Ameliorative Effect of Cardamom Aqueous Extract on Doxorubicin-Induced Cardiotoxicity in Rats

Doxorubicin (DOX) is a cytotoxic anthracycline antibiotic, and it is commonly used in the treatment of a wide range of cancers, including many types of solid tumors, soft tissue sarcomas, and hematological malignancies [Vatsyayan et al., 2009]. The principal anticancer mechanism of DOX is through DNA chelation, which further inhibits the progression of topoisomerase II and then produces free radicals to kill cancer cells. However, this effect is not selective for cancer cells alone as healthy (normal) cells can also be affected by the same mechanism. Indeed, some dangerous side effects were reported for DOX, such as cardiotoxicity, electrocardiographic changes, and ultimately fatal congestive heart failure [Christiansen and Autschbach, 2006], which restrict the clinical applicability of DOX. The pathogenesis of DOX-related cardiotoxicity and heart failure remains unclear. However, some mechanisms, such as oxidative stress, excessive calcium, and cytokine release were recorded. Among these possible mechanisms, the oxidative stress hypothesis is still the cornerstone [Bruynzeel et al., 2007; Saeed et al., 2015; El-Agamy et al., 2016; Abdel-Daim et al., 2017; Shahidullah et al., 2017]. Induction of free radicals and other toxic nonradicals released from cells by anthracyclines can be neutralized by generating endogenous antioxidants or by introducing exogenous antioxidants in nutritional supplements [Abushouk et al., 2017]. These interventions may prevent or ameliorate the toxic effects of anthracyclines. The natural antioxidants are numerous in herbs and spices. However, it is not easy to find promising natural antioxidants that can decrease the incidence of DOX-induced cardiotoxicity, especially after failure of some antioxidants to relieve DOX-induced cardiotoxicity [Bruynzeel et al., 2007]. To be an effective anticancer drug, it is necessary to find other new therapeutic agents to act as adjuvants to decrease the cardiotoxicity of DOX.

As a member of Zingiberaceae family, cardamom (CAR) is a sweet, aromatic, commonly used spice that is usually called “the queen of spices” as it occupies the third place in world trade and is high priced [Goyal et al., 2015]. Its dried fruit is used as a flavoring agent and in medical preparations. In addition, CAR has been used in traditional medicine to treat throat infections, high blood pressure, kidney disorders, and some cardiovascular diseases [Das et al., 2012]. CAR extracts also possess antifungal, antimicrobial, antidiabetic, anti-hyperlipidemic, anticancer, and antioxidant effects [reviewed in Rahman et al., 2017]. Moreover, CAR enhanced the activity of antioxidant enzymes in the heart of hypercholesterolemic rats [Nagashree et al., 2017].

The protective role of CAR on DOX-induced cardiac toxicity has not been fully assessed. Only 1 very recent study by Shahidullah et al. [2017] reported the ability of a high-dose CAR extract (500 mg/kg body weight [BW]) to reduce lipid profile parameters and total chloride levels in DOX-induced cardiac injury in rats. However, this previous study lacked important details concerning the associated mechanism by which CAR can relieve DOX side effects on the heart. Therefore, the aim of the present work was to study the possible cardioprotective effects of CAR on DOX-induced myocardial toxicity through investigation of histological alterations and the associated oxidative stress, apoptosis, and inflammation in rat myocardium.

Cardamom fruits were purchased from the local market of agricultural herbs, spices, and medicinal plants in Mansoura, Egypt. Following grinding of plant seeds, the resultant powder (10 g) was mixed thoroughly with distilled water (100 mL). The obtained solution was filtered twice through a Whatman filter No.1 and finally centrifuged at 5,000 rpm for 15 min. The concentration of the filtrate (aqueous extract) was adjusted by saline to reach a final concentration of 100 mg/mL [Kaushik et al., 2010].

The present study was carried out on 30 adult male albino rats, aged 3–4 months and with an average weight of 200–250 g. Animals were studied according to recommendations done by the Animal Welfare Act and Guide for the Care Use of Experimental Animals (Mansoura University, Egypt). The experimental protocol was approved by an independent ethical committee before the study. Animals were housed in suitable clean ventilated cages. Conventional food and drinking water were provided ad libitum.

