Introduction

Atherosclerosis is the primary cause of cardiovascular diseases (CVD), which are the top cause of death worldwide [1, 2]. Although the precise mechanisms of atherosclerosis have not been fully elucidated, oxidative stress and dyslipidemia have been implicated in its pathogenesis [3, 4].

Nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, a multi-subunit complex, is a primary source of reactive oxygen species (ROS) in the blood vessels, particularly in the presence of atherosclerosis. Several studies have demonstrated that the expression of its subunits, such as p22phox, p67phox, and p47phox, is significantly increased in the vessels of humans with coronary atherosclerosis [5] and that inhibition of NADPH oxidase by apocynin attenuates the progression of atherosclerosis [6]. Heme oxygenase-1 (HO-1), a crucial mediator of antioxidant and tissue-protective actions, also plays a critical role in protecting against atherogenesis [7–9] and increasing HO-1 expression in RAW 264.7 macrophages, which effectively decreases NADPH oxidase activity [10]. Additionally, the regulation of lipid metabolism, which involves cellular enzymes, membrane transport proteins and nuclear receptors, is very complicated [11]. Peroxisome proliferator-activated receptor-α (PPARα), one of the transcription factors, is a promising target for the development of novel antiatherogenic treatments due to its ability to regulate genes that are involved in lipid metabolism, such as carnitine palmitoyltransferase 1A (CPT1A) and acyl CoA oxidase (ACOX1) [12, 13]. In particular, CPT1A catalyses the transfer of long chain fatty acyl groups from CoA to carnitine for translocation across the mitochondrial inner membrane [14], whereas ACOX1 is the first enzyme of peroxisomal fatty acid β-oxidation, which catalyses the dehydrogenation of acyl-CoA thioesters to the corresponding trans-2-enoyl-CoA [15].

In recent years, accumulating evidence has shown that the low density lipoprotein receptor (LDLr) and scavenger receptor class B type I (SR-BI), the receptors of LDL and HDL, respectively, also play pivotal roles in lipid metabolic processes and atherosclerosis [16, 17]. Both oxidative damage and lipid metabolic disorders are critical and common in the pathogenesis of atherosclerosis. Therefore, studies of atherosclerosis in the fields of food and nutritional science have focused on the search for extracts of herbal plants and/or functional food ingredients that can prevent or ameliorate oxidative stress and dyslipidemia.

Lutein is one of the most prevalent carotenoids, accounting for the brilliant color pigment of many dark green leafy vegetables such as spinach and kale [18]. As an efficient quencher of singlet oxygen atoms and free radicals, lutein is a powerful antioxidant [19, 20] and is effective in preventing lipid peroxidation [21–24]. In addition, lutein reduces ROS in cultured vascular smooth muscle cells [25] and increases HO-1 mRNA expression in liver tissue, which results in the protection of mice from oxidative stress induced by d-gal [26]. Furthermore, evidence from animal studies suggests that lutein prevents cholesterol accumulation in aortic tissue in atherosclerotic guinea pigs [27]. Epidemiological data support the finding that an increase in serum lutein after supplementation reduces the serum levels of low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) [28]. Furthermore, progression of the intima-media thickness (IMT) of the common carotid arteries over 18 months was related to plasma lutein levels among a randomly sampled cohort of utility employees aged 40–60 years [29]. This result is supported by data from other studies showing an inverse association between serum levels of lutein and carotid IMT [30–33]. However, the protective effects of lutein against atherosclerosis and its association with NADPH oxidase, HO-1, PPARα, LDLr and SR-BI have not been well established.

Therefore, the objectives of the present study were to explore the effects of lutein on atherosclerosis prevention in vivo using apoE-deficient mice, a well established animal model used for studying atherosclerosis, and to better define the underlying mechanisms involved in oxidative stress related molecules (HO-1 and NADPH oxidase) and lipid metabolism-related molecules (PPARα, LDLr and SR-BI).

