and L respectively. Bax/Bcl-2 ratio was also calculated and represented in Fig. 6M. Moreover, immunohistochemical examination of cytochrome c was also performed. Both control and chrysin only treated groups were just minimally stained (Fig. 7A and E) respectively. Contrariwise, DOX induced a marked increase in the expression of cytochrome c which was evident from the intense brown color (Fig. 7B). While pretreatment of intoxicated animals with chrysin at both the lower (Fig. 7C) and the higher dose (Fig. 7D) markedly

diminished this elevated expression as manifested by the faint brown staining. The immunohistochemical staining was quantified as OD of the stained regions using the image analysis software, and the results are represented in Fig. 7F. Apoptosis was further confirmed by assessing caspase-3 activity. DOX significantly increased it by 58% compared to the control group. On other hand, pretreatment of intoxicated animals with chrysin at both doses exhibited anti-apoptotic effects by significantly decreasing caspase 3 activity compared to DOX group. Treatment of rats with chrysin alone showed no significant variation in caspase 3 activity compared to the control group (Fig. 7G).

Please cite this article as: Mantawy, E.M., et al., Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.01.065i

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Fig. 6. Expression of Bax and Bcl-2 by immunohistochemical staining (200  ). Photomicrographs of histological sections of heart depicting (A) and (F) expression of Bax and Bcl-2, respectively in the control group, (B) and (G) expression of Bax and Bcl-2, respectively in the group treated with DOX (15 mg/kg), (C) and (H) expression of Bax and Bcl-2, respectively in the group treated with chrysin (25 mg/kg)þ DOX (15 mg/kg), (D) and (I) expression of Bax and Bcl-2, respectively in the group treated with chrysin (50 mg/kg)þ DOX (15 mg/kg), (E) and (J) expression of Bax and Bcl-2, respectively in the group treated with chrysin (50 mg/kg). Scale bar, 50 mm. (K) and (L) Quantitative image analysis for immunohistochemical staining of Bax and Bcl-2 respectively expressed as optical densities (OD) across 10 different fields for each rat section. (M) The ratio of Bax to Bcl-2 expression in the heart of different groups. Values are given as mean7S.D. for groups of 10 rats. *Po0.05 statistically significant from control group and #Po0.05 statistically significant from DOX group using one-way analysis of variance (ANOVA) followed by Tukey–Kramer as a post-hoc test. For immunohistochemical analyses, brown color (positive) indicates specific immunostaining of Bax/Bcl-2 and light blue color (negative) indicates hematoxylin staining. DOX intoxication caused a significant increase in Bax expression as indicated by intense brown staining (arrows) and a significant decrease in Bcl-2 expression compared to the control group. Chrysin pretreatment at both doses significantly reduced Bax expression and increased Bcl-2 expression as indicated by intense brown staining (arrows) compared to DOX group. However there was no significant difference in the expression of both Bax and Bcl-2 in chrysin only treated group compared to the control group. Moreover, chrysin pretreatment at both doses significantly reduced the DOX-induced elevation in Bax/Bcl-2 ratio. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4. Discussion In this study we evaluated the potential of chrysin in ameliorating DOX-induced acute cardiotoxicity in rats and elucidated the underlying molecular mechanisms. DOX-induced cardiotoxicity was assessed by ECG, biochemical analysis of serum cardiotoxicity indices and histopathological examination of heart tissue. ECG tracing of DOX-treated rats showed bradycardia, QRS widening and prolongation of both QT and PR intervals. These changes reflected arrhythmias, conduction abnormalities and attenuation of left ventricular function. Similar ECG changes have been reported by other studies (Elberry et al., 2010). Additionally, DOX-induced myocardial injury was further manifested by the significant elevation in activities of serum CK-MB and LDH enzymes; which are released from damaged myocytes and

sensitive indicators of cardiac injury (Herman et al., 1971). The increase in activities of these enzymes is in agreement with previous studies (Ibrahim et al., 2009). The aforementioned ECG abnormalities and biochemical data were further confirmed by histopathological examination of the cardiac tissues. As DOX intoxication caused marked myocardial degeneration in the form of myofibrillar loss, inflammatory cell infiltration, cytoplasmic vacuole formation, interstitial edema and hemorrhage. These histopathological changes have been previously reported in acute DOX-induced cardiotoxicity (Fouad and Yacoubi, 2011). On the other hand, chrysin pretreatment significantly ameliorated DOXinduced ECG changes and inhibited elevations of CK-MB and LDH enzymes activities. Furthermore, it almost preserved the normal architecture of the heart. This implies that chrysin effectively protected against DOX- induced cardiotoxicity.

