Mechanistic insights to the cardioprotective effect of blueberry nutraceutical extract in isoprenaline-induced cardiac hypertrophy

Accepted Manuscript

Mechanistic insights to the cardioprotective effect of Blueberry Nutraceutical Extract in Isoprenaline-Induced Cardiac Hypertrophy Radwa A. Eladwy , Eman M. Mantawy , Wesam M. El-Bakly , Mohamed Fares , Laila A. Ramadan , Samar S. Azab PII: DOI: Reference:

S0944-7113(18)30529-4 https://doi.org/10.1016/j.phymed.2018.10.009 PHYMED 52712

To appear in:

Phytomedicine

Received date: Revised date: Accepted date:

16 June 2018 30 September 2018 9 October 2018

Please cite this article as: Radwa A. Eladwy , Eman M. Mantawy , Wesam M. El-Bakly , Mohamed Fares , Laila A. Ramadan , Samar S. Azab , Mechanistic insights to the cardioprotective effect of Blueberry Nutraceutical Extract in Isoprenaline-Induced Cardiac Hypertrophy, Phytomedicine (2018), doi: https://doi.org/10.1016/j.phymed.2018.10.009

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Mechanistic insights to the cardioprotective effect of Blueberry Nutraceutical Extract in Isoprenaline-Induced Cardiac Hypertrophy

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Radwa A. Eladwya , Eman M. Mantawyb , Wesam M. El-Baklyc , Mohamed Faresd, Laila A. Ramadana and Samar S. Azab b* Department of Pharmacology and Toxicology, Faculty of Pharmacy, Egyptian Russian

University, Badr City, Cairo 11829, Egypt b

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University,

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Cairo 11566, Egypt

Department of Pharmacology and Therapeutics, Faculty of Medicine, Ain Shams University,

Cairo 11566, Egypt

School of Chemistry, University of Wollongong, Wollongong 2522, New South Wales,

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Australia

Samar S. Azab

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* Correspondence: All correspondence should be addressed to:

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Egypt

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Pharmacology & Toxicology Department, Faculty of Pharmacy, Ain Shams University, Cairo 11566,

Mobile : +2-01003814389 Fax : +202- 24051107 E-mail address: [email protected]

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Abstract Background: Lowbush blueberry extract (Vaccinium angustifolium) is abundant with polyphenols (such as chlorogenic acid ) with high antioxidant profile. It has received great interest due to its protective role in many disorders such as heart diseases and neurological

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disorders. Hypothesis: We hypothesized that blueberry leaf extract might have a protective effect against cardiac hypertrophy via suppressing oxidative stress, inflammation and fibrosis.

Method: Blueberry leaf nutraceutical extract was administered orally to male albino rats at three different doses (25, 50 and 100 mg/kg/day of the extract, equivalent to 3.4, 6.8 and 13.6 mg of

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chlorogenic acid, respectively) once daily for 28 consecutive days against a dose of isoprenaline (ISO) (5 mg/kg) for 14 days.

Results: The results indicated that isoprenaline induced significant myocardial damage, characterized by conduction abnormalities, increased heart-to-body weight ratio, increased serum CKMB, AST, c-TnI and LDH. Pretreatment with blueberry extract at a dose of 50 mg/kg/day

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(equivalent to 6.8 mg chlorogenic acid) protected against ISO-induced ECG changes, leakage of cardiac enzymes and histopathological changes. Also, ISO caused significant glutathione

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depletion, lipid peroxidation and reduction in activities of antioxidant catalase enzyme. These effects were prevented by pretreatment with blueberry extract. Additionally, ISO elicited inflammatory effects by increasing the expression of NF-κB, COX-2, TNF-α and IL-6 while

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pretreatment with blueberry extract significantly inhibited these inflammatory responses. Furthermore, ISO induced fibrosis by increasing the level of TGF-β while pretreatment with

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blueberry extract significantly reduced it. Conclusion: These findings indicate that blueberry leaf extract possessed a potent protective

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effect against ISO-induced cardiac hypertrophy via suppressing oxidative stress, inflammation and fibrosis. Keywords:

Cardiac hypertrophy; Isoprenaline; Blueberry; Nutraceuticals; Inflammation.

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List of Abbreviations:

Analysis of Variance (ANOVA); Aspartate aminotransferase (AST); Blue berry (BB); Bovine serum albumin (BSA); Cardiac hypertrophy (CH); Cardiac troponin I (c-TnI); Catalase (CAT);

2); Electrocardiography (ECG); Isoprenaline

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Chlorogenic acid (CGA); creatine kinase isoenzyme-MB (CK-MB), Cyclooxygenase-2 (COXreduced Glutathione (GSH); horseradish peroxidase (HRP);

(ISO); Interleukin-1β (IL-1β) and Interleukin-6 (IL-6); Lactate dehydrogenase

(LDH); Malondialdehyde (MDA); Nuclear factor κB (NF-κB); Subcutaneous (S.C); standard

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Deviation (SD); thiobarbituric acid reactive substances (TBARS); 3,3',5,5'-tetramethylbenzidine (TMB); Trichloroacetic acid (TCA) and Thiobarbituric acid (TBA); Transforming growth factor

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beta 1 (TGF-β1); Tumor necrosis factor-α (TNF-α).

