Research A rticle
The SGLT-2 Inhibitor Dapagliflozin Has a Therapeutic
Effect on Atherosclerosis in Diabetic ApoE
−/−
Mice
Weiling Leng,
1
Xinshou Ouyang,
2
Xiaotian Lei,
1
Mingxia Wu,
1
Liu Chen,
1
Qinan Wu,
1
Wuquan Deng,
1
and Ziwen Liang
1
1
Department of Endocrinology, e First Aliated Hospital of ird Military Medical University, Chongqing 400038, China
2
Department of Internal Medicine, Section of Digestive Diseases, Yale University of Medicine, New Haven, CT 06520, USA
Correspondence should be addressed to Ziwen Liang; ziwenliang@.com
Received August ; Revised November ; Accepted December
Academic Editor: Anshu Agrawal
Copyright © Weiling Leng et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. Our study aimed to observe the eect of sodium glucose cotransporter- (SGLT) inhibitor dapagliozin on diabetic
atherosclerosis and investigate the subsequent mechanism. Methods. Aortic atherosclerosis was induced in streptozotocin induced
diabetic ApoE
−/−
mice by feeding with high-fat diet, and dapagliozin was administrated intragastrically for weeks as treatment.
Eects of dapagliozin on indices of glucose and fat metabolism, IL-𝛽, IL-, NLRP protein levels, and the reactive oxygen species
(ROS) were measured. e atherosclerosis was evaluated by oil red O and hematoxylin-eosin staining. e eects of dapagliozin
on the IL-𝛽 production in culturing primary macrophages of wild type and NLRP
−/−
knockout mice were investigated for
mechanism analyses. Results. Dapagliozin treatment showed favorable eects on glucose and fat metabolism, partially reversed
the formation of atherosclerosis, inhibited macrophage inltration, and enhanced the stability of lesion. Also, reduced production
of IL-𝛽, IL-, NLRP protein, and mitochondrial ROS in the aortic tissues was detected with dapagliozin treatment. In vitro,
NLRP inammasome was activated by hyperglucose and hyperlipid through ROS pathway. Conclusions. Dapagliozin may be of
therapeutic potential for diabeti c atherosclerosis induced by high-fat diet, and these benets may depend on the inhibitory eect
on the secretion of IL-𝛽 by macrophages via the ROS-NLRP-caspase- pathway.
1. Introduction
Previous evidence from epidemiological studies has indicated
that cardiovascular diseases (CVDs) have become the most
serious complications of diabetes mellitus (DM), which is the
leading cause of mortality and morbidity for DM patients.
It has been well understood that pathophysiological changes
in patients with DM, mainly the toxicity of hig h blood
glucose to the endothelium and other cells of the vessels, may
lead to the pathogenesis of atherosclerosis and subsequent
CVDs. Although the key pathophysiological mechanisms
underlying the so-called diabetic atherosclerosis were not
fully determined, chronic inammatory response, and asso-
ciated lipid deposition, macrophage inltration and smooth
muscle cell proliferation in DM have been suggested to play
important roles [–].
Recently, interleukin-𝛽 (IL-𝛽)andinterleukin-
(IL-) have been recognized as two inammatory
cytokines which contribute to the development of diabetic
atherosclerosis [], and the underlying mechanisms
downstream of these cytokines have been e valuated. e
inherent immunity has been linked to the activation
of inammatory response of the body. e inherent
immunity recognizes damage-associated molecular patterns
(DAMPs) and pathogen-associated molecular patterns
(PAMPs) via pattern recognition receptors (PRRs) and
thus excites the inammatory reaction and starts the
defense mechanism []. NLRP, a NOD-like receptor
andalsoanintracellularPRR,hasbeensuggestedto
play a key role during the above pathophysiological
process. e NLRP can be activated by several dierent
exogenous and endogenous stimulation signals to form a
multiprotein complex known as NLRP inammasome.
