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Phytotherapy Research
Vitis vinifera (grape) seed extract and resveratrol alleviate

bisphenol-A-induced metabolic syndrome: biochemical and
molecular evidences
Journal: Phytotherapy Research
Manuscript ID PTR-18-0999.R2
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Wiley - Manuscript type: Research Article
Date Submitted by the

13-Nov-2018
Author:
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Complete List of Authors: Rameshrad, Maryam; School of Pharmacy, Mashhad,

razavi, Bibi Marjan; 2- Targeted Drug Delivery Research Center,
Pharmacodynamy and Toxicology
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Imenshahidi, Mohsen; School of Pharmacy, ;

Hosseinzadeh, Hossein; School of Pharmacy, Pharmaceutical Research
Center
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Bisphenol-A, Dyslipidemia, Grape, Hypertension, Metabolic syndrome,

Keyword:
Resveratrol
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http://mc.manuscriptcentral.com/ptr
Page 1 of 52 Phytotherapy Research
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3 Vitis vinifera (grape) seed extract and resveratrol alleviate bisphenol-A-induced
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6 metabolic syndrome: biochemical and molecular evidences
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8 Maryam Rameshrad1, Bibi Marjan Razavi2, Mohsen Imenshahidi3, Hossein Hosseinzadeh1*
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12 1Pharmaceutical
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Research Center, Department of Pharmacodynamics and Toxicology, School
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15 of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
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17 2Targeted Drug Delivery Research Center, Department of Pharmacodynamics and Toxicology,
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School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
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3Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University

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24 of Medical Sciences, Mashhad, Iran
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26 *Corresponding author: Pharmaceutical Research Center, Pharmaceutical Technology
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29 Institute, Mashhad University of Medical Sciences, Mashhad, Iran, Tel.: +98 51 38819042;
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31 fax: +98 51 38823251, E-mail address: hosseinzadehh@mums.ac.ir
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33 Short title: Grape seed and resveratrol against BPA- metabolic syndrome
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Phytotherapy Research Page 2 of 52
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3 Abstract
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6 The mechanisms of bisphenol-A (BPA) induced metabolic syndrome as well as the protective
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8 role of grape seed extract (GSE) and resveratrol were investigated. Rats were treated with BPA
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10 (0 and 35 mg/kg/day, gavage) plus resveratrol (25, 50 and 100 mg/kg/day, ip) or GSE (3, 6, 12
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mg/kg/day, ip) or vitamin E (200 IU/kg/every other day, ip). After 2 months, mean systolic
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15 blood pressure (MSBP), serum lipid profile, glycaemia and fat index were examined. By
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17 ELISA, the serum concentrations of insulin, leptin, adiponectin and paraoxonase 1, and by real
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time-PCR as well as western blotting, key liver elements in cholesterol hemostasis (ldlr,
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22 cyp7a1, abcg5 and 8) and insulin signaling (p-Akt/Akt and p-PI3K/PI3K) were measured. BPA
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24 increased MSBP, total cholesterol and LDL-C, and reduced paraoxonase1, and the hepatic
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26 expression of both abcg5 and abcg8. It increased the body fat index, leptin, adiponectin, insulin
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29 and glycaemia level and decreased the hepatic protein expression of p-Akt/Akt and p-
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31 PI3K/PI3k. GSE, resveratrol or vitamin E co-administration along with BPA restored the
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33 detrimental effects of BPA in some levels.
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Herein, the predisposing effects of BPA induced metabolic syndrome were restored by GSE
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38 and resveratrol, linked to the regulation of insulin signaling, abcg8 expression and their
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40 antioxidant properties.
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Key words: Bisphenol-A; Dyslipidemia; Grape; Hypertension; Metabolic syndrome;
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45 Resveratrol
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3 1. Introduction
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6 Metabolic syndrome that is characterized by the coincidence of hypertension, dyslipidemia,
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8 abdominal obesity and hyperglycemia, is a main predisposing factor of cardiovascular diseases.
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10 Diet, sedentary lifestyle as well as exposing to endocrine disturbing chemicals as the case
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bisphenol-A (BPA) have promoting impacts on the characteristic feature of the metabolic
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15 syndrome (Stojanoska et al., 2017).
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17 BPA is a diphenylmethane derivative which is used excessively in polycarbonates
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products and epoxy resins (Fiege et al., 2000). Humans are exposed to it via contaminated
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22 foods, water and dental sealants (Koch and Calafat, 2009, Vandenberg et al., 2007).
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24 There are many pieces of evidence that proved detrimental effects of BPA in
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26 hepatotoxicity (Vahdati Hassani et al., 2017, Vahdati Hassani et al., 2018), infertility,
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29 neurological disorders (Wetherill et al., 2007) and polycystic ovary syndrome (Vandenberg et
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31 al., 2007). Furthermore, BPA exposure could evoke various constituents in metabolic
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33 syndrome including obesity, insulin resistance (Wang et al., 2012), diabetes (Shankar and
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Teppala, 2011), hypertension, heart attack, vascular diseases and atherosclerosis (Gao and
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38 Wang, 2014, Shankar and Teppala, 2012, Lind and Lind, 2011).
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40 So many plants and their constituents downregulate various adverse health problems
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related to metabolic disorders. In this case, berberine (Tabeshpour et al., 2017a), green tea
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45 (Razavi et al., 2017), avocado (Tabeshpour et al., 2017b), cinnamon (Mollazadeh and
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47 Hosseinzadeh, 2016), saffron (Razavi and Hosseinzadeh, 2017), rosemary (Hassani et al.,
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49 2016), garlic (Hosseini and Hosseinzadeh, 2015), Garcinia mangostana (Tousian Shandiz et
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52 al., 2017), milk thistle (Tajmohammadi et al., 2018) and grape (Akaberi and Hosseinzadeh,
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54 2016) have been introduced for their beneficial effects on metabolic syndrome to replace with
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56 synthetic medicines.
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3 Grape with the scientific name of Vitis vinifera from Vitaceae family is one of the most
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6 important members of the genus Vitis. The main active constituents of grapes are phenolic
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8 compounds as stilbenoids (resveratrol), phenolic acids (gallic acid), flavan-3-
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10 ols/proanthocyanidins (catechin), procyanidins, anthocyanins, flavonols and leucocyanidines.
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Besides, grape seeds, the waste product of grape juice, are a source of polyphenols such as
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15 procyanidins, flavonoids, catechins and procyanidins (Akaberi and Hosseinzadeh, 2016).
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17 The pharmacological effects of V. vinifera (grape) and resveratrol, have been described
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as the antioxidant, anti-inflammation, antibacterial, antiviral, antifungal, anticancer,
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22 antidiabetic, cardioprotective, neuroprotective and hepatoprotective (Nassiri-Asl and