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1. Introduction: Cardiac hypertrophy (CH) is a recognized risk factor in many cardiovascular disorders and is globally considered as one of the most prevalent causes of morbidity and mortality (Althurwi et al., 2013; Li et al., 2014). CH is an adaptive response of the cardiac myocytes to different stimuli

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such as pathological disorders including myocardial infarction, hypertension and certain chemicals, for instance the non-selective β-adrenergic receptor agonist isoprenaline (ISO) (Li et al., 2014; Liao et al., 2004; Lin et al., 2008). This response would eventually affect the differentiation of the cardiomyocytes leading to increase in size without undergoing cell division (Balakumar and Singh, 2006; Rose et al., 2007). Although the hypertrophic response can be

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adapted by the body in the beginning of the pathogenesis, enduring hypertrophy may hasten cardiac fibrosis, contractile dysfunction and heart failure (Selvetella and Lembo, 2005). Macroscopically CH is characterized by myocardial muscle mass increase (Selvetella and Lembo, 2005). On the cellular level, the exact mechanism of the CH is not completely understood. It is broadly accepted that the molecular mechanism for the development of CH is

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linked with the regeneration of capillaries in the heart (Batra et al., 1991). The development of apoptosis in the cardiac tissues and increased oxidative stress has also been linked to the

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development of CH (Liu et al., 2010). Others argue that inflammation due to oxygen free radicals or pressure overload plays a vital role in the development of CH (Oka et al., 2012). Isoprenaline is a β-adrenergic agonist, when administered S.C, it mimicked catecholamine`s action on heart

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tissues (Katare et al., 2017). Complete understanding of the β-adrenergic agonist induced cardiotoxicity is yet unclear, however, there are some reports that oxidative stress, inflammation

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and fibrosis plays an important role in the development of the pathogenesis of cardiac hypertrophy (Gayathri et al., 2011). A recent study showed that, ISO-induced cardiac

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hypertrophy is associated with increased cardiac oxidative stress and abnormalities in ECG (Katare et al., 2017). There is a substantial body of evidence supporting that increased oxidative stress activates

transcription regulator nuclear factor-κB (NF-κB) (Rahman et al., 2002). NF-κB is reported to be implicated with the pathogenesis of CH including, vascular remodeling and oxidative stress (Li et al., 2004). NF-κB pathway is considered a proinflammatory signaling pathway as it increase the

expression of proinflammatory genes including cytokines, chemokines, and adhesion 4

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molecules (Ali and Mann, 2004). Transcription of inflammatory mediators, such as interleukin1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α) and COX-2 are regulated by NF-κB and is linked to CH (Ghosh et al., 1998). Furthermore, increased oxidative stress is linked to the development of fibrosis, and subsequently inducing the expression of profibrogenic cytokines such as TGF-β (Liu and Pravia, 2010).

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Nutraceuticals are dietary components or food derivatives that are proved to be beneficial to health and can be used for prophylaxis or treatment for certain medical conditions (Garcia-Rios et al., 2013). Nutraceuticals are considered essential to prevent and treat cardiovascular diseases and also play a pivotal role in maintaining healthy cardiovascular conditions (Garcia-Rios et al., 2013). Different nutraceuticals taken as fruits, extracts and crude formulations are reported to

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alleviate heart disorders. Blueberries in particular have a high antioxidant profile, which is due to the presence of several compounds including polyphenols, flavonoids and anthocyanins (Ahmet et al., 2009; Prior and Cao, 2000). It has been stated that blueberry extract and blueberry enriched diets can alleviate cognitive impairment, oxidative stress, heart diseases and neurological disorders (Ahmet et al., 2009; Song et al., 2013). Chlorogenic acid (CGA) is an

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abundant polyphenol compound in the blueberry leaf extract that possesses anti-inflammatory, antioxidant and antihypertensive effects. The CGA anti-inflammatory effect maybe due to its

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ability to inhibit Cox2 and NF-κB that can suppress inflammatory cytokines release (Li et al., 2014; Shan et al., 2009). Previous research has also indicated CGA ability to attenuate chronic

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ventricular remodeling after myocardial ischemia via inhibition of macrophage infiltration (Kanno et al., 2013). Thus, the primary objective of the current study is to investigate the

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potential cardioprotective effect of the blueberry leaf extract against ISO-induced cardiac hypertrophy in rats and to elucidate the potential molecular mechanisms by studying its effect on

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different cardiac indices, oxidative stress, inflammatory and fibrotic markers.

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Material and Methods: 2.1. Drugs and chemicals Isoprenaline (ISO) was purchased as isoprenaline hydrochloride (5 gm isoprenaline hydrochloride) from Sigma-Aldrich Co. (Missouri, USA). The lowbush (Vaccinium

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angustifolium) blueberry leaf extract (1 mL is equivalent to 106 mg of dry herb) was purchased from Stakich, Inc. Royal Oak, (Michagin, USA, Lot# 36257IH) and chlorogenic acid (CGA) was purchased from AK scientific (Union City, CA 94587, USA). Ellman’s reagent [5,5-dithio-bis (2-nitrobenzoic acid); DTNB], trichloroacetic acid (TCA) and thiobarbituric acid (TBA) were purchased from Biodiagnostic Co. (Giza, Egypt). N-Butanol, dipotassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4) and bovine serum albumin were

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purchased from El-Nasr Chemical Co (Cairo, Egypt). Rat transforming growth factor beta 1 (TGF-β1) and tumor necrosis factor alpha (TNF-α) ELISA kits were purchased from cloud-clone corp. (Texas, USA) and rat interleukin 6 (IL-6) ELISA kit was purchased from ImmunoBiological Laboratories IBL (Minneapolis, USA). All other chemicals were of the highest purity

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grade commercially available.

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2.2. Animals

Male albino rats (150-250 g) were obtained from Nile Co. for Pharmaceutical and Chemical

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industries (Cairo, Egypt) and acclimated for two weeks before experimentation. All animals were housed in an air-conditioned atmosphere with alternatively 12-hour light and dark cycles and

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maintained on food and water available ad libitum. Standard diet pellets were purchased from ElNasr, Abu Zaabal, Egypt including not less than 5% fiber, 20% protein, 3.5% fat, 6.5% ash and a vitamin supplement. The experimental protocol including procedures of laboratory animal care

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in research were carried out according to the Arrive guidelines and in accordance with U.K. Animals Act, 1986 and approved by the Research Ethics Committee, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt under the memorandum no. 54. All efforts were made to minimize animal suffering and reduce the number of animals used.

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2.3.

