The Journal of Nutrition
Nutrition and Disease
Dietary Blueberries Attenuate Atherosclerosis
in Apolipoprotein E-Deficient Mice by
Upregulating Antioxidant Enzyme Expression1–4
Xianli Wu,5,6* Jie Kang,5,6Chenghui Xie,5,6Ramona Burris,5Matthew E. Ferguson,5
Thomas M. Badger,5,6and Shanmugam Nagarajan5,7
5USDA Arkansas Children’s Nutrition Center,6Department of Physiology and Biophysics, and7Department of Immunology, University
of Arkansas for Medical Sciences, Little Rock, AR 72202
Protective effects of blueberries (BB) against atherosclerosis and potential underlying mechanisms in reducing oxidative
stress were examinedin apoE-deficient(apoE2/2) mice. ApoE2/2mice were fed an AIN-93G diet (CD) or CD formulated to
contain 1% freeze-dried whole BB for 20 wk. The mean lesion area for apoE2/2mice fed BB was reduced by 39% (P ,
0.001) in theaorta sinus and 58% (P , 0.001)in the descending aorta comparedwithCD-fedmice. These atheroprotective
effects were independent of the serum lipid profile or total antioxidant capacity (as measured by oxygen radical
absorbance capacity). The concentration of a biomarker of lipid peroxidation, F2-isoprostane, was lower in liver of BB-fed
mice (P , 0.05). Genes analyzed by RT-PCR array showed that 4 major antioxidant enzymes in aorta [superoxide
dismutase (SOD) 1, SOD2, glutathione reductase (GSR), and thioredoxin reductase 1] were upregulated in BB-fed mice.
EnzymeactivitiesofSODand GSRweregreater(P ,0.05) in liverand/orserumof BB-fedmicethanthoseofCD-fedmice.
In addition, serum paraoxonase 1 activity in serum of BB-fed mice was also greater than that of CD-fed mice (P , 0.05) at
the end of the study. These results suggest a protective effectiveness of BB against atherosclerosis in this apoE2/2
mouse model. The potential mechanisms may involve reduction in oxidative stress by both inhibition of lipid peroxidation
and enhancement of antioxidant defense.J. Nutr. 140: 1628–1632, 2010.
It has long been recognized that diets rich in fruits and vegetables
have protective effects against cardiovascular diseases (1–3).
However, scientific studies on the effects of individual fruits or
vegetables are still limited. In recent years, evidence has emerged
suggestingthat somephytochemicals inplant foods,polyphenols
in particular, may be largely responsible for their antiatherogenic
activities, mainly through antioxidant and antiinflammatory
activities. Dark-colored berries, such as blueberries (BB),8were
found to contain extremely high levels of polyphenols and ex-
hibit strong antioxidant capacities (4–7). Prevention of cardio-
vascular diseases by BB has been implicated (8) yet still lacks
direct evidence for protection against atherosclerosis.
LDL oxidation is a key step in early stages of atherosclerosis
development. In addition to causing LDL oxidation, oxidative
stress is also involved in many other aspects of the pathogenesis
of atherosclerosis and various risk factors (9). A recent study
(10) reported that plasma oxidized LDL (oxLDL) levels were
elevated in apoE-deficient (apoE2/2) mice by 10 wk and fatty
streak lesions developed after 10 wk, which suggested that
oxLDL and oxLDL-mediated inflammatory responses appear to
play a role in the initiation of atherosclerotic processes. BB has
been shown to inhibit human LDL and liposome oxidation in
cell culture and to significantly reduce lipid hydroperoxides in
humans (11–13). These studies suggest that reducing oxidative
stress might be one underlying mechanism for BB’s potential
The apoE2/2mouse model has been widely used in cardio-
vascular research (14,15). ApoE deficiency in mice leads to the
(16). Several studies (17–20) demonstrated that, in parallel with
high cholesterol levels in the blood, apoE deficiency promotes
increased oxidative stress and depressed antioxidant defenses.
In this study, the atheroprotective effects of BB and potential
underlying mechanisms were evaluated using the apoE2/2
1Supported by the USDA (6251-51000-005-02S).
2Author disclosures: X. Wu, J. Kang, C. Xie, R. Burris, M. E. Ferguson, T. M.
Badger, and S. Nagarajan, no conflicts of interest.
3Supplemental Tables 1 and 2 are available with the online posting of this paper
4Presented in part at Experimental Biology 10, April 2010, Anaheim, CA [Wu X,
Kang J, Xie C-H, Ferguson ME, Badger TM, Nagarajan S. 2010. Blueberries
reduce lipid peroxidation and boost antioxidant enzymes in apoE knockout mice.
FASEB J. 24:564.20 (abstract)].
8Abbreviations used: ApoE2/2, apolipoprotein E deficient; BB, blueberries; CD,
AIN-93G diet; GSR, glutathione reductase; m/z, mass:charge ratio; ORAC,
oxygen radical absorbance capacity; oxLDL, oxidized LDL; PON1, paraoxonase 1;
SOD, superoxide dismutase; Txnrd1, thioredoxin reductase 1.
* To whom correspondence should be addressed: E-mail: email@example.com
Phone: 501-364-2813 Fax: 501-364-3161.
ã 2010 American Society for Nutrition.
Manuscript received March 10, 2010. Initial review completed April 19, 2010. Revision accepted June 25, 2010.
First published online July 21, 2010; doi:10.3945/jn.110.123927.
by guest on December 21, 2015
Supplemental Material can be found at: