Neuroprotective Effect of Hydroxytyrosol In Experimental Diabetes

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Neuroprotective Effect of Hydroxytyrosol In Experimental Diabetes Mellitus José Julio Reyes, Beatriz Villanueva, Juan Antonio López-Villodres, José Pedro De La Cruz, Lidia Romero, María Dolores Rodríguez-Pérez, Guillermo RodríguezGutiérrez, Juan Fernández-Bolaños, and José Antonio González-Correa J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02945 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 18, 2016

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Neuroprotective Effect of Hydroxytyrosol In Experimental Diabetes Mellitus

José Julio Reyes1, Beatriz Villanueva2, Juan Antonio López-Villodres1, José Pedro De La Cruz1, Lidia Romero1, María Dolores Rodríguez-Pérez1, Guillermo RodriguezGutierrez3, Juan Fernández-Bolaños3, José Antonio González-Correa1

1

Departmento de Farmacología, Facultad de Medicina, Instituto de Investigación

Biomédica (IBIMA), Universidad de Málaga. 2Universidad Metropolitana de Puerto Rico (UMET), 3Instituto de la Grasa, Consejo Superior de Investigaciones Científicas (CSIC), Ctra. Utrera Km 1, Campus Universitario Pablo de Olavide, Edificio 46, Seville, Spain.

Author for correspondence: J.A. González-Correa, M.D., Department of Pharmacology, School of Medicine, University of Málaga, Campus de Teatinos s/n, 29071 Málaga, Spain Tel: +34-952131567; Fax: +34-952131568; E-mail: [email protected]

Running title: Hydroxytyrosol and neuroprotection in diabetes

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Abstract

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The aim of the study was to analyze the possible neuroprotective effect of

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hydroxytyrosol (HT) in diabetic animals in a model of hypoxia-reoxygenation. Rats (10

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animals/group) were distributed in five groups: nondiabetic rats, control diabetic rats

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(DR), and DR rats treated for 2 months with 1, 5 or 10 mg/kg/day p.o. HT. At the end of

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follow-up an experimental model of hypoxia-reoxygenation in brain slices was tested.

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The DR group showed increased cell death, oxidative and nitrosative stress and an

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increase in brain inflammatory mediators. These alterations were significantly greater in

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DR than normoglycemic animals. HT significantly reduced oxidative (38.5-52.4% lipid

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peroxidation) and nitrosative stress (48.0-51.0% nitric oxide and 43.9-75.2%

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peroxynitrite concentration) and brain inflammatory mediators (18.6-40.6%

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prostaglandin E2 and 17.0-65.0% interleukin 1ß concentration). Cell death was reduced

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by 25.9%, 37.5% and 41.0% after the administration of 1, 5 or 10 mg/kg/day. The

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administration of HT in rats with experimental diabetes thus had a neuroprotective

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effect.

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Keywords: Hydroxytyrosol. Diabetes mellitus. Neuroprotection. Oxidative stress.

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Inflammatory mediators.

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Introduction

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Chronic hyperglycemia is the first step in the evolution of the long-term complications

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of diabetes mellitus. Diabetic macroangiopathy and microangiopathy are highly

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prevalent in the population of patients with diabetes mellitus1. In these vascular

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complications oxidative stress plays a key role from the earliest stages, participating

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directly in the development of endothelial dysfunction and upregulating other

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biochemical pathways that facilitate cell damage resulting from chronic hyperglycemia2.

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The main consequence of diabetic vasculopathy is the loss of antithrombotic

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endothelial function, which increases the risk of ischemic processes1. Patients with

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diabetes mellitus have a higher prevalence of acute coronary syndrome, cerebral

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ischemic stroke and peripheral arterial disease than the nondiabetic population3.

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Concerning cerebrovascular complications of diabetes, animal experiments have shown

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that brain tissues in diabetic animals are more sensitive to ischemic damage because of

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increased brain oxidative stress compared to normoglycemic animals4,5. Therefore,

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oxidative stress is involved not only in the development of diabetic vasculopathy but

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also in the increased sensitivity of brain tissue to ischemic insult.

