Açaí (Euterpe oleracea Mart.) seed extract protects against hepatic steatosis and fibrosis in high-fat diet-fed mice: Role of local renin-angiotensin system, oxidative stress and inflammation

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Açaí (Euterpe oleracea Mart.) seed extract protects against hepatic steatosis and fibrosis in high-fat diet-fed mice: Role of local renin-angiotensin system, oxidative stress and inflammation Matheus Henrique Romão, Graziele Freitas de Bem, Izabelle Barcellos Santos, Ricardo de Andrade Soares, Dayane Teixeira Ognibene, Roberto Soares de Moura, ⁎ Cristiane Aguiar da Costa, Ângela Castro Resende Department of Pharmacology, Institute of Biology, Rio de Janeiro State University, Rio de Janeiro, RJ, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Euterpe oleracea Mart. Polyphenols Obesity Hepatic steatosis Fibrosis Renin-angiotensin system

The Euterpe oleracea Mart. (açaí) seed extract (ASE), rich in polyphenols, has been evidenced as a potential regulator of body mass with antioxidant and anti-inflammatory actions. We aimed to assess the effects of ASE and enalapril (ENA) on the hepatic steatosis and fibrosis and related overexpressed RAS, oxidative stress and inflammation in C57BL/6 mice fed a high-fat diet (HFD). The animals were fed a standard diet (10% fat, Control), 60% fat (HF), HF + ASE (300 mg kg−1 d−1) and HF + ENA (30 mg kg−1 d−1) for 12 weeks. Our results demonstrate that ASE prevented the obesity and related hepatic steatosis and fibrosis in HFD-fed mice. The negative modulation of RAS in hepatic tissue may contribute to these beneficial effects of ASE by favoring the reduction of the oxidative stress and inflammation, highlighting a greater antioxidant activity of ASE compared to ENA.

1. Introduction Nonalcoholic fatty liver disease (NAFLD) is the most common obesity-related disease in the world and has a significant association with dyslipidemia, and insulin resistance (Schetz et al., 2019). The HFD and the positive energy balance result in a large stock of triacylglycerol (TG) in the liver leading to NAFLD, which begins with accumulation of triacylglycerol (steatosis) (Jennison, Patel, Scorletti, & Byrne, 2019), contributing to the dysfunction of hepatic cells, and progress to more aggressive liver injury, inflammation, and fibrosis in the form of nonalcoholic steatohepatitis (NASH) (Farrell & Larter, 2006). The renin-angiotensin system (RAS) is an important physiological regulator of blood pressure, electrolyte balance, and fluid homeostasis. Angiotensin II (ANG II) is the major effector of the RAS and is related to chronic tissue structural and functional changes through its profibrotic effects (Souza-Mello, 2017). Both circulating and local RAS components are up-regulated in obese individuals and animals (Frigolet, Torres, &

Tovar, 2013; Kalupahana & Moustaid-Moussa, 2012), and liver activation of the classical RAS axis [angiotensin-converting enzyme (ACE)/ ANG II/ANG II type 1 receptor (AT1r)] seems to be involved in the development of steatosis, inflammation and fibrosis of the liver (Bataller & Brenner, 2005; Shim, Eom, Kim, Kang, & Baik, 2018; SouzaMello, 2017). Furthermore, the activation of the AT1r causes the upregulation of NADPH oxidase activity, generating reactive oxygen species (ROS) and leading to insulin resistance, fat deposing and accumulation in the liver, contributing to the progression to NASH (Moreira de Macêdo, Guimarães, Feltenberger, & Sousa Santos, 2014; Souza-Mello, 2017). Therefore, the modulation of the hepatic RAS seems to be a potential pharmacological target to prevent the hepatic steatosis and fibrosis. Several studies have attributed to polyphenols a broad range of biological activities including anti-inflammatory, antioxidant, cardiovascular protective actions, as well as hepatoprotective effects (de Oliveira et al., 2015; Guo et al., 2012; van der Heijden et al., 2016). The

Abbreviations: ACE, angiotensin-converting enzyme; ANG 1-7, angiotensin 1-7; ANG II, angiotensin II; ANOVA, one-way analysis of variance; ASE, Açaí seed extract; AT1r, angiotensin II type 1 receptor; AT2r, angiotensin II type 2 receptor; CAT, catalase; CEUA, ethics Committee for Experimental Animals Use and Care; ENA, enalapril; GPx, glutathione peroxidase; HFD, high-fat diet; HPLC, high- performance liquid chromatography; MDA, malondialdehyde; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; NF-kB, nuclear factor-kB; RAS, renin-angiotensin system; ROS, reactive oxygen species; SEM, standard error measure; SOD, superoxide dismutase; TG, triacylglycerol ⁎ Corresponding author at: Department of Pharmacology, Institute of Biology, Rio de Janeiro State University. Av. 28 de Setembro, 87, Rio de Janeiro, RJ, Brazil. E-mail address: [email protected] (Â.C. Resende). https://doi.org/10.1016/j.jff.2019.103726 Received 6 September 2019; Received in revised form 18 November 2019; Accepted 30 November 2019 1756-4646/ © 2019 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Matheus Henrique Romão, et al., Journal of Functional Foods, https://doi.org/10.1016/j.jff.2019.103726

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plant Euterpe oleracea Mart. (Aracaceae family) and its fruit “açaí”, are widely found in the Amazon region of Brazil. Studies carried out by our group have shown that the hydroalcoholic extract of the “açaí” seeds (ASE) rich in polyphenols, such as catechin, epicatechin and polymeric proanthocyanidins (de Oliveira et al., 2015) has an antihypertensive effect in experimental models of spontaneous and renovascular hypertension (da Costa et al., 2012; da Silva Cristino Cordeiro et al., 2018). Interestingly, this antihypertensive effect of ASE is accompanied by the reduction of plasma renin levels (da Costa et al., 2012; da Silva Cristino Cordeiro et al., 2018), suggesting that ASE may exert some interference with RAS, possibly contributing to its beneficial cardiovascular effects. In addition, our group also demonstrated that this extract prevents hyperglycemia, dyslipidemia, and accumulation of fat in the liver of mice fed a HFD (de Oliveira et al., 2010, 2015). However, no previous studies have evaluated the pharmacological effects of ASE on the local RAS and inflammation associated with hepatic steatosis and fibrosis in obese mice. Therefore, the present study aimed to evaluate and compare the effects of treatment with ASE and the ACE inhibitor, ENA on the metabolic and hepatic changes in a diet-induced obesity experimental model. The potential mechanism for the protection of hepatic steatosis and fibrosis was also investigated by focusing on hepatic RAS components, the fibrosis-associated oxidative stress and inflammation.

light/12 h dark, lights on at 6 a.m.), and the ambient temperature was kept at 23 ± 2 °C. The animals had free access to water and food. Afterward, mice were randomly divided into 4 groups (n = 10 for each group): Control group was fed standard diet (10% energy from lipids, 76% from carbohydrate, and 14% from protein; total 3.8 kcal/g), and HF, HF + ASE, and HF + ENA groups, were fed HF diet (60% energy from lipids, 26% from carbohydrate, and 14% from protein; total 5.4 kcal/g). The mice in HF + ASE group were given 300 mg.kg-1.d-1 of ASE by oral gavage for 12 weeks, HF + ENA group were given 30 mg.kg-1.d-1 of enalapril by oral gavage for 12 weeks and the mice in the Control and HF groups received only the vehicle (water). The dose of ASE was based on previous studies that showed significant changes in programming metabolic syndrome (de Oliveira et al., 2010). The chronic treatment (12 weeks) with ASE was based on the time necessary to induce the metabolic alterations in the C57BL/6 mice fed a HF diet (de Oliveira et al., 2010). The diets were elaborated by Rhoster (São Paulo, Brazil) following the standard recommendations for rodents in the maintenance state of the American Institute of Nutrition (AIN-93M) (Reeves, Nielsen, & Fahey, 1993). 2.4. Food consumption, energy intake and body weight measurements Food consumption of the mice was estimated, daily, by subtracting the amount of food left on the grid and amount of spilled food from the initial weight of food supplied. Energy intake was calculated on the basis of 3.8 kcal/g for the control diet and 5.4 kcal/g for the high-fat diet. Body weight of all groups was measured weekly using a digital balance (precision of 0.01 g). The average weight of all groups was calculated through the 12 weeks of treatment. Body weight was measured every 15 days and body mass gain was expressed considering the weight (grams) before treatment and 90 days at the end of treatment.

2. Materials and methods 2.1. Preparation of Açaí (Euterpe oleracea Mart.) seed extract (ASE) Euterpe oleracea Mart. fruits were obtained from the Amazon Bay (Pará State, Brazil). The plant was identified at the Goeldi Museum (Belém do Pará, Brazil), where a voucher specimen was deposited under number MG 205222. The hydroalcoholic extract was obtained as previously described (Rocha, Carvalho, & Sousa, 2007). First, we separated the pulp from the seed and then weighed 200 g of seed, which were boiled in 400 ml of water for 5 min. After the boiling, 400 ml of ethanol were added. The extract was stored at a dark bottle in refrigerator temperature and shook 2 h a day for 10 days. Then, the extract was filtered through a Whatman n°1 filter paper, and the ethanol was evaporated under low pressure at 55 °C. The extract was then lyophilized at −30 to −40 °C temperature in a vacuum of 200 mmHg. The obtained extract was stored at room temperature for posterior use. The polyphenol content was measured using the Folin-Ciocalteu procedure and its vasodilator potential was measured through vascular reactivity.

