Ginkgo Biloba El Boghdady 2012 Antiapoptosis

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Antioxidant and antiapoptotic effects of proanthocyanidin and ginkgobiloba extract against doxorubicin-induced cardiac injury in rats

Noha Ahmed El Boghdady*

Biochemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt

Grape seed proanthocyanidins (GSPE) and ginkgo biloba extract (EGb761) are considered to have protective effects against several diseases.The cardiotoxicity of doxorubicin (DOX) has been reported to be associated with oxidative damage. This study was conducted to evaluatethe cardioprotective effects of GSPE and EGb761 against DOX-induced heart injury in rats. DOX was administered as a single i.p. dose(20 mgkg1) to adult male rats. DOX-intoxicated rats were orally administered GSPE (200 mg kg1 day1) or EGb761 (100 mg kg1 day1)

for 15 consecutive days, starting 10 days prior DOX injection. DOX-induced cardiotoxicity was evidenced by a significant increase in serumaspartate transaminase (AST), creatine phosphokinase isoenzyme (CK-MB), lactate dehydrogenase (LDH), total cholesterol (TC) andtriglyceride (TG) activities and levels. Increased oxidative damage was expressed by the depletion of cardiac reduced glutathione (GSH),elevation of cardiac total antioxidant (TAO) level and accumulation of the lipid peroxidation product, malondialdehyde (MDA). Significantrises in cardiac tumour necrosis factor-alpha (TNF-a) and caspase-3 levels were noticed in DOX-intoxicated rats. These changes wereameliorated in the GSPE and EGb761-treated groups. Histopathological analysis confirmed the cardioprotective effects of GSPE and EGb761.In conclusion, GSPE and EGb761 mediate their protective effect against DOX-induced cardiac injury through antioxidant, anti-inflammatoryand antiapoptotic mechanisms. Copyright 2012 John Wiley & Sons, Ltd.

key wordscaspase-3; doxorubicin; ginkgo biloba extract; grape seed proanthocyanidins; heart injury; oxidative stress; tumour necrosisfactor-a

INTRODUCTION

Doxorubicin (DOX) is a powerful anthracycline antibioticused to treat a multitude of human neoplasms. However,studies have recognized severe cardiotoxicity after an acute,as well as cumulative, dose of DOX, which compromisesthe clinical usefulness of the drug.1 The chronic side effectsof DOX including the development of cardiomyopathy areirreversible and can lead to a potentially congestive heartfailure.2 The cause of DOX cardiotoxicity is multifactorial,but most DOX-induced cardiotoxicity can be attributed tothe formation of reactive oxygen species (ROS), whichultimately results in myocyte apoptosis.3 An alternativeapproach is the use of cardioprotective drugs as adjuvantsto DOX therapy. Agents that prevent cardiotoxicity would

allow us to exploit the full therapeutic potential of DOX,making a tremendous impact on cancer therapy.Polyphenols, present abundantly in vegetables and fruits,

have been recognized as functionally active moleculespossessing a novel spectrum of biological, therapeutic andchemopreventive properties.4,5 Proanthocyanidins are themost abundant phenolic compounds in grape seeds and have

been reported to exert antibacterial, antiviral, anticarcino-

genic, antimutagenic, anti-inflammatory, antiallergic, andvasodilatory actions.5 Grape seed proanthocyanidins(GSPE) were provided to be highly bioavailable and providesignificantly greater protection against damage of oxidativestress than vitamins C, E and b-carotene.6 Proanthocyani-dins have been reported to inhibit lipid peroxidation, plateletaggregation, capillary permeability and fragility, and alsomodulate the activity of some enzyme systems includingphospholipase A2, cyclooxygenase, and lipoxygenase.5 Alarge number of reports demonstrated that proanthocyani-dins could both enhance the activity of chemotherapeuticagents and diminish their normal tissue toxicity.6,7

Extracts from the leaves of ginkgo biloba have beenwidely used therapeutically in China and Western countries

for years. Standard ginkgo biloba extract (EGb761) contains24% flavone glycosides (kaempferol, quercetin andisorhamnetin) and 6% terpenoid (diterpene lactones,namely, ginkgolides and bilobalide and the biflavonesginkgetin, isoginkgetin, bilobetin).8 The extracts have beenfound to possess cardioprotective, antiasthmatic, antidia-betic, hepatoprotective and potent CNS activities.911

