Bio-Medical Materials and Engineering 27 (2016) 577587 577

DOI 10.3233/BME-161610
IOS Press

Effect of curcumin on inhibiting

atherogenesis by down-regulating
lipocalin-2 expression in apolipoprotein E
knockout mice
Qiang Wan a,b, , Zhong-yong Liu a , Yu-ping Yang a and Shi-ming Liu b
a
Department of Medical Cardiology, Affiliated Hospital, Jiangxi University of Traditional Chinese
Medicine, Nanchang, China
b
Institute of Cardiovascular Disease, Second Affiliated Hospital, Guangzhou Medical University,
Guangzhou, China

Received 26 July 2016

Accepted 17 November 2016

Abstract.
BACKGROUND: Curcumin possesses significant anti-atherosclerosis properties. Lipocalin-2 (LCN2) is already known as one
of the most promising biomarkers of atherosclerosis. However, research on the effect of curcumin on regulating LCN2 expres-
sion in atherogenesis is very limited. The aim of the study was to investigate whether curcumin could alleviate atherosclerosis
in ApoE/ mice by down-regulating LCN2 expression.
METHODS: Fifty apolipoprotein E knockout (ApoE/ ) mice were fed with a western diet for 12 weeks and randomly divided
into five groups: model group (Mod), positive control group (Lov, with 30 mg/kg/d lovastatin), three curcumin groups (CurL,
CurM and CurH, with 40, 60 and 80 mg/kg/d curcumin), while 10 C57BL/6J mice were fed with a standard mouse chow diet as
a control group (Con). LCN2 in serum and aorta, biomarkers of serum lipid and inflammation were determined and the plaque
area of the atherosclerosis lesions was quantified.
RESULTS: CurM and CurH group were significantly lower than Mod group in terms of serum LCN2, lipid and inflammatory
cytokine levels, plaque area and LCN2 expression in atherosclerotic lesions, similar to lovastatin treatment.
CONCLUSIONS: Our findings indicate that dietary curcumin ameliorates western diet-induced atherosclerosis in ApoE/
mice, which is related to LCN2 down-regulation, anti-hyperlipidemia effect as well as the inhibition of inflammation.
Keywords: Curcumin, atherosclerosis, lipocalin-2 (LCN2), inflammation, anti-hyperlipidemia, apolipoprotein E knockout
(ApoE/ ) mice

1. Introduction

Atherosclerosis is a complex chronic inflammatory and metabolic disease, which is a major contrib-
utor of morbidity and mortality in the world. In addition to arterial lipid accumulation and lipid dys-
function, adipose tissue inflammation and perturbation of adipokine secretion have been increasingly
* Corresponding author: Qiang Wan. E-mail: [email protected].

0959-2989/16/$35.00 2016 IOS Press and the authors. All rights reserved
578 Q. Wan et al. / Effect of curcumin on inhibiting atherogenesis

