Lithium induced oxidative damage and inflammation in the rat’s heart: Protective effect of grape seed and skin extract

Biomedicine & Pharmacotherapy 95 (2017) 1103–1111

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Lithium induced oxidative damage and inflammation in the rat’s heart: Protective effect of grape seed and skin extract

MARK

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Ali Meznia,b, , Hanène Aouaa,b, Olfa Khazria,b, Ferid Limama, Ezzeddine Aouania,b a b

Laboratoire des Substances Bioactives (LSBA), Centre de Biotechnologie de BorjCedria, BP-901, 2050 Hammam-Lif, Tunisie Université de Carthage, Faculté des Sciences de Bizerte, 7021 Jarzouna, Tunisie

A R T I C L E I N F O

A B S T R A C T

Keywords: Lithium GSSE Heart Rat Oxidative stress Inflammation

Lithium (Li) is a relevant mood stabilizer metal for the treatment of bipolar disorder (BD), as it protects from both depression and mania and reduces the risk of suicide. However, Lihas some clinical concerns as a narrow therapeutic index requiring routine monitoring of the serum level. The present study was designed to analyze the cardio-toxic side effect of Li and the ability of grape seed and skin extract (GSSE) to protect the heart against such toxicity. After 30 days of exposure to Li (0, 2, 5 and 100 mg/kg bw) and prevention with GSSE (4000 mg/kg bw), rats were killed by decapitation and their heart processed for Li-induced oxidative stress. Data mainly showed that Li increased lipoperoxidation and protein carbonylation, it decreased superoxide dismutase and glutathione peroxidase activities, altered acetylcholinesterase (AChE) activity and increased the pro-inflammatory cytokine interleukin 6 (IL-6). Interestingly, GSSE efficiently alleviated all the deleterious effects of Li especially in low therapeutic doses. Based on our results, GSSE could be proposed as a nutritional supplement to mitigate the cardiotoxic side effects of lithium.

1. Introduction Lithium (Li) is one of the most effective long-term treatments for bipolar disorder, protecting against both depression and mania and reducing the risk of suicide [1]. Lithium has some clinical concerns as a narrow therapeutic index requiring routine monitoring of serum levels [2], but it is still commonly used despite numerous side effects [3]. The most common alterations occur in the kidney, endocrine glands, gastrointestinal tract and in the heart [3]. Electrocardiographic changes in patients receiving Li therapy have been reported, even when Li is used in low therapeutic doses [4]. Li treatment is associated with variable electrocardiography (ECG) changes including QT prolongation, ST segment and T wave changes, cardiac arrhythmias, trio-ventricular dysfunction and myocardial infarction (MI) [5]. In addition, lithium attenuates cardiac sympathetic re-innervation after MI [6] and Reactive Oxygen Species (ROS)are highly implicated in ventricular remodeling following MI [7]. Although some previous works highlighted the involvement of oxidative stress and inflammation in Li-induced toxic side effects in several organs such as the kidney [8] or liver [9], data concerning the heart of rats exposed to Li are scarce. Interestingly, Li has recently been shown to induce an oxidative stress into the heart of male rats, which was alleviated using a Malva Sylvestris polyphenol extract

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[10]. Grape seed and skin extract (GSSE) is a complex mixture of bioactive compounds including proanthocyanidins, flavonoids and stilbenes [11]. Flavonoids, highly present in grape seeds, are mainly composed of monomeric catechins and flavonols such as quercetin [12]. GSSE has numerous beneficial health effects, mainly via its antioxidant and antiinflammatory properties [13], it is neuro-protective, reno-protective, hepato-protective and cardio-protective [14]. The present study was designed to investigate the ability of a high dose of GSSE to protect the heart against the toxic side effects of various doses of Li ranging from therapeutic to non-lethal doses with a special emphasis on Li-induced oxidative stress and inflammation and on the anti-oxidative and antiinflammatory role of GSSE. 2. Material and methods 2.1. Chemicals Thiobarbituric acid (TBA); 2,6,-di-tert-butyl-4-hydroxy-toluene (BHT); trichloroacetic acid (TCA); hydrogen peroxide (H2O2); 2-methoxyphenol (gaiacol); bovine catalase and 4-(1-Hydroxy-2-methylaminoethyl)-benzene-1,2-diol (epinephrine), 2,4,dinitrophenyl hydrazine

Corresponding author at: Université de Carthage, Faculté des Sciences de Bizerte, 7021 Jarzouna, Tunisie. E-mail address: [email protected] (A. Mezni).

http://dx.doi.org/10.1016/j.biopha.2017.09.027 Received 20 May 2017; Received in revised form 5 September 2017; Accepted 6 September 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.

