Cardioprotective effect of embelin on isoproterenol-induced myocardial injury in rats: Possible involvement of mitochondrial dysfunction and apoptosis Bidya Dhar Sahu, Harika Anubolu, Meghana Koneru, Jerald Mahesh Kumar, Madhusudana Kuncha, Sunder R. Shyam, Ramakrishna Sistla PII: DOI: Reference:

S0024-3205(14)00455-X doi: 10.1016/j.lfs.2014.04.035 LFS 14022

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

20 February 2014 23 April 2014 25 April 2014

Please cite this article as: Sahu Bidya Dhar, Anubolu Harika, Koneru Meghana, Kumar Jerald Mahesh, Kuncha Madhusudana, Shyam Sunder R., Sistla Ramakrishna, Cardioprotective effect of embelin on isoproterenol-induced myocardial injury in rats: Possible involvement of mitochondrial dysfunction and apoptosis, Life Sciences (2014), doi: 10.1016/j.lfs.2014.04.035

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ACCEPTED MANUSCRIPT Cardioprotective effect of embelin on isoproterenol-induced myocardial injury in rats: possible involvement of mitochondrial dysfunction and apoptosis

Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical

Technology (IICT), Hyderabad-500 007, India.

CSIR-Centre for Cellular and Molecular Biology (CCMB), Hyderabad-500 007, India.

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b

Faculty of Pharmacy, Osmania University, Hyderabad-500 007, India.

*

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c

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a

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Madhusudana Kunchaa, Shyam Sunder Rc, Ramakrishna Sistlaa*

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Bidya Dhar Sahua, Harika Anubolua, Meghana Konerua, Jerald Mahesh Kumarb,

Corresponding Address:

Dr Ramakrishna Sistla, PhD

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Principal Scientist

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Medicinal Chemistry & Pharmacology Division Indian Institute of Chemical Technology (IICT)

Phone: +91-40-27193753 Fax: +91-40-27193189 Email: [email protected]

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Hyderabad- 500 007, India

Abbreviations used

AST, Aspartate transaminase; CAT, catalase; CDNB, 1-Chloro-2, 4-dinitrobenzene; CK-MB, creatine kinase-MB isoenzyme; DCIP, 2, 6-dichlorophenolindophenol; DTNB, 5, 5-dithio-bis (2-nitrobenzoic acid); GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSH, oxidized glutathione; GST, glutathione S-transferase; HDL, high density lipoprotein; ISO, isoproterenol; LDH, lactate dehydrogenase; LDL, low density lipoprotein; MTT,

3-(4,

5-dimethylthiazol-2-yl)-2,

5-diphenyltetrazolium

bromide;

NADH,

β-

nicotinamide adenine dinucleotide hydrate; NADPH, β-nicotinamide adenine dinucleotide 3phosphate reduced form; NBT, nitro blue tetrazolium salt; NQO1, NAD (P) H: quinine oxidoreductase 1; PARP, poly (ADP-ribose) polymerase; SOD, superoxide dismutase; TBA, 2-thiobarbituric acid; TBARS, thiobarbituric acid reactive substance; VLDL, very low density lipoprotein. 1

ACCEPTED MANUSCRIPT Abstract Aims: Preventive and/or therapeutic interventions using natural products for ischemic heart

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disease have gained considerable attention worldwide. This study investigated the

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cardioprotective effect and possible mechanism of embelin, a major constituent of Embelia

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ribes Burm, using isoproterenol (ISO)-induced myocardial infarction model in rats. Materials and Methods: Rats were pretreated for three days with embelin (50 mg/kg, p.o) before inducing myocardial injury by administration of ISO (85 mg/kg) subcutaneously at an

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interval of 24h for 2 consecutive days. Serum was analyzed for cardiac specific injury

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biomarkers, lipids and lipoproteins content. Heart tissues were isolated and were used for histopathology, antioxidant and mitochondrial respiratory enzyme activity assays and western

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blot analysis.

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Key findings: Results showed that pretreatment with embelin significantly decreased the elevated levels of serum specific cardiac injury biomarkers (CK-MB, LDH and AST), serum

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levels of lipids and lipoproteins and histopathological changes when compared to ISOinduced controls. Exploration of the underlying mechanisms of embelin action revealed that

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embelin pretreatment restored the myocardial mitochondrial respiratory enzyme activities (NADH dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and mitochondrial redox activity), strengthened antioxidant status and attenuated ISO-induced myocardial lipid peroxidation. Immunoblot analysis revealed that embelin interrupted mitochondria dependent apoptotic damage by increasing the myocardial expression of Bcl-2 and down regulating the expression of Bax, cytochrome c, cleaved-caspase-3 & 9 and PARP. Histopathology findings further strengthened the cardioprotective findings of embelin. Significance: Result suggested that embelin may have a potential benefit in preventing ischemic heart disease like myocardial infarction. Keywords: Embelin, Isoproterenol, Oxidative stress, Apoptosis, Mitochondrial dysfunction. 2

ACCEPTED MANUSCRIPT Introduction Myocardial infarction refers to a condition in which a portion of the myocardium undergoes

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damage due to lack of oxygen for certain period of time followed by reperfusion leading to

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irreversible damage (Thygesen et al. 2012). Ischemia of the myocardium causes development

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of arrhythmias and may also lead to cardiac necrosis. The etiology of many cardiovascular disorders depends on a number of interlinked factors such as changing lifestyle, stress, aging,

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food habits and other socioeconomic determinants of developing nations (Vendrame et al. 2013). Excess generation of free radicals and associated oxidative and apoptotic damage due

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to ischemia play a major role during ischemic heart diseases by causing qualitative and quantitative alterations in the myocardium (Wang et al. 2009; Prince et al. 2011; Li et al.

