Naringin Mitigates Cardiac Hypertrophy by Reducing

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peritoneal injection of 65 mg/kg BW of streptozotocin. Groups III, ... of ligands with membrane receptors, intermediary molecules, ... Received for publication May 3, 2015; accepted September 9, 2015. From the ... were considered diabetic and included in the study (Table1). .... 160 6 3.46. 207.2 6 ..... Mol Nutr Food Res. 2005 ...
ORIGINAL ARTICLE

Naringin Mitigates Cardiac Hypertrophy by Reducing Oxidative Stress and Inactivating c-Jun Nuclear Kinase-1 Protein in Type I Diabetes A. Olubunmi Adebiyi, MSc, Oluwafeysetan O. Adebiyi, MSc, and Peter M. O. Owira, PhD

Abstract: Cardiac hypertrophy (CH) in type 1 diabetes mellitus is attributed to increased oxidative stress–associated activation of c-Jun Nuclear Kinase (JNK). We investigated the effects of naringin on hyperglycemia-associated oxidative stress, activation of JNK-1, and CH. Male Sprague-Dawley rats (225–250 g) (n = 7) were divided into 6 groups. Groups I and II were orally treated with distilled water [3.0 mL/kg body weight/day (BW)] and naringin (50 mg/kg BW), respectively. Groups III-VI were rendered diabetic by a single intraperitoneal injection of 65 mg/kg BW of streptozotocin. Groups III, IV, and V were further treated with insulin (4.0 I.U, s.c, twice daily), naringin (50 mg/kg BW), and ramipril (3.0 mg/kg BW), respectively. After 56 days, the animals were sacrificed and then plasma and cardiac tissues obtained for further analysis. Naringin treatment of diabetic rats significantly reversed oxidative stress, lipid peroxidation, proteins oxidation, CH indices, and JNK protein activation compared with untreated diabetic animals. Our results do suggest that naringin mitigates CH by inhibiting oxidative stress leading to inactivation of JNK-1. Naringin supplements could therefore ameliorate CH in diabetic patients. Key Words: diabetes, oxidative stress, cardiac hypertrophy, naringin (J Cardiovasc Pharmacol  2016;67:136–144)

INTRODUCTION Cardiac hypertrophy (CH) is a compensatory response of the myocardium to stressors aimed at normalizing and optimizing cardiac output.1,2 CH is characterized by enhanced cardiomyocyte growth, increased protein synthesis, sarcomere reorganization, and increased fetal genes expression.1,2 CH has previously been reported in type 1 diabetic patients and is a major cause of increased cardiovascular morbidity and mortality in the diabetic population.3–6 Received for publication May 3, 2015; accepted September 9, 2015. From the Department of Pharmacology, Discipline of Pharmaceutical Sciences, School of Health Sciences, University of KwaZulu-Natal, Durban, South Africa. Supported in part by The Medical Research Council of South Africa provided. The authors report no conflicts of interest. Reprints: Peter M. O. Owira, PhD, Department of Pharmacology, Discipline of Pharmaceutical Sciences, School of Health Sciences, University of KwaZulu-Natal, Westville Campus, Private Bag X5400 Durban 3629, South Africa (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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The development and progression of CH is dependent on pro-hypertrophic factors and antihypertrophic factors.2 It involves complex molecular processes requiring interactions of ligands with membrane receptors, intermediary molecules, and generation of transcription factors culminating in increased cardiomyocyte growth.1 Other noted contributors to the hypertrophic process include adaptive changes induced by increased hemodynamic stress,7 reduction of functional contractile cardiomyocytes,8 and increased activation of neuro-humoral factors.9 The general mechanism of cardiac hypertrophy requires an initiating hypertrophic stimulus (endothelin-1, angiotensin-II (Ang. II), norepinephrine, volume overload, and pressure overload).2 Hyperglycemia induces renin– angiotensin–aldosterone system, which increases the local generation of Ang. II that contributes significantly to the hypertrophic process.1,2,10 The hypertrophic stimulus through activation of NADPH oxidase and increased mitochondrial generation of reactive oxygen species (ROS) activates hypertrophic signaling kinases such as protein kinase C and mitogen-activated protein kinases [such as Extracellular signal-related kinases and Jun nuclear kinase (JNK)],11,12 which enhance transcription of hypertrophic factors.1,13 NADPH oxidase–dependent ROS signaling is central to the development of hypertrophic changes in the myocardium,6 acting as second messengers in the hypertrophic signaling pathways.1 Additionally, ROS induce oxidative damage in the myocardium through the interaction with biomolecules, including lipids, proteins, and nucleic acids.14 Enzymatic antioxidants such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) detoxify these ROS, thus limiting oxidative damage.6 However, these enzymatic antioxidants exhibit reduced expression and activity under diabetic conditions6,15 because of increased oxidative stress triggered by hyperglycemia.16 Hyperglycemia triggered–oxidative stress occurs through multiple mechanisms, including increased glucose autoxidation, increased metabolism of glucose through alternative metabolic pathways, increased formation of advanced glycation endproducts, and local activation of renin–angiotensin–aldosterone system.6 Bioactive chemical compounds that possess antioxidant activities coupled with ability to suppress maladaptive signaling might be of therapeutic benefit in oxidative stress–induced cardiac hypertrophy. Naringin (4,5,7-trihydroxyflavanone-7rhamnoglucoside), a dietary bioflavonoid derived from grapefruit and related citrus species, has been shown to possess

