Comparison of lycopene and fluvastatin effects on atherosclerosis induced by a high-fat diet in rabbits

Nutrition 24 (2008) 1030 –1038 www.elsevier.com/locate/nut

Basic nutritional investigation

Comparison of lycopene and fluvastatin effects on atherosclerosis induced by a high-fat diet in rabbits Min-Yu Hu, Ph.D.a, Yi-Lin Li, M.S.a, Chong-He Jiang, Ph.D.b, Zhao-Qian Liu, Ph.D.a, Shu-Lin Qu, M.D.b,*, and Yi-Ming Huang, M.S.a a

Department of Nutrition and Food Hygiene, School of Public Health, Central South University, Changsha, China b Medical College of Hunan Normal University, Changsha, China Manuscript received September 23, 2007; accepted May 5, 2008.

Abstract

Objective: We evaluated the antiatherogenic effect of lycopene in rabbits fed a high-fat diet. Methods: Forty adult male rabbits were divided into five groups that were fed a standard diet, a high-fat diet, a high-fat diet plus 4 mg/kg of lycopene, a high-fat diet plus 12 mg/kg of lycopene, and a high-fat diet plus 10 mg/kg of fluvastatin, respectively. Lycopene and fluvastatin were administered intragastrically. The level of serum total cholesterol, total triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, total antioxidant capacity, and malondialdehyde were measured before and after 4 and 8 wk of experimental treatment. In addition, plasma levels of lycopene, oxidized low-density lipoprotein, serum nitric oxide, and interleukin-1 were measured after the experiment. The area of atherosclerotic plaque and pathologic changes of the aorta were evaluated. Results: Compared with the control, levels of total cholesterol, total triacylglycerol, low-density lipoprotein cholesterol, malonaldehyde, oxidized low-density lipoprotein, and interleukin-1 were increased and total antioxidant capacity and nitric oxide were decreased in the animals with a high-fat diet (P ⬍ 0.05). Intragastric administration of lycopene counteracted the change in these parameters (P ⬍ 0.05). In this case, the data showed that lycopene in the used dose was better than the fluvastatin intervention. Morphologic analysis revealed that lycopene and fluvastatin markedly reduced the formation of atherosclerotic plaques in the aorta compared with the situation in rabbits on a high-fat diet alone. Conclusion: Lycopene, like fluvastatin, significantly attenuated atherogenesis in rabbits fed a high-fat diet. © 2008 Elsevier Inc. All rights reserved.

Keywords:

Lycopene; Fluvastatin; Atherogenesis; Rabbit

Introduction Atherosclerosis is one of the most widespread conditions that threaten human health and survival [1–3]. The basic pathogenesis of atherosclerosis involves an insult to the endothelial and smooth muscle cells of the arterial wall by various harmful factors such as viral infection, mechanical damage, and dyslipidemia, especially abnormal oxidized low-density lipoprotein (ox-LDL), leading to an excessive

This project was supported by grant 00SSY1013-86 from the Society Development Foundation of Hunan. Shu-Lin Qu and Yi-Ming Huang were equal contributors. * Corresponding author. Tel./fax: ⫹86-731-863-0333. E-mail address: [email protected] (S.-L. Qu). 0899-9007/08/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2008.05.006

chronic inflammatory/fibroproliferative response. The pathologic process results in a progressive accumulation of lipids and fibrous elements in the large arteries [1– 4]. Based on the effect of oxidation and modification of LDL on the development of atherosclerosis, it might be possible to prevent the initiation and progression of lesions by therapy with antioxidants. Recent investigations have shown that all categories of high-risk patients benefit from LDLlowering therapy with statins [5–7]. Fluvastatin, one of the generally accepted drugs for treatment of coronary atherosclerosis, bases its effects on antioxidation and suppression of LDL levels, but not without sizable costs and risk for side effects [8]. Thus, it would be valuable to search for substances or ingredients with antioxidative effects in our daily food.

M.-Y. Hu et al. / Nutrition 24 (2008) 1030 –1038

Lycopene is an open-chain unsaturated carotenoid reported to be an efficient antioxidant. It is the pigment molecule that imparts the red color to some fruits and is found in high concentration in tomatoes and tomato products, watermelons, red grapefruits, and guava [9 –14]. Epidemiologic investigations have reported that lycopene consumption can reduce and prevent the development of cardiovascular disease [15–20]. Experimental studies have shown that it has many important bioactivities, e.g., quenching singlet oxygen, eliminating reactive oxygen species, blocking lipid peroxidation, suppressing cell reproduction, reinforcing immunity, and inducing gap junction intercellular communication [10,12–14]. It is one of the most popular research topics in nutrition and pharmaceutics [12,20,21]. However, few quantitative experimental studies of the preventive effect of lycopene on atherosclerosis have been reported, especially in vivo. The present study was designed to elucidate the effects of lycopene on the development of atherosclerosis in rabbits on a high-fat diet. Comparing the results with the antiatherosclerotic effect of fluvastatin would provide laboratory evidence for the potential of lycopene as a therapeutic agent in atherosclerosis.

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LDL and interleukin-1 (IL-1) enzyme-linked immune assay reagent kits were purchased from Dingguo Biology Limited Company (Shanghai, China). Experimental design

The standard diet for rabbits consisted of 10% wheat, 40% grass powder, 12% soybean cake, 20% corn, 10% wheat bran, 3% fish flour, 1% salt, 3% bone meal, and 1% multivitamins (percent weight). The diet contained 3.5% fat, 20% protein, and 25% carbohydrates and was purchased readymade from the Department of Zoology, Xiangya School of Medicine, Central South University. The high-fat experimental diet consisted of 5% lard, 1% cholesterol, and 94% standard diet.

Forty male New Zealand white rabbits with a body weight of 1800 ⫾ 300 g were used in this study. After 1 wk of adaptation to the standard diet, blood samples were collected from the rabbit ear artery to measure basal lipid levels. Based on the obtained level of TC, the rabbits were stratified into five groups of eight animals with similar distributions of pretreatment cholesterol levels. Group C (normal control) was fed the standard diet during the experimental period; group F received the high-fat diet; group FL1 (low-dose lycopene) the high-fat diet plus 4 mg of lycopene · kg⫺1 · d⫺1; group FL2 (high-dose lycopene) the high-fat diet plus 12 mg of lycopene · kg⫺1 · d⫺1; group FF (fluvastatin) the high-fat diet plus 10 mg of fluvastatin · kg⫺1 · d⫺1. Both drugs were dissolved in 1% sodium carboxymethyl cellulose to a final solvent volume of 2 mL/kg of body weight. The drugs were administered intragastrically, once per day, by a 12-Fr catheter inserted into the stomach. Groups C and F received the same amount of solvent without the drugs. The duration of treatment was 8 wk for each animal. They were kept two together in each cage with daylight illumination and access to water ad libitum. The food intake was recorded everyday and, based on the consumption of normal rabbits in a pilot experiment, restricted to 150 ⫾ 10 g of the compositions above. The temperature in the animal room was controlled at 18 ⫾ 4°C, and humidity was kept at 60 – 65%. The general health and activity of rabbits were monitored closely. All experimental procedures were conducted in accordance with the guidelines of the animal ethical committee for animal experimentation in China.

Pharmaceuticals and reagents

Assessment of plasma lycopene

Lycopene was purchased from the Huabei Pharmaceuticals Limited Company (Shijiazhuang, China; purity 90%, batch no. 041202). The standard lycopene preparation was purchased from Sigma Chemical Company (St. Louis, MO, USA; purity 95%). The fluvastatin-sodium capsule was purchased from the Nuohua Pharmaceuticals Limited Company (Beijing, China; batch no. X0006). Cholesterol was a product from the Tongxin Biochemistry Factory (Qidong, Hunan, China; batch no 050422). Total cholesterol (TC), total triacylglycerol (TG), LDL cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) kits were purchased from Dongou Biological Engineering Limited Company (Wenzhou, Zhejiang, China). Total antioxidant capacity (T-AOC), malonaldehyde (MDA), and nitric oxide (NO) kits were obtained from Jiancheng Biological Engineering Institution (Nanjing, China). Ox-

The lycopene standard solution was prepared by dissolving 1 mg of standard lycopene in 5 mL of isopropyl alcohol (0.2 mg/mL). This solution was further diluted to final concentrations of 2, 1, 0.5, 0.25, 0.125, 0.063, 0.031, 0.016, 0.008, and 0.004 ␮g/mL to obtain standard lycopene highperformance liquid chromatographic curves. The area under the lycopene peak was then plotted against the corresponding concentration, revealing good linearity in the range of interest (y ⫽ ⫺0.0006 ⫹ 8.2E ⫺ 7x, R2 ⫽ 0.9997). To determine plasma lycopene concentration, 400 ␮L of rabbit plasma was mixed with 200 ␮L of ethanol and vortexmixed for 20 min; 500 ␮L of water was added followed by 500 ␮L of N-hexane and the mixture was vortex-mixed for another minute. After centrifugation for 10 min at 1900 ⫻ g, the upper phase was removed and, by adding 1 mL of N-hexane, a second upper phase was obtained. Both extracts

Materials and methods Composition of diets

M.-Y. Hu et al. / Nutrition 24 (2008) 1030 –1038

were combined and dried under nitrogen and then dissolved in 200 ␮L of ethanol. The sample was analyzed by highperformance liquid chromatography under the following conditions: chromatographic column, Shimadzu Shim-pack VP-ODS 150 mm ⫻ 4.6 mm; mobile phase; isopropanol: methanol, 3:7, v/v; column temperature 40°C; flow rate 1.0 mL/min; detection wavelength 472 nm; sample injection volume 10 ␮L.

Lipid-related parameters and aortic atherosclerosis determinations The levels of TC, TG, HDL-C, LDL-C, T-AOC, and MDA in serum were measured, after 12 h of fasting, on the day before the start of the experiment and after 4 and 8 wk of treatment. Blood samples (5 mL) were collected from the rabbit central ear artery and divided equally into two different disposable sodium citrate anticoagulant tubes, one for serum and another for plasma. Serum TG and TC were analyzed by enzymatic methods of glycerol phosphate oxidase-peroxidase-4-aminoantipyrine (GPOPAP) and cholesterol oxidase-peroxidase-4-aminoantipyrine (CHOD-PAP) respectively) [22]. Concentrations of HDL-C were determined in the supernatant after precipitation of lipoprotein-B using phosphototungstic acid/Mg2⫹ (PTA/ Mg2⫹), and the concentrations of LDL-C were calculated as described by Friedewald et al. [23]. T-AOC was measured as described by Miller et al. [24]. MDA was measured using the thiobarbituric acid color reaction as described by Wills [25] and the absorbance was detected at the optical wavelength of 532 nm. Ox-LDL in plasma was measured by the Mercodia Oxidized LDL enzyme-linked immunosorbent assay kit using a Bio-Rad autoanalyzer (Roche, Switzerland) [26]. The levels of NO were determined photometrically at 550 nm using the nitrate reductase method [27]. IL-1 was determined by a biotin-avidin– based enzyme-linked immunosorbent assay kit from Sun Biomedical Technology (Beijing, China; available at: http://www.sunbio.com.cn) [28]. At the end of experiment, the animals were sacrificed by an overdose of 3% sodium pentobarbital (100 mg/kg, intraperitoneally); the aorta from the aortic arch to the aortoiliac bifurcation was dissected out by opening the chest and abdominal cavity. After fat staining by means of Sudan IV, the harvested aorta was opened up and gently spread on a white paper for gross examination and photography. The outlines of the aortic wall and atherosclerotic plaques were traced on transparent graph paper. To calculate the ratio of plaque area to that of the aorta, the atherosclerotic plaque tracings were cut out of the transparent paper and weighed separately from the remaining trace of the aorta. In addition, a 50-mm specimen of aorta was taken from the initial segment of the aortic arch and fixed in 10% formalin for 12 h, embedded in paraffin, and cut into serial 5-␮m sections. The sections were dewaxed with dimethylbenzene

and stained with hematoxylin and eosin for evaluation of morphologic changes under the light microscope. Statistical analysis Data were analyzed using SPSS 10.0 for Windows (SPSS Inc., Chicago, IL, USA). All data are presented as mean ⫾ standard deviation. Analysis of variance was used for multigroup comparison and Student-Newman-Keuls test for two-group comparison. P ⬍ 0.05 was considered statistically significant. Results Weight changes of the rabbits There was no significant difference in mean body weight (kilograms) of the rabbits in the control and experimental groups before the experiment (C, 1.880; F, 1.890; FL1, 1.960; FL2, 1.965; FF, 1.730). All groups showed a slow but significant increase in weight during the 8-wk experimental period. However, animals on the high-fat diet gained about twice as much weight as animals in the control group on the standard diet (P ⬍ 0.01; Fig. 1). There were no significant differences between groups on the high-fat diet at any stage of the experiment. Plasma concentration of lycopene at the end of the experiment After 8 wk of treatment, mean plasma concentrations of lycopene were 0.19 ⫾ 0.13 ␮mol/L in the low-dose lyco150

