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Apple flavonols and n-3 polyunsaturated fatty
acid–rich fish oil lowers blood C-reactive protein
in rats with hypercholesterolemia and
acute inflammation
Satvir Sekhon-Loodu a , Adriana Catalli b , Marianna Kulka b , Yanwen Wang b ,
Fereidoon Shahidi c , H.P. Vasantha Rupasinghe a,⁎
a
b
c
Department of Environmental Sciences, Faculty of Agriculture, Dalhousie University, P.O. Box 550, Truro, Nova Scotia B2N 5E3, Canada
Aquatic and Crop Resource Development, National Research Council of Canada, Charlottetown, Prince Edward Island C1A 4P3, Canada
Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, A1B 3X9 Canada
ARTI CLE I NFO
A BS TRACT
Article history:
Both quercetin glycosides and omega-3 polyunsaturated fatty acids (n-3 PUFA) are well
Received 23 December 2013
established for their individual health benefits in ameliorating metabolic disease. However,
Revised 12 April 2014
their combined effects are not well documented. It was hypothesized that the beneficial
Accepted 6 May 2014
properties of quercetin glycosides can be enhanced when provided in combination with n-3
PUFA. Therefore, the aim of the present study was to investigate the effects of apple flavonols (AF)
Keywords:
and fish oil (FO), alone and in combination, on proinflammatory biomarkers and lipid profiles in
Apple flavonols
rats fed a high-fat diet. Sixty male Wistar rats were randomly divided into 5 groups (n = 12) and fed
Fish oil
a high-fat diet for 4 weeks. One of the 5 groups of rats was used as the high-fat control. The other 4
Cytokines
groups of rats were injected with lipopolysaccharide (LPS) (5 mg/kg body weight)
C-reactive protein
intraperitoneally, 5 hours before euthanization. One of these 4 groups was used as the
Inflammation
hypercholerolemic and inflammatory control (high-fat with lipopolysaccharide [HFL]), and the
Hyperlipidemia
other 3 received AF (HFL + 25 mg/kg per day AF), FO (HFL + 1 g/kg per day FO), or the combination
Rats
(HFL + AF + FO). Compared to the HFL group, the AF, FO, and AF + FO groups showed lower serum
concentrations of interleukin-6 and C-reactive protein (CRP) levels. The AF, FO, and AF + FO also
had lowered serum triacylglycerol and non–high-density lipoprotein cholesterol (HDL-C)
concentrations, but higher HDL-C levels relative to the HFL group. An additive effect was
observed on serum CRP in the AF + FO group as compared with the AF or FO groups. The results
demonstrated that AF and FO inhibited the production of proinflammatory mediators and
showed an improved efficacy to lower serum CRP when administered in combination, and they
significantly improved blood lipid profiles in rats with diet-induced hyperlipidemia and LPSinduced acute inflammation.
© 2014 Elsevier Inc. All rights reserved.
Abbreviations: AF, apple flavonols; ANOVA, analysis of variance; CE, cholesteryl ester; CRP, C-reactive protein; CVD, cardiovascular
disease; ELISA, enzyme-linked immunosorbant assay; FC, free cholesterol; FO, fish oil; HDL-C, high-density lipoprotein cholesterol; HFC,
high-fat control; HFL, high-fat with lipopolysaccharide; IFN-γ, interferon-γ; IL, interleukin; LDL-C, low-density lipoprotein cholesterol;
LPS, lipopolysaccharide; NF-κB, nuclear factor-kappa B; PUFA, polyunsaturated fatty acids; ROS, reactive oxygen species; TAG,
triacylglycerols; TC, total cholesterol; TNF-α, tumor necrosis factor α.
⁎ Corresponding author. Tel.: +1 902 893 6623; fax: +1 902 893 1404.
E-mail address: [email protected] (H.P.V. Rupasinghe).
http://dx.doi.org/10.1016/j.nutres.2014.05.002
0271-5317/© 2014 Elsevier Inc. All rights reserved.
536
1.
N U TR IT ION RE S EA RCH 3 4 ( 2 0 14 ) 53 5 –5 43
Introduction
The prevalence of obesity in Western populations has been
postulated to be the major cause of metabolic syndrome,
including insulin resistance, type 2 diabetes, hyperlipidemia,
and hypertension [1]. Accumulating evidence suggests that a
long-standing high-fat diet might have contributed to metabolic
endotoxemia and chronic inflammation [2,3]. Hyperlipidemia is
commonly characterized by elevated circulating levels of total
cholesterol (TC), low-density lipoprotein cholesterol (LDL-C),
triacylglycerol (TAG) and reduced high-density lipoprotein cholesterol (HDL-C) [4]. At the initial stage of most metabolic
disorders, the circulating levels of C-reactive protein (CRP),
tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and adiponectin are altered, thus increasing risk for developing cardiovascular disease (CVD) and insulin resistance [5].