2.5. Measurement of blood glucose Blood glucose concentration was determined with a glucometer (Accu-Chek ® Active; Roche, Mannheim, Germany) based on the reaction of glucose-glucose oxidase. Mice have previously fasted for 6 h, and blood samples were then collected by cutting a small area of the animals’ tail. 2.6. Euthanasia and tissue extraction After 12 weeks of treatment, the animals were deeply anesthetized (thiopental sodium, 70 mg/Kg i.p). The liver was removed and weighted and samples were prepared for light microscopy and stereology or frozen at −80 °C for further biochemical analysis. The fat pads (epididymal, retroperitoneal and subcutaneous) were carefully dissected and weighted for calculating the adiposity index determined as the ratio between the sum of all pads masses divided by the total body mass, and represented as a percentage.

2.2. Polyphenol analyses The analysis of the aqueous fraction residue from ASE by highperformance liquid chromatography (HPLC) and MALDI-TOF mass spectrum was previously reported by our group (de Moura et al., 2012; de Oliveira et al., 2015). The HPLC analysis of ASE revealed that it is composed by proanthocyanidins (88% of the total area) and in a minor extent catechin and epicatechin. The chemical and spectrometric analysis revealed that ASE is composed predominantly of polymeric procyanidins, heteropolymers with one gallocatechin unit and, a minor extent, of galloylated procyanidins (de Oliveira et al., 2015).

2.7. Hepatic cholesterol and triglycerides Frozen mouse liver samples (50 mg) were homogenized in an ultrasonic processor with 1 ml of isopropanol, and centrifugated at 2000g for 10 min at 4 °C. The aliquots of the supernatant were used for measuring TG and cholesterol in a semiautomatic biochemical analyzer using a commercial kit (Bioclin, Belo Horizonte, Brazil).

2.3. Animals and diet All experimental procedures were approved by the Ethics Committee for Experimental Animals Use and Care (CEUA) of Roberto Alcântara Gomes Institute of Biology / Rio de Janeiro State University (protocol: CEUA n° 034/2015). Male mice of the C57BL/6 strain (n = 40) were obtained from ANILAB, laboratory of animals (São Paulo, São Paulo, Brazil) at 4 weeks of age. After 1 week of adaptation and at 5 weeks of age, the animals were housed in individual cages in a controlled environment room, with light/dark cycle conditions (12 h

2.8. Western blotting The expressions of renin, angiotensin conversing enzyme (ACE), ACE2, AT1 receptor (AT1r), AT2 receptor (AT2r), Mas receptor (MASr) and nuclear factor-kB (NF-kB) were evaluated in liver homogenates. Liver samples were homogenized in cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% SDS, 5 mM EDTA, 50 mM NaF and 1% Triton 2

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2.12. Immunohistochemistry

X-100) containing Complete Protease Inhibitor Cocktail Tablets (Roche, Basel, Switzerland) using an Ultra-Turrax homogenizer (IKA Werke GmbH & Co. KG, Staufen, Germany). The total protein content was determined by the BCA protein assay kit (Pierce, Rockford, IL, USA). Samples (20 μg total protein) were electrophoresed in 10% tris–glycine sodium dodecyl sulfate–polyacrylamide gels. Proteins were transferred to polyvinylidene fluoride membranes (Hybond ECL; Amersham Pharmacia Biotech, London, UK). The blots were blocked with 5% bovine albumin (Sigma-Aldrich Co., St. Louis, MO, USA) in T-TBS (0.02 M Tris/0.15 M NaCl, pH 7.5, containing 0.1% Tween 20) at room temperature for 1 h and incubated with primary antibodies (renin (1:500), ACE (1:1000), AT1r (1:1000), AT2r (1:500), MAS receptor (MASr, 1:500), ACE-2 (1:1000), NF-kB (1:500), pNF-kB (1:500) and β-actin (1:500), Santa Cruz Biotechnology, Santa Cruz, CA, EUA) overnight at 4 °C. After washing with T-TBS, blots were incubated with corresponding secondary conjugated antibodies at 1:5000 and 1:10000 concentrations for 1 h. We also incubated all membranes with β-actin antibody (1:500) to avoid possible inconsistency in protein loading and/or transfer. Antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Blots were developed with enhanced chemiluminescence (ECL; Amersham Biosciences Inc., Piscataway, NJ, USA). The signals were visualized by ChemiDoc Resolutions System and determined by quantitative analysis of digital images of gels using Adobe Photoshop version 13.0 (Adobe System Incorporated).

Liver tissue sections were initially dewaxed and rehydrated by serial passages through xylene and graded alcohols. To block endogenous peroxidase activity, they were placed in 3% H2O2, and to reduce nonspecific protein binding, they were incubated in phosphate-buffered saline supplemented with 5% bovine serum albumin for 20 min. Then, the slides were incubated with primary antibodies for 8-isoprostane, IL6 and TNF-α at a dilution of 1:100 (polyclonal antibody; Oxford Biomedical Research, Oxford, MI, USA), at 4 °C overnight in a humidified chamber. After that, the samples were rinsed with phosphate buffered saline and incubated with biotinylated linked antibody and peroxidase-labeled streptavidin, according to the product datasheet instructions (LSAB kit; Universal Dako Cytomation, Glostrup, Denmark). The peroxidase activity was revealed by 3,30′-diaminobenzidine tetrachloride (K3466, DAB; Universal Dako Cytomation, Glostrup, Denmark). Sections were counterstained with Mayer's hematoxylin, rinsed and then mounted. Digital images from the liver were obtained and studied by image analysis. A selection tool was used to identify the liver area with positive immunoreactions, and this selection was segmented in a black-and-white image, where white shows the immunostained area. The liver was delimited using an irregular AOI tool, and inside this delimited area, the tissue area occupied by white color was quantified using the image histogram tool. It was expressed as density stained per liver (%) using Image-Pro Plus version 7.0 (Media Cybernetics, Silver Spring, MD, USA).