C (standard diet) F (high-fat diet) FL1 (high-fat, low dose lycopene)

140

Increase in body weight (%)

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**

FL2 (high-fat, high dose lycopene) FF (high-fat, fluvastatin)

130

120

110

100

90

80

Before

1

2

3

4

5

6

7

8

Time (week) Fig. 1. Time course of body weight changes in all groups of rabbits (see MATERIALS AND METHODS). Data were normalized and expressed as the percentage of the group mean value obtained before start of the experiment (mean ⫾ SD, n ⫽ 8/group, **P ⬍ 0.01).

M.-Y. Hu et al. / Nutrition 24 (2008) 1030 –1038

pene (FL1) group and 0.24 ⫾ 0.15 ␮mol/L in the high-dose (FL2) group. No lycopene was detected in animals of groups C, F, and FF, which received no lycopene intervention. Serum lipid parameters at different experimental stages Before the experiment, serum concentrations of TC, TG, LDL-C, and HDL-C were similar in all groups of rabbits (Fig. 2). After 4 wk on the high-fat diet, levels of TC, TG, and LDL-C were dramatically increased in group F (TC from 1.54 ⫾ 0.25 to 21.21 ⫾ 4.25 mmol/L; TG from 0.53 ⫾ 0.19 to 1.46 ⫾ 0.36 mmol/L; LDL-C from 0.59 ⫾ 0.22 to 15.82 ⫾ 2.5 mmol/L) and remained unchanged in the control group. Serum concentrations of these lipids were even higher after 8 wk on the high-fat diet (TC 48.57 ⫾ 1.48 mmol/L; TG 2.39 ⫾ 0.45 mmol/L; LDL-C 31.21 ⫾ 6.09 mmol/L). There was no significant change in the level of HDL-C in these two groups (C and F) at any stage of the experiment. Hyperlipidemia was also induced by the high-fat diet in the rabbits receiving lycopene and fluvastatin (groups FL1,

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FL2, and FF). Compared with control group C, their TC levels were significantly higher after 4 wk and further increased after 8 wk of treatment (P ⬍ 0.05; Fig. 2A). However, the increase was much less than in high-fat nonintervention group F. After 8 wk, the mean TC values in groups FL1, FL2, and FF were 72%, 73%, and 76% of group F (P ⬍ 0.05; Fig. 2A). The same was true for serum concentrations of TG and LDL-C, with mean values in groups FL1, FL2, and FF being 52%, 50%, and 65% for TG and 63%, 63%, 62% for LDL-C of those in group F after 8 wk (P ⬍ 0.01). In parallel with this suppression effect of the drugs, there was an increase in the level of HDL-C, although this change only reached significance for the fluvastatin treatment group (FF). Serum T-AOC There were no differences in the level of serum T-AOC among groups before the experiment (Fig. 3A). After 4 and 8 wk there was a progressive decrease in the level of T-AOC in the rabbits of group F compared with those in group C, but an increase in the rabbits of groups FL1 and

Fig. 2. Comparison of serum lipid levels before and after 4 and 8 wk of experimental treatments: (A) TC, (B) TG, (C) LDL-C, (D) HDL-C. Data are presented as mean ⫾ SD (n ⫽ 8/group). Bars without a common superscript differ significantly (P ⬍ 0.05). HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TC, total cholesterol; TG, total triacylglycerol.