Furthermore, this extract has been shown to modulateexpression of apoptotic related genes, reduce generation offree radicals and increase activity of antioxidant enzymesin various types of animal tissues and cells,12,13 implying

*Correspondence to: Noha Ahmed El Boghdady, Biochemistry Depart-ment, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.E-mail: [email protected]

Received 20 June 2012Revised 22 July 2012Accepted 10 September 2012Copyright 2012 John Wiley & Sons, Ltd.

cell biochemistry and function

Cell Biochem Funct (2012)

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/cbf.2907


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that it is a promising cytoprotective agent against a range ofexogenous toxic stimuli.

Therefore, the present study was undertaken to evaluatethe cardioprotective effects of GSPE and EGb761 on cardiacinjury induced by DOX in rats via monitoring the changesof different biochemical parameters such as serum aspartate

transaminase (AST), lactate dehydrogenase (LDH), creatinephosphokinase isoenzyme (CK-MB), total cholesterol (TC)and triglycerides (TG); endogenous cardiac antioxidants asreduced glutathione (GSH) as well as total antioxidant level(TAO) and lipid peroxides expressed as malondialdehyde(MDA). Moreover, the cardiac tissue damage marker,tumour necrosis factor-a (TNF-a), and the apoptotic marker,caspase-3, were measured.

MATERIALS AND METHODS

Chemicals and kits

DOX (AdriblastinaW

) was obtained from Pharmacia (Milan,Italy). GSPE, commercially known as NoxylifeW, was obtainedfrom Pharco Pharmaceuticals Company (Alexandria, Egypt).GSPE is a standardized waterethanol extract from grapeseeds. The extract was supplied in the form of standardized95% oligomeric proanthocyanidins. EGb761, commerciallyknown as TanakanW, was obtained from Amriya for Pharma-ceutical Industries (Alexandria, Egypt). The biochemical kitfor estimation of serum AST was supplied by Quimica ClinicaAplicada (Spain). Serum TC, TG, LDH and CK-MB kits werepurchased from Stanbio (Texas). Cardiac TAO level was mea-sured by using a commercial kit supplied by Bio-diagnostic(Egypt). Cardiac TNF-a ELISA kit and caspase-3 colouri-metric assay kit were purchased from Quantikine, R&DSystems (Minneapolis, MN). All other chemicals used in theexperiment were of the finest analytical grade and obtainedfrom Sigma-Aldrich Co. (USA).

Animals and experimental design

Thirty-two adult male Wistar albino rats weighing 200250gwere used in this study. They were obtained from the animalhouse of the Faculty of Medicine, Cairo University, Egypt.They were kept under controlled conditions, fed standardchow diet, and provided with free access to food and water.The study was approved by the Ethical Committee forAnimal Experimentation of the Faculty of Pharmacy, Cairo

University. After 1 week of acclimatization, animals wererandomly divided into four groups of eight rats each. GroupI (NC): Normal control untreated rats received orally anequivalent volume of normal saline based on body weight.Group II (DOX group): Rats were injected with a single doseof DOX dissolved in normal saline (20mg kg1 i.p.). Thisdose is well documented to induce cardiotoxicity in rats.14

Group III (DOX + GSPE): Rats were administered GSPE(200mgkg1 day1), orally7 dissolved in normal saline for10 days. Group IV (DOX + EGb761): Rats were administeredEGb761 (100 mg kg1 day1), orally15 for 10 days. Followinga single dose of DOX on the 10th day of pre-treatment, either

GSPE or EGb761 was administered for the next 5 days.At the end of the experimental period, rats were overnightfasted. Then the animals were sacrificed by decapitation;blood was collected and centrifuged at 800g at 30 C for15 min. The separated serum was used to estimate TC, TG,AST, LDH and CK-MB. The hearts were dissected out

immediately, washed with ice-cold saline and blotted dryusing filter paper. Tissue samples from the heart were takenfor histopathology. A 20% of heart homogenate wasprepared in ice-cold double distilled water. The obtainedheart homogenate was aliquoted and immediately frozenat80C for biochemical analysis.