recognized as essential reasons in the pathogenesis of atherosclerosis. Lipocalin-2 (LCN2), also termed
neutrophil gelatinase-associated lipocalin (NGAL) or siderocalin, mainly released from adipocytes, is a
small 25 kDa protein which was first discovered in human neutrophils [1,2]. LCN2 originally identified
as an inflammatory factor released by activated neutrophils, is also expressed in hepatocytes and various
human epithelial tissue, such as trachea, colon, stomach and kidney in response to various inflammatory
signals and different types of injury [3]. Recently LCN2 has emerged as one of the most reliable and
attractive biomarkers for the diagnosis and risks tratification of patients suffering from cardiovascular
diseases (CVD) [4,5]. Elevated serum LCN2 level is an independent predictor of CVD events in men
in a population-based cohort [6]. Recent data also indicated that LCN2 is closely related to atheroge-
nesis, the underlying pathology for CVD. Patients with subclinical atherosclerosis which is assessed
by intima-media thickness (IMT) at carotid and femoral arteries with ultrasound showed significantly
higher circulating concentrations of LCN2 compared with those without, besides, serum LCN2 level was
positively correlated with carotid IMT [7]. In addition to its circulating level, enhanced LCN2 content
has been detected in unstable human atherosclerotic plaque when intra plaque hemorrhage or luminal
thrombus is present [8]. Based on these pieces of evidence, we hypothesized that agents suppressing
LCN2 expression would be potential therapeutic agents that prevent against atherosclerosis.
Curcumin, the active constituent in the dietary spice turmeric (Curcuma longa Linn), is a pharma-
cologically effective and safe agent that plays a key role in anti-atherosclerosis processes. Curcumin
may prevent the formation of atherosclerotic lesions in rabbits by attenuating adhesion molecules and
matrix metalloproteinase (MMPs) expression [9]. Curcumin modulated macrophage polarization and
inhibited inflammatory cytokines secretion through the inhibition of the toll-like receptor 4 expression
and its signaling pathway [10]. The anti-atherogenic property of curcumin also could be linked to its
effect on gene networks and cell functions related to leukocyte adhesion and transendothelial migration
via nuclear factor-B pathways [11]. Although beneficial effects of curcumin on atherosclerosis have
been suggested, the underlying mechanisms responsible for the amelioration of atherosclerosis have
not been fully elucidated. Whether curcumin could ameliorate the development of atherosclerosis by
down-regulating LCN2 expression is currently unknown.
On the basis of these findings, we hypothesized that curcumin could prevent against atherogenesis by
down-regulating LCN2 expression. Apolipoprotein E knockout (ApoE/ ) mice is a genetically modi-
fied strain of mice that is commonly used as the animal model for spontaneous atherosclerosis. There-
fore, we evaluated the effect of curcumin on high fat diet-induced atherogenesis in ApoE/ mice and
investigated the mechanisms underlying curcumin-mediated modulation of atherosclerosis.

2. Materials and methods

2.1. Animals and diets

Fifty male 6-week-old ApoE/ mice with a genetic C57BL/6J background and 10 male 6-week-old
C57BL/6J mice were purchased from Vital River Experimental Animal Technology Co. Ltd (Beijing,
China) and housed in SPF grade Experimental Animal House at Jiangxi University of Traditional Chi-
nese Medicine (Jiangxi, China) in environmentally controlled conditions (23 2C, 55% 10% relative
humidity, with a 12-h light/dark cycle) with ad libitum access to food and water for 1 week. All ApoE/
mice were randomly divided into five groups (n = 10/group): a model group (Mod), a positive control
group (Lov), three curcumin groups (CurL, CurM and CurH) and were fed with a western diet (21%
 Q. Wan et al. / Effect of curcumin on inhibiting atherogenesis 579

fat and 0.15% cholesterol) from Experimental Animal Center of Nanchang University (Jiangxi, China)
for consecutive 12 weeks to establish an animal model of atherosclerosis, while 10 C57BL/6J mice
were fed with a standard mouse chow diet as a control group (Con). Mice in CurL, CurM, CurH or
Lov groups were treated with curcumin (40 mg/kg, purity 98%, Sigma, USA), curcumin (60 mg/kg),
curcumin (80 mg/kg) or lovastatin (30 mg/kg, purity 98%, Sigma, USA), respectively. All drugs were
dissolved in pure water and were administered by gavage once a day for 12 weeks. Mice in Mod and
Con groups were treated with the same volume of normal saline. All animal experiments were approved
by the Ethics Committee of Jiangxi University of Traditional Chinese Medicine and were conducted in
accordance with international guidelines.

2.2. Biochemical tests of serum

All mice were sacrificed by collecting whole blood via the abdominal aorta under ether euthanasia at
the last day of experiment after 12-h fasting. Serum were isolated from blood by centrifuge and were
stored at 80C until required for analysis. Serum levels of total cholesterol (TC), triglycerids (TG),
high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) were
assayed using commercially available kits (Invitrogen, USA). The circulating levels of serum LCN2,
interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-) and high-sensitivity C-reactive protein (hs-
CRP) were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturers
protocols of ELISA kits (eBioscience, USA).

