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Cardioprotective effect of cannabidiol in rats exposed to doxorubicin toxicity
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 6 ( 2 0 1 3 ) 347–357
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/etap
Cardioprotective effect of cannabidiol in rats exposed to doxorubicin toxicity Amr A. Fouad a,∗ , Waleed H. Albuali b , Abdulruhman S. Al-Mulhim c , Iyad Jresat d a
Department of Biomedical Sciences, Pharmacology Division, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia Department of Pediatrics, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia c Department of Surgery, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia d Department of Biomedical Sciences, Pathology Division, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia b
a r t i c l e
i n f o
a b s t r a c t
Article history:
The potential protective effect of cannabidiol, the major non-psychotropic Cannabis con-
Received 9 January 2013
stituent, was investigated against doxorubicin cardiotoxicity in rats. Cardiotoxicity was
Received in revised form
induced by six equal doses of doxorubicin (2.5 mg kg−1 i.p., each) given at 48 h intervals
7 April 2013
over two weeks to achieve a total dose of 15 mg kg−1 . Cannabidiol treatment (5 mg kg−1 /day,
Accepted 21 April 2013
i.p.) was started on the same day of doxorubicin administration and continued for four
Available online 10 May 2013
weeks. Cannabidiol significantly reduced the elevations of serum creatine kinase-MB
Keywords:
calcium ion levels, and attenuated the decreases in cardiac reduced glutathione, sele-
and troponin T, and cardiac malondialdehyde, tumor necrosis factor-␣, nitric oxide and Cannabidiol
nium and zinc ions. Histopathological examination showed that cannabidiol ameliorated
Doxorubicin cardiotoxicity
doxorubicin-induced cardiac injury. Immunohistochemical analysis revealed that cannabid-
Oxidative stress
iol significantly reduced the expression of inducible nitric oxide synthase, nuclear factor-B,
Inflammation
Fas ligand and caspase-3, and increased the expression of survivin in cardiac tissue of
Rats
doxorubicin-treated rats. These results indicate that cannabidiol represents a potential protective agent against doxorubicin cardiac injury. © 2013 Elsevier B.V. All rights reserved.
1.
Introduction
Doxorubicin is an anthracycline anticancer antibiotic commonly used for treatment of hematological malignancies and solid tumors. Despite the broad therapeutic effectiveness, the clinical use of doxorubicin is often limited because of its dosedependent cardiotoxic adverse effects which frequently lead to congestive heart failure (Singal and Iliskovic, 1998; Minotti et al., 2004). Oxidative stress and inflammation play an important role in the pathogenesis of doxorubicin cardiotoxicity; indeed, several antioxidants and anti-inflammatory agents were proved effective in protecting against cardiac tissue
damage induced by doxorubicin (Andreadou et al., 2007; Jiang et al., 2008; Ammar et al., 2011; Xin et al., 2011). Cannabidiol is the major non-psychoactive cannabinoid component derived from the plant Cannabis sativa. It possesses powerful antioxidant and anti-inflammatory activities, although the exact mechanisms of action of cannabidiol remain obscure. In contrast to the other cannabinoids, cannabidiol is known to have a very low affinity for the cannabinoid CB1 and CB2 receptors. The antioxidant and anti-inflammatory activities of cannabidiol may be due to its direct action or mediated through a new cannabinoid, nonCB1 and non-CB2 , receptor (Begg et al., 2005; De Petrocellis and Di Marzo, 2010). Cannabidiol may also exert its beneficial
∗ Corresponding author at: Department of Biomedical Sciences, Pharmacology Division, College of Medicine, King Faisal University, Al-Ahsa 31982, Saudi Arabia. Tel.: +966 501776517. E-mail address: [email protected] (A.A. Fouad). 1382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2013.04.018
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effects by inhibiting adenosine uptake and activating transient receptor potential vanilloid-1 (Bisogno et al., 2001; Carrier et al., 2006). Previous reports showed that cannabidiol may have therapeutic utility in a number of conditions involving inflammation and oxidative stress, including diabetes mellitus, rheumatoid arthritis, neurodegenerative disorders and ischemia/reperfusion tissue injury (Blake et al., 2006; Durst et al., 2007; Iuvone et al., 2009; Rajesh et al., 2010). However, to the best of our knowledge, the protective effect of cannabidiol against doxorubicin cardiotoxicity was not yet investigated. Hence, we have conducted the present study to evaluate the potential cardioprotective effect of cannabidiol in rats which received doxorubicin, and to investigate the possible mechanisms underlying this protective effect.
2.
Materials and methods
2.1.
Animals
Male Sprague-Dawley rats, weighing 250 ± 10 g were obtained from the Animal House, College of Medicine, King Faisal University. The animals were kept at standard housing facilities (24 ± 1 ◦ C, 45 ± 5% humidity and 12 h light/dark cycle). They were supplied with standard laboratory chow and water ad libitum, and left to acclimatize for 1 week before the experiments. The experimental protocol was approved by the Ethical Committee, Deanship of Scientific Research, King Faisal University (approval number: 130243). The experimental procedures were carried out in accordance with international guidelines for care and use of laboratory animals.
2.2.
Drugs
Doxorubicin hydrochloride (Sigma-Aldrich Co., USA) was dissolved in normal saline, and cannabidiol (Cayman Chemical Company, USA) was prepared in 1% aqueous solution of Tween 80. The doses of doxorubicin and cannabidiol used in this study were selected based on our preliminary experiments and in accordance with previous reports (Durst et al., 2007; Ibrahim et al., 2009).
2.3.
Experimental design
The rats were randomly assigned to four equal groups (n = 8, each). The first group (control group) received six i.p. injections of normal saline (vehicle of doxorubicin) at 48 h intervals over a period of two weeks. Rats of the second and third groups received six equal doses of doxorubicin (2.5 mg kg−1 i.p., each) given at 48 h intervals over two weeks to achieve a total dose of 15 mg kg−1 which is well documented to produce cardiotoxicity. The animals of the second and third groups received a daily i.p. injection of the vehicle of cannabidiol (1% aqueous solution of Tween 80) or cannabidiol at a dose of 5 mg kg−1 , respectively, starting on the same day of doxorubicin administration and continued for four weeks. The fourth group animals received cannabidiol for four weeks without induction of doxorubicin cardiotoxicity.
2.4.