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24 Hosseinzadeh, 2009, Nassiri-Asl and Hosseinzadeh, 2016). Akaberi and Hosseinzadeh
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26 reviewed that grape polyphenols increase lipolysis and decrease proliferation and lipogenesis
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29 in adipose tissue and increase fatty acid oxidation in skeletal muscle. They possess
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31 cardiovascular protective effects by modulation of cellular redox state, improvement of
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33 endothelial cell function, inhibition of platelet aggregation and regulation of hypertension.
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Grape polyphenols decrease insulin resistance and increase insulin sensitivity (Akaberi and
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38 Hosseinzadeh, 2016).
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40 There is a direct association between body fat mass and metabolic syndrome. Both
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adiponectin and leptin are secreted from adipose tissue to the bloodstream. It has been found
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45 that leptin directly correlates with body weight and adiponectin has an inverse association
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47 (Ailhaud, 2006). The liver has a fundamental role in cholesterol hemostasis. LDL receptor
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49 (LDLR) mediates reverse cholesterol transport to the liver. 7α-hydroxylase (CYP7A1) is
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52 important in the transformation of cholesterol into bile acids and ATP-binding cassette sub-
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54 family G member 5 and 8 (ABCG5 and ABCG8) have a key role in the secretion of cholesterol
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56 to the bile (Ikonen, 2006). Besides, paraoxonase 1 (PON1), one of the main components of
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HDL, decreases the risk of LDL oxidation and atherosclerosis (Fülöp et al., 2016). Insulin that
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3 is secreted via beta cells in the pancreas regulates glucose hemostasis. By binding to insulin
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6 receptors in insulin-sensitive tissues (liver, muscles and adipose tissues), it operates in
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8 sequence to activation of several downstream enzymes, including PI3K-Akt pathway, that
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10 leads to glucose uptake and gluconeogenesis suppression (Pahlavani et al., 2017).
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In this regards, we examined the protective effects of grape seed extract (GSE) and
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15 resveratrol on the metabolic disorders caused by BPA chronic exposure in rats received the
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17 normal diet (not high-fat diet) by evaluating the effects on the blood pressure, lipid profile,
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body weight, hepatic gene expression of cyp7a1, abcg5, abcg8 and ldlr, and serum paraoxonase
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22 1, leptin and adiponectin concentrations. Furthermore, the impact of BPA chronic exposure
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24 along with GSE or resveratrol on the insulin signaling was judged by assessing fasting blood
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26 sugar (FBS), serum insulin concentration and phospho-Akt/Akt and phospho-PI3K/PI3K level
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29 in the liver.
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33 2. Material and methods
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2.1. Materials
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38 The chemicals and materials were as follow: BPA (Sigma, USA), sodium orthovanadate
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40 (Na3VO4; Sigma, Madhya Pradesh, India), β-glycerol phosphate (Sigma, Germany), sodium
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deoxycholate (Sigma, New Zealand), phenylmethanesulfonyl fluoride (PMSF; Sigma,
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45 Germany), NaF (Sigma, USA), bromophenol blue (Merck, Darmstadt, Germany), Tris (Merck,
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47 Darmstadt, Germany), ethylenediaminetetraacetic acid (EDTA; Pars Tous Biotechnology,
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49 Mashhad, Iran ), ethylene glycol tetraacetic acid (EGTA; Sigma, USA), 2-mercaptoethanol (2-
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52 ME; Merck, Hohenbrunn, Germany), sodium dodecyl sulfate (SDS; Merck, Hohenbrunn,
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54 Germany), glycerol (Merck, Darmstadt, Germany), dry skim milk (Quetlab, UK) and dimethyl
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56 sulfoxide (DMSO; Merck, Darmstadt, Germany).
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3 Protease and phosphatase inhibitor cocktail and Pierce™ ECL Western Blotting
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6 Substrate were from Thermo Fisher Scientific, USA, and protein assay kit (Bradford reagent)
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8 and polyvinylidene difluoride (PVDF) were from Bio-Rad, USA.
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10 Rabbit polyclonal antibodies against Akt, phospho-Akt (Ser473), PI3K, phospho-PI3K
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p85 (Tyr458)/p55 (Tyr199) and mouse monoclonal antibody against β-actin, were obtained
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15 from Cell Signaling Technology, USA. Peroxidase-conjugated goat anti-rabbit and rabbit anti-
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17 mouse secondary antibodies were obtained from Cell Signaling Technology, USA. The other
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reagents were of a commercial analytical grade.
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22 Resveratrol was purchased from China and Vitamin E was from OSVE Pharmaceutical
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24 Co., Tehran, Iran. The grapes, V. vinifera Var. black were obtained in February from local
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26 market of Kashmar, Iran. The botanical identification was made by Dr. Mahmoud Shoor
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29 (Horticultural Department, College of Agriculture, Ferdowsi University, Mashhad, Iran).

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33 2.2. Grape seed extract preparation
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Grape seed extraction was done based on Vayupharp and Laksanalamai (2012) with some
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38 modifications (Vayupharp and Laksanalamai, 2012). After washing with distilled water, grape
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40 seeds were dried at room temperature and grounded. Then, 100 g of the prepared samples were
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soaked in n-hexane, in dark at room temperature and the mixture was filtered after 24 h. The
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45 remained powder on the filter paper was dried at room temperature and extracted with ethanol
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47 (50%, 400 ml×3) by maceration at 50 °C for 6h. After that, the mixture was filtered and the
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49 solvent removed at 60 °C using a rotary evaporator. The extract then freezes dried and kept at
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52 -20 °C in an airtight bottle for the further analysis.
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54 It is important to notice that this GSE-polyphenols has been standardized according to
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56 gallic acid (equals to 176 mg of gallic acid in one g of deride extract) and based on the HPLC
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method its identified antioxidant compounds were gallic acid, ellagic acid, epigallocatechin
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3 gallate, epicatechin gallate, epigallocatechin, epicatechin, catechin and their derivatives
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6 (Rameshrad et al., 2018).
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10 2.3. Animal studies
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Male albino Wistar rats (230-260 g) in this study were provided by the Animal Center, School
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15 of Pharmacy, Mashhad University of Medical Sciences, Iran. All procedures performed in this
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17 study were in accordance with the Guide for the Care and Use of Laboratory Animals of
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Mashhad University of Medical Sciences, Mashhad-Iran (931122; January 2015). The animals
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22 were given food and water ad libitum, housed at ambient temperature (25 ± 2°C) with relative
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24 humidity (50 ± 10%) in standard polypropylene cages, six per cage, under a 12 h light/dark
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26 cycle. The study was conducted on thirteen groups, each consisting of six animals. Vitamin E
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29 was used as the positive control group.

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31 1- Control: BPA vehicle (sesame oil and ethanol (2.4% v/v sesame oil)), 200 µl, gavage +
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33 vehicle of both resveratrol and GSE (normal saline and DMSO (15% v/v normal saline)),
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1 ml, i.p
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38 2 and 3- Resveratrol (25 and 100 mg/kg/day, ip) + BPA vehicle, gavage
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40 4 and 5- GSE (3 and 12 mg/kg/day, ip) + BPA vehicle, gavage
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6- BPA: (35 mg/kg/day, gavage) + vehicle of both resveratrol and GSE, ip
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45 7, 8 and 9- BPA (35 mg/kg/day, gavage) + resveratrol (25, 50 and 100 mg/kg/day, ip,
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47 separately)
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49 10, 11 and 12- BPA (35 mg/kg/day, gavage) + GSE (3, 6 and 12 mg/kg/day, ip, separately)
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52 13- BPA (35 mg/kg/day, gavage) + vitamin E (200 IU/kg/every other day, ip) (Razavi et al.,
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54 2014))
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56 Animals were treated once daily (around 11 am-14 pm), for 2 months on a normal diet.
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The solvents and dose selections were selected according to references (Vahdati Hassani et al.,
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3 2017, Rameshrad et al., 2018). The doses were adjusted according to the mean of animals’
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6 body weight in each group, weekly.
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8 After 2-month treatment, the mean systolic blood pressure (MSBP) was measured using
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10 the tail-cuff method. The next, the animals were left fasting overnight (10- 12 h) with free
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access to water and then were sacrificed by decapitation. The blood was collected into gel and
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15 clot activator tube for biochemistry analysis. The liver, inguinal and visceral epididymal
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17 adipose fat depot were removed and prepared for further analysis.
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22 2.4. Tail-cuff method for non-invasive blood pressure measurement