A high-fat diet is lipogenic and induces hyperlipidemia in
rats [6,7] and excessive body weight, which serves as an
experimental model of diet-induced obesity [8]. In addition to
a healthy lifestyle, nutritional intervention has long been
recommended for maintaining healthy lipid profiles [9].
Polyphenols have been shown to decrease the risk of
degenerative diseases such as CVD and cancer [10]. Apples,
especially the peel (skin), are a great source of dietary
polyphenols [11,12]. It is reported that apple flavonoids
possess anti-inflammatory and lipid-lowering properties [13,14]. The major flavonols in apple peel are quercetin
glycosides, which contribute approximately 70% of all flavonols in the North American diet [15]. Quercetin is well
documented for its antioxidant and anti-inflammatory effects
[16]. In addition to flavonoids, omega-3 polyunsaturated fatty
acids (n-3 PUFA) are another class of naturally occurring
biomolecules that are recognized for their anti-inflammatory
and hypotriglyceridemic benefits [9,17–20]. However, n-3
PUFA is highly susceptible to lipid oxidation due to a high
degree of unsaturation. Flavonoids (as antioxidants) may
inhibit oxidation, thus protecting n-3 PUFA [21] and enhancing
quercetin efficacy in vivo [22].
The potential benefits of combining flavonoids and n-3 PUFA
have not been well described. Herein, we hypothesized that the
lipid-lowering and anti-inflammatory properties of apple quercetin glycosides can be enhanced by supplementing a diet with
n-3 PUFA. Accordingly, the objective of this study was to
determine the effect of apple flavonols (AF) and fish oil (FO),
alone and in combination, on proinflammatory markers and
lipid profiles in rats with diet-induced hyperlipidemia and
lipopolysaccharide (LPS)–induced acute inflammation.
2.
Methods and materials
2.1.
Chemicals and apparatus
All dietary ingredients for animal feed, except AF and FO, were
purchased from Dyets Inc. (Bethlehem, PA, USA). The eicosapentaenoic acid–rich FO that was provided by DSM (formerly
Ocean Nutrition Canada, Dartmouth, NS, Canada) contained
75.7% total PUFA (n-3 and n-6), and 70.6% were n-3 PUFA (82%
eicosapentaenoic acid and 14% docosahexaenoic acid). Rat
cytokine flex kits and CRP enzyme-linked immunosorbant
assay (ELISA) kit was purchased from BD Biosciences (Mississauga, ON, Canada), and rat ELISA kits (IL-6 and adiponectin)
were obtained from Invitrogen (Burlington, ON, Canada).
Reagents for liver and serum lipid reagents were obtained
from BioVision (Milpitas, CA, USA) and BioPacific Diagnostic
Inc. (North Vancouver, BC, Canada). Lipopolysaccharide
lyophilized powder (from Escherichia coli) was purchased
from Sigma-Aldrich (Oakville, ON, Canada).
2.2.
Ultraperformance liquid chromatography–tandem
mass spectrometry analysis of apple peel phenolic compounds
The phenolic compounds were extracted and fractionated using
reversed-phase chromatography, as previously described [21].
The fractions containing high amounts of quercetin-3-O-glycosides were pooled and freeze-dried to form AF concentrate.
Analyses of the major individual phenolic compounds present in
apple peel extract were performed according to the method
previously reported [23]. All analyses were conducted using
ultrapressure liquid chromatography (Waters, Milford, MA, USA),
coupled with Micromass Quattro micro API MS/MS system, and
controlled with Mass lynx V4.0 data analysis system (Micromass,
Cary, NC, USA).
2.3.
