Flaxseed oil containing flaxseed oil ester of plant sterol attenuates high-fat diet-induced hepatic steatosis in apolipoprotein-E knockout mice

journal of functional foods 13 (2015) 169–182

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Flaxseed oil containing flaxseed oil ester of plant sterol attenuates high-fat diet-induced hepatic steatosis in apolipoprotein-E knockout mice Hao Han a,b, Hongfei Ma a,b, Shuang Rong a,b, Li Chen a,b, Zhilei Shan a,b, Jiqu Xu c, Yunjian Zhang d,*, Liegang Liu a,b,** a

Department of Nutrition and Food Hygiene, Hubei Key Laboratory of Food Nutrition and Safety, Huazhong University of Science and Technology, Wuhan 430030, China b Key Laboratory of Environment and Health, Ministry of Education, Ministry of Environmental Protection, State Key Laboratory of Environmental Health (Incubating), School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China c Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Hubei Key Laboratory of Oilcrops Lipid Chemistry and Nutrition, Huazhong University of Science and Technology, Wuhan 430030, China d Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

A R T I C L E

I N F O

A B S T R A C T

Article history:

The prevalence of non-alcoholic fatty liver disease (NAFLD) has dramatically increased glob-

Received 21 October 2014

ally during recent decades. Dietary flaxseed oil and plant sterol exert potential benefit to

Received in revised form 18

NAFLD. Present study was designed to evaluate the effects of flaxseed oil containing flax-

December 2014

seed oil ester of plant sterols (FO-PS) on hepatic steatosis induced by high fat diet (HFD)

Accepted 23 December 2014

and investigate the underlying molecular mechanisms. C57BL/6 mice were administered a

Available online 19 January 2015

regular diet (RG) and apoE−/− mice were given HFD alone or plus 5% flaxseed oil with or without 3.3% FO-PS for 18 weeks. Our data showed that HFD induced NAFLD while dietary

Keywords:

flaxseed oil fortified with FO-PS offered a synergistically effective strategy for improving hepatic

Hepatic steatosis

steatosis as well as optimizing overall lipid levels, reducing oxidative stress and inhibiting

Flaxseed oil ester of plant sterols

system inflammation. The expression levels of hepatic genes involved in cholesterol efflux

Lipid metabolism

(ABCA1, LXR, SR-BI) and triacylglycerol catabolism (PPARα) were also increased signifi-

Oxidative stress

cantly by intervention of flaxseed oil plus FO-PS. In addition, combination treatment reduced

Inflammation

ROS production and down-regulated inflammatory markers (IL-6, TNF, MCP-1 and ICAM-1) in liver. Thus, dietary supplementation of flaxseed oil containing FO-PS altered the expressions of genes related to lipid metabolism and inflammation, and thus ameliorated hepatic steatosis. © 2014 Elsevier Ltd. All rights reserved.

* Corresponding author. Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022, China. Tel.: +86 13995609051; fax: +86 27 85726670. E-mail address: [email protected] (Y. Zhang). ** Corresponding author. Department of Nutrition & Food Hygiene, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China. Tel.: +86 13487080378; fax: +86 27 83650522. E-mail address: [email protected] (L. Liu). http://dx.doi.org/10.1016/j.jff.2014.12.046 1756-4646/© 2014 Elsevier Ltd. All rights reserved.

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1.

journal of functional foods 13 (2015) 169–182

Introduction

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease encompassing a wide spectrum of liver damage stages ranging from isolated hepatic steatosis without liver inflammation to non-alcoholic steatohepatitis (NASH) or even cryptogenic cirrhosis and hepatocellular carcinoma (Adams & Lindor, 2007). In recent years, NAFLD has emerged not only as a new feature of the metabolic syndrome but also as one of the main underlying risk factors for cardiovascular disease (Stepanova & Younossi, 2012; Targher, Marra, & Marchesini, 2008). Although physiopathology and natural history of these conditions have not been completely elucidated, according to the commonly cited “2 hits hypothesis” of NAFLD progression, dyslipidaemia, oxidative stress and inflammatory injury are closely linked to the pathogenesis of NAFLD (Day & Saksena, 2002). Therefore, investigation of effective substances from dietary sources that are able to lower lipid concentrations, enhance antioxidant defence and mitigate systemic inflammatory response is warranted. Plant sterols (PS) with similar structure to cholesterol, one kind of natural compound, have long been reported to decrease circulating cholesterol concentrations by suppressing intestinal absorption of cholesterol (Jones et al., 2000). An intake of plant sterols ester (around 2 g/day) can achieve reduction in low density lipoprotein cholesterol (LDL-C) in the order of 10–15% in about 90% of individuals (Madsen, Jensen, & Schmidt, 2007; Nestel, 2002). Additionally, several studies have further demonstrated the beneficial impacts of PS preparations on lipid peroxidation (De Jong et al., 2008), endothelial markers (AbuMweis et al., 2006), and inflammation markers (Clifton, Mano, Duchateau, van der Knaap, & Trautwein, 2008). Therefore, it is likely that supplementation of PS in diet may prevent or ameliorate NAFLD. However, purified PS has low intestinal bioavailability and poor effect on the level of triacylglycerol (TG) (Ostlund, 2002). The most common process used to enhance the bioavailability and solubility of PS is esterifying PS with n-6 polyunsaturated fatty acids (PUFAs), such as soyabean oil and sunflower oil (SO) fatty acids. Recently, emerging new approaches consist of esterifying PS to n-3 PUFAs associated with additional health benefits, such as fish oil fatty acids (Demonty, Chan, Pelled, & Jones, 2006). Flaxseed oil is a particularly rich source of α-linolenic acid (ALA, C18:3n-3) which is the major n-3 fatty acid and the precursor of longer chain n-3 PUFA (EPA and DHA). Consumption of flaxseed oil displayed significant hypotriglyceridaemic effect with concurrent modifications of other risk factors for cardiovascular disease, including pro-inflammatory mediators and oxidative stress (Xu et al., 2012). In addition, dietary flaxseed oil was found to lower hepatic lipid levels and GSH depletion thus attenuating NAFLD (Yang, Tseng, Chang, & Chen, 2009). Lipid aberrations rarely occur in isolation. Usually, they highly interact with oxidative stress and inflammation in the process of NAFLD, hence, simultaneous management is required. Considering the beneficial effects of PS and flaxseed oil mentioned above, it is possible that flaxseed oil containing flaxseed oil fat acid ester of plant sterols (FO-PS) may offer a synergistic and complementary strategy for NAFLD amelioration. In

addition, as one of the highly frequent and largely consumed in Chinese daily cooking, flaxseed oil is a good source for PS supplementation. Therefore, the present study was designed to investigate the effects of dietary supplementation of flaxseed oil containing FO-PS on NAFLD induced by high-fat diet in apolipoprotein-E knockout (apoE-KO) mice, a well-established animal model used for studies of atherogenic hypercholesterolaemia, and investigate the underlying molecular mechanisms.

2.

Materials and methods

2.1.