Estimation of cardiac function enzymes activities, TG andTC levels

Spectrophotometric diagnostic kits were used for the estima-tion of AST, LDH and CK-MB activities,1618 as well asTG19 and TC20 levels. Serum enzyme activity and lipid profilewere expressed as U l1 and mg dl1 respectively.

Estimation of cardiac MDA

An aliquot of the homogenate was mixed with equal vol-ume of 2.3% KCl and centrifuged at 600g for 15 minat 4 C. The supernatant was used for the determinationof malondialdehyde (MDA), identified as the product oflipid peroxidation that reacts with thiobarbituric acid (TBA)to produce a pink-coloured complex that can be measuredspectrophotometrically at 532 nm and 520 nm using tetra-methoxypropane as a standard.21 The difference betweenthe two determinations was calculated as TBA value andexpressed as nmolmg1 protein.

Estimation of cardiac GSH

A second aliquot of the homogenate was mixed with equalvolume of 7.5% sulfosalicylic acid. The contents were mixedwell for complete precipitation of proteins and centrifuged at600g at 4 C for 10 min. The protein-free supernatant wasused for the estimation of the reduced glutathione (GSH) levelbased on the reaction of GSH with 5,5-dithiobis-2-nitrobenzoicacid, forming a product that has a maximal absorbance at412 nm. The results were expressed as mg gm1 tissue.22

Estimation of cardiac TAO

Another aliquot of the homogenate was mixed with equal

volume of cold phosphate buffer (5 mM potassium phos-phate, pH 7.4, containing 0.9% sodium chloride and 0.1%glucose) and subsequently centrifuged at 800g for 15min at 4 C. The obtained supernatant was used for thedetermination of TAO level based on the ability of the anti-oxidant molecules in the sample to react with a definedamount of exogenously provided hydrogen peroxide (H2O2)causing its decomposition. The residual H2O2 is determinedby an enzymatic reaction that involves the conversion of3,5-dichloro-2-hydroxybensulphonate to a coloured productmeasured colourimetrically at 505 nm. TAO level wasexpressed as mmol mg1 protein.23

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Estimation of cardiac TNF-a and caspase-3 levels

A portion of the heart was weighed (~50 mg) and homo-genized in 0.8 ml lysis buffer, pH 7.4. The lysate wascentrifuged at 10 000g fo r 1 5 m i n at 4 C, and thesupernatant was taken for estimation of the TNF-a and

caspase-3 levels. TNF-a was measured by solid phasesandwich ELISA using two kinds of high specificantibodies. Tetramethyl benzidine was used as chromogen.The strength of colour measured at 450 nm was propor-tional to the quantities of rat TNF-a, which was expressedas pg mg1 protein.24

The caspase-3 colourimetric assay was based on thespectrophotometric detection of the chromophore p-nitroanilide(pNA) after cleavage from the labelled substrate DEVD-pNA(acetyl-Asp-Glu-Val-Asp p-nitroanilide). The pNA can bequantified using a spectrophotometer at 405 nm.25 The caspaselevel was expressed as optical density.

Estimation of protein in cardiac homogenate

The protein content of the different fractions, resulting fromultracentrifugation of heart homogenate was determined bythe method of Lowry et al.26 using bovine serum albuminas the standard.

Heart histological examination

Heart specimens were fixed in 10% formaldehyde andsubsequently embedded in paraffin and sliced into slices of4-mm thickness followed by staining with hematoxylin andeosin (H&E) and examined under light microscope.27

Statistical analysis

Statistical analysis was performed by GraphPad InStatsoftware. Means and standard error of means were calculated,and statistical significance was tested by one-way ANOVA.

Table 1. Effect of GSPE and EGb761 on serum AST, LDH, CK-MB, TC and TG in DOX-induced cardiac injury in rats

NC DOX DOX + GSPE DOX + EGb761

AST (U l1) 6.5 0.27 8.5 0.42 ** 6.75 0.25 ## 6.88 0.35 ##

LDH (U l1) 247 17.5 472 42.7 *** 265 19.99 ### 243 23.09 ###

CK-MB (U l1) 49 4.5 101 8 *** 46 4.2 ### 46 3.8 ###

TC (mg dl1) 56 6.7 153 10.9 *** 64 4.4 ### 69 7.4 ###

TG (mg dl1) 54 5.4 124 10.8 *** 48 3.9 ### 53 4.3 ###

Data represent the meansSEM (n =8).**P0.01, as compared with the normal control group.##

P 0.01, as compared with DOX-intoxicated rats.***P 0.001, as compared with the normal control group.###P0.001, as compared with DOX-intoxicated rats.