After 1 week of acclimatization, rats were randomized to 3 groups (n = 10/group). Table 1 shows the experimental design, animal groups, and treatment regimen (dose, route, and duration) of CAR and DOX. In group I (control), rats received 1 mL saline orally by intraperitoneal injection for 3 weeks. In group II (DOX), rats were injected with DOX (2.5 mg/kg BW i.p.; EIMC, United Pharmaceuticals, Egypt) every other day for 2 weeks from day 8 to day 21 of the experiment [Shivakumar et al., 2012]. In group III (CAR+DOX), rats received CAR extract (200 mg/kg BW) orally for 3 weeks [Goyal et al., 2015], and 1 week later they were intraperitoneally injected with DOX with the same dose and for the duration as group II. DOX and CAR were dissolved in appropriate volumes of saline depending on their concentration, and each rat was given 1 mL per day. DOX-treated groups did not receive any other cotherapies (adjuvants) to counteract DOX-induced cardiotoxicity.

At the end of the experiment (on day 21), blood samples were collected, and sera were separated as described previously [Alzahrani et al., 2018]. The rats were then euthanized by cervical dislocation under light ether anesthesia, and the heart of each animal was immediately dissected and sliced into 2 parts. One part was kept in 10% neutral buffered formalin for histological examination, and the second was homogenized using cold PBS, followed by centri-fugation at 5,000 g for 15 min at 4°C; the obtained supernatant was used for biochemical evaluation.

Serum levels of cardiac damage markers (lactate dehydrogenase [LDH], creatine kinase [CK], and cardiac troponin T [cTnT]) were determined using commercially available diagnostic kits (Stanbio Laboratory Boerne, USA, and Roche Diagnostics, Mannheim, Germany) as described previously [El-Magd et al., 2017]. The concentrations of the oxidative stress marker nitric oxide (NO) and the lipid peroxidation marker malondialdehyde (MDA) as well as the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined in heart homogenates using locally available, commercial kits (Biodiagnostic, Egypt), as previously described [Abdelhadya et al., 2017; El-Magd et al., 2017]. Caspase 3 activity was determined on heart homogenate using caspase 3 fluorogenic substrate (Ac-DEVD-AMC; Alexis Biochemicals) as described previously [Badawy et al., 2018]. Nuclear factor κB (NFκB) levels were measured in heart homogenate using the rat NFκB ELISA kit (ab210613; Abcam) according to the manufacturer’s instructions.

Specimens from the left-ventricular muscle were extracted, fixed in 10% neutral buffered formalin (HT501128; Sigma Aldrich) for paraffin block preparation and were sectioned at 5-µm thickness for histological staining (H&E and Mallory trichrome [MT]) [Bancroft and Gamble, 2008]. MT staining was used to determine collagen fiber deposition (trichrome stain kit, ab150686; Abcam).

For immunohistochemistry, paraffin sections were stained with the avidin-biotin peroxidase complex (Vector Laboratories) technique to assess immunoreactivity to caspase 3 (apoptotic marker) and vascular endothelial growth factor (VEGF) (ThermoFisher Scientific; Fremont, CA, USA). Slides were then counterstained with hematoxylin. Caspase 3- and VEGF-expressing cells demonstrated brown cytoplasmic labeling. In order to detect unintended background staining, the regular immunohistochemical staining protocol was also applied on rat myocardial specimens but omitting the primary antibody step (negative control).

MT-stained sections and immunohistochemical reactions were analyzed morphometrically using an image analysis system (Leica Q500 DMLB; Leica, Cambridge, UK) at the Image Analysis Unit in the National Research Center, Egypt. Five different nonoverlapping fields from 5 different sections of different rats were examined in each group at a magnification of ×400 for measuring the mean area of collagen fibers (%) and positive immune reaction for caspase 3 and VEGF.

All data were collected, revised, and statistically analyzed using one-way analysis of variance (ANOVA) with GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA). Mean values were compared with the Tukey HSD (honestly significant difference) test. Data were expressed as means ± SEM. Means with different letters (a [higher mean value] – c [lower mean value]) are significantly different at p < 0.05.