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24 Systolic blood pressure and heart rate were measured by non-invasive blood pressure method
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26 according Lorenz with some modification (Lorenz, 2002). The blood pressure and heart rate in
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29 animals were measured between 8 am-12 pm. The day before scarification the animals were
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31 anesthetized with ketamine and xylazine mixture (65 and 13 mg/kg, respectively). The rat tail
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33 was cleaned and warmed to increase blood flow. After that, a pulse transducer/pressure cuff
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(MLT125/R rat pulse transducer and tail cuff, ADInstruments, Australia) was placed around
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38 the proximal end of the tail. Then the cuff was inflated by pressing the related button on the
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40 NIBP (ML125 NIBP controller, ADInstruments, Australia) controller apparatus and SBP was
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calculated from the lab chart software (LabChart 7, ADInstruments, Australia). The mean
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45 values of 5 BPs readings were used for each animal.
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49 2.5. Body weight and fat peripheral index
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52 The body weight of the control and treated animals was measured at the beginning of the study
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54 followed by weekly measurement. The body weight –change of each animal was calculated
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56 every week. Besides, after the decapitation inguinal and visceral epididymal adipose fat depot
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isolated (Mann et al., 2014), washed in ice-cold saline, blotted between tow filter papered, and
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3 weighed. The fat index was estimated from the ratio of total fat weight (inguinal and epididymal
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6 fat weight) to the body weight of the animal at the end of the study. Fat mass index= (total fat
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8 weight/ body weight) × 100
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2.6. Biochemical blood tests
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15 At the end of the experiment, the fasted animals (overnight, 10-12 h) were decapitated and the
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17 thorax blood was collected into the gel and clot activated tube. After 15 min standing in the
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RT, the tubes were centrifuged at 3500 RPM for 15 min. The serum was collected in tubes and
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24 The serum samples were analyzed for the lipid profile (total cholesterol (TC), low-density
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26 lipoprotein (LDL-C), high-density lipoprotein (HDL-C) and triglyceride (TG)) and fasting
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29 blood sugar (FBS) using standard diagnostic kits with a clinical spectrophotometer (Hitachi,
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2.7. Measurement of cyp7a1, ldl receptor, abcg5 and abcg8 mRNA expression
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38 in the liver by quantitative real-time polymerase chain reaction
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40 Herein, we evaluated the effects of tested compounds on the hepatic expression of cyp7a1, ldl
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receptor, abcg5 and abcg8.
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45 TRI reagent (TriPure isolation reagent, Roche, USA) was used for total RNA extraction
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47 according to the manufacturer's instructions. Optical density measurement (A260/A280 ratio)
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49 with a Nanodrop instrument (Thermo scientific NanoDrop 2000 spectrophotometer, USA) was
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52 used to determine the purity of total RNA. Real-time PCR was performed using EXPRESS
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54 One-Step SYBER® GreenER® SuperMix Kit for one-step qRT-PCR according to the
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56 manufacturer's instructions and a StepOne® Real-Time PCR system (ABI, USA). Data were
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3 analyzed using the ΔΔCt method (Livak and Schmittgen, 2001). β-actin gene expression was
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6 used as the endogenous control.
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8 The cycling conditions were as follow: activation of reverse transcriptase and cDNA
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10 synthesis in one cycle at 50° C for 5 min, PCR activation in one cycle at 95° C for 2 min,
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denaturation in 40 cycles at 95° C for 15s for each and annealing/extension in one cycle at 60°
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15 C for 1 min.
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17 The primers were designed for detection of cyp7a1, abcg5, abcg8 (Wang et al., 2014),
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ldl receptor, and β-actin (Lari et al., 2014) gene expression, as given in table 1.
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24 2.8. Western blot analysis for phospho-Akt/Akt and phospho-PI3K/PI3K level
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26 in the liver
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29 Western blotting was performed for evaluating the effect of tested compounds on the protein
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31 expression of Akt and PI3K, as previously described with minor modifications (Vahdati
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33 Hassani et al., 2017). Following the experimental procedure, the liver was removed and
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immediately deep-frozen in liquid nitrogen. The tissue samples were homogenized in ice-cold
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38 solution (pH 7.4) containing Tris–HCl 50 mM (pH: 7.4), 2 mM EDTA, 10 mM NaF, 1 mM
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40 Na3VO4, 10 mM β-glycerol phosphate, 0.2% w/v sodium deoxycholate, 1 mM PMSF, and
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complete protease inhibitor cocktail using Polytron homogenizer (POLYTRON® PT 10–35,
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45 Kinematica, Switzerland). Following homogenization in lysis buffer, to completely destruct
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47 the cell membrane, samples were sonicated for 40 Sec. on ice using a probe sonicator (UP100H,
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49 Germany). Homogenized samples were centrifuged at 10000 RPM at 4˚C for 20 min
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52 (Hettich®, universal 320R Germany). The supernatant was aliquoted and stored at -70˚C for
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54 further analysis. The Bradford Protein Assay kit was used to evaluate the protein concentrations
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56 in the supernatant. The samples were mixed with loading buffer (100 mM Tris-base, 10% V/V
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2-ME, 4% W/V SDS, 20% V/V glycerol and 02% W/V bromophenol blue) at equal volume
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3 and subsequently incubated in boiling water for 5 min. An equal amount of proteins were
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6 loaded on the 8% SDS-polyacrylamide gel using a Min‑Protean Tetra Cell system (Bio‑Rad
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8 Laboratories, Inc., Hercules, CA, USA) to be separated by electrophoresis at 120 mA in
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10 running buffer. Separated proteins were transferred to an Immobilon‑P membrane in transfer
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buffer at 350 mAmp for 45 min. The next, for phosphorylated and non-phosphorylated
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15 antibodies the membranes separately were blocked in BSA or 5% non‑fat milk in wash buffer
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17 at room temperature (RT) with gentle shaking, for 2 h. After that, the membranes were soaked
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in the wash buffer at RT, with gentle shaking, for 5 min, 3 times. The membranes were
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22 incubated with the primary antibodies against phospho‑Akt (Ser473), Akt, phospho-PI3K p85
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24 (Tyr458)/p55 (Tyr199), PI3K or β-actin in 1:1000 dilution factor at 4˚C, with gentle shaking,
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26 overnight. The membranes were washed and incubated with the peroxidase‑conjugated goat
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29 anti‑rabbit and rabbit anti‑mouse secondary antibodies (1:3000), at RT, with gentle shaking,
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31 for 90 min.
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33 Finally, protein bands were visualized using PierceTM ECL Western Blotting Substrate
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(Thermo Fischer Scientific, USA) and Alliance 4.7 gel doc (UK) and quantified using UVIB
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38 and Map software (UVITC, UK). The densitometry values of p‑Akt were normalized to Akt
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40 and in the case of p-PI3K to PI3K. Besides, in each membrane, the blots were normalized to
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their β-actin blots.
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47 2.9. Enzyme-linked immunosorbent assay (ELISA) for leptin, adiponectin,
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49 paraoxonase 1 and insulin concentrations in the serum
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52 We quantified serum levels of leptin (rat ELISA kit, Abcam, Germany), adiponectin (rat
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54 ELISA kit, Abcam, Germany), paraoxonase 1 (rat ELISA kit, Aviva system biology, USA) and
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56 insulin (mouse/rat insulin enzyme immunoassay kit, Cayman Chemical, USA) accordance to
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the manufacturer‫׳‬s instructions to use of ELISA kits.
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3 The concentration of all proteins is expressed as ng/ml of the serum except for leptin that is
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6 expressed as pg/ml of the serum.
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10 2.10. Statistics:
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Data are presented as the mean ± SEM. One-way analysis of variance (ANOVA) was used for
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15 comparison among the groups. If the ANOVA analysis indicated significant differences, the
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17 LSD post-hoc test was performed. p<0.05 was considered to indicate a statistically significant
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difference.
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24 3. Results:
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26 3.1. The effects on the mean systolic blood pressure
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29 As demonstrated in figure 1, in BPA treated rats, MSBP significantly increased in comparison

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31 with the control group (p<0.01). Co-administration with resveratrol at 50 mg/kg/day decreased
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33 the effects of BPA in a significant manner. However, co-treatment with protective agents did
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not modify the increase in MSBP induced by BPA, significantly.
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40 3.2. The effects on the lipid profile and hepatic gene expression of cyp7a1,
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abccg5, abcg8
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45 As presented in table 2, chronic administration of BPA increased the serum level of total
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47 cholesterol (p<0.01) and LDL-C (p<0.001), while there were no significant differences
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49 between the serum levels of HDL-C and TG concentrations. Co-administration with resveratrol
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52 decreased concentration of total cholesterol (50 and 25 mg/kg/day, p<0.01) and LDL-C (25
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54 mg/kg/day, p<0.01). Co-treatment of GSE with BPA had no improving effects on the level of
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56 total cholesterol, in comparison with the BPA receiving group. However, it (3 mg/kg/day)
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decreased the level of LDL-C to near the normal level, significantly (p<0.01), in comparison
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3 with the BPA group. Furthermore, vitamin E co-administration restored the levels of both total
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6 cholesterol and LDL-C near the normal (p<0.01). It is important to notice that in comparison
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8 with the normal group, administration of just GSE (without BPA) with 12 mg/kg/day increased
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10 the level of total cholesterol, LDL-C and HDL-C, significantly (p<0.05), where with the lowest
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dose (3 mg/kg/day) there were no significant differences.
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15 Since co-treatment with lowest doses of both GSE and resveratrol regulated lipid profile
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17 more than the other doses, the effects on the hepatic gene expression of cyp7a1, abccg5, abcg8
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and ldlr were evaluated with lowest doses. In comparison with the control group,
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21
Fo
22 administration of BPA for 2 months significantly decreased the hepatic expression of both
23
24 abcg5 (p<0.01) and abcg8 (p<0.05). However, it had no effects on the expression of cyp7a1
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25
26 and ldl receptor in the liver tissue. It should be considered that GSE (3 mg/kg/day) but not
27
28
ee
29 resveratrol (25 mg/kg/day) decreased the hepatic expression of abcg5 in a significant manner
30
31 (p<0.01) (figure 2).
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32
33 About the hepatic level of abcg5 expression, protective co-treatments did not modify
34
35
ev
36
the decrease in expression of abcg5 induced by BPA, significantly.
37
38 Co-administration of BPA with resveratrol (25 mg/kg/day) or vitamin E (200
iew
39
40 IU/kg/every other day) increased the hepatic expression of abcg8, significantly (p<0.01 and
41
42
p<0.001, respectively). Furthermore, a non-significant (p=0.247) rise in hepatic expression of
43
44
45 abcg8 was seen in co-administration with GSE. abcg8 expression was increased by GSE
46
47 (3mg/Kg/day) alone compared with the control.
48
49
50
51
52 3.3. The effects on the body weight gain, fat mass index, paraoxonase 1, leptin
53
54 and adiponectin serum concentrations
55
56 In this study, there was no significant difference in weight gain between control and BPA
57
58
59
treated groups (data are not included). However, BPA induced a marked elevation in the body
60
14
1
2
3 fat index comparing control (p<0.01). Notably, co-treating BPA with highest dose of GSE (12
4
5
6 mg/kg/day, p<0.01) and resveratrol (100 mg/kg/day, p<0.001; 50 and 25 mg/kg/day, p<0.01)
7
8 showed an outstanding reduction in the fat index compared to BPA (figure 3).
9
10 According to the ELISA results, chronic exposure with BPA decreased the serum level
11
12
13
of PON1 from 44.34 ± 4.9 to 18.27 ± 3.7 ng/ml (p<0.05). In comparison with the BPA group,
14
15 the PON1 concentration significantly (p<0.05) increased to 62 ± 14.7 and 56.8 ± 9 ng/ml in co-
16
17 administration of BPA with GSE 12 and 3 mg/kg/day, respectively. Co-administration of BPA
18
19
with resveratrol 50 mg/kg/day profoundly (p<0.001) increased the serum level of PON1 to
20
21
Fo
22 120.5 ± 13.7 ng/ml. Furthermore, PON1 concentration in the rats received BPA with the lowest
23
24 dose of resveratrol (25 mg/kg/day) or vitamin E (200IU/kg/every other day) increased
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25
26 significantly (p<0.05) (figure 4). Co-administration with resveratrol at 100 mg/kg/day
27
28
ee
29 increased the concentration of PON1 to 48.9 ± 22 but not significantly (p=0.1).