Materials

The flaxseed oil was manufactured by Inner Mongolia Caoyuan Kanghen Food Co. Ltd. (Hohhot, China) and contained 57.82% ALA (% of total fatty acids). Fatty acid of flaxseed oil (αlinolenic acid 80%, linoleic acid 15%, oleic acid 5%) was purchased from Henan Linuo Biochemical CO. LTD (Anyang, Henan, China). PS (β-sitosterol 77%, campesterol 17%, stigmasterol 5%) was provided by BASF Co. Ltd. (Shanghai, China). The plant sterol esters were synthesized by Oil Crops Research Institute, Chinese Academy of Agricultural Sciences (Wuhan, China) by using polyunsaturated fatty acid and PS mentioned above. The main kind of plant sterol esters is β-sitosterol ester of α-linolenic acid which accounts for about 88% in the purified products (Deng et al., 2011).

2.2.

Animals and treatments

Male apoE-KO mice and wild type C57BL/6 mice (4 weeks), purchased from Peking University Resources Centre (Beijing, China) with body weight of 15–19 g, were cared for according to the Guiding Principles in the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health. After one week of acclimatization period, 10 wild type C57BL/6 mice were assigned to the control group and given normal chow. Thirty male apoE-KO mice were randomly divided into high-fat diet (HFD), flaxseed oil-treated (FO) and flaxseed oil combined with its ester of plant sterols-treated (FOPS) groups matched with their mean body weight and plasma total cholesterol levels as previously published (Moghadasian, McManus, Godin, Rodrigues, & Frohlich, 1999). HFD group were given a high-fat diet containing 21% fat and 0.15% cholesterol (Table 1) (Nakashima, Plump, Raines, Breslow, & Ross, 1994). This high-fat diet was further supplemented with 5% (w/w) flaxseed oil for FO group. A combination of 3.3% (w/w) flaxseed oil ester of plant sterols mixture with amount of flaxseed oil which provided equivalent ALA to FO group was added to the high-fat diet and used for FO-PS group. After 18 weeks, the animals were fasted for 12 h and sacrificed. Blood samples were rapidly obtained by cardiac puncture. Serum was prepared from blood centrifuged at 3000 g for 10 min at 4 °C and mononuclear cells were isolated using ficoll density gradient centrifugation. Liver tissues were frozen immediately in liquid nitrogen and stored at −80 °C, or fixed in 4% buffered formalin.

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Table 1 – Composition of the experimental diets (g/kg). Components

Control group

HFD group

FO group

FO-PS group

Cornstarch Casein Dextrinized cornstarch Sucrose Soybean oil Fiber Mineral mix Vitamin mix L-Cystine Choline bitartrate Lard Cholesterol Flaxseed oil FO-PS Energy, kJ

397.5 200 132 100 70 50 35 10 3 2.5 0 0 0 0 12,735.1

321.3 200 106.7 80.8 56.6 40.4 28.3 8.1 2.4 2 153.4 1.5 0 0 19,063.7

321.3 200 106.7 80.8 56.6 40.4 28.3 8.1 2.4 2 103.4 1.5 50 0 19,063.7

321.3 200 106.7 80.8 56.6 40.4 28.3 8.1 2.4 2 127.9 1.5 25.5 33 19,063.7

HFD: high fat diet; FO: HFD + 5% flaxseed oil; FO-PS: HFD + 2.55% flaxseed oil + 3.3% flaxseed oil ester of plant sterol.

2.3.

Histopathological examination

Fresh samples from the same position of liver were divided into two parts. One part of samples was fixed in 4% paraformaldehyde and embedded in paraffin. Cross-sections (5 µm thick) obtained from the paraffin blocks were stained with haematoxylin eosin (H&E). The other part of samples was embedded in tissue freezing medium and sectioned into consecutive 5 µm thick sections. Every sixth section was stained with Oil red O and digitally photographed under ×200 magnification.

2.4. Determination of lipid parameters in serum and liver The concentrations of TC, TG, LDL-C and HDL-C in serum and liver were measured by enzymatic colorimetric assays using commercially available detection kits (Biosino Biotechnology CO., Ltd, Beijing, China).

2.5.

Evaluation of ROS levels in liver of mice

Dihydroethidium (DHE) (Molecular Probes, Eugene, OR, USA) was used for in situ detection of reactive oxygen species (ROS) in liver of mice. Fresh cross-sections (5 µm) of unfixed but frozen liver were immediately incubated with 5M DHE at 37 °C for 15 min in a humidified chamber. Fluorescence level was then visualized with a fluorescence microscope. Fluorescence intensities in randomly selected areas of the images were quantified by using the Image-Pro Plus (IPP) image analysis software.

2.6.

Assay of lipid peroxidation and antioxidant capacity

Malondialdehyde (MDA) in serum and liver was measured by using thiobarbituric acid (TBA) colorimetry slightly modified by Ohkawa, Ohishi, and Yagi (1979). Glutathione (GSH) was measured according to its reaction with 5,5′-dithiobis-2-nitrobenzoic

acid (DTNB) into 2-nitro-5-thiobenzoic acid (TNB), following deproteinization with 5% trichloroacetic acid (Eady, Orta, Dennis, Stratford, & Peacock, 1995). The MDA and GSH contents were standardized by protein concentration measured by the protein assay kit (Bio-Rad, Hercules, CA, USA).

2.7. Measurement of serum biomarkers for liver injury Serum aspartate (AST) and alanine aminotransferases (ALT) were measured with enzymatic kinetic method by Mindray BS-200 automatic biochemistry analyzer (Shenzhen, China) with matching kits. Results are expressed as units per litre (U/L).

2.8. Detection of inflammation cytokines in plasma The levels of interleukin-6 (IL-6), tumour necrosis factor-α (TNFα), monocyte chemoattractant protein-1 (MCP-1) and soluble inter-cellular adhesion molecule-1 (sICAM-1) in plasma were measured by enzyme-linked immunosorbent assays according to the manufacturer’s instructions of commercially available detection kits (R&D Systems, Minneapolis, MN, USA).

2.9.

Real-time RT-PCR analysis

Total RNA was isolated from the stored frozen liver using the TRIzol reagent. (Invitrogen, Carlsbad, CA, USA). Messenger RNA (mRNA) expression was quantified by using specific oligo primers and SYBR green-based qRT-PCR kit (TaKaRa Biotechnology CO., Ltd, Dalian, China) in 7900HT instrument (Applied Biosystems, Foster City, CA, USA). The specificity of the product was assessed from melting curve analysis. Gene expressions were determined using the 2−ΔΔCt method. The mRNA of β-actin was quantified as an endogenous control. Gene expressions are presented as fold change relative to control.