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2012). Over production of catecholamines due to adrenergic overstimulation is believed to be

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a major cause of stress-induced cardiac dysfunction (Shao et al. 2013). It has been reported that supraphysiological plasma concentrations of catecholamines produce myocardial

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dysfunction through cardiomyocyte apoptosis or necrosis and also have a great influence on myocardial energy metabolism through perturbations of lipid metabolism in heart (Radhiga et

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al. 2012; Shao et al. 2013). Isoproterenol (ISO), a β-adrenergic agonist and synthetic catecholamine is employed at sub-maximal dose as a non-invasive method to induce myocardial lesions in rodents (Li et al. 2012). Out of several mechanisms proposed for understanding ISO-induced myocardial injury, production of highly cytotoxic free radicals through auto-oxidation is widely accepted. It induces severe stress and loss of myocardial integrity through hypoxia, calcium overload and increased free radical production (Roy and Prince 2013). Phytomedicine is being used for the prevention of many cardiac ailments such as heart failure, coronary insufficiency and atherosclerosis from times immemorial. Many epidemiological studies suggest the protective effect of specific groups of fruits and 3

ACCEPTED MANUSCRIPT vegetables against cardiovascular disorders (Yu et al. 2013). Natural compounds rich in antioxidants are of paramount importance in dealing with various pathological conditions.

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Recently they have been used as adjuvants to treat nephro-, neuro-, hepato- and

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cardiovascular related disorders (Azofeifa et al. 2013). Embelin (2, 5-dihydroxy-3-undecyl-1,

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4-benzoquinone), an alkyl substituted benzoquinone bioactive molecule and represents the major constituents of Embelia ribes Burm belonging to the family of Myrsinaceae (Schaible et al. 2013). Embelin was found to possess a wide spectrum of medicinal and

anxiolytic,

antibacterial,

anticonvulsant

and

antidepressant

properties

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antidiabetic,

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pharmacological properties including anti-inflammatory, analgesic, antitumor, antioxidant,

(Thippeswamy et al. 2011). In a previous study, Embelia ribes seed extract has been reported

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to elicit cardioprotective effect in rats (Bhandari et al. 2008). However, it is not clear, in this

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study, whether the cardioprotective effect is because of embelin or other active ingredients of Embelia ribes seed extract. Inspired by the observed cardioprotective effect of Embelia ribes

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extract on one hand and the role of oxidative stress in cardiovascular disorders on the other, the current study was designed to study the cardioprotective effect of embelin as a natural

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antioxidant and to elucidate the mechanism of protection by employing ISO-induced myocardial infarction model in rats. Materials and Methods Chemicals Embelin (Purity: 98%), isoproterenol, Bradford reagent, cytochrome c, catalase, reduced glutathione

(GSH),

oxidized

glutathione

(GSSG),

glutathione

reductase,

2,

6-

dichlorophenolindophenol (DCIP), 2-thiobarbituric acid (TBA), 5, 5-dithio-bis (2nitrobenzoic acid) (DTNB), β-nicotinamide adenine dinucleotide hydrate (NADH), βnicotinamide adenine dinucleotide 3-phosphate reduced form (NADPH), 1-chloro-2,4dinitrobenzene (CDNB), nitro blue tetrazolium salt (NBT), hydroxylamine hydrochloride, 4

ACCEPTED MANUSCRIPT MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide were purchased from Sigma-Aldrich Co, St Louis, MO, USA. Antibodies against Bax, Bcl-2, cytochrome c,

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cleaved caspase-3, caspase-9, cleaved PARP, β-actin and HRP-conjugated secondary anti-

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rabbit and anti-mouse antibody were obtained from Cell Signaling Technology (Boston,

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MA). All other chemicals and reagents were from Sigma-Aldrich Co, St Louis, MO, USA unless otherwise stated.

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Animals

Male Sprague-Dawley rats, weighing between 150 and 180g, were obtained from National

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Institute of Nutrition (NIN), Hyderabad, India. Rats were housed under optimal conditions of temperature, humidity and light-cycle (12L: 12D) and were allowed to acclimatize for 1 week

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prior to initiation of the experiment (food and water ad libitum). All experimental procedures

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were approved by the Institutional Animal Ethics Committee (IAEC) of the institute (Permission No: IICT/PHARM/SRK/FEB/2013/11) and the study was conducted according

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to the ethical norms approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

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Induction of myocardial infarction and experimental design As reported in earlier literature, experimental myocardial injury in rats was induced by administering isoproterenol (Isoproterenol was dissolved in normal saline and was administered at the dose of 85 mg/kg body weight) subcutaneously at an interval of 24h for 2 consecutive days (Wang et al. 2009; Li et al. 2012). The experimental animals were randomly divided in to four groups consisting 8 rats in each. Group I (Vehicle control, control): Rats received 2% gum acacia suspension (2 ml/kg/day, orally) for 5 consecutive days and normal saline (1 ml/kg/day, s.c) on 4th and 5th days. Group II (Embelin control, Emb): Rats received 50 mg/kg embelin suspension in 2% gum acacia (2 ml/kg/day, orally) for 5 consecutive days and normal saline (1 ml/kg/day, s.c) 5

ACCEPTED MANUSCRIPT on 4th and 5th days. Group III (ISO control, ISO): Rats received 2% gum acacia suspension (2 ml/kg/day, orally) for 5 consecutive days and 85 mg/kg ISO dissolved in normal saline (1

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ml/kg/day, s.c) on 4th and 5th days. Group IV (ISO + Emb): Rats received 50 mg/kg embelin

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suspension in 2% gum acacia (2 ml/kg/day, orally) for 5 consecutive days and 85 mg/kg ISO

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dissolved in normal saline (1 ml/kg/day, s.c) on 4th and 5th days. The dose of the embelin (50 mg/kg) was selected based on previous studies (Thippeswamy et al. 2011; Kumar et al. 2011) and duration of pretreatment (5 days) of embelin was based on our pilot study (data not

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shown).