2.3. Histologic analysis

Right atrium was incised and the heart was perfused by PBS (10 mM, pH = 7.4) through the apex of
the left ventricle at a constant pressure of 100 mm Hg followed by 4% paraformaldehyde (pH = 7.4)
after the thoracic cavity was opened. For each mouse, one part of aorta was used for histological exami-
nation and the other part was used for western blotting. The aortic root was separated from aortic arc at
the right subclavical branching point and fixed in 10% zinc-formalin, embedded in paraffin, sliced into
4 m-thick sections and stained with Hematoxylin and eosin (H&E). Serial sections were cut from the
proximal 1 mm of the aortic root. Five sections were collected at 80-m intervals starting at a 100-m
distance from the appearance of the aortic valves. The intima of the aorta thickness was analyzed un-
der a microscope (Olympus, Japan). Images of the aorta were captured using a digital camera (Olympus,
Japan) and analyzed for plaque area quantification using ImageJ software (National Institutes of Health).
For each animal a mean lesion area was calculated from five sections, reflecting the cross-section area
covered by atherosclerosis.

2.4. Immunohistochemistry

After antigen retrieval by boiling in 0.01 M sodium citrate for 10 min, deparaffinized sections were
quenched in 0.3% hydrogen peroxide for 30 min, followed by incubated in 1% BSA in PBS for 30 min.
Sections were labeled with goat anti-mouse LCN2 antibody (Santa Cruz, USA, 1:200 dilution) at 37C
for 45 min and then overnight at 4C. After washing, the bound antibodies were conjugated with sec-
ondary antibodies at 37C for 1 h, and then the DAB substrate was administrated and incubated for
1 min. The sections were counterstained with hematoxylin and the result was acquired with Image-Pro
Plus 5.0 Analysis Software (Media Cybernetics, USA).
580 Q. Wan et al. / Effect of curcumin on inhibiting atherogenesis

2.5. Western blotting

The mice aortas were homogenized in lysis buffer containing 1% NP-40, 50 mM Tris (pH = 7.4),
150 mM NaCl and 1 mM PMSF for 30 min on ice. After centrifugation at 14,000 rpm for 20 minutes
at 4C, the protein concentrations were quantified by BCA protein assay kit (Invitrogen, USA). For
immunoblots, 50 g of protein per sample was separated by 12% SDSPAGE and transferred to PVDF
membrane (Millipore, USA). Membranes were blocked with 5% skim milk in PBS with 0.1% Tween 20
(PBST) for 1 h at 37C. Goat anti-mouse LCN2 polyclonal antibody (Santa Cruz, USA, 1:1000 dilution)
in PBST were incubated with membranes overnight at 4C. The membranes were washed thoroughly for
60 min with PBST before incubation with IgG-horseradish peroxidase-conjugated secondary antibody
(Santa Cruz, USA, 1:2000 dilution) for 1 h. Proteins were visualized with ECL Plus (GE Healthcare,
Sweden) on Kodak 2000MM. Densitometric analysis was conducted by PDI Imageware System (Bio-
Rad, USA).

2.6. Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from the treated mice descending aortas using RNeasy Plus Micro
Kit (Qiagen, Germany) according to the operating instruction. The first-strand cDNA was synthe-
sized from the total RNA to establish a reverse transcription system by PrimeScript RT Mas-
ter Mix Sample kit (Takara, Japan). Amplified DNA was separated on 1.5% agarose gels and vi-
sualized under UV light. qRT-PCR was performed using an Applied Biosystems Prism sequence
detection system (Applied Biosystems, USA) according to the manufacturers protocols. Primer
sequences for qRT-PCR were: LCN2: forward 5 -TGACAGCCCACCTCCCT-3 and reverse 5 -
CCACTTCCATCCTGGATGGT-3 ; -actin, forward 5 -CAAGAGATACGGCCGGCTGCT-3 and re-
verse 5 -TCCTTCTGCTGCATCTCGGCA-3 .