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Table 2 – Effect of FO-PS on body and liver weight in each group throughout the feeding period. Parameters

Groups Control

HFD

FO

FO + ALA-PS

Initial body weight (g) Final body weight (g) Body weight gain (g) Liver weight (g/100 g body weight)

13.08 ± 1.48 29.26 ± 0.34 16.18 ± 0.31 3.82 ± 0.22

13.00 ± 1.08 32.98 ± 2.11a 19.98 ± 1.74a 5.21 ± 0.36a

13.05 ± 0.88 33.90 ± 1.81a 20.85 ± 2.17a 4.82 ± 0.36a,b

13.10 ± 1.11 34.05 ± 1.23a 10.95 ± 1.31a 4.45 ± 0.59a,b,c

Values are given as means ± SEM (n = 10). a P < 0.05 versus the control. b P < 0.05 versus the HFD group. c P < 0.05 versus the FO group. HFD: high fat diet; FO: HFD + 5% flaxseed oil; FO-PS: HFD + 2.55% flaxseed oil + 3.3% flaxseed oil ester of plant sterol.

Gene

Forward primer

Reverse primer

SR-BI

5′–TGGCAAGCCCC TGAGCACGTT-3′

5′–TAGTGTCTTCAGGA CCCTGGCTGC-3′

LDLr

5′–CACACAGCCTAGA GAAGTCGACAC-3′

5′–CTGTGCTTCGGT GGCCTGGTA-3′

LXR

5′–GCTGGGATTAGGGT GGGGGTGAC-3′

5′–AATGGGCCAAGGCGT GACGC-3′

ABCA1

5′–TGTGCTGCCGTACG AAGCCG-3′

5′–TCCCCAGCCAAGCAA GGGGT-3′

PPARα

5′–GGAGTGCAGCCTCA GCCAAGTT-3′

5′–AGGCCACAGAGCGCT AAGCTGT-3′

SREBP-1c

5′–TCCTTAACGTGGG CCTAGTCCGAAG-3′

5′–GCTCGAGTAACCC AGCACGGG-3′

ACC

5′–CGTTGGCCAAAACT CTGGAGCTA-3′

5′–CCCACATGGCCTGGC TTGGAG-3′

VCAM-1

5′–GATAGACAGCCCA CTAAACG-3′

5′–CAATGACGGGAGT AAAGGT-3′

TNF-α

5′–TCTCATTCCTGC TTGTGG-3′

5′-ACTTGGTGGTTTG CTACG-3′

IL-6

5′–ATTTCCTCTGG TCTTCTGG-3′

5′–TGGTCTTGGTCCT TAGCC-3′

MCP-1

5′–GCAGGTGTCCC AAAGAA-3′

5′–GGTGGTTGTGGA AAAGG-3′

ICAM-1

5′–CCATCACCGTGT ATTCGT-3′

5′–CTGGCGGCTCAGTATCT-3′

IL-1β

5′–CTTTCCCGTGG ACCTT-3′

5′–ATCTCGGAGCCTG TAGTG-3′

2.10.

(Amersham Biosciences, Little Chalfont, UK) according to the manufacturer’s instructions. Quantitative analysis of the relative density of the bands in Western blots was performed by Quantity One 4.62 software (Bio-Rad). Data were corrected for background standardized to β-actin as optical density (OD/mm2). Primary antibodies were as follows: monoclonal anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA), Rabbit anti-SR-BI (LSBio, Seattle, WA, USA), mouse anti-LDLr (R&D Systems), rabbit antiLXR alpha (Abcam, Cambridge, MA, USA), rabbit anti-ABCA1 (Abcam), rabbit anti-PPARα (Abcam), rabbit antibody antiSREBP1 (Abcam), mouse anti-ACC (R&D Systems), mouse antiTNF alpha (Abcam), rabbit anti-IL-6 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-MCP-1 (Abcam), rabbit antiICAM1 (Abcam), mouse anti-IL-1β (Cell Signaling Technology), anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology) and anti-mouse IgG HRP-linked antibody (Cell Signaling Technology).

2.11.

Statistical analysis

Results are expressed as means ± SEM, and P < 0.05 was considered significant. Statistical analyses of data were performed using one-way analysis of variance with SPSS 12.0 software package.

3.

Results

3.1. Effect of flaxseed oil added FO-PS on body and liver weight

Western blot analysis

Liver tissues were homogenized and lysed in RIPA Lysis Buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS). Total protein was determined according to the method previously described by Hartree (1972). Tissue lysates with equal protein amounts were subjected to western blotting. The protein was separated by 10% SDS-polyacrylamide gel and then transferred onto a PVDF membrane. The membranes were incubated with specific primary antibodies overnight at 4 °C after being blocked with 5% nonfat milk solution. Then the target proteins were incubated with the species-specific second antibodies conjugated to horseradish peroxidase. Immunoreactive bands were detected by means of an ECL plus Western Blotting Detection System

Treatment of flaxseed oil added FO-PS significantly decreased body and liver weight increased by HFD (Table 2).

3.2. Effects of flaxseed oil containing FO-PS on liver morphology H&E and Oil red O staining of lipid deposition in liver were analyzed to evaluate the effect of flaxseed oil containing FO-PS on hepatic steatosis. As representative results shown in Fig. 1A and B, the mice given high-fat diet were characterized by a large number of macrovesicular steatosis. The circular lipid droplets were markedly reduced in livers of mice fed flaxseed oil and even not present in those of mice given flaxseed oil

journal of functional foods 13 (2015) 169–182

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Fig. 1 – Dietary flaxseed oil containing FO-PS improved hepatic steatosis in mice. (A) H&E and (B) Oil-Red O staining of lipid droplets in the livers of mice in each group (magnification ×200). (C) Quantitative analysis of hepatic fat accumulation. Data represent means ± SEM and are normalized to % of field area (n = 6 mice). a, P < 0.05 versus the control; b, P < 0.05 versus the HFD group; c, P < 0.05 versus the FO group.

containing FO-PS. Quantitative analysis of the lipid droplets in the liver of mice (Fig. 1C) demonstrated statistically smaller lipid droplet size in FO-PS-treated animals than that in FOtreated mice.

3.3. Dietary flaxseed oil plus FO-PS synergistically improved lipid metabolism in mice As shown in Fig. 2, high levels of TG in serum and liver induced by HFD were significantly reduced after being exposed to flaxseed oil, and this depression effect was further improved by adding FO-PS. Although FO intervention elevated serum HDLC, it had no effect on TC and LDL-C. Addition of FO-PS to flaxseed oil not only substantially decreased TC and LDL-C but also elevated the level of HDL-C. In an effort to further investigate molecular pathways of lipid disorder induced by HFD and the concomitant protection exerted by flaxseed oil plus FOPS on this process, we also examined the key proteins involved in lipid metabolism. Results showed that flaxseed oil added FOPS apparently reversed the decreased mRNA and protein expressions of hepatic scavenger receptor class B type I (SRBI), Liver X receptor alpha (LXRa) and Adenosine triphosphatebinding cassette transporter A1 (ABCA1) induced by HFD, but had no effect on expression level of low density lipoprotein receptor (LDLr) (Fig. 3). It was also observed that administrations of flaxseed oil with FO-PS significantly up-regulated mRNA and protein expressions of hepatic proliferator activated receptor alpha (PPARα) and down-regulated sterol regulatory element binding transcription factor 1c (SREBP-1c) and acetyl CoA Carboxylase (ACC) (Fig. 4).

