European Journal of Pharmacology xxx (xxxx) xxx–xxx

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Full length article

Anti-atherosclerotic effect of hesperidin in LDLr−/− mice and its possible

mechanism
⁎
Ye-Zi Suna, , Jian-Fei Chenb, Li-Min Shena, Ji Zhoua, Cui-Fang Wangc
a
Department of Endocrinology, Zhang Jia Gang First People's Hospital, Zhang Jia Gang, People's Republic of China
b
Department of Traditional Chinese Medicine, Zhang Jia Gang First People's Hospital, Zhang Jia Gang, People's Republic of China
c
Department of Endocrinology, Affiliated Hospital of Nantong University, Nantong, People's Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: Hesperidin, a citrus bioflavonoid, exerts numerous pharmacological activities. However, its protective effect
Hesperidin against atherosclerosis in vivo remains poorly understood. In the present study, we aimed to observe the effects
Atherosclerosis of hesperidin on high fat diet (HFD)-induced atherosclerosis using LDL receptor deficient (LDLr-/-) mice. After 12
Hepatic steatosis weeks of treatment, the animals were sacrificed. The blood samples were collected for further analysis. Mouse
peritoneal macrophages were collected. Hepatic lipid content, quantification of atherosclerosis, assessment of
oxidative stress and inflammation, gene expressions were performed on liver and aorta samples. The data
showed that hesperidin ameliorated HFD-induced weight gain, improved insulin resistance and ameliorated
hyperlipidemia. Hesperidin suppressed HFD-induced hepatic steatosis, atherosclerotic plaque area and macro-
phage foam cell formation. Further study showed that hesperidin down-regulated expressions of acetyl coen-
zyme A carboxylase alpha (ACCα) and fatty acid synthase (FAS) which are two key enzymes in fatty acid and
triglyceride synthesis in liver; and upregulated expression of hepatic ATP-binding cassette transporters G8
(ABCG8), macrophage ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) which are transporters
involved in the process of reverse cholesterol transport. Hesperidin also reduced oxidative stress by normalizing
activities of antioxidant enzymes and inflammation in HFD-fed LDLr−/− mice. These findings suggest that he-
speridin reduced atherosclerosis via its pleiotropic effects, including improvement of insulin resistance, ameli-
oration of lipid profiles, inhibition of macrophage foam cell formation, anti-oxidative effect and anti-in-
flammatory action.

1. Introduction redox status and increased levels of circulating proinflammatory factors

Atherosclerosis is a chronic vascular disease process that is char-
acterized by focal narrowing and thickening of the lumen of blood
vessels due to the formation of lipid-laden plaques in the luminal sur-
face of arterial wall (Weber et al., 2017). Dyslipidemia, oxidative stress
and persistent inflammation are well-known risk factors in the devel-
opment of atherosclerosis (Hurtubise et al., 2016). In addition, ather-
osclerosis is usually associated with other metabolic disorders such as
insulin resistance and fatty liver (Xu et al., 2015; Zeadin et al., 2013).
Insulin has been reported to induce forceful effects on lipid accumula-
tion as well as expressions of genes involved in lipogenesis (Zeadin
et al., 2013). The liver plays a critical metabolic role in regulating lipid
homeostasis through many processes including uptake of cholesterol
from the peripheral tissues, synthesis of cholesterol and triglycerides,
and excretion of cholesterol into the bile. Hepatic dysfunction can ac-
celerate atherosclerosis via deterioration of dyslipidemia, imbalanced
(Xu et al., 2015; Targher et al., 2010). Thus, manipulation of hepatic
lipid metabolism, oxidative stress and inflammation is a commonly
pursued strategy to inhibit the progression of atherosclerosis.
Reverse cholesterol transport (RCT) is a cholesterol metabolism
pathway ensuring the effux of cholesterol from lipid-laden macro-
phages in the artery wall and its transport back to the liver for eventual
excretion (Favari et al., 2015). RCT is believed to be an important de-
fense mechanism in atherogenesis. Cholesterol eflux from the macro-
phage is the first step in RCT, which is mediated by various membrane
transporters, such as ATP-binding cassette transporters G1 and A1
(ABCG1 and ABCA1) on the membrane of the macrophage. ABCG1
mediates the effux of cholesterol and phospholipids to HDL particles,
while ABCA1 induces cholesterol effux to lipid-poor apolipoprotein A-I
(apoA-I). After uptaking of HDL/apoA-I-accociated cholesterol or cho-
lesterol ester into hepatocytes by hepatic scavenger receptor class B
type I (SR-BI), secretion of cholesterol and bile acids into the bile is