2.9. Determination of oxidative damage: Malondialdehyde and carbonyl protein

2.13. Statistical analysis Values are expressed as the mean ± standard error measure (SEM). Statistical analysis was determined using one-way analysis of variance (ANOVA), with Tukey post hoc test. P values less than or equal to 0.05 accepted as statistically significant. All data were analyzed in GraphPad Prism version 6 software (GraphPad Software, La Jolla, USA).

The lipid membrane damage was determined in liver homogenates by formation of products of lipid peroxidation (malondialdehyde-MDA) concentration, using the thiobarbituric acid reactive substances method, as previously described (Draper & Hadley, 1990). Briefly, the samples were mixed with 1 ml of 10% trichloroacetic acid and 1 ml of 0.67% thiobarbituric acid. They were then, heated in a boiling water bath for 30 min. The absorbance of the organic phase containing the pink chromogen was measured by spectrophotometry at 532 nm. MDA equivalents were expressed in nmol/mg protein. The protein carbonylation was determined from the formation of the carbonyl group by reaction with 2,4-dinitrophenylhydrazine (2,4DNPH) according to the method described by Levine et al. (1990).

3. Results 3.1. Body weight and mass gain, adiposity index, food and energy intake The body weight of the animals from the four groups was the same at the beginning of the study (Fig. 1). At the end of the 12 weeks’ treatment period, the increase in the body weight, body mass gain (Fig. 1) and adiposity index (Table 1) induced by HF diet were prevented by treatment with ASE and ENA (p ≤ 0.05; n = 10). The amount of food consumed was similar between HF and Control groups, while the energy intake was higher (p ≤ 0.05) in HF compared to the Control group (Table 1). The treatment with ASE did not change the food and energy intakes in the HF + ASE compared with the HF group, but the energy intake of the HF + ASE group was not different from the Control. These parameters were reduced by ENA in the HF + ENA group compared with the HF group (p ≤ 0.05) (Table 1).

2.10. Antioxidant enzyme activity Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities were determined in liver homogenates. SOD activity was assayed by measuring the inhibition of noradrenaline autooxidation as absorbance at 480 nm (Bannister & Calabrese, 1987). Catalase activity was measured in terms of the rate of decrease in hydrogen peroxide at 240 nm (Aebi, 1984). GPx activity was measured by monitoring the oxidation of NADPH at 340 nm in the presence of hydrogen peroxide (Flohé & Günzler, 1984).

3.2. Blood glucose, lipid profile, hepatic steatosis and fibrosis 2.11. Light microscopy and stereology At the beginning of the study, the glycemic levels were similar between the different groups studied (Table 1). At the end of the experimental protocol, the HF group developed hyperglycemia (p ≤ 0.05; n = 10), whereas the HF + ASE group remained normoglycemic (p ≤ 0.05). Treatment of HF diet-fed mice with ENA reduced (p ≤ 0.05) the hyperglycemia, and the levels were not different from the HF + ASE and Control groups (Table 1). The liver cholesterol and TG levels were higher (p ≤ 0.05; n = 10) in the HF compared to the Control group at the end of the study period (Table 1). The increase in lipid levels was prevented by ASE and ENA (p ≤ 0.05) in the HF + ASE and HF + ENA compared with the HF group (Table 1). The liver weight was higher (p ≤ 0.05) in the HF group

Liver samples were fixed in formalin (freshly prepared 1.27 mol/L formaldehyde, 0.1 mol/L phosphate-saline buffer pH 7.2) and embedded in Paraplast plus (Sigma-Aldrich, St Louis, MO, USA), sectioned (5 μm), and then placed in glass slides to stain with hematoxylin/eosin. Ten digital images per animal were studied to assess the volume density of hepatic steatosis by point counting, as previously described (Fraulob, Souza-Mello, Aguila, & Mandarim-de-Lacerda, 2012). Liver fibrosis was examined by staining with Picrosirius red. The morphological study was performed utilizing randomized digital images (Olympus, Tokyo, Japan). The analyses were performed with the program Image-Pro Plus version 7.0 (Media Cybernetics, Silver Spring, MD, USA). 3

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Fig. 1. Body weight and body mass gain. Effects of ASE and ENA on body weight analyzed weekly in HFD-fed mice. Values are presented as means ± SEM, n = 10 for all groups. *Significantly different from the control group (p ≤ 0.05). + Significantly different from the corresponding HF group (p ≤ 0.05). $Significantly different from the HF + ASE group (p ≤ 0.05).