(C)

Figure 1. Effect of GSPE and EGb761 on cardiac (A) MDA, (B) GSH and (C) TAO levels of normal control, DOX-intoxicated, DOX + GSPE and DOX +EGb761 rats. Data represent the means SEM (n =8) . *P0.05, **P0.01, and ***P0.001, as compared with normal control group. #P0.05,##P0.01, ###P


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The strength of association between pairs of variables wasassessed by Pearson correlation coefficient. The level ofsignificance was set atP0.05.

RESULTS

Biochemical results

Testing of the cardiac function and the lipid profile followinga single injection of DOX (20 mgkg1 i.p.) in normalrats revealed significant elevations in serum AST, LDH,CK-MB, TC and TG, which were restored by either GSPEor EGb761 treatment (Table 1). A considerable elevation oflipid peroxides (MDA) and TAO levels in cardiac tissuewas demonstrated following DOX injection. The elevatedMDA was ameliorated, but the elevated TAO was apparentlyreduced following treatment with either GSPE or EGb761(Figure 1). Further evidence for DOX-induced toxicity in thecardiac tissue was the profound reduction in the GSH level.

GSPE and EGb761 have succeeded in elevating the reducedcardiac GSH level (Figure 1). Both TNF-a and caspase-3levels showed about four- and 1.5-fold elevation, respectively,following DOX injection. These rises were significantlyameliorated by GSPE and EGb761 (Figure 2).

Histopathological results

As shown in Figure 3, the heart of normal rats revealed nohistopathological alteration. Normal histological structureof the myocardium was recorded (A). By contrast, heartsections from rats receiving DOX showed hyalinization of

the myocardium in a focal manner (B and C), in associationwith inflammatory cells infiltration in pericardium withoedema in both pericardium and in between the myocar-dium (D). Concurrent administration of GSPE (E) andEGb761 (F) improved the alterations in heart morphologyas evidenced by decreased and even abolished myocardialhyalinization in GSPE and EGb761, respectively. However,congestion in myocardial blood vessels and mild vacuolizationin the myocardial cells were still detected in GSPE andEGb761, respectively (Table 2).

DISCUSSION

Effective anticancer therapy with DOX and other quinineanthracyclines is severely limited by acute and chronic sideeffects such as myelosuppression and cardiotoxicity.28 Thepresent study was planned to investigate the cardioprotec-tive effects of GSPE and EGb761 against DOX-inducedcardiomyopathy and apoptotic changes in rats. Interestingly,the cytotoxic activity of DOX is partly related to its quininestructure. DOX is converted into its semiquinone form inthe cardiac myocyte by myocardial cytochrome P450 andflavin monoxygenases. The semiquinone form is a toxic,short-lived metabolite and interacts with molecular oxygenand initiates a cascade of reaction, producing ROS.29

Another reported mechanism of DOX-induced oxidativestress is the formation of an anthracycline-iron (Fe2+) free

radical complex.30 The latter reacts with hydrogen peroxideto produce hydroxyl (OH) radical. ROS react with lipids,protein and other cellular constituents to cause damage tomitochondria and cell membranes of the heart muscle cells.The DOX-induced cardiotoxicity, which was observed5 days after treatment, was documented by the significantincrements in the activities of serum AST, LDH, andCKMB, which may attributed to the myocardial membranedamage produced by DOX and the leakage of theseenzymes from cardiac cytosol into blood. The current resultswere consistent with previous studies.14,31 These changeswere corrected to normalcy upon oral administration ofeither GSPE or EGb761 to DOX-intoxicated rats.