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a myriad of pharmacological actions that include antioxidant, free radical scavenging actions, antimicrobial, antiinflammatory, antiapoptotic, and antimutagenic amongst others.17 Naringin has been suggested to exert its reported cardio-protective effects by limiting oxidative stress in isoproterenol-provoked myocardial infarction and doxorubicin-induced cardiotoxicity.18,19 Antioxidant effects of naringin have previously been attributed to its radical scavenging actions, which are due to the presence of phenolic moiety in its chemical structure.20 Furthermore, naringin exhibits vitamin E–sparing effects and increases antioxidant enzyme gene expression.21–23 There is dearth of information on the effect of naringin on diabetes-induced cardiac hypertrophy and the downstream hypertrophic process. We therefore sought to investigate if antioxidant effects of naringin could reverse cardiac hypertrophy in type I diabetes mellitus by reducing oxidative stress–associated JNK-1 activation.

METHODS Experimental Animals The research was approved by the Biomedical Research Ethics committee of the University of KwaZulu-Natal (008/ 14/Animal), and institutional protocols on the care and handling of animals were strictly adhered to. Forty-Two male Sprague-Dawley rats (Rattus Norvegicus) (235 6 15 g) obtained from the Biomedical Resource Unit were studied. They were randomly allotted into 6 groups (groups I-VI, n = 7), housed in well-ventilated standard rat cages at the animal holding unit of the Biomedical Resource Unit, and were allowed 1 week to acclimatize to their new environment before commencement of the study. They were maintained under controlled conditions [Temperature (To) = 23 6 28C, controlled humidity 55 6 5%, and 12 hours dark/light cycle (05.00–17.00)] and were allowed free access to standard commercial rat chow and drinking water ad libitum.

Chemicals Naringin, n-butanol, phosphoric acid, thiobarbituric acid, butylated hydroxytoluene, and D-glucose were purchased from Sigma Aldrich, Johannesburg, South Africa. Ramipril (Novartis, South Africa), insulin (Novo Nordisk, Norway), superoxide dismutase assay kit, glutathione peroxidase assay kit, and carbonyl stress assay kit were obtained from Cayman Chemicals, USA. Insulin detection kit (DRG, Germany) and standard glucometer (one touch, CA), (Cayman chemicals).

Methods Animal Treatment Age- and weight-matched controls (groups I and II) received 0.2 mL of 0.1 M fresh citrate buffer alone by a single intraperitoneal injection. Type I Diabetes mellitus was induced by intraperitoneal injection of streptozotocin (STZ) [65 mg/kg body weight (BW)] dissolved in 0.2 mL of 0.1 M freshly prepared citrate buffer (pH 4.5) after an overnight fast in groups III, IV, V, and VI. Diabetes was confirmed 72 hours after STZ injection by measuring fasting blood glucose using Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

Naringin Reduces Cardiac Hypertrophy

a standard glucometer (one Touch select, Milpitas, CA) through tail prick sampling after an overnight fast. STZtreated rats with fasting blood glucose levels over 8.0 mmol/L were considered diabetic and included in the study (Table1). Rats in groups I and II were subsequently treated with 3.0 mL/kg of distilled water and 50 mg/kg of naringin,24 respectively by oral gavage daily for 8 weeks. The diabetic rats, groups III, IV, V, and VI were also treated with subcutaneous insulin (4 IU/kg twice daily), orally administered naringin (50 mg/kg), distilled water (3.0 mL/kg), and ramipril (3.0 mg/kg) respectively over 8 weeks. The rats were weighed daily and at the end of the study (day 57) after an overnight fast, FBG was measured and animals euthanized by halothane overdose. Blood was obtained by cardiac puncture, and the plasma was separated by centrifugation of blood at 3000 rpm for 10 minutes and aliquots of the supernatant stored at 270 8C for biochemical analysis. The hearts were excised, washed in ice-cold phosphate buffered saline, blotted dry, and weighed. The left ventricles were dissected free and weighed. The heart tissues were subsequently rapidly snap-frozen in liquid nitrogen and stored at 270 8C for further analysis.

Fasting Plasma Insulin Assay Fasting plasma insulin levels were assayed by EnzymeLinked Immunosorbent Assay (ELISA) using commercially available ultrasensitive rat insulin ELISA kit (DRG diagnostics, Germany), according to the manufacturer’s instructions.

Thiobarbituric Acid Reactive Substances Assay Plasma and cardiac tissue thiobarbituric acid reactive substances assay were carried out as previously described by Halliwell and Chirico25 with some slight modifications. Briefly, 100 mg of left ventricular tissue was homogenized in 500 mL of 0.2% phosphoric acid (H3PO4) and centrifuged at 1600g for 5 minutes at 48C. Five hundred micro litter of 2% phosphoric acid, 400 mL of 7% phosphoric acid, and 400 mL of BHT/TBA solution were added sequentially to 200 mL of plasma samples or cardiac tissue supernatant in a set of clean glass test tubes. The reactions were initiated with 400 mL of 1.0 M HCl. All tubes were incubated in vigorously boiling water bath (1008C) for 15 minutes and cooled at room temperature. n-butanol (1.5 mL) was added to each tube and thoroughly mixed and allowed to settle down. Two hundred microliters (200 mL) of the top phase was transferred to a 96-well microplate in triplicates and read at 532 nm and 600 nm using a Nanostar microplate reader (CA). The difference in absorbances measured at 600 nm and 532 nm was determined and used in calculating the plasma and cardiac concentration of malondialdehyde (MDA) using an extinction coefficient of 1.56 · 105 cm21 M21, and the MDA concentrations were expressed in nanomolar unit.

Carbonyl Stress Assay The plasma and left ventricular tissue carbonyl stress assay were carried out by using commercial carbonyl assay kit (Cayman chemical) according to the manufacturer’s instruction. The carbonyl protein concentrations were expressed as nmol/mg. www.jcvp.org |

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Adebiyi et al

TABLE 1. Animal treatment protocol Groups I (CTR) II (NRN) III (INS) IV (NRN-DM) V (DM) VI (RMP-DM)

Streptozotocin (mg/kg)

Citrate Buffer (mLs)

Distilled Water (mL/kg per BW day)

NRN (mg/kg BW/day)

Insulin (I.U/day)

Ramipril (mg$kg21$d21)

— — 65 65 65 65

0.2 0.2 0.2 0.2 0.2 0.2

3.0 — — — — —

— 50 — 50 — —

— — 8.0 — — —

— — — — — 3.0

CTR, control; DM, diabetes mellitus; INS, insulin; NRN, naringin; RMP, ramipril.

Superoxide Dismutase Tissue Activity Assay The total SOD (cytosolic and mitochondrial) was assayed using commercially available ELISA kit (Cayman chemicals). Activity was expressed in mM/mL.

Glutathione Peroxidase Tissue Activity Assay GPx tissue activities were also assayed by using commercially available ELISA kit (Cayman chemicals). The cardiac tissue activity was expressed in nmol/mL per minute.