Fig. 2. Hepatic histopathological analysis. (A) Photomicrographs of the liver structure and (B) hepatic steatosis (%). Sections were stained with hematoxylin and eosin, and each photomicrograph is shown at the same magnification. The usual liver appearance in the control group; macro- and micro-vesicular steatosis (black arrows) in the HF group was prevented in the HF + ASE and HF + ENA groups. Magnification 100x. Values are presented as means ± SEM, n = 5 for all groups. *Significantly different from the control group (p ≤ 0.05). +Significantly different from the corresponding HF group (p ≤ 0.05).

than in the Control group (Table 1). The HF group showed macro- and microvesicular steatosis within hepatocytes, indicative of hepatic steatosis (Fig. 2) and consistent with the increased levels of hepatic TG. Both treatments prevented (p ≤ 0.05, n = 5) the increase in liver weight and hepatic steatosis (Table 1 and Fig. 2). Staining with PicroSirius Red to visualize collagen fibers (fibrosis) in liver sections revealed an increase (p ≤ 0.05, n = 5) in liver fibrosis

Table 1 Effects of HF diet, ASE and ENA on food and energy intake, glycemia, liver parameters and visceral adipose mass in C57BL/6 mice. Control

HF

HF + ASE

HF + ENA

Food intake (g/day) Energy intake (Kcal) Initial glucose (mg/dL) Final glucose (mg/dL)

2.67 ± 0.2 10.1 ± 0.9 124 ± 4.9 129 ± 2.7

2.80 ± 0.1 15.1 ± 0.9* 131 ± 5.7 192 ± 8.9*

2.48 ± 0.2 13.4 ± 1.1 123 ± 3.6 135 ± 5.4+

2.03 ± 0.08+ 10.9 ± 0.5+ 118 ± 4.8 150 ± 4.9+

Adipose Tissue Visceral (g) Visceral/body weight (g) Body adiposity index (%)

0.24 ± 0.01 0.011 ± 0.001 3.57 ± 0.15

1.14 ± 0.12* 0.027 ± 0.002* 8.71 ± 0.92*

0.46 ± 0.02+ 0.012 ± 0.002+ 5.30 ± 0.49+

0.22 ± 0.03+ 0.008 ± 0.001+ 2.87 ± 0.28+

Liver Weight (g) Liver/body weight (g) Triglycerides (mg/dL) Cholesterol (mg/dL)

1.2 ± 0.03 0.043 ± 0.001 84.5 ± 1.5 7.2 ± 0.6

1.7 ± 0.08* 0.048 ± 0.001 291 ± 57.0* 20.1 ± 3.7*

1.3 ± 0.06+ 0.039 ± 0.001+ 118 ± 7.9+ 10.9 ± 0.9+

1.03 ± 0.01+$ 0.041 ± 0.001+ 88.2 ± 10.1+ 6.16 ± 0.6+

Data are means ± SEM. One-way ANOVA. *Significantly different from the Control (p ≤ 0.05). (p ≤ 0.05). $Significantly different from the HF + ASE group (p ≤ 0.05). 4