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After 48 h of first dose of ISO (i.e. on 6th day), body weight of all experimental animals was recorded and blood samples were collected through retro-orbital plexus. Serum

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was separated by centrifugation at 4000g for 15min and stored at -80ºC for biochemical

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analyses. Then animals were sacrificed, heart tissue was removed and homogenized in ice cold phosphate buffer saline (50mM, pH 7.4) to obtain a 10% (w/v) tissue homogenate. Heart

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tissue homogenate of different experimental animals was centrifuged at 12000g for 45 min at 4 ºC and the supernatant obtained was used for estimation of various biochemical parameters.

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Total protein concentration in tissue samples was measured using Bradford reagent (SigmaAldrich) against bovine serum albumin (BSA) as standard. Estimation of serum myocardial injury markers Serum levels of CK-MB (creatine kinase-MB isoenzyme), LDH (lactate dehydrogenase) and AST (Aspartate transaminase) were estimated using respective commercial kits (Siemens, India) and employing auto blood analyzer (Siemens, Dimension Xpandplus, USA). The relative weight of heart for each experimental animal was also recorded as indices of cardiac hypertrophy. Estimation of serum lipids and lipoproteins

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ACCEPTED MANUSCRIPT Serum levels of total cholesterol, triglycerides and HDL-C were estimated using commercially available enzymatic kits (Siemens, India) and employing auto blood analyzer

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(Siemens, Dimension Xpandplus , USA). Serum LDL-cholesterol was calculated by the

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Friedewald formula: LDL-C = Total cholesterol–{HDL-C+ (Triglycerides / 5)}. Similarly,

Measurement of myocardial antioxidant status

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VLDL-C was calculated as follows: VLDL-C = Triglycerides/5.

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Myocardial content of various enzymatic and non-enzymatic antioxidants were estimated in heart tissue of all experimental animals. Total superoxide dismutase (SOD) activity was

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estimated by using SOD assay kit (Sigma-Aldrich Co, St Louis, MO, USA) and the activity was expressed as U/mg protein. Catalase (CAT) activity was assayed by monitoring the

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disappearance of H2O2 at 240 nm and was expressed as U/mg protein (Aebi 1974). The

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activity of glutathione reductase (GR) was measured by following the decrease in absorbance

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due to the oxidation of NADPH utilized in the reduction of oxidized glutathione at 340nm for 3 min at an interval of 30 s (Carlberg and Mannervik 1975). Glutathione reductase activity was calculated using the extinction coefficient of 6.22 mM-1cm-1 and expressed as U/mg

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protein. Similarly, glutathione peroxidase (GPx) activity was assessed spectrophotometrically at 340nm and was expressed as U/mg protein (Paglia and Valentine 1967). Phase II detoxifying enzymes such as glutathione S-transferase (GST) and NAD (P) H: quinine oxidoreductase 1 (NQO1) were also determined in cardiac tissue homogenate. Glutathione Stransferase activity was measured at 340nm using 1-chloro-2, 4-dinitrobenzene (CDNB) as substrate, calculated at molar extinction coefficient of 0.0096µM−1cm−1 and was expressed as nmol of CDNB conjugated/min/ml (Habig et al. 1974). NAD (P) H: quinine oxidoreductase 1 activity in heart homogenate was determined using reaction mixture contained 50mM Tris– HCl, pH7.5, 0.08% Triton X-100, 0.25mM NADPH, 80µM 2,6-dichlorophenolindophenol (DCIP) in the presence or absence of 60µM dicumarol as described earlier (Zhu et al. 2005). 7

ACCEPTED MANUSCRIPT The reaction was started by addition of sample, and the reduction of DCIP was monitored at 600nm, for 3min. The dicumarol-inhibitable NQO1 activity was calculated using the

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extinction coefficient of 21.0mM-1cm-1 and expressed as nmol of DCIP reduced per min per

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mg of protein. Cardiac tissue levels of reduced glutathione (GSH) were estimated

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spectrophotometrically at 412 nm using Ellman reagent. Reduced glutathione (Sigma-Aldrich Co, St Louis, MO, USA) was used for preparation of standard curve and the content of GSH

Assay for myocardial lipid peroxidation

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was expressed as µg/g of wet tissue (Ellman 1959).

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Thiobarbituric acid reactive substances (TBARS), which serve as lipid peroxidation marker was determined in heart tissue homogenate of all experimental animals according to the

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method described previously by Ohkawa et al. (1979). The pink color developed due to

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reaction with thiobarbituric acid was measured spectrophotometrically at 532nm and

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calculated as MDA equivalent using an extinction coefficient of 1.56 × 105 M-1 cm-1. Values were expressed as nmole of MDA eq. /g wet tissue.