Animal study
Six-week-old male Wistar rats (Charles River, Montreal, QC,
Canada), weighing 200 to 250 g, were housed individually in cages
with a 12-hour dark/light cycle, and free access to regular rodent
chow and water. After a week of adaptation, the rats were
weighed and randomly divided into five groups (n = 12 per group),
and fed their respective diets for four additional weeks. One group
was fed the AIN-93G diet, modified to contain 23% fat, 1.9%
cholesterol, and 0.5% cholic acid (Table 1) to induce hyperlipidemia and other obesity-related characteristics (high-fat control
[HFC]). Among the 4 high-fat with lipopolysaccharide (HFL)
groups, 1 group was used as an obese and inflammatory control
(HFL), and the other 3 were supplemented in the diet with 25 mg/
kg per day of AF (HFL + AF), 1 g/kg per day of FO (HFL + FO), or the
combination of the same amounts of AF and FO (HFL + AF + FO).
The diets were prepared weekly and stored at 4°C. The amount of
AF and FO in the diet was estimated, respectively, on the basis of
the body weight and food intake [24]. Weekly body weight and
daily food intake were obtained, and the animals were also
observed for any abnormal behaviors throughout the study
period. Five hours before euthanization, the HFL, HFL + AF,
HFL + FO, and HFL + AF + FO groups were injected intraperitoneally with 5 mg/kg body weight of LPS to induce acute
inflammation The animal use and experimental procedures
were approved by the Joint Animal Care and Research Ethics
Committee of the National Research Council Canada–Institute for
Nutrisciences and Health and the University of Prince Edward
Island, Canada. The study was conducted in accordance with the
guidelines of the Canadian Council on Animal Care.
2.4.
Collection and storage of blood and tissue samples
At the end of the study, the animals were anesthetized with
isoflurane inhalation and killed by exsanguination. Blood was
537
N U TR IT ION RE S E ARCH 3 4 ( 2 0 14 ) 53 5 –5 4 3
Table 1 – Ingredient composition of the diet fed to rats
Ingredients
Composition (g/kg)
Casein
Corn starch
Sucrose
Fat a
Cellulose
DL-<ethionine
Mineral mix b
Vitamin mix c
Choline bitartrate
Butylated hydroxytoluene
Cholesterol
Cholic acid
229.0
269.8
121.4
229.0
63.5
3.8
44.3
12.6
2.5
0.2
19.1
4.7
2.6.
Determination of serum CRP
The serum CRP was quantified using a solid-phase sandwich
ELISA kit (BD Biosciences) following the supplier's instructions
[26]. The samples were read for absorbance at 460 nm on a
BioTek Powerwave microplate reader (Winooski, VT, USA).
2.7.
a
Composed of 96% of lard and 4% of sunflower oil.
Mineral mix contained the following: 5000 mg Ca, 1561 mg P, 3600
mg K, 1019 mg Na, 1571 mg Cl, 300 mg S, 507 mg Mg, 35 mg Fe, 6 mg
Cu, 10 mg Mn, 30 mg Zn, 1 mg Cr, 0.2 mg I, 0.15 mg Se, 1 mg F, 0.5 mg
B, 0.15 mg Si, 0.5 mg Ni, 0.1 mg Li, and 0.1 mg V per kilogram of the
mineral mix.
c
The composition of vitamin mix was 20 mg thiamine HCl, 15 mg
riboflavin, 7 mg pyridoxine HCl, 90 mg niacin, 40 mg calcium
pentothenate, 2 mg folic acid, 0.6 mg biotin, 10 mg cyanocobalamin
(B12, 0.1%), 4 mg menadione sodium bisulfate, 5000 IU vitamin A
palmitate, 50 IU vitamin E acetate, 2400 IU vitamin D3, and 100 mg
inositol per kilogram of the vitamin mix.
b
collected by cardiac puncture into serum tubes (BD Vacutainers, Franklin Lakes, NJ, USA). The serum was separated by
centrifugation at 650g for 15 min using an Allegra 25R
centrifuge (Beckman Coulter, Mississauga, ON, Canada), and
stored in a −80°C freezer for later analyses. Liver, spleen, and
adipose tissue were dissected, washed with saline solution,
frozen in liquid nitrogen, and stored at −80°C.
2.5.
tions [25]. The samples were analyzed on a BD FACSArray
Bioanalyzer, and the data analysis was performed using the
FCAP Array software (BD Biosciences). The concentration of each
cytokine was presented as picogram per milliliter.
Analysis of serum cytokines
The serum concentration of cytokines IL-10, TNF-α, and interferon-γ (IFN-γ) were quantified using cytometric bead array rat
flex kits (BD Biosciences) following the manufacturer's instruc-
Determination of serum IL-6 and adiponectin
The serum IL-6 and adiponectin concentrations were measured using ELISA kits supplied by Invitrogen, according to the
manufacturer's instructions [26]. The optical density was read
at 450 nm on a Varioskan Flash plate reader (Thermofisher
Scientific, Nepean, ON, Canada).