Experimental design

The duration of the experiment was 28 days, isoprenaline ISO was dissolved in saline (final volume = 0.25 ml per rat) at 5 mg/kg/day and distilled water was used to dilute the blueberry alcohol-free extract. Male Albino rats (150-200 g) were randomly segregated into six groups (ten animals per groups A and C-F and 20 animals per group B). The control group (Group A)

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received 5 ml/kg of distilled water through oral gavage once daily for 28 consecutive days. The ISO group (Group B) were given a single subcutaneous injection of ISO (5 mg/kg) per day for fourteen days and started from the 15th day of the experiment until the 28th day (Khatua et al., 2016). Each of groups C, D, and E were maintained on oral doses of blueberry extract (equivalent to 25, 50 and 100 mg /kg of dry blueberry leaf, respectively) from day 1 to day 28.

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After 1 h of the blueberry treatment, a subcutaneous injection of ISO (5 mg/kg) was given daily starting from day 15 until day 28. Group F received a single oral dose of blueberry extract (equivalent to 100 mg /kg/ dry blueberry leaf) during all the experiment period. Thereafter, animals were anesthetized with urethane and subjected to ECG recording. Blood

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samples were obtained from the retro-orbital plexus and allowed to clot. Then, samples were centrifuged, and serum collected for biochemical analyses. Animals were euthanized and tail

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lengths, body and heart weights were measured. Heart tissues were collected and homogenized in saline with the homogenate used for different biochemical parameters assessment. Heart specimens from different groups were fixed in 10% buffered formalin for histopathological and

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immunohistochemical examination. Samples were immediately frozen in liquid nitrogen and

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stored at -80º C until analysis. Time line for the experimental work flow is outlined in Figure 1.

Electrocardiography (ECG):

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ECG was recorded using Bioscience ECG recorder (Bioscience, Washington, USA). Needle

electrodes were inserted beneath the skin for the limb lead at position II. Analysis of ECG waves was done to calculate heart rate (beats/min), QRS duration (ms), QT interval (ms), which was corrected for heart rate using the formula [QTc = QT/ (square root of RR interval)] and PR interval (ms). For each parameter, measurements were done at three non-consecutive, randomly chosen points every 5 minutes recording. The results are reported as mean of the three randomly selected segments. 7

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2.5.

Determination of cardiotoxicity indices:

Heart indices were assessed using the formula: (heart weight/body weight) × 100. Serum aspartate aminotransferase (AST), creatine kinase isoenzyme-MB (CK-MB) and lactate dehydrogenase (LDH) activities were determined spectrophotometrically following the standard procedures using commercial kits obtained from Spectrum diagnostics, Cairo, Egypt (Reitman

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and Frankel, 1957). Enzyme-linked immunosorbent assay (ELISA) of the serum level of cardiac troponin I (c-TnI) was determined according to the manufacturer’s instructions developed by Life Diagnostics Inc. West Chester PA, USA.

Assessment of oxidative stress markers:

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2.6.

Extent of lipid peroxidation in myocardial tissues was evaluated by determining the level of malondialdehyde (MDA) using a thiobarbituric acid reactive substances (TBARS) assay kit (Mihara and Uchiyama, 1978). 1,1,3,3-Tetraethoxypropane was used as a standard and the MDA results were expressed as nmol of MDA/g of heart tissue. To further assess the values of the

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soluble thiols in homogenate, the reduced glutathione (GSH) was estimated according to the literature (Ellman, 1959). Briefly, in a tube 0.5 mL homogenate and 0.5 mL of 10% TCA were

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mixed thoroughly for 10 min at the room temperature followed by centrifugation of the reaction tube at 3000 rpm for 15 min. In another tube, 0.5 mL of the resultant supernatant was added to 1.0 mL phosphate buffer and 0.1 mL Ellman's reagent. The reaction tube was mixed thoroughly,

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and the absorbance recorded at 405 nm within 5-10 min against the blank and the results were expressed as mmol/g tissue. Catalase (CAT) activities in the cardiac homogenate were assessed

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per the method described by Aebi (Aebi, 1984) and expressed as U/g protein. The principle of

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determination was based on evaluation of the H2O2 decomposition rate at 240 nm.

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Histopathological examination

Autopsy samples were taken from the rats’ heart from different groups and fixed with 10%

formal saline for twenty-four hours. Washing was done with tap water then serial dilutions of alcohol were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56 ºC in hot water oven for twenty-four hours. Paraffin bees wax tissue blocks were prepared

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for sectioning at 4 μm thickness by the slide microtome. The obtained tissue sections were collected on glass slides, deparaffinized, stained by hematoxylin and eosin stains for routine examination and Masson's trichrome for demonstration of collagen fibers and further examined through the light electric microscope (Olympus BX-50 Olympus Corporation, Tokyo, Japan)

2.8.

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(32).

Assessment of inflammatory markers:

The concentration of the inflammatory markers, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were assessed in the tissue homogenates using ELISA kits according to the manufacturer’s instructions (Boulanger et al., 2003; Gao et al., 2016). The levels of both TNF-α

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and IL-6 were normalized to total protein and expressed as pg/mg protein.

Generally, quantification of both markers started with the first incubation of the pre-coated substrate specific antibody, the standards and samples onto 96-well plates. A second incubation of the biotin linked detection antibody completes the sandwich. Next, washing of the enzymatic

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reaction using a buffer and adding of 3,3',5,5'-tetramethylbenzidine (TMB) to interact with horseradish peroxidase (HRP) and visualize the interaction by producing a blue colored product that changed into yellow after adding acidic stop solution. The concentration of the captured

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analyte in the plate is proportional to the produced yellow color. The recorded optical density absorbance is read at 450 nm in a microtiterplate reader and the samples are then quantified by

Immunohistochemical detection of NF-κB(p65) and COX-2.

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2.9.

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comparing their O.D. to the standard curve.