Histology

Left ventricular (LV) sections fixed in 10% neutral buffered formalin were embedded in paraffin, cut into 5 mmthick sections, and stained with hematoxylin and eosin (H&E). The sections were scanned on the Olympus compound microscope X451 (Hitachi, Japan) and analyzed using NIS-300 software (Version 3). Histological analysis of cardiomyocyte diameters were carried at ·400 magnification using 10 fields per slide (n = 4). Results are expressed in micromolar unit.

c-Jun Nuclear Kinase-1 Protein Expression The expression of JNK protein was determined by Western blot analysis. Briefly, 100 mg of LV tissue homogenized in 900 mL of ice-cold RIPA buffer on ice was centrifuged at 12,000 rpm at 48C for 20 minutes. The total protein concentrations in the supernatant were determined by the Bradford protein assay method.25 Samples were adjusted for equal loading and diluted with SDS sample buffer containing 5% b-mercaptoethanol (1:1) and denatured at 958C for 5 minutes on a dry heat block. Equal amounts of protein (20 mg) were loaded on 10% SDS polyacrylamide gel and were allowed to run at 150 V for 90 minutes. The separated

samples on the gel were transferred to 0.45 mM nitrocellulose membranes in a wet transfer system (100 V for 1 hour). Nitrocellulose membranes were blocked with TBS-T containing 3% BSA for 1 hour at room temperature with constant agitation. The membranes were incubated with a rabbit polyclonal JNK-1 antibody (1:200 dilution) and a p-JNK-1 antibody (Santa Cruz biotechnology, Santa Cruz) overnight at 48C. The membranes were washed thrice and incubated with a secondary horseradish peroxidase–linked antirabbit antibody (1:5000 dilution) (Santa Cruz biotechnology, Santa Cruz) for 1 hour. The membrane was further washed 3 times with TBS-T and visualized in Chemi-Doc (Biorad, Hercules) after development with Biorad Lumiglo reagent. The relative densities of the bands were analyzed with Image Lab software.

Statistics

All data were presented as mean 6 SEM. Statistical analyses between groups were determined by Graphpad statistical software version 5 using either unpaired student t-test or 1-way analysis of variance followed by multiple comparison Student Newman–Keuls test. A P value of 5% (P , 0.05) was considered statistically significant.

RESULTS Effects on Body Weight and Blood Glucose Regulation

The untreated diabetic rats were significantly (P , 0.05) hyperglycemic, hypoinsulinemic, and they exhibited significant (P , 0.05) weight loss compared with controls (Table 2). However, naringin and insulin treatment of diabetic rats showed significant (P , 0.05) reduction in the FBG, increased in plasma insulin concentrations, and improvement

TABLE 2. Fasting blood sugar, plasma insulin, and BW after 56 days of treatment with 50 mg/kg of naringin FBS (mmol/L) FPI (ng/L) BW (g)

Control

Control/NRN

DM/INS

DM/NRN

DM

DM/RMP

6.3 6 0.52 160 6 3.46 365.2 6 5.97

5.63 6 0.42 207.2 6 7.09† 366.2 6 13.10

25.07 6 0.99* 64.8 6 28.9* 342 6 14.4*

28.59 6 1.97* 57.4 6 2.2* 200.4 6 14.83*

34.47 6 0.72† 8.0 6 1.37† 173 6 9.357†

33.41 6 0.44 35.4 6 5.80* 195.6 6 8.56

*P , 0.05 (Compared to DM). †P , 0.05 (compared to Control) (n = 7 per group). DM, diabetes mellitus; FPI, fasting plasma insulin; FBS, fasting blood sugar; NRN, Naringin; INS, Insulin; RMP, Ramipril.

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in BW compared with the untreated diabetic rats, respectively.

Reduction of Cardiac Hypertrophy Indices CH is defined by increased cardiac mass. To determine the effects of naringin on cardiac hypertrophy, the rats’ heart weight (HW), and left ventricular weights (LVWs) normalized with final BW and tibia length were assessed. We observed that the diabetic rats HWs were significantly (P , 0.05) reduced compared with the controls (Fig. 1A). Furthermore, we found that all diabetic rats exhibited significantly (P , 0.05) increased (49.8%) HW/BW ratios compared with controls (Fig. 1B). Interestingly, naringin, ramipril, and insulin all significantly (P , 0.05) reduced HW/BW ratios by 24.6%, 17.8%, and 28.8%, respectively, compared with untreated diabetic rats (Fig. 1B). Similarly, naringin and ramipril significantly (P , 0.05) reduced the HW/tibia length

Naringin Reduces Cardiac Hypertrophy

ratio by 32.9% and 28.2%, respectively in the diabetic rats compared with untreated diabetic controls (Fig. 1C). The LVW/BW ratios assessment also revealed a similar picture, where naringin and ramipril significantly (P , 0.05) reduced LVW/BW ratios by 20% and 25% (P , 0.05) in the diabetic rats compared with untreated diabetic controls (Fig. 1D), respectively.