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(DNPH) were obtained from Sigma-Aldrich Co (Germany).

Table 1 Phenolics levels from carignan GSSE.

2.2. Preparation and composition of grape seed and skin extract GSSE was processed from the grape cultivar Carignan of Vitisvinifera from northern Tunisia. Seeds and skin were dried and grounded separately with an electric mincer (FP3121 Moulinex) until a fine powder was obtained. Whole GSSE was prepared in a high dosage (4 g/kg bw) by mixing grape seed and skin powders (50/50) in 10% ethanol in the dark, then vigorously vortexed for 10 min, centrifuged at 10000g for 15 min at 4 °C for debris elimination, and supernatant containing soluble polyphenols was used. The total phenolic content was determined by the folin-Ciocalteau colorimetric method [15], flavonoids and condensed tannins according to Dewanto et al. [16] and Sun et al. [17] respectively. The GSSE composition was established by an HPLC–MS/MS analysis. Briefly, liquid chromatography was performed using a Perkin Elmer system series 200 equipped with a binary micro-pump. Analyses were carried out on a C18 column (Zorbax Eclipse XDB-C18, 4.6 × 150 mm, particle size 5 μm). Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. Elution was performed at a flow rate of 1 mL min−1 and an injection volume of 20 μL. Tandem mass spectrometry (MS/MS) was carried out using a 3200 QTRAP mass spectrometer (Applied Biosystems/MDS Sciex Forster city USA) equipped with an electrospray ionization (ESI) interface. Data were acquired and processed with Analyst 1.5.1 software. The detector was set in the negative ion mode and the ion trap mass spectrometer was operated in the m/z 50–1700 mass range.

Phenolics

Seed

Total phenolics (mg/g extract) Total flavonoids (mg/g extract) Non flavonoids (mg/g extract) Condensed tannins (mg/g extract) Total anthocyanins(μg/g extract)

67.0 16.0 51.0 1.22 1.00

Skin ± ± ± ± ±

1.5 0.9 0.4 0.13 0.01

51.0 14.0 37.0 3.43 0.96

± ± ± ± ±

3.8 1.3 4.8 0.16 0.01

extinction coefficient of 22000 M−1 cm−1 and the results were expressed as nmol carbonyl protein/mg protein. The total protein content was determined according to Hartree [22] with bovine serum albumin as standard. Non-proteinthiols were determined spectrophotometrically according to Ellman [23]. Heart homogenates were also used for endogenous antioxidant enzyme activities as glutathione peroxidase (GPx) (E.C. 1. 11. 1. 9) [24], catalase (CAT) (E.C. 1. 11. 1. 6) [25] and superoxide dismutase (SOD) (E.C.1.15.1.1) [26]. SOD isoform activities were performed using KCN (3 mM), which inhibited Cu/Zn-SOD or H2O2 (5 mM) affecting both Cu/Zn-SOD and Fe-SOD whereas Mn-SOD was insensitive to both inhibitors. 2.5. Intracellular mediators The free iron in the heart was determined according to Leardi et al. [27] using a commercially available kit from Biomaghreb, Tunisia. At an acidity of pH 4.8, all Fe3+ released by transferrin is reduced by ascorbic acid into Fe2+, which is composed of ferrozine,of complex of a purple color measurable at 560 nm. Briefly, heart homogenates were added to a reaction mixture containing ascorbic acid (5 g/L) and ferrozine (40 mM) and incubation performed at 37 °C for 10 min. Ionizable calcium was determined according to Stern and Lewis [28] using a commercially available kit from Biomaghreb. At a basic pH value, calcium with cresolphtalein are a purple complex measurable at 570 nm. Heart homogenates were added to the reaction mixture containing a 2-amino-2-methyl 1-propanol buffer (500 mmol/L), cresolphtalein (0.62 mmol/L) and hydroxy-8 quinoline (69 mmol/L), and incubation was carried out at room temperature for 5 min assuming that the complex was stable for 1 h. The magnesium content was determined according to Gindler [29] using a commercially available kit from Biomaghreb. Magnesium forms a purple colored complex when treated with calmagite at an alkaline pH in the presence of EGTA. The intensity of the purple color measured at 520 nm is proportional to the magnesium concentration. H2O2 was determined enzymatically according to Kakinuma et al. [30] using a commercially available kit from Biomaghreb. Briefly, in the presence of peroxidase, H2O2 reacts with 4-amino-antipyrine and phenol to give a red colored quinine imine which was absorbed at 505 nm and results expressed as mmol H2O2/mg protein. The nitrite (NO2−) level was measured as an index of NO production using the Griess reagent. Heart homogenate was precipitated with 200 μL 30% sulphosalicylic acid, vortexed for 30 min, and centrifuged at 3000g. Equal volumes of the supernatant and the Griess reagent, containing 1% sulphanilamide in 5% phosphoricacid/0.1% naphthalene ethylenediamine dihydrochloride were mixed, incubated for 10 min in the dark, and absorbance measured at 543 nm. Nitrite levels were calculated using sodium nitrite as standard (Green et al., 1982) [31].