First, heart tissue sections of 3 μm thickness were processed for paraffin embedding followed

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by rehydration in xylene and serial ethyl alcohol dilutions. Next, slides were blocked with 5% bovine serum albumin (BSA) in tris buffered saline (TBS) for 2 h. Sections were immunostained with one of the following primary antibodies; rabbit polyclonal anti COX-2 antibody (Thermo Fisher Scientific, Cat No. RB-9072-R7) or rabbit polyclonal anti NF-κB p65 antibody (Thermo Fisher Scientific, Cat No. RB-9034-P) overnight at 4º C. After incubation with the secondary antibody,

sections

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washed

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TBS,

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3,3-diaminobenzidine

tetrahydrochloride and counterstained with hematoxylin. Slides were visualized under a light 9

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microscope and the immunohistochemical quantification was performed using image analysis software (Image J, 1.46a, NIH, USA).

2.10. Evaluation of fibrosis: The tissue homogenates level of TGF-β was quantified by rat immunoassay kits according to

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the manufacturer’s instructions (cloud-clone corp. Texas, USA) and similar to the detection of TNF-α and IL-6 in section 2.7 (Cao et al., 2012). The extent of fibrosis in the heart tissues was further assessed using Masson's trichome staining for collagen fiber detection.

Determination of protein content

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2.11.

The content of protein in myocardial tissue homogenates was assessed using Lowry protein assay and bovine albumin as a standard (Lowry et al., 1951).

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2.12. Standardization of CGA in the blueberry extract:

Chlorogenic acid CGA content in the blueberry extract was analyzed using Shimadzu Nexera

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X2 series HPLC system (Shimadzu Corp, Tokyo, Japan) equipped with a photo diode array (PDA) detector and an auto-sampler using a modified method (Rodriguez-Mateos et al., 2012). The column was Luna C18 column (250 × 4.6 mm; 5 μm particle size; 23 °C; Phenomenex,

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Australia). The mobile phase consisted of (A) methanol/water/5 M HCl (5:94.9:0.1, v/v/v) and (B) acetonitrile/water/5 M HCl (50:49.9:0.1, v/v/v). The flow rate was set at 1.00 mL/min and

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the linear gradient profile was (min/% mobile phase B) 0/30, 5/40, 20/70, 39.9/75, 40/100, 50/30, 60/30. Detection of the eluent was recorded by the photodiode array at 320 nm with spectra

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obtained between 200 and 600 nm. Quantification was accomplished using a four-point regression curve (R2 ≥ 0.989) based on a genuine CGA sample. Chlorogenic acid purity was checked prior to use using NMR and ESI mass spectrophotometry (Supplementary Figs S1, S2 & S3). The standard curve was constructed utilizing peak area data, and linear least-squares regression was used to estimate the slope, intercept, and correlation coefficient (r2).

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2.13. Statistical analysis: Data are presented as mean ± SD. Multiple comparisons were performed using one-way ANOVA followed by Tukey-Kramer as a post-hoc test. When P < 0.05, a result was considered statistically significant. All statistical analyses were performed using Instat version 3 software package and graphs were sketched using GraphPad Prism (ISI® software, USA) version 5

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software. Results

3.1.

Cardiotoxicity indices:

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3.1.1. Electrocardiography (ECG), heart index and heart weight/tail length ratio:

ECG tracing showed normal properties in the control and BB only treated rats. Rats in ISPintoxicated group showed ECG changes including bradycardia and taller R and T waves compared to the control group. BB 50 mg co-treatment prevented bradycardia and tall waves

Biochemical cardiotoxicity markers:

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3.1.2.

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(Figure 2). No significant changes between groups in the other parameters.

Heart index % (ht index%) and weight/tail length ratio (wt/t) (gm/cm) significantly increased

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in the ISO-intoxicated group by 48% and 59% respectively, compared to the control group (Table 1). Pre-treatment with 25 mg/kg blueberry leaf extract slightly decreased the ht index% and wt/t by 10% compared to the ISO-intoxicated group. Meanwhile, pretreatment of intoxicated

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animals with (50 mg/kg) blueberry leaf extract significantly decrease both ht index% and wt/t by 20% and 25%, respectively (Table 1). Higher doses of blueberry 100 mg/kg maintained the ht

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index% at a comparable level of the ISO-intoxicated group, however, it showed a decrease in the wt/t compared to the ISO only treated group (Table 1). Blueberry alone did not modify the Heart index % (ht index%) and weight/tail length ratio (wt/t) relative to the control group. The AST, CKMB, LDH and troponin activities of the four groups were assessed and are presented in Table 1. The serum activities of AST, CK-MB LDH and troponin I were elevated significantly in the ISO-treated group by 113%, 157%, 53% and 169% respectively as compared

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with the control group. However, 25 mg/kg pretreatment of the blueberry extract lowered the isoprenaline-mediated induction of AST, CK-MB, LDH and c-TnI by 28%, 13%, 9% and 19% respectively as compared with the ISO-intoxicated group (Table 1). The 50 mg/kg blueberry leaf extract pretreated group showed substantial decreases in the serum levels of AST, CKMB, LDH and troponin I 52%, 53%, 43% and 46% respectively, as compared with ISO-intoxicated group.

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The 100 mg/kg blueberry group showed 59%, 12%, 8% and 41% inhibition of the activities of AST, CK-MB, LDH and c-TnI, respectively when compared with ISO-treated group as presented in Table 1. There was no significant change in the levels of all the enzymes among the blueberry-only-treated group when compared to the control group. Histopathological examination

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3.1.3.

To further assess the protective effect of the blueberry extract in the cardiac hypertrophy, the histopathological examination of the rat heart tissues was performed for all the groups. Control group and the blueberry only treated group showed normal histological structure of the myocardium (Figure 3A and 3F, respectively). Hematoxylin and eosin–stained heart sections

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from the ISO-treated group showed an increased cardiomyocyte cross-sectional area as well as induction of interstitial fibrosis (Figure 3). Pre-treatment of 25 mg/kg and 50 mg/kg blueberry

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displayed normal myocardial bundles with no evidence of heart tissues injuries (Figure 3C and 3D respectively). Nevertheless, pre-treatment of 100 mg/kg blueberry with ISO showed variable degenerative changes in cardiac muscles fibers focal inflammatory cells infiltration and

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haemorrhage (Figure 3E).