3.4. Application of flaxseed oil fortified with FO-PS alleviated oxidative stress in mice Increased ROS production indicated liver oxidative stress status. As shown in Fig. 5, combination treatment mitigated the rise of ROS induced by high-fat diet beyond what was observed with flaxseed oil feeding alone. In addition, MDA, an indicator of lipid peroxidation, was increased in high-fat diet-fed mice compared with the control mice, and significantly suppressed by the combined treatment of flaxseed oil with FO-PS. Moreover, dietary flaxseed oil containing FO-PS prevented the decrease of GSH in serum and liver.

3.5. Concerted intervention with flaxseed oil and FO-PS attenuated inflammation and ameliorated serum transaminases As shown in Fig. 6A and B, compared with control, mice fed on a high-fat diet exhibited significantly higher serum levels of ALT and AST, which indicated hepatic injury. A significant reduction of ALT and AST were noted after flaxseed oil treatment, and the combination treatment was even more effective in diminishing serum transaminases. In addition, flaxseed oil supplementation on its own had no effect on plasma inflammatory cytokines initially increased by high-fat diet except descending IL-6. When fortified with FO-PS, flaxseed oil was capable of declining the levels of IL-6, TNF-a, MCP-1 and sICAM-1 in plasma (Fig. 6C–F). To seek an explanation for the improved inflammation, we assessed the expression levels of the markers of inflammation in liver and circulating monocytes.

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Fig. 2 – Effects of flaxseed oil plus FO-PS on lipid profiles in mice. (A) Serum TG, (B) Serum TC, (C) Serum LDL-C, (D) Serum HDL-C, (E) Hepatic TG and (F) Hepatic TC. Each bar or point denotes mean ± SEM (n = 10 mice). a, P < 0.05 versus the control; b, P < 0.05 versus the HFD group; c, P < 0.05 versus the FO group.

The results clearly displayed that flaxseed oil intervention in the presence of FO-PS dramatically reversed the increased mRNA and protein expressions of IL-6, TNF-a, MCP-1 and ICAM-1 induced by HFD in liver (Fig. 7A–H). In circulating monocytes, up-regulated MCP-1, interleukin-1 beta (IL-1β) and IL-6 in response to high-fat diet were almost completely counteracted by flaxseed oil with FO-PS. However, flaxseed oil intervention alone only significantly modified the mRNA and protein expression of IL-1β (Fig. 8A–F).

4.

Discussion

NAFLD, the hepatic manifestation of the metabolic syndrome, is a common cause of chronic liver disease, and its worldwide prevalence continues to increase with the growing obesity epidemic (Vernon, Baranova, & Younossi, 2011). As

NAFLD can progress to liver cirrhosis, its pathogenesis and treatment are attracting greater attention. Reports have demonstrated that the pathogenesis of NAFLD is closely associated with dyslipidaemia, oxidative stress and inflammation (Yoon & Cha, 2014). Increased serum levels of lipids as a result of high fat diet can cause fat accumulation in the liver, as well as oxidative stress and inflammation, which leads to hepatic steatosis. This study provides the first evidence, to our knowledge, of dietary flaxseed oil containing FO-PS that synergistically and complementarily improved hepatic steatosis induced by HFD via restoring impaired lipid metabolism, reducing oxidative stress and inhibiting inflammation response. Dysregulation of the lipid metabolism which leads to cholesterol accumulation and fat deposition in the liver are closely linked to the pathogenesis of hepatic steatosis (Musso, Gambino, & Cassader, 2009). Therapeutic options targeting hepatic lipid metabolism are therefore crucial to the management ofNAFLD.Recently, a meta-analysis of randomized

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Fig. 3 – Effects of flaxseed oil fortified with FO-PS on mRNA and protein expressions of hepatic SR-BI, LDLr, LXR-α, and ABCA1 in mice. Quantitative real-time RT-PCR and western blot analysis of the mRNA and protein expressions of (A–B) SR-BI, (C–D) LDLr, (E–F) LXR-α, and (G–H) ABCA1 in the liver of mice in each group. Fold changes of mRNA levels were determined after normalization to internal control β-actin RNA levels. Blotting with anti-β-actin was used as a protein loading control. Protein expressions were presented as fold change relative to control. Representative immunoblots are shown. Each bar denotes mean ± SEM (n = 4 mice). a, P < 0.05 versus the control; b, P < 0.05 versus the HFD group; c, P < 0.05 versus the FO group.

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Fig. 4 – Effects of flaxseed oil supplemented with FO-PS on mRNA and protein expressions of hepatic PPARα, SREBP-1c and ACC in mice. Quantitative real-time RT-PCR and western blot analysis of the mRNA and protein expressions of (A–B) PPARα, (C–D) SREBP-1c and (E–F) ACC in the liver of mice in each group. Fold changes of mRNA levels were determined after normalization to internal control β-actin RNA levels. Blotting with anti-β-actin was used as a protein loading control. Protein expressions were presented as fold change relative to control. Representative immunoblots are shown. Each bar denotes mean ± SEM (n = 4 mice). a, P < 0.05 versus the control; b, P < 0.05 versus the HFD group; c, P < 0.05 versus the FO group.

controlledstudies of LDL-cholesterol-lowering effect of PS showed that a daily intake of 3 g/day of PS resulted in an average reduction in LDL cholesterol of up to 12% (Ras, Geleijnse, & Trautwein, 2014). Meanwhile, it has been well established that increased consumption of ALA was associated with improvements in the levels of TG and HDL-C (Riediger et al., 2008; Wilkinson et al., 2005). In the present study, we found that concerted intervention of flaxseed oil with FO-PS offered a more comprehensive strategy for optimizing overall lipid levels including LDL-C, HDL-C and TG, which indicated that combination of PS and ALA might have a synergistic effect on lipid-decreasing. This result was consistent with previous research that reported that the combined supplementation with phytosterols and n-3 PUFAs had both synergistic and complementary lipid-lowering effects in hyperlipidaemic men and women (Micallef & Garg, 2008). To better explain the effects, at a molecular level, of FO-PS on the lipid, we next assayed the mRNA and protein expressions of factors in charge of hepatic lipid metabolism. Substantial evidence has shown that lipoprotein receptors SR-BI and LDLr play pivotal roles in lipid metabolic processes (Leiva et al., 2011; Pieper-Fürst & Lammert, 2013). In detail, the first molecularly well-defined and physiologically relevant HDL receptor to be characterized was the SR-BI, a cell surface glycoprotein, predominantly expressed in liver and

steroidogenic tissues, where it mediates the selective uptake of cholesteryl ester from HDL (Acton et al., 1996). Murine models have supported that the up-regulation of SR-BI in liver improved NAFLD induced by high fat diet (Jourdan et al., 2010; Xin et al., 2013). LDLr, a transmembrane glycoprotein, mediates the binding and endocytosis of lipoproteins containing apolipoprotein B and E, particularly LDL. In humans, the liver is the most LDLr-abundant organ and accounts for more than 70% of the total LDL clearance in plasma (Spady, 1992). Wang et al. proved that the decrease of LDLr resulted in fatty acid metabolism disequilibrium and lipidoses in liver, which is one of the reasons for NASH morbidity (Wang, Meng, & Zhang, 2012). Since activity changes of these lipoprotein receptors will alter the rates of LDL-C and HDL-C uptake by the liver with corresponding transformation of plasma cholesterol level, hepatic expressions of SR-BI and LDLr were detected in present research. In response to the flaxseed oil and FO-PS addition in diet, mice manifested a higher mRNA and protein expressions of SR-BI than those in high-fat diet fed animals, which indicated that lower cholesterol effect of FO-PS was partly due to promoting cholesterol uptake. Our finding was supported by data from other studies showing that amount of hepatic SRBI was significantly higher in hamsters fed the ALA-rich diet (Morise et al., 2004). However, no significant differences in the mRNA and protein expressions of hepatic LDLr were observed