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Because of the importance of oxidative stress in diabetes mellitus and its

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complications, the administration of antioxidants has been postulated to prevent both

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cardiovascular and cerebral alterations6. The Mediterranean Diet provides a variety of

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antioxidants, and virgin olive oil (VOO) contains polyphenols that have shown clear

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antioxidant effects in animal models and in humans6,7. Hydroxytyrosol (HT) is the main

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polyphenol present in its free form in VOO.

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Experimental studies have shown that the administration of VOO or isolated HT

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has a neuroprotective effect in rat brain tissue, affecting major biochemical pathways of 3


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brain damage in ischemia7. However these studies were done in normoglycemic

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animals. Therefore, the main objective of this study was to analyze the possible

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neuroprotective effect of HT in diabetic animals, and to search for evidence of a

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possible effect of HT on brain oxidative and nitrosative stress and the production of

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inflammatory mediators.

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Materials and Methods

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Chemicals

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Prostaglandin E2, interleukin-1ß and 3-nitrotyrosine enzyme immunoassay kits were

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from GE Healthcare UK Limited (Little Chalfont, Buckinghamshire, UK). The

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nitrite/nitrate ELISA kit was obtained from Cayman Chemical (Ann Arbor, MI, USA).

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All other reagents were from Sigma Chemical Corp. (St. Louis, MO, USA).

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Hydroxytyrosol was isolated from the liquid phase obtained from alperujo

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treated hydrothermally at 160 °C for 60 min8. Alperujo is the by-product of the two-

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phase olive oil extraction system. The liquid was extracted by chromatography

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fractionation in two steps, with a final yield of 99.6% purity referred to dry matter,

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following the process described by Fernández-Bolaños et al.9. For HPLC analysis, the

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standard compound of tyrosol was obtained from Fluka (Buchs, Switzerland) and HT

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was obtained from Extrasynthese (Lyon Nord, Geney, France). The phenols were

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quantified in a Hewlett-Packard 1100 HPLC system with an ultraviolet-visible light

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detector. A Mediterranea sea C18 analytical column (250 × 4.6 mm i.d.; particle size =

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5 µm) (Teknokroma, Barcelona, Spain) was used at room temperature. The system was

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equipped with Rheodyne injection valves (20 µL loop). The mobile phases were 0.01%

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trichloroacetic acid in water and acetonitrile, at the following gradient over a total run

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time of 55 min: 95% initially, 75% at 30 min, 50% at 45 min, 0% at 47 min, 75% at 50

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min, and 95% at 52 min until the run was complete. Quantification was carried out by

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integration of peaks at 280 nm wavelength with reference to calibrations made with

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external standards. The main compounds detected were HT (99.6% purity) and tyrosol

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(0.1% purity referred to dry matter).

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Study design

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Animals were adult male Wistar rats (body weight 200–250 g). Spanish legislation for

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animal care (EDL 2013/80847, BOE-A-2013-6271) was taken into account. The Guide

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for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985)

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were followed, as well the Spanish Law on the Protection of Animals. The study

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protocol was approved by the University of Malaga Ethics Committee for the Use of

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Animals.

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Animals (10 rats/group) were distributed in five groups: 1) control nondiabetic

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rats, 2) control diabetic rats (DR), 3) DR rats treated with HT 1 mg/kg/day p.o., 4) DR

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rats treated with HT 5 mg/kg/day p.o., 5) DR rats treated with HT 10 mg/kg/day p.o..

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Previous results with HT in healthy animals were the rationale to choose the dose of HT

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used in this study7. Hydroxytyrosol was given (endogastric cannula) once per day for 7

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days before diabetes was induced, and then daily until the end of the diabetic period (2

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months).

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Experimental diabetes was induced with streptozotocin (50 mg/kg i.v.). Blood

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glucose concentration was measured with a Glucocard Memory II glucosimeter

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(Menarini, SA, Barcelona, Spain). When blood glucose was higher than 200 mg/dL

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animals were defined as diabetic. Rats in the nondiabetic control group were treated 5


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with saline, and glucose concentration was also measured. Blood glucose was measured

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at 9:00 a.m.