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Plasma ox-LDL and serum NO and IL-1 Plasma ox-LDL and serum NO and IL-1 were measured only after 8 wk of the experiment (Table 1). The plasma level of ox-LDL increased significantly in groups F, FL1, FL2, and FF compared with group C. However, the increase was much less in the rabbits with lycopene intervention (groups FL1 and FL2) than in those on the high-fat diet alone (group F). In rabbits on fluvastatin (group FF) there was no significant difference in the level of ox-LDL compared with that in group F. The serum NO content was reduced in the rabbits on the high-fat diet. This reduction was completely counteracted by the lycopene intervention (groups FL1 and FL2). Thus, both doses of lycopene were sufficient to preserve serum NO at its normal level for rabbits. This protective effect on serum NO was less pronounced in the rabbits of group FF, treated with fluvastatin, indicating that lycopene is better than fluvastatin with respect to this parameter. With respect to serum IL-1, the results showed that the production of IL-1 is increased in hyperlipidemic rabbits but maintained close to its normal level by lycopene treatment (Table 1). Morphologic evaluations

Fig. 3. Serum levels of TAO-C (A) and MDA (B) measured before and after 4 and 8 wk of experimental treatments. Data are presented as mean ⫾ SD (n ⫽ 8/group). Bars without a common superscript letter differ significantly (P ⬍ 0.05). MDA, malondialdehyde; TAO-C, total antioxidant capacity.

FL2. As for serum lipids, there was no significant difference between groups on low and high doses of lycopene (FL1 and FL2). This outcome implies that serum T-AOC was depressed by the high-fat diet and that lycopene intervention counteracted this effect and enhanced the antioxidative capacity of rabbit serum. Fluvastatin had no such effect (Fig. 3A). Serum MDA

Gross observation The aortic intima in the rabbits of the control group looked smooth and neat with no corpus alienum. In the hyperlipidemic rabbits of group F, there were variable degrees of gray-yellow lipid plaques spreading over most of the tunica intima, notably in the area of the aortic root and at branch points of the arterial tree. These atherosclerotic plaques were present as strips or focal lesions scattered as irregular patches with a clearcut margin. They extruded slightly from the endothelial layer with no cracks or ulcerations at the surface. Such pathologic alterations were fewer in the rabbits of group FL1 and were found in the segment of the aortic arch. Even less pathologic changes were seen in the rabbits of groups FL2 and FF with only scattered spotlike lesions. Table 1 Plasma ox-LDL, serum NO, and IL-1 in different groups after 8 wk of treatment* Group

The content of MDA was similar among groups at the beginning of the experiment (Fig. 3B). After 4 wk, levels of serum MDA in groups F, FL1, FL2, and FF were increased compared with that in group C. The increase was somewhat less in groups FL1 and FL2 than in group F. This difference became significant after 8 wk of treatment, suggesting that a longer period of treatment would be needed for lycopene to present its inhibitory effect on the production of MDA in rabbits on the high-fat diet. Lycopene was in this aspect better than fluvastatin (P ⬍ 0.05).

C F FL1 FL2 FF

n

ox-LDL (␮g/mL)

NO (␮mol/L)

IL-1 (ng/mL)

8 8 8 8 8

23.51 ⫾ 4.68 47.35 ⫾ 5.00c 35.77 ⫾ 3.47b 33.45 ⫾ 2.24b 45.44 ⫾ 3.17c

19.12 ⫾ 2.39 7.84 ⫾ 2.08b 22.07 ⫾ 4.07a 25.80 ⫾ 9.05a 13.88 ⫾ 6.87c

0.04 ⫾ 0.01a 0.10 ⫾ 0.02b 0.05 ⫾ 0.02a 0.06 ⫾ 0.02a 0.10 ⫾ 0.01b

a

a,c

C, normal diet; F, high-fat diet; FF, high-fat diet plus fluvastatin; FL1, high-fat diet plus low-dose lycopene; FL2, high-fat diet plus high-dose lycopene; IL-1, interleukin-1; ox-LDL, oxidized low-density lipoprotein; NO, nitric oxide * Values are means ⫾ SDs. Different superscript letters in each column indicate significant differences between groups (P ⬍ 0.05).

M.-Y. Hu et al. / Nutrition 24 (2008) 1030 –1038

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structure of the superficial tunica media. These morphologic alternations, typical for atherosclerotic lesions, were less pronounced in the rabbits treated with low-dose lycopene (group FL1) in which foam cells in some part of the intima were diminished and fibroplasias not observed. The changes were even less obvious in the rabbits treated with high-dose lycopene (group FL2); the foam cells in the intima were much fewer and even lacking in some part of the aorta with morphologic features close to that of the control rabbits of group C. The changes in group FF rabbits were similar to those in group FL2, which indicated a similar preventive effect of lycopene and fluvastatin in the development of atherosclerosis in this experimental model.