2.3. Animals and experimental design Forty-eight male Wistar rats (210–230 g) from the Pasteur Institute (Tunis) were used in agreement with the NIH guidelines [18]. They were provided with food and water ad libitum and maintained in an animal house at a controlled temperature of 22 ± 2 °C with a 12 h light–dark cycle. Rats were randomly divided into eight groups of six animals each and received daily intraperitoneal (IP) injections of 0, 2, 5, or 100 mg of lithium chloride per kilogram of body weight (bw) with or withoutGSSE (4 g/kg bw). The experiments were carried out for 30 days. Doses of Li were chosen in the therapeutic range (2 and 5 mg/ kg bw) or in the toxic but non-lethal amounts (100 mg/kg bw) according to available literature [19]. At the end of the exposure period, rats were killed by decapitation, their heart collected, weighed and homogenized in a phosphate buffer saline solution with pH 7.4 with an ultra-Turrax homogenizer. After centrifugation (10 min at 10.000g, 4 °C), a supernatant was used for the evaluation of Li-induced cardiac oxidative stress status, transition metals and associated enzymes alteration and intracellular mediators of inflammation. 2.4. Heart analysis Malondialdehyde (MDA), a marker of lipid peroxidation was determined according to Draper and Hadley [20]. Heart homogenate was mixed with a butylatedhydroxyltoluene/trichloroacetic acid (BHT/ TCA) solution containing 1% (w/v) BHT dissolved in 20% TCA (w/v) and centrifuged at 4000g for 15 min at 4 °C. The supernatant was blended with 0.6 N HCl and 120 mmol L−1thiobarbituric acid in 26 mmol L−1 Tris buffer, heated at 80 °C for 10 min, cooled, and the absorbance was measured at 532 nm and the MDA level calculated using the absorbance coefficient of the MDA-TBA complex 1,56 105 cm−1 M−1. Protein carbonylation was measured in the heart homogenates according to Levine et al. [21]. Briefly, after protein precipitation with 20% TCA and dissolution in 2,4dinitrophenylhydrazine (DNPH)-containing buffer, absorbance was measured at 366 nm using the molar

2.6. Heart enzymology Acetylcholinesterase activity (AChE; EC 3.1.1.7) was determined according to the method of Ellman et al. [32] using acetylthiocholine (ATCI). The reaction mixture (3.0 mL), in a 0.1 M phosphate buffer of 1104

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Table 2 LC–MS/MS data of phenolics composition of carignan GSSE. Compounds

m/z negative mode [M−H]−

MS2 fragment

Relative abundance (%) Seed

Catechin Epicatechin Procyanidin dimmer Procyanidin trimer Quercetin Resveratrol Rutin Vanillin Gallic acid p-Coumaric acid Rosmarinic acid 2,5dihydroxybenzoic acid Caffeic acid Chlorogenic acid Ferulic acid

289 289 577 865 301 227 609 151 169 163 359 152 179 353 193

245/108.8/122.8 245/108.8/122.8 289.3/407.4 577 150.8/120.9 184.6/143 300.1 135.7/108.1 124.7/78.9 119/93 160.8/197.1 108.7/90.7 135 191 134/89