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During the follow-up period, diabetic animals were treated with 4 IU/day s.c. of

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long-acting insulin (Levemir®). Control animals received the same volume of isotonic

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saline solution s.c.

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All rats were anesthetized with pentobarbital sodium (40 mg/kg i.p.), and were

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then decapitated with a guillotine.

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Hypoxia–reoxygenation procedure

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We use this method in order to assess possible brain alterations due to the diabetes

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model excluding the influence of vascular disturbances of diabetes. Thus the

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modifications that may induce diabetes in brain tissue and possible prevention with

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hydroxytyrosol is valued. Brains tissue (except cerebellum and brain stem) was cut

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transversally into 1-mm slices with a vibrating microtome (Campden Instruments, San

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Francisco, CA, USA). The slices were placed in buffer (composition in mmol/L: 100

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NaCl, 0.05 KCl, 24 NaHCO3, 0.55 KH2PO4, 0.005 CaCl2, 2 MgSO4, 9.8 glucose, pH

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7.4) and perfused with a mixture of 95% O2 and 5% CO2. After 30 min the brain slices

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were placed in fresh buffer with no glucose and a concentration of CaCl2 of 3 mmol/L

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and MgSO4 of 0.001 mmol/L. A mixture of 95% N2 and 5% CO2 for 20 min (hypoxia)

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was perfused. Then the slices were placed in a buffer with glucose and perfused with a

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mixture of 95% O2 and 5% CO2 (reoxygenation).

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Brain samples were analyzed before hypoxia period, after 20 min of hypoxic

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incubation, and after 180 min of reoxygenation. Brain samples were frozen in liquid

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nitrogen and stored at –80 °C. All the analytical techniques were done not more than 7

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days after the sample was frozen. 6



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Analytical techniques

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All techniques were run in a single-blind manner, i.e., the persons who did the assays

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were unaware of the origin and nature of the samples.

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Lipid peroxidation

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Thiobarbituric acid reactive substances (TBARS) in brain cell membrane-enriched

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fractions were measured. Briefly, samples was diluted (1:10 wt/vol) in 0.1 M NaCl, 5 ×

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10-4M KCl, 3.1 × 10-3M CaCl2, 1 × 10-3 M MgSO4, 4.9 × 10-3M glucose, 2.4 × 10-2M

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Na2CO3, 5.5 × 10-4M PO4H2K and 0.32 M sucrose. Then diluted samples were

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homogenized and centrifuged (10 000 g, 15 min, 4°C), and the supernatant was

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collected and centrifuged (12 000 g, 20 min, 4°C). The resulting pellet was resuspended

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in the same buffer without sucrose at a proportion appropriate for the determination of

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lipid peroxide production.

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Absorbance was determined spectrophotometrically at 532 nm (FLUOstar-

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POLARstar, BMG Labtech, Offenburg, Germany). The results were expressed as nmol

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TBARS per mg protein.

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Glutathione levels

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We used a spectrofluorometrically method to measure total content of glutathione. Brain

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slices were homogenized in 0.1 M sodium phosphate buffer (pH 8.0) with 25%

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phosphoric acid (1:20), then centrifuged (13 000 gm 15 min, 4°C). Cuvettes were

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prepared with sodium phosphate buffer, the supernatant for each sample, and o-

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phthaldehyde. To determine the proportions of oxidized (GSSG) and reduced (GSH)

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glutathione 4 vinylpyridine was added to each sample, then proceeded as for as total

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glutathione.

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Glutathione peroxidase activity (GSHpx)

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This enzymatic activity was determined spectrophotometrically. Brain samples were

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diluted in 0.1 M phosphate-buffered saline (pH 7.0) and 25% phosphoric acid. Diluted

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samples were homogenized and centrifuged (13 000 g, 15 min, 4°C). Protein content

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was determined in supernatants after 0.1 N NaOH was added. Twenty-five µg protein

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and 0.1 M phosphate-buffered saline was added (880 µL); 53 µL glutathione reductase,

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133 µL GSH, 100 µL nicotinamide-adenine dinucleotide phosphate (NADPH) and 100

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µL terbutyl-hydroperoxide were also added to the cuvette. Samples were read at 340 nm

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and the decrease in absorbance was recorded every 30 s for 5 min.