2.8.
Lipid profile analysis in rat serum
The serum TC, HDL-C, and TAG were analyzed on a Pointe-180
chemistry analyzer (Pointe Scientific Inc., Canton, MI, USA),
with all reagents provided by the manufacturer. The HDL-C
was measured after the precipitation of apolipoprotein B
containing lipoproteins with dextran sulfate and magnesium
chloride. Non-HDL cholesterol was calculated by subtracting
HDL-C from TC [24].
2.9.
Analysis of liver lipids
The liver cholesterol was extracted by homogenizing 100 mg
of liver in 1 mL of chloroform/isopropanol/nonidet P-40
(octylphenoxypolyethoxyethanol) (7:11:01, v/v/v). After centrifugation, 200 μL of supernatant was dried under nitrogen.
For liver TAG analysis, 100 mg of liver was homogenized in 5%
nonidet P-40 in water, heated for 5 minutes in the water bath
at 90°C, and cooled down to room temperature. The liver TC,
Table 2 – Concentrations of phenolic compounds of Northern Spy AF concentrate prepared using reverse-phase
chromatography
Phenolic compounds
Quercetin-3-O-galactoside
Quercetin-3-O-rhamnoside
Quercetin-3-O-glucoside
Quercetin-3-O-rutinoside
Quercetin
Phloridzin
Phloritin
Chlorogenic acid
Caffeic acid
Catechin
Epicatechin
Total phenolics by UPLC-MS/MS
Concentration
104.6
99.98
10.89
3.66
2.88
66.78
0.69
16.55
0.77
6.12
37.86
350.7
±
±
±
±
±
±
±
±
±
±
±
±
a
(mg/g DW)
21.1
20.0
1.80
1.18
0.70
9.09
0.07
4.58
0.08
0.99
8.75
67.7
Composition (%)
29.8
28.5
31.0
1.04
0.82
19.0
0.19
4.71
0.21
1.74
10.7
The polyphenolics extracted in absolute ethanol were fractioned using 20 to 100% ethanol gradient by reverse-phase chromatography. The
fractions were analyzed using UPLC-MS/MS and the fractions rich in flavonols were concentrated and dried into powder. Abbreviation:
UPLC-MS/MS, ultraperformance liquid chromatography–tandem mass spectrometry.
a
Data presented as mean (SEM), n = 3.
538
N U TR IT ION RE S EA RCH 3 4 ( 2 0 14 ) 53 5 –5 43
Table 3 – Food intake, body weight gain, liver weight and spleen weight of the 5 treatment groups of hyperlipidemic rats a
Dietary treatments
Measurements
Food intake, g/d
Body weight gain, g
Liver weight, g
Spleen weight, g
a
b
HFC
23.89
286.4
39.28
1.54
±
±
±
±
b
0.81
9.62
1.40
0.10
HFL
23.71
297.4
41.10
1.68
±
±
±
±
0.80
10.0
1.41
0.12
HFL + AF
HFL + FO
24.20
287.9
41.41
1.72
23.81
287.7
38.51
1.68
±
±
±
±
0.82
8.21
1.30
0.15
±
±
±
±
0.71
8.30
0.61
0.10
HFL + AF + FO
24.24
298.2
41.90
1.70
±
±
±
±
0.70
8.90
1.82
0.10
Values are expressed as mean (SEM), (n = 12).
HFL, high fat + LPS; HFL + AF, high fat + LPS + AF; HFL + FO, high fat + LPS + FO; HFL + AF + FO, high fat + LPS + AF + FO.
free cholesterol (FC), cholesteryl esters (CEs), and TAG were
determined using the commercial kits (BioVision), as reported
previously [24].
2.10.
Statistical analyses
All data analyses were performed using SAS software version
8 (Cary, NC, USA). The normality of the residuals was tested
using the Anderson-Darling test, and the data were logtransformed for serum CRP, TC, and TAG. Treatment effects
were determined using one-way analysis of variance
(ANOVA). When a significant treatment effect was detected,
the differences among the groups were determined using the
Duncan test. Results are presented as mean (SEM) (n = 12). The
significance level was set at P < .05.
3.
Results
3.1.
Composition of flavonol-rich apple peel concentrate
The total polyphenols content in the freeze-dried apple peel
concentrate was 350 mg/g. Quercetin-3-glycosides were the
predominant component, at 63%, that is, 221.9 mg/g (Table 2).