Reduction of Myocardial Lipid Peroxidation and Protein Oxidation The plasma MDA and carbonyl protein concentrations in the untreated diabetic rats were significantly (P , 0.0001) elevated compared with their respective vehicle-treated controls (Figs. 2A, 3A). Similarly, cardiac tissue MDA concentrations and protein carbonyls were significantly (P , 0.01) increased by 54.8% and by 31.9%, respectively in the untreated diabetic group compared with their respective vehicle-treated

FIGURE 1. A, Heart weights, (B) Cardiac hypertrophy index calculated as HW:BW ratio, (C) Heart weight to tibia length ratio (HW/ TL), and (D) Left ventricular weight to body weight ratio (LVW/BW). BWs were measured as terminal live weights. ***P , 0.0001, **P , 0.01, and *P , 0.05 (compared to DM); aP , 0.05 (compared to control). CTR, control; DM, diabetes mellitus. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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controls (Figs. 2B, 3B). Naringin, insulin, and ramipril treatment significantly (P , 0.05) reduced plasma and cardiac MDA concentrations by 71% and 60%, respectively in diabetic rats compared with the untreated diabetic controls. Furthermore, naringin and insulin significantly (P , 0.05) reduced both plasma and cardiac tissue carbonyl proteins while ramipril significantly reduced only cardiac tissue carbonyl proteins compared with untreated diabetic controls.

Effects on Cardiac Superoxide Dismutase and Glutathione Peroxidase

GPx activity was significantly (P , 0.001) reduced in diabetic untreated rats compared with controls, but treatment with insulin, naringin, and ramipril significantly (P , 0.05) increased GPx activity in the diabetic rats (Fig. 4A). Furthermore, there was a 48-fold increase in GPx activity in the insulin-treated group and a 5-fold increase in naringin- and ramipril-treated diabetic rats, respectively compared with untreated diabetic controls (Fig. 4A). Surprisingly, GPx activity was significantly (P , 0.001) reduced in naringin-treated nondiabetic rats compared with the vehicle-treated controls (Fig. 4A). Similarly, the total SOD activities in the diabetic untreated group were significantly (P , 0.05) lower in comparison with those in the control group but naringin and insulin treatment significantly (P , 0.05) increased total SOD activities in diabetic rats (Fig. 4B), respectively.

Effects on Cardiomyocyte Size One of the defining characteristics of cardiac hypertrophy is increased cardiac myocyte size; to evaluate changes in cardiomyocyte size, an assessment of cardiomyocyte diameters was carried out by measuring cardiomyocyte sizes by using 10 randomly picked fields per slide. We noted that the untreated diabetic rats exhibited significantly (P , 0.05) increased cardiomyocyte diameter compared with the vehicle-treated controls (Figs. 5A–G). However, diabetic rats treated with naringin, insulin, and ramipril significantly (P , 0.001) reduced cardiomyocyte diameter among diabetic rats. Surprisingly, naringin significantly increased cardiomyocyte diameter among normal nondiabetic population.

Inhibition of the Activation of Cardiac Jun Nuclear Kinase

The untreated diabetic rats showed significantly (P , 0.05) increased expression of p-JNK-1 and JNK-1 compared with the vehicle-treated rats (Fig. 6A, B). There was an approximately 20-fold and 10-fold increase in the levels of expression of p-JNK-1 and JNK-1, respectively in untreated diabetic rats relative to the controls. Interestingly, naringin significantly down-regulated the expression of both p-JNK1 and JNK-1 in the diabetic rats compared with the untreated diabetic rats (Fig. 6A, B). As expected, insulin and ramipril also significantly down-regulated the expression of p-JNK-1 and JNK-1 in the diabetic rats relative to the untreated diabetic rats. We observed that the p-JNK/JNK ratio was significantly (P , 0.05) increased in the diabetic untreated compared with control rats but naringin significantly (P , 0.0001) reduced this ratio by 41% (Fig. 6C). Ramipril and insulin also significantly (P , 0.0001) decreased the ratio by 59% and 63%, respectively relative to untreated diabetic rats.