Sample preparation and biochemical studies
Twenty-four hours following the last dose of cannabidiol, the rats were sacrificed. Blood samples were collected, left for 60 min to clot, and then centrifuged for 10 min at 5000 rpm to obtain clear sera which were stored at −80 ◦ C. Subsequently, serum creatine kinase-MB (CK-MB) level was assayed using a commercial kit following the instructions of the manufacturer (Stanbio Laboratory, USA). Also, serum troponin T level was measured by enzyme-linked immunosorbent assay (ELISA) using rat troponin T immunoassay kit according to the recommendations of the manufacturer (Uscn Life Science Inc., China). The heart was removed from each animal and its fresh weight was recorded. The isolated hearts were kept at −80 ◦ C and subsequently homogenized in cold potassium phosphate buffer (0.05 M, pH 7.4). The homogenates were centrifuged at 5000 rpm for 10 min at 4 ◦ C. The resulting supernatant was used for determination of malondialdehyde (MDA) and reduced glutathione (GSH) levels using colorimetric assay kits according to the recommendations of the manufacturer (Biodiagnostic, Egypt). The level of nitric oxide (NO) was assayed using colorimetric assay kit as indicated by the manufacturer (Cayman Chemical Company, USA). Also, the level of tumor necrosis factor-␣ (TNF-␣) in cardiac homogenates was determined by enzyme-linked immunosorbent assay (ELISA) using rat TNF-␣ immunoassay kit according to the recommendations of the manufacturer (R&D Systems, USA). In addition, parts of the cardiac tissue were dried overnight at 80 ◦ C and the dry weight was recorded. The samples were then digested with equal volumes of 30% (w/v) H2 O2 and 70% (w/v) nitric acid, and the clear digest was diluted with ultrapure water (1:3). Calcium, selenium and zinc ion concentrations were analyzed using inductively coupled plasma optical emission spectrometer (Optima 2100 DV, PerkinElmer, USA) at 317.93, 196.02 and 206.2 nm, respectively, with samplebased standards.
2.5.
Histopathological examination
Parts of the cardiac tissue obtained from each animal were fixed in 10% formalin solution, dehydrated in ascending grades of alcohol and embedded in paraffin. Sections at 4 m thickness were taken, stained with hematoxylin and eosin (H&E), and examined under light microscope by a pathologist unaware of the treatment protocol.
2.6.
Immunohistochemical examinations
Four m thick sections were deparaffinised, rehydrated, and endogenous peroxidase activity was blocked with H2 O2 in methanol. Sections were pre-treated in citrate buffer (pH 6.0) in a microwave. Sections were incubated at room temperature with rabbit polyclonal antibodies specific for the rat targets. These antibodies used were anti-inducible nitric oxide synthase (iNOS), anti-nuclear factor-B (NF-B), anti-Fas ligand (FasL), anti-survivin and anti-caspase-3 antibodies (Thermo Scientific, USA, dilution 1:1000). Sections were incubated with biotinylated goat anti-polyvalent, then with streptavidin peroxidase and finally with diaminobenzedine plus chromogen.
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Table 1 – Effects of cannabidiol (CBD) treatment on serum creatine kinase-MB (CK-MB) and troponin T, and cardiac tissue levels of malondialdehyde (MDA), reduced glutathione (GSH), tumor necrosis factor-␣ (TNF-␣) and nitric oxide (NO) in rats exposed to doxorubicin (DOX) cardioxicity. Control CK-MB (U/L) Troponin T (ng/ml) MDA (nmol/g tissue) GSH (mol/g tissue) TNF-␣ (pg/100 mg tissue) NO (mol/g tissue)
68.59 ± 4.36 ND 102.55 ± 9.79 0.81 ± 0.05 ND 99.02 ± 8.71
Vehicle + DOX 315.23 1.79 419.73 0.25 47.39 296.43
± ± ± ± ± ±
38.61a 0.12 a 38.36a 0.02a 5.41a 25.68a
CBD + DOX 159.74 0.64 176.18 0.64 23.63 162.95
± ± ± ± ± ±
9.93a ,b 0.07a ,b 13.85b 0.06b 2.47a ,b 11.46a ,b
CBD 78.17 ± 8.14 ND 124.06 ± 7.74 0.71 ± 0.08 ND 108.41 ± 8.25
All the values are expressed as mean ± S.E.M., n = 8 in each group. ND = non-detectable. p < 0.05 vs. control group. b p < 0.05 vs. vehicle + DOX group. a
Slides were counterstained with hematoxylin, visualized under light microscope and the extent of cell immunopositivity was assessed by using a semi-quantitative analysis. The number of immunopositive cells was counted in 5 separate microscopic fields in each slide and the mean number for each slide was obtained, then the mean ± S.E.M. was calculated for each group (8 slides). The same procedures were repeated using normal rabbit serum instead of the primary antibody to have negative control to indicate the specificity of the antibody.
2.7.
Statistical analysis
All values are expressed as mean ± S.E.M. The results were analyzed by one-way analysis of variance (ANOVA) followed by Tukey test for multiple comparisons using SPSS for Windows (version 18). Differences were considered significant at p < 0.05.
3.
Results
3.1. Effects of cannabidiol on the measured biochemical parameters Significant increases of serum CK-MB and troponin T, and cardiac tissue levels of MDA, TNF-␣, and NO, associated with a significant reduction in cardiac GSH resulted from doxorubicin administration as compared to the control values (p < 0.05). Cannabidiol treatment significantly attenuated the elevations of serum CK-MB and troponin T, and suppressed lipid peroxidation, reduced the elevations of TNF-␣ and NO,
and prevented the depletion of GSH in cardiac tissue as compared to the doxorubicin group non-treated with cannabidiol (p < 0.05) (Table 1). The levels of serum CK-MB and troponin T were reduced by 49.33 and 64.25%, respectively, while cardiac MDA, TNF-␣, and NO were reduced by 58.03, 50.14 and 45.03%, respectively, and cardiac GSH level was increased by 156% in the doxorubicin cannabidiol-treated group as compared with the corresponding values in the doxorubicin group non-treated with cannabidiol. Also, rats that received doxorubicin showed a significant increase in calcium ion concentration, associated with significant reductions in selenium and zinc ions in the cardiac tissue as compared to the control animals (p < 0.05). The cardiac calcium ion level was increased by 115.93%, while selenium and zinc ions were decreased by 62.23 and 58.01%, respectively, than the corresponding control values. However, cannabidioltreated rats had a significantly lower cardiac calcium level and higher selenium and zinc levels in comparison with the doxorubicin cannabidiol non-treated group (p < 0.05) (Table 2). The cardiac calcium level was reduced by 25.58%, while cardiac selenium and zinc levels were increased by 104.63 and 79.88%, respectively, in the doxorubicin cannabidiol-treated group as compared with the corresponding values in the doxorubicin group non-treated with cannabidiol.
3.2.
Effect of cannabidiol on cardiac histopathology
Doxorubicin caused extensive damage of cardiac tissue in the form of myofibrillar loss, cytoplasmic vacuolization, coagulative necrosis, edema and hemorrhages. However, cannabidiol treatment markedly ameliorated the doxorubicin-induced
Table 2 – Effects of cannabidiol (CBD) treatment on cardiac calcium, selenium and zinc ion concentrations in rats exposed to doxorubicin (DOX) cardiotoxicity.