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Isolation of mitochondria from heart tissue Mitochondria rich fraction was isolated from heart tissue of different experimental animal by differential centrifugation as described in earlier reported literature (Johnson and Lardy 1967). Briefly, a part of the heart tissue from each experimental animal was minced and a 10% (w/v) heart tissue homogenate was prepared in ice-cold isolation buffer (10 mM Tris, 250 mM Sucrose, 1 mM EGTA, pH 7.2) using Teflon Potter homogenizer. The homogenate samples were centrifuged at 600g for 10 min to separate cellular and nuclear debris and the resultant supernatant was centrifuged at 10, 000 g for 10 min to obtain mitochondria rich pellet. The obtained pellet was resuspended in wash buffer containing 10mM Tris-HCl (pH 7.2) and 250mM sucrose (without EGTA) and once again centrifuged at 10, 000 g for 10 min.

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ACCEPTED MANUSCRIPT The obtained mitochondrial fraction was adjusted in same buffer to give a protein concentration of 1–2 mg/ml. The whole isolation procedure was performed at 4°C and the

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concentration of mitochondrial protein was estimated using Bradford reagent (Sigma-

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Assay of mitochondrial respiratory enzyme activities

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Aldrich) against bovine serum albumin (BSA) as standard.

The activity of mitochondrial NADH dehydrogenase (NDH) in heart tissue was determined

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spectrophotometrically by recording the change in optical density at 550nm for 180 s at 15 s interval as described earlier (King and Howard 1967). The detection of NADH

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dehydrogenase enzyme activity method involves oxidation of NADH to NAD+ with subsequent reduction of cytochrome C. The enzyme activity was expressed as nmol of

of

heart

tissue

from

different

experimental

group

was

estimated

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fraction

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NADH oxidized/min/mg protein. Succinate dehydrogenase (SDH) activity in mitochondrial

spectrophotometrically at 420nm using kinetic readings as described earlier (King 1967). The

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method involves oxidation of succinate in the presence of an artificial electron acceptor, potassium ferricyanide and was expressed as U/mg protein. The activity of cytochrome c

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oxidase was assayed in isolated mitochondria using cytochrome c oxidase assay kit (Sigma– Aldrich Co., St. Louis, MO, USA) and the activity was expressed as U/mg protein. MTT [3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] reduction rate at 580 nm was used to examine the mitochondrial redox activity in isolated mitochondria by the method as described earlier (Liu et al. 1997). Western blot analysis Whole

protein

extract

from

left

ventricular

heart

tissue

was

prepared

using

Radioimmunoprecipitation (RIPA) buffer (Pierce Biotechnology, Rockford, IL, USA) containing 1% protease inhibitor cocktail (Sigma-Aldrich) at 4°C. The protein concentration was determined using Bicinchoninic acid (BCA) protein estimation kit (Pierce 9

ACCEPTED MANUSCRIPT Biotechnology, Rockford, IL, USA). Samples were mixed with Lammelie buffer and boiled at 95°C for 5min. Equal amount of protein samples (40µg/lane) were loaded and separated by

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SDS-PAGE and then transferred to polyvinylidine difluoride (PVDF) membranes (Pierce,

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Rockford, IL, USA). The membranes were blocked at room temperature for 1h using 3%

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bovine serum albumin in TBS- 0.05% Tween 20 (TBST) solution. The membranes were washed once with TBST solution and incubated with respective primary antibody for overnight at 4°C. Primary antibodies such as cleaved caspase-3 (rabbit monoclonal 1:1000,

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Cell Signaling Technology), caspase-9 (mouse monoclonal 1:1000, Cell Signaling

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Technology), Bax (rabbit monoclonal 1:1000, Cell Signaling Technology), Bcl-2 (rabbit monoclonal 1:1000, Cell Signaling Technology), cytochrome c (rabbit monoclonal 1:1000,

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Cell Signaling Technology) and cleaved PARP (rabbit monoclonal 1:1000, Cell Signaling

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Technology) were used to assess the expression of cleaved caspase-3, caspase-9, Bax, Bcl-2, cytochrome c and cleaved-PARP protein respectively. β-actin (rabbit monoclonal 1:1000,

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Cell Signaling Technology) was used as loading control for equal loading of samples. After overnight incubation at 4°C, the membranes were washed with TBST solution thrice and

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incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:4000, Cell Signaling Technology) for 2h at room temperature. The membranes were once again washed with TBST for three times and the blots were visualized using enhanced chemiluminescence (ECL) reagent (Pierce Biotechnology, Rockford, IL, USA). Image J software (NIH) was used for densitometry analysis of immunoblots. Histopathology of heart tissue For histopathological evaluation, tissue samples from the left ventricles were fixed in 10% neutral buffered formalin. The specimens embedded in paraffin were processed, sectioned at 5μm thickness and stained with hematoxylin- eosin (H&E) for light microscopy. The stained

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ACCEPTED MANUSCRIPT sections were examined for myocardial changes like any necrotic foci, myofibrillar architecture, degenerative changes and infiltration of inflammatory cells.

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Statistical analysis

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All statistical analyses were performed using one way ANOVA with the Graph Pad Prism,

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version 5.0 software. Comparisons between groups were performed by applying Dunnett's multiple comparison procedure. Results were expressed as Mean ± S.E.M or as percent

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activity when compared to control animals. Statistical significance was considered at p< 0.05.

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Results

Effect of embelin on ISO-induced changes in cardiac injury markers

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We assessed the effect of embelin on ISO-induced myocardial injury by measuring the serum levels of CK-MB, LDH and AST in all control and experimental groups of animals (Fig. 1).