30
31 Furthermore, the serum level of leptin and adiponectin was assessed by ELISA method.
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32
33 As demonstrated in figure 5 a, the serum level of leptin was profoundly increased by BPA from
34
35
ev
36
360 ± 30 to 618 ± 21 pg/ml (p<0.001). The serum level of leptin in the rats who had received
37
38 BPA with GSE decreased significantly to 387 ± 23 (p<0.001), 395 ± 72 (p<0.001) and 495 ±
iew
39
40 21 (p<0.01) pg/ml, respectively with 12, 6 and 3 mg/kg/day. Besides, co-treatment BPA with
41
42
resveratrol 50 and 25 mg/kg/day significantly (p<0.001) decreased leptin level to 379 ± 60 and
43
44
45 347 ± 35 pg/ml, respectively.
46
47 It is important to notice that the highest dose of resveratrol alone group (100 mg/kg/day)
48
49 showed a slight increase in the level of leptin in comparison with the control group (p<0.05).
50
51
52 Compared with the control group, the serum concentration of adiponectin (figure 5 b)
53
54 was significantly elevated from 6020 ± 1030 to 9410 ± 2020 ng/ml (p<0.05). This rise was
55
56 significantly (p<0.01) reduced by adding GSE (p<0.01), resveratrol or vitamin E (p<0.001) to
57
58
59
BPA.
60
15
1
2
3 It should be considered that the highest dose of resveratrol alone group showed
4
5
6 significant (p<0.01) lower concentration of adiponectin compared with the control.
7
8
9
10 3.4. The effects on the fasting blood sugar, serum insulin concentration and
11
12
13
phospho-Akt/Akt and phospho-PI3K/PI3K levels in the liver
14
15 Biochemical analysis (figure 6) showed that FBS concentration in the BPA treated rats
16
17 increased significantly from 89.7 ± 2.7 to 100.9 ± 3.4 mg/dl (p<0.05). This was accompanied
18
19
by an increase in the serum level of insulin (figure 7) where BPA increased its concentration
20
21
Fo
22 from 2.4 ± 0.09 to 3.3 ± 0.1 ng/ml (p<0.01).

23
24 In comparison with the BPA group, adding GSE to BPA regime maintained the insulin
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25
26 concentration at the level of BPA but decreased the FBS level significantly (12 mg/kg/day,
27
28
ee
29 p<0.05 and 3 mg/kg/day, p<0.01). Besides, administration of resveratrol with the highest dose
30
31 (100 mg/kg/day) along with BPA decreased profoundly both insulin level (p<0.001) and FBS
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32
33 concentration (p<0.01). Co-administration of BPA with resveratrol 25 mg/kg/day and vitamin
34
35
ev
36
E had no effects on the level of insulin but decreased FBS concentration significantly
37
38 (p<0.001).
iew
39
40 Although co-administration of BPA with resveratrol 50 mg/kg/day, decreased insulin
41
42
level significantly (p<0.01), however, it had no diminishing effects on the level of FBS.
43
44
45 It is important to notice that GSE alone at the highest dose (12 mg/kg/day) increased
46
47 FBS level (p<0.05) slightly without a significant increase in the serum level of insulin.
48
49 Resveratrol alone at the highest dose (100 mg/kg/day) increased the serum concentration of
50
51
52 insulin (p<0.05) in comparison with the control. Both GSE and resveratrol at lowest doses had
53
54 no significant differences in the level of FBS and insulin compared with the control group.
55
56 Since the lowest doses of GSE and resveratrol showed better results compared to the
57
58
59
others, for evaluating the effects on the signaling of insulin, the lowest doses were chosen. As
60
16
1
2
3 demonstrated in figure 8, chronic exposure with BPA significantly decreased the level of
4
5
6 phospho (p)-Akt/Akt and phospho (p)-PI3K/PI3K (p<0.05). Co-administration of GSE (3
7
8 mg/kg/day) along with BPA had no significant improving effect on p-Akt/Akt level but
9
10 restored p-PI3K/PI3K (p<0.05). Co-administration of resveratrol (25 mg/kg/day) along BPA
11
12
13
improved p-Akt/Akt ratio (p<0.001) with no significant effects on the p-PI3K/PI3K level.
14
15 Furthermore, administration of vitamin E along with BPA had no improving effects on the p-
16
17 Akt/Akt level but increased p-PI3K/PI3K ratio (p<0.01).
18
19
20
21
Fo
22 4. Discussion
23
24 4.1. Effect of BPA, resveratrol and GSE on blood pressure
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25
26 Findings of our study implied that BPA chronic administration increased blood pressure in
27
28
ee
29 comparison with the control group. Comparing with BPA group, co-treatment with the GSE or
30
31 resveratrol or vitamin E diminished blood pressure but not significantly. However, co-
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32
33 administration with resveratrol 50 mg/kg/day decreased it significantly.
34
35
ev
36
Previous studies proved BPA effects on the arterial wall and atherosclerosis, the main
37
38 reasons for hypertension. Administration of BPA (40 mg/kg/day, gavage, 8 weeks) to
iew
39
40 hyperlipidemic rabbits induced atherosclerosis, infiltration of inflammatory cells to the
41
42
myocardium, myocardial infarction and calcification (Fang et al., 2015). BPA exposure (0.4
43
44
45 mg/kg/day, gavage, 12 weeks) in hyperlipidemic rabbits enhanced atherosclerosis in both aorta
46
47 and left coronary arteries (Fang et al., 2014). In another study, BPA administration (50 mg/kg,
48
49 in diet, for 12 weeks) to PXR-humanized ApoE deficient mice increased atherosclerosis in the
50
51
52 aortic root and brachiocephalic artery (Sui et al., 2014). Furthermore, BPA (50 µg/kg/day in
53
54 drinking water) with the high-fat diet in apolipoprotein E knockout mice increased
55
56 atherosclerotic lesion in the aorta (1.7 fold) (Kim et al., 2014).
57
58
59
60
17
1
2
3 Hypertension is a risk factor for metabolic syndrome. Different studies proved the
4
5
6 beneficial activity of grape polyphenols in lowering blood pressure. For examples, in isolated
7
8 rat aorta, endothelium-dependent relaxation of grape extract has been proved. This effect was
9
10 linked to the NO-cGMP pathway and prostacyclin-cAMP pathway via muscarinic and
11
12
13
histamine receptors (Ha et al., 2016). In adult (age: 90 and 180 days) offspring of rats had been
14
15 fed with a high-fat-diet during lactation (21 days in weaning period), grape skin extract
16
17 significantly decreased high fat-fed dams induced offspring hypertension during sucking in age
18
19
90 and 180 days. In this study, antihypertensive effects of grape skin extract were attributed to
20
21
Fo
22 induction of NO synthesis and antioxidant action (Emiliano et al., 2011).