DOX-injected rats (group II) showed significantly elevated serum levels of the cardiac damage markers LDH, CK, and cTnT compared to the control group (Table 2). In contrast, CAR-treated rats (group III) exhibited a significant reduction in the levels of these markers compared to group II, but levels were significantly higher than in group I. This indicates that CAR can reduce the increase in cardiotoxic markers induced by DOX treatment.

Examination of H&E-stained longitudinal sections of the control group showed a normal histological structure of the left-ventricular myocardium where cardiac muscle fibers appeared branching and anastomosing with acidophilic cytoplasm, central oval vesicular nuclei (arrowhead; Fig. 1a), cross striations (thin arrow), intercalated disks (thick arrows), and congested blood vessels (curved arrow; Fig. 1a). MT-stained sections revealed the presence of few collagen fibers between the muscle fibers (arrow; Fig. 1b). In contrast, DOX-treated rats (group II) exhibited marked histological alterations where most cardiac myocytes exhibited disorganization and discon tinuity with decreased sarcoplasmic eosinophilia (thin arrows; Fig. 1c), marginated and pyknotic nuclei (thick arrows), as well as numerous interstitial mononuclear cellular infiltration (arrowhead), and congested blood vessels (V) between the widely separated bundles (Fig. 1c). Notable increases in the amount of collagen fibers deposited was noticed between cardiac muscle fibers (arrows; Fig. 1d). On the other hand, CAR-treated rats (group III) depicted a nearly normal myocardial structure with very limited mononuclear cell infiltration (arrow; Fig. 1e) and an apparent decrease in the amount of collagen fibers between cardiac myocytes compared to group II (Fig. 1f). Histomorphometric results confirmed the previous histological findings and showed a significant increase in the mean area of collagen fibers deposited between cardiac muscle fibers in the DOX-treated group compared to the control group (Fig. 1g). This elevated score was significantly decreased in CAR-treated rats to a level comparable to, but still significantly higher than that of the control group (Fig. 1g). Collectively, these findings indicate that CAR pretreatment can restore the disrupted histological structure of myocardium induced by DOX.

The cardiac levels of the oxidative stress marker NO, the lipid peroxidation marker MDA, and the inflammation-related marker NFκB were significantly increased in group II compared to group I (Table 2). However, group II had significantly lower levels of the antioxidant enzymes SOD, CAT, and GPx than group I. On the other hand, group III showed significantly lower levels of NO, MDA, and NFκB and significantly higher levels of SOD, CAT, and GPx than group II (Table 2). In general, the levels of all these biochemical parameters were significantly different in group III compared to group I, with higher NO, MDA, and NFκB levels and lower SOD, CAT, and GPx levels in group III than group I. These results indicate that CAR can attenuate DOX-induced oxidative stress and inflammation.

The cardiac level of the apoptotic marker caspase 3 was significantly increased in DOX-intoxicated rats (group II) compared to the control group (Table 2). In contrast, administration of CAR (group III) showed a significant decrease in caspase 3 levels in the heart compared to group II. For further validation, caspase 3 immuno-reactivity was determined in the myocardium, and the obtained results showed negative immunoreactivity (Fig. 2a), strong positive immunoreactivity (Fig. 2b), and moderate positive immunoreaction (Fig. 2c) in the sarcoplasm of cardiac muscle fibers of group I, group II, and group III, respectively. The histomorphometric results confirmed the aforementioned histological findings and exhibited a significantly increased mean area percentage of caspase 3 immunostaining in group II compared to group I (Fig. 2d). This elevated score was significantly decreased in rats treated with CAR before and during DOX treatment to a level comparable to but significantly lower than that of the control group (Fig. 2d). Thus, CAR can decrease DOX-triggered apoptosis.

In DOX-treated rats, myocardial sections immuno-stained with the angiogenesis-related marker VEGF showed an apparent increase in the positive immunoreaction in the cytoplasm of endothelial cells of some blood vessels compared to the control group (Fig. 3a, b). Interestingly, pretreatment with CAR (group III) led to a significant increase in the VEGF-positive immunoreaction compared to group II (Fig. 3c). In agreement, quantification results also revealed a significant increase in the mean area percentage of VEGF immunostaining in the treated groups (groups II and III), which was highest in group III (Fig. 3d). In general, these findings indicate that CAR can increase VEGF immunostaining, thereby, suggesting a role for CAR in angiogenesis.