Calcium ion (g/g tissue) Selenium ion (g/g tissue) Zinc ion (g/g tissue)
Control
Vehicle + DOX
CBD + DOX
64.21 ± 6.94 4.58 ± 0.35 25.46 ± 2.14
138.65 ± 12.82a 1.73 ± 0.18a 10.69 ± 1.03a
103.18 ± 8.77a ,b 3.54 ± 0.29b 19.23 ± 1.49a ,b
All the values are expressed as mean ± S.E.M., n = 8 in each group. p < 0.05 vs. control group. b p < 0.05 vs. vehicle + DOX group. a
CBD 73.46 ± 5.36 3.96 ± 0.41 23.57 ± 1.45
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Fig. 1 – Photomicrographs of rat heart (H&E) from: (A, 200×) control group showing normal architecture of cardiac tissue; (B, 200× and C, 400×) doxorubicin group without cannabidiol treatment showing widespread damage in the form of cytoplasmic vacuolization, loss of muscle fibers, edema, hemorrhages (B, black arrow) and coagulative necrosis (C, white arrow); (D, 200×) doxorubicin plus cannabidiol group showing a histological picture comparable to that of the control group.
pathological changes in the cardiac tissue and maintained the normal histological picture of the myocardium (Fig. 1).
staining at all indicating the specificity of the used antibodies (figures not shown).
3.3. Effects of cannabidiol on cardiac immunohistochemistry
4.
Doxorubicin administration resulted in significant increases in the immunoreactivity of iNOS, NF-B, FasL and caspase-3, associated with a significant decrease in survivin expression in the cardiomyocytes as compared to the control group (p < 0.05). However, cannabidiol-treated rats showed significant reductions in the immunostaining for iNOS, NF-B, FasL and caspase-3, accompanied with a significant increase in survivin immunoreactivity in the cardiac tissue as compared to the doxorubicin cannabidiol non-treated group (p < 0.05) (Figs. 2–6). The slides from the doxorubicin group without cannabidiol treatment which were incubated with normal rabbit serum instead of the primary antibodies showed no
Discussion
The present study revealed that cannabidiol treatment for four weeks provided a significant protective effect against doxorubicin-induced cardiotoxicity in rats. Also, the present results, in accordance with previous studies, showed that oxidative stress, depletion of antioxidant defenses and increased production of inflammatory mediators are implicated in the pathogenesis of doxorubicin cardiotoxicity (Andreadou et al., 2007; Jiang et al., 2008; Ammar et al., 2011; Xin et al., 2011). In addition, it was reported that doxorubicin caused a significant elevation of cardiac NO levels due to increased expression of iNOS in the cardiac tissue (SayedAhmed et al., 2001; Andreadou et al., 2007). This may be due to the ability of TNF-␣ to up-regulate the iNOS gene (Morris and
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Fig. 2 – Immunohistochemical staining of inducible nitric oxide synthase (iNOS) in rat heart (200×) from: (A) control group showing no expression of iNOS; (B) doxorubicin group without cannabidiol treatment showing a significant increase in iNOS immunoreactivity in the cardiomyocytes (white arrows); (C) doxorubicin plus cannabidiol group showing a significant reduction in iNOS immunostaining. Brown color indicates iNOS positivity; (D) percentage expression of iNOS, data is mean ± S.E.M. of 8 rats, ND = non-detectable, *p < 0.05 vs. control group, • p < 0.05 vs. doxorubicin (DOX) group without cannabidiol (CBD) treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Billiar, 1994). Excess NO reacts with superoxide anion to produce peroxynitrite radical which causes further cardiac tissue damage by oxidizing and nitrating cellular macromolecules. Also, excess NO depletes intracellular GSH increasing the susceptibility to oxidative stress (Clancy and Abramson, 1995). Cannabidiol has been shown to have prominent antioxidant and antinitrative properties in several disease models. It inhibits the generation of reactive oxygen species, scavenges lipid peroxidation products during free radical reactions, and suppresses excess NO production preventing nitrosative ˜ et al., 2011). In stress (Pan et al., 2009; Ruiz-Valdepenas addition, cannabidiol exhibits anti-inflammatory activity by reducing the release of proinflammatory cytokines as TNF-␣ (Costa et al., 2004; Rajesh et al., 2010).
It was also demonstrated that doxorubicin treatment resulted in NF-B activation with subsequent inflammatory reactions responsible for myocardial dysfunction and apoptosis (Kim et al., 2007; Li and Yu, 2008). Increased generation of ROS is well-known to induce NF-B signaling pathway and cannabidiol has the ability to inhibit the activation of NF-B signaling pathway which promotes the transcription of TNF␣ and iNOS genes (Rajesh et al., 2010). This is in accordance with the present results which revealed that cannabidiol treatment significantly suppressed lipid peroxidation, maintained GSH level, attenuated the production of TNF-␣ and NO, and reduced the expression of NF-B and iNOS in the cardiac tissue of rats exposed to doxorubicin cardiotoxicity. Also, the disturbance in myocardial Ca2+ homeostasis induced by doxorubicin is implicated as one of its possible
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Fig. 3 – Immunohistochemical staining of nuclear factor-B (NF-B) in rat heart (200×) from: (A) control group showing no expression of NF-B; (B) doxorubicin group without cannabidiol treatment showing a significant increase in NF-B immunoreactivity in the cardiomyocytes (white arrows); (C) doxorubicin plus cannabidiol group demonstrating a significant reduction in NF-B immunostaining. Brown color indicates NF-B positivity; (D) percentage expression of NF-B, data is mean ± S.E.M. of 8 rats, ND = non-detectable, *p < 0.05 vs. control group, • p < 0.05 vs. doxorubicin (DOX) group without cannabidiol (CBD) treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
mechanisms of cardiotoxicity. Doxorubicin increases cytosolic Ca2+ level in cardiac cells by releasing Ca2+ from the sarcoplasmic reticulum through ROS-mediated opening of ryanodine receptor (Kim et al., 2006). The increased cytosolic Ca2+ activates the Fas/FasL pathway. Cross-linking of FasL to its Fas cell surface receptor triggers cell apoptosis by inducing recruitment of the Fas-associated protein with death domain, which eventually leads to activation of the caspase family of proteases and myocardial apoptotic cell death (Kalivendi et al., 2005; Choudhary et al., 2006). The survivin gene, a new member of the anti-apoptotic protein family, is generally accepted as the most powerful antiapoptotic gene in vivo. Its mechanism of regulating apoptosis involves various complex factors. One relatively clear mechanism is that survivin inhibits the activation of caspase3, an executioner of cell apoptosis (Altieri, 2006; Ouhtit et al.,