Materials and Methods

Animals

Male apoE knockout mice (C57/BL6 background, 8 weeks of age, 18–25 g) purchased from the Animal Center of Medical College of Peking University. The animals were maintained in constant temperature-controlled rooms (25 ± 2 °C) with controlled lighting (12 h light–dark cycles). The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and was approved by the Tongji Medical College Council on Animal Care Committee.

Experimental Procedures

The mice were randomly allocated to a control group (n = 7), a high fat diet group (HFD) (n = 12) or three lutein groups (n = 12 for each). The control group was fed normal chow, and the HFD group was given a high fat diet containing 21 % fat (5.66 % soybean oil and 15.34 % lard) and 0.15 % cholesterol by weight [27]. The three lutein groups were given the same high fat diet mixed with 0.01, 0.02 or 0.04 % lutein by weight (equivalent to 25, 50 and 100 mg/kg body weight, respectively) for 24 weeks (lutein was provided by InnoBio Co., Ltd, Dalian, China). At the end of the experiment, the animals were food deprived for 8 h, anesthetized with ketamine HCl (50 mg/kg)/xylazine (10 mg/kg), and subsequently killed by cervical dislocation. Blood was collected, centrifuged for 20 min at 5,000g at 4 °C and then stored at −80 °C. The heart, aorta and liver were excised from the mice, immediately frozen in liquid nitrogen, and stored at −80 °C until use. Three animals in each group were randomly selected for aortic sinus, aortic arch and liver Oil red O staining and hematoxylin and eosin (H&E) staining to observe any pathological changes and lipid deposition.

Assessment of Atherosclerotic Lesion Formation

Aortic arch samples fixed with 4 % paraformaldehyde, embedded in paraffin and sectioned into consecutive 8-μm thick sections. Every sixth section was stained with H&E and digitally photographed under magnification 200× for histological examination.

The heart samples were embedded in tissue freezing medium optimum cutting temperature compound (OCT) and sectioned into consecutive 8-μm thick sections at −20 °C. The distal end of the aortic sinus was recognized by the disappearance of the three aortic valve cusps as previously described [34]. Every sixth section was stained with Oil red O and digitally photographed under magnification 40×.

Atherosclerotic lesions in the aortic sinus stained with Oil red O were evaluated using IPP image analysis software. The lesion area index was calculated as the percentage of the aortic lumen area covered by atherosclerotic lesions.

Histology and Morphometry Evaluations of Lipid Deposition in Liver Tissue

Fresh samples from the same position of the liver were divided into two groups. One group of samples was fixed in 4 % paraformaldehyde and embedded in paraffin. Tissue sections (5 μm thick) were stained with H&E. The other group of samples was embedded in tissue freezing medium OCT and sectioned into consecutive 5-μm thick sections. Every sixth section was stained with Oil red O and digitally photographed under magnification 200×. Lipid deposition in the liver tissue stained with Oil red O was evaluated using IPP image analysis software.

Measurement of Lipid Parameters in Serum and Liver

The concentrations of TC, triacylglycerol (TAG), LDL-C and high density lipoprotein cholesterol (HDL-C) in serum and liver tissue were measured by enzymatic colorimetric assays using commercially available detection kits (Biosino Biotechnology Co., Ltd, Beijing, China). The atherogenic index of plasma (AIP = TAG/HDL-C) and non-high density lipoprotein cholesterol (NHDL-C = TC-HDL-C) were calculated.

Determination of Oxidative Stress Parameters in Serum and the Liver

The concentration of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in serum and the liver were measured with enzymatic colorimetric assays using commercially available detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Assay of HO-1 and NADPH Oxidase in Serum

The levels of HO-1 and NADPH oxidase in serum were determined with enzyme-linked immunosorbent assays using commercially available detection kits (R&D Systems, USA).

Evaluation of ROS levels In Mouse Aortas

Dihydroethidium (Molecular Probes, Eugene, OR, USA) was used for in situ detection of ROS in mouse aortas [35, 36]. Fresh cross sections (5 μm) of unfixed but frozen aorta were immediately incubated with 5 M DHE at 37 °C for 15 min in a humidified chamber. The fluorescence level was then visualized with a fluorescence microscope. Fluorescence intensities in randomly selected areas of the images were quantified using IPP image analysis software.