23
24 Recently Rameshrad and her colleagues identified GSE antioxidant compounds and
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25
26 concluded that it is rich in polyphenols. They evaluated malondialdehyde (MDA) level both in
27
28
ee
29 the liver and aorta in a similar model with our study. MDA, a thiobarbiturate reactive substance,
30
31 is a characteristic feature of oxidative stress. That study showed, BPA treatment (35 mg/kg/day,
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32
33 gavage, 2-monts) increased the MDA level significantly in the liver and aorta (p<0.001) and
34
35
ev
36
co-treatment with GSE (3, 6, 12 mg/kg/day, ip, 2 months), resveratrol (25, 50, 100 mg/kg/day,
37
38 ip, 2 months) or vitamin E (200 IU/kg per every other day, ip), significantly decreased its level
iew
39
40 both in the liver and aorta (p<0.001).They showed BPA predisposing effects in atherosclerosis
41
42
and protective effects of GSE and resveratrol on it, linked to their antioxidant properties
43
44
45 (Rameshrad et al., 2018).
46
47
48
49 4.2. Effect of BPA, resveratrol and GSE on lipid profile and obesity
50
51
52 In the current investigation, there was a positive significant correlation between BPA exposure
53
54 and body fat index, total cholesterol, LDL-C, leptin and adiponectin level while a negative one
55
56 between BPA and PON1 concentration, hepatic abcg5 and 8 expressions. Co-administration of
57
58
59
GSE or resveratrol with BPA reversed BPA aforementioned effects.
60
18
1
2
3 Our results were in line with the previous study that BPA administration to rats (0.5
4
5
6 mg/kg/day, gavage, for 30 days) had no effects on the body weight but increased total
7
8 cholesterol and LDL-C (Vahdati Hassani et al., 2017). The other study reported that BPA
9
10 exposure (2 mg/kg/day, ip, for 4 weeks) increased total cholesterol and LDL-C concomitant
11
12
13
with an increase in the body weight in male mice (Moghaddam et al., 2015). Fang and his
14
15 colleagues (2014) reported administration of BPA (0.4 mg/kg/day, gavage, 12 weeks) to
16
17 hyperlipidemic rabbits increased adiposity and induced adipocytes hypertrophy with no effects
18
19
on the body weight (Fang et al., 2014). Previously, it has been reported that BPA by affecting
20
21
Fo
22 on the expression of some genes evokes an important role in adipocytes differentiation,

23
24 function, white adipose tissue weight and obese phenotype in rodents. Exposure with BPA
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25
26 during developmental stage increases the expression of the proadipogenic transcription factors
27
28
ee
29 as the case CAAT enhancer binding protein alpha, peroxisome proliferator-activated receptor-γ
30
31 (PPAR), sterol regulatory element binding protein-1C, and lipogenic enzymes such as
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32
33 lipoprotein lipase, fatty acid synthase and stearoyl-CoA desaturase 1 (Somm et al., 2009).
34
35
ev
36
BPA has an important role in endocrine adipose tissue function by an effect on the
37
38 secretion of leptin and adiponectin (Menale et al., 2016). Leptin, a hormone produced by
iew
39
40 adipose cells, by influence on the CNS and hypothalamus, has a main role in regulating body
41
42
weight. There is a direct association between body fat mass and leptin level. Obese people have
43
44
45 higher blood concentration of leptin than normal weight people (Ailhaud, 2006). Adiponectin,
46
47 the other secreted hormone from adipocytes, decreases hepatic glucose production (Berg et al.,
48
49 2001) and enhances skeletal muscle glucose uptake and fatty oxidation (Yamauchi et al., 2002).
50
51
52 In obesity, type II diabetes, coronary artery disease and hypertension, the level of adiponectin
53
54 decreases, while in chronic inflammation, chronic left ventricular systolic heart failure, type I
55
56 diabetes, systemic lupus erythematosus, rheumatoid arthritis and irritable bowel syndrome, its
57
58
59
level increases (Aprahamian and Sam, 2011). Although a decrease in adiponectin level with an
60
19
1
2
3 increase in leptin concentration is seen in obesity, the increase in both could be seen in chronic
4
5
6 kidney disease (Lim et al., 2015). It is supposed to evaluate the possible effects of BPA on the
7
8 kidney function in future studies.
9
10 Contrary to our expectations, this study found that chronic exposure to BPA increases
11
12
13
adiponectin level accompany with leptin increase and body fat index that could be attributed
14
15 to its detrimental effects on the heart or kidney, a hypothesis that should be evaluated in future
16
17 studies.
18
19
Pon1 antiatherogenic effects are mediated by inhibiting LDL oxidation, decreasing
20
21
Fo
22 oxidized-LDL uptake by macrophages and stimulating cholesterol efflux from these cells.
23
24 Mostly, there is an inverse association between Pon1 activity and obesity markers as the case
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25
26 leptin level (Fülöp et al., 2016).
27
28
ee
29 The liver has a fundamental role in cholesterol hemostasis. Totally in the liver,
30
31 cholesterol synthesis is mediated especially by 3-hydroxy-3-methyl-glutaryl –CoA reductase
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32
33 (HMGCR), cholesterol output is mainly performed by apolipoprotein B (ApoB), reverse
34
35
ev
36
cholesterol transport to the liver is facilitated with scavenger receptor B1 (SR-B1) and LDLR,
37
38 and cholesterol transformation into bile acids is achieved by CYP7A1. In hepatocytes, biliary
iew
39
40 secretion of cholesterol into bile is mediated by ATBCG5 and ATBCG8 (Ikonen, 2006). In this
41
42
regards, any decrease in the reverse transport of cholesterol to the liver, cholesterol conversion
43
44
45 to the biliary acids or cholesterol secretion to the bile causes an increase in the blood cholesterol
46
47 level. In our study, BPA increased the level of total cholesterol and LDL-C accompanied with
48
49 a decrease in hepatic expression of both abcg5 and abcg8, a novel result that is reported for the
50
51
52 first time.
53
54 There is some evidence that has proved lipid-lowering and anti-hyperlipidemic effects
55
56 of GSE and resveratrol. Co-administration of GSE (250 and 500 mg/kg, orally) reduced the
57
58
59
effect of high-fat emulsion on serum TG and cholesterol levels in the rat. This was attributed
60
20
1
2
3 to GSE inhibitory effects on pancreatic lipase and cholesterol esterase. Besides, it has been
4
5
6 shown that GSE inhibits the formation of cholesterol micelles and diminishes the binding
7
8 capacity of cholesterol to bile acids (Adisakwattana et al., 2010).
9
10 GSE decreased cellular [3H] cholesterol uptake in HT29, HepG2 and HuTu80 cells
11
12
13
(Leifert and Abeywardena, 2008). In vitro, GSE induces inhibitory effects on pancreatic lipase
14
15 and lipoprotein lipase (Moreno et al., 2003). Resveratrol inhibited copper- and irradiation-
16
17 induced LDL and HDL oxidation. It significantly decreased cholesterol influx and increased
18
19
cholesterol efflux from J774 cells (Berrougui et al., 2009). In hepatoma-bearing rats, feeding
20
21
Fo
22 with resveratrol (50 ppm in the diet, 20 days) decreased the serum TG and VLDL plus LDL
23
24 level. Resveratrol hypolipidemic action was linked to an increase in excretion of neutral sterols
rP
25
26 and bile acids into feces (Miura et al., 2003).
27
28
ee
29 Herein, the protective effects of highest dose of GSE or resveratrol is weaker than the
30
31 lowest doses. It has been discussed that overdose of antioxidants may result in toxic and pro-
rR
32
33 oxidant effects instead of protective effects that is related to their relative antioxidant capacity,
34
35
ev
36
concentration and bioavailability (Bouayed and Bohn, 2010, Nie et al., 2002, Hou et al., 2008,
37
38 Chung et al., 2003).
iew
39
40
41
42
4.3. Effect of BPA, resveratrol and GSE on glycaemia and insulin resistance
43
44
45 Herein, we found that BPA treatment increases FBS accompanied by an increase in insulin
46
47 concentration and a decrease in hepatic pAkt/Akt and pPI3K/PI3K ratio. Co-administration of
48
49 GSE or resveratrol in some levels by increasing insulin concentration decreased FBS and
50
51
52 improved the hepatic ratio of pAKT/AKT and pPI3K/PI3K.
53
54 It is important to notice that in western blotting section, the expression level of β-actin
55
56 (housekeeping protein) was evaluated as the loading control and internal standard. In this
57
58
59
60
21
1
2
3 regards, the ratio of expression of target protein in each PVDF membrane was normalized to
4
5
6 its related β-actin protein expression (Dennis-Sykes et al., 1985).
7
8 Previous studies showed hyperglycemic effects of BPA both in rat (Vahdati Hassani et
9
10 al., 2017) and mice (Moghaddam et al., 2015). Oxidative stress, inflammation (Pahlavani et
11
12
13
al., 2017) and insulin resistance, that is seen in diabetes or metabolic syndrome, is accompanied
14
15 with impaired insulin-stimulated proteins (PI3K and pAkt) (da Costa et al., 2017).
16
17 BPA induces insulin intolerance and hyperglycemia with decrease in Akt signaling.
18
19
Exposure of rats with BPA (20 and 200 mg/kg/day, orally for one month) increased serum
20
21
Fo
22 insulin level, decreased hepatic glucose oxidation, insulin receptor expression, Akt, p-Akt and
23
24 glycogen level, in a dose-dependent manner with no effect on the fasting blood sugar
rP
25
26 (Jayashree et al., 2013). Administration of BPA with 10 and 100 µg/kg/day, subcutaneously,
27
28
ee
29 for 4 days to adult mice induced hyperinsulinemia and feature of insulin resistance (Alonso-
30
31 Magdalena et al., 2006).
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32
33 Many studies proved antidiabetic properties of grapes and resveratrol by different
34
35
ev
36
mechanisms. Protective effects of grape seed-skin extract ( 4g/kg) in the diabetic rat have been
37
38 attributed to its antioxidant properties (Oueslati et al., 2016). Administration of grapes
iew
39
40 polyphenols decreased high-fat adverse effects. It decreased percentages of body fat content,
41
42
TG, liver weight, insulin resistance and inflammation. Grapes polyphenols decreased insulin
43
44
45 resistance and improved glucose tolerances in male mice (Collins et al., 2016). Administration
46
47 of resveratrol (20 mg/kg, ip, 4 weeks) to diabetic rats decreased hepatic level of glucose and
48
49 TG with an increase in the hepatic level of insulin and pi3k and akt gene expression (Sadi et
50
51
52 al., 2015).
53
54 Since we included the healthy animals with the same source and age, we did not evaluate
55
56 mentioned items at the start of study. Furthermore, based on our unpublished studies, this
57
58
59
model seems to be a repeatable model for induction of metabolic syndrome. Nevertheless, it is
60
22
1
2
3 important to evaluate these parameters not only after 2-month of treatment, but also in the
4
5
6 baseline and at the start of the study. Some limitations in finance made us exclude this part of
7
8 study.
9
10
11
12
13
5 Conclusion
14
15 Herein, we showed detrimental effects of BPA (35 mg/kg/day, gavage, 2 months) on blood
16
17 pressure, hepatic expression of abcg5 and abcg 8 and lipid profile. Besides, chronic exposure
18
19
to BPA decreased the serum concentration of pon1 and increased the serum concentration of
20
21
Fo
22 leptin, adiponectin and body fat index. It impaired insulin signaling by an increase in FBS,
23
24 along with serum insulin concentration and a decrease in hepatic p-Akt/Akt and p-PI3K/PI3K
rP
25
26 ratio. The present study provides evidence for the protective effects of GSE (3 mg/kg/day, ip,
27
28
ee
29 2 months) and resveratrol (25 mg/kg/day, ip, 2 months) against BPA induced metabolic
30
31 disorders. Likewise, vitamin E (200 IU/kg/every other day, ip, 2 months) as a potent
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32
33 antioxidant restored the detrimental effects of BPA on metabolic factors, on some levels. In
34
35
ev
36
this regards, the protective effects of GSE and resveratrol could be attributed to effects on
37
38 hepatic abcg8 expression, improving insulin signaling and their antioxidant properties. It
iew
39
40 proposes GSE and resveratrol as an adjuvant treatment for chemical or drug-induced metabolic
41
42
disorders.
43
44
45
46
47
48
49 Acknowledgments
50
51
52 We thank the Research Vice Chancellors of Mashhad University of Medical Sciences
53
54 (NO.941389) and Iran National Science Foundation (NO.95004557) for financial supported.
55
56
57
58
59
Conflict of interest
60
23
1
2
3 The authors declare that they have no conflict of interest.
4
5
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8 Adisakwattana S., Moonrat J., Srichairat S., Chanasit C., Tirapongporn H., Chanathong B., . .
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15 Ailhaud G. 2006. Adipose tissue as a secretory organ: from adipogenesis to the metabolic
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6 (BPA) concentration associates with obesity and insulin resistance. J Clin Endocrinol
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17 Yamauchi T., Kamon J., Minokoshi Y., Ito Y., Waki H., Uchida S., . . . Kadowaki T. 2002.
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Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-
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22 activated protein kinase. Nat Med, 8(11), 1288-1295.