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Lactate dehydrogenase assay

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Lactate dehydrogenase (LDH) efflux was measured as an indirect representation of cell

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death. Enzyme activity was measured spectrophotometrically at 340 nm with a

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commercial kit (Cytotoxicity Detection Kit, Roche Applied Science, Barcelona, Spain)

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according to the manufacturer’s instructions.

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Nitrite+nitrate concentration

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Nitrite+nitrate levels were measured as an index of brain nitric oxide production. A part

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of buffer was filtered (Ultrafree MC microcentrifuge filters) to remove high-molecular-

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weight substances released by cells. The nitrite+nitrate level was measured with a

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commercial kit (Cayman Chemical Company). Levels of nitrite/nitrate were determined

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spectrophotometrically at 540 nm and compared with a standard curve obtained with

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sodium nitrite.

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3-nitrotyrosine

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Brain slices were homogenized (1:10 wt/vol) in 100 mM KH2PO4/K2HPO4 and 0.1%

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digitonin (pH 7.4). Then they were centrifuged (5000 g, 10 min, 4°C). The

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concentration of 3-nitrotyrosine in the supernatant was measured according to the

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manufacturer’s instructions for the enzyme immunoassay kit (Cayman Chemical

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Company).

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Prostaglandin E2

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This prostaglandin is chosen in order to asses a possible role of cyclooxygenase activity

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in this experimental model. Brain slices were homogenized (1:10 wt/vol) in 15%

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methanol with 0.1 N phosphate-buffered saline (pH 7.5), then centrifuged (37 000 g, 15

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min, 4°C). The supernatant was run through a C18 column (Bio-Rad Laboratories,

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Hercules, CA, USA) activated with absolute methanol and distilled water. Then, the

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column was washed with 15% methanol in distilled water followed by petroleum ether.

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Prostaglandins were eluted with methylformate, and the samples were then dried at

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room temperature and reconstituted with phosphate-buffered saline. The concentration

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of prostaglandin E2 (PGE2) was measured with a commercial enzyme immunoassay

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(GE Healthcare UK Limited, Little Chalfont, Buckinghamshire, UK).

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Interleukin 1ß

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Brain slices were homogenized in 50 mM Tris, 1 mM EDTA, 6 mM MgCl2, 1 mM

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phenylmethylsufonyl fluoride, 5 µg/mL leupeptin, 1 mg/mL antipain, 1 mg/mL

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aprotinin and 1 mg/mL soybean trypsin inhibitor (4 °C, pH 7.2). The samples were

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sonicated (VC-50T Vibracell, Sonics Materials, Newtown, CT, USA) for 5–10 s and

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centrifuged (14 000 g, 10 min, 4°C). The supernatant was processed according to the

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instructions for commercial kits for interleukin 1ß (IL1ß) determination (GE Healthcare 9


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UK Limited, Little Chalfont, Buckinghamshire, UK). (GE Healthcare UK Limited,

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Little Chalfont, Buckinghamshire, UK).

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Statistical methods

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Each result represent the mean ± SEM of 10 independent experiments or rats. Statistical

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analyses were done with the Statistical Package for Social Sciences v. 23.0 (SPSS Co.,

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Chicago, IL, USA). One-way analysis of variance followed by Bonferroni

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transformation and unpaired Student’s t tests were used.

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Results and Discussion

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The only statistically significant difference between nondiabetic and diabetic animals

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was in blood glucose levels after 2 months of follow-up (Table 1). Mean blood glucose

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levels before the diabetes mellitus was induced with streptozotocin was 98.6 ± 3.9

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mg/dL. In diabetic groups blood glucose higher than 200 mg/dL was measured after 4.2

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± 0.2 days of intravenous streptozotocin administration. Treatment with HT did not

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change any of the parameters, including blood glucose levels. The absence of

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differences between groups is important in order to rule out that the effects of HT might

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be due to a possible antidiabetic effect, as postulated previously10. All of the doses of

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HT used here did not modify blood glucose levels after 2 months of treatment. Animals

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in a previous study10 had blood glucose levels of approximately 280–300 mg/dL,

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whereas in the present study blood glucose values approached 400 mg/dL. These values

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may be an obstacle to the capacity of HT treatment to reduce circulating glucose.