2.7. Statistical analysis

Data were expressed as means SD. Statistical analyses were performed using one-way ANOVA and
a value of P < 0.05 was considered statistically significant.

3. Results

3.1. Curcumin decreased serum lipid profiles

To determine whether curcumin altered the serum lipid profiles, we measured serum TC, TG, LDL-C
and HDL-C levels in each group at the conclusion of the study (Table 1). Comparing with Con group,
serum levels of TC, TG and LDL-C in Mod group were significantly increased, indicating occurrence
of hyperlipidemia. After treatments of lovastatin or curcumin, serum lipid profiles were remarkably
improved. TC, TG and LDL-C levels in Lov, CurM and CurH groups were significant reduced compared
to Mod group, while curcumin supplementation did not affect HDL-C levels compared to Mod group.
 Q. Wan et al. / Effect of curcumin on inhibiting atherogenesis 581

Table 1
Effects of curcumin on serum lipid profiles in mice (n = 10, mmol/L)
Group TC TG HDL-C LDL-C
Con 3.96 0.44## 0.35 0.05## 3.16 0.25## 0.69 0.09##
Mod 5.14 0.46** 0.46 0.07** 2.38 0.39** 0.89 0.13**
Lov 4.47 0.71**## 0.37 0.05*## 3.02 0.26**## 0.71 0.11*##
CurL 5.08 0.53## 0.45 0.06## 2.39 0.36** 0.88 0.17##
CurM 4.72 0.67**## 0.41 0.07**## 2.40 0.26** 0.79 0.12**##
CurH 4.52 0.61**## 0.40 0.06**## 2.37 0.35** 0.74 0.15**##
Values are mean SD. * P < 0.05, ** P < 0.01 vs Con group; # P < 0.05, ## P < 0.01 vs Mod group.
Con, C57BL/6J mice were fed with a standard mouse chow diet; Mod, ApoE/ mice were fed with a western diet; Lov,
ApoE/ mice gavaged with lovastatin (30 mg/kg) were fed with a western diet; CurL, ApoE/ mice gavaged with curcumin
(40 mg/kg) were fed with a western diet; CurM, ApoE/ mice gavaged with curcumin (60 mg/kg) were fed with a western
diet; CurH, ApoE/ mice gavaged with curcumin (80 mg/kg) were fed with a western diet.

Table 2
Effects of curcumin on serum levels of LCN2, IL-6, TNF- and hs-CRP in mice (n = 10)
Group LCN2 (ng/mL) IL-6 (pg/mL) TNF- (pg/mL) hs-CRP (ng/mL)
Con 50.72 10.34## 42.81 3.72## 169.85 8.91## 98.92 31.03##
Mod 103.22 12.26** 88.63 5.37** 437.52 48.91** 504.53 87.65**
Lov 59.51 9.27**## 57.42 6.06**## 237.43 45.35**## 188.28 51.48**##
CurL 100.53 11.39** 85.57 5.51** 434.48 53.63** 499.59 78.72**
CurM 70.84 10.53**## 60.45 5.76**## 277.62 46.64**## 297.92 64.26**##
CurH 57.27 9.69**## 55.92 6.15**## 254.97 52.59**## 172.45 55.82**##
Values are mean SD. * P < 0.05, ** P < 0.01 vs Con group; # P < 0.05, ## P < 0.01 vs Mod group.
Con, C57BL/6J mice were fed with a standard mouse chow diet; Mod, ApoE/ mice were fed with a western diet; Lov,
ApoE/ mice gavaged with lovastatin (30 mg/kg) were fed with a western diet; CurL, ApoE/ mice gavaged with curcumin
(40 mg/kg) were fed with a western diet; CurM, ApoE/ mice gavaged with curcumin (60 mg/kg) were fed with a western
diet; CurH, ApoE/ mice gavaged with curcumin (80 mg/kg) were fed with a western diet.