2.27 2.85 0.47 ND 0.64 0.14 1.51 10.7 50.3 ND ND 30.6 ND ND 0.55

Skin ± 0.07 ± 0.18 ± 0.03 ± ± ± ± ±

0.02 0.01 0.01 0.2 0.5

± 0.5

± 0.02

0.36 ± 0.04 0.37 ± 0.01 ND ND 0.47 ± 0.02 ND 0.50 ± 0.02 7.75 ± 0.39 32.8 ± 1.2 0.38 ± 0.03 0.75 ± 0.02 52.0 ± 0.2 2.8 ± 0.1 0.34 ± 0.02 1.46 ± 0.11

Fig. 1. Effect of GSSE on Li-induced brain lipoperoxidation (a), carbonylation (b) and non protein thiols NPSH (c). Rats were treated daily for 4 weeks with various doses of Li (2, 5 and 100 mg/kg bw) with or without GSSE (4 g/kg bw). Results are expressed as a mean ± SEM. (n = 6) p < 0.05 was considered significant: *for lithium vs. control, and §for lithium + GSSE vs. lithium.

absorption spectroscopy. Tyrosinase activity (E.C.1.14.18.1.) was determined according to Worthington Biochemical Corporation [34] using L-tyrosine as substrate. Heart homogenate (50 μL ≈ 150 μg protein) was mixed with a 50 mM potassium phosphate buffer of pH 6.5 containing 1 mM L-tyrosine in 3 mL as final volume at 25 °C and absorbance followed at 280 nm every min for 10 min. One unit of tyrosinase activity increased the absorbance by 0.001 per min at 280 nm. Lactate dehydrogenase (LDH) activity (E.C.1.1.1.1.) was assayed spectrophotometrically according to Bergmeyer [35] using a commercial kit from Biomaghreb Tunisia.

pH 7.4 containing heart homogenate, 0.33 mM DTNB and 0.5 mM ATCI, was incubated for 30 min at 37 °C and absorbance measured at 412 nm. Activity of AChE was expressed as nmol/min/mg protein. Xanthine oxidase (XO) (EC 1.1.3.22) was determined by measuring a uric acid formation from xanthine according to [33]. An aliquot of xanthine solution (250 mM) was added to a test tube containing heart homogenate and 0.54 mL potassium oxonate (1 mM) in 50 mM sodium phosphate buffer pH 7.4 that was previously pre-incubated at 35 °C for 15 min. The reaction was stopped after 30 min by adding 0.1 mL 0.6 M HCl, centrifuged at 3000g for 5 min and absorbance measured at 295 nm. XO activity was expressed as μmol uric acid/min/mg protein.

2.8. Heart inflammation 2.7. Transition metals and associated enzymes Heart homogenates were diluted with a calibrator solvent and centrifuged, and the IL-6 containing supernatant was determined using a Quantikine IL-6 R immunoassay (R & D Systems, Minneapolis, MN).

Tissue samples were wet washed in nitric acid (15.5 mol/L), diluted, and filtered for copper, zinc, and manganese determination by atomic 1105

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Fig. 2. Effect of GSSE on Li-induced brain SOD (a), CAT (b) and GPx (c) activities. Rats were treated for 4 weeks and various doses of Li were administered daily (2, 5 and 100 mg/kg bw) with or without GSSE (4 g/kg bw). Results are expressed as a mean ± SEM. (n = 6) p < 0.05 was considered significant: *for lithium vs. control, and §for lithium + GSSE vs. lithium.

The heart was cut into 0.5 μm pieces, mounted on slides, stained with hematoxylin and eosin (H & E) and examined in a blinded fashion under a light microscope. One representation micrography of heart tissue from each group is shown in Fig. 7 (n = 6).

activity by −11% at 2 mg/kg, by −43% at 5 mg/kg and by −86% (p = 0.000183) at 100 mg/kg (Fig. 2C). GSSE efficiently restored these oxidative stress biomarkers close to control level at therapeutic doses of Li (2 and 5 mg/kg) and only partially at 100 mg/kg, reaching 20% protection for SOD, 90% for CAT but was ineffective in restoring GPx activity.

2.10. Statistical analysis

3.3. Intracellular mediators

The data were analyzed using the unpaired Student’s t-test or the one-way analysis of variance (ANOVA) and expressed as the means ± standard error of the mean (S.E.M.). All statistical tests were 2-tailed, and p < 0.05 was considered significant.