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Fig. 5 – Effects of flaxseed oil added FO-PS on aortic ROS production and the levels of MDA and GSH in serum and liver of mice. (A) ROS in the aorta of the mice was detected by using DHE which reacts with ROS and forms ETH that binds to DNA and produces red fluorescence signal, visualized with fluorescence microscope (×200) and quantified. (B) Fluorescence intensities in randomly selected areas of the images were quantified by using the IPP image analysis software. Data represent means ± SEM and are normalized to % of field area (n = 6 mice). (C) Serum MDA (D) Hepatic MDA (E) Serum GSH and (F) Hepatic GSH. For panels C–F, each bar or point denotes mean ± SEM (n = 10 mice). a, P < 0.05 versus the control; b, P < 0.05 versus the HFD group; c, P < 0.05 versus the FO group.

in each group suggesting that LDLr did not mediate the FOPS dependent ameliorating of NAFLD and decrease of LDL-C in serum and liver may be not regulated by LDLr but by other pathways. Excessive accumulation of lipids within hepatocytes has been considered an important factor for hepatic steatosis, therefore, efficient transportation or delivery of lipids may be crucial to NAFLD prevention and treatment (Dowman, Armstrong, Tomlinson, & Newsome, 2011). ABCA1 is an integral membrane protein that mediates the transport of cellular cholesterol (Wang & Tall, 2003). LXRa is a nuclear receptor that has previously been shown to regulate the metabolic conversion of cholesterol to bile acids (Venkateswaran et al., 2000). Because of the ability to increase ABCA1-dependent reverse cholesterol transport, LXRa is considered an attractive therapeutic target in the treatment of NAFLD (Higuchi et al., 2008). Plat et al. found that plant sterols and stanols from the 4-desmethylsterol family could increase cholesterol efflux by activating both LXRa and LXRb (Plat, Nichols, & Mensink, 2005). The latest report indicates that biological activities of schottenol and spinasterol, two natural phytosterols present in argan oil and in cactus pear seed oil, modulated cholesterol metabolism in murine microglial BV2 cells through up-regulation of LXR-α and its target gene ABCA1 (El Kharrassi et al., 2014). Meanwhile, the increased cholesterol efflux following n-3 PUFA treatment also was observed

in another study (Marmillot, Rao, Liu, Chirtel, & Lakshman, 2000). Here, compared with control, remarkably higher gene and protein expressions of LXRa and ABCA1 accompanying lower level of TC in liver were observed in FO-PS treatment group, which indicated that one of the mechanisms by which FO-PS decreased TC and improved NAFLD may be increasing cholesterol efflux. NAFLD is characterized by excessive fat acid accumulation in hepatocytes. Fatty acid oxidation and de novo lipogenesis are considered as the two key metabolic pathways that control the hepatic lipid metabolism, and two transcription factors, PPARα and SREBP-1c, play a very important role in these pathways, respectively (Musso et al., 2009). Specifically, PPARα, one member of the ligand-activated nuclear hormone receptors super family, is regarded as the principal regulator in betaoxidation of fatty acids (Giby & Ajith, 2014). SREBP-1c that belongs to the family of SREBPs has a crucial effect on hepatic de novo lipogenesis through stimulating the expression of lipogenic genes such as ACC (Yang et al., 2014). A large number of evidence suggests that the hepatic steatosis is associated with either decreased expression of PPARα or increased expression of SREBP-1c mediated pathway (Ferre & Foufelle, 2010; Giby & Ajith, 2014), and the activation of PPARα and/or inhibition of SREBP-1c can reduce the cellular fat accumulation both in vivo and in vitro (Pawlak et al., 2014; Quan et al., 2013). Aloe

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Fig. 6 – Effects of flaxseed oil containing FO-PS on serum transaminases and plasma inflammatory cytokines in mice. (A) Serum ALT, (B) Serum AST, (C) Plasma IL-6, (D) Plasma TNF-α, (E) Plasma MCP-1 and (F) Plasma sICAM-1. Each bar or point denotes mean ±SEM (n = 10 mice). a, P < 0.05 versus the control; b, P < 0.05 versus the HFD group; c, P < 0.05 versus the FO group.

vera phytosterols were reported to activate PPAR transcription in a dose-dependent manner by which they improve fatty acid metabolism in the livers of mice with diet-induced obesity (Nomaguchi et al., 2011) and decrease hepatic protein expression of SREBP-1c in zucker diabetic fatty rats (Misawa et al., 2012). In addition, ALA has been well demonstrated to be a natural ligand of PPAR-a (Forman, Chen, & Evans, 1997), which could bind and activate this key transcriptional regulator of lipid metabolism to stimulate beta-oxidation of fatty acids, thereby reducing intrahepatic fat deposition (Davidson, 2006; Murase, Aoki, & Tokimitsu, 2005). Research by Devarshi et al. showed that dietary flaxseed oil improved lipid metabolism through up-regulation of PPARα and down-regulation of SREBP1c in streptozotocin-nicotinamide induced diabetic rats (Devarshi, Jangale, Ghule, Bodhankar, & Harsulkar, 2013). As we expected, in the present study, combination administrations of flaxseed oil and FO-PS reduced mRNA and protein expressions of hepatic SERPB-1c and its target gene ACC as well as up-regulated PPARα. Since a recent report showed that enhancement in the liver SREBP-1/PPARα ratio, a condition that may favour lipogenesis over fatty acid oxidation, is associated with obesity, insulin resistance, and steatosis (Pettinelli et al., 2009), we suggest that improvement effect of flaxseed oil fortified with FO-PS on hepatic steatosis may link to regulating PPARα, SREBP-1c and ACC. The “two-hit hypothesis” is a key concept of NAFLD pathogenesis. In fatty livers, simple hepatic steatosis (first hit) sensitizes the liver to inflammatory cytokines or oxidative stress