3.2.
Food intake, body weight, and liver and spleen weights
There were no significant differences in the weekly and final
body weights (Table 3) among the 5 groups, after 4 weeks of
treatment. Similarly, the treatments had no effect on food
intake. No effect was observed on the liver and spleen weights
among the 5 groups (Table 3). Previous studies demonstrated
that after 4 weeks of high-fat diet in rats, they developed
characteristics of obesity [7,8]. To minimize the use of rats, a
normal diet control group was not used as advised by the
Animal Care and Research Ethics Committee.
treatment groups (Fig. 1C). The serum concentration of IFN-γ
in the HFL group was increased to 99-fold of the HFC group.
The supplementation of FO lowered serum concentrations
of IFN-γ by 45%; however, AF or AF + FO did not show a
significant effect (Fig. 1D). The serum IL-6 concentration was
raised by nearly twofold after LPS injection in the HFL groups,
compared with the HFC group. As compared with the HFL
group, the supplementation of AF, FO, and AF + FO reduced IL-6
serum concentrations by 41, 25, and 35%, respectively (Fig. 1E).
The IL-10 concentration in the HFL group was 18-fold of
the HFC group (P < .05). The IL-10 concentration was further
elevated with AF and AF + FO treatment (Fig. 1F).
3.4.
The effects of the treatments on serum lipids are summarized
in Fig. 2. There were no differences between the HFC and HFL
groups in all parameters measured (Fig. 2A-D). The AF, FO, or
AF + FO decreased the serum TAG by 35, 39, and 36%,
respectively, as compared with the HFL group (Fig. 2A).
Similarly, the three treatments decreased the serum non–
HDL-C levels. Rats of the AF, FO, and AF + FO groups showed
53, 51, and 49% lower levels of serum non–HDL-C than the HFL
group, respectively (Fig. 2D). In contrast, the AF, FO, and AF +
FO increased serum HDL-C by 56, 46, and 43% relative to the
HFL group (Fig. 2C). There were no differences among the 5
groups in the serum TC.
The concentrations of liver TAG (Fig. 3A) and CEs (Fig. 3D)
did not differ among the 5 groups. In comparison to the HFL
group, the AF and AF + FO groups reduced liver TC by 15
and 18%, respectively (Fig. 3B). The liver FC concentrations
were decreased by 37, 38, and 37% in rats treated with AF,
FO, and AF + FO, respectively, as compared with the HFL
control (Fig. 3C).
4.
3.3.
Serum and liver lipids
Discussion
Serum inflammatory biomarkers
The serum adiponectin levels did not show any difference
among all groups (Fig. 1A). There was no significant difference
in the serum CRP concentrations between HFC and HFL.
However, significant reductions were observed following the
4-week treatment with AF, FO, and AF + FO (11, 27, and 29%,
respectively), which was lower than the HFL group (Fig. 1B).
The serum TNF-α concentrations of the HFL group were
increased to 24-fold of the HFC group. Nevertheless, there
were not differences between the HFL and each of the three
A moderate dose of LPS is known to trigger signal transduction
associated with inflammation, mimicking the natural response of the human body under inflammatory conditions.
High-fat and cholesterol diet–induced hyperlipidemia, in
combination with LPS-induced acute inflammation, produces
characteristics that are shared by metabolic diseases, such as
CVD and insulin resistance [2]. There is a significantly
increased public interest in seeking products derived from
natural resources [23]. The results of the current study
demonstrated that dietary supplementation of flavonols and
539
N U TR IT ION RE S E ARCH 3 4 ( 2 0 14 ) 53 5 –5 4 3
B
A
14
Adiponectin
800
NS
C-reactive protein
ab
a
b
c
600
10
CRP (μg/mL)
Adiponectin (μg/mL)
12
8
6
4
d
400
200
2
0
0
C
HF
L
HF
F
+A
L
HF
O
FO
+F
F+
+A
L
HF
C
HF
L
HF
L
HF
O
AF +FO
+F
L
AF
HF
L+
HF
L+
HF
C
TNF- α
10000
D
7000
a
IFN-γ
a
6000
IF N-γ (pg/mL)
TNF-α (pg/mL)
ab
ab
6000
b
4000
2000
5000
3000
2000
0
0
C
bc
4000
1000
c
HF
ab
ab
8000
L
HF
F
+A
L
HF
O
+F
L
HF
C
HF
O
+F
F
+A
d
L
HF
L
HF
AF
L+
HF
FO
FO
F+
+A
L
HF
L+
HF
F
E
a
a
40000
I L- 6 (pg/mL)
IL-10
8000
IL-6
b
bc
c
30000
c
20000
ab
6000
I L-10 (pg/mL)
50000
ab
b
4000
2000
10000
c
0
0
C
HF
L
HF
O
AF +FO
+F
L
AF
HF
L+
HF
L+
HF
C
HF
L
HF
O
AF +FO
+F
L
AF
HF
L+
HF
L+
HF
Fig. 1 – Serum adiponectin (A), CRP (B), TNF-α (C), IFN-γ (D), IL-6 (E), and IL-10 (F) in 5 groups of high-fat diet–fed rats. Except
control 1 (HFC), LPS (5 mg/kg) was injected in control 2 (HFL) along with 4 treatment groups, 5 hours before euthanization.