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Isoproterenol administration produced a significant increase in serum levels of CK-MB (Fig. 1A, p< 0.05), LDH (Fig. 1B, p< 0.05) and AST (Fig. 1C, p< 0.001) when compared to

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vehicle control rats. In addition, rats administered with ISO showed a significant increase in relative weight of heart when compared to vehicle control rats (Fig. 1D, p< 0.001). Pretreatment with embelin followed by ISO administration not only significantly (p< 0.05: CK-MB and LDH, p< 0.01: AST) decreased the serum levels of CK-MB, LDH and AST but also significantly (p< 0.01) reduced the relative weight of heart when compared to only ISO treated rats. Effect of embelin on ISO-induced changes in myocardial antioxidants As shown in Fig. 2, the myocardial content of reduced glutathione (GSH) (Fig. 2A) and its associated antioxidant enzyme activities of glutathione peroxidase (GPx) (Fig. 2B) and glutathione S-transferase (GST) (Fig. 2C) were significantly (p< 0.01: GSH and GST, p< 11

ACCEPTED MANUSCRIPT 0.05: GPx) decreased in ISO per se administered rats. The myocardial glutathione reductase (GR) level was markedly lower in ISO administered rats when compared to vehicle control

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rats (Fig. 2D). However, pretreatment with embelin followed by ISO significantly increased

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the levels of myocardial GSH content and the activities of GPx, GR and GST (p< 0.05: GSH,

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GPx and GR; p< 0.01: GST) when compared with only ISO administered rats. Myocardial content of superoxide dismutase (SOD) (Fig. 3A), catalase (CAT) (Fig. 3B) and NAD (P) H: quinine oxidoreductase 1 (NQO1) (Fig. 3C) activities were significantly (p< 0.05: SOD and

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NQO1, p< 0.01: CAT) decreased in the ISO control group when compared with the vehicle

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control group, while the thiobarbituric acid reactive substances (TBARS) level (Fig. 3D) in the ISO control group was significantly (p< 0.05) higher than that in the vehicle control

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group. Pretreatment with embelin (50 mg/kg) was found to significantly (p< 0.05) increased

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the activities of SOD, CAT, and NQO1 and significantly (p< 0.05) decreased the myocardial TBARS content when compared to ISO control group. No significant (p> 0.05) difference of

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GSH, GPx, GR, GST, SOD, CAT, NQO1 and TBARS levels could be seen in heart from the rats treated with embelin only, when compared with vehicle control.

activities

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Effect of embelin on ISO-induced changes in myocardial mitochondrial enzyme

As shown in Fig. 4, the mitochondrial enzyme activities such as NADH dehydrogenase (NDH) (Fig. 4A), succinate dehydrogenase (SDH) (Fig. 4B), mitochondrial redox activity (Fig. 4C) and cytochrome c oxidase (COX) (Fig. 4D) were significantly (p< 0.05: NDH, COX and redox activity; p< 0.01: SDH) decreased in the ISO control group of rats when compared with the vehicle control rats. Pretreatment with embelin significantly (p< 0.05) increased the mitochondrial NDH, COX and redox activity when compared with ISO control group. However, pretreatment with embelin could not produce any significant change to SDH

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ACCEPTED MANUSCRIPT activity when compared with the ISO control group. Embelin per se treated rats had no effect on the above mitochondrial enzyme activities when compared to vehicle control.

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Effect of embelin on ISO-induced changes in myocardial apoptotic proteins

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As shown in Fig. 5, the amount of pro-apoptotic related protein cytochrome c and Bax were

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significantly (p< 0.05) increased and anti-apoptotic protein, Bcl-2 was significantly (p< 0.01) decreased in ISO control group when compared to vehicle control group. Pretreatment with

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embelin (50 mg/kg) followed by ISO significantly decreased (p< 0.01) the left ventricular protein expression of cytochrome c and Bax when compared to ISO control group. The

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protein expression of anti-apoptotic protein Bcl-2 was significantly (p< 0.01) increased in embelin pretreated ISO-induced rats compared to only ISO administered rats. In addition, as

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shown in Fig. 6, ISO administration also caused a significant increase in amount of cleaved

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caspase-3, cleaved caspase-9 and cleaved PARP protein compared to vehicle control group. Embelin pretreatment significantly attenuated the caspases activation as evident by reduced

control group.

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amount of cleaved caspase-3, cleaved caspase-9 and cleaved PARP when compared to ISO

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Effect of embelin on ISO-induced changes in lipids and lipoproteins As shown in Table 1, ISO treated rats showed a significant (p< 0.05) increase in the serum total cholesterol and triglyceride levels when compared to vehicle control rats. Pretreatment with embelin (50 mg/kg) significantly (p< 0.05) decreased these cholesterol and triglyceride levels near to normal. Compared to vehicle control rats, the serum levels of VLDL-C was significantly (p< 0.05) increased and the serum level of HDL-C and LDL-C were unaltered in only ISO administered rats. Embelin pretreatment followed by ISO showed a significant (p< 0.05) decrease in the levels of serum VLDL-C without altering the serum levels of HDL-C and LDL-C when compared to ISO control rats (Table 1).

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ACCEPTED MANUSCRIPT Histopathological findings As shown in Fig. 7, left ventricular heart tissue sections from vehicle (Fig. 7A) and embelin

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(Fig. 7B) treated rats showed no obvious pathological abnormalities and have intact cardiac

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morphology. When compared with the vehicle control, left ventricular tissue sections of rats

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administered with ISO showed extensive myocardial alterations. Focal necrosis, myocardial degeneration, loss of myofibrillar alignment, severe cytoplasmic vacuolization and

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infiltration of inflammatory cells were prominent in tissue sections from ISO treated group of rats (Fig. 7C). Pretreatment with embelin followed by ISO, dramatically reduced the ISO-

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induced severe vacuolization, necrosis and infiltration of inflammatory cells when compared to ISO treated rats (Fig. 7D). However, mild myocardial degeneration was observed without

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any necrotic foci in embelin pretreated ISO-induced group when compared to ISO control

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group.