⁎
Corresponding author.
E-mail address: sunyezi1976@163.com (Y.-Z. Sun).

http://dx.doi.org/10.1016/j.ejphar.2017.09.010
Received 20 June 2017; Received in revised form 30 August 2017; Accepted 8 September 2017
0014-2999/ © 2017 Published by Elsevier B.V.

Please cite this article as: Sun, Y.-Z., European Journal of Pharmacology (2017), http://dx.doi.org/10.1016/j.ejphar.2017.09.010
Y.-Z. Sun et al.
regulated by the respective hepatic transporters ABCG5 and ABCG8
(Zhang et al., 2016).
Hesperidin, a specific flavonoid glycoside, can be isolated in large
amounts from the rinds of some citrus species. Preclinical studies and
clinical trials have demonstrated hesperidin has anti-inflammatory,
anti-oxidant, lipid-lowering and insulin-sensitizing properties, con-
tributing to its therapeutical effects in various diseases (Li and
Schluesener, 2017; Mulvihill et al., 2016). But, until now, no study has
reported the direct effect of hesperidin on atherosclerosis using LDL
receptor deficient (LDLr-/-) mice, which is a well established animal
model with many characteristics of the metabolic syndrome. Thus, the
aim of the present study was to determine the potential effects of he-
speridin on atherosclerosis and its associated metabolic disorders in
LDLr-/- mice, and also explore the possible mechanisms involved.
2. Materials and methods
2.1. Animal experiment
Male LDLr−/− mice on C57BL/6JNju were purchased from Nanjing
Biomedical Research Institute of Nanjing University. All procedures
were approved by the animal care and use committee of Nantong
University. The mice were housed under standard laboratory conditions
with a 12-h light/dark cycle and free access to water and food. Starting
from 8 weeks, the mice were randomly separated into four groups: a
control group, a high fat diet (HFD) model group, 100 mg/kg/day he-
speridin-treated group and 200 mg/kg/day hesperidin-treated group.
The dose of hesperidin used in this study was based on previous reports
of its beneficial pharmacological effects in animals with different dis-
ease (Jung et al., 2004; Mahmoud et al., 2012; Çetin et al., 2016).
Hesperidin (purity > 98%) was provided by XI’AN natural field bio-
technioue Co., LTD, China. The control group was fed a standard chow
diet. The HFD group was given a high fat diet containing 21% fat and
0.21% cholesterol (D12079B, Open Source Diets, Research Diets, Inc).
The two hesperidin treatment groups were given the same high fat diet
and dosed daily via intragastric gavage with 100 and 200 mg/kg/day
hesperidin by weight for 12 weeks. Hesperidin was suspended in 0.5%
carboxymethyl cellulose (CMC). Mice in control and HFD model group
received the same volume of 0.5% CMC vehicle gastrically. At the end
of the study, animals were fasted overnight and were sacrificed. The
blood samples and tissues were collected for further analysis.
2.2. Biochemical measurements
Fasting insulin was determined using a commercial mouse insulin
elisa kit (Millipore, MA). Fasting glucose were measured with a mul-
tifunctional biochemistry analyzer Olympus AU2700 (Olympus, Tokyo,
Japan). Insulin resistance was evaluated by homeostasis model assess-
ment (HOMA) index as described previously for mice (Mulvihill et al.,
2009). Serum high density lipoprotein cholesterol (HDL-c), low density
lipoprotein cholesterol (LDL-c), triglyceride (TG) and total cholesterol
(TC) were detected using the biochemical kits (Zhongsheng Bio-tech
Co., Ltd, Beijing, China). Activities of superoxide dismutase (SOD) and
glutathione peroxidase (GSH-Px) were measured with enzymatic col-
orimetric assays using commercial kits (Nanjing Jiancheng Bioengi-
neering Institute, Nanjing, China). Serum levels of ox-LDL TNF-α and
IL-6 were measured using elisa kits (Shanghai Westang Biotechnology
Co. Ltd, Shanghai, China).
2.3. Determination of hepatic lipid content
For Oil Red O staining, frozen liver tissues were embedded in
Tissue-Tek OCT compound, sectioned at 8 µm, and stained with Oil Red
O (Sigma, St. Louis, USA) to detect lipid deposition. For hepatic lipid
content measurement, total lipids were extracted from frozen hepatic
tissue (approximately 50 mg) according to the modified Folch method