2007). Survivin is detectable in nonproliferating differentiated myocardial tissues. It is also induced after myocardial infarction in humans and in the hearts of spontaneously hypertensive rats (Santini et al., 2004; Abbate et al., 2006). Survivin plays an important role in controlling cardiomyocyte number during embryonic development and adult life through its profound impact on cardiomyocyte replication. It was demonstrated that cardiac-specific deletion of survivin caused premature cardiac cell death resulting in increased hemodynamic load per cell with progressive heart failure in cardiomyocyte-specific survivin-deficient mice (Levkau et al., 2008). The present study revealed that cannabidiol treatment significantly reduced the elevation of Ca2+ concentration and caspase-3 expression and cardiac cell apoptosis resulted from doxorubicin. The antiapoptotic activity observed with
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Fig. 4 – Immunohistochemical staining of Fas ligand (FasL) in rat heart (200×) from: (A) control group showing no expression of FasL; (B) doxorubicin group without cannabidiol treatment showing a significant increase in FasL immunoreactivity in the cardiomyocytes (white arrows); (C) doxorubicin plus cannabidiol group demonstrating a significant reduction in FasL immunostaining. Brown color indicates FasL positivity; (D) percentage expression of FasL, data is mean ± S.E.M. of 8 rats, ND = non-detectable, *p < 0.05 vs. control group, • p < 0.05 vs. doxorubicin (DOX) group without cannabidiol (CBD) treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
cannabidiol can be attributed to inhibition of FasL expression and induction of survivin protein. This is in accordance with a recent study which showed that cannabidiol reduced FasL expression and up-regulated survivin expression, and therefore protected against hepatocellular apoptosis in rats exposed to ischemia/reperfusion liver injury (Fouad and Jresat, 2011). Also, free radical scavenging activity, anti-inflammatory action with reduced TNF-␣ production and inhibition of NF-B may contribute to the antiapoptotic effect of cannabidiol. On the other hand, it was recognized that down-regulation of survivin prevents chemoresistance of cancer cells and enhances the antitumor activity to doxorubicin (Zou et al., 2010; Yang et al., 2011). However, the point of whether the induction of survivin by cannabidiol can interfere with the anticancer effect of doxorubicin or not warrants further investigation.
In the present study, cannabidiol significantly attenuated the reductions in cardiac selenium and zinc concentrations resulted from doxorubicin administration. Selenium is an essential component of glutathione peroxidase, while zinc acts as a cofactor for superoxide dismutase. Also, both elements preserve GSH which has powerful antioxidant properties (Satoh et al., 2000; Rooney, 2007). It can be speculated that cannabidiol through its antioxidant effect prevented the depletion of selenium and zinc ions in the heart tissue which results in an additional cardioprotective effect. Previous studies showed that cannabidiol treatment provided a significant cardioprotective effect in different models of oxidative stress and inflammatory injuries of the cardiac muscle. A previous study which showed that cannabidiol administration (5 mg kg−1 i.p., pre-ischemic and for 7 days post-ischemic) significantly reduced myocardial infarct size
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Fig. 5 – Immunohistochemical staining of survivin in rat heart (200×) from: (A) control group showing that survivin is expressed in normal cardiomyocytes (white arrows); (B) doxorubicin group without cannabidiol treatment showing a significant reduction in survivin immunoreactivity in the cardiomyocytes; (C) doxorubicin plus cannabidiol group showing a significant increase in survivin immunostaining. Brown color indicates survivin positivity; (D) percentage expression of survivin, data is mean ± S.E.M. of 8 rats, ND = non-detectable, *p < 0.05 vs. control group, • p < 0.05 vs. doxorubicin (DOX) group without cannabidiol (CBD) treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
and myocardial inflammation resulted from ligation of left anterior descending coronary artery (Durst et al., 2007). Another study showed that acute pre-ischemic treatment with cannabidiol (10 and 50 g kg−1 i.v.) given 10 min either pre-ischemic or pre-reperfusion significantly suppressed the total number of ischemia-induced arrhythmias, and reduced the cardiac infarct size in a dose-dependent manner (Walsh et al., 2010). Also, cannabidiol treatment (20 mg kg−1 i.p., for 11 weeks) significantly decreased left ventricular dysfunction, myocardial fibrosis, myocardial oxidative and nitrative stress, and inflammation in a mouse model of diabetic cardiomyopathy (Rajesh et al., 2010). These studies suggest that the cardioprotective effect of cannabidiol may be due to direct effect or through systemic antioxidant and anti-inflammatory effects. Therefore, further investigations are required to
measure the plasma and cardiac tissue levels of cannabidiol to indicate whether its carioprotective effect is a direct or a general one. Also, recent studies demonstrated that cannabidiol is a safe and well tolerated agent even with chronic use in large doses and has no adverse effect on the physiologic cardiac parameters (Bergamaschi et al., 2011; Martin-Santos et al., 2012). However, the present work is the first one to investigate the protective effect of cannabidiol against doxorubicin-induced cardiotoxicity. Therefore, further studies are required to evaluate its protective effect and also possible adverse effects at different doses. The present results indicate that cannabidiol treatment significantly ameliorated doxorubicin-induced cardiotoxicity in rats. The antioxidant and anti-inflammatory activities are
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Fig. 6 – Immunohistochemical staining of caspase-3 in rat heart (200×) from: (A) control group showing no expression of caspase-3; (B) doxorubicin group without cannabidiol treatment showing a significant increase in caspase-3 immunoreactivity in the cardiomyocytes (white arrows); (C) doxorubicin plus cannabidiol group showing a significant reduction in caspase-3 immunostaining. Brown color indicates caspase-3 positivity; (D) percentage expression of caspase-3, data is mean ± S.E.M. of 8 rats, ND = non-detectable, *p < 0.05 vs. control group, • p < 0.05 vs. doxorubicin (DOX) group without cannabidiol (CBD) treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
the main factors responsible for the cardioprotective effect of cannabidiol. Therefore, cannabidiol can be considered a potential option to minimize doxorubicin cardiotoxicity which is a major clinical challenge. However, it was reported that cannabidiol can inhibit platelet aggregation when given to rats at a dose of 50 g kg−1 i.v., prior to coronary artery occlusion (Walsh et al., 2010). Therefore, further studies are required to evaluate the effect of the cannabidiol dose used in the present work on platelet aggregation and if there is a possibility for adverse interaction with antiplatelet agents.
Conflict of interest statement The authors declare that there is no conflict to disclose.
Acknowledgments Special thanks to the Deanship of Scientific Research, King Faisal University for continuous encouragement and support. Also, special thanks to Dr Mohamed A. Morsy for his valuable advices.