Clinical use of DOX is limited by the development of life-threatening cardiomyopathy [Christiansen and Autschbach, 2006]. CAR is a well-known spice that is widely used in traditional medicine due to its antioxidant and anti-inflammatory properties [Zhihua et al., 2014]. However, little is known about whether CAR can be used as a cardioprotective adjuvant to decrease DOX side effects, and its underlying mechanism of action has not been elucidated yet. Collectively, our results showed that treatment with CAR is able to relieve myocardial damage (including structural and functional disruptions) induced by DOX, at least in part, through inhibition of reactive oxygen species (ROS) release, reduction in the activity and expression of caspase 3 (apoptotic marker) and NFκB (a marker of inflammation) in addition to initiation of angiogenesis via induction of VEGF.

In the present study, DOX induced histological alterations in rat myocardium (including disorganized muscle fibers, decreased cytoplasmic acidophilia, pyknotic dark peripheral nuclei, interstitial mononuclear cellular infiltration, and congested blood vessels) similar to those previously reported [Kim et al., 2006; El-Agamy et al., 2016]. This indicates induction of cardiotoxicity by DOX. The wide separation of myocardial fibers present in the current study was attributed to disruption of the connection between the sarcolemma and the muscle fibrils and the forceful systolic tugs by intact fibers adjacent to the nonviable fibers, leading to their stretching and separation [Panjrath et al., 2007].

The cardiotoxic effect of DOX was also confirmed by significantly elevated serum levels of cardiac damage markers (LDH, CK, and cTnT) and increased concentrations of oxidative stress (NO) and lipid peroxidation markers (MDA), as well as decreased SOD, CAT, and GPx activity in the rat heart. Thus, DOX induces cardiac oxidative stress mediated by ROS production, thereby leading to cardiotoxicity. The widely accepted mechanism to explain the role of DOX in ROS generation is an enzymatic-mediated pathway through conversion of the DOX quinine moiety to the corresponding DOX semiquinone form by NADH dehydrogenase and cytochrome P₄₅₀ reductase [Kalishina et al., 2003]. The molecular oxygen oxidizes the DOX semiquinone form to its quinine form with superoxide radical production [Kaiserova et al., 2007]. This redox reaction involves the reaction of Fe³⁺ with DOX, where the iron atom gains an electron and the Fe²⁺-DOX free radical complex that reduces O₂ to H₂O₂ and other active oxygen species [Kalishina et al., 2003].

In agreement with our results, Abdel-Daim et al. [2017], Ayaz et al. [2004], and El-Agamy et al. [2016] also showed a significant increase in the cardiac MDA level following DOX treatment. As the phospholipid is the main component of the cell membrane, MDA elevation results in increased lipid peroxidation which leads to cardiomyocyte cell membrane destruction and LDH, CK, and cTnT release into the circulation. Moreover, DOX has a very high affinity to cardiolipin phospholipids that is normally present in cardiac mitochondrial membranes thereby leading to DOX accumulation in cardiac cells [Kaiserova et al., 2007]. DOX can also induce Ca²⁺ ion overload in the myocardium which can activate phospholipase, leading to decomposition of membrane phospholipids [Kim et al., 2006]. The body reacts to excessive ROS through activation of the endogenous antioxidant enzymes. However, this defense mechanism was also targeted by DOX. The fall in the antioxidant enzymes indicates the inability of cells to scavenge free radicals which consequently leads to oxidative damage to cardiomyocytes [Tokarska-Schlattner et al., 2006]. Goyal et al. [2015] and Tokarska-Schlattner et al. [2006] also found a similar effect for DOX on the antioxidant/ROS status of the heart. Consistent with our data, Abdel-Daim et al. [2017] and Pinto et al. [2007] also reported a significant elevation in inducible NO synthase expression and NO production after DOX induction. NO is an essential mediator of vascular homeostasis, and its increase correlates with dilated cardiomyopathy and congestive heart failure [Pinto et al., 2007]. This can explain the congestion and dilatation of blood vessels in the myocardium of DOX-treated rats in our study.