(second hit), leading to development of steatohepatitis (Machado et al., 2008). Reactive oxygen species (ROS) is generated during the metabolism of free fatty acids in microsomes, peroxisomes, and mitochondria (Mitsuyoshi, Itoh, & Okanoue, 2006). The second hit triggers such as diet, smoke and pollutants leading to mitochondrial dysfunction which impairs fat homeostasis in liver and also leads to an overproduction of ROS. These ROS may cause membrane lipid peroxidation, cell degeneration, proinflammatory cytokine expression and liver stellate cell fibrogenesis which play an important role in inducing lethal hepatocyte injury associated with NAFLD (Paradies, Paradies, Ruggiero, & Petrosillo, 2014). Equilibrium between production and detoxification of ROS in cells is guaranteed by the concerted action of the antioxidant enzyme systems. Depletion of mitochondrial GSH has been implicated in the development of alcoholic liver disease in that GSH participates in pathways responsible for ROS detoxification (García-Ruiz et al., 1994), whereas, increased reactive lipid peroxidation products such as MDA can further potentiate oxidative stress, leading to the formation of new sources of oxidants. In the present study we first found that FO-PS was able to reduce ROS, MDA and elevate GSH. Thus, FO-PS can be considered as new antioxidant, which may play a protective role on NAFLD by the inhibition of oxidative stress. In advanced stage of NAFLD, increased hepatic cholesterol and fatty acids resulted from an imbalance between uptake and export of lipids by hepatocytes as well as oxidative stress will further activate inflammatory responses, which

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Fig. 7 – Supplementation of flaxseed oil combined with FO-PS decreased mRNA and protein expressions of hepatic inflammatory cytokines in mice. Quantitative realtime RT-PCR and western blot analysis of the mRNA and protein expressions of (A) TNF-α, (B) IL-6, (C) MCP-1, (D) ICAM-1 in the liver of mice in each group. Fold changes of mRNA levels were determined after normalization to internal control β-actin RNA levels. Blotting with anti-β-actin was used as a protein loading control. Protein expressions were presented as fold change relative to control. Representative immunoblots are shown. Each bar denotes mean ± SEM (n = 4 mice). a, P < 0.05 versus the control; b, P < 0.05 versus the HFD group; c, P < 0.05 versus the FO group.

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Fig. 8 – Flaxseed oil containing FO-PS decreased mRNA and protein expressions of inflammatory cytokines in circulating monocytes of mice. Quantitative real-time RT-PCR and western blot analysis of the mRNA and protein expressions of (A) IL-1β, (B) IL-6, (C) MCP-1 in circulating monocytes of mice in each group. Fold changes of mRNA levels were determined after normalization to internal control β-actin RNA levels. Blotting with anti-β-actin was used as a protein loading control. Protein expressions were presented as fold change relative to control. Representative immunoblots are shown. Each bar denotes mean ± SEM (n = 4 mice). a, P < 0.05 versus the control; b, P < 0.05 versus the HFD group; c, P < 0.05 versus the FO group.

ultimately causes hepatic injury and fibrosis (Tilg & Moschen, 2010). Consistent with this pathological progression of NAFLD/ NASH, a growing body of evidence showed that the patients with NAFLD/NASH have elevated concentrations of TNF-α, IL-6, MCP-1 and sICAM-1 (Haukeland et al., 2006; Sookoian et al., 2010; Valenti et al., 2002; Wieckowska et al., 2008). In our study, we found the higher levels of TNF-α, IL-6, MCP-1 and sICAM-1 accompanying elevated levels of AST and ALT in HFD group than those in control, which may indicate ongoing liver injury and steatohepatitis. Dietary FO-PS significantly reduced plasma levels of TNF-α, IL-6, MCP-1 and sICAM-1 initially increased by high-fat diet, and similar results were also observed in mRNA and protein expression levels in aorta. At the cellular level, FO-PS also exhibited an inhibition effect on protein expressions of IL-1β, IL-6 and MCP-1 in circulating monocytes. These data suggest that better function in NAFLD prevention of FO-PS is at least partly through perfecting inflammatory response. In conclusion, our data are supportive of the improvement effects of flaxseed oil containing FO-PS supplementation on overall lipid, oxidative stress and systemic inflammation which may result in further amelioration in NAFLD. In the future, well-designed randomized controlled trials of adequate size and duration are needed to assess long-term safety and efficacy of dietary flaxseed oil fortified with FO-PS for the treatment of NAFLD patients.

Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgements This work was supported by the BASF Newtrition™ Asia Research Grant and the National Natural Science Foundation of China (31000777).

REFERENCES

AbuMweis, S. S., Vanstone, C. A., Ebine, N., Kassis, A., Ausman, L. M., Jones, P. J., & Lichtenstein, A. H. (2006). Intake of a single morning dose of standard and novel plant sterol preparations for 4 weeks does not dramatically affect plasma lipid concentrations in humans. The Journal of Nutrition, 136, 1012– 1016. Acton, S., Rigotti, A., Landschulz, K. T., Xu, S., Hobbs, H. H., & Krieger, M. (1996). Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science, 271, 518–520. Adams, L. A., & Lindor, K. D. (2007). Nonalcoholic fatty liver disease. Annals of Epidemiology, 17, 863–869.

journal of functional foods 13 (2015) 169–182

Clifton, P. M., Mano, M., Duchateau, G. S., van der Knaap, H. C., & Trautwein, E. A. (2008). Dose-response effects of different plant sterol sources in fat spreads on serum lipids and C-reactive protein and on the kinetic behavior of serum plant sterols. European Journal of Clinical Nutrition, 62, 968–977. Davidson, M. H. (2006). Mechanisms for the hypotriglyceridemic effect of marine omega-3 fatty acids. The American Journal of Cardiology, l98, 27–33. Day, C., & Saksena, S. (2002). Non-alcoholic steatohepatitis: Definitions and pathogenesis. Journal of Gastroenterology and Hepatology, (Suppl. 3), S377–S384. De Jong, A., Plat, J., Bast, A., Godschalk, R. W., Basu, S., & Mensink, R. P. (2008). Effects of plant sterol and stanol ester consumption on lipid metabolism, antioxidant status and markers of oxidative stress, endothelial function and lowgrade inflammation in patients on current statin treatment. European Journal of Clinical Nutrition, 62, 263–273. Demonty, I., Chan, Y. M., Pelled, D., & Jones, P. J. (2006). Fish-oil ester of plant sterols improve the lipid profile of dyslipidemic subjects more than do fish-oil or sunflower oil ester of plant sterols. The American Journal of Clinical Nutrition, 84, 1534–1542. Deng, Q. C., Pin, Z., Huang, Q. D., Huang, F. H., Fang, W., Zheng, M. M., Yu, X., Zhou, Q., & Zheng, C. (2011). Chemical synthesis of phytosterol esters of polyunsaturated fatty acids with ideal oxidative stability. European Journal of Lipid Science and Technology: EJLST, 113, 441–449. Devarshi, P. P., Jangale, N. M., Ghule, A. E., Bodhankar, S. L., & Harsulkar, A. M. (2013). Beneficial effects of flaxseed oil and fish oil diet are through modulation of different hepatic genes involved in lipid metabolism in streptozotocin-nicotinamide induced diabetic rats. Genes & Nutrition, 8, 329–342. Dowman, J. K., Armstrong, M. J., Tomlinson, J. W., & Newsome, P. N. (2011). Current therapeutic strategies in non-alcoholic fatty liver disease. Diabetes, Obesity & Metabolism, 13, 692–702. Eady, J. J., Orta, T., Dennis, M. F., Stratford, M. R., & Peacock, J. H. (1995). Glutathione determination by the Tietze enzymatic recycling assay and its relationship to cellular radiation response. British Journal of Cancer, 72, 1089–1095. El Kharrassi, Y., Samadi, M., Lopez, T., Nury, T., El Kebbaj, R., Andreoletti, P., El Hajj, H. I., Vamecq, J., Moustaid, K., Latruffe, N., El Kebbaj, M. S., Masson, D., Lizard, G., Nasser, B., & Cherkaoui-Malki, M. (2014). Biological activities of Schottenol and Spinasterol, two natural phytosterols present in argan oil and in cactus pear seed oil, on murine miroglial BV2 cells. Biochemical and Biophysical Research Communications, 446, 798– 804. Ferre, P., & Foufelle, F. (2010). Hepatic steatosis: A role for de novo lipogenesis and the transcription factor SREBP-1c. Diabetes, Obesity and Metabolism, 12(Suppl. 2), 83–92. Forman, B. M., Chen, J., & Evans, R. M. (1997). Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator activated receptors alpha and delta. Proceedings of the National Academy of Sciences of the United States of America, 94, 4312–4317. García-Ruiz, C., Morales, A., Ballesta, A., Rodés, J., Kaplowitz, N., & Fernández-Checa, J. C. (1994). Effect of chronic ethanol feeding on glutathione and functional integrity of mitochondria in periportal and perivenous rat hepatocytes. The Journal of Clinical Investigation, 94, 193–201. Giby, V. G., & Ajith, T. A. (2014). Role of adipokines and peroxisome proliferator-activated receptors in nonalcoholic fatty liver disease. World Journal of Hepatology, 6, 570–579. Hartree, E. F. (1972). Determination of protein: A modification of the Lowry method that gives a linear photometric response. Analytical Biochemistry, 48, 422–427. Haukeland, J. W., Damås, J. K., Konopski, Z., Løberg, E. M., Haaland, T., Goverud, I., Torjesen, P. A., Birkeland, K., Bjøro, K.,