Real-Time RT-PCR Analysis

Total RNA was extracted from aorta and liver tissue using TRIzol reagent. (Invitrogen, 154 Carlsbad, CA, USA). A SYBR green-based qRT-PCR kit (TaKaRa Biotechnology Co., Ltd, Dalian, China) was used according to the manufacturer’s instructions in a 7900HT instrument (Applied Biosystems, Forster, CA, USA). The specificity of the product was assessed from melting curve analysis. Gene expression levels were determined using the 2−ΔΔCt method. Gene expression levels are presented as the fold-change relative to the control. The mRNA of β-actin was quantified as an endogenous control. Quantitative real-time PCR primers were as follows:

Gene

Forward primer

Reverse primer

HO-1

5′-TCACGGTCTCCAGTCGCCTCC-3′

5′-CGGGCTATGCTCGAGACGGC-3′

p22phox

5′-GTGTGCGCAGGGTCCTCGTC-3′

5′-TCCAACCTGTGGCCGCTCCT-3′

p47phox

5′-GGTCGACCATCCGCAACGCA-3′

5′-GCGGCGATAGGTGTCCTGGC-3

PPARα

5′-GGAGTGCAGCCTCAGCCAAGTT-3′

5′-AGGCCACAGAGCGCTAAGCTGT-3

CPT1A

5′-AAGAACATCGTGAGTGGCGTC-3′

5′-AGCACCTTCAGCGAGTAGCG-3′

ACOX1

5′-GCCTTTGTTGTCCCTATCCGT-3′

5′-CTTCAGGTAGCCATTATCCATCTCT-3′

LDLr

5′-CACACAGCCTAGAGAAGTCGACAC-3′

5′-CTGTGCTTCGGTGGCCTGGTA-3′

SR-BI

5′-TGGCAAGCCCCTGAGCACGTT-3′

5′-TAGTGTCTTCAGGACCCTGGCTGC-3′

β-actin

5′-TTCGTTGCCGGTCCACACCC-3′

5′-GCTTTGCACATGCCGGAGCC-3′

Western Blot Analysis

Thoracic aorta and liver tissues were homogenized and lysed in RIPA Lysis Buffer (1 % Triton X-100, 1 % deoxycholate, 0.1 % SDS). Total protein was determined as previously described [8]. Tissue lysates with equal protein amounts were subjected to Western blot analysis and probing with specific primary antibodies overnight at 4 °C after blocking. Then, the target proteins were secondarily labeled with species-specific second antibodies conjugated to horseradish peroxidase. Immunoreactive bands were detected by means of an ECL plus Western Blotting Detection System (Amersham Biosciences, Little Chalford, UK) according to the manufacturer’s instructions. The relative density of the bands in Western blots was quantified with Quantity One 4.62 software (Bio-Rad, Hercules, CA, USA). Data were corrected for the background standardized to β-actin as the optical density (OD/mm2). The mouse monoclonal anti-β-actin antibody and rabbit anti-CPT1A antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). The rabbit polyclonal anti-HO-1 antibody, purified rabbit polyclonal anti-LDLr antibody, rabbit polyclonal anti-SR-BI antibody, rabbit polyclonal anti-PPAR alpha antibody and rabbit polyclonal anti-ACOX1 antibody were purchased from Abcam Limited (Cambridge, UK). The rabbit polyclonal anti-p47phox antibody was purchased from Merck Millipore Corporation (Billerica, MA, USA). The rabbit polyclonal anti-p22phox antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling (Beverly, MA, USA).

Statistical Analysis

All data are expressed as means ± SD. Statistical analyses of data were performed using one-way analysis of variance with SPSS 12.0 software package (SN: 59245 46841 40655 89389 09859 21671 21957 29589 12). The results were considered statistically significant at P < 0.05.

Results

Effect of Lutein Treatment on Body Weight and Fat Accumulation

Lutein treatment significantly decreased body weight as well as abdominal, perirenal, epididymis and total adipose tissue, which were increased by HFD (Table 1).