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(Folch et al., 1957). Triglyceride and total cholesterol concentrations
were determined using the commercial enzyme kits (Zhongsheng Bio-
tech Co., Ltd, Beijing, China) and were normalized to liver protein
content.
2.4. Quantification of atherosclerosis
Atherosclerotic lesion severity was assessed by en face analysis of
the aorta as previously described (Liu et al., 2017). In brief, the as-
cending aorta and thoracic aorta were opened longitudinally, and fixed
with 4% paraformaldehyde and then stained with Oil Red O (Sigma, St.
Louis, MO). Lesion and total areas were determined using Image-Pro
Plus software 6.0.
2.5. Isolation of mouse peritoneal macrophages and studies of macrophage
foam cell formation
In a separate study, after 12 weeks, male LDLr−/− mice fed with
standard chow, HFD or HFD with hesperidin as described above were
injected intraperitoneally with 1 ml of 10% thioglycolate. Three days
later, Isolation of mouse peritoneal macrophages was performed as
described previously (Mo et al., 2014). The cells were resuspended in
RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin and
100 mg/ml streptomycin for use in subsequent experiments. Upon
fixation with paraformaldehyde (4%), the peritoneal macrophages from
mice were stained with 0.5% oil red O and hematoxylin was used as
counterstaining. After alcohol extraction, density of the total cellular
neutral lipid content was determined by a microplate reader (absor-
bance at 540 nm). The intracellular cholesterol was quantified using the
Amplex Red cholesterol assay kit (Invitrogen, CA, USA) and were nor-
malized to cell total protein which was determined by the BCA protein
assay (Beyotime Biotech, China).
2.6. Cholesterol efflux assay
Peritoneal macrophages from untreated LDLr−/− mice at 8 weeks
old were equilibrated with 22-NBD-cholesterol (Life Technologies,
Carlsbad, CA, USA) (1 μg/ml) for 24 h. Then cells were washed with
PBS and incubated with 0.1, 1 and 10 µM of hesperidin for an addi-
tional 18 h. 50 μg/ml HDL (Luwen Biotechnologies, China) or ApoA1
(Sigma, USA) was added as the receptor protein to start the efflux ex-
periment for 6 h. The fluorescence-labeled cholesterol released from
cells into the medium was measured using a microplate reader (synergy
H1, BioTek, USA). Cholesterol efflux was expressed as a percentage of
fluorescence in the medium relative to the total amounts of fluores-
cence detected in cells and the medium.
2.7. RNA analysis
mRNA expressions of targeted genes were quantified by real-time
PCR with forward and reverse primers (Supplementary Table 1). Total
RNA was extracted using Trizol reagent and was reverse-transcribed
into cDNA using the PrimeScript RT Master Mix Kit (Takara, TaKaRa
Biotechnology, Dalian, China). The real-time PCR were carried out on
StepOne Plus Real-Time PCR System (Applied Biosystems) using SYBR
Green PCR master mix (Applied Biosystems) with the resulting cDNAs.
Housekeeping gene 18 s was used as a reference. Data were analyzed
using the cycle threshold (Ct) method.
2.8. Western blot analysis
Equal amounts of protein were separated and electrophoretically
transferred to nitrocellulose membranes (Bio-Rad, USA). After blocking,
the membranes were incubated with the first antibodies (all obtained
from Abcam, USA) overnight. And then the membranes were incubated
with the appropriate IRDye 680RD secondary antibodies (1:10000) in
Y.-Z. Sun et al.
Fig. 1. Effects of hesperidin on HFD-induced metabolic syndrome in LDLr−/− mice. (A) Body weight of LDLr−/− mice fed a chow, HFD or HFD with hesperidin diet for 0 and 12 weeks.
(B) Serum glucose level. (C) Serum insulin level. (D) HOMA index. (E) Serum TC concentrations. (F) Serum TG concentrations. (G) Serum LDL-c concentrations. (H) Serum HDL-c
concentrations. n = 12 per group. Results are presented as the mean ± S.D. #P < 0.05 vs. control group; *P < 0.05 vs. HFD alone group; ΔP < 0.05 vs. HFD+H100 group. H100,
hesperidin 100 mg/kg/day; H200, hesperidin 200 mg/kg/day.
the dark. The blot was imaged and quantitated using the Odyssey in-
frared imaging system (LI-COR Biosciences, Inc.).
2.9. Statistical analysis