DISCUSSION CH is one of the major mechanisms of cardiac remodeling that characterizes diabetic cardiomyopathy, an important cause of morbidity and mortality in diabetic patients.4 CH is characterized by increased cardiomyocyte growth stemming from enhanced protein synthesis and is often identified by increased cardiac mass.1 In this study, we observed an increase in cardiomyocyte diameter and cardiac hypertrophy indices in untreated diabetic rats indicating development of cardiac hypertrophy. Increased hemodynamic stresses coupled with enhanced neuro-humoral activation have been shown to predispose diabetic patients to the development of cardiac hypertrophy.7,9 Furthermore, we observed that naringin significantly reduced the increases in cardiomyocyte diameters by 34.6% and cardiac hypertrophy indices by 24.6% and 32.9%, respectively, in diabetic rats compared with the untreated diabetic controls indicating that naringin retarded the development of CH in these diabetic rats. To understand the possible mechanism by which naringin

FIGURE 2. A, Plasma MDA concentrations. B, Cardiac tissue MDA concentrations. MDA concentrations were measured as an index of lipid peroxidation. ***P , 0.001, **P , 0.01, and *P , 0.05 (compared to DM) and aP , 0.05 (compared to control). CTR, control; DM, diabetes mellitus.

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Naringin Reduces Cardiac Hypertrophy

FIGURE 3. Carbonyl protein concentration in (A) plasma and (B) cardiac tissue. ***P , 0.0001, **P , 0.001, *P , 0.05 (compared to DM) and aP , 0.001, bP , 0.05 (compared to CTR). CTR, control; DM, diabetes mellitus.

produced these observed effects, we investigated the effect of naringin on the generation of ROS in the diabetic myocardium because much of naringin’s cardio-protective actions have previously been associated with its antioxidant effects.18,19,26 In addition, increased ROS have been shown to contribute significantly to the development of CH through induction and activation of signaling molecules, particularly the mitogen-activated protein kinases that participate in the hypertrophic process.1,11,12 In this study, we observed that there were marked increases in lipid peroxidation and protein oxidation products (known as biomarkers of oxidative stress) in diabetic rats relative to vehicle-treated controls. However, naringin reduced these observed elevations in lipid peroxidation and protein oxidation by 60% and 26%, respectively, suggesting that the significant reduction in cardiomyocyte size and cardiac hypertrophy indices might have been mediated by naringin’s antioxidant actions. In this study, we also observed that naringin seems to reduce lipid peroxidation products more than carbonyl proteins, it is not clear what is responsible for this but a plausible explanation may be that lipid peroxidation results from a certain subspecies of ROS that are different from the ones associated with protein oxidation.27

Diabetes mellitus produces profound oxidative stress and attendant oxidative damage in tissues.6,28 Oxidative stress is composed of an unregulated increase in ROS and a simultaneous depletion of enzymatic antioxidants in tissue. In this study, there was marked reduction in GPx activity among the untreated diabetic rats relative to controls. In diabetes, decreased expression and activity of enzymatic antioxidants have been clearly demonstrated.6,29 Our findings therefore suggest that the GPx was probably depleted or its production was overwhelmed by increased generation of ROS. This line of thought was further supported by our subsequent observation that naringin significantly boosted the activity of GPx among diabetic rats compared with untreated diabetic controls. It is likely that naringin by mopping up excess free oxygen radicals reduced the burden placed on GPx and allowed for its replenishment and increased activity, because it is an inducible enzyme.6 Conversely, in the normal rats treated with naringin, GPx activity was reduced, this is possibly due to lessened ROS burden, and hence the inducing influence on GPx was not present. GPx is an important antioxidant enzyme that regulates the ratio of oxidized/reduced glutathione in tissues including heart tissues.6,30 Glutathione is very

FIGURE 4. A, Cardiac tissue GPx activity, (B) Cardiac tissue total SOD Activity. *P , 0.05, **P , 0.01, ***P , 0.001 (compared to DM) and a,bP , 0.05 (compared to CTR). CTR, control; DM, diabetes mellitus. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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FIGURE 5. A–F: Cross-sectional H&E micrographs of the left ventricular myocardium, X200 Scale Bar (50 mM). A, Diabetic untreated rats B, Naringin-treated diabetic rats. C, Ramipril-treated rats. D, Naringin-treated normal rats. E, Insulin-treated diabetic rats. F, Control rats. G, Cardiomyocyte diameter in micromolar taken from 50 fields. CTR, control; DM, diabetes mellitus; NRN (naringin), INS (insulin), RMP (ramipril), and DM [untreated Diabetic, ***P , 0.0001 (compared to DM) and aP , 0.0001 (compared to CTR)].