Results are expressed as mean (SEM) (n = 12). Data were analyzed by ANOVA, and statistical significance by Duncan test using
the general linear model. The values with different letters are significantly different, *P < .05. NS, not significant.
n-3 PUFA significantly improved blood lipid profiles and
inflammatory biomarkers in rats of hyperlipidemia and
acute inflammation. The results are in agreement with
previous studies where quercetin-rich diets alleviated hyperlipidemia in obese Wistar rats after 4 weeks of treatment at a
dose range of 19 to 35 mg/kg per day [27,28], and where omega3 PUFA has demonstrated lower blood lipids at a daily intake
of 1-4 g per day in humans [29].
Numerous studies in the past decade demonstrated that
high blood TAG, low HDL-C levels, and high LDL-C levels are
associated with increased blood levels of inflammatory
cytokines, such as CRP, TNF-α, and IL-6, and that they form
a cascade for endothelial dysfunction [30,31]. In hypercholesterolemic rats, intraperitoneal injection of LPS significantly
increased circulating levels of proinflammatory cytokines,
TNF-α, IFN-γ, and IL-6. This observation was in accordance
with previous reports [32,33]. The insignificant change of
serum TAG after the LPS injection was also in agreement with
a previous study showing that serum lipid profiles were not
altered after 18 hours of LPS injection in rats [33]. It may be
suggested that chronic, rather than acute inflammation,
increases blood TAG and cholesterol levels [32].
540
N U TR IT ION RE S EA RCH 3 4 ( 2 0 14 ) 53 5 –5 43
Serum Triacylglycerols
A
200
180
140
Serum Total Cholesterol
B
a
NS
120
a
100
140
120
b
b
TC (mg/dL)
TAG (mg/dL)
160
b
100
80
60
80
60
40
40
20
20
0
0
C
HF
C
F
A
L+
HF
O
FO
+F
L+
AF
HF
L+
F
H
HF
C
Seum HDL-C
ab
b
40
c
20
80
L
HF
O
AF
FO
+F
L+
AF
HF
L+
F
H
L+
HF
D
a
a
60
c
100
non-HDL-C (mg/dL)
HDL-C (mg/dL)
80
L
HF
Serum Non-HDL-C
ab
bc
60
c
c
40
20
0
0
C
HF
L
HF
O
AF
FO
+F
L+
AF
HF
L+
F
H
H
+
FL
C
HF
L
HF
O
AF
FO
+F
L+
AF
HF
L+
HF
L+
HF
Fig. 2 – Serum TAG (A), serum TC (B), HDL-C, (C) and non–HDL-C (D) levels in 5 groups of high-fat diet–fed rats. Except control 1
(HFC), LPS (5 mg/kg) was injected in control 2 (HFL) and the 3 treatment groups, 5 hours before euthanization.Results are
expressed as mean (SEM) (n = 12). Data were analyzed by ANOVA, and statistical significance by Duncan test using the general
linear model. The values with different letters are significantly different, *P < .05. NS, not significant.
The proinflammatory cytokines measured in the present
study are considered to initiate a variety of pathophysiological
processes and cause acute inflammation [31]. A body of evidence
demonstrates that consumption of n-3 PUFA reduces blood TAG
while having an inconsistent effect on cholesterol [34,35]. It is
reported that n-3 PUFA lowers TAG levels by increasing
mitochondrial fatty acid oxidation, which may increase the
leakage of reactive oxygen species (ROS) from the electron
transport chain [36]. The ROS causes oxidative stress, which is
associated with chronic inflammation [31]. Antioxidants, such as
flavonols, could counteract the negative effects of fatty acid
oxidation by scavenging ROS [21]. Although the beneficial effects
of n-3 PUFA and polyphenols on hyperlipidemia and inflammation are reported, little is known if a better effect could be
achieved when they are provided in combination. Among the
proinflammatory cytokines tested in this study, TNF-α and IL-6
play important roles in inducing an acute phase reaction.