We further sought to determine the effect of Li on intracellular mediators as free iron (Fig. 3A), Ca++ (Fig. 3B), Mg++ (Fig. 3C), H2O2 (Fig. 3D) and NO metabolites (Fig. 3E). Li significantly increased free iron (+112%, p = 0.00217) at 100 mg/kg, which was partially corrected by GSSE (−20%). Li slightly increased Ca++ (+23%) at 5 mg/kg and significantly (+51%, p = 0.00322) at 100 mg/kg, and GSSE was only partially preventive (−20%). Li also affected cardiac Mg++ by +57% at 2 mg/kg, +60% at 5 mg/kg and +83% (p = 0.000214) at 100 mg/kg and GSSE was protective at the lowest therapeutic dose of 2 mg/kg but inefficient at higher doses. Li affected the H2O2 level by +59% at 2 mg/kg, +99% at 5 mg/kg and +145% (p = 0.00199) at 100 mg/kg and GSSE efficiently prevented from this alteration at all Li doses. Li also disturbed NO metabolites by +33% at 5 mg/kg and +77% (p = 0.000245) at 100 mg/kg and GSSE brought them to near control level whatever the dose of Li.

2.9. Histopathology

3. Results 3.1. Grape seed and skin extract composition Phenolics level found into in seed and skin powders from carignan cultivar are shown in Table 1. Total phenolics and flavonoids are slightly higher into in seeds, whereas condensed tannins are much higher into in skins (three fold). Among phenolics compounds identified within seeds and skins, 2,5dihydroxybenzoic, caffeic and ferulic acid were more abundant into in skins (Table 2). 3.2. Heart oxidative stress Li increased lipoperoxidation by +43% (p = 0.001544) at the highest dose of 100 mg/kg (Fig. 1A), protein carbonylation by +13% at 2 mg/kg, +33% at 5 mg/kg, and by +91% (p = 0.0231) at 100 mg/kg (Fig. 1B) and decreased NPSH by −26% (p = 0.181) at 100 mg/kg (Fig. 1C). All these Li-induced alterations were efficiently corrected to control the level upon GSSE treatment even at the toxic, but non-lethal, dose of 100 mg/kg. In addition, Li exposure affected the antioxidant enzyme activities, depressing SOD activity by −10% at 2 mg/kg, −17% at 5 mg/kg and −65% (p = 0.00017) at 100 mg/kg (Fig. 2A), CAT activity by −8% at 5 mg/kg and by −39% (p = 0.017) at 100 mg/kg (Fig. 2B) and GPx

3.4. Transition metals and associated enzymes Li provoked the accumulation of zinc in the heart (Fig. 4A) by +34% (p = 0.0332) at 100 mg/kg and of the zinc containing enzyme, LDH, (Fig. 4B) by +32% at 5 mg/kg and +110% (p = 0.00024) at 100 mg/kg. GSSE efficiently protected the heart at low therapeutic doses of Li and only partially at the toxic non-lethal dose of 100 mg/kg. Li induced a gradual accumulation of copper (Fig. 4C) reaching +49% at 2 mg/kg, +189% at 5 mg/kg and +314% (p = 0.00118) at 100 mg/kg and of the copper dependent enzyme, tyrosinase, (Fig. 4D) 1106

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Fig. 3. Effect of GSSE on Li-induced intracellular mediators as iron (A),calcium (B), magnesium (C), H2O2 (D), NO (E).Rats were treated for 4 weeks and various doses of Li (2, 5 and 100 mg/kg bw) were administered daily with or without GSSE (4 g/kg bw). Results are expressed as a mean ± SEM. (n = 6) p < 0.05 was considered significant: *for lithium vs. control, and §for lithium + GSSE vs. lithium.

3.7. Histopathology

reaching +35% at 5 mg/kg and +113% (p = 0.001) at 100 mg/kg. A high dosage of GSSE efficiently brought copper and tyrosinase to near control level at all Li doses.

The effect of various doses of Li on the myocardial architecture is shown in Fig. 7. Li induced morphological alterations such as the enlargement of myocardial fibers with inflammatory mononuclear collection, edema, and myocardial necrosis (Fig. 7C, E and F). GSSE protected the heart from Li injury especially at the therapeutic doses of 2 and 5 mg/kg (Fig. 7D and F) but was inefficient at 100 mg/kg.