The DOX-induced hyperlipidemia seen in this study hasalso been reported by others.32,33 Hyperlipidemia wasindicated by approximately twofold and threefold increasesin serum TG and TC, respectively, in the DOX group.DOX is reported to cause the development of chronicglomerulonephritis, leading to progressive glomerulosclero-sis associated with the nephrotic syndrome.33 Typically,nephrotic syndrome is characterized by the presence ofpersistent proteinuria, hypoalbuminemia, hyperlipidemia,and lipiduria.32,33 Thus, it is likely that the hyperlipidemiaobserved due to DOX treatment is basically the result ofthe DOX-induced nephrotic syndrome.33,34 It is suggested

###

Figure 2. Effect of GSPE and EGb761 on cardiac (A) TNF-a and (B)caspase-3-activity of normal control, DOX-intoxicated, DOX + GSPE andDOX+EGb761 r ats. Data r epresent the meansSEM (n =8).***P0.001, as compared with the normal control group. ##P0.01,###P0.001, as compared with DOX-intoxicated rats

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that hyperlipidemia is deleterious for heart function andappears to contribute to the DOX-induced heart failure. By

treating rats with GSPE or EGb761, TG and TC levels weredecreased to a level near that of normal control group.Similar studies have found that EGb761 lowers circulatingfree cholesterol in ageing rats35 and inhibits the elevationof serum TG in high cholesterol diettreated rats.36 Thehypolipidemic action of EGb761 seems to be principallyderived from the flavonoids. Epidemiological data showedthat total intake offlavonoids, or a single quercetin compo-nent, was inversely correlated with the plasma TC and low-density lipoprotein cholesterol (LDL-C).37 Moreover, as amajorflavonoid component of EGb761, quercetin has beenidentified as a potent hypolipidemic in experimental

studies.38,39 Quercetin promotes an increase in faecalsterols, which in turn leads to a decreased absorption ofdietary cholesterol, as well as lower plasma and hepaticcholesterol by inactivating b-hydroxy-methyl-glutaryl CoA(HMG CoA) reductase, a rate-limiting enzyme in choles-

terol biosynthesis.39

On the other side, GSPE was found toinhibit the elevation of serum TC and TG in high cholesteroldiet treated rats.40 Previous research showed that themicellar solubility of cholesterol in vitro is reducedsignificantly by proanthocyanidins, suggesting that proantho-cyanidins inhibit the absorption of cholesterol.41

Oxidative stress injury was monitored by MDA, TAOand GSH levels. In the present study, DOX caused aconsiderable increase in myocardial levels of MDA andTAO capacity along with a marked depression of GSH.These results were in agreement with several studies.14,42

A close association of GSH depletion and lipid peroxidesformation has been reported previously.43 The significant

increase in cardiac lipid peroxides of DOX-treated ratsexplains the observed leakage of cellular AST, LDH andCK-MB to circulation due to cardiac injury. On the otherhand, the superoxide anions generated by the semiquinonestructure of DOX, stimulated the activity of antioxidantenzymes, as evidenced here by the obvious stimulation ofTAO in cardiac tissue, leading to activation of glutathioneperoxidase (GSHPx) in response to the accumulatedperoxides, which can subsequently lead to the formation ofhydroxyl radicals in the presence of metal ion catalysts.44

The latter can react with polyunsaturated phospholipids thatultimately lead to the formation of a great variety of

(A) (B) (C)

(D) (E) (F)

Figure 3. Photomicrographs of heart sections from (A) normal control rats (H&E, 80): normal histological structure of the myocardium (my). (B, C and D)DOX-treated rats (H&E, 80, 160, 160 respectively): hyalinization of the myocardium in focal manner (h), inflammatory cells infiltration in pericardium (m)with oedema in both pericardium (o) and in between the myocardium (o). (E) DOX + GSPE (H&E, 160): congestion of myocardial blood vessels (v) with

focal hyalinization in the myocardium in few manner (h). (F) DOX + EGb761 (H&E, 160): mild vacuolization in some myocardial cells (!)

Table 2. The severity of the reaction in heart according to the

histopathological alterations in different groups

Gp 1 Gp 2 Gp 3 Gp 4

Contr ol DOX

DOX +

GSPE

DOX+

EGb761

Myocardial hyalinization +++ + Inflammatory cellsinfiltration in pericardium

++

Oedema in myocardium +++ Oedema in pericardium +++ Congestion in blood vessels + Myocardial vacuolization +

++++, very severe; +++, severe; ++, moderate; +, mild; , nil.

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diffusible aldehydes represented by MDA.45 The stimula-tion of GSHPx and the overproduction of free radicals canbe detoxified by the endogenous antioxidants, causing theircellular stores to be depleted.46 This is in agreement withour results as revealed by GSH depression.