Table 1 Effect of lutein supplementation on body weight and fat accumulation in each group throughout the feeding period
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Lutein Administration Ameliorated HFD-Induced Atherosclerosis in Mice

As indicated in Fig. 1a, representative images of the aortic arch cross section stained by H&E showed that mice fed the control diet did not exhibit appreciable atherosclerotic plaque formation. A HFD induced plaque coverage on most of the luminal surface of the aortic vessel, while smaller lesions were observed in the lutein supplement groups. Lipid deposition was assessed in cross sections of the aortic sinus using Oil red O. The results show that little lipid deposition was detected in control tissue. The extensive lipid deposits induced by a HFD were reduced by the addition of lutein in the diet (Fig. 1b). Quantitative analyses show that lutein inhibited this atherogenesis in a dose-dependent manner (Fig. 1c).

Fig. 1
figure 1

Dietary lutein reduced atherosclerosis in HFD-fed apoE-deficient mice. Representative images of cross sections taken from the aortic sinuses and aortic arch obtained from apoE-deficient mice in each group. The aortic arch sections were stained with H&E (200× magnification) (a), and the aortic sinus sections were stained with Oil red O (40× magnification) for lipid deposition (red) and counterstained with hematoxylin (blue) (b). Quantitative analysis of lipid accumulation is shown (c). Data represent the means ± SD and are normalized to % of field area. *P < 0.05, **P < 0.01 vs. control; # P < 0.05, ## P < 0.01 vs HFD. n = 3

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The Administration of Lutein Alleviated Oxidative Stress in Mice

As shown in Fig. 2 and Table 2, a HFD induced ROS production in the aorta and increased concentrations of MDA and reduced activity of SOD in the serum and liver; these effects were significantly prevented by the concomitant addition of lutein. In addition, the lutein intervention apparently decreased NADPH oxidase activity in the serum and down-regulated the mRNA and protein expression levels of aortic p47phox and p22phox. Furthermore, protein expression of HO-1 in the aorta was statistically increased in lutein-treated animals compared with HFD-fed animals. However, lutein attenuated an increased mRNA expression level of aortic HO-1 in atherosclerotic mice (Fig. 3).

Fig. 2
figure 2

Effects of lutein on oxidative stress induced by HFD in mice. Lutein prevented increases in ROS production in the aortas of mice fed a HFD. ROS in the aortas of the mice was detected with DHE, which reacts with ROS and forms ETH; this compound binds to DNA and produces a red fluorescence signal, which can be visualized with a fluorescence microscope (×200) and quantified (a–b). Fluorescence intensities in randomly selected areas of the images were quantified with IPP image analysis software. Results are presented as the mean ± SD. *P < 0.05, **P < 0.01 vs control; # P < 0.05, ## P < 0.01 vs HFD. n = 3

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Table 2 Effect of lutein treatment on oxidative stress in mice
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Fig. 3
figure 3

Effects of lutein on the mRNA and protein expression levels of HO-1 (a–b), p47phox (c–d) and p22phox (e–f) in mouse aortas. Total RNA was extracted from the aortas of mice by Trizol. HO-1, p47phox and p22phox mRNA expression levels were analyzed by real-time RT-PCR. The mRNA of β-actin was quantified as an endogenous control. Aortic lysates were prepared and immunoblotted with the corresponding antibodies. Blotting with anti-β-actin was used as a protein loading control. HO-1, p47phox and p22phox are presented as the fold-change relative to the control. Results are presented as the mean ± SD. *P < 0.05, **P < 0.01 vs control; # P < 0.05, ## P < 0.01 vs HFD. n = 4

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Lutein Treatment Improved Lipid Metabolism in Mice