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Moreover this effect has been postulated in type-2 diabetes model10 in which other

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metabolic profile distinct to the streptozotocin-induced diabetes could influence a

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possible antidiabetic effect of HT. Therefore, a possible glucose lowering effect of HT

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in our study can be ruled out in favor of a direct effect of this polyphenol on the

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biomarkers we analyzed.

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In brain tissues from diabetic animals we documented an increase in the

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concentration of lipid peroxides and inflammatory mediators (IL1ß and PGE2) before

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the brain slices were subjected to hypoxia-reoxygenation (Table 2). Under these

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baseline conditions, the values for variables relevant to the glutathione system and

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nitrosative stress did not change significantly in diabetic animals (p < 0.1). These results

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agree with earlier findings by several authors of higher concentrations of lipid peroxides

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in diabetes, either in streptozotocin-diabetic animals4 or in human patients with type I11

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or type II12 diabetes mellitus. The higher levels of lipid peroxides were especially

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evident in people with poor control of blood glucose levels, a situation which was

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reproduced in our experimental model of diabetes. Increased lipid peroxide production

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may be due to the well known increase in free radical production in diabetes, as a

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consequence of glycation end products or metabolic activation of the sorbitol pathway2.

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A higher lipid peroxide concentration in the brain of diabetic than normoglycemic

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animals indicates that this tissue is more susceptible to oxidative damage in diabetes.

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Under these circumstances, HT administration (1, 5 and 10 mg/kg/day)

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significantly reduced the concentration of TBARS in the brains of diabetic animals

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(Table 2). The antioxidant effect of HT has been widely described both in chemical and

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biological models, including in brain tissue13. Here we show evidence that HT also

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normalized brain lipid peroxidation in diabetic animals.

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The model of hypoxia-reoxygenation in brain slices from diabetic animals

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caused alterations in all variables analyzed here (Table 3): increased oxidative stress

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(increased TBARS and reduced glutathione levels) and nitrosative stress (nitric oxide

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and 3-nitrotyrosine production), and an increase in the brain inflammatory mediators

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PGE2 and IL1ß. These alterations were significantly greater in diabetic than in

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normoglycemic animals (Table 3). Therefore, brain tissues from diabetic animals

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showed a greater biochemical response after the hypoxia-reoxygenation injury than

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control tissues from normoglucemic animals. These changes have been found

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previously in models of ischemia-reperfusion injury in diabetic animals, especially for

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oxidative and nitrosative stress and inflammatory mediators4,14. In the experimental

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model used in this study we saw a greater increase in oxidative and nitrosative stress in

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diabetic than in normoglycemic animals4,15.

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In healthy animals used in this experimental model, HT had a beneficial effect

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on the variables studied here7,13. These results led us to investigate whether HT also had

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this pleiotropic effect in diabetic animals, in which brain biochemical damage was

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greater. Hydroxytyrosol significantly reduced the exacerbated oxidative stress in

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diabetic animals, acting primarily on lipid peroxidation rather than on the antioxidant

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glutathione system (Table 3). HT administration reduced brain TBARS concentration

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before hypoxia-reoxygenation, although not prevent the production of lipid peroxides

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after reoxygenation which was reduced only 10-18%. However, the absolute values of

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TBARS after reoxygenation were statistically lower than those of untreated diabetic

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animals with HT (Table 2 and 3).