Discussion

Fig. 4. Relative area of aortic wall surface covered by atherosclerotic plaques in the different groups. Data are presented as mean ⫾ SD (n ⫽ 8/group). Bars without a common superscript letter differ significantly (P ⬍ 0.05). C, normal diet; F, high-fat diet; FF, high-fat diet plus fluvastatin; FL1, high-fat diet plus low-dose lycopene; FL2, high-fat diet plus high-dose lycopene.

Area of atherosclerotic plaques After staining by Sudan IV, the scarlet atherosclerotic lesions of the aorta were easily visualized. The ratio of the area covered by such atherosclerotic plaques to the total aortic surface area was determined as described in MATERIALS AND METHODS and presented as a mean percentage for each group of animals (Fig. 4). There were no lesions in the aorta in the control rabbits of group C. All other groups of rabbits on the high-fat diet exhibited a variable degree of atherosclerotic lesions. The most affected rabbits were those in group F, with atherosclerotic lesions accounting for almost half the aortic surface (48 ⫾ 11%). The rabbits in groups FL1, FL2, and FF were much less affected, with atherosclerotic lesions covering 15%, 5%, and 5%, respectively, of the surface area (P ⬍ 0.05). The high dose of lycopene had a stronger effect on plaque formation than the low dose (P ⬍ 0.05) being comparable to that of fluvastatin. Morphologic changes of rabbit aorta stained with hematoxylin and eosin Representative aortic sections stained with hematoxylin and eosin from each group are shown in Figure 5. The aortic walls in the control rabbits of group C were smooth and intact. The structures of intima, medial, and adventitial layers were clearly distinguishable with no pathologic changes. In the rabbits of group F, the arterial lumen was irregular and the tunica intima much thickened. The arterial wall presented large numbers of diffuse bulges composed of foam cells, piled up in the intima and subintimal layers, some reaching the medial layer and causing a disorganized

Extensive epidemiologic and experimental studies have supported the notion that lycopene possesses strong antioxidative capacity and that the incidence of cardiovascular disease can be reduced by the consumption of foods rich in lycopene [12,14,18,19,29,30]. To date, such studies and intervention experiments have mainly focused on the lycopene content in meals or on lycopene supplementation. Based on the hypothesis of an interaction of lycopene with other carotenoids, increased intake of foods containing lycopene has been recommended, assuming that it would act synergistically with biologically active compounds of other ingested fruits or vegetables. However, the antiatherogenic effect of lycopene contained in meals has been questioned recently [31]. A possible explanation could be that various compounds in the food may interact with each other and thus affect the bioavailability of lycopene and possibly its antiatherogenic effect. Obviously, the mechanism(s) and efficacy of lycopene in lowering lipid levels need to be further clarified [10,20,32]. There are many factors that may affect lycopene absorption and bioavailability including dietary sources, the composition and structure of the food, and food processing including cooking or heating [21,33]. Thus, it is difficult to estimate the actual absorption of lycopene from the diet. To minimize such complications, we used a pure lycopene compound administrated intragastrically once a day in conjunction with standard meals. Rabbits do not absorb intact carotenoids efficiently and therefore we had to use high doses of lycopene (4 and 12 mg/kg of body weight) to produce measurable plasma levels (0.19 ⫾ 0.13 and 0.24 ⫾ 0.15 ␮mol/L for the lower and higher doses, respectively). This corresponds to low plasma levels of lycopene in humans who may achieve five-fold higher levels already with the intake of only 0.3 mg/kg of body weight [34]. Fluvastatin, a hydroxy-3-methylglutaryl coenzyme A reductase inhibitor with antioxidative property, is a generally accepted drug with lipid-lowering and antiatherogenic effects [35,36]. The antiatherogenic effect of fluvastatin was

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C

A

D

B

E

Fig. 5. Morphologic changes of the rabbit aorta stained by hematoxylin and eosin using a light microscope (magnification 120⫻). (A) Rabbits fed the standard diet (control); (B) rabbit fed the high-fat diet; (C) rabbits fed the high-fat diet plus 4 mg of lycopene · kg⫺1 · d⫺1; (D) rabbits fed the high-fat diet plus 12 mg of lycopene · kg⫺1 · d⫺1; (E) rabbits fed the high-fat diet plus 10 mg of fluvastatin · kg⫺1 · d⫺1.