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3.2.1. Oxidative stress markers: Isoprenaline-induced cardiotoxicity in rats was evaluated by determination of the levels of GSH, lipid peroxidase and catalase levels with comparing to the control group. As displayed in Table 2, ISO-intoxicated group displayed a substantial reduction in GSH by 52% and in CAT by 35%, however, MDA level increased by 26% compared to control group. Pre-treatment with 50 mg/kg blueberry together with isoprenaline maintained GSH, MDA and CAT to a very similar level in

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the control group. In addition, animals treated with blueberry only elicited statistically no difference when compared to the control group (Table 2).

3.2.2. Assessment of Inflammatory mediators:

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The levels of IL-6 and TNF-α were assessed to address the effect of the isoprenaline-induced cardiotoxicity on the inflammatory mediators (Figure 4A, 4B). Isoprenaline-intoxicated group showed around 2 and 3-fold increase in IL-6 and TNF-α, respectively when compared to control group (Figure 4A, 4B). Pre-treatment with 50 mg/kg blueberry leaf extract, compared to isoprenaline-intoxicated group, decreased the tissue levels of IL-6 and TNF-α significantly and maintained comparable levels of the control group. Expression of IL-6 and TNF-α were not

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altered in the blueberry-only treated group compared to control values.

3.2.3. Immunohistochemical detection of NF-κB (p65) and COX-2:

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Detection and quantification of the cardiac tissues NF-κB were evaluated using immunohistochemical staining by detecting the activated p65 subunit (Figure 5A-E). The

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control group indicated low level of p65, however ISO-intoxicated group showed 272% rise in p65 levels compared to the control group (Figure 5E). Pre-treatment with 50 mg/kg Blue berry (BB) inhibited the p65 content by 53%, compared to the ISO-intoxicated group (Figure 5C).

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Blue berry (BB) only treated group showed no change when compared to the control group (Figure 5D). Immunohistochemical detection and quantitation of the proinflammatory mediator

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COX-2 were assessed and the results are depicted in Figure 5F-J. Control group displayed minimal immunostaining for COX-2 (Figure 5F), while ISO group showed intense brown

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staining of COX-2 with an increase of 390% of the myocardial COX-2 expression (Figure 5G), when compared to control group (Figure 5J). COX-2 immunohistochemical staining in the 50 mg/kg blueberry pre-treated group showed increase in the COX-2 expression by 25%, when compared to ISO-intoxicated group (Figure 5H). No change in the enzyme expression was observed in the group treated with BB alone (Figure 5I).

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3.2.4. Evaluation of fibrosis: Evaluation of tissue TGF-β in animals showed a 3-fold increase in ISO-intoxicated group when compared to control group (Figure 6F). Concomitant treatment with 50 mg/kg/day blueberry extract prevented this elevation and maintained tissue TGF-β near to control levels (Figure 6F). On the other hand, there was no significant change in cardiac TGF-β content in animals treated

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with the extract. Liver fibrosis was further assessed using Masson's trichome staining for collagen fiber detection and the stained cardiac hypertrophic sections showed distinct blue collagen fibers and red myocytes Figure 6A-D. Masson staining showed normal collagen fibers appearance in control and blueberry groups (Figure 6A and 6D). In contrast, ISO-group (Figure 6B) revealed a marked collagen accumulation and fibrosis in the heart stained tissues, while

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administration of blueberry 50 mg/kg dose reduced the degree of cardiac fibrosis (Figure 6C). Morphologic analysis of ISO-intoxicated group elicited a 150% increase in the mean density of collagen fibers/μm2 surface area of cardiac tissues compared to control group (Figure 6E). Pretreatment of 50 mg/kg blueberry along with ISO induced a significant decrease in the mean fibre density by 38% as compared to the ISO-intoxicated group (Figure 6E). Treatment of animals

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control group (Figure 6E).

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with BB alone showed no significant effect on mean density of collagen as compared to the

3.2.5. Standardization of CGA in the blueberry extract:

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The blueberry leaf extract was analysed by HPLC and recoveries were calculated based on peak areas of the chlorogenic acid (Figure 7). The chlorogenic acid standard was used to identify the

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peak of the chlorogenic acid and to construct the calibration curve (elution time:43.86 min). Representative chromatograms of chlorogenic acid standard and chlorogenic acid content in the

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blueberry extract are illustrated in Figure 7 A and B, respectively. Quantitation of chlorogenic acid in the used BB leaf extract was calculated with a yield of 13.6±0.04 mg CGA per 100 mg leaf extract. The LOD and LOQ of HPLC analysis were calculated by the residual standard deviation of a regression line and were found to be 0.034 and 0.103 mg/ml, respectively.

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4.

Discussion: Our aim in the current study was to investigate the effect of pre-treatment of the blueberry

nutraceutical leaf extract in ISO-induced cardiac hypertrophy in rats and to elucidate the underlying molecular mechanisms. The cardioprotective mechanisms of the blue berry (BB)

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extract were investigated including its effect on different oxidative stress, inflammatory as well as fibrotic markers. Firstly, ISO-induced cardiac hypertrophy was assessed by ECG, biochemical analysis of serum cardiotoxicity indices and histopathological examination of heart tissues. It was reported that, the model of cardiac hypertrophy is associated with an ECG pattern characterized by increased voltage, widened QRS complex, prolonged QTc interval, and LV

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strain (T-wave inversion and ST depression) (Sorensen et al., 2016).

Several ECG abnormalities in the ISO-intoxicated group were noticed in the form of bradycardia, increase R-voltage, but pre-treatment with BB 50 mg/kg significantly reduced ISO induced ECG abnormalities.