H&E and Oil red O staining of lipid deposition in liver tissues were analyzed to evaluate the effect of lutein on hepatic steatosis. As illustrated in Fig. 4, the control group had a small, single fat vacuole. A great number of vacuoles were seen in the HFD group, while the lutein groups had fewer vacuoles than that in HFD group. In addition, the lutein intervention significantly decreased the concentrations of TC and TAG in serum and the liver as well as the serum level of LDL-C. Meanwhile, API and NHDL-C were markedly decreased in the lutein groups compared with that in HFD group. However, there was no apparent difference in the serum HDL-C level among the groups (Table 3). To examine the potential molecular signaling pathways of lutein against HFD-induced dyslipidemia, the main proteins involved in lipid metabolism were evaluated. The results showed that administrations of lutein increased mRNA and protein expressions of PPARα as well as CPT1A and ACOX1 in liver (Fig. 5). In addition, the decreased mRNA and protein expression levels of hepatic LDLr and SR-BI in the mice with atherosclerosis were significantly reversed after intervention with lutein (Fig. 6).

Fig. 4
figure 4

The lutein intervention ameliorated hepatic steatosis in apoE-deficient mice induced by HFD. Representative images of liver sections from apoE-deficient mice in each group; the sections were stained with H&E (200× magnification) (a). The lipid droplets in the hepatocytes were stained red by Oil red O staining (200× magnification) (b). Quantitative analysis of hepatic fat accumulation is shown (c). The data represent the mean ± SD and are normalized to % of field area. *P < 0.05, **P < 0.01 vs control; # P < 0.05, ## P < 0.01 vs HFD. n = 3

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Table 3 Effect of lutein intervention on lipid profiles in mice
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Fig. 5
figure 5

Effects of lutein on the mRNA and protein expression levels of hepatic PPAR-α (a–b), CPT1A (c–d) and ACOX1 (e–f) in mice. After being treated with lutein for 24 weeks, total RNA was extracted from the livers of mice by Trizol. The PPAR-α, CPT1A and ACOX1 mRNA expression levels were analyzed using real-time RT-PCR. The mRNA of β-actin was quantified as an endogenous control. Hepatic lysates were prepared and immunoblotted with the corresponding antibodies. Blotting with anti-β-actin was used as a protein loading control. PPARα, CPT1A and ACOX1 are presented as the fold-change relative to the control. Results are presented as the mean ± SD. *P < 0.05, **P < 0.01 vs control; # P < 0.05, ## P < 0.01 vs HFD. n = 3

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Fig. 6
figure 6

Effects of lutein on the mRNA and protein expression levels of hepatic LDLr (a–b) and SR-BI (c–d) in mice. After being treated with lutein for 24 weeks, total RNA was extracted from the livers of mice by Trizol. The LDLr and SR-BI mRNA expression levels were analyzed using real-time RT-PCR. The mRNA of β-actin was quantified as an endogenous control. Hepatic lysates were prepared and immunoblotted with the corresponding antibodies. Blotting with anti-β-actin was used as a protein loading control. LDLr and SR-BI are presented as the fold-change relative to the control. Results are presented as the means ± SD. *P < 0.05, **P < 0.01 vs control; # P < 0.05, ## P < 0.01 vs HFD. n = 3

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Discussion

Lutein may act as a preventive agent in atherosclerosis, although the exact mechanism of such a protection is unclear. In the present study, lutein displayed efficacy in protecting against HFD-induced atherosclerosis in apoE-deficient mice through improving lipid metabolism and antioxidant defense. Our results provide the first evidence, to our knowledge, that the attenuated oxidative stress in response to lutein treatment was due to the increased HO-1 expression and decreased NADPH oxidase expression in the aorta. The data also demonstrated that lutein had hypotriglyceridemic and hypocholesterolemic effects that were potentially mediated by up-regulating hepatic PPARα, LDLr and SR-BI.

According to previous publications, lutein dosage used in randomized clinical trial for prevent atherosclerosis or cardiovascular diseases are generally 10 or 20 mg per day, which average equivalent to 0.25 mg/kg bw [37, 38]. The doses of lutein (25, 50, 100 mg/kg bw) used in the present study were based on the surface area of the mice which calculated by the Meeh–Rubner formula, and were equivalent to 10, 20 and 40 times the recommended human daily intake of lutein in a 60 kg person [39]. Also, the lutein dosages provided are almost consistent with a previous study by Mai et al. [26].