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1
2
3 Table 1: Primer sequences for RT-PCR:
4
5
6 1 cyp7a1, forward 5’ GGAAAAGCTGGCTGAGGGAT 3’
7
8 cyp7a1, reverse 5’ TCAAAGGTGGAGAGCGTGTC 3’
9
10 2 ldl receptor, forward 5’ GACTGCAAGGACAAGTCGGA 3’
11
12
13 ldl receptor, reverse 5’ GCACTGGGTCACATTGATGC 3’
14
15 3 atbcg5, forward 5’ GGGAAGTGTTTGTGAACGGC 3’
16
17
atbcg5, reverse 5’ GTGTATCTCAGCGTCTCCCG 3’
18
19
20 4 abcg8, forward 5’ ACGTGGACTTGACGAGCATT 3’
21
Fo
22 abcg8, reverse 5’ GTGTCCTGTGTGAGGGTCTG 3’

23
24
5 β-actin, forward 5’ GGGAAATCGTGCGTGACATT 3’
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26
27 β-actin, reverse 5’ GCGGCAGTGGCCATCTC 3’
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ee
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31
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38
iew
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1
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3 Table 2: Serum level of total cholesterol (TC), low-density lipoprotein (LDL-C), high-density
4
5
6 lipoprotein (HDL-C) and triglyceride (TG) in rat following 2 months treatment with: control
7
8 (BPA co-solvent, gavage; GSE and resveratrol co-solvents, ip), GSE12 and 3 (GSE 12 and 3
9
10 mg/kg/day, ip; BPA co-solvent, gavage), R100 (resveratrol 100 mg/kg/day, ip; BPA co-
11
12
13
solvent, gavage), BPA (BPA 35 mg/kg/day, gavage; GSE and resveratrol co-solvents, ip), BPA
14
15 + GSE (BPA 35 mg/kg/day, gavage; GSE 3, 6 and 12 mg/kg/day, ip), BPA + RES (BPA 35
16
17 mg/kg/day, gavage; resveratrol 25, 50 and 100 mg/kg/day, ip), B + E (BPA 35 mg/kg/day,
18
19
gavage; vitamin E 200 IU/kg/every other day, ip). Data are expressed as the mean ± SEM of
20
21
Fo
22 the six independent experiments. The significant of results was tested by one-way ANOVA
23
24 with LSD post hoc test.
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26 TC (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) TG (mg/dl)
27
28
ee
29 Control 68.7 ± 2.5 28.7 ± 1.4 20.5 ± 1.16 88.6 ± 5.1

30
31 GSE 12 mg/kg/day 79.6 ± 3.9 + 38.6 ± 2.5 + 26.6 ± 1.25 ++/*** 71.8 ± 5.8
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33
GSE 3 mg/kg/day 78.0 ± 5.7 38.8 ± 4.6 22.0 ± 1.0 ** 86.0 ± 4.0
34
35
ev
36 Resveratrol 100 68.0 ± 2.8 * 24.9 ± 2.9 *** 23.0 ± 1.4 85.2 ± 9.8
37
38 mg/kg/day
iew
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40
BPA 35 mg/kg/day 81.4 ± 2.6 ++ 42.5 ± 2.6 +++ 19.5 ± 0.4 87.9 ± 5.1
41
42
43 BPA 35 + GSE 12 85.2 ± 2.3 ++ 37.7 ± 1.4 27.4 ± 0.7 ++/*** 97.0 ± 11.7
44
45 mg/kg/day
46
47 BPA 35 + GSE 6 82.6 ± 5.1 + 40.9 ± 3.2 + 27.2 ± 1.0 ++/*** 85.4 ± 12.1
48
49
50 mg/kg/day
51
52 BPA 35 + GSE 3 87.0 ± 6.4 ++ 28.6 ± 4.1 ** 27.8 ± 1.5 +++/*** 105.8 ± 6.5
53
54 mg/kg/day
55
56
57 BPA 35 + RES 100 76.0 ± 5.2 33.4 ± 2.5 22.75 ± 1.2 99.2 ± 9.0
58
59 mg/kg/day
60
33
1
2
3 BPA 35 + RES 50 64.0 ± 5.8 ** 33.7 ± 3.9 18.8 ± 1.5 57.4 ± 4.8 ++/**
4
5
6 mg/kg/day
7
8 BPA 35 + RES 25 62.0 ± 1.8 ** 27.4 ± 4.5 ** 24.0 ± 1.4 * 59.2 ± 10.3 +/*
9
10 mg/kg/day
11
12
13 BPA + vitamin E 58.0 ± 5.6 31.2 ± 5.3 ** 23.9 ± 2.0 +/** 69.2 ± 3.5
14
15 +/***
16
17 +p<0.05, ++p<0.01 and +++p<0.001 from respective control value; *p<0.05, **p<0.01 and
18
19 ***p<0.001
20 as compared with the BPA group.
21
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22
23
24
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25
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ee
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31
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iew
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34
1
2
3 Figure 1: Mean systolic blood pressure (MSBP) following 2 months treatment with: control
4
5
6 (BPA co-solvent, gavage; GSE and resveratrol co-solvents, ip), GSE (GSE 12 mg/kg/day, ip;
7
8 BPA co-solvent, gavage), R (resveratrol 100 mg/kg/day, ip; BPA co-solvent, gavage), BPA
9
10 (BPA 35 mg/kg/day, gavage; GSE and resveratrol co-solvents, ip), BPA + GSE (BPA 35
11
12
13
mg/kg/day, gavage; GSE 3,6 or 12 mg/kg/day, ip), BPA + RES (BPA 35 mg/kg/day, gavage;
14
15 resveratrol 25, 50 or 100 mg/kg/day, ip), B + E (BPA 35 mg/kg/day, gavage; vitamin E (200
16
17 IU/kg/every other day, ip). Data are expressed as the mean ± SEM of the six independent
18
19
experiments. The significant of results was tested by one-way ANOVA with LSD post hoc test.
20
21
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++p<0.01 from respective control value; *p<0.05 as compared with the BPA group.
22
23
24
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26 Figure 2: Hepatic expression of cyp7a1 (a), abcg5 (b), abcg8 (c) and ldl receptor (d) following
27
28
ee
29 2 months treatment with: control (BPA co-solvent, gavage; GSE and resveratrol co-solvents,
30
31 ip), GSE3 (GSE 3 mg/kg/day, ip; BPA co-solvent, gavage), R25 (resveratrol 25 mg/kg/day, ip;
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33 BPA co-solvent, gavage), BPA (BPA 35 mg/kg/day, gavage; GSE and resveratrol co-solvents,
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35
ev
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ip), BPA + GSE (BPA 35 mg/kg/day, gavage; GSE 3 mg/kg/day, ip), BPA + RES (BPA 35
37
38 mg/kg/day, gavage; resveratrol 25 mg/kg/day, ip), BPA + vitE (BPA 35 mg/kg/day, gavage;
iew
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40 vitamin E 200 IU/kg/every other day, ip). Data are expressed as the mean ± SEM of the 5
41
42
independent experiments. The significant of results was tested by one-way ANOVA with LSD
43
44
45 post hoc test. +p<0.05, ++p<0.01 and +++p<0.001 from respective control value; *p<0.05,
46
47 **p<0.01 and ***p<0.001 as compared with the BPA group.
48
49
50
51
52 Figure 3: Fat index (100×(inguinal and visceral epididymal adipose fat weight)/body weight)
53
54 following 2 months treatment with: control (BPA co-solvent, gavage; GSE and resveratrol co-
55
56 solvents, ip), GSE12 (GSE 12 mg/kg/day, ip; BPA co-solvent, gavage), R100 (resveratrol 100
57
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59
mg/kg/day, ip; BPA co-solvent, gavage), BPA (BPA 35 mg/kg/day, gavage; GSE and
60
35
1
2
3 resveratrol co-solvents, ip), BPA + GSE (BPA 35 mg/kg/day, gavage; GSE 3, 6 and 12
4
5
6 mg/kg/day, ip), BPA + RES (BPA 35 mg/kg/day, gavage; resveratrol 25, 50 and 100
7
8 mg/kg/day, ip). Data are expressed as the mean ± SEM of the 5 independent experiments.
9
10 The significant of results was tested by one-way ANOVA with LSD post hoc test. ++p<0.01
11
12
13
from respective control value; **p<0.01 and ***p<0.001 as compared with the BPA group.
14
15
16
17 Figure 4: Serum concentration of paraoxonase 1 (Pon1) following 2 months treatment with:
18
19
control (BPA co-solvent, gavage; GSE and resveratrol co-solvents, ip), GSE12 and GSE3 (GSE
20
21
Fo
22 12 and 3 mg/kg/day, ip; BPA co-solvent, gavage), R100 and R25 (resveratrol 25 and 100
23
24 mg/kg/day, ip; BPA co-solvent, gavage), BPA (BPA 35 mg/kg/day, gavage; GSE and
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31 mg/kg/day, ip) and B + E (BPA 35 mg/kg/day, gavage; vitamin E 200 IU/kg/every other day,
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33 ip). Data are expressed as the mean ± SEM of the 5 independent experiments. The significant
34
35
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36
of results was tested by one-way ANOVA with LSD post hoc test. +p<0.05 and +++p<0.001
37
38 from respective control value; *p<0.05 and ***p<0.001 as compared with the BPA group.
iew
39
40
41
42
Figure 5: Serum concentration of leptin (a) and adiponectin (b) following 2 months treatment
43
44
45 with: control (BPA co-solvent, gavage; GSE and resveratrol co-solvents, ip), GSE12 and GSE3
46
47 (GSE 12 and 3 mg/kg/day, ip; BPA co-solvent, gavage), R100 and R25 (resveratrol 25 and 100
48
49 mg/kg/day, ip; BPA co-solvent, gavage), BPA (BPA 35 mg/kg/day, gavage; GSE and
50
51
53
55
56 mg/kg/day, ip) and B + E (BPA 35 mg/kg/day, gavage; vitamin E 200 IU/kg/every other day,
57
58
59
ip). Data are expressed as the mean ± SEM of the 5 independent experiments.
60
36
1
2
3 The significant of results was tested by one-way ANOVA with LSD post hoc test. +p<0.05,
4
5 ++p<0.01
6 and +++p<0.001 from respective control value; **p<0.01 and ***p<0.001 as compared
7
8 with the BPA group.
9
10
11
12
13
Figure 6: Serum concentration of fasting blood sugar (FBS) following 2 months treatment
14
15 with: control (BPA co-solvent, gavage; GSE and resveratrol co-solvents, ip), GSE12 and 3
16
17 (GSE 12 and 3 mg/kg/day, ip; BPA co-solvent, gavage), R100 and 25 (resveratrol 100 and 25
18
19
mg/kg/day, ip; BPA co-solvent, gavage), BPA (BPA 35 mg/kg/day, gavage; GSE and
20
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Fo
23
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26 mg/kg/day, ip), B + E (BPA 35 mg/kg/day, gavage; vitamin E 200 IU/kg/every other day, ip).
27
28
ee
29 Data are expressed as the mean ± SEM of the 4 independent experiments. The significant of
30
31 results were tested by one-way ANOVA with LSD post hoc test. +p<0.05 and ++p<0.01 from
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33 respective control value; *p<0.05, **p<0.01 and ***p<0.001 as compared with the BPA group.
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35
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36
37
38 Figure 7: Serum concentration of insulin following 2 months treatment with: control (BPA co-
iew
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40 solvent, gavage; GSE and resveratrol co-solvents, ip), GSE12 and 3 (GSE 12 and 3 mg/kg/day,
41
42
ip; BPA co-solvent, gavage), R100 and 25 (resveratrol 100 and 25 mg/kg/day, ip; BPA co-
43
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45 solvent, gavage), BPA (BPA 35 mg/kg/day, gavage; GSE and resveratrol co-solvents, ip), BPA
46
47 + GSE (BPA 35 mg/kg/day, gavage; GSE 3, 6 and 12 mg/kg/day, ip), BPA + RES (BPA 35
48
49 mg/kg/day, gavage; resveratrol 25, 50 and 100 mg/kg/day, ip), B + E (BPA 35 mg/kg/day,
50
51
52 gavage; vitamin E 200 IU/kg/every other day, ip). Data are expressed as the mean ± SEM of
53
54 the 4 independent experiments. The significant of results were tested by one-way ANOVA
55
56 with LSD post hoc test. +p<0.05 and ++p<0.01 from respective control value; **p<0.01 and
57
58 ***p<0.001
59
as compared with the BPA group.
60
37
1
2
3
4
5
6 Figure 8: Representative immunoblots of p-AKT at residue serine 473 to Akt protein
7
8 expression ratio (a and b) and p-PI3K at residue 458 to PI3K protein expression ratio (c and d)
9
10 in the hepatic tissue following 2 months treatment with: control (BPA co-solvent, gavage; GSE
11
12
13
and resveratrol co-solvents, ip), GSE3 (GSE 3 mg/kg/day, ip; BPA co-solvent, gavage), R25
14
15 (resveratrol 25 mg/kg/day, ip; BPA co-solvent, gavage), BPA (BPA 35 mg/kg/day, gavage;
16
17 GSE and resveratrol co-solvents, ip), BPA + GSE (BPA 35 mg/kg/day, gavage; GSE 3
18
19
mg/kg/day, ip), BPA + RES (BPA 35 mg/kg/day, gavage; resveratrol 25 mg/kg/day, ip), BPA
20
21
Fo
22 + vitE (BPA 35 mg/kg/day, gavage; vitamin E 200 IU/kg/every other day, ip). Data are
23
24 expressed as the mean ± SEM of the 4 independent experiments. The significant of results was
rP
25
26 tested by one-way ANOVA with LSD post hoc test. +p<0.05 and ++p<0.01 from respective
27
28
ee
29 control value; *p<0.05, **p<0.01 and ***p<0.001 as compared with the BPA group.
30
31
rR
32
33
34
35
ev
36
37
38
iew
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
38
1
2
3
4
5
6 100
7
8
++
9
10
80 *
MSBP (mmHg)
11
12
13
14
60
15
16
17
18
40
19
20
21 20
Fo
22
23
24
0
rP
25
26
27
6
3
12
50
25
12
0
G ol
E
0
10
10
28
B+
BP
r
ee
SE
nt
29
R
co
30
31 BPA+GSE BPA+RES
rR
32
33
34
Fig 1
35
ev
36
37
38
iew
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
39
1
2
3
4
5
6
cyp7a1 mRNA/ actin mRNA ratio
7 2.0
8
9
10
11
12 1.5
13
14
15
16
17 1.0
18
19
20
21
Fo
22
23
0.5
24
rP
25
26
27
28
0.0
ee
29
3
25
A
l
SE
ES
itE
ro
SE
30
BP
R
nt
+G
31 +R
+v
G
rR
co
32
A
A
33 BP
BP
BP
34
35
ev
36
37 (a)
38
iew
39
40
41 Fig 2a
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
40
1
2
3
4
5
6
abcg5 mRNA/ actin mRNA 1.5
7
8
9
10
11
12
13 1.0 * +
14
15
++ +
16
17 ++
18
19
20 0.5 +++
21
Fo
22
23
24
rP
25
26
27
28 0.0
ee
29
3
25
A
l
SE
30
ES
itE
ro
SE
BP
31
R
nt
+G
+R
+v
rR
G
32
co
A
A
A
33
BP
BP
BP
34
35
ev
36
37 (b)
38
iew
39
40
41 Fig 2b
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
41
1
2
3
4
5
6
abcg8 mRNA/ -actin mRNA 3 +
7
8 ***
9
10
11
** **
12
13
2
14
15
16
17
18
19
20
1
21
+
Fo
22
23
24
rP
25
26
27
0
28
3
ee25
A
l
SE
ES
itE
ro
SE
29
BP
R
nt
+G
+R
+v
30
G
co
31
A
A
A
rR
32
BP
BP
BP
33
34
35 (c)
ev
36
37
38
iew
39 fig 2c
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
42
1
2
3
4
5
6
ldl receptor mRNA/ -actin mRNA
7 2.0
8
9
10 ***
11
12 1.5
13
14
15
16
17 1.0
18
19
20
21
0.5
Fo
22
23
24
rP
25
26
27 0.0
28
ee
29
3
25
A
l
SE
ES
itE
ro
SE
BP
30
R
nt
+G
+R
+v
G
31
co
rR
A
32
A
BP
BP
BP
33
34
35
ev
36 (d)
37
38
iew
39
40 Fig 2d
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
43
1
2
3
4
5
2.5
6
7 ++
8
9
10
2.0
Fat index (%)
11
12 ** ** **
13
14
1.5 ***
15
16
17
18
1.0
19
20
21
0.5
Fo
22
23
24
rP
25
26
0.0
27
6
3
12
50
25
12
0
G ol
28
10
10
BP
r
ee
SE
nt
29
R
co
30
31 BPA+GSE BPA+RES
rR
32
33
34
Fig 3
35
ev
36
37
38
iew
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
44
1
2
3
4
5
6
Pon1 concentration (ng/ml) 150 +++
7
8 + ***
9
10
11
12 100
13
14
15
* *
16 * *
17
18
19 50
20
21
+
Fo
22
23
24
rP
25
26
0
27
6
3
R 3
12
50
B+ 5
G 12
BP 5
A
0
G rol
E
0
28
SE
2
2
10
10
ee
SE
R
nt
29
co
30
31 BPA+GSE BPA+RES
rR
32
33
34
35 Fig 4
ev
36
37
38
iew
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
45
1
2
3
4
5
6
Leptin concentration (pg/ml) 800
7
8
9
+++
10
11
600 +
12
13
14
*** ** ***
*** ***
15
16
400
17
18
19
20
21
200
Fo
22
23
24
rP
25
26
0
27
6
3
R 3
12
50
B+ 5
G 12
BP 5
A
0
G rol
E
0
SE
2
28
2
10
10
ee
SE
R
nt
29
co
30
31 BPA+GSE BPA+RES
rR
32
33
34
(a)
35
ev
36
37 Fig 5a
38
iew
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
46
1
2
3
4
5 Adiponectin concentration (ng/ml)
6
7 15000
8
9
10
11
+
12
13 10000
14
15
16
17 **
** **
18
19 5000
20 + ******
21 ++ ***
Fo
22
23
++ ***
24
rP
25
26
0
27
6
3
R 3
12
50
25
G 12
25
A
0
G rol
E
0
SE
10
10
28
B+
BP
SE
R
ee
nt
29
co
30
31
BPA+GSE BPA+RES
rR
32
33 (b)
34
35
ev
36
37 Fig 5b
38
iew
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
47
1
2
3
4
5
6 150
7
8
9
10 + + +
11
FBS (mg/dl)
12
13
100 * * ** ** ++ ***
14
15
***
16
17
18
19 50
20
21
Fo
22
23
24
rP
25
26
0
27
6
3
R 3
12
50
25
G 12
25
A
0
G rol
E
0
28
SE
10
10
ee
B+
BP
SE
R
nt
29
co
30
31 BPA+GSE BPA+RES
rR
32
33
34
35 Fig 6
ev
36
37
38
iew
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
48
1
2
3
4
5
6 5
7
8
9 ++
10 4
+ + ++ ++
Insulin (ng/ml)
11
12 ++
13
14
3 **
15
16 +
17
18 ***
19 2
20
21
Fo
22
23 1
24
rP
25
26
27
28
0
ee
29
6
3
R 3
12
50
25
G 12
25
A
0
G rol
E
0
30
SE
10
10
B+
BP
SE
31
nt
rR
32
co
33
34
BPA+GSE BPA+RES
35
ev
36
37 Fig 7
38
iew
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
49
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Fo
22
23
24
rP
25
26
27
28
ee
29
30
31
rR
32
33
34
35
ev
36
37
38
iew
39
40
41
42
43
44
Fig 8a
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
50
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Fo
22
23
24
rP
25
26
27
28
ee
29
30
31
rR
32
33
34
35
ev
36
37
38
iew
39
40
41
42
43 Fig 8b
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
51
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Fo
22
23
24
rP
25
26
27
28
ee
29
30
31
rR
32
33
34
35
ev
36
37
38
iew
39
40
41
42
43 Fig 8c
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
52
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Fo
22
23
24
rP
25
26
27
28
ee
29
30
31
rR
32
33
34
35
ev
36
37
38
iew
39
40
41
42
43 Fig 8d
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60