Surprisingly, heart rate in the ISO-intoxicated group was lower compared to the control

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group. This observation is in line with previous findings generating cardiac hypertrophy models in rats using isoprenaline (15 μg/g/day, s.c. for 7 days) (Gava et al., 2004; Heap et al., 1996). A

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baroreceptor mediated reflex bradycardia could be the cause of the decreased heart rate in the ISO-intoxicated group. This reflex bradycardia might be initiated by the release of noradrenaline

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in the isolated tissue, which subsequently stimulates cardiac contractility and ultimately increases heart rate and blood pressure. This rise in blood pressure induces a reflex rise in vagal activity by

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stimulating baroreceptors, resulting in reflex bradycardia, which could be sufficient to counteract the local action of noradrenaline on the heart (Finkel et al., 2009). Down regulation of adrenoceptors under these conditions has been reported in some studies (Hogikyan and Supiano,

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1994) but the exact mechanism responsible for the decreased heart rate remains unclear. ISO intoxicated group showed tall T wave, this can be explained by the myocardial strain secondary to hypertrophy as in some cases with acute ST elevation myocardial infarction (MI), the T wave is initially tall and is called hyperacute T wave changes. This is in contrast to other studies which showed inversion of T wave in rat ECG after injection of isoprenaline (5-80 mg/kg for 2 days or several weeks) (Hill et al., 1960).

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Additionally, ISO-induced myocardial injury was further manifested by significant elevation in the activities of serum CK-MB, c-TnI, AST and LDH enzymes; which are released from damaged myocytes and considered as sensitive indicators of cardiac injury. The increase in activity of these enzymes in ISO-administered rats is in agreement with previous studies (Akila and Vennila, 2016; Ghule et al., 2009). In the current study, ECG changes were dose

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dependently disappearing, reaching the optimal effect with the dose of 50 mg/kg blueberry. In addition, the only dose of the blueberry leaf extract that caused a significant reduction in serum CK-MB, c-TnI, AST and LDH levels was 50 mg/kg. The mentioned ECG abnormalities and biochemical data were further supported by the histopathological examination of cardiac tissues.

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Extensive damage was observed in cardiac tissue of the ISO-intoxicated group in the form of a focal hyalinization with inflammatory cell infiltration in the myocardium and a focal area, which showed proliferation of myofibroblasts to replace the infarction. These histopathological changes were reported to typically be present in ISO-induced cardiac hypertrophy of rats (Kannan and Quine, 2013). On the other hand,

in the current study blueberry extract

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pretreatment significantly ameliorated ISO-induced ECG changes and significantly inhibited elevations of serum CK–MB, c-TnI, AST and LDH enzymes activities. The severity of cardiac

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changes was gradually ameliorated in intoxicated groups pretreated with different doses of blueberry, reaching preserved normal myocardium architecture at the dose of 50 mg/kg. This

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data suggests blueberries have a protective role against ISO-induced cardiac hypertrophy and the most appropriate dose was 50 mg/kg as it almost preserved the normal architecture of the heart.

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Thus, dose 50 mg/kg/day was selected for further investigation in the mechanistic study. In the next step, we explored the cardioprotective mechanisms of blueberries by studying

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different markers of oxidative stress, inflammation and myocardial fibrosis. One of the main contributing factors in the development of myocardial hypertrophy is oxidative stress (Maulik and Kumar, 2012; Zong et al., 2013). Oxidative stress is fundamentally due to generation of cytotoxic free radicals that stimulate lipid peroxidation in heart tissue and subsequently initiate peroxidation of membrane bound polyunsaturated fatty acids. This would lead eventually to damage of the structural and functional integrity of the myocardium with consequent changes in membrane permeability (Dhalla et al., 1992). Further, enhanced free radical formation and lipid 16

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peroxide accumulation have been proposed as a possible biochemical mechanism for myocardial damage and cardiac hypertrophy (Ennis et al., 2003). It was reported that ISO-induced cardiotoxicity is due to its imitation of the oxidative stress cascade (Katare et al., 2017). Oxidative stress originating from ISO injection is mediated primarily by β1-adrenergic receptors (Katare et al., 2017). The most abundant reactive oxygen species generated in living

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cells are superoxide anion and its derivatives, particularly the highly reactive and damaging hydroxyl radical, which induces peroxidation of lipid cell membranes (Hemnani and Parihar, 1998). Furthermore, stimulation of β1-adrenergic receptors rapidly generates ROS as well as depresses total cellular antioxidant enzymes capacity (Wheatley et al., 1985). Significant reductions in cardiac tissue levels of protective endogenous antioxidant enzymes, such as CAT,

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have been observed in ISO-induced cardiac disease and therefore, reducing the ability of cardiac cells to inactivate ROS (Choudhary et al., 2006; Remião et al., 2001; Wheatley et al., 1985). This is presumably because CAT is considered as the first line of cell defense against oxidative stress mediated cardiac injury, where CAT subsequently converts hydrogen peroxide to water (Chaiswing et al., 2005).

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In the current study, administration of ISO was associated with a significant increase of lipid peroxidation products (MDA) in cardiac tissues, depleted GSH levels and decreased antioxidant

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enzyme activities suggesting an increase in oxidative stress. On the other hand, daily blueberry supplementation at 50 mg/kg dose for 14 days effectively reduced these alterations in ISO-

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induced cardiac pathophysiology. This strongly supports the antioxidant and tissue protective effect of the blueberry leaf extract. These results are consistent with previous studies that

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reported the involvement of oxidative stress and lipid peroxidation in ISO-induced cardiac hypertrophy and cardiotoxicity (Banerjee et al., 2003; Rathore et al., 1998). Administration of blueberry leaf extract improved the biochemical markers, indicating decreased oxidative stress,

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manifested as increased GSH concentration and CAT activity with decreased MDA concentration compared with ISO treatment alone. The current results are in line with the work of other investigators (Gayathri et al., 2011; Kannan and Quine, 2013; Panda and Naik, 2009). Oxidative stress can also activate redox sensitive transcription factors, such as NF-κB which can eventually develop cellular inflammatory responses (Rahman et al., 2002). It was reported that NF-κB activation plays a vital role in the development of cardiac hypertrophy through 17