Please cite this article as: Mantawy, E.M., et al., Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.01.065i

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Fig. 7. Expression of cytochrome c by immunohistochemical staining (200  ) and cardiac caspase-3 activity expressed as pmol pNA/min/mg protein. Photomicrographs of histological sections of heart depicting (A) control group, (B) DOX (15 mg/kg) treated group, (C) chrysin (25 mg/kg) þ DOX (15 mg/kg) treated group, (D) chrysin (50 mg/kg)þ DOX (15 mg/kg) treated group, (E) chrysin (50 mg/kg) treated group. Scale bar, 50 mm. (F) Quantitative image analysis for immunohistochemical staining expressed as optical densities (OD) across 10 different fields for each rat section. (G) Caspase-3 activity in cardiac tissues. Values are given as mean 7 S.D. for groups of 10 rats for each. *Po 0.05 Statistically significant from control group and #Po 0.05 statistically significant from DOX group using one-way analysis of variance (ANOVA) followed by Tukey–Kramer as a post-hoc test. For immunohistochemical analyses, brown color (positive) indicates specific immunostaining of cytochrome c and light blue color (negative) indicates hematoxylin staining. DOX intoxication caused a significant increase in both cytochrome c expression as indicated by intense brown staining (arrows) and caspase-3 activity compared to the control group. Chrysin pretreatment at both doses significantly reduced cytochrome c expression and caspase-3 activity compared to DOX group. However there was no significant difference in the cytochrome c expression and caspase-3 activity chrysin only treated group compared to the control group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

In the next step, we explored the cardioprotective mechanisms of chrysin by assessing different markers of oxidative stress, inflammation and apoptosis. Existing evidence points to a multitude of molecular mechanisms involved in DOX-induced cardiotoxicity (Olson and Mushlin, 1990). In this context, oxidative stress is considered a major contributor in triggering and progressing DOXinduced myocardial dysfunction (Simunek et al., 2009). The semiquinone form of DOX is a toxic short-lived metabolite which interacts with molecular oxygen and initiates a cascade of reactions producing reactive oxygen species including superoxide and hydroxyl radicals and hydrogen peroxide (Davies and Doroshow, 1986).

These radicals can alter the structural and functional integrity of cells by a variety of mechanisms, including lipid peroxidation and proteolysis. This causes diverse oxidative damage of critical cellular components and cellular and mitochondrial membranes leading finally to cardiomyocyte cell death (Sarvazyan, 1996). Furthermore, DOX increases cardiomyocyte susceptibility to reactive oxygen species by reducing activities of antioxidant enzymes such as CAT and SOD and thereby, reducing the ability of cardiac cells to inactivate them (Doroshow et al., 1980). In this regards, cardiac tissue is more vulnerable to oxidative damage compared to other organs because of higher oxidative metabolism due to abundance of

Please cite this article as: Mantawy, E.M., et al., Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.01.065i

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mitochondria in cardiomyocytes and inherent decreased antioxidant defenses (Herman et al., 2000). In the current study, increased oxidative damage was observed in DOX-treated rats, as shown by markedly increased lipid peroxidation products (MDA), depleted GSH levels and decreased antioxidant enzyme activities; CAT and SOD. On the other hand, chrysin supplementation effectively reduced the extent of DOXinduced oxidative stress whereas it has potent free radical scavenging properties which are associated with the hydroxyl substitution in the fifth and seventh positions that directly sequestrates free radicals (Sathiavelu et al., 2009). Additionally, chrysin could indirectly repress oxidative stress via upregulating antioxidant enzyme activities (Rehman et al., 2013). Beside its direct deleterious effect, oxidative stress can also induce inflammatory responses via activation of redox sensitive transcription factors, such as NF-κB (Rahman, 2002). NF-κB is a transcription factor consisting of p65 and p50 subunits of Rel protein family that regulates the host inflammatory and immune responses (Ali and Mann, 2004). NF-κB is retained within the cytoplasm in an inactive form through association with one of the IκB inhibitory proteins. Upon activation by different extracellular stimuli such as infection, inflammation and oxidative stress, IκB is ubiquitinated and degraded by proteasome unmasking the NF-κB nuclear localization signal. This leads to transport of NF-κB from the cytoplasm to the nucleus, where it binds to specific target genes to induce their transcription (Hoffmann et al., 2006). NF-κB as a critical mediator of inflammatory response, regulates the expression of a battery of distinct proinflammatory mediators such as TNF-α, COX-2 and nitric oxide (Elsharkawy and Mann, 2007). Several studies have reported that DOX provokes a series of inflammatory reactions in the vicinity of heart tissues by upregulating NF-κB and stimulating subsequent proinflammatory cytokines production (Abd El-Aziz et al., 2012). These inflammatory cytokines lead to profound pathological changes in the form of cardiomyopathy, transmural myocarditis and biventricular fibrosis (Bozkunt et al., 1998). In the present study, DOX intoxication significantly increased NF-κB p65, iNOS and COX-2 expressions as well as TNF-α and nitric oxide levels reflecting amplified inflammatory responses. On the contrary, chrysin pretreatment significantly reduced the expression of NF-κB and hence inhibited the downstream inflammatory cascade as evidenced by decreasing the expression of iNOS and COX-2 as well as the levels of TNF-α and nitric oxide, so chrysin provides satisfactory anti-inflammatory effects. Our results coincided with previous studies that have reported that chrysin is a potential inhibitor of NF-κB and can diminish the downstream inflammatory cytokines production (Dou et al., 2013). Growing evidence implicates that DOX-induced apoptotic death of cardiomyocytes is the most direct cause of DOX cardiotoxicity (Kalay et al., 2006). As the quinine moiety of DOX is prone to generation of reactive oxygen species through enzymatic mechanisms in mitochondrial respiratory chain producing hydroxyl and superoxide radicals, triggering the intrinsic mitochondrial dependent apoptotic pathway (Green and Leeuwenburgh, 2002). Reactive oxygen species produced in the mitochondria lead to damage of the membrane phospholipids resulting in loss of mitochondrial membrane potential and hence, release of cytochrome c (Sabbah, 2000). Released cytochrome c binds to apoptosis protease activation factor-1 and forms complex which recruits and activates the initiator caspase-9. This in turn activates the effector, caspase-3 that initiates the apoptotic degradation phase (Nicholson and Thornberry, 2003). During the above signal conduction, apoptosis is regulated by a series of regulating proteins. Members of the Bcl-2 family of proteins are major regulators of apoptosis (Kunisada et al., 2002). This family includes pro-apoptotic (e.g. Bax and Bid) and