3.5. Heart enzymology Li slightly decreased the AChE activity at low therapeutic doses reaching-18% at 5 mg/kg and significantly (p = 0.006) reaching −42% at 100 mg/kg versus control (Fig. 5A), while GSSE corrected these deficiencies till control even at the highest dose of 100 mg/kg. Li also gradually increased XO activity reaching +65% (p = 0.00028) at 100 mg/kg (Fig. 5B) and GSSE efficiently protected against low therapeutic doses of Li and only partially at 100 mg/kg.

3.8. Discussion Li is a first-line treatment for bipolar disorder, with a narrow therapeutic window that is toxic to humans and the environment [36]. Li is known to have a variety of cardiovascular effects in humans and experimental animals [37]. Although Li cardiotoxicity is increasingly recognized, the cellular mechanisms involved are poorly understood [38] and no robust preventive or healing agent is available. The purpose of the current study was to investigate whether Li induced an oxidative stress and inflammation in the rat’s heart and whether treatment with GSSE could be cardioprotective. Li provoked an increased oxidative stress status, evidenced by high lipoperoxidation and protein carbonylation, but with a low NPSH level and affected antioxidant enzyme activities as CAT, Gpx and SOD. The findings obtained from this study revealed that, even if used at low

3.6. Heart inflammation Li increased the heart pro-inflammatory IL6 cytokine reaching +45% at 5 mg/kg and +322% (p = 0.00057) at the highest dose of 100 mg/kg. GSSE corrected Li disturbances especially at low therapeutic doses (2 and 5 mg/kg) but was inefficient at the highest toxic dose of 100 mg/kg (Fig. 6).

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Fig. 4. Effect of GSSE on Li-induced transition metals and associated enzymes such as zinc (A) and LDH (B), copper (C) and tyrosinase (D). Rats were treated for 4 weeks and various doses of Li (2, 5 and 100 mg/kg bw) were administered daily with or without GSSE (4 g/kg bw). Results are expressed as a mean ± SEM. p < 0.05 was considered significant. *p < 0.05 for Li vs. control. §p < 0.05 for GSSE + LivsLi.

Fig. 5. Effect of GSSE on Li-induced AChE (A) and XO (B) activity. Rats were treated for 4 weeks and various doses of Li (2, 5 and 100 mg/kg bw) were administered daily with or without GSSE (4 g/kg bw). Results are expressed as a mean ± SEM. (n = 6) p < 0.05 was considered significant: *for lithium vs. control, and §for lithium + GSSE vs. lithium.

therapeutic doses, Li has cardiotoxic side effects. Like many drugs, Li may directly or indirectly induce oxidative stress through the mitochondria via redox cycling, or promoting iron accumulation and oxidative/nitrative modifications of essential mitochondrial proteins; and by this way, it plays a crucial role in the development of myocardial dysfunction [39]. Many reports have shown that long term lithium treatment leads to severe oxidative stress and increases lipoperoxidation that could result from the drop in antioxidant enzyme activities [40]. Reactive Oxygen Species (ROS) are constantly generated in-vivo for physiological purposes. However, excessive ROS production beyond the detoxifying ability of antioxidant system induces oxidative damage to lipids, proteins and nucleic acid, resulting in oxidative stress. Our data showed a decrease in SOD, CAT and GPx activities in Li groups and similar observations have previously been reported [41,42]. The reduction in SOD activity in Li-treated hearts may be due to the enhanced production of superoxide radical anions. Catalase scavenges H2O2 generated by free radicals or by SOD in the removal of superoxide anions [43]. Our data also showed a decrease in the cytosolic isoform, Cu/Zn SOD, that was correlated with altered transition metals. Indeed, Li increased heart copper and zinc, as well as their associated enzymes

Fig. 6. Effect of GSSE on Li-induced cardiac IL-6 level. Rats were treated for 4 weeks and various doses of Li (2, 5 and 100 mg/kg bw) were administered daily with or without GSSE (4 g/kg bw). Results are expressed as a mean ± SEM. (n = 6) p < 0 0.05 was considered significant: *for lithium vs. control, and §for lithium + GSSE vs. lithium.