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Discussion

Ischemic heart disease, especially myocardial infarction, account for a major portion of cardiovascular diseases. Epidemiological studies have shown that diets rich in phyto-

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chemicals and/or phyto-nutrients reduce the risk of cardiovascular diseases (Prince and Roy 2013). We investigated the effect of embelin, a benzoquinone ring containing bioactive molecule from Embelia ribes on oxidative stress, mitochondrial dysfunction and apoptosis induced by ISO in rat heart. Number of studies reported that pathophysiological, morphological and metabolic changes that occur in heart of experimental animals following isoproterenol administration are similar to those observed in humans (Wang et al. 2009; Li et al. 2012). Increased serum levels of CK-MB, LDH and AST, increase in heart to body weight ratio with abnormal histo-architecture of heart tissue indicated ISO-induced myocardial damage. Studies have shown that increased serum levels of these cardiac injury biomarkers as an indicative measure of leakage and loss of functional integrity and/or necrotic damage of 14

ACCEPTED MANUSCRIPT cell membrane (Roy and Prince 2013). Pretreatment with embelin followed by ISO significantly decreased not only the serum levels of CK-MB, LDH and AST but also

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decreased the heart to body weight ratio when compared to only ISO treated rats (Fig. 1).

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Moreover, embelin pretreatment followed by ISO considerably decreased the pathological

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changes such as loss of myofibrillar alignment, myocardial degeneration, severe cytoplasmic vacuolization and infiltration of inflammatory cells when compared to only ISO administered rats (Fig. 7). These primary findings of our study indicate the cardioprotective effect of

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embelin.

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It has been widely reported that excessive generation of ROS and subsequent oxidative stress is not only directly associated with cardiovascular diseases but also with

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many cardiovascular risk factors such as hypertension, diabetes mellitus and chronic renal

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failure. Furthermore, it has also been documented that heart tissue is highly susceptible to oxidative stress than other tissues, as the activity of antioxidant enzyme is lower in the heart

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tissues (Angeloni et al. 2009). In the body, there are two major antioxidant defense systems (enzymatic such as SOD, CAT, GPx, GR, GST and NQO1 and non-enzymatic such as GSH,

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vitamin C and vitamin E) plays a key role in scavenging and/or by detoxifying the free radicals. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide anions to oxygen and H2O2, which can be further detoxified by GPx and CAT to water. NQO1 plays a major role in detoxifying of H2O2 and superoxide via its ability to maintain the cellular levels of vitamin E and ubiquinol. GSH redox system, which includes GSH and its related antioxidant enzymes, GPx, GR and GST, plays an important role in scavenging of ROS and lipid peroxidation. The results of the present study showed that embelin pretreatment followed by ISO significantly restored the activities of these antioxidants near to normal. (Fig. 2 and 3). Thus, we presume that embelin effectively scavenges the excess production of ISO-induced free radicals and reduces the ISO-induced myocardial damage in rats. Reactive

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ACCEPTED MANUSCRIPT oxygen species (ROS) can also affect the pathophysiology directly by affecting to DNA, lipid and protein molecules. Lipid peroxidation, a ROS-mediated mechanism and increase in tissue

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thiobarbituric acid reactive substances (TBARS) such as malondialdehyde (MDA) have been

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implicated in the pathogenesis of cardiovascular disorders (Radhiga et al. 2012). Results of

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our study showed that embelin pretreatment significantly restored the myocardial antioxidant defense and attenuated the ISO-induced lipid peroxidation in heart tissue.

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Mitochondrial dysfunction plays an important role in the pathogenesis of many cardiovascular diseases. In the heart about 45% of the myocardial volume is taken up by

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mitochondria (Finsterer and Ohnsorge 2013). Literature suggests that ISO damage the heart by various different mitochondrial mechanisms. Out of them, increase of mitochondrial ROS

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production, induction of apoptosis, decrease in antioxidative capacity and alteration of

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mitochondrial respiratory chain function through down regulation of specific mitochondrial respiratory enzyme activities are some of pathological events that are involved in ISO-

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induced mitochondrial dysfunction (Finsterer and Ohnsorge 2013). In the present study, the activity of enzymes that are involved in mitochondrial respiratory chain process such as

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NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase activities were significantly decreased in ISO administered group of rats when compared to those of vehicle control (Fig. 4). The results of our study corroborate with earlier studies and we speculate that the decreased activities of these respiratory chain enzymes could be due to the enhanced phospholipid degradation resulting in the non-availability of cardiolipin for their functional activity (Sangeetha and Quine 2009). In addition, literature also points out that the decrease in these enzymes might be due to the marked deficiency in one or more electron transport chain components. For example, the decrease in the activities of these enzymes due to depletion of reducing equivalent such as NADH and NADPH, which are utilized for the formation of GSH to counter oxidative damage of mitochondrial components (Punitharathi et 16

ACCEPTED MANUSCRIPT al. 2010). We also investigated the mitochondrial redox activity of different experimental groups by using MTT assay. MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium

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bromide] is reduced by intact mitochondria in living cell and is a measure of viable cells.

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Depending upon the mitochondrial intactness, yellow colored MTT was reduced to purple

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colored formazan. The absorbance of purple colored formazan at 580nm thus formed was used to represent the mitochondrial redox activity (Sahu et al. 2014). Embelin pretreatment significantly increased the MTT reduction activity when compared to only ISO treated rats.