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/etap
Cardioprotective effect of cannabidiol in rats exposed to doxorubicin toxicity Amr A. Fouad a,∗ , Waleed H. Albuali b , Abdulruhman S. Al-Mulhim c , Iyad Jresat d a
Department of Biomedical Sciences, Pharmacology Division, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia Department of Pediatrics, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia c Department of Surgery, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia d Department of Biomedical Sciences, Pathology Division, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia b
a r t i c l e
i n f o
a b s t r a c t
Article history:
The potential protective effect of cannabidiol, the major non-psychotropic Cannabis con-
Received 9 January 2013
stituent, was investigated against doxorubicin cardiotoxicity in rats. Cardiotoxicity was
Received in revised form
induced by six equal doses of doxorubicin (2.5 mg kg−1 i.p., each) given at 48 h intervals
7 April 2013
over two weeks to achieve a total dose of 15 mg kg−1 . Cannabidiol treatment (5 mg kg−1 /day,
Accepted 21 April 2013
i.p.) was started on the same day of doxorubicin administration and continued for four
Available online 10 May 2013
weeks. Cannabidiol significantly reduced the elevations of serum creatine kinase-MB
Keywords:
calcium ion levels, and attenuated the decreases in cardiac reduced glutathione, sele-
and troponin T, and cardiac malondialdehyde, tumor necrosis factor-␣, nitric oxide and Cannabidiol
nium and zinc ions. Histopathological examination showed that cannabidiol ameliorated
Doxorubicin cardiotoxicity
doxorubicin-induced cardiac injury. Immunohistochemical analysis revealed that cannabid-
Oxidative stress
iol significantly reduced the expression of inducible nitric oxide synthase, nuclear factor-B,
Inflammation
Fas ligand and caspase-3, and increased the expression of survivin in cardiac tissue of
Rats
doxorubicin-treated rats. These results indicate that cannabidiol represents a potential protective agent against doxorubicin cardiac injury. © 2013 Elsevier B.V. All rights reserved.
1.
Introduction
Doxorubicin is an anthracycline anticancer antibiotic commonly used for treatment of hematological malignancies and solid tumors. Despite the broad therapeutic effectiveness, the clinical use of doxorubicin is often limited because of its dosedependent cardiotoxic adverse effects which frequently lead to congestive heart failure (Singal and Iliskovic, 1998; Minotti et al., 2004). Oxidative stress and inflammation play an important role in the pathogenesis of doxorubicin cardiotoxicity; indeed, several antioxidants and anti-inflammatory agents were proved effective in protecting against cardiac tissue
damage induced by doxorubicin (Andreadou et al., 2007; Jiang et al., 2008; Ammar et al., 2011; Xin et al., 2011). Cannabidiol is the major non-psychoactive cannabinoid component derived from the plant Cannabis sativa. It possesses powerful antioxidant and anti-inflammatory activities, although the exact mechanisms of action of cannabidiol remain obscure. In contrast to the other cannabinoids, cannabidiol is known to have a very low affinity for the cannabinoid CB1 and CB2 receptors. The antioxidant and anti-inflammatory activities of cannabidiol may be due to its direct action or mediated through a new cannabinoid, nonCB1 and non-CB2 , receptor (Begg et al., 2005; De Petrocellis and Di Marzo, 2010). Cannabidiol may also exert its beneficial
∗ Corresponding author at: Department of Biomedical Sciences, Pharmacology Division, College of Medicine, King Faisal University, Al-Ahsa 31982, Saudi Arabia. Tel.: +966 501776517. E-mail address: [email protected] (A.A. Fouad). 1382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2013.04.018
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effects by inhibiting adenosine uptake and activating transient receptor potential vanilloid-1 (Bisogno et al., 2001; Carrier et al., 2006). Previous reports showed that cannabidiol may have therapeutic utility in a number of conditions involving inflammation and oxidative stress, including diabetes mellitus, rheumatoid arthritis, neurodegenerative disorders and ischemia/reperfusion tissue injury (Blake et al., 2006; Durst et al., 2007; Iuvone et al., 2009; Rajesh et al., 2010). However, to the best of our knowledge, the protective effect of cannabidiol against doxorubicin cardiotoxicity was not yet investigated. Hence, we have conducted the present study to evaluate the potential cardioprotective effect of cannabidiol in rats which received doxorubicin, and to investigate the possible mechanisms underlying this protective effect.
2.
Materials and methods
2.1.
Animals
Male Sprague-Dawley rats, weighing 250 ± 10 g were obtained from the Animal House, College of Medicine, King Faisal University. The animals were kept at standard housing facilities (24 ± 1 ◦ C, 45 ± 5% humidity and 12 h light/dark cycle). They were supplied with standard laboratory chow and water ad libitum, and left to acclimatize for 1 week before the experiments. The experimental protocol was approved by the Ethical Committee, Deanship of Scientific Research, King Faisal University (approval number: 130243). The experimental procedures were carried out in accordance with international guidelines for care and use of laboratory animals.
2.2.
Drugs
Doxorubicin hydrochloride (Sigma-Aldrich Co., USA) was dissolved in normal saline, and cannabidiol (Cayman Chemical Company, USA) was prepared in 1% aqueous solution of Tween 80. The doses of doxorubicin and cannabidiol used in this study were selected based on our preliminary experiments and in accordance with previous reports (Durst et al., 2007; Ibrahim et al., 2009).
2.3.
Experimental design
The rats were randomly assigned to four equal groups (n = 8, each). The first group (control group) received six i.p. injections of normal saline (vehicle of doxorubicin) at 48 h intervals over a period of two weeks. Rats of the second and third groups received six equal doses of doxorubicin (2.5 mg kg−1 i.p., each) given at 48 h intervals over two weeks to achieve a total dose of 15 mg kg−1 which is well documented to produce cardiotoxicity. The animals of the second and third groups received a daily i.p. injection of the vehicle of cannabidiol (1% aqueous solution of Tween 80) or cannabidiol at a dose of 5 mg kg−1 , respectively, starting on the same day of doxorubicin administration and continued for four weeks. The fourth group animals received cannabidiol for four weeks without induction of doxorubicin cardiotoxicity.
2.4.