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Respect to glutathione system in the diabetic animals the amount of GSH

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decreased more than non-diabetic animals after the hypoxia-reoxygenation (54.5% in

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DR with respect to 31.8% in NDR). In parallel, the percentage of glutathione in

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oxidized form (GSSG) increased significantly, possibly as an attempt antioxidant

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response to increased lipid peroxidation after reoxygenation, a fact corroborated by the

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increase in GSHpx activity (enzyme that converts GSH to GSSG). HT had a poor effect 12



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on these changes in the glutathione system (only 10 mg/kg/day exerted a significant

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effect), however significantly reduced the content of lipid peroxides, for that reason it

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can be postulated a direct effect of HT on the production of lipid peroxides instead an

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effect through a stimulus of the antioxidant glutathione system.

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Nitrosative stress was also reduced, as shown by the lower total amount of nitric

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oxide (5 and 10 mg/kg/day) and peroxynitrite produced (1, 5 and 10 mg/kg/day) after

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reoxygenation in brain slices (Table 3). Hydroxytyrosol also significantly reduced the

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production of tissue inflammatory mediators PGE2 and IL1ß (1, 5 and 10 mg/kg/day)

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(Table 3).

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The antioxidant effect of HT has been amply demonstrated in both chemical

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experiments and biological media16. Some studies, especially in vitro and after chemical

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stimulation of oxidative stress in cell cultures, have shown that HT can recover reduced

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levels of glutathione after oxidative induction17. However, in diabetic animals we saw

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no significant effect of HT on the glutathione system after cerebral hypoxia-

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reoxygenation. The greater effect of HT on lipid peroxidation than on the glutathione

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system has been reported previously in this experimental model in healthy animals13.

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Thus the antioxidant effect of this polyphenol in diabetic animals seems to target

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inhibition of brain lipid peroxidation.

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Regarding the effect of HT on nitrosative stress, some authors have reported that

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this phenolic compound inhibits the expression and activity of inducible nitric oxide

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synthase in human blood leukocytes18. It has been suggested that the increased nitric

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oxide concentration in brain slices from diabetic rats may be due in part to an increase

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in the inducible nitric oxide synthase isoform4.

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Regarding the effect of HT on biochemical components of tissue inflammation

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in our experimental model, the effect on PGE2 depends on the expression and activity of

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cyclooxygenase type 1 (COX-1) and 2 (COX-2). The specific inhibition of COX-2 was

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shown to correlate much better with a neuroprotective effect that the inhibition of COX-

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1 or nonspecific COX-1/COX-219. Moreover, in human blood samples it was found that

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HT inhibits both COX-1 and COX-218.

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In human blood samples it was also reported that HT inhibits leukocyte IL1ß

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induced with lipopolysaccharide18. Moreover, in human leukocyte cultures HT inhibits

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the expression of stimulatory factors for cytokine synthesis (NFkB, STAT-1α, IRF-1)

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and most inflammatory mediators20. In this connection, Maiuri et al.21 demonstrated in

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activated macrophages that HT does not directly inhibit the activity of inducible

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enzymes (iNOS and COX-2) but negatively regulates transcription factors for the

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synthesis and expression of these enzymes. These findings may explain the relationship

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between the effect of HT on IL1ß production and the antioxidant capacity of these

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compounds, as free radicals are primarily responsible for the activation of these

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transcription factors.

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These biochemical alterations may partly explain the greater neuronal death after

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hypoxia-reoxygenation in diabetic than in normoglycemic animals (Figure 1). This

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increased sensitivity to cell death after ischemic injury has been documented in

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experimental in vivo models of ischemia-reperfusion5 and hypoxia-reoxygenation in

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brain slices from diabetic rats4.

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Treatment with HT in diabetic animals decreased LDH efflux in brain slices after the model of hypoxia-reoxygenation (Figure 1). The percentage rates of reduction

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in neuronal death were 25.9%, 37.5% and 41.0% after the administration of 1, 5 and 10

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mg/kg/day of HT, respectively. This study is the first report, to our knowledge, of a neuroprotective effect of HT

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in a model of hypoxia-reoxygenation in brain slices from diabetic rats after oral

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treatment with this polyphenol, present in VOO. Earlier research on metabolic status or

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peripheral neuropathy in models of diabetes mellitus also used HT for experimental

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treatment22. Regarding the possible use of HT to treat disorders that affect the nervous

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system, some polyphenols have shown a neuroprotective effect, such as derivatives of

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curcumin, silibinin, green tea polyphenols and other extracts from various plants23-25.