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stimulation of the cardiomyocytes hypertrophic effect (Li et al., 2004; Pagani et al., 2003; Yamamoto et al., 2001). NF-κB is a transcription heterodimer factor that usually consists of p65 and p50 subunits of the Rel protein family that maintains the inflammatory and immune responses within the host cells (Ali and Mann, 2004). Extracellular stimuli such as inflammation, oxidative stress and infection can activate NF-κB dimer subunits in the cytoplasm via

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proteasome degradation of the inhibitory protein IκB. The activated NF-κB can transport from the cytoplasm to the nucleus where it induces the transcription of cellular genes including IL-6, TNF-α and COX-2, which are pivotal for the inflammatory response (Ali and Mann, 2004; Elsharkawy and Mann, 2007; Hoffmann et al., 2006). ISO was reported to develop a series of inflammatory reactions in the myocardial cells by promoting NF-κB expression and enhancing

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proinflammatory cytokines production. These cytokines can lead to distinctive pathological disorders including biventricular fibrosis and development of cardiac myopathy (Li et al., 2014). In the current study, ISO-intoxication induced a significant increase of cardiac NF-κB, TNF-α and COX-2 expression together with significant elevation in tissue levels of IL-6 indicating amplified inflammatory responses. On the contrary, pretreatment of intoxicated animals with

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blueberry extract significantly decreased expression of NF-κB and therefore inhibited the downstream inflammatory cascade as evidenced by the reduced expression of COX-2 as well as

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cardiac levels of TNF-α and IL-6, so CGA in blueberry extract can provide satisfactory antiinflammatory effects. Our results coincided with previous studies, which have reported CGA is a

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potential inhibitor of NF-κB and can further impede the production of downstream inflammatory cytokines (Lau et al., 2007; Li et al., 2014).

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Transforming Growth Factors (TGF-βs) are pleiotropic cytokines that play a pivotal role in cardiac remodeling and fibrosis in hypertrophic cardiomyopathy (Dobaczewski et al., 2011).

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Previous in vivo experiments in transgenic mice indicated overproduction of TGF-β, which leads finally to myocardial hypertrophy and fibrosis (Leask, 2010). β-adrenoreceptor stimulators such as ISO were reported to induce TGF-β as a causal factor in development of hypertrophic responsiveness (Schluter et al., 1995). The result of this study indicated an increase in the level of TGF-β in ISO-intoxicated group when compared to control group. However, concomitant treatment with blueberry leaf extract (50 mg/kg/day) maintained the TGF-β level to control level. Masson's trichromic stain histopathological examination of collagen fibers supported these 18

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findings. ISO induced myocardial fibrosis is reported via increasing of the collagen accumulation in the heart tissues (Benjamin et al., 1989; Lin et al., 2016) and this is in line with our observation. Our results suggest that BB can ameliorate the effect of the collagen accumulation induced by ISO may be by possessing anti-inflammatory activity and increasing of the myocardial antioxidant capacity.

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In conclusion, the present study has indicated for the first time the protective role of blueberry nutraceutical leaf extract against ISO-induced cardiac hypertrophy. The data indicated that pretreatment of ISO-intoxicated rats with BB extract alleviates the ISO-induced cardiotoxicity as evidenced by lowering myocardial injury markers and preventing ECG abnormalities and histopathological changes. The underlying cardioprotective effect of the BB

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extract proceeded more likely via controlling the cardiac oxidative stress by maintaining the oxidative stress markers to their normal values. In addition, the protective effect of the BB extract was possibly due to suppressing ISO-induced inflammatory responses and abrogating the resultant fibrosis. Our study implies that BB effectively protects against ISO-induced

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Disclosure of Conflict of interest

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hypertrophy by regulating oxidative stress and the resulting inflammation and fibrosis.

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Funding

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The authors have read the journal's policy on disclosure of potential conflicts of interest and they all wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Acknowledgment

The authors would like to thank Mr Nicholas M. Butler, School of Chemistry, University of Wollongong, Wollongong, Australia for proofreading the manuscript.

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5.

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Wheatley, A.M., Thandroyen, F.T., Opie, L.H., 1985. Catecholamine-induced myocardial cell damage: catecholamines or adrenochrome. J. Mol. Cell. Cardiol. 17, 349-359. Yamamoto, K., Ohki, R., Lee, R.T., Ikeda, U., Shimada, K., 2001. Peroxisome proliferator-activated receptor γ activators inhibit cardiac hypertrophy in cardiac myocytes. Circulation 104, 1670-1675. Zong, J., Deng, W., Zhou, H., Bian, Z.-y., Dai, J., Yuan, Y., Zhang, J.-y., Zhang, R., Zhang, Y., Wu, Q.-q., 2013. 3, 3′-Diindolylmethane protects against cardiac hypertrophy via 5′-adenosine monophosphateactivated protein kinase-α2. PLoS One 8, e53427.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Disclosure: The authors declare no conflict of interest.

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Figure Legends:

Figure 1. Time line for the experimental work flow

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Figure 2. Effect of the blueberry leaf extract on ECG pattern, in ISO-intoxicated rats. (A)

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ECG graph. (B) Heart rate (beat/min), (C) T wave amplitude (mV), (D) R wave amplitude (mV). Data are represented as mean ± SD. * or #: Statistically significant from the control or ISO

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intoxicated group respectively at P < 0.05 using one-way analysis of variance ANOVA followed

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by Tukey-Kramer as a post-hoc test.

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Figure 3. Effect of the blueberry leaf extract on ISO-induced histological alterations of the heart tissue (x 100).