Sample preparation and biochemical studies
Twenty-four hours following the last dose of cannabidiol, the rats were sacrificed. Blood samples were collected, left for 60 min to clot, and then centrifuged for 10 min at 5000 rpm to obtain clear sera which were stored at −80 ◦ C. Subsequently, serum creatine kinase-MB (CK-MB) level was assayed using a commercial kit following the instructions of the manufacturer (Stanbio Laboratory, USA). Also, serum troponin T level was measured by enzyme-linked immunosorbent assay (ELISA) using rat troponin T immunoassay kit according to the recommendations of the manufacturer (Uscn Life Science Inc., China). The heart was removed from each animal and its fresh weight was recorded. The isolated hearts were kept at −80 ◦ C and subsequently homogenized in cold potassium phosphate buffer (0.05 M, pH 7.4). The homogenates were centrifuged at 5000 rpm for 10 min at 4 ◦ C. The resulting supernatant was used for determination of malondialdehyde (MDA) and reduced glutathione (GSH) levels using colorimetric assay kits according to the recommendations of the manufacturer (Biodiagnostic, Egypt). The level of nitric oxide (NO) was assayed using colorimetric assay kit as indicated by the manufacturer (Cayman Chemical Company, USA). Also, the level of tumor necrosis factor-␣ (TNF-␣) in cardiac homogenates was determined by enzyme-linked immunosorbent assay (ELISA) using rat TNF-␣ immunoassay kit according to the recommendations of the manufacturer (R&D Systems, USA). In addition, parts of the cardiac tissue were dried overnight at 80 ◦ C and the dry weight was recorded. The samples were then digested with equal volumes of 30% (w/v) H2 O2 and 70% (w/v) nitric acid, and the clear digest was diluted with ultrapure water (1:3). Calcium, selenium and zinc ion concentrations were analyzed using inductively coupled plasma optical emission spectrometer (Optima 2100 DV, PerkinElmer, USA) at 317.93, 196.02 and 206.2 nm, respectively, with samplebased standards.
2.5.
Histopathological examination
Parts of the cardiac tissue obtained from each animal were fixed in 10% formalin solution, dehydrated in ascending grades of alcohol and embedded in paraffin. Sections at 4 m thickness were taken, stained with hematoxylin and eosin (H&E), and examined under light microscope by a pathologist unaware of the treatment protocol.
2.6.
Immunohistochemical examinations
Four m thick sections were deparaffinised, rehydrated, and endogenous peroxidase activity was blocked with H2 O2 in methanol. Sections were pre-treated in citrate buffer (pH 6.0) in a microwave. Sections were incubated at room temperature with rabbit polyclonal antibodies specific for the rat targets. These antibodies used were anti-inducible nitric oxide synthase (iNOS), anti-nuclear factor-B (NF-B), anti-Fas ligand (FasL), anti-survivin and anti-caspase-3 antibodies (Thermo Scientific, USA, dilution 1:1000). Sections were incubated with biotinylated goat anti-polyvalent, then with streptavidin peroxidase and finally with diaminobenzedine plus chromogen.
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Table 1 – Effects of cannabidiol (CBD) treatment on serum creatine kinase-MB (CK-MB) and troponin T, and cardiac tissue levels of malondialdehyde (MDA), reduced glutathione (GSH), tumor necrosis factor-␣ (TNF-␣) and nitric oxide (NO) in rats exposed to doxorubicin (DOX) cardioxicity. Control CK-MB (U/L) Troponin T (ng/ml) MDA (nmol/g tissue) GSH (mol/g tissue) TNF-␣ (pg/100 mg tissue) NO (mol/g tissue)
68.59 ± 4.36 ND 102.55 ± 9.79 0.81 ± 0.05 ND 99.02 ± 8.71
Vehicle + DOX 315.23 1.79 419.73 0.25 47.39 296.43
± ± ± ± ± ±
38.61a 0.12 a 38.36a 0.02a 5.41a 25.68a
CBD + DOX 159.74 0.64 176.18 0.64 23.63 162.95
± ± ± ± ± ±
9.93a ,b 0.07a ,b 13.85b 0.06b 2.47a ,b 11.46a ,b
CBD 78.17 ± 8.14 ND 124.06 ± 7.74 0.71 ± 0.08 ND 108.41 ± 8.25
All the values are expressed as mean ± S.E.M., n = 8 in each group. ND = non-detectable. p < 0.05 vs. control group. b p < 0.05 vs. vehicle + DOX group. a
Slides were counterstained with hematoxylin, visualized under light microscope and the extent of cell immunopositivity was assessed by using a semi-quantitative analysis. The number of immunopositive cells was counted in 5 separate microscopic fields in each slide and the mean number for each slide was obtained, then the mean ± S.E.M. was calculated for each group (8 slides). The same procedures were repeated using normal rabbit serum instead of the primary antibody to have negative control to indicate the specificity of the antibody.
2.7.
Statistical analysis
All values are expressed as mean ± S.E.M. The results were analyzed by one-way analysis of variance (ANOVA) followed by Tukey test for multiple comparisons using SPSS for Windows (version 18). Differences were considered significant at p < 0.05.
3.
Results
3.1. Effects of cannabidiol on the measured biochemical parameters Significant increases of serum CK-MB and troponin T, and cardiac tissue levels of MDA, TNF-␣, and NO, associated with a significant reduction in cardiac GSH resulted from doxorubicin administration as compared to the control values (p < 0.05). Cannabidiol treatment significantly attenuated the elevations of serum CK-MB and troponin T, and suppressed lipid peroxidation, reduced the elevations of TNF-␣ and NO,
and prevented the depletion of GSH in cardiac tissue as compared to the doxorubicin group non-treated with cannabidiol (p < 0.05) (Table 1). The levels of serum CK-MB and troponin T were reduced by 49.33 and 64.25%, respectively, while cardiac MDA, TNF-␣, and NO were reduced by 58.03, 50.14 and 45.03%, respectively, and cardiac GSH level was increased by 156% in the doxorubicin cannabidiol-treated group as compared with the corresponding values in the doxorubicin group non-treated with cannabidiol. Also, rats that received doxorubicin showed a significant increase in calcium ion concentration, associated with significant reductions in selenium and zinc ions in the cardiac tissue as compared to the control animals (p < 0.05). The cardiac calcium ion level was increased by 115.93%, while selenium and zinc ions were decreased by 62.23 and 58.01%, respectively, than the corresponding control values. However, cannabidioltreated rats had a significantly lower cardiac calcium level and higher selenium and zinc levels in comparison with the doxorubicin cannabidiol non-treated group (p < 0.05) (Table 2). The cardiac calcium level was reduced by 25.58%, while cardiac selenium and zinc levels were increased by 104.63 and 79.88%, respectively, in the doxorubicin cannabidiol-treated group as compared with the corresponding values in the doxorubicin group non-treated with cannabidiol.
3.2.
Effect of cannabidiol on cardiac histopathology
Doxorubicin caused extensive damage of cardiac tissue in the form of myofibrillar loss, cytoplasmic vacuolization, coagulative necrosis, edema and hemorrhages. However, cannabidiol treatment markedly ameliorated the doxorubicin-induced
Table 2 – Effects of cannabidiol (CBD) treatment on cardiac calcium, selenium and zinc ion concentrations in rats exposed to doxorubicin (DOX) cardiotoxicity.