reproduced in the present in vivo study by a reduction in the aortic surface area covered by atherosclerosis plaques and by less pronounced morphologic changes compared with untreated rabbits on the same atherogenic diet. A similar effect was achieved in rabbits treated with lycopene, particularly in those on the high lycopene dose. It follows that lycopene, like fluvastatin, has an antiatherogenic effect and may play a similar role in preventing or delaying the initiation and progression of atherosclerotic lesions. Among plasma lipoproteins, LDL has a crucial role in inducing atherosclerosis and it has been identified as an independent risk factor for coronary heart disease [3,37– 39]. The results of our experiment in the high-fat diet rabbit model showed that lycopene and fluvastatin lowered serum levels of TC and LDL-C, improved lipid metabolism, and reduced the amount of TG. The biochemical and biological properties of ox-LDL differs from non– ox-LDL, having a higher density, faster electrophoresis, and higher content of

free cholesterol [32,40]. Lycopene intervention reduced the increase in ox-LDL levels in rabbits on the high-fat diet, whereas fluvastatin did not show such an effect. The cause of this difference is at present not known, although the result speaks in favor of lycopene. Total AOC in serum represents the total level of enzymatic and non-enzymatic antioxidants. The serum level of MDA manifests the degree of lipid peroxidation and reflects indirectly the state of the cells affected by free radicals [41]. We found that lycopene enhanced the activity of serum T-AOC and inhibited the increase of serum MDA induced by the high-fat diet. Both results are in keeping with the idea that lycopene would lower the level of oxidative stress in rabbits on the high-fat diet. These effects of lycopene seem helpful in raising the AOC and facilitating the clearance of free radicals. Thus lycopene may block the oxidative modification of LDL and counteract inflammatory reactions. In comparison, fluvastatin presented a lesser effect in this respect.

M.-Y. Hu et al. / Nutrition 24 (2008) 1030 –1038

Vascular endothelial cells are not only an important barrier between the blood and the vascular smooth muscle but also an endocrine organ, which synthesizes and secretes important vasoactive substances, such as NO and endothelin. These regulatory substances respond to various pathophysiologic stimuli to protect the integrity of the blood vessels. In addition to the control of vascular tone, NO reduces platelet adhesion and aggregation, lowers the expression of endothelial cell adhesion molecules, induces feedback inhibition of endothelin synthesis and release, and increases clearance of free radicals, functions that are essential for antiatherogenesis [1,42]. Our study showed that high and low doses of lycopene raised the level of NO and maintained it at the normal level in rabbits on the high-fat diet and, as a consequence, protected the normal function of the artery. Compared with lycopene, fluvastatin showed less effect on the maintenance of NO levels in the same situation. Recently, it has been realized that atherosclerosis involves a chronic inflammatory process [2– 4]. The secretion of inflammatory mediators such as IL-1, IL-6, and tumor necrosis factor and recruitment of leukocytes to the intima contribute to the early formation of atherosclerotic lesions. IL-1 is a key factor in this inflammatory reaction. It can induce the synthesis of other cytokines, including chemotactic factors, adhesion molecules, and acute-phase proteins [1,27,43,44]. In the present study, we found that the production of IL-1, induced by the high-fat diet, was lower in the rabbits with lycopene intervention. Thus, lycopene seems to have an anti-inflammatory effect by inhibiting IL-1 secretion, whereas fluvastatin had no such effect in our experimental model. In summary, lycopene significantly attenuated atherogenesis in rabbits on the high-fat diet. In terms of changes in plasma and serum lipid parameters, the effect of lycopene was superior to that of fluvastatin, although the morphologic analysis showed comparable effects of high-dose lycopene and fluvastatin. It is assumed that the antiatherogenic effect of lycopene is associated with improved lipid homeostasis, antioxidation, inhibition of ox-LDL modification, protection of the vascular endothelium, and anti-inflammation. These findings provide a theoretical rationale for the use of lycopene as a preventive and therapeutic drug in atherosclerosis.

Acknowledgments The authors thank Professor Sivert Lindström, Department of Biomedicine and Surgery, University of Linköping, Sweden, for a constructive discussion of this report.

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