3.2. Curcumin decreased serum levels of inflammatory cytokines

In order to evaluate whether curcumin altered the serum levels of inflammatory cytokines, serum IL-6,
TNF- and hs-CRP levels were detected by ELISA (Table 2). Western diet fed in Mod group had re-
markably higher serum IL-6, TNF- and hs-CRP levels than those of Con group, whereas administration
with lovastatin significantly decreased the alterations compared with the Mod group. Curcumin in CurM
and CurH group showed a similar trend of lovastatin effect on serum levels of IL-6, TNF- and hs-CRP.

3.3. Curcumin down-regulated the expression of LCN2

We measured serum LCN2 level, LCN2 protein and mRNA expression in the aorta in each group by
ELISA, Western blotting and qRT-PCR assay, respectively. Serum LCN2 level (Table 2), LCN2 protein
(Fig. 1(A)) and mRNA expression (Fig. 1(B)) in the aorta in Mod group were much higher than in Con
group. Serum LCN2 level, LCN2 protein and mRNA expression in Lov, CurM and CurH groups were
significant reduced compared to Mod group. To identify the distribution of LCN2 in mice aorta, we
determined its level by immunohistochemistry assay. Immunohistochemical staining result (Fig. 1(C))
showed that significantly more LCN2 was detected from Mod group compared with Con group, and
less LCN2 were detected from Lov, CurM and CurH groups compared with Mod group. These results
582 Q. Wan et al. / Effect of curcumin on inhibiting atherogenesis

Fig. 1. Effect of curcumin on LCN2 expression in ApoE/ mice fed a western diet for 12 weeks. (A) The protein expression of
LCN2 in mice aorta. (B) The mRNA expression of LCN2 in mice aorta. (C) Representative images of immunohistochemistry
for LCN2 in mice aorta (400). Values are mean SD. ** P < 0.01 vs Con group; ## P < 0.01 vs Mod group. Con, C57BL/6J
mice were fed with a standard mouse chow diet; Mod, ApoE/ mice were fed with a western diet; Lov, ApoE/ mice gavaged
with lovastatin (30 mg/kg) were fed with a western diet; CurL, ApoE/ mice gavaged with curcumin (40 mg/kg) were fed
with a western diet; CurM, ApoE/ mice gavaged with curcumin (60 mg/kg) were fed with a western diet; CurH, ApoE/
mice gavaged with curcumin (80 mg/kg) were fed with a western diet.
 Q. Wan et al. / Effect of curcumin on inhibiting atherogenesis 583

indicated that curcumin supplementation in a dose-dependent manner suppressed LCN2 expression in

the progression of atherosclerosis.

3.4. Curcumin suppressed the formation of atherosclerotic lesions

We investigated whether curcumin attenuated western diet-induced atherosclerotic lesions in mouse

aortas. Plaque area was measured using cross-sectional slices of the ascending aorta to estimate the
progression of atherosclerosis. The analysis of representative images from the sections after staining
with H&E (Fig. 2(A)) showed that atherosclerotic lesions in Mod group were markedly larger than those
of Con group, plaque was predominantly observed in the medial and intimal areas of the arterial wall
but less so in the adventitial layers, whereas small, sparse plaques were observed in the Lov group.
Supplementing with curcumin in CurM group and CurH group reduced lesion development significantly
in spite of high fat diet intake compared to Mod group. The percentages of aortic surface area occupied
by the atherosclerotic lesions were significantly reduced in CurM group and CurH group compared with
Mod group. Mice fed the low curcumin concentration in CurL group showed no suppressive effect.
Findings also revealed significantly greater neointimal thickening in Mod group compared with Con
group. However, the curcumin supplement in CurM group and CurH group reduced the neointima for-
mation (Fig. 2(B)). Taken together, these results indicated that curcumin suppressed the development of
atherosclerotic lesions in aortas of western diet-fed mice.