Calcium ion (g/g tissue) Selenium ion (g/g tissue) Zinc ion (g/g tissue)
Control
Vehicle + DOX
CBD + DOX
64.21 ± 6.94 4.58 ± 0.35 25.46 ± 2.14
138.65 ± 12.82a 1.73 ± 0.18a 10.69 ± 1.03a
103.18 ± 8.77a ,b 3.54 ± 0.29b 19.23 ± 1.49a ,b
All the values are expressed as mean ± S.E.M., n = 8 in each group. p < 0.05 vs. control group. b p < 0.05 vs. vehicle + DOX group. a
CBD 73.46 ± 5.36 3.96 ± 0.41 23.57 ± 1.45
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Fig. 1 – Photomicrographs of rat heart (H&E) from: (A, 200×) control group showing normal architecture of cardiac tissue; (B, 200× and C, 400×) doxorubicin group without cannabidiol treatment showing widespread damage in the form of cytoplasmic vacuolization, loss of muscle fibers, edema, hemorrhages (B, black arrow) and coagulative necrosis (C, white arrow); (D, 200×) doxorubicin plus cannabidiol group showing a histological picture comparable to that of the control group.
pathological changes in the cardiac tissue and maintained the normal histological picture of the myocardium (Fig. 1).
staining at all indicating the specificity of the used antibodies (figures not shown).
3.3. Effects of cannabidiol on cardiac immunohistochemistry
4.
Doxorubicin administration resulted in significant increases in the immunoreactivity of iNOS, NF-B, FasL and caspase-3, associated with a significant decrease in survivin expression in the cardiomyocytes as compared to the control group (p < 0.05). However, cannabidiol-treated rats showed significant reductions in the immunostaining for iNOS, NF-B, FasL and caspase-3, accompanied with a significant increase in survivin immunoreactivity in the cardiac tissue as compared to the doxorubicin cannabidiol non-treated group (p < 0.05) (Figs. 2–6). The slides from the doxorubicin group without cannabidiol treatment which were incubated with normal rabbit serum instead of the primary antibodies showed no
Discussion
The present study revealed that cannabidiol treatment for four weeks provided a significant protective effect against doxorubicin-induced cardiotoxicity in rats. Also, the present results, in accordance with previous studies, showed that oxidative stress, depletion of antioxidant defenses and increased production of inflammatory mediators are implicated in the pathogenesis of doxorubicin cardiotoxicity (Andreadou et al., 2007; Jiang et al., 2008; Ammar et al., 2011; Xin et al., 2011). In addition, it was reported that doxorubicin caused a significant elevation of cardiac NO levels due to increased expression of iNOS in the cardiac tissue (SayedAhmed et al., 2001; Andreadou et al., 2007). This may be due to the ability of TNF-␣ to up-regulate the iNOS gene (Morris and
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Fig. 2 – Immunohistochemical staining of inducible nitric oxide synthase (iNOS) in rat heart (200×) from: (A) control group showing no expression of iNOS; (B) doxorubicin group without cannabidiol treatment showing a significant increase in iNOS immunoreactivity in the cardiomyocytes (white arrows); (C) doxorubicin plus cannabidiol group showing a significant reduction in iNOS immunostaining. Brown color indicates iNOS positivity; (D) percentage expression of iNOS, data is mean ± S.E.M. of 8 rats, ND = non-detectable, *p < 0.05 vs. control group, • p < 0.05 vs. doxorubicin (DOX) group without cannabidiol (CBD) treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Billiar, 1994). Excess NO reacts with superoxide anion to produce peroxynitrite radical which causes further cardiac tissue damage by oxidizing and nitrating cellular macromolecules. Also, excess NO depletes intracellular GSH increasing the susceptibility to oxidative stress (Clancy and Abramson, 1995). Cannabidiol has been shown to have prominent antioxidant and antinitrative properties in several disease models. It inhibits the generation of reactive oxygen species, scavenges lipid peroxidation products during free radical reactions, and suppresses excess NO production preventing nitrosative ˜ et al., 2011). In stress (Pan et al., 2009; Ruiz-Valdepenas addition, cannabidiol exhibits anti-inflammatory activity by reducing the release of proinflammatory cytokines as TNF-␣ (Costa et al., 2004; Rajesh et al., 2010).
It was also demonstrated that doxorubicin treatment resulted in NF-B activation with subsequent inflammatory reactions responsible for myocardial dysfunction and apoptosis (Kim et al., 2007; Li and Yu, 2008). Increased generation of ROS is well-known to induce NF-B signaling pathway and cannabidiol has the ability to inhibit the activation of NF-B signaling pathway which promotes the transcription of TNF␣ and iNOS genes (Rajesh et al., 2010). This is in accordance with the present results which revealed that cannabidiol treatment significantly suppressed lipid peroxidation, maintained GSH level, attenuated the production of TNF-␣ and NO, and reduced the expression of NF-B and iNOS in the cardiac tissue of rats exposed to doxorubicin cardiotoxicity. Also, the disturbance in myocardial Ca2+ homeostasis induced by doxorubicin is implicated as one of its possible
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Fig. 3 – Immunohistochemical staining of nuclear factor-B (NF-B) in rat heart (200×) from: (A) control group showing no expression of NF-B; (B) doxorubicin group without cannabidiol treatment showing a significant increase in NF-B immunoreactivity in the cardiomyocytes (white arrows); (C) doxorubicin plus cannabidiol group demonstrating a significant reduction in NF-B immunostaining. Brown color indicates NF-B positivity; (D) percentage expression of NF-B, data is mean ± S.E.M. of 8 rats, ND = non-detectable, *p < 0.05 vs. control group, • p < 0.05 vs. doxorubicin (DOX) group without cannabidiol (CBD) treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
mechanisms of cardiotoxicity. Doxorubicin increases cytosolic Ca2+ level in cardiac cells by releasing Ca2+ from the sarcoplasmic reticulum through ROS-mediated opening of ryanodine receptor (Kim et al., 2006). The increased cytosolic Ca2+ activates the Fas/FasL pathway. Cross-linking of FasL to its Fas cell surface receptor triggers cell apoptosis by inducing recruitment of the Fas-associated protein with death domain, which eventually leads to activation of the caspase family of proteases and myocardial apoptotic cell death (Kalivendi et al., 2005; Choudhary et al., 2006). The survivin gene, a new member of the anti-apoptotic protein family, is generally accepted as the most powerful antiapoptotic gene in vivo. Its mechanism of regulating apoptosis involves various complex factors. One relatively clear mechanism is that survivin inhibits the activation of caspase3, an executioner of cell apoptosis (Altieri, 2006; Ouhtit et al.,