Herein, we found that CAR administration did not only reduce the oxidative stress triggered by DOX but also elevated the activities of endogenous antioxidant enzymes (SOD, CAT, and GPx). In agreement, Qiblawi et al. [2015] also reported an antioxidant effect for CAR through free radical scavenging, thereby protecting body organs from oxidative damage. This antioxidant activity of CAR was attributed mainly to its contents of 1,8-cineole, protocatechualdehyde, α-terpineol, and protocatechuic acid [Elguindy et al., 2016]. This antioxidant effect is not restricted to the heart or DOX toxicity as CAR can also suppress lipid peroxidation and increase antioxidant enzyme activities in gentamicin-induced nephrotoxicity in rats [Elkomy et al., 2015] and in the liver of obese rats [Elguindy et al., 2016]. CAR treatment before and during DOX led to a significant reduction in serum levels of cardiac damage markers (LDH, CK, and cTnT) compared to treatment with DOX alone. These results are in accordance with those reported by Qiblawi et al. [2015] and indicate the ability of CAR to preserve the integrity of the cardiomyocyte membrane (due to prevention of lipid peroxidation) that prevents the leakage of these cardiac markers. Moreover, histopathological examination of myocardial tissue in CAR+DOX animals confirmed the biochemical results and showed intact cell membranes.

In the present study, the interstitial mononuclear cell infiltration could be attributed to the free radicals that have the great potential to trigger inflammatory cascades through cytokines and regulate leukocyte trafficking to the cardiac lesion site [Anjos Ferreira et al., 2007]. Indeed, we and El-Agamy et al. [2016], Saeed et al. [2015], and Wang et al. [2002] also found a significant increase in the inflammatory marker NFκB in the heart following DOX treatment. ROS can also activate NFκB that further stimulates the production of inflammatory cytokines such as TNFα, COX2, and NO [Abd El-Aziz et al., 2012]. This can also explain the increased cardiac level of NO in DOX-treated animals in our study. Interestingly, CAR did not only significantly decrease NFκB levels, it also reduced the NO production in the heart of DOX-intoxicated rats. These data confirm the anti-inflammatory effect of CAR through inhibition of NFκB that was previously reported in rats with chemically induced hepatocellular carcinoma [Elguindy et al., 2016]. Overall, it is likely that the protective effects of CAR against DOX cardiotoxicity are mediated, at least in part, by ROS inhibition, which further prevents the activation of NFκB and the production of its downstream proinflammatory cytokines, thereby reducing tissue damage.

In the present study, the increased deposition of collagen fibers between cardiac myocytes in DOX-treated rats may be due to interstitial mononuclear cell infiltration and their inflammatory markers such as IL1β, TNFα, and NFκB, which generally contribute to extracellular matrix deposition in tissue and fibrosis initiation [Passino et al., 2015]. Monocytes, which represent a great subset of heart-infiltrating cells at the lesion site, can support angiogenesis and collagen deposition [Kania et al., 2009]. In contrast, CAR-treated rats demonstrated an apparent decrease in the amount of collagen fiber deposition compared to rats treated with DOX only.

In the present study, induction of apoptosis by DOX was evidenced by histological examination and biochemical assay. Histologically, DOX caused cellular changes similar to those occurring during apoptosis, such as pyknosis, condensed chromatin, and marginated nuclei. Condensed nuclei are a distinctive feature of apoptosis and attributed to a caspase-targeted protein in the nuclear lamina (i.e., between peripheral chromatin and the inner nuclear membrane) which when cleaved causes chromatin condensation and nuclear shrinkage [Jang et al., 2004]. Induction of apoptosis by DOX was also confirmed biochemically and immunohistochemically by increased caspase 3 activity and immunoreaction, respectively, in DOX-treated rats. Consistent with our data, several previous studies also reported a significant increase in caspase 3 activity and protein expression, and downregulation of antiapoptotic proteins following DOX exposure [Soni et al., 2011; El-Agamy et al., 2016; Widyaningsih et al., 2017]. Moreover, ROS initiated by DOX can cause Bax translocation to the outer mitochondrial membrane and subsequently leads to cytochrome C release from the mitochondria into the cardiomyocyte cytoplasm, which further activates caspase 3-dependent apoptosis [Darwish and Abd El Azime, 2013]. NFκB can also stimulate DOX-induced apoptosis in cardiac cells [Wang et al., 2002]. Caspase 3 could cleave cardiac myofibrillar proteins, e.g., ventricular α actin, myosin light chain, troponin T, and α actinin, thereby reducing myocyte contractile function [Ueno et al., 2006]. In contrast to DOX-treated rats, a significant decrease in caspase 3 immunostaining was demonstrated in rats treated with CAR before and during DOX treatment. As oxidative stress causes damage to the DNA and the antioxidants inhibit DNA fragmentation and apoptosis [Communal et al., 2002], it is possible that CAR has an antiapoptotic and antioxidant effect probably through modulating the caspase pathway in response to oxidative stress.