+

Significantly different from the corresponding HF group

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3.4. Hepatic oxidative damage and antioxidant enzyme activity Oxidative damage markers, such as MDA, carbonyl and 8-isoprostane were measured in hepatic tissue from the different groups (Fig. 5). The MDA and carbonyl levels (n = 10), as well as, the immunostaining of 8-isoprostane (n = 5) were markedly increased (p ≤ 0.05) in the HF compared with the Control group, which was prevented by treatment with ASE and ENA (Fig. 5). The antioxidant enzyme activities (SOD, CAT, and GPx) were reduced (p < 0.05, n = 10) in the liver homogenate of the HF group (Fig. 6), as compared to the Control group. The treatment with ASE, but not ENA prevented (p ≤ 0.05) the reduction of SOD and catalase activities in HF + ASE compared with the HF group (Fig. 6A and B). The treatments of ASE and ENA were effective in preventing (p ≤ 0.05) the reduction of the antioxidant activity of GPx (Fig. 6C). 3.5. Hepatic inflammation and fibrosis In the present study, the HF group had increased hepatic staining of the inflammatory markers, TNF-α and IL-6 compared with the Control group (p ≤ 0.05, n = 5, Fig. 7). Both ASE and ENA treatments prevented the increase in the inflammatory markers. The activation of the transcriptional factor NF-κB is closely related to the process of liver inflammation and fibrosis. Accordingly, we measured the protein expression of NF-κB and pNF-κB in the liver homogenates of the different groups. As depicted in Fig. 8A, protein expression of NF-kB was not different between the different groups. However, the protein expression of pNF-kB and pNF-kB/NF-kB was increased in HF compared with the Control group (Fig. 8B and C, n = 5). While ASE prevented (p ≤ 0.05) the overexpression of both pNF-kB and pNF-kB/NF-kB in HF + ASE compared with HF group, ENA prevented (p ≤ 0.05) the increased expression of pNF-kB in HF + ENA compared with the HF group (Fig. 7B). No difference was observed in the pNF-kB/NF-kB expression between HF + ENA and HF groups, and between HF + ENA and Control groups (Fig. 7C). 4. Discussion Fig. 3. Assessment of hepatic fibrosis. (A) Representative Sirius Red staining showed hepatic fibrosis (red, arrow) in liver sections of mice from HF group and a marked decrease of collagen accumulation in HF + ASE and HF + ENA groups. (B) % Collagen in liver sections of control, HF, HF + ASE and HF + ENA groups. Magnification 100x. Values are presented as means ± SEM, n = 5 for all groups. *Significantly different from the control group (p ≤ 0.05). + Significantly different from the corresponding HF group (p ≤ 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Obesity-related NAFLD is currently the major cause of morbidity in the world (Drescher, Weiskirchen, & Weiskirchen, 2019), requiring new strategies for prevention and medical treatment. In the current study, we demonstrated that diet-induced obesity, hepatic steatosis and fibrosis were prevented by simultaneous administration of ASE. Besides the improvement of hepatic lipid profile, we showed that the prevention of the increased renin expression, the stimulation of ACE-2-MASr axis, as well as the reduction of the oxidative stress and inflammation may contribute to the protective effect of ASE against hepatic steatosis and fibrosis in HFD-fed mice. In the present study, changes such as weight gain, hyperglycemia and increased adiposity associated with hepatic steatosis were similar to previous studies in HFD-fed mice (de Oliveira et al., 2015; Fraulob et al., 2012). We found that ASE prevented the body weight gain and hepatic steatosis in mice from HF + ASE group, correlating with the reduction of adipose and liver masses, which may contribute to ASEmediated decrease in body weight. Additionally, the reduction of liver masse by ASE was associated with a marked reduction in the hepatic levels of TG and cholesterol, demonstrating a positive effect of ASE on the altered lipid profile, which may also be attributed to a decrease in hepatic lipogenesis and an increased cholesterol excretion, as previously reported by our group in the same experimental model (de Oliveira et al., 2015). The presence of polyphenols in ASE (de Oliveira et al., 2015) may be responsible for this beneficial effects of ASE, since studies from our group and others have reported that polyphenols from ASE and other sources reduce serum TG, cholesterol, glucose and ameliorate hepatic lipid profile (Chiva-Blanch et al., 2013; de Oliveira et al., 2010, 2015; Snoussi et al., 2014).

of the HF group compared to the Control group. The treatments with ASE and ENA prevented this alteration (Fig. 3). 3.3. Protein expression of RAS components in the liver To investigate the effects of ASE on the local hepatic RAS, the expressions of renin, ACE, ACE-2, AT1r, AT2r, and MASr were assessed in the liver of the different groups. As shown in Fig. 4 A and C, the hepatic expression of renin and AT1r was increased (p ≤ 0.05, n = 4–5) in the HF group compared with the Control group. The treatments with ASE and ENA prevented the increase (p ≤ 0.05) in renin expression and did not change the increased expression of AT1r in HF group, but these expressions were not significantly different from the Control group. We did not observe any differences in ACE and AT2r expression between the studied groups. As shown in Fig. 4B and E, the ACE-2/ACE and MASr expressions were reduced (p ≤ 0.05, n = 4–5) in HF compared with the Control group, and the treatments with ASE and ENA increased both ACE-2/ACE and MASr expressions in HF + ASE and HF + ENA groups compared with the HF group. 5

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Fig. 4. Expression of RAS components in the liver. Effects of ASE and ENA on the expression of renin (A), ACE2/ACE (B), AT1 (C), AT2 (D), and MAS (E) receptors in hepatic tissue from HFD-fed mice. Values are presented as means ± SEM, n = 4–5. *Significantly different from the Control group (p ≤ 0.0.05). +Significantly different from the corresponding HF group (p ≤ 0.05).

Almost all of the RAS components are expressed in the liver, and local RAS activation has been associated with liver lesion pathophysiology in humans, and similarly, diet-induced obese rodent model shows overexpression of hepatic RAS components (Bataller & Brenner, 2005; Graus-Nunes & de Santos, 2019; Lubel, Herath, Burrell, & Angus, 2008). Intrahepatic activation of RAS favors NAFLD onset as it elicits greater triglycerides accumulation due to impaired beta-oxidation in conjunction with a significant fall in VLDL secretion. These conditions comply with the increase of de novo lipogenesis (Lubel et al., 2008; Wu et al., 2016). In the present study, we showed an increased hepatic expression of renin and AT1r and reduced expression of ACE-2/ACE and MASr, which is in line with a recent study in the liver of HFD-fed mice (Graus-Nunes & de Santos, 2019), reinforcing the overexpression

Similarly, previous studies in HFD-induced obesity model have shown that ACE inhibitors reduce the body weight, food and caloric intake (Frantz, Penna-de-Carvalho, & de Batista, 2014; Weisinger et al., 2009), as well as hepatic steatosis (Souza-Mello, 2017). Although the mechanism is not fully elucidated, the ACE inhibition increases plasma adiponectin expression, and also the oxidation of fatty acids, favoring the reduction of weight gain (Kalupahana & Moustaid-Moussa, 2012). In addition, the ACE inhibitors reduce the AT1r-mediated actions, such as inflammation and insulin resistance (Karnik et al., 2015), favoring the activation of alternative axis of the RAS system, such as ACE2/ANG 1-7/MAS and ACE2/ANG 1-9/AT2, leading to an improvement of the insulin resistance and prevention of the hepatic steatosis (Souza-Mello, 2017).