2007). Survivin is detectable in nonproliferating differentiated myocardial tissues. It is also induced after myocardial infarction in humans and in the hearts of spontaneously hypertensive rats (Santini et al., 2004; Abbate et al., 2006). Survivin plays an important role in controlling cardiomyocyte number during embryonic development and adult life through its profound impact on cardiomyocyte replication. It was demonstrated that cardiac-specific deletion of survivin caused premature cardiac cell death resulting in increased hemodynamic load per cell with progressive heart failure in cardiomyocyte-specific survivin-deficient mice (Levkau et al., 2008). The present study revealed that cannabidiol treatment significantly reduced the elevation of Ca2+ concentration and caspase-3 expression and cardiac cell apoptosis resulted from doxorubicin. The antiapoptotic activity observed with
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Fig. 4 – Immunohistochemical staining of Fas ligand (FasL) in rat heart (200×) from: (A) control group showing no expression of FasL; (B) doxorubicin group without cannabidiol treatment showing a significant increase in FasL immunoreactivity in the cardiomyocytes (white arrows); (C) doxorubicin plus cannabidiol group demonstrating a significant reduction in FasL immunostaining. Brown color indicates FasL positivity; (D) percentage expression of FasL, data is mean ± S.E.M. of 8 rats, ND = non-detectable, *p < 0.05 vs. control group, • p < 0.05 vs. doxorubicin (DOX) group without cannabidiol (CBD) treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
cannabidiol can be attributed to inhibition of FasL expression and induction of survivin protein. This is in accordance with a recent study which showed that cannabidiol reduced FasL expression and up-regulated survivin expression, and therefore protected against hepatocellular apoptosis in rats exposed to ischemia/reperfusion liver injury (Fouad and Jresat, 2011). Also, free radical scavenging activity, anti-inflammatory action with reduced TNF-␣ production and inhibition of NF-B may contribute to the antiapoptotic effect of cannabidiol. On the other hand, it was recognized that down-regulation of survivin prevents chemoresistance of cancer cells and enhances the antitumor activity to doxorubicin (Zou et al., 2010; Yang et al., 2011). However, the point of whether the induction of survivin by cannabidiol can interfere with the anticancer effect of doxorubicin or not warrants further investigation.
In the present study, cannabidiol significantly attenuated the reductions in cardiac selenium and zinc concentrations resulted from doxorubicin administration. Selenium is an essential component of glutathione peroxidase, while zinc acts as a cofactor for superoxide dismutase. Also, both elements preserve GSH which has powerful antioxidant properties (Satoh et al., 2000; Rooney, 2007). It can be speculated that cannabidiol through its antioxidant effect prevented the depletion of selenium and zinc ions in the heart tissue which results in an additional cardioprotective effect. Previous studies showed that cannabidiol treatment provided a significant cardioprotective effect in different models of oxidative stress and inflammatory injuries of the cardiac muscle. A previous study which showed that cannabidiol administration (5 mg kg−1 i.p., pre-ischemic and for 7 days post-ischemic) significantly reduced myocardial infarct size
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Fig. 5 – Immunohistochemical staining of survivin in rat heart (200×) from: (A) control group showing that survivin is expressed in normal cardiomyocytes (white arrows); (B) doxorubicin group without cannabidiol treatment showing a significant reduction in survivin immunoreactivity in the cardiomyocytes; (C) doxorubicin plus cannabidiol group showing a significant increase in survivin immunostaining. Brown color indicates survivin positivity; (D) percentage expression of survivin, data is mean ± S.E.M. of 8 rats, ND = non-detectable, *p < 0.05 vs. control group, • p < 0.05 vs. doxorubicin (DOX) group without cannabidiol (CBD) treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
and myocardial inflammation resulted from ligation of left anterior descending coronary artery (Durst et al., 2007). Another study showed that acute pre-ischemic treatment with cannabidiol (10 and 50 g kg−1 i.v.) given 10 min either pre-ischemic or pre-reperfusion significantly suppressed the total number of ischemia-induced arrhythmias, and reduced the cardiac infarct size in a dose-dependent manner (Walsh et al., 2010). Also, cannabidiol treatment (20 mg kg−1 i.p., for 11 weeks) significantly decreased left ventricular dysfunction, myocardial fibrosis, myocardial oxidative and nitrative stress, and inflammation in a mouse model of diabetic cardiomyopathy (Rajesh et al., 2010). These studies suggest that the cardioprotective effect of cannabidiol may be due to direct effect or through systemic antioxidant and anti-inflammatory effects. Therefore, further investigations are required to
measure the plasma and cardiac tissue levels of cannabidiol to indicate whether its carioprotective effect is a direct or a general one. Also, recent studies demonstrated that cannabidiol is a safe and well tolerated agent even with chronic use in large doses and has no adverse effect on the physiologic cardiac parameters (Bergamaschi et al., 2011; Martin-Santos et al., 2012). However, the present work is the first one to investigate the protective effect of cannabidiol against doxorubicin-induced cardiotoxicity. Therefore, further studies are required to evaluate its protective effect and also possible adverse effects at different doses. The present results indicate that cannabidiol treatment significantly ameliorated doxorubicin-induced cardiotoxicity in rats. The antioxidant and anti-inflammatory activities are
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Fig. 6 – Immunohistochemical staining of caspase-3 in rat heart (200×) from: (A) control group showing no expression of caspase-3; (B) doxorubicin group without cannabidiol treatment showing a significant increase in caspase-3 immunoreactivity in the cardiomyocytes (white arrows); (C) doxorubicin plus cannabidiol group showing a significant reduction in caspase-3 immunostaining. Brown color indicates caspase-3 positivity; (D) percentage expression of caspase-3, data is mean ± S.E.M. of 8 rats, ND = non-detectable, *p < 0.05 vs. control group, • p < 0.05 vs. doxorubicin (DOX) group without cannabidiol (CBD) treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
the main factors responsible for the cardioprotective effect of cannabidiol. Therefore, cannabidiol can be considered a potential option to minimize doxorubicin cardiotoxicity which is a major clinical challenge. However, it was reported that cannabidiol can inhibit platelet aggregation when given to rats at a dose of 50 g kg−1 i.v., prior to coronary artery occlusion (Walsh et al., 2010). Therefore, further studies are required to evaluate the effect of the cannabidiol dose used in the present work on platelet aggregation and if there is a possibility for adverse interaction with antiplatelet agents.
Conflict of interest statement The authors declare that there is no conflict to disclose.
Acknowledgments Special thanks to the Deanship of Scientific Research, King Faisal University for continuous encouragement and support. Also, special thanks to Dr Mohamed A. Morsy for his valuable advices.
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