In the present study, immunoreactivity of VEGF, a potent growth factor that acts as a marker for angiogenesis and is consequently involved in tissue repair, was significantly increased in cytoplasmic endothelial cells of some blood vessels in DOX-treated rats compared to the control group. Interestingly, rats treated with CAR revealed a significantly higher VEGF immunoreactivity than the DOX-treated group. The mononuclear cells, especially activated T cells, enhance VEGF production in injured cardiac cells to promote healing [Beckman et al., 2013]. Thus, CAR could accelerate angiogenesis through induction of VEGF, which eventually could induce faster healing of the injured cardiac muscle. In agreement with our results, obese rats treated with CAR showed increased VEGF immunoreactivity and subsequently enhanced liver damage [Mor et al., 2004]. Induction of VEGF by CAR could be due to the antioxidant effect of CAR that might bind with superoxide radicals which could increase VEGF expression and subsequently increase vascular density, regional blood flow in cardiac myocytes [Rahman et al., 2017], adhesion between cardiomyocytes and the extracellular membrane, and endothelial cell adhesion [Elguindy et al., 2018].

The cardioprotective effect of CAR was attributed to its components, including flavonoids, saponins, terpenes, glycosides, and steroids, which can reduce low-density lipoprotein cholesterol and total chloride levels [Shahidullah et al., 2017], inhibit ROS, apoptosis, and inflammation (our study), and exert hypotensive, fibrinolytic, vasorelaxant, and antiplatelet properties [Suneetha and Krishnakantha, 2005]. Thus, it is reasonable to presume that regular consumption of CAR may play a protective role in the treatment of patients with ischemic heart disease. Surprisingly, an anticancer effect was also reported for CAR [Bhattacharjee et al., 2007]. Thus, we administered CAR as a safe, cheap, easily available adjuvant to DOX treatment not only to decrease the cardiotoxicity of DOX but also to be probably able to enhance its anticancer effect. However, further studies are first required to check the combined effect of DOX and CAR on cancer progression. Studying the role of individual CAR components is crucial for pharmaceutical preparations employing the CAR mechanisms of action, which could not be addressed in this study. Instead, we focused on the overall effect of all CAR components rather than using one alone to mimic the effect after CAR consumption.

The current investigation showed that administration of CAR extract before and during DOX treatment ameliorated, to a great extent, the toxic effect of DOX on rat myocardium. The CAR extract reduces structural disruptions (decreasing discontinuity and disorganization of cardiac muscle fibers, mononuclear cellular infiltration, and collagen fiber deposition) and functional disturbances (via lowering serum LDH, CK, and cTnT levels) induced by DOX. This ameliorative effect occurred probably through inhibition of ROS (via reducing NO and MDA and elevating SOD, CAT, and GPx), apoptosis (by decreasing caspase 3 activity and immunostaining), and inflammation (decreasing the NFκB level), stimulation of angiogenesis (increasing VEGF immunostaining), and probably tissue regeneration. Therefore, CAR could be a promising cytoprotective agent against DOX cardiotoxicity. Although our study may open a new avenue of research for using cardamom in relieving DOX-induced myocardial injuries, further investigations are necessarily needed to find out its benefit in a clinical setup.

The experimental protocol was approved by an independent ethical committee before the study.

The authors have no conflicts of interest to disclose.