4. Discussion

ApoE/ mice are unable to produce the key glycoprotein ApoE essential for transport and metabolism
of lipids. Fed with normal chow, ApoE/ mice start to develop atherosclerosis lesions at the age of 1
month to 2 months [12]. Therefore, we chose 6-week-old ApoE/ mice on a high-fat diet for 12 weeks
in this study to examine the development of atherosclerosis at initial stages. As our expectation, the sizes
of atherosclerotic lesions within aorta in Mod group were remarkably increased as compared with Con
group.
Adipose tissue is no longer considered as an inert tissue mainly devoted to energy storage but is
emerging as a huge gland which participates actively in regulating physiologic and pathologic processes,
including inflammation and immunity. Recently it has become clear that many adipokines which are
secreted by adipose tissue, are mediators in the adipo-cardiovascular axis, the cross talk between
adipose tissue and cardiovascular disease.
LCN2, mainly released from adipocytes, is an adipokine belonging to the lipocalin superfamily whose
members generally bind a range of small hydrophobic molecules, specific cell-surface receptors and
forms complexes with soluble macromolecules [13]. Recent studies seem to suggest a potential involve-
ment of this adipokine in the genesis and progression of atherosclerosis. LCN2 level was positively
correlated with lesion severity and complexity of coronary artery disease in patients with non-ST ele-
vation acute coronary syndrome, and serum LCN2 levels on admission are associated with increased
burden of atherosclerosis in these patients compared to control subjects. LCN2 also plays a critical role
in the development and disruption of atherosclerotic plaques. Increased LCN2 is able to inhibit MMP-9
inactivation by forming a complex with MMP-9, thereby prolonging the enzymatic activity leading to
degraded collagen content, weakening the atherosclerotic plaque structure, and making it more prone to
rupture, which is the majority of acute clinical manifestations of atherosclerosis [14]. Thus, increased
LCN2 results in an increase in plaque instability. From our results, serum LCN2 level, LCN2 protein in
584 Q. Wan et al. / Effect of curcumin on inhibiting atherogenesis

Fig. 2. Effect of curcumin on atherosclerotic lesions formation in ApoE/ mice fed a western diet for 12 weeks. (A) Repre-
sentative photographs of aortic arch stained with H&E (40). (B) Representative images of aorta neointimal thickness stained
with H&E (200). Arrows indicate neointimal thickness. Values are mean SD. ** P < 0.01 vs Con group; ## P < 0.01 vs
Mod group. Con, C57BL/6J mice were fed with a standard mouse chow diet; Mod, ApoE/ mice were fed with a western
diet; Lov, ApoE/ mice gavaged with lovastatin (30 mg/kg) were fed with a western diet; CurL, ApoE/ mice gavaged with
curcumin (40 mg/kg) were fed with a western diet; CurM, ApoE/ mice gavaged with curcumin (60 mg/kg) were fed with a
western diet; CurH, ApoE/ mice gavaged with curcumin (80 mg/kg) were fed with a western diet.
 Q. Wan et al. / Effect of curcumin on inhibiting atherogenesis 585