Elevation of the circulating IL-6 and TNF-α up-regulates the
expression of CRP in the liver [31], resulting in an increase of CRP
concentration in the blood stream. In return, CRP amplifies the
production of proinflammatory cytokines and induces endothelial dysfunction [37]. Thus, the serum concentration of CRP is a
crucial biomarker of systemic and chronic inflammation and a
risk factor for CVD. Because there was no significant difference in
serum CRP between the HFC and HFL groups, this may suggest
that the 4-week feeding of the high-fat high-cholesterol diet
might have induced obesity-related subclinical inflammation in
rats; and thus, there was no further increase after the LPS
injection. It is possible that a longer period is required to elevate
CRP gene and protein expression after LPS injection through other
cytokines such as IL-6 and TNF-α. This further supports the
previous observation that obesity itself is an inflammatory
condition [38]. It has been reported that flavonoid intake is
inversely associated with serum CRP concentrations in human
adults [39]. In line with this, in this study, dietary supplementation of AF significantly reduced the serum concentration of CRP.
Moreover, a synergistic effect among multipolyphenols may be
involved in lowering CRP production [40]. Fish oil showed a
similar effect on CRP production, and interestingly, when they
were combined, an additive effect was observed. Both the AF and
FO lowered the serum levels of IL-6, but no additive effect was
seen when supplemented together. Flavonols and n-3 PUFA
possibly share a common molecular mechanism to downregulate the production of CRP and IL-6 by inhibiting the
expression of nuclear factor-κB (NF-κB) [41–43]. It is well known
that NF-κB is the primary transcriptional regulator of proinflammatory mediator expression, including IL-6, TNF-α, cyclooxygenase-2, phospholipase, and inducible nitric oxide synthase [44,45].
Numerous in vitro and in vivo studies have shown the antiinflammatory [32,46] and antioxidant effects [21,47] of apple
541
N U TR IT ION RE S E ARCH 3 4 ( 2 0 14 ) 53 5 –5 4 3
A
2.5 Liver Triacylglycerols
2.5
B
NS
Liver Total Cholesterol
a
1.5
1.0
0.5
C
HF
L
HF
b
1.5
1.0
0.0
O
AF
FO
+F
L+
AF
HF
L+
F
H
C
HF
L+
HF
C
Liver Free Cholesterol
1.6
a
D
L
HF
O
AF
FO
+F
L+
AF
HF
L+
F
H
L+
HF
Liver Cholestryl Ester
NS
1.4
a
0.8
1.2
b
b
b
0.6
0.4
CE (mg/g)
FC (mg/g)
ab
b
0.5
0.0
1.0
a
2.0
TC (mg/g)
TAG (mg/g)
2.0
1.0
0.8
0.6
0.4
0.2
0.2
0.0
0.0
C
HF
L
HF
F
+A
L
HF
O
+F
L
HF
O
+F
F
+A
FL
H
C
HF
L
HF
AF
FO
FO
L+
F+
+A
HF
L
HF
L+
HF
Fig. 3 – Liver TC (A), TAG (B), FC, (C) and CE (D) levels in 5 groups of high-fat diet–fed rats. Except control 1 (HFC), LPS (5 mg/kg) was
injected in control 2 (HFL) and the 3 treatment groups, 5 hours before euthanization.Results are expressed as mean (SEM) (n = 12). Data
were analyzed by ANOVA, and statistical significance by Duncan test using the general linear model. The values with different letters
are significantly different, *P < .05. NS, not significant.
peel extract. Quercetin glycosides are a major group of
flavonols in apple peel and are reported to suppress NF-κB
expression and function [48]. Apple peel also contains a high
concentration of phloridzin, which has been shown to have
similar biological properties as flavonols [49]. An in vitro study
showed the anti-inflammatory effects of an apple extract
fraction containing phloridzin, quercetin glycosides, and
epicatechin [32]. A decrease in the circulating concentrations
of adiponectin and a high TNF-α level are considered to be
predictive markers of CVD and diabetes and are strongly
correlated with visceral fat accumulation [5,50]. The AF, FO,
and AF + FO did not show any significant effect on adiponectin
and TNF-α concentrations, contrary to previous reports
[42,51]. The significant decreases in other proinflammatory
cytokines in the present study might suggest that TNF-α
expression is regulated by a pathway that is different from the
NF-κB–dependent signaling [33,52]. Fish oil significantly
lowered the serum IFN-γ concentrations. The AF and AF +
FO showed a trend to reduce IFN-γ but did not reach the
significance level. The stimulation of IL-10 expression and
production by LPS and quercetin was reported earlier [53,54].