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Respect to silibinin, it provides DNA protection and reduces oxidative stress in a brain

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specific area, in part via the activation of the heme oxygenase (HO-1) system25. In

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previous work with the same experimental model used here, HT obtained from VOO

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had a neuroprotective effect in diabetic animals15. The effect of HT in our experimental

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model of diabetes thus confirms, at least in part, the effects previously obtained with

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VOO.

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A neuroprotective effect after an ischemic insult has been described previously

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only for resveratrol14 and some catechins26. Hydroxytyrosol has been studied as a

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neuroprotective agent after ischemia in healthy animals13,27, but not in experimental

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animal models of diabetes.

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In conclusion, we show here that HT had a neuroprotective effect in an

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experimental model of diabetes induced in rats. The biochemical pathways by which

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HT produces this effect appear to be the same as in normoglycemic animals, although

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these pathways are more impaired in diabetic animals before. However, these

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biochemical pathways should be studied in greater depth in brain tissues from animals

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with diabetes mellitus treated with hydroxytyrosol.

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Abbreviations Used

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cNOS, constitutive nitric oxide synthase; COX-1, cyclooxygenase type 1; COX-2,

350

cyclooxygenase type 2; DR, diabetic rats; GSH, reduced glutathione; GSSG, oxidized

351

glutathione; GSHpx, glutathione peroxidase; HO-1, type-1 heme oxygenase; HT,

352

hydroxytyrosol; IL1ß, interleukin 1ß; iNOS, indlucible nitric oxide synthase; i.p.,

353

intraperitoneal; IRF-1, interferon regulatory factor 1; IU, international units; LDH,

354

lactate dehydrogenase; NADPH, nicotinamide-adenine dinucleotide phosphate; NDR,

355

non-diabetic rats; NFkB, nuclear factor kappa B; NO, nitric oxide; NO2- + NO3-,

356

nitrites + nitrates; PGE2, prostaglandin E2; STAT-1α, type 1α, signal transducer and

357

activator of transcription protein; TBARS, thiobarbituric acid reactive substances;

358

VOO, virgin olive oil.

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359

360

Safety

361

The research reported here does not involve any special safety considerations.

362

363

Acknowledgements

364

We thank A. Pino for excellent technical assistance and K. Shashok for improving the

365

use of English in the manuscript.

366

367

Supporting Information description

368

No supporting information provided.

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370 371

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Malaguarnera, M.; Galvano, F.; Nicolosi, A.; Li Volti, G. Neuroprotective effect of

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silibinin in diabetic mice. Neurosci. Lett. 2011, 504, 252-256.

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462

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463

464

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465

Figure captions

466

Figure 1. Lactate dehydrogenase efflux (LDH) measured after reoxygenation of brain

467

slices in nondiabetic rats (NDR) and in diabetic rats (DR) (N = 10 per group) treated

468

with saline (DR) or hydroxytyrosol (HT) at 1, 5 and 10 mg/kg/day p.o. *p < 0.01 with

469

respect to NDR, †p < 0.05 with respect to DR.

470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490

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Table 1. Mean values (± SEM) of body weight, blood glucose and blood cell counts in nondiabetic rats (NDR) and diabetic rats without treatment (DR) or treated with hydroxytyrosol (HT) (1, 5 and 10 mg/kg/day p.o.) after 2 months of follow-up. N = 10 rats per group.