181

& Aukrust, P. (2006). Systemic inflammation in nonalcoholic fatty liver disease is characterized by elevated levels of CCL2. Journal of Hepatology, 44, 1167–1174. Higuchi, N., Kato, M., Shundo, Y., Tajiri, H., Tanaka, M., Yamashita, N., Kohjima, M., Kotoh, K., Nakamuta, M., Takayanagi, R., & Enjoji, M. (2008). Liver X receptor in cooperation with SREBP1c is major lipid synthesis regulator in nonalcoholic fatty liver disease. Hepatology Research, 38, 1122–1129. Jones, P. J., Raeini-Sarjaz, M., Ntanios, F. Y., Vanstone, C. A., Feng, J. Y., & Parsons, W. E. (2000). Modulation of plasma lipid levels and cholesterol kinetics by phytosterol versus phytostanol ester. Journal of Lipid Research, 41, 697–705. Jourdan, T., Djaouti, L., Demizieux, L., Gresti, J., Vergès, B., & Degrace, P. (2010). CB1 antagonism exerts specific molecular effects on visceral and subcutaneous fat and reverses liver steatosis in diet-induced obese mice. Diabetes, 59, 926–934. Leiva, A., Verdejo, H., Benítez, M. L., Martínez, A., Busso, D., & Rigotti, A. (2011). Mechanisms regulating hepatic SR-BI expression and their impact on HDL metabolism. Atherosclerosis, 217, 299–307. Machado, M. V., Ravasco, P., Jesus, L., Marques-Vidal, P., Oliveira, C. R., Proença, T., Baldeiras, I., Camilo, M. E., & Cortez-Pinto, H. (2008). Blood oxidative stress markers in nonalcoholic steatohepatitis and how it correlates with diet. Scandinavian Journal of Gastroenterology, 43, 95–102. Madsen, M. B., Jensen, A. M., & Schmidt, E. B. (2007). The effect of a combination of plant sterol-enriched foods in mildly hypercholesterolemic subjects. Clinical Nutrition: Official Journal of the European Society of Parenteral and Enteral Nutrition, 26, 792– 798. Marmillot, P., Rao, M. N., Liu, Q. H., Chirtel, S. J., & Lakshman, M. R. (2000). Effect of dietary omega-3 fatty acids and chronic ethanol consumption on reverse cholesterol transport in rats. Metabolism: Clinical and Experimental, 49, 508–512. Micallef, M. A., & Garg, M. L. (2008). The lipid-lowering effects of phytosterols and (n-3) polyunsaturated fatty acids are synergistic and complementary in hyperlipidemic men and women. The Journal of Nutrition, 138, 1086–1090. Misawa, E., Tanaka, M., Nomaguchi, K., Nabeshima, K., Yamada, M., Toida, T., & Iwatsuki, K. (2012). Oral ingestion of aloe vera phytosterols alters hepatic gene expression profiles and ameliorates obesity-associated metabolic disorders in zucker diabetic fatty rats. Journal of Agricultural and Food Chemistry, 60, 2799–2806. Mitsuyoshi, H., Itoh, Y., & Okanoue, T. (2006). Role of oxidative stress in non-alcoholic steatohepatitis. Nippon Rinsho. Japanese Journal of Clinical Medicine, 64, 1077–1182. Moghadasian, M. H., McManus, B. M., Godin, D. V., Rodrigues, B., & Frohlich, J. J. (1999). Proatherogenic and antiatherogenic effects of probucol and phytosterols in apolipoprotein E-deficient mice: Possible mechanisms of action. Circulation, 99, 1733–1739. Morise, A., Sérougne, C., Gripois, D., Blouquit, M. F., Lutton, C., & Hermier, D. (2004). Effects of dietary alpha linolenic acid on cholesterol metabolism in male and female hamsters of the LPN strain. The Journal of Nutritional Biochemistry, 15, 51–61. Murase, T., Aoki, M., & Tokimitsu, I. (2005). Supplementation with alpha-linolenic acid-rich diacylglycerol suppresses fatty liver formation accompanied by an up-regulation of beta-oxidation in Zucker fatty rats. Biochimica et Biophysica Acta, 1733, 224– 231. Musso, G., Gambino, R., & Cassader, M. (2009). Recent insights into hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD). Progress in Lipid Research, 48, 1–26. Nakashima, Y., Plump, A. S., Raines, E. W., Breslow, J. L., & Ross, R. (1994). ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arteriosclerosis, Thrombosis, and Vascular Biology, 114, 133–140.