Previous studies indicated that ligand-binding NLRP
can promote the formation of inammasome and activate
caspase-, eventually facilitating the maturation and
Hindawi Publishing Corporation
Mediators of Inflammation
Volume 2016, Article ID 6305735, 13 pages
http://dx.doi.org/10.1155/2016/6305735
Mediators of Inammation
secretion of pro-IL-𝛽 and pro-IL- [, ]. Re cent studies
have conrmed that NLRP inammasome is involved
in inammatory response during the pathogenesis of
atherosclerosis. Indeed, activation of NLRP inammasome
in macrophages has been involved in the cholesterol
crystals formation in the pathogenesis of atherosclerosis [].
Moreover, silence of NLRP gene has been found to delay
the progression of atherosclerosis in mice []. Although
IL-𝛽 [, ] and IL- [] have been suggested to be
proatherosclerosis, whether they functions via regulation of
NLRP inammasome remains to be determined.
Sodium glucose cotransporters- (SGLT-) inhibitors are
a new class of antidiabetic medications targeting against renal
glucose reabsorption. D apagliozin, as a SGLT- inhibitor,
was marketed in . e glucose lowering eect of
dapagliozin has been conrmed in many randomized con-
trolled clinical trials which showed distinguished lowering
eects of dapagliozin for glycosylated hemoglobin (HbAc),
fasting blood glucose, and postprandial blood glucose [, ].
Moreover, besides the antidiabetic eect, dapagliozin also
seemed to be cardioprotective, by lowering blood pressure
[], reducing body weight [], triglyceride and uric acid
[, ], and improving insulin resistance []. Recently,
a few studies indicated that SGLT- inhibitors may exert
their cardiometabolic benets via anti-inammatory eects
[, ]. However, the overall eects of dapagliozin on
atherosclerosis in DM and the potential benets involved,
for example, their eects on IL-𝛽 and IL- cytokines
and NLRP inammasome systems, have not been eval-
uated. Randomized controlled clinical t rials have proven
that dapagliozin can be used alone to treat the patients
with type diabetes mellitus and it has good eects in
decreasing glycosylated hemoglobin (HbAc), fasting blood
glucose, and postprandial blood glucose [, ]. Interestingly,
dapagliozin can improve cardiovascular diseas es in diabetes
mellitus by slightly lowering blood pressure [], reducing
body weight [], triglyceride and uric acid [, ], and
improving insulin resistance []. In addition, there are trials
showing that SGLT- inhibitors can reduce inammatory
markers in the serum and organs an d thus decrease the degree
of arteriosclerosis in the diabetic animal mo dels [, ].
However, the eects of SGLT - inhibitors on inammatory
markers, including NLRP inammasome and IL-𝛽,IL-
cytokines have not been completely claried yet. It is unclear
that whether SGLT- inhibitors can aect atherosclerosis
progression or not. Accumulated knowledge ab out eect of
dapagliozin on atherosclerosis and its underlying mecha-
nisms can bring us more information about its safety and
practicability in clinic.
e aims of the present study were to explore the eects
of dapagliozin on aortic atherosclerosis in diabetic versus
nondiabetic ApoE
−/−
mice and detect the possible underlying
mechanism, particularly their inuence on the ROS-NLRP-
caspase- pathway.
2. Methods
2.1. Mice. e spontaneous atherosclerotic lesions in
apolipoprotein E knockout (ApoE
−/−
)miceaerthe
hypercholesterolemia have been widely used as a mouse
model of atherosclerosis in previous studies [].
Consistently, we used this atherosclerotic mouse model
in our study. Briey, male ApoE
−/−
mice ( weeks old,
Beijing HFK Bioscience Co., Ltd.) and the male CBL/J
mice ( weeks old, e Experimental Animal Center of
the ird Military Medical University) were all bred in the
animal rooms of the First Aliated Hospital of the ird
Military Medical University. e specic conditions were as
follows: a SPF environment, temperature (–
∘
C), relative
humidity (%), and h light/ h dark cycle. e animal
cages and drinking bottles were disinfected regularly, the
beddings were sterilized in an autoclave, and the animal
rooms were disinfected with ultraviolet lamps perio dica lly.