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Fig. 5. Assessment of hepatic oxidative damage. Effects of ASE and ENA on carbonyl (A) and malondialdeyde (B) levels in liver homogenate, and % 8-isoprostane (C) and immunostaning (D, in light brown) in hepatic sections. The increased malondialdeyde, carbonyl and immunostaining of 8-isoprostane in HF group was markedly reduced in HF + ASE and HF + ENA. Values are presented as means ± SEM, n = 10 for A and B; n = 5 for C and D. *Significantly different from the Control group (p ≤ 0.0.05). +Significantly different from the corresponding HF group (p ≤ 0.05).

Fig. 6. Hepatic antioxidant enzymatic activity. Effect of ASE and ENA on SOD (A); Catalase (B), and GPx (C) activities in the liver from HFD-fed mice. Values are presented as means ± SEM, n = 10 for all groups. *Significantly different from the Control group (p ≤ 0.0.05). +Significantly different from the corresponding HF group (p ≤ 0.05). $Significantly different from the HF + ASE group (p ≤ 0.05).

Based on elevated levels of several RAS components during the progression of hepatic fibrosis, the role of RAS in hepatic fibrogenesis has also been attributed to the classical RAS axis [ACE/ANG II/ AT1r] (Hirose et al., 2007; Shim et al., 2018; Zhang, Miao, Li, Wang, & Zhang, 2013). Similar to ENA, we found that ASE reduced the renin expression and increased the ACE-2/ACE, as well as MASr expression, suggesting

of local RA in obesity. Intrahepatic RAS overexpression triggers extracellular matrix synthesis and impairs LDL receptor function, leading to adverse liver remodeling (Y. Zhang, Ma, Ruan, & Liu, 2016). These events are mediated by AT1r activation by ANG II and predispose the subject to adverse hepatic remodeling and hepatic steatosis (Lubel et al., 2008). 7

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Fig. 7. Assessment of hepatic inflammation. Effects of ASE and ENA on TNF-α (A, B) and IL-6 (C, D) immunostaning (in light brown) in hepatic sections. The increased immunostaining of TNF-α (A) and IL-6 (C) and respective % (B and D) in the HF group were prevented in HF + ASE and HF + ENA. Values are presented as means ± SEM, n = 10 for A and B; n = 5 for C and D. *Significantly different from the Control group (p ≤ 0.0.05). +Significantly different from the corresponding HF group (p ≤ 0.05).

(Ramalingam et al., 2017). Overall evidences in humans suggest that when steatosis is associated with hepatocellular damage and necroinflammation in the presence of hyperglycemia, the fibrosis progression rate is faster (Ekstedt et al., 2006; McPherson et al., 2015; Singh, Allen, Wang, Prokop, Murad, & Loomba, 2015). In particular, activation of the RAS, directly favors steatosis, inflammation and fibrogenesis via enhanced activation of hepatic stellate cells, whereas RAS inhibitors attenuate this process (Hirose et al., 2007; Moreno et al., 2010; Wu et al., 2016). Supporting a role for inflammation in fibrosis, we found in HF group increased immunostaining for IL-6 and TNF-α in hepatic sections associated with increased oxidative stress and expression of RAS components. The results shown here indicate that ANG II by activating AT1r increases the oxidative stress and inflammation that may be involved in the development of NAFLD from steatosis to hepatic fibrosis (Tan, Shi, Li, Zhong, & Kang, 2015). Both ASE and ENA prevented the increases in hepatic inflammatory markers, suggesting an important mechanism by which ASE and ACE inhibitors protect against steatosis and fibrosis. This result reinforces the anti-inflammatory action of ASE, recently shown by our group in an experimental model of diabetic nephropathy (da Silva Cristino Cordeiro et al., 2018). Based on previous studies showing that RAS inhibitors, as well as, Ang-(1-7) infusion reduce the inflammation and improve hepatic fibrosis (Cao et al., 2016; Moreira de Macêdo et al., 2014; Souza-Mello, 2017), we suggest that ASE could prevent the inflammation by decreasing the renin expression and increasing the ACE-2/ANG 1-7/MASr axis. Recent studies have found that with increasing severity of NAFLD, NF-κB activity is enhanced, and closely related to the process of liver inflammation and fibrosis (Tan et al., 2015). ANG II induces NF-κB,