important in limiting oxidative damage in tissues; it interacts with hydroperoxides and lipid hydroperoxides (products in lipid peroxidation) preventing them from further damage to membrane lipids. Similarly, naringin increased total SOD

activities in diabetic rats by over 50% compared with the untreated diabetic rats suggesting that depression of activity of SOD is associated with events leading up to the development of CH in diabetic rats. SOD enzymes are regarded as

FIGURE 6. Western blot assay of JNK-1 protein expression in the left ventricle tissue (A). Densitometry scans of phosphorylated (B) and nonphosphorylated (C) JNK-1 relative to b-actin and (D) p-JNK-1/JNK-1 ratio: ***P , 0.0001, **P , 0.01 (compared to DM), and aP , 0.0001 (compared to control). CTR, control; DM, diabetes mellitus.

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primary antioxidants because they neutralize superoxide anions, which are produced firstly in the ROS cascade.6,30 SOD converts superoxide anions to hydrogen peroxide and molecular oxygen.6 Many studies have reported depletion in the SOD enzymes in type 1 diabetes mellitus, it is generally accepted that increased ROS is negatively correlated with the cellular content of natural enzyme antioxidants such as SOD and GPx.6 Superoxide anions are important signaling molecules in the hypertrophic process1; hence, SOD provide a first line of defense against increased and detrimental signaling that culminates in the hypertrophy of the myocardium. To understand the effects of naringin on mitogen activated kinases involved in the hypertrophic process, we studied the effects of naringin on the expression of p-JNK and JNK-1 in diabetic rats. JNK and extracellular signal-related kinases are important signaling molecules in the hypertrophic process in the myocardium.1,6 They are activated through ROS-mediated signaling.1 Activation of JNK in CH has previously been linked with increased ROS levels in tissues.6 In this study, we observed significantly increased expression of p-JNK-1 (20-fold) in untreated diabetic rats relative to vehicle-treated controls. We also noted that a ten-fold increase in JNK-1 and a 2.7-fold increase in p-JNK/JNK ratio in the untreated diabetic rats compared with the controls. These findings suggest that diabetes is associated with increased activation of JNK-1 and enhancement of the development of CH in diabetes, which likely explains the increased CH observed in this group of rats. Interestingly, naringin treatment of diabetic rats significantly and markedly reduced the levels of expression p-JNK-1 and JNK-1 as well as p-JNK/JNK ratio, suggesting that the increased activation of JNK-1 in diabetes mellitus might be under the influence of increased ROS. Naringin possibly through its radical scavenging action, vitamin E–sparing effects, and enhanced antioxidant gene expression limited the burden imposed by unregulated ROS levels in diabetes mellitus and therefore retarded the activation of JNK-1, consequently limiting the development and progression of CH in the diabetic rats.21–23 This finding also is consistent with the previous observations of reduction in CH in probucol- and N-acetylcysteine-treated experimental animals.26 Taken together, these observations suggest that naringin through limitation of oxidative stress seems to inhibit JNK-1 to mediate the reduction of CH in naringin-treated diabetic rats. There is no known toxicity of naringin to either humans or animals. Naringin has poor solubility in water, and 50 mg/kg BW was the maximum dispensable in 1.0 mL allowable for gastric gavage in the animal weight category of 250–300 g as per the institutional guidelines that granted ethical approval for the study. These effects of naringin could as well be attributed to plasma naringenin glucuronide, a naringin metabolite generated by intestinal bacteria metabolism and absorbed by an active process and then biotransformed in the liver.31,32 Naringin has been shown to exhibit two plasma concentration peaks at 1 hour and 6.7 hours due to enterohepatic recycling, as well as delayed plasma clearance, all possibly contributing to the cumulative effects of naringin in cardiac tissue.31 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

Naringin Reduces Cardiac Hypertrophy

CONCLUSIONS Naringin therefore seems to hold promise in limiting the effects of cardiac hypertrophy through its antioxidant actions particularly by influencing c-JNK-1, which is involved in the cardiac hypertrophy process.

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