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We believe that, during ischemia, the subsequent generation of ROS or lipid peroxides

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produced an inhibition of respiratory chain function might have caused mitochondrial dysfunction.

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Multiple lines of evidence demonstrate that apoptosis has been observed in a number

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of cardiovascular diseases including myocardial infarction, hypertrophy and in heart failure (Thygesen et al. 2012). It has been reported that administration of isoproterenol in rats

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produced death of cardiomyocyte in heart by inducing apoptosis (Radhiga et al. 2012; Prince and Roy 2013). Bcl-2 family proteins and caspases are checkpoints of the apoptosis

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pathways. The up and down regulation of Bax/Bcl-2 protein usually determine the susceptibility of apoptosis whether the cell survives or is going to die. Increased level of ROS induces Bax to permeabilize the external mitochondrial membrane, leading to the release of cytochrome c and the activation of caspases and finally PARP. However this process is blocked by anti-apoptotic protein Bcl-2. To explore the effect of embelin on myocardial apoptosis, we analyzed the expression levels of various proteins related to apoptosis. In the present study, ISO administered rats displayed a reduced levels of myocardial protein expression of Bcl-2 and increased myocardial levels of Bax, cytochrome c, cleaved caspase3, cleaved caspase-9 and cleaved PARP when compared to those of vehicle control (Fig. 5). We speculate that embelin pretreatment prevented cytochrome c release by stabilizing 17

ACCEPTED MANUSCRIPT mitochondrial outer membrane caused by ISO and thereby interrupted mitochondrial dependent intrinsic apoptotic process (Fig. 6) (Carotenuto et al. 2013). The insignificant

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differences among various parameters such as serum cardiac injury biomarkers (CK-MB,

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LDH and AST), histo-architecture of heart tissue, antioxidant and mitochondria specific

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enzymes and apoptosis related protein markers between vehicle control and embelin per se treated rats revealed that embelin itself has no effect when compared to those of vehicle

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control.

Lipids play an important role in cardiovascular disease, not only by developing

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atherosclerosis, but also by modifying the composition, structure and stability of cellular membranes. It has been also reported that catecholamine at higher concentration cause

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perturbations of cardiac lipid metabolism and cardiac dysfunction through β-adrenoreceptor

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stimulation (Shao et al. 2013). It is documented that the biochemical changes on isoproterenol administration; in particular, lipid metabolism and these changes are

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comparable to those taking place in humans (Meeran et al. 2012). In the present study, the observed increase in levels of cholesterol and triglycerides in the serum of ISO administered

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rats may be due to elevated flux of fatty acids or due to enhanced lipid biosynthesis by cardiac cyclic adenosine monophosphate (Meeran et al. 2012). Thus, administration of embelin modulated the levels of lipids and lipoproteins and exhibited cardioprotective effect (Table 1). In summary, we demonstrated the cardioprotective effect of embelin by using isoproterenol-induced myocardial injury model in rats. Pretreatment with embelin enhanced myocardial antioxidant status and there by attenuated ISO-induced myocardial oxidative stress and associated cardiomyocyte apoptosis. The results of the present study also demonstrate that pretreatment with embelin effectively attenuated the ISO-induced dyslipidemia and mitochondrial dysfunction. Hence, it may be concluded that embelin pre18

ACCEPTED MANUSCRIPT treatment might be beneficial for prevention of ischemic heart disease (IHD). However, therapeutic benefit of such a treatment in already established IHD is unknown.

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Conflict of interest

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The authors declare no competing financial interest. Acknowledgments

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This work was supported in part by CSIR 12th 5-year plan project ‘SMiLE’ (CSC 0111). Bidya Dhar Sahu thanks Council of Scientific and Industrial Research (CSIR), New Delhi for

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financial assistance in the form of Senior Research Fellowship. We thank the Director, CSIR-

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IICT, Hyderabad for providing necessary facilities.

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[21] Punithavathi VR, Shanmugapriya K, Stanely Mainzen Prince P. Protective effects of Rutin on mitochondrial damage in isoproterenol-induced cardiotoxic rats: An in vivo and in vitro study. Cardiovasc Toxicol 2012; 10:181–189. [22] Radhiga T, Rajamanickam C, Sundaresan A, Ezhumalai M, Pugalendi KV. Effect of ursolic acid treatment on apoptosis and DNA damage in isoproterenol-induced myocardial infarction. Biochimie 2012; 94:1135-1142.

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Schuster D, Werz O. Potent inhibition of human 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 by the anti-carcinogenic and anti-inflammatory agent embelin. Biochem Pharmacol 2013; 86:476–486. [28] Shao Y, Redfors B, Mattson-Hulten L, Tang MS, Daryoni E, Said M, Omerovic E. Adenosine prevents isoprenaline-induced cardiac contractile and electrophysiological dysfunction. Eur J Pharmacol 2013; 718:475-483. [29] Thippeswamy BS, Mahendran S, Biradar M I, Raj P, Srivastava K, Badami K, Veerapur VP. Protective effect of embelin against acetic acid induced ulcerative colitis in rats. Eur J Pharmacol 2011; 654:100–105.

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[31] Vendrame S, Daugherty A, Kristo AS, Klimis-Zacas D. Wild blueberry (Vaccinium

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angustifolium)-enriched diet improves dyslipidaemia and modulates the expression of

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salvianolic acid A on isoproterenol-induced myocardial infarction in rats. Eur J Pharmacol 2009; 615:125–132.