Lipopolysaccharide induces both Th1- and Th2-mediated
inflammations [55]. The serum concentration of IL-10 was
increased after LPS injection, and it was further elevated by
the supplementation of AF and marginally by the AF + FO.
Interleukin-10 exerts an array of host defence responses by
inducing the expression of a wide range of anti-inflammation
genes [56].
Fish oil and AF lowered the serum TAG and elevated HDL-C
levels. Feeding n-3 PUFA lowers TAG by reducing hepatic
synthesis of TAG-rich very low-density lipoprotein and
removing TAG from the circulating lipoproteins because of a
higher activity of hormone-sensitive lipase [20,43]. The
increase of serum HDL-C concentrations by FO is in line with
a previous report [57]. Non–HDL-C predicts the risk of CVD as
equally effective as LDL-C or even better than LDL-C [58]. The
guidelines and recommendations for the treatment of hyperlipidemia consider non–HDL-C as the secondary target if TAG
levels remain elevated after treating LDL-C levels [59]. Clinical
studies have reported that n-3 PUFA, combined with statins,
reduce the risk of coronary events without any significant
changes in LDL-C and HDL-C, suggesting other mechanisms
than LDL-C might be involved [59,60]. The hepatic cholesterollowering effect of apple flavonoids might be attributed to the
modulation of hepatic cholesterol 7α-hydroxylase, thus
leading to the conversion of cholesterol into bile acids and
the increase of fecal excretion of bile acids and neutral
steroids [14,61]. By suppressing the sterol regulatory element-binding protein-1 mRNA expression, n-3 PUFA could
encounter the hepatic lipid synthesis [62].
542
N U TR IT ION RE S EA RCH 3 4 ( 2 0 14 ) 53 5 –5 43
The results of this study partially support the hypothesis that
AF and FO could have complementary effects on the production
of CRP, which can further influence the development of
inflammation and hyperlipidemia. However, these results did
not show that acute inflammation influences lipid profile under
hyperlipidemia. One of the limitations of the present study in rats
is the period of treatment after the induction of acute inflammation by LPS injection. The anti-inflammatory effects of AF and FO
observed are more relevant to the prevention of acute inflammation rather than the treatment of chronic inflammation. If a
longer time had been given after the induction of acute
inflammation, an increase of serum CRP and significant treatment effects might have been achieved in rats. Another limitation
is the lack of normal fat control and treatments of such rats to
determine whether the observed beneficial effects are applicable
to lower fat levels in rats. We were also limited since the potential
molecular mechanism underlying the anti-inflammatory ability
of AF and FO remains unclear. Finally, future research into lipid
metabolism and related enzymes would be helpful to understand
the hypolipidemic effects of AF and FO.
In conclusion, LPS injection markedly increased the serum
concentration of several proinflammatory biomarkers, such as IL6, IFN-γ, and TNF-α, as well as anti-inflammatory biomarker IL10, whereas no effect was noted on CRP in rats fed a high-fat diet.
The supplementation of AF, FO, or AF + FO decreased or reversed
the serum concentration of these proinflammatory cytokines,
and by contrast, increased the anti-inflammatory cytokine. The
AF, FO, and/or AF + FO group of rats had lower serum TAG and
non-HDL-C, while increasing HDL-C levels and lower liver TC
and FC contents. When AF and FO were provided in combination
to rats, they showed an improved effect on serum CRP. The
combination therapies of two or more drugs, especially those
that involve different mechanisms, have been increasingly used
in treating metabolic diseases. Apple flavonols and n-3 PUFA–rich
FO should be further investigated for their complementary effects
on proinflammatory cytokines and lipids, particularly in a
metabolic syndrome model with chronic inflammation.
Acknowledgment
The authors acknowledge the funding provided by the Discovery
Grant program of the Natural Sciences and Engineering Research
Council of Canada and the Atlantic Innovation Funds Program of
the Atlantic Canada Opportunities Agency.
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