that the inhibition of renin and the activation of the ACE-2/ANG 1-7/ MASr axis in the liver may contribute to the protective effect of ASE against hepatic steatosis and fibrosis. The pathogenesis of NAFLD is driven by oxidative stress and lipid peroxidation damage, leading to the dysfunction of liver cells and mitochondrial activity (García-Berumen et al., 2019). The increased production of ROS by ANG II and the raised expression of pro-inflammatory cytokines contribute to the progression to NASH (Moreira de Macêdo et al., 2014; Wei et al., 2008). These effects are mainly mediated by higher expression of ACE, ANG II, and AT1r concomitant to reduced ACE-2 tissue expression in the hepatocytes of obese mice (Souza-Mello, 2017). Here, we showed that ASE and ENA prevented the increase in hepatic oxidative damage markers MDA, carbonyl and 8isoprostane in HFD-fed mice. However, ASE was more effective than ENA in preventing the reduced antioxidant activity of SOD and catalase in the HF group. These findings suggest an important protection of ASE against the oxidative stress that may contribute to its protective effect against hepatic steatosis and fibrosis. The antioxidant effect of ASE has been reported by our group in obese mice (de Oliveira et al., 2015), and this effect may be due to the major constituents of ASE, such as catechin, epicatechin, and polymeric procyanidins. As previously reported to the inhibitors of the RAS (Cao et al., 2016; Moreira de Macêdo et al., 2014), the upregulation of the ACE-2/ANG 1-7/MASr axis by ASE could contribute to the prevention of the oxidative stress associated with the hepatic alterations in the HF group. Increasing evidences have shown that ANG II can exacerbate inflammation (Pahlavani, Kalupahana, Ramalingam, & Moustaid-Moussa, 2017) and associated metabolic disease by increasing oxidative stress 8

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Fig. 8. Hepatic expression of nuclear transcriptional factor (NF-kB). Effects of ASE and ENA on the expression of NF-kB (A), pNF-kB (B), and pNF-kB/NF-kB in hepatic tissue from HFD-fed mice. Values are presented as means ± SEM, n = 4–5. *Significantly different from the Control group (p ≤ 0.0.05). +Significantly different from the corresponding HF group (p ≤ 0.05).

guidelines).

resulting in the activation of a large number of target genes, such as cytokines, chemokines, adhesion molecules, and TNF-α, which increase liver inflammation, thereby maintaining HSC activation and eventually leading to the occurrence of liver fibrosis (Tan et al., 2015). Indeed, ACE inhibitors and ANG II receptor blockers may play an antiliver fibrosis role by inhibiting NF-κB (Saber, Goda, El-Tanbouly, & Ezzat, 2018). Consistent with previous finding in obese animal (Tan et al., 2015), we demonstrated in this study, increased expression of pNF-κB/ NF-κB in HF group, which was prevented by both ASE and ENA. Taken together these findings suggest that one mechanism by which ASE protects the liver from fibrosis induced by HFD is by inhibiting the ANG II-mediated increase in NF-κB expression and inflammation. In conclusion, we have demonstrated that supplementation with ASE protects mice fed an HFD from obesity-associated hepatic steatosis and fibrosis. The modulation of intrahepatic RAS with up-regulation of ACE2/MASr axis by ASE may contribute to the prevention of hepatic remodeling by favoring the reduction of the oxidative stress, NF-κB expression and inflammation. ENA was effective as ASE in preventing the HFD-related disorders, interfering with the RAS and inflammation, but less effective as an antioxidant. These findings support ASE as a useful option for obese patients to control hepatic steatosis and fibrosis. Ethics Statements All experimental procedures were approved by the Ethics Committee for Experimental Animals Use and Care (CEUA) of Roberto Alcântara Gomes Institute of Biology / Rio de Janeiro State University (protocol: CEUA n° 034/2015), according to International Guidelines (ARRIVE and EU Directive 2010/63/EU for animal experiments

CRediT authorship contribution statement Matheus Henrique Romão: Methodology, Investigation, Formal analysis, Writing - original draft. Graziele Freitas de Bem: Methodology, Investigation, Formal analysis, Writing - original draft. Izabelle Barcellos Santos: Visualization, Investigation. Ricardo de Andrade Soares: Visualization, Investigation. Dayane Teixeira Ognibene: Data curation, Writing - review & editing. Roberto Soares de Moura: Supervision. Cristiane Aguiar da Costa: Data curation, Writing - review & editing. Ângela Castro Resende: Conceptualization, Writing - review & editing, Resources, Funding acquisition. Declaration of Competing Interest Roberto Soares de Moura is the inventor of a patent (PCT/ BR0200038) that supported the development of a new patent application (PCT/BR2007/000178). The other authors state no declaration of interest. Acknowledgements This work was funded by the National Council of Scientific and Technological Development (CNPq, n° 444983/2014-7), Rio de Janeiro State Research Agency (FAPERJ, n° E-26/202.913/2017), and Coordination for the Improvement of Higher Education Personnel 9

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(CAPES, n° 155625/2018-7).

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