Data were represented as mean ± S.D. Statistical differences were
assessed by one-way ANOVA analysis of Tukey's multiple comparison
test using Graphpad Prism 5 software, and P < 0.05 was considered
statistically significant.
3. Results
3.1. Hesperidin ameliorated HFD-induced metabolic syndrome in LDLr−/−
mice
Compared with standard chow fed mice, LDLr-/- mice fed a HFD for
12weeks had significantly higher body weight, obvious insulin re-
sistance and hyperlipidemia (Fig. 1). Administration with hesperidin at
200 mg/kg/day dosage markedly decreased body weight, fasting glu-
cose level, fasting insulin level and HOMA index (Fig. 1A-D), suggesting
that insulin resistance caused by HFD had been alleviated. Notably,
hesperidin treatment (100 and 200 mg/kg/day) markedly decreased
the serum levels of TC, TG and LDL-c and increased HDL-c level com-
pared with HFD alone group (Fig. 1E-H).
3.2. Hesperidin attenuated HFD-induced hepatic steatosis in LDLr−/− mice
Analyse of fat content on liver sections stained with Oil-red-O
showed an accumulation of many micro and macro-lipid droplets in the
livers of HFD-fed LDLr-/- mice (Fig. 2A). Consistently, hepatic TC and
TG levels were significantly increased in HFD-fed LDLr-/- mice (Fig. 2B
and C). However, livers from HFD-fed LDLr-/- mice treated with he-
speridin displayed a dramatic reduction in the amount of lipid droplets
and hepatic TC and TG levels compared with HFD-fed alone group
(Fig. 2A-C). To further elucidate the underlying mechanisms, the mRNA
expression levels of several key genes related to lipid synthesis and
metabolism were determined. The mRNA levels of acetyl coenzyme A
carboxylase alpha (ACCα) and fatty acid synthase (FAS), two key en-
zymes in fatty acid synthesis were significantly higher in the livers from
HFD-fed LDLr-/- mice. But these increases were significantly reversed by
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hesperidin administration (Fig. 2D). The mRNA abundance of genes
promoting fatty acid oxidation (peroxisome proliferator-activated re-
ceptor-alpha, PPARα; camitine palmitoyltransferase 1 alpha, CPT-1α)
and cholesterol biosynthesis (3-hydroxy-3-methylglutaryl-coenzyme A
reductase, HMGCR; 3-hydroxy-3-methylglutaryl-coenzyme A synthase,
HMGCS) were not significantly altered by hesperidin supplementation
compared with HFD-fed alone group. Among the three hepatic genes
ABCG5, ABCG8 and SR-BI which are involved in RCT, hesperidin sig-
nificantly increased ABCG8 mRNA expression but has little effect on
ABCG5 and SR-BI mRNA expression compared with HFD-fed alone
group (Fig. 2D). The alteration of mRNA expressions in ACCα, FAS and
ABCG8 by hesperidin administration was confirmed at protein levels
(Fig. 2E-G).
3.3. Hesperidin inhibited atherosclerotic lesion formation in HFD-fed
LDLr−/− mice
The potential effects of hesperidin in HFD-induced atherosclerosis in
LDLr−/− mice were then investigated. The percentage of plaque area
was significantly higher in HFD-fed LDLr−/− mice, and hesperidin
treatment significantly attenuated HFD-induced atherosclerotic lesion
formation (Fig. 3A and B). Furthermore, the expressions of ABCA1 and
ABCG1 which are responsible for RCT in aorta were measured. The
results showed that compared to the HFD alone group, the expressions
of ABCA1 and ABCG1 both at mRNA and protein levels were markedly
up-regulated in hesperidin administration group (Fig. 3C-F).
3.4. Hesperidin inhibited macrophage foam cell formation in HFD-fed
LDLr−/− mice
Peritoneal macrophages were collected from mice and analyzed for
foam-cell formation. The results of Oil-red-O staining and intracellular
cholesterol content showed that lipid droplet staining and cholesterol
content were significantly increased in peritoneal macrophages from
HFD-fed LDLr-/- mice, which indicated that foam cells were formed
(Fig. 4A-C). But administrated with hesperidin decreased the accumu-
lation of cholesterol and foam cell formation in peritoneal macrophages
compared with HFD-fed alone group. In addition, the expressions of
ABCA1 and ABCG1 both at mRNA and protein levels in peritoneal
macrophages were significantly up-regulated in hesperidin
Y.-Z. Sun et al. European Journal of Pharmacology xxx (xxxx) xxx–xxx