Growing evidence indicates that the chronic and acute overproduction of ROS in pathophysiological conditions is an important etiological factor in the development of atherosclerosis [40, 41]. The modification of LDL into its oxidized form (oxLDL) by ROS is an initial step in the formation of an atheroma [42]. Researches investigated the role of dietary lutein supplementation on CVD prevention, due to their antioxidant effects [43–45]. Lutein is a beneficial antioxidant [46]. In details, lutein is not only a scavenger of reactive oxygen species (singlet molecular oxygen and peroxyl radicals), but also an effective deactivators of electronically excited sensitizer molecules. Additionally, lutein could protect cellular membranes and lipoproteins against oxidative damage, especially, improve LDL resistance to oxidation. Furthermore, lutein has a cooperative synergistic effect with other antioxidants (i.e., vitamin C and vitamin E) in scavenging reactive nitrogen species [47]. In the current study, lutein-treated mice showed lower levels of ROS and MDA as well as higher activities of SOD. These findings are consist with the results of previous studies that showed that lutein inhibited ROS production in vascular smooth muscle cells [48] and decreased MDA concentrations in the liver and eyes of guinea pigs fed a hypercholesterolemic diet [49]. In summary, the lutein-mediated prevention of atherosclerosis induced by a HFD diet is tightly linked to the lutein-mediated antioxidant defense, but the molecular mechanisms involved are not clear.

In the context of CVD, the oxidative stress that leads to increased ROS production is largely produced by NADPH oxidase, a multi-component enzyme that includes a membrane-associated heterodimer composed of two subunits (Nox2 and p22phox) and four cytosolic proteins (p47phox, p67phox, Rac, and p40phox) [50–52]. Among these subunits, p47phox and p22phox play a crucial role in increasing the activity of NADPH oxidase and closely relate to the development of atherosclerosis [5]. Previous studies showed that apoE−/− mice lacking p47phox have a marked reduction of atherosclerosis in the descending aorta [53], and increased expression of p22phox contributes to the development of atherosclerosis [54]. Considering the key role of NADPH oxidase in ROS production and atherosclerosis development, we speculate that the downregulating effects of lutein on the aortic p47phox and p22phox expression levels seen in this study may account for the low levels of ROS in lutein-treated groups. Certainly, further study is necessary to evaluate the precise molecular mechanisms.

HO-1, an antioxidant stress response protein, plays a very important role in protecting against oxidative stress [55, 56]. Epidemiological evidence has shown that the expression of HO-1 is reduced in patients with coronary atherosclerosis, which indicates that a reduced ability to induce HO-1 may be involved in the mechanism of coronary atherosclerosis [57]. A study by Wang et al. [58] showed that lycopene (a major carotenoid from tomatoes) reduces oxidative stress and is associated with increasing HO-1 protein expression in the livers of HFD-fed rats. Similarly, our data showed that lutein supplements induced HO-1 protein expression in the aorta. However, in contrast to the change in protein expression, the HO-1 mRNA level in the aorta was lower in the high dose lutein group than in the HFD group. According to previous findings that the induction of HO-1 gene expression is correlated with the production of ROS [59, 60], we postulated that the increased mRNA expression of HO-1 induced by HFD feeding acted as an adaptive response to increased oxidative stress but is not sufficient to counteract the generation of free radicals. While inhibiting oxidative stress by lutein may initially make HO-1 activation unnecessary in the absence of extra oxidant stimulus, the increased effect of lutein on the protein expression of HO-1, but not mRNA abundance, suggests that the regulation process may occur at the post-transcriptional level. However, the exact mechanism underlying this phenomenon needs further investigation. Taken together, our results suggest that lutein could mediate its protective effects against atherosclerosis by regulating inherent oxidative stress systems.