NDR

Body weight (g)

DR

HT

HT

HT

1

5

10

mg/kg/day

mg/kg/day

mg/kg/day

415±9

392±12

389±15

385±14

391±15

99.8±4.2*

485±15

479±11

489±15

480±11

8.5±0.2

8.7±0.3

8.5±0.2

8.6±0.3

8.5±0.2

Leukocytes (×106/L)

6.0±0.2

6.3±0.3

6.0±0.3

6.3±0.2

6.0±0.3

Platelets

900±35

915±40

899±36

903±45

893±58

Hemoglobin (g/L)

16.0±0.3

15.9±0.3

16.1±0.2

15.8±0.4

16.2±0.3

Hematocrit (%)

46.9±0.9

46.5±0.8

46.5±0.7

46.8±0.6

46.2±1.0

Blood glucose (mg/dL) Red blood cells (×1012/L)

(×106/L)

*p < 0.0001 with respect to all diabetic groups

23


Page 25 of 27


Table 2. Mean values (± SEM) of variables determined before the hypoxiareoxygenation experiment in nondiabetic rats (NDR) and diabetic rats without treatment (DR) or treated with hydroxytyrosol (HT) (1, 5 and 10 mg/kg/day p.o.)

TBARS

NDR

DR

HT-1

HT-5

HT-10

4.4±0.3

6.8±0.5**

3.9±0.4†

3.8±0.3†

3.3±0.4†

12.9±0.8

12.3±1.3

12.1±1.0

12.0±1.5

12.4±1.3

9.2±0.9

9.1±0.8

9.1±1.0

9.2±1.1

9.1±0.9

3.5±0.6

3.3±0.7

3.1±0.5

3.3±0.6

3.5±0.5

11.2±1.3

11.7±1.8

11.8±1.6

11.7±0.9

12.0±1.5

2.1±0.05

2.1±0.2

2.0±0.4

1.9±0.3

2.2±0.4

19.0±1.0

31.2±4.0**

33.8±3.5

30.7±4.1

29.6±3.8

17.4±1.5

21.6±1.7*

20.5±1.6

19.3±1.5

19.0±1.6

(nmol/mg protein) GSH (µmol/g tissue) % GSSG vs GSH + GSSG GSHpx (µmol/min/mg protein) NO2- + NO3(pmol/0.1 g tissue) 3nitrotyrosine (nmol/0.1 g tissue) PGE2 (pg/0.1 g tissue) IL1ß (pg/0.1 g tissue)

*p < 0.05, **p < 0.0001 with respect to NDR. †p < 0.0001 with respect to DR. GSSG, oxidized glutathione

24



Page 26 of 27

Table 3. Mean values (± SEM) of variables determined after the hypoxia-reoxygenation experiment in nondiabetic rats (NDR) and diabetic rats without treatment (DR) or treated with hydroxytyrosol (HT) (1, 5 and 10 mg/kg/day p.o.)

TBARS

NDR

DR

HT-1

HT-5

HT-10

8.7±0.5

14.3±0.8**

8.8±0.7†

7.3±0.6††

6.8±0.8††

8.8±0.3

5.6±0.3*

5.6±0.4

5.9±0.5

6.5±0.3†

13.9±0.2

21.1±0.8**

22.1±0.8

10.9±1.0

19.6±0.7

6.6±0.4

11.5±0.4**

9.7±0.2

9.0±0.5†

8.9±0.5†

17.8±1.1

26.2±1.3**

24.1±0.8

13.6±1.3††

12.6±1.5††

1.9±0.2

6.9±0.8**

3.9±0.4††

2.0±0.3††

1.7±0.07††

56.2±1.3

105±9**

85.4±7.5††

85.2±9.6††

62.3±8.0††

35.4±2.3

59.8±1.5**

49.6±1.7†

33.9±1.5††

20.9±0.6††

(nmol/mg protein) GSH (µmol/g tissue) % GSSG vs GSH + GSSG GSHpx (µmol/min/mg protein) NO2- + NO3(pmol/0.1 g tissue) 3nitrotyrosine (nmol/0.1 g tissue) PGE2 (pg/0.1 g tissue) IL1ß (pg/0.1 g tissue)

*p < 0.05, **p < 0.0001 with respect to NDR. †p < 0.05, ††p < 0.0001 with respect to DR. GSSG, oxidized glutathione

25


Page 27 of 27


Figure 1

500

*

LDH (IU/L)

400

+

300

+

+

5

10

200 100 0 NDR

DR

1

HT (mg/kg/day p.o.)

26


Neuroprotective Effect of Hydroxytyrosol In Experimental Diabetes

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