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ACCEPTED MANUSCRIPT Figure legends: Fig. 1. Effect of embelin and/or ISO on (A) Creatine kinase-MB (CK-MB), (B) Lactate

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dehydrogenase (LDH), and (C) Aspartate transaminase (AST) in serum. (D) Effect of

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embelin on ISO-induced changes in heart to body weight ratio. Values are expressed as Mean

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± S.E.M, N=8. Where, control, vehicle control; Emb, embelin; ISO, isoproterenol; ISO+Emb, embelin pretreated ISO-induced rats.

p< 0.05, ***p< 0.001 vs. vehicle control; #p< 0.05, **p< 0.01 vs. ISO control.

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Fig. 2. Effect of embelin on ISO-induced changes in myocardial (A) reduced glutathione

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(GSH) and it’s linked antioxidant enzymes, (B) Glutathione peroxidase (GPx), (C) Glutathione S-transferase (GST) and (D) Glutathione reductase (GR) activities. Values are

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expressed as Mean ± S.E.M, N=8. Where, CDNB, 1-chloro-2, 4-dinitrobenzene; control,

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vehicle control; Emb, embelin; ISO, isoproterenol; ISO+Emb, embelin pretreated ISO-

#

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induced rats.

p< 0.05, **p< 0.01 vs. vehicle control; *p< 0.05, $p< 0.01 vs. ISO control.

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Fig. 3. Effect of embelin and/or ISO on (A) Superoxide dismutase (SOD), (B) Catalase (CAT), and (C) NAD (P) H: quinine oxidoreductase 1 (NQO1) and (D) Thiobarbituric acid reactive substances (TBARS) levels. Values are expressed as Mean ± S.E.M, N=8. Where, DCIP, 2, 6-dichlorophenolindophenol; control, vehicle control; Emb, embelin; ISO, isoproterenol; ISO+Emb, embelin pretreated ISO-induced rats. #

p< 0.05, **p< 0.01 vs. vehicle control; *p< 0.05 vs. ISO control.

Fig. 4. Effect of embelin on ISO-induced changes in myocardial mitochondrial respiratory enzyme activities, (A) NADH dehydrogenase, (B) Succinate dehydrogenase, (C) Mitochondrial redox activity and (D) Cytochrome c oxidase activities. Values are expressed 24

ACCEPTED MANUSCRIPT as Mean ± S.E.M, N=8. Where, control, vehicle control; Emb, embelin; ISO, isoproterenol; ISO+Emb, embelin pretreated ISO-induced rats. p< 0.05, **p< 0.01 vs. vehicle control; *p< 0.05 vs. ISO control.

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#

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Fig. 5. Representative immunoblot showing left ventricular heart tissue protein expression of (A) cytochrome C (Cyt C) and (B) Bax and Bcl-2. Graphical representation of the intensity of (C) Cyt C and (D) Bax/Bcl-2 protein expression in different experimental groups. Data

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represent the means (4 animals per group), with their standard error (S.E.M) represented by vertical bars. β–actin was used as an internal control for equal loading of protein. Where,

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control, vehicle control; Emb, embelin; ISO, isoproterenol; ISO+Emb, embelin pretreated ISO-induced rats.

p< 0.05, #p< 0.01 vs. vehicle control; **p< 0.01 vs. ISO control.

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*

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Fig. 6. Representative immunoblot showing left ventricular protein expression of cleaved caspase-9, cleaved caspase-3 and cleaved PARP. Immunoblot represent the protein expressions of three independent experiments. β–actin was used as an internal control for

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equal loading of protein. Where, control, vehicle control; Emb, embelin; ISO, isoproterenol; ISO+Emb, embelin pretreated ISO-induced rats. Fig. 7. Light microscopic examination of left ventricular heart tissue stained with hematoxylin-Eosin (H & E) (Magnification 20X). Representative photomicrograph of left ventricular heart tissue sections from vehicle control (control) and embelin control (Emb) showing intact cardiac morphology. Tissue sections from rats treated with ISO showing myocardial degeneration, loss of myofibrillar alignment, inflammatory cells infiltration and severe cytoplasmic vacuolization (ISO). Isoproterenol-induced rats pretreated with embelin

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ACCEPTED MANUSCRIPT (ISO+Emb) showing mild myocardial degeneration without vacuolization when compared to

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ISO-induced rats.

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ACCEPTED MANUSCRIPT Table 1: Effect of embelin on isoproterenol-induced changes in serum lipids and lipoproteins.

Control

Emb

Total cholesterol (mg/dl)

62.5±3.13

63.9±2.91

Triglycerides (mg/dl)

37.6±3.31

35.8±4.6

HDL-C (mg/dl)

52.8±2.24

56.3±2.8

LDL-C (mg/dl)

8.0±1.98

7.9±0.97

VLDL-C (mg/dl)

7.52±0.66

ISO

ISO+Emb

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Parameters estimated

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80.4±2.79#

80.6±7.86***

48.0±6.78**

45.6±3.29

49.0±3.92

9.9±1.29

8.28±2.41

16.11±1.57***

9.4±1.87**

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7.16±1.11

62.7±5.13*

All data were expressed as mean ± S.E.M, N=8. Where, Control, vehicle control; ISO, isoproterenol; Emb, embelin; ISO+Emb, embelin

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pretreated ISO-induced rats; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; VLDL-C, very low-

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density lipoprotein-cholesterol. Where, #p< 0.05, ***p< 0.001 vs. vehicle control; *p< 0.05, **p< 0.01 vs. ISO control.

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