The oral administration of GSPE and EGb761 prior to the

DOX injection significantly reduced the extent of lipidperoxidation and TAO and increased GSH levels, suggest-ing that these agents scavenged free radicals and decreasedcellular injury. These outcomes were consistent withhistopathologic scores. Because GSPE and EGb761 areflavonoids, it is known thatflavonoids have a potential func-tion in the transfer of electrons between their isoalloxazinegroup and cell reactants in oxidationreduction reactions torender them less reactive.4,47 They can also chelate metalslike iron involved in free radical formation47,48 and inhibitmany enzymes, including cyclooxygenase, lipoxygenase,49

and phospholipase A2.4,50 In addition, the molecularstructure of flavonoids allows them to accumulate at lipid

interfaces

50

and/or to penetrate membranes,

48

so there aredecreased lipid peroxidation and subsequent protection ofcells. Moreover, in vitro investigation of antioxidantpotential of GSPE shows that it is a strong antioxidant, upto five times more effective than vitamin C or E.51

In order to evaluate the cardiac inflammation andapoptosis, TNF-a and caspase-3 were measured. DOX, inthe present study, caused marked increase in myocardialTNF-a and caspase-3 levels, which were consistent withprevious reports.52,53 It is possible that increased oxidativestress following DOX administration caused expression ofTNF-a. Oxidative stress is known to activate P38-mitogenactivated protein kinase and nuclear factor kappa B and thusplays a role in the sequence of signalling events involved in

myocardial TNF-a production.54,55 On the other hand,increased oxidative stress caused disturbances in mitochon-drial membrane permeability, causing leakage of freeradicals and cytochrome c from the mitochondria to thecytosol. Once cytochrome c is released into the cytosol, itbinds to another protein, Apaf-1, and promotes activationof the caspase cascade, leading to cell death.56 From theresults of the present study, it was seen that alteration inlipid profile, increased free-radical generation and lipidperoxidation caused damage to the myocardium. Thesechanges affect the cellular membrane, thereby releasingmitochondrial cytochrome c into the cytosol and activatingcaspase-3, which may further lead to apoptosis. GSPE as

well as EGb761 treatment signifi

cantly reduced the elevatedlevels of myocardial TNF-a and caspase-3 observed in theDOX group an effect that might be attributed to reductionin oxidative stress.

Histopathological effects of DOX show severe hyaliniza-tion of the myocardium, associated with moderate focalinflammatory cells infiltration in the pericardium, as wellas severe oedema in both pericardium and myocardium.Thus, histopathological analysis has confirmed that adminis-tration of a single dose of DOX (20 mg kg1 i.p.) producedsignificant cardiomyopathy in rats. This is in line withprevious literature.57,58 Evidence suggests that EGb761

(100mgkg1 day1) pre-treatment significantly protects themyocardium from DOX-induced toxicity while GSPE(200mgkg1 day1) partially protects the myocardium, asevidenced by fewer hyalinized myocardium as compared tothe DOX group.

There are some limitations in the present study. First,

GSPE and EGb761 are specific and complex productsprepared from grape seed and ginkgo leaves respectively.Grape seeds extract contained other flavonoids as gallicacid, catechin, epicatechin, gallocatechin, and epigallocate-chin besides the proanthocyanidins. EGb761 contained24% ginkgo-flavonole glycosides and 6% terpenoids.Which component produces the protective effect or doesmore work in protecting against DOX-induced cardiacinjury in the present study remains to be elucidated. Second,data relating to arterial blood pressure and heart rates werenot recorded. Finally, more prospective studies are neededto evaluate the efficacy and optimum dosage of GSPE andEGb761 to be used as a protective agent against the side

effects of chemotherapeutics.In conclusion, the present study indicates that GSPEand EGb761 have a protective effect on cardiac injuryinduced by DOX, which may be related to antioxidative,anti-inflammatory and antiapoptotic properties. They appearto be an effective protective agent against cardiomyopathy,although their mechanisms remain to be elucidated.

CONFLICT OF INTEREST

The author has declared that there is no conflict of interest.

ACKNOWLEDGEMENT

The author is thankful to Dr. Adel Bakeer, HistologyDepartment, Faculty of Veterinary Medicine, Cairo University,for performing the histopathological examination.

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