Photomicrographs of haematoxylin and eosin stained sections of heart depicting (A) control

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group, (B) ISO treated group (5 mg/kg), (C) BB (25 mg/kg) + ISO (5 mg/kg) treated group, (D) BB (50 mg/kg) + ISO (5 mg/kg) treated group, (E) BB (100 mg/kg) + ISO (5 mg/kg) treated group, (F) BB treated group (100 mg/kg) (A) & (F) show normal histoarchitecture of the rat heart. (B) show that ISO induced cytoplasmic vacuolization (arrowheads) and inflammatory cell infiltration (solid arrows) (D) shows that BBpretreatment prevented the cardiomyocyte damage induced by ISO and ameliorated

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inflammatory cell infiltration but the higher dose of BB (E) and the lower one (C) where there

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was still some inflammatory cell infiltration.

Figure 4. Effect of the blueberry leaf extract on the IL-6 (A) and TNF-α (B) of the ISO-

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induced cardiotoxicity in rats. Data are the mean ± SD (n = 10). * or #: Data are represented as mean ± SD. * or #: Statistically significant from the control or ISO intoxicated group respectively at P < 0.05 using one-way analysis of variance ANOVA followed by Tukey-Kramer as a post-hoc test. IL-6, Interleukin 6; TNF-α, Tumor necrosis factor-alpha.

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Figure 5. Expression of nuclear factor kappa B (NF-κB) and cycloxygenase-2 (COX-2) by immunoistochemical staining: Photomicrographs of histological sections of heart depicting (A)

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and (E) expression of NF-κB and COX-2 respectively in the control group (B) and (F) expression of NF-κB and COX-2 respectively in the ISO treated group (5 mg/kg), (C) and (G)

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expression of NF-κB and COX-2 respectively in the BB (50 mg/kg) + ISO (5 mg/kg) treated group, (D) and (H) expression of NF-κB and COX-2 respectively in the BB treated group (100 mg/kg), (I) and (J) Quantitative image analysis for immunohistochemical staining of NF-κB and

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COX-2, respectively, expressed as optical densities (OD) across 10 different fields for each rat section. Data are represented as mean ± SD. * or #: Statistically significant from the control or

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ISO intoxicated group respectively at P < 0.05 using one-way analysis of variance (ANOVA) followed by Tukey-Kramer as a post-hoc test. Pretreatment of BB extract (50 mg/kg) reduced

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both NF-κB and COX-2 expression as compared to ISO group, significantly. However, there was no statistically difference in the NF-κB and COX-2 expression levels in the BB only treated group as compared to the control group.

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Figure 6. Effect of BB extract on ISO-induced histological alterations of the heart tissue stained by Masson's trichrome (A-E) and effect of the blueberry leaf extract on the

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myocardial TGF-β (F). (A) control group, (B) ISO treated group (5 mg/kg), (C) BB (50 mg/kg) + ISO (5 mg/kg) treated group, (D) BB treated group (100 mg/kg), (E) The mean density of

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collagen fibers distribution/μm2 surface area of heart tissues in different studied groups, and (F) TGF-β content pg/mg protein. Data are represented as mean ± SD. * or #: Statistically significant from the control or ISO intoxicated group respectively at P < 0.05 using one-way analysis of

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factor beta.

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variance (ANOVA) followed by Tukey-Kramer as a post-hoc test. TGF-β, Transforming growth

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Figure 7. HPLC chromatograms of (A) chlorogenic acid standard, and (B) blueberry leaf extract chlorogenic acid. The HPLC-UV detection wavelength was set at 320 nm for profiles A and B.

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Graphical Abstract. Schematic diagram of potential markers involved in ISO-induced cardiac

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hypertrophy mechanism

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Tables Table 1. Effect of pre-treatment of 25, 50 and 100 mg/kg blueberry on AST, CK-MB, LDH and c-TnI levels in isoprenaline-induced cardiotoxicity in rats.

CK-MB (IU/L)

0.31 ± 0.03b

ISO

0.46 ± 0.04a

0.051 ± 0.006a 128 ± 32a

816 ± 182a 1002 ± 53a

0.722 ± 0.074a

BB (25) + ISO

0.44 ± 0.05a

0.046 ± 0.008a

92 ± 22b

714 ± 96a

908 ± 58a

0.588 ± 0.102a

BB (50) + ISO

0.37 ± 0.02b

0.038 ± 0.006b

62 ± 10b

380 ± 78b

659 ± 97b

0.390 ± 0.056b

BB (100) + ISO

0.45 ± 0.07a

0.040 ± 0.007b

52 ± 12b

716 ± 117a

923 ± 52a

0.428 ± 0.065a,b

BB

0.32 ± 0.03b

0.035 ± 0.004b

589 ± 63b

0.394 ± 0.063b

62 ± 14b

LDH (IU/L)

c-TnI (ng/mL)

657 ± 58b

0.268 ± 0.044b

CR IP T

Control

Heart wt./Tail AST length (IU/L) (gm/cm) 0.032 ± 0.007b 60 ± 6b

317 ± 63b

AN US

Heart index (%)

390 ± 55b

ED

M

Data are the mean ± SD (n = 10). a or b: Significantly different from the control or ISO group respectively at P < 0.05 using one-way ANOVA followed by Tukey-Kramer as a post-hoc test.GSH, reduced glutathione; MDA, malondialdehyde; CAT, catalase.

Table 2: Oxidative stress markers of rats pretreated with 50 mg/Kg blueberry extract. MDA nmol/g tissue

CAT U/g protein

0.52 ± 0.065b

30.19 ± 6.73b

0.086 ± 0.008b

0.28 ± 0.029a

40.78 ± 5.57a

0.056 ± 0.003a

BB (50 mg /kg) + ISO

0.54 ± 0.046b

29.38 ± 4.59b

0.075 ± 0.007b

BB

0.47 ± 0.078b

26.05 ± 5.02b

0.093 ± 0.007b

Control

AC

CE

ISO

PT

GSH mmol/g tissue

Data are the mean ± SD (n = 10). a or b: Significantly different from the control or ISO group respectively at P < 0.05 using one-way ANOVA followed by Tukey-Kramer as a post-hoc test.GSH, reduced glutathione; MDA, malondialdehyde; CAT, catalase.

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