Fig. 2. Effects of hesperidin on HFD-induced hepatic steatosis in LDLr−/− mice. (A) Representative images of liver sections stained with Oil Red O. (B) Liver TC content (n = 12). (C)
Liver TG content (n = 12). (D) mRNA expression of ACCα, FAS, PPARα, CPT-1α, HMGCR, HMGCS, ABCG5 and ABCG8 in liver (n = 6). (E-G) Protein expression of ACCα (E), FAS (F) and
ABCG8 (G) in liver (n = 6). Results are presented as the mean ± S.D. #P < 0.05 vs. control group; *P < 0.05 vs. HFD alone group; ΔP < 0.05 vs. HFD+H100 group. H100, hesperidin
100 mg/kg/day; H200, hesperidin 200 mg/kg/day.

administration group compared with HFD-fed alone group (Fig. 4D-G).
We subsequently evaluated the effect of hesperidin on cholesterol efflux
from peritoneal macrophages in vitro. As shown in Fig. 4H and I,
treatment peritoneal macrophages from untreated LDLr−/− mice at 8
weeks old with hesperidin (0.1–10 μM) increased NBD-cholesterol ef-
flux to both HDL and ApoA1 in a concentration-dependent manner.
3.5. Hesperidin reduced oxidative stress in HFD-fed LDLr−/− mice
LDLr−/− mice fed a HFD developed high ox-LDL serum level and
low serum levels of SOD and GSH-Px, and these effects were sig-
nificantly reversed by treatment with hesperidin (Fig. 5A-C). In addi-
tion, hesperidin at high dosage evidently increased SOD and GSH-Px

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Fig. 3. Effect of hesperidin on atherosclerotic lesion formation in HFD-fed LDLr−/− mice. (A) Representative images of Oil Red O staining of en face preparations of aortas and (B)
quantification of the atherosclerotic surface area of the entire aorta (n = 6). (C and D) mRNA expression of ABCA1 and ABCG1 in aorta (n = 6). (E and F) Protein expression of ABCA1
and ABCG1 in aorta (n = 6). Results are presented as the mean ± S.D. #P < 0.05 vs. control group; *P < 0.05 vs. HFD alone group. H100, hesperidin 100 mg/kg/day; H200, hesperidin
200 mg/kg/day.