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Phytotherapy Research

Vitis vinifera (grape) seed extract and resveratrol alleviate

Journal: Phytotherapy Research

Date Submitted by the

Complete List of Authors: Rameshrad, Maryam; School of Pharmacy, Mashhad,

Imenshahidi, Mohsen; School of Pharmacy, ;

Bisphenol-A, Dyslipidemia, Grape, Hypertension, Metabolic syndrome,

3Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University

22 antidiabetic, cardioprotective, neuroprotective and hepatoprotective (Nassiri-Asl and

29 (Horticultural Department, College of Agriculture, Ferdowsi University, Mashhad, Iran).

29 was used as the positive control group.

22 2.4. Tail-cuff method for non-invasive blood pressure measurement

22 stored at -70° C for further analysis.

29 As demonstrated in figure 1, in BPA treated rats, MSBP significantly increased in comparison

29 increased the concentration of PON1 to 48.9 ± 22 but not significantly (p=0.1).

22 from 2.4 ± 0.09 to 3.3 ± 0.1 ng/ml (p<0.01).

22 induction of NO synthesis and antioxidant action (Emiliano et al., 2011).

22 on the expression of some genes evokes an important role in adipocytes differentiation,

22 therapy of the metabolic syndrome. Phytother Res, 30(4), 540-556.

29 resistance. Environ Health Perspect, 114(1), 106-112.

22 Arch Pharmacol, 389(9), 931-949.

22 chronic kidney disease in Asian adults. PLoS One, 10(3), e0122009.

29 of US adults. J Environ Public Health, 2012.

29 male rats exposed to bisphenol A: Mechanism of hepatotoxicity and biomarker

22 activated protein kinase. Nat Med, 8(11), 1288-1295.

22 abcg8, reverse 5’ GTGTCCTGTGTGAGGGTCTG 3’

29 Control 68.7 ± 2.5 28.7 ± 1.4 20.5 ± 1.16 88.6 ± 5.1

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