Obesity is a risk factor for cardiovascular events and mortality from all causes [61]. Though the mechanisms by which obesity increases vascular risk are unclear, excessive visceral adipose tissue is independently associated with cardiovascular events [62] and has been identified as an indicator of cardiometabolic risks [63]. A cross-sectional analysis of a nationally representative sample of American adults showed that the serum lutein level is negatively correlated with the odds ratio of obesity among both sexes [64]. However, little research has explored the effect of lutein supplementation on obesity and visceral adipose tissue. Here, our results show that lutein supplementation not only alleviated excessive body weight gain and visceral adipose tissue, but also ameliorated lipid deposition in the liver. Moreover, reductions in serum TAG, TC and LDL-C levels were observed in the lutein-treated groups. These results are consist with the data from a cross-sectional, case–control and case study by Renzi et al. [65] that showed that serum lutein was significantly related to the serum concentrations of TC and HDL-C; additionally, a recent report demonstrated that lutein supplementation reduces circulating total and LDL cholesterol levels in an early atherosclerotic population [33]. Because lipid metabolism disorder is a major risk factor for atherosclerosis [11, 12], the lipid-lowering effects of lutein are likely the main contributing factor to its antiatherogenic potential; however, the molecular mechanism by which lutein regulates lipid metabolism in the liver has not been fully investigated. Therefore, the key molecules involved in the lipid metabolism pathways that play pivotal roles in atherosclerosis formation were assessed in this study.

PPARα, which belongs to the super family of ligand-activated nuclear hormone receptors is thought to be the principal regulator in the fatty acid oxidation through modulation of CPT1A and ACOX1, the rate-limiting enzymes in mitochondrial and peroxisomal fatty acid oxidation respectively [66]. In addition, PPARα was reported to play a very important role in inhibiting atherosclerosis in LDL receptor-deficient mice [67]. It has been published that lipopolysaccharide injections decrease PPARα mRNA in the liver and thymus tissues of chickens during an inflammatory response, and this decrease is partially reversed by increasing the dietary lutein content to 50 mg/kg feed [68]. In addition, lutein also induces PPAR expression in bovine adipose tissue [69]. Recently, β-cryptoxanthin, a carotenoid, was discovered to ameliorate HFD-induced obesity and abdominal adipose tissue weight gain via inducing PPARα expression in rats [70]. This evidence made us speculate that increased β-oxidation of fatty acids may be responsible for the TAG-lowering effect of lutein displayed in this study based on the significantly higher mRNA and protein expression levels of PPARα as well as CPT1A and ACOX1.in the livers of lutein-treated animals.

LDLr, a transmembrane glycoprotein, mediates the binding and endocytosis of lipoproteins containing apolipoprotein B and E, particularly LDL [16]. SR-BI, which is considered the receptor of HDL, is predominantly expressed in the liver and steroidogenic tissues, where it mediates the selective uptake of cholesteryl ester from HDL [17]. Evidence suggests that decreases in LDLr result in fatty acid metabolism disequilibrium and lipidosis in the liver tissue [71]. Mice deficient in both SR-BI and apolipoprotein E develop early, occlusive, atherosclerotic coronary artery diseases and die prematurely at 6–8 weeks of age [72, 73]. A previous study conducted with apoE-KO knockout mice determined that astaxanthin, which is similar in structure to lutein, might improve cholesterol metabolism through increasing mRNA abundance of hepatic LDLr, which in turn enhances the uptake of LDL from the circulation, and up-regulating intestinal SR-BI mRNA, thereby reducing cholesterol absorption [74]. Similarly, the present study showed for the first time that lutein up-regulated the mRNA and protein expression levels of LDLr and SR-BI in the liver. Because LDLr and SR-BI play key roles in mediating cholesterol, the favorable impact of lutein treatment on TC and LDL-C exhibited in this study may result partly from the modulation of LDLr and SR-BI.

In conclusion, this study demonstrated that lutein plays a role as a regulator of the expression of genes involved in oxidative stress (NADPH oxidase, HO-1) and lipid metabolism (PPARα, CPT1A, ACOX1, LDLr and SR-BI), thereby mitigating the progression of atherosclerosis. The present findings regarding the beneficial effects of lutein illustrate that in the prevention of CVD, lutein administration could represent an adjunct or alternative therapeutic strategy.