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anti-apoptotic (e.g. Bcl-2 and Bcl-xL) members. (Golstein, 1997). Bax is a pore-forming cytoplasmic protein that in response to increased oxidative stress, translocates to the outer mitochondrial membrane, influencing its permeability and inducing cytochrome c loss from the intermembrane space of the mitochondria and subsequent release into the cytosol (Crompton, 2000). The antiapoptotic Bcl-2 is associated with the outer mitochondrial membrane where it stabilizes the membrane permeability, thus preserving mitochondrial integrity and suppressing the release of cytochrome c (Yang et al., 1997). Cell survival in the early phases of apoptotic cascade depends mostly on the balance between the pro and anti-apoptotic proteins of the Bcl-2 family (Desagher and Martinou, 2000). Our results indicated that DOX intoxication induced marked apoptosis of cardiomyocytes as shown by the significant increase of cytochrome c expression and caspase 3 activity. Hence, the apoptotic effect of DOX could be mainly attributed to triggering the intrinsic mitochondrial dependent apoptotic pathway through the generation of reactive oxygen species as shown by the severe oxidative stress observed in the DOX-treated group. Our results are in accordance with previous studies that reported mitochondrial dysfunction due to DOX-induced oxidative stress (Childs et al., 2002). Our study also showed that DOX has profound effects on the Bcl-2 family proteins. While it upregulated Bax expression, it also downregulated Bcl-2 expression. Consequently, the ratio of the pro-apoptotic Bax to the antiapoptotic Bcl-2 was significantly increased upon treatment with DOX. This is also in agreement with other studies showing an increase in Bax and a decrease in Bcl-2 protein expression in cardiomyocytes following DOX treatment (Xin et al., 2009). In addition, NF-κB has been reported to promote DOX-induced apoptosis in vascular cells and myocytes (Wang et al., 2002). The proapoptotic character of NF-κB might be due to down-regulating the expression of some anti-apoptotic factors, e.g. Bcl-XL (Hettmann et al., 1999). Interestingly, DOX-induced alteration in the expression of cytochrome c, Bax and Bcl-2 proteins and caspase-3 activity were almost restored to normal levels with chrysin supplementation. Furthermore, a significant decrease in the Bax to Bcl-2 ratio is likely to be one of the factors responsible for the inhibitory effect of chrysin on DOX-induced apoptosis. This indicates that chrysin abrogated DOX-induced apoptosis by decreasing the expression of Bax and cytochrome c, increasing that of Bcl-2 and finally inhibiting caspase-3 activity. The antiapoptotic effect of chrysin can be also attributed to a free radical scavenging capability. This finding is consistent with previous studies reporting the antiapoptotic effects of chrysin (Khan et al., 2012a, 2012b). In conclusion, the present study revealed for the first time a protective role of chrysin against DOX-induced acute cardiotoxicity. The main mechanism underlying this cardioprotective effect could be primarily through suppressing the two major upstream apoptotic signals induced by DOX (one mitochondrial dependent and the other oxidative stress related). Secondarily, abrogating DOX-induced inflammatory responses also contributes to the cardioprotective effect of chrysin. Thus, adjuvant administration of chrysin with DOX could provide a new promising solution to the very serious and potentially fatal cardiac complication of DOX.

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Please cite this article as: Mantawy, E.M., et al., Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.01.065i