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Fig. 7. Histopathology of the rat’s heart. Rats were treated daily for one month with various doses of Li (2, 5 and 100 mg/kg bw) with or without GSSE (4 g/ kgbw). Staining with hematoxylin-eosin (HE). 0 Li (A), 0 Li + GSSE (B), 2 mg/kg Li (C), 2 mg/kg Li + GSSE (D), 5 mg/kg Li (E), 5 mg/kg Li + GSSE (F), 100 mg/kg Li (G), 100 mg/kg Li + GSSE (H).

between the heart and the brain [51]. The most relevant result drawn from the current study is the cardioprotection provided by GSSE against Li toxicity. GSSE has been successfully used in the protection of the heart subjected to various biotic or abiotic alterations such as doxorubicin [52], arsenic [53], cadmium [54], cyclosporine A [55], isoproterenol [56] or cisplatin [57] and our data fully corroborate all these findings. To the best of our knowledge, our data are the first to demonstrate a potent protection of GSSE against Li cardiotoxicity especially at a therapeutic dosage of Li. In addition, the high dosage of GSSE of 4 g/kg used in the present study has been reportedly shown as highly safe and multi-organ protective in various experimental settings, for instance in renal protection after neoplastic treatment using doxorubicin [58], lung subjected to bleomycin [59], pancreas subjected to fat treatment [60] or in-brain protection after Li exposure [61]. We do not know at the present time the exact mechanism of action of GSSE, and which kind of polyphenol is specifically involved in such cardio-protection, having in mind the complex composition and synergism that likely occurs between the numerous bioactive compounds contained in GSSE. Owing to its anti-oxidant, anti-inflammatory and free radical scavenging abilities, a high dosage of GSSE constitutes a real and firstchoice therapeutic option in the prevention and treatment of a broad spectrum of degenerative diseases (reviewed in [14]). In conclusion, GSSE exerts a potent protection against therapeutic doses of Li-induced cardiotoxicity in healthy rats. GSSE should be considered as a safe adjuvant to Li therapy to avoid cardio-toxic side effects and will be essential in future experiments to ascertain that GSSE does not hinder the therapeutic effect of Li.

such as tyrosinase and LDH activity respectively. Li-induced heart oxidative stress could be linked to a concomitant increase of H2O2 and free iron, which, most likely, via the Fenton chemistry, provoked a burst of calcium. Increases in mitochondrial and cytoplasmic Ca2+ favor each other in a vicious relationship that amplifies the damaging processes [44]. In addition, Li increased NO which is also related tomyocardial Ca2+ and a well-known inducer of apoptosis in many different cell types in-vitro and in-vivo [45]. The increase in free radicals such as H2O2 and NO induced by the Li treatment, in turn provoked the increase of IL-6, the pro-inflammatory action of which is central in controlling cardiac and systemic oxidative stress and promoting cellular rescue leading to apoptosis [46]. We also found that Li increases XO activity, a free-radical producing enzyme that generates superoxide and hydrogen peroxide by using molecular oxygen as an electron acceptor [47]. It should be noted that XO activity is high in many cardiovascular diseases, including hypertension, heart failure, and metabolic syndrome [48]. Another biochemical marker used in the present study to assess heart function is AChE. This enzyme is an important component of the heart’s cholinergic system known to regulate the cardiac parasympathetic responses by controlling the acetylcholine levels [49]. Indeed, AChE rapidly catalyzes acetylcholine hydrolysis, thereby terminating its signaling action at the cholinergic neuro-effector junctions of the heart [50]. In our case, Li reduced AChE activity in the rat’s heart, which is probably related to Li-induced oxidative stress and particularly to the increased level of H2O2. However, such a result is in disagreement with a recent study reporting the dose-dependent increase in AChE activity exerted by H2O2 in brain cells [51]; such a discrepancy could be partly explained by the differential cellular environment 1109

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Conflict of interest [28]

The authors have no conflict of interest to disclose. [29]

Acknowledgments

[30]

This work was supported through grants from the Ministry of Higher Education, Carthage University, the Faculty of Sciences of Bizerte, the Laboratory of Bioactive Substances (LSBA) and the Centre de Biotechnologie de BorjCedria, BP 901, 2050 Hammam-Lif, Tunisia. Mr. Ridha Charrada, head of the Domaine Neferis Grombalia, is thanked for kindly providing grape pomace.

[31]

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