Aer one-week accommodation, the ApoE
−/−
mice were fed
with a high-fat diet (HFD, general diet .% + fat (oil)
% + custard powder % + cholesterol .%) to induce
atherosclerosis of the aorta, while the CBL/J mice were fed
with a general diet. e animal diets were purchased from the
Experimental Animal Center of the ird Aliated Hospital
of the ird Military Medical University. Male ApoE
−/−
mice ( weeks old, to g) were intraperitoneally
injected STZ (Sigma-Aldrich, St. Louis, MO) at the dose
of mg/kg dissolved in mM citrate buer (pH.)
to induce DM, while the controls received buered saline
alone. Aer one week, we measured the blood glucose levels
of all mice using a glucometer (Abbott Diabetes Care Inc.
Optium Xceed) by tail vein puncture blood s ampling. Aer
weeks, the mice with blood glucose > . mmol/l served
as diabetic group according to the previous studies [], and
the non-STZ-injected ApoE
−/−
mice served as nondiabetic
group. e diabetic and nondiabetic mice were randomly
assigned to treatment group (𝑛=12) and control group
(𝑛=8) separately. e mice in treatment group received
intragastrically dapagliozin (AstraZeneca) . mg/kg/d
for -week treatment, while CBL/J mice and mice in
control group were intragastrically given vehicle . mg/kg/ d.
All animals received human care and all study protocols
were approved by the Institutional Animal Care and Use
Committee of the ird Military Medical University before
performance.
2.2. Metabolic Measurement. Aer intraperitoneal injection
ofSTZ,thebloodglucoseandbodyweightofmicewere
measured weekly. A t an age of weeks, all mice were subject
to – h fasting and then anesthetized by intraperitoneal
injection of % chloral hydrate. ereaer, the blood was
collected via orbital vein and then centrifuged (
∘
C, r
× min) to separate the serum. A part of serum was used
to detect the serum concentration of total cholesterol (TCH),
triglyceride (TG), high density lipoprotein cholesterol (HDL-
c), low density lipoprotein cholesterol (LDL-c), and free
fatty acid (FFA) at Clinical Laboratory of the First Aliated
HospitaloftheirdMilitaryMedicalUniversity,andtherest
were preserved at −
∘
Cforthemeasurementsoftheserum
levels of NLRP, IL-𝛽, and IL- with ELISA kits (Shanghai
Jinglai Biotech, shanghai, China) according to t he methods
and procedures provided by the reagent manufacturer .
Mediators of Inammation
2.3. Quantication of Atherosclerotic Lesion Area. Aer blood
collection via orbital vein, the aorta of the mice was dissected
with fat removed under a surgery microscope. e aortic
rootwasembeddedwithOCTandthenmadeinto𝜇m
frozen sections. ereaer, the sections were dried, soaked in
% isopropanol for min, stained with oil red O (Sigma-
Aldrich, O, USA) working uid ( : ) for min, dif-
ferentiatedinisopropanolfor–stilltheinterstitiumwas
clear,andthenwashedwithwater.eobtainedsectionswere
counter-stained with Mayer hematoxylin, dierentiated in %
Hcl-alcohol for s, blued with running tap water, carefully
dried with lter papers, and mounted with glycogelatin. e
upper segments of aortas were xed in % paraformaldehyde
solution, embedded in paran, and sectioned at 𝜇m. Aer
dehydration, sections were stained with hematoxylin-eosin
(HE, Beyotime, Shanghai, China). Aer removal of adjacent
fat, the upper segments of thoracic aorta and abdominal
aorta were atly placed on the slides, dierentiated in %
isopropanol for min, stained in oil red O working uid
(:) for h, dierentiated in % isopropanol for -
times till the background color became white, and then
photographed. e area of lesion in the sections of aorta
and aortic root was calculated using Image-pro plus .
(MediaCybernetics, Inc.).
2.4. Immu nouor escent Staining. e frozen sections of aor -
tic root were xed with ice-acetone for min, added with
.%TritonX-tomakecellmembranepermeabilized,
and blocked with % BSA at room temperature for h. en
they were blocked at
∘
C overnight with primary antibodies
(MOMA- antibody : , Abcam; 𝛼-SMA antibody : ,
Sigma), and incubated at room temperature for h using
secondary antibodies ( : and : ) protecting from
light for coloration by antigen-antibody binding. Finally they
were counterstained for nuclei with DAPI and mounted with
the uorescer. Macrophages and smooth muscle actin were
observed under confocal uorescence microscope (Zeiss
LSM ) and the positively stained macrophages and
smooth muscle cells were automatically analyzed and quan-
tiedonImage-proplus..
2.5. ROS Assay. To investigate aortic atherosclerotic
lesions ROS levels, isolated arteries were lo aded with DHE
( 𝜇mol/L) for minutes at
∘
C. e ROS were observed
under confocal uorescence microscope (Zeiss LSM ),
andDHEturnreduorescentuponoxidation.
2.6. Isolation and Culture Macrophages from Bone Marrow.
WT and NLRP
−/−
KO male mice bone marrow-derived
macrophages (BMDMs) were obtained by dierentiating
bone marrow. Briey, bone marrow cells were ushed out of
femurs and tibias and the remaining cells were maintained in
dierent medium. BMDMs were maintained in macrophage
dierentiating medium (DMEM and M-CSF ( ng/mL,
Sigma)) for days. e medium was supplemented with
% FBS (Hyclone Laboratories), m M L-glutamine, peni-
cillin ( U/mL), and streptomycin ( 𝜇g/mL). To test
the direct action of dapagliozin on macrophages, the WT
macrophageswereplatedintothesterile-wellplate,
stimulated with LPS ( ng/mL, Sigma) for h, and then,
respectively, cultured with . M palmitate (PA, Sigma), hig h
glucose (HG, mmol/L), and PA+HG DMEM medium
for h. Finally the medium was added with dapagliozin
(. 𝜇M) and new-prepared ATP ( mM) for min. en
we collected the supernatant of cell culture to assay the
content of IL-𝛽 with ELISA kits (Shanghai Jinglai Biotech,
shanghai, China). To test the action pathway of PA or HG on
IL-𝛽 production, the WT and NLRP
−/−
KO macrophages
were plated into -well plate, stimulated with LPS for h, and
then, respectively, cultured with . M PA, HG ( mmol/L),
and PA+HG DMEM medium for h and collected cell
lysates to test NLRP and caspase- expression with Western
blot.
2.7. Western Blot. e total aorta tissue was homogenized.
e proteins of tissues and cell lysates were then separated
by SDS-PAGE and further blotted using following specic
antibodies: (anti-NLRP antibody : , anti-ASC antibody
: , anti-caspase- antibody : , anti-IL-𝛽 antibody
: , and anti-IL- antibody : ) (Novus biological,
Littleton, Co., USA). e membranes were scanned with
Typhoon (Pharmacia, USA) and quantitated using Quality
One. e experiments were repeated for times.
2.8. Statistical Analysis. All data were presented as means ±
standard deviation (M ± SD). e nonparametric rank sum
test or analysis of variance (ANOVA) was applied to evaluate
the dierences among the groups. A 𝑝 < 0.05 indicates a
statistically signicant dierence. All statistical analyses were
performed on SPSS (., Inc., Chicago, IL, USA).
3. Results
3.1. Changes of Metabolic Parameters aer Dapagliozin Treat-
ment. e fasting body weight, blood glucose, and blood
lipid levels of mice in each group measured during the
study were shown in Table . No signicant changes of body
weight were detected in DM or non-DM ApoE
−/−
mice t hat
received dapagliozin. However, the levels of blood glucose,
TCH, TG, and FFA of diabetic ApoE
−/−
mice were markedly
increased as compared with nondiabetic ApoE
−/−
mice. More
importantly, aer treatment with dapagliozin, the fasting
blood glucose decreased by % (𝑝 < 0.01)intheDM
ApoE
−/−
mice (𝑝 < 0.05), wh ile HDL-c, TC, and LDL-c levels
were merely aected (Figure ).
3.2. Changes of Serum NLRP3 Inammasome, IL-1𝛽,and
IL-18 aer Dapagliozin Treatment. As shown in Table ,
compared with those of the non-DM mice, DM mice had
signicantly increased serum levels of NLRP (𝑝 < 0.01),
IL-𝛽 (𝑝 < 0.05), and IL- (𝑝 < 0.05). Moreover, serum
IL-𝛽 and IL- levels in nondiabetic control group were
also signicantly increased as compared with those of the
CBL/J group (𝑝 < 0.01). More importantly, aer -week
treatment with dapagliozin in DM mice, the serum levels of
NLRP (𝑝 < 0.01), IL-𝛽 (𝑝 < 0.05), and IL- (𝑝 < 0.05)
proteins were all signicantly reduced, while NLRP and IL-
levels were not signicantly aected (Figure ).