activities in livers compared with HFD-fed alone group (Fig. 5D and E).
3.6. Hesperidin reduced inflammation in HFD-fed LDLr−/− mice
LDLr-/- mice fed a HFD developed high serum level of pro-in-
flammatory cytokine TNF-α and IL-6 compared with standard chow fed
mice (Fig. 6A and D). Hepatic and aortic mRNA expressions of TNF-α
and IL-6 were consistent with this finding (Fig. 6B, C, E and F). But
treatment with hesperidin significantly reduced this increase of TNF-α
in serum level, hepatic and aortic mRNA expression (Fig. 6A-C).
Hesperidin at high dosage significantly reduced the IL-6 serum level
and hepatic IL-6 mRNA expression compared with HFD-fed alone group
(Fig. 6D-E). In addition, hesperidin had trends in decreasing aortic IL-6
mRNA expression compared with HFD-fed alone group, yet there was
no statistical significant difference (Fig. 6F).
4. Discussion
In the present study, LDLr-/- mice were used as an experimental
model of atherosclerosis. LDLr−/− mice fed a diet rich in saturated fats,

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Fig. 4. Effect of hesperidin on macrophage foam cell formation in HFD-fed LDLr−/− mice. (A-G) are results from LDLr−/− mice fed with standard chow diet, HFD or HFD with two
dosages of hesperidin. (A) Representative images of Oil Red O staining of mice peritoneal macrophages and (B) the density of the lipid content by measuring the absorbance at 540 nm (n
= 6). (C) Cholesterol content in mice peritoneal macrophages (n=6). (D and E) mRNA expression of ABCA1 and ABCG1 in peritoneal macrophages (n = 6). (F and G) Protein expression
of ABCA1 and ABCG1 in peritoneal macrophages (n = 6). Results are presented as the mean ± S.D. #P < 0.05 vs. control group; *P < 0.05 vs. HFD alone group. H100, hesperidin
100 mg/kg/day; H200, hesperidin 200 mg/kg/day. (H and I) Cholesterol efflux assay in vitro were performed on peritoneal macrophages from untreated LDLr−/− mice at 8 weeks old
with the indicated concentrations of hesperidin. (H) Cholesterol efflux to ApoA1 (n = 6). (I) Cholesterol efflux to HDL (n = 6). Results are presented as the mean ± S.D. aP < 0.05 vs.
untreated cell group; bP < 0.05 vs. 0.1 µM hesperidin treated cell group; cP < 0.05 vs. 1 µM hesperidin treated cell group.

showing a human ‘‘like’’ lipoprotein profile, develop hepatic steatosis
and advanced atherosclerotic lesions combined with high-fat diet-in-
duced obesity and insulin resistance (Emini et al., 2017; Liang et al.,
2014). This is the first study show that administration of hesperidin can
attenuate hepatic steatosis and atherosclerosis in HFD-fed LDLr-/- mice.
Contributions of hyperglycemia and insulin resistance in the de-
velopment of atherosclerosis have been identified recently. Insulin re-
sistance is associated with various risk factors for atherosclerosis, in-
cluding lipid accumulation, inflammation and obesity, which
subsequently promote the development of atherosclerosis (Stöhr and
Federici, 2013; Yoon et al., 2017). In the present study, administration
of hesperidin to HFD-fed LDLr-/- mice prevented the weight gain, in-
creases of glucose and insulin serum levels, and HOMA index, sug-
gesting that hesperidin may ameliorate insulin resistance in HFD-fed
LDLr-/- mice. Dyslipidemia is a significant risk factor leading to ather-
osclerosis. Administration of hesperidin has been shown to improve
lipid profile in streptozotocin-induced marginal type 1 diabetic rats
(Akiyama et al., 2010) and high-cholesterol-diet fed rats (Wang et al.,
2011). Consistent with these previous findings, this study demonstrated
that administration of hesperidin increased level of HDL-c and de-
creased the serum levels of TG, TC and LDL-c in HFD-fed LDLr-/- mice,
suggesting that hesperidin might benefit atherosclerosis by reducing
lipid levels. Liver is the organ directly responsible for lipid and lipo-
protein metabolism, not only through lipid synthesis and apolipopro-
tein production, but also through the final elimination of excess cho-
lesterol from the body. Disturbance of lipid homeostasis in liver by
overloading this metabolic organ with high levels of fat and cholesterol,
can lead to the pathology of fatty liver disease and peripheral lipid

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Fig. 5. Effect of hesperidin on oxidative stress in HFD-fed LDLr−/− mice. (A) serum level of ox-LDL (n = 11–12). (B) Serum level of SOD (n = 11–12). (C) Serum level of GSH-Px (n =
11–12). (D) SOD activity in liver (n = 6). (E) GSH-Px activity in liver (n = 6). Results are presented as the mean ± S.D. #P < 0.05 vs. control group; *P < 0.05 vs. HFD alone group;
Δ
P < 0.05 vs. HFD+H100 group. H100, hesperidin 100 mg/kg/day; H200, hesperidin 200 mg/kg/day.

accumulative diseases such as atherosclerosis (Xu et al., 2015). It has
been shown that LDLr-/- mice fed a high-fat-high-cholesterol diet ra-
pidly develop hepatic steatosis and suggested that in the setting of LDL
deficiency non-alcoholic fatty liver disease precedes atherosclerosis
(Wouters et al., 2008; Teupser et al., 2003). The present study, for the
first time, showed that administration of hesperidin to LDLr -/- mice not
only prevented aortic fatty streak development but also notably re-
duced lipid accumulation and steatosis in the liver. Consistent with our
study, Wang et al. have reported the protective effects of hesperidin
against hypercholesterolemia and fatty liver in high-cholesterol diet in
rats (Wang et al., 2011). Next, the key molecules involved in the lipid
metabolism pathways such as fatty acid synthesis, fatty acid oxidation,

Fig. 6. Effect of hesperidin on inflammation in HFD-fed LDLr−/− mice. (A) serum level of TNF-α (n = 11–12). mRNA expression levels of TNF-α in liver (B) and aorta (C) (n = 6). (D)
serum level of IL-6 (n = 11–12). mRNA expression levels of IL-6 in liver (E) and aorta (F) (n = 6). Results are presented as the mean ± S.D. #P < 0.05 vs. control group; *P < 0.05 vs.
HFD alone group; ΔP < 0.05 vs. HFD + H100 group. H100, hesperidin 100 mg/kg/day; H200, hesperidin 200 mg/kg/day.