182

journal of functional foods 13 (2015) 169–182

Nestel, P. (2002). Cholesterol-lowering with plant sterols. Medical Journal of Australia, 176, S122. Nomaguchi, K., Tanaka, M., Misawa, E., Yamada, M., Toida, T., Iwatsuki, K., Goto, T., & Kawada, T. (2011). Aloe vera phytosterols act as ligands for PPAR and improve the expression levels of PPAR target genes in the livers of mice with diet-induced obesity. Obesity Research & Clinical Practice, 5, 190–201. Ohkawa, H., Ohishi, N., & Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry, 95, 351–358. Ostlund, R. E. (2002). Phytosterols in human nutrition. Annual Review of Nutrition, 22, 533–549. Paradies, G., Paradies, V., Ruggiero, F. M., & Petrosillo, G. (2014). Oxidative stress, cardiolipin and mitochondrial dysfunction in nonalcoholic fatty liver disease. World Journal of Gastroenterology, 20, 14205–14218. Pawlak, M., Baugé, E., Bourguet, W., De Bosscher, K., Lalloyer, F., Tailleux, A., Lebherz, C., Lefebvre, P., & Staels, B. (2014). The transrepressive activity of peroxisome proliferator-activated receptor alpha is necessary and sufficient to prevent liver fibrosis in mice. Hepatology (Baltimore, Md.), 60, 1593–1606. Pettinelli, P., Del Pozo, T., Araya, J., Rodrigo, R., Araya, A. V., Smok, G., Csendes, A., Gutierrez, L., Rojas, J., Korn, O., Maluenda, F., Diaz, J. C., Rencoret, G., Braghetto, I., Castillo, J., Poniachik, J., & Videla, L. A. (2009). Enhancement in liver SREBP-1c/PPAR-a ratio and steatosis in obese patients: Correlations with insulin resistance and n-3 long chain polyunsaturated fatty acid depletion. Biochimica et Biophysica Acta, 1792, 1080–1086. Pieper-Fürst, U., & Lammert, F. (2013). Low-density lipoprotein receptors in liver: Old acquaintances and a newcomer. Biochimica et Biophysica Acta, 1831, 1191–1198. Plat, J., Nichols, J. A., & Mensink, R. P. (2005). Plant sterols and stanols: Effects on mixed micellar composition and LXR (target gene) activation. Journal of Lipid Research, 46, 2468– 2476. Quan, H. Y., Kim do, Y., Kim, S. J., Jo, H. K., Kim, G. W., & Chung, S. H. (2013). Betulinic acid alleviates non-alcoholic fatty liver by inhibiting SREBP1 activity via the AMPK-mTOR-SREBP signaling pathway. Biochemical Pharmacology, 85, 1330–1340. Ras, R. T., Geleijnse, J. M., & Trautwein, E. A. (2014). LDLcholesterol-lowering effect of plant sterols and stanols across different dose ranges: A meta-analysis of randomised controlled studies. The British Journal of Nutrition, 112, 214–219. Riediger, N. D., Othman, R., Fitz, E., Pierce, G. N., Suh, M., & Moghadasian, M. H. (2008). Low n-6:n-3 fatty acid ratio, with fish- or flaxseed oil, in a high-fat diet improves plasma lipids and beneficially alters tissue fatty acid composition in mice. European Journal of Clinical Nutrition, 47, 153–160. Sookoian, S., Castaño, G. O., Burgueño, A. L., Rosselli, M. S., Gianotti, T. F., Mallardi, P., Martino, J. S., & Pirola, C. J. (2010). Circulating levels and hepatic expression of molecular mediators of atherosclerosis in nonalcoholic fatty liver disease. Atherosclerosis, 209, 585–591. Spady, D. K. (1992). Hepatic clearance of plasma low density lipoproteins. Seminars in Liver Disease, 12, 373–385. Stepanova, M., & Younossi, Z. M. (2012). Independent association between nonalcoholic fatty liver disease and cardiovascular disease in the US population. Clinical Gastroenterology and Hepatology, 10, 646–650.

Targher, G., Marra, F., & Marchesini, G. (2008). Increased risk of cardiovascular disease in non-alcoholic fatty liver disease: Causal effect or epiphenomenon? Diabetologia, 51, 1947–1953. Tilg, H., & Moschen, A. R. (2010). Evolution of inflammation in nonalcoholic fatty liver disease: The multiple parallel hits hypothesis. Hepatology (Baltimore, Md.), 52, 1836–1846. Valenti, L., Fracanzani, A. L., Dongiovanni, P., Santorelli, G., Branchi, A., Taioli, E., Fiorelli, G., & Fargion, S. (2002). Tumor necrosis factor α promoter polymorphisms and insulin resistance in nonalcoholic fatty liver disease. Gastroenterology, 122, 274–280. Venkateswaran, A., Laffitte, B. A., Joseph, S. B., Mak, P. A., Wilpitz, D. C., Edwards, P. A., & Tontonoz, P. (2000). Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha. Proceedings of the National Academy of Sciences of the United States of America, 97, 12097–12102. Vernon, G., Baranova, A., & Younossi, Z. M. (2011). Systematic review: The epidemiology and natural history of nonalcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. Alimentary Pharmacology & Therapeutics, 34, 274–285. Wang, L., Meng, X., & Zhang, F. (2012). Raspberry ketone protects rats fed high-fat diets against nonalcoholic steatohepatitis. Journal of Medicinal Food, 15, 495–503. Wang, N., & Tall, A. R. (2003). Regulation and mechanisms of ATPbinding cassette transporter A1-mediated cellular cholesterol efflux. Arteriosclerosis, Thrombosis, and Vascular Biology, 23, 1178–1184. Wieckowska, A., Papouchado, B. G., Li, Z., Lopez, R., Zein, N. N., & Feldstein, A. E. (2008). Increased hepatic and circulating interleukin-6 levels in human nonalcoholic steatohepatitis. The American Journal of Gastroenterology, 103, 1372–1379. Wilkinson, P., Leach, C., Ah-Sing, E. E., Hussain, N., Miller, G. J., Millward, D. J., & Griffin, B. A. (2005). Influence of alphalinolenic acid and fish-oil on markers of cardiovascular risk in subjects with an atherogenic lipoprotein phenotype. Atherosclerosis, 181, 115–124. Xin, P., Han, H., Gao, D., Cui, W., Yang, X., Ying, C., Sun, X., & Hao, L. (2013). Alleviative effects of resveratrol on nonalcoholic fatty liver disease are associated with up regulation of hepatic low density lipoprotein receptor and scavenger receptor class B type I gene expressions in rats. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association, 52, 12–18. Xu, J., Yang, W., Deng, Q., Huang, Q., Yang, J., & Huang, F. (2012). Flaxseed oil and α-lipoic acid combination reduces atherosclerosis risk factors in rats fed a high-fat diet. Lipids in Health and Disease, 11, 148. Yang, S. F., Tseng, J. K., Chang, Y. Y., & Chen, Y. C. (2009). Flaxseed oil attenuates nonalcoholic fatty liver of hyperlipidemic hamsters. Journal of Agricultural and Food Chemistry, 57, 5078– 5083. Yang, Y., Li, W., Liu, Y., Sun, Y., Li, Y., Yao, Q., Li, J., Zhang, Q., Gao, Y., Gao, L., & Zhao, J. (2014). Alpha-lipoic acid improves highfat diet-induced hepatic steatosis by modulating the transcription factors SREBP-1, FoxO1 and Nrf2 via the SIRT1/ LKB1/AMPK pathway. The Journal of Nutritional Biochemistry, 25, 1207–1217. Yoon, H. J., & Cha, B. S. (2014). Pathogenesis and therapeutic approaches for non-alcoholic fatty liver disease. World Journal of Hepatology, 6, 800–811.