the aorta and the distribution of LCN2 in the atherosclerotic plaque in Mod group were much higher than
in Con group [15]. These results indicated that LCN2 may play a significant role in the pathogenesis of
atherosclerosis.
Hyperlipidemia, as a result of accumulation of lipids in blood has come to issue on the therapy for
the atherosclerosis. Oxidized phospholipids contribute to inflammation within the artery wall, initiat-
ing atherogenic chemokine expression that leads to monocyte adhesion [16]. Therefore, lipids can be
regarded as triggers of the inflammatory process in atherosclerosis. Clinical and epidemiologic observa-
tions have consistently documented that LDL-C concentration is positively correlated with atherosclero-
sis [17]. In this study, we found that lovastatin or curcumin treatment partly recovered high serum lipid
profile induced by western diet and their anti-hyperlipidemia effects were comparable [18].
Activation of pro-inflammatory cytokines play a major role in the progression of atherosclerosis by in-
creasing monocyte adhesion, smooth muscle cell proliferation, endothelial dysfunction, oxidative stress,
and vascular calcification. In atherosclerosis patients marked elevated circulating levels of IL-6, TNF-
and hs-CRP were observed. IL-6 induces oxidative stress and endothelial dysfunction by over-expression
of the angiotensin II type 1 (AT1) receptor in the atherosclerotic process [19]. TNF- may play an
atherogenic role by up-regulating the expressions of monocyte chemotactic protein-1 (MCP-1) in the
vascular wall, and by inducing oxidized LDL (Ox-LDL) uptake and scavenger receptor class A expres-
sion in macrophages [20]. Hs-CRP is a classical acute-phase reactant, which binds to phosphocholine
expressed on the surface of dead or dying cells and activates the complement system in atherosclerotic
plaques via the C1q complex [21]. LCN2 was originally identified as an inflammatory cytokine released
by activated neutrophils, and it could inhibit the secretion of anti-inflammatory factors such as perox-
isome proliferator-activated receptor (PPAR ) and adiponectin in both adipocytes and macrophages
[3,22]. In the present study, treatment with curcumin remarkably reduced the plaque area, LCN2 expres-
sion in atherosclerotic lesions and serum levels of LCN2, IL-6, TNF- and hs-CRP in ApoE/ mice
compared with Mod group, and such interactions may help to explain the anti-atherosclerosis properties
of curcumin.
Consumption of curcumin was shown to be beneficial to prevent atherosclerosis. A 6-month random-
ized controlled trial showed that curcumin intervention in type 2 diabetic population could lower the
atherogenic risks by increasing serum level of adiponectin and decreasing serum level of leptin [23].
The key initiating step of the earliest stage of atherosclerosis is sub-endothelial accumulation of choles-
terol and monocyte-derived macrophages, leading to chronic inflammation. Curcumin significantly sup-
pressed MCP-1 production and up-regulated cholesterol efflux via suppressing the c-Jun N-terminal
kinase (JNK) pathway, which indicated that curcumin could have the effect of anti-atherosclerosis via
inflammation-related pathway [24]. In addition, curcumin increased the stabilization of atherosclerotic
plaque by inhibiting MMP-9 expression through Protein Kinase C (PKC) signaling pathway in phorbol
myristate acetate (PMA)-induced macrophages [25]. Our results indicated that the western diet signifi-
cantly accelerated atherosclerosis and up-regulated adipokine LCN2 and several inflammatory cytokines
such as IL-6, TNF- and hs-CRP in ApoE/ mice, dietary curcumin attenuated atherosclerosis by re-
ducing LCN2 and inflammatory cytokines production.
Although the findings of our study could give new insights to the understanding of atherogene-
sis, in association with LCN2 expression, other adipokine should not be ignored in contributing to
atherosclerosis, such as adipocyte fatty acid-binding protein (A-FABP). Curcumin was reported to alle-
viate atherosclerosis in LDL receptor deficient mice through suppression of A-FABP [26], the beneficial
effects of curcumin in attenuating atherosclerosis may due to not only down-regulating LCN2 expression
but also suppression of A-FABP.
586 Q. Wan et al. / Effect of curcumin on inhibiting atherogenesis

5. Conclusion

In summary, our findings provide a potential biological basis for the association between an
atherosclerosis-related biomarker and a Chinese herb extract, this presents the high beneficial effects
of curcumin on lipid metabolism and cardiovascular health, particularly when these findings need to
be translated for human use. Further studies are needed to add additional insights into the molecular
mechanisms that drive down-regulating lipocalin-2 expression of curcumin in treating atherosclerotic
lesions.

Acknowledgements

This work was supported by grants from the National Nature Science Foundation of China
(No. 81660770), the National Nature Science Foundation of Jiangxi Province (No. 20161BAB215256),
the Science and Technology Planning Project of Guangdong Province (No. 2016A020226023) and the
China Postdoctoral Science Foundation (No. 2016M592476).

Conflict of interest

The authors have no conflict of interest to report.

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