7
Y.-Z. Sun et al.
cholesterol biosynthesis and cholesterol output were assessed to find
out by which hesperidin regulates lipid metabolism in the liver. Our
data showed that administration of hesperidin significantly decreased
expression of ACCα and FAS which are two key enzymes in fatty acid
and triglyceride synthesis in liver (Strable and Ntambi, 2010). Our re-
sults also demonstrated that hesperidin markedly up-regulated the ex-
pression of hepatic ABCG8 which play a critical role in the eflux of
cholesterol into the bile (Yu et al., 2013). Hence, the down-regulation of
ACCα and FAS and upregulation of ABCG8 protein expressions in liver
by hesperidin treatment could partly explain the observed beneficial
effects of hesperidin on lipid profiles, hepatic steatosis and athero-
sclerosis in HFD-fed LDLr-/- mice.
Both ABCG1 and ABCA1 play an important role in facilitating excess
cholesterol removal from macrophages or foam cells to extracellular
cholesterol acceptor such as HDL and apolipoprotein A-I (apoA-I) in the
initial step of RCT (Chistiakov et al., 2016). The development of foam
cells, derived mainly from the accumulation of excess cholesterol in
macrophages, leads to the formation of fatty streak, complex athero-
sclerotic lesion, and finally plaque rupture (Mo et al., 2014). Our data
showed that hesperidin significantly increased ABCA1 and ABCG1 ex-
pressions in the aortas with atherosclerotic lesions, suggesting that
hesperidin prevented lipid accumulation and atherosclerosis of HFD-fed
LDLr-/- mice partly through upregulating the expression of ABCA1 and
ABCG1 in the aorta. Since macrophages and macrophage-derived foam
cells are the main cell types that lead to the accumulation of cholesterol
in the arterial wall with atherosclerotic lesions, it is reasonable to
speculate that hesperidin promotes lesional macrophage cholesterol
efflux. To test this hypothesis, we isolated and cultured peritoneal
macrophages of mice, which is commonly used substitute for lesional
macrophages (Lin et al., 2015), for further experiments in the present
study. Our data showed that hesperidin administration resulted in less
cholesterol content associated with an enhancement of cellular cho-
lesterol efflux capacity. Meanwhile, the expressions of ABCA1 and
ABCG1 in peritoneal macrophages were upregulated in hesperidin-
treated mice. In accordance with our finding, Iio et al. reported that
hesperetin can upregulates ABCA1 expression and promotes cholesterol
efflux from THP-1 Macrophages. Besides macrophage transporters,
several hepatic transporters are also involved in the process of RCT,
including SR-BI responsible for the direct selective uptake of HDL-c by
the liver and ABCG5/ABCG8 mediating the secretion of cholesterol into
the bile. However, in the present study, the expression of hepatic SR-BI
and ABCG5 were not altered in hesperidin-treated mice. These finding
suggest that hesperidin may exert anti-atherosclerotic effects by upre-
gulating the expression of transporters involved in the process of RCT,
including hepatic ABCG8, macrophage ABCA1 and ABCG1.
Oxidative stress and inflammation play crucial roles in the patho-
genesis of atherosclerosis. Oxidative stress is defined as a state of cel-
lular redox imbalance in which pro-oxidants overwhelm antioxidant
capacity, leading to increased ROS production. Excessive ROS cause
multiple pathophysiological actions in the vessel wall, including pro-
moting oxidative modification of LDL to form ox-LDL which aggravates
subendothelial lipid accumulation, accelerates inflammation and pro-
motes atherosclerotic lesion formation (Ahotupa, 2017). Hesperidin, as
a member of flavonoids, has been proved to possess potent anti-oxi-
dative and anti-inflammatory properties (Li and Schluesener, 2017). In
the present study, hesperidin administration increased antioxidant en-
zymes SOD and GSH-Px both in serum and liver of HFD-fed LDLr-/-
mice, which may strengthen the endogenous defense against oxidative
stress in vivo. Consistently, an obvious decrease in serum ox-LDL level
in hesperidin-treated mice was observed. In addition, administration of
hesperidin showed significant reductions in serum levels of IL-6 and
TNF-α as well as mRNA expression of these two pro-inflammatory cy-
tokines in liver and aorta. Taken together, the present study indicated
that administration of hesperidin can alleviate oxidative stress and in-
flammation, which may contribute to its inhibitory effects on athero-
sclerosis.
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European Journal of Pharmacology xxx (xxxx) xxx–xxx
In summary, the present study showed for the first time that he-
speridin can inhibit atherosclerosis via its pleiotropic effects, including
improvement of insulin resistance, amelioration of lipid profiles, in-
hibition of macrophage foam cell formation, anti-oxidative effect and
anti-inflammatory action. Therefore, the present study suggested a
possible therapeutic application of hesperidin in atherosclerosis and
atherosclerosis associated with metabolic dysregulation.
Financial support
The research was supported by Jiangsu Provincial Maternal and
Child Health Research Program (F201512).
Conflict of interest statement
The authors declare no conflict of interest.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.ejphar.2017.09.010.
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