Mediators of Inammation
DM+dapa
DM+con
NDM+dapa
NDM+con
C57
0
10
20
30
Glucose (mmol/l)
17
18
19
20
21
22
23
24
25
26
27
28
16
Weeks age
(a)
DM+dapa
DM+con
NDM+dapa
NDM+con
C57
20
25
30
35
Weight (g)
17
18
19
20
21
22
23
24
25
26
27
28
16
Weeks age
(b)
##
##
##
∗
∗∗
DM+dapa
DM+con
NDM+dapa
NDM+con
C57
0
10
20
30
Glucose (mmol/l)
16 w
28 w
(c)
DM+dapa
DM+con
NDM+dapa
NDM+con
C57
0
10
20
30
40
Weight (g)
16 w
28 w
(d)
∗∗
∗∗
DM+dapa
DM+con
NDM+dapa
NDM+con
C57
0
10
20
30
40
50
TCH (mmol/l)
(e)
∗∗∗∗
DM+dapa
DM+con
NDM+dapa
NDM+con
C57
0
1
2
3
TG (mmol/l)
(f)
F : Continued.
Mediators of Inammation
∗
C57
DM+con
DM+dapa
NDM+con
NDM+dapa
0
1
2
3
4
5
HDL-C (mmol/l)
(g)
∗∗
C57
DM+con
DM+dapa
NDM+con
NDM+dapa
0
10
20
30
40
LDL-C (mmol/l)
(h)
∗
∗∗
C57
DM+con
DM+dapa
NDM+con
NDM+dapa
0.0
0.5
1.0
1.5
2.0
FFA (mmol/l)
(i)
F : Changes of metabolic parameters in mice from each group. (a) and (c) Compared with non-DM group, the blood glucose in DM
group was signicantly increased; and dapagliozin treatment of wks signicantly decreased blood glucose in both groups; (b) and (d) no
signicant changes of body weight were detected before and aer dapagliozin treatment in each group; (e), (g), and (h) the levels of cholesterol
were signicantly increased in the ApoE
−/−
mice fed with a high-fat diet, part icularly in DM ApoE
−/−
mice. Dapagliozin treatment did not
reduce the levels of TCH, HDL-c, or LDL-c; (f) and (i): dapagliozin treatment decreased the levels of TG and FFA. ∗, 𝑝 < 0.05; ∗∗, 𝑝 < 0.01;
, 𝑝 < 0.01.
T : Data on body weight, fasting blood glucose, and blood lipid prole in all groups.
DM+dapa DM+con NDM+dapa NDM+con C
BW (g) . ± . . ± . . ± . . ± . . ± .
FBG (mmol/l) 13.69 ± 2.58
##
. ± . 9.17 ± 2.28
∗
. ± . . ± .
TC (mmol/l) . ± . . ± . . ± . . ± . 2.48 ± 0.41
&&
TG (mmol/l) 0.87 ± 0.22
##
. ± . . ± . . ± . . ± .
HDL-c (mmol/l) . ± . . ± . . ± . . ± . . ± .
LDL-c (mmol/l) . ± . . ± . . ± . . ± . 0.45 ± 0.14
&&
FFA (mmol/l) 1.02 ± 0.22
#
. ± . . ± . . ± . . ± .
Diabetic mice that received dapagliozin (DM+dapa), diabetic mice that received vehicle (DM+con), nondiabetic mice that received dapagliozin
(NDM+dapa), nondiabetic mice that received vehicle (NDM+con), and wild-type (C/BL) mice were measured. BW: body weight; FBG: fasting blood
glucose; TC: total cholesterol; TG: triglycerides; HDL-c: HDL cholesterol; LDL-c: LDL cholesterol; FFA: free fat acid; values are expressed as mean ± SD, per
group. #, 𝑝 < 0.05; ##, 𝑝 < 0.01 versus DM+con; ∗, 𝑝 < 0.05 versus NDM+con; &&, 𝑝 < 0.01 versus NDM+con.