Biochemical and Biophysical Research Communications 531 (2020) 602e608

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Biochemical and Biophysical Research Communications

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Toll-like receptor 4 plays a key role in advanced glycation end

products-induced M1 macrophage polarization
Zhongwei Liu a, Yanpeng Ma a, Qianwei Cui a, Jing Xu a, Zhiguo Tang a, Yuan Wang b, *,
Chunhui He c, **, Xi Wang d, ***
a
Department of Cardiology, Shaanxi Provincial People’s Hospital, Xi’an, Shaanxi, China
b
Department of Medical Prevention, Shaanxi Provincial People’s Hospital, Xi’an, Shaanxi, China
c
Department of Cardiology, Fuwai Hospital, National Center for Cardiovascular Diseases, Peking Union Medical College, Chinese Academy of Medical
Sciences, Beijing, China
d
Department of Obstetrics and Gynecology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China

a r t i c l e i n f o a b s t r a c t

Article history: Objective: This study was aimed to investigate the role of Toll-like receptor 4 (TLR4) in advanced gly-
Received 4 August 2020 cation end products (AGEs)- induced macrophage polarization toward M1.
Accepted 6 August 2020 Methods: Isolated primary macrophages were exposed to prepared AGEs at concentrations of 0, 2.5, 5
Available online 16 August 2020
and 10 mmol/L. Macrophages were also exposed to hydrogen peroxide (H2O2) which provided exogenous
reactive oxygen species (ROS). Receptor for AGEs (RAGE) was over-expressed by a vector. Specific siRNA
Keywords:
silencing TLR4 and inhibitor TAK-242 were used to pre-treat the macrophages. Intracellular ROS was
Macrophage polarization
determined by DCFH-DA. Immunofluorescence staining was used to evaluate the expression of inducible
Advanced glycation end products
Toll-like receptor 4
nitric oxide synthase (iNOS) which is the marker of M1 macrophage phenotype. Real-time PCR was used
Signal transducers and activators of to assess the mRNA expression level of TLR4 and RAGE. Protein expression levels of cytoplasmic RAGE,
transcription 1 TLR4, nuclear signal transducers and activators of transcription 1 (STAT1) and phosphorylation levels of
cytoplasmic STAT1 were evaluated by Western blotting. ELISA was used to measure concentrations of
interleukin 6 (IL6), IL12 and tumor necrosis factor (TNF)a in supernatant of cell culture medium of
macrophages.
Results: AGEs significantly elevated intracellular ROS generation, expression levels of iNOS, cytoplasmic
RAGE, TLR4, nuclear STAT1, phosphorylation levels of cytoplasmic STAT1, as well as IL6, IL12 and TNFa
contents in a concentration-dependent manner. TLR4 silencing and inhibitor pre-treatment reduced
expression levels of cytoplasmic RAGE, TLR4, phosphorylation of STAT1 and nuclear STAT1 in AGEs-
exposed macrophages without affecting RAGE expression and intracellular ROS production levels.
RAGE over-expression elevated both ROS and TLR4 expression levels in macrophages. TLR4 expression
elevation was also found in H2O2-treat macrophages.
Conclusion: AGEs induced macrophage polarization toward M1 via activating RAGE/ROS/TLR4/STAT1
signaling pathway.
© 2020 Elsevier Inc. All rights reserved.

1. Introduction

* Corresponding author. Department of Medical Prevention, Shaanxi Provincial

People’s Hospital, 256, Youyi Xi Rd, Xi’an, 710068, China.
** Corresponding author. Department of Cardiology, Fuwai Hospital, National
Center for Cardiovascular Diseases, Peking Union Medical College and Chinese
Academy of Medical Sciences, 167 Beilishi Rd, Beijing, 100037, China.
*** Corresponding author. Department of Obstetrics and Gynecology, The Second
Xiangya Hospital, Central South University, 139 Renminzhong Rd, Changsha,
410011, China.
E-mail addresses: ooozkb@sina.com (Y. Wang), hechunhuiyisheng@163.com
(C. He), wangxi825@csu.edu.cn (X. Wang).
Macrovascular complications are believed as major contributors
to the mortality of diabetes mellitus (DM). Atherosclerosis (AS) is
the characterized pathological manifestation which is found
accelerated under circumstance of uncontrolled hyperglycemia in
DM patients [1]. It has been supposed that the underlying mech-
anism associating DM and AS is advanced glycation end products
(AGEs) which are produced by sets of non-enzymatic glycation
reactions of proteins, nucleic acids and lipids [2].

https://doi.org/10.1016/j.bbrc.2020.08.014
0006-291X/© 2020 Elsevier Inc. All rights reserved.
 Z. Liu et al. / Biochemical and Biophysical Research Communications 531 (2020) 602e608
AS could be recognized as the chronic inflammation of arterial
wall. Many immune cells are involved. Macrophages are dominant
immune cell type in AS and participated in formation and devel-
opment of AS lesions [3]. Generally, there are M1 and M2 pheno-
types of macrophages. M1 macrophages are related with
aggravating AS lesions due to their pro-inflammatory roles [4].
Thus, it aroused our interesting to investigate the possibly under-
lying mechanisms of AGEs- induced macrophage M1 polarization.
As harmful stimuli, AGEs are linked with pathological outcomes
by interacting with their receptor RAGE, which is considered as an
important mediator of oxidative stress. Upon activation, RAGE
further mediates NADPH oxidase to generate reactive oxygen spe-
cies (ROS) [5]. According to ours and others’ previous in-
vestigations, ROS was potent to activate Toll-like receptor 4
signaling [6,7], which took responsibility in polarizing macro-
phages toward M1 phenotype [8]. TLR4 further facilitates activation
and nuclear translocation of its down-stream effecter signal
transducers and activators of transcription 1 (STAT1) which facili-
tates macrophage M1 polarization [9,10].
In the current investigation, primary peritoneal macrophages
were isolated and exposed to AGEs. Moreover, in order to study the
role of TLR4 in AGEs- induced M1 polarization, small interference
RNA (siRNA) silencing TLR4 or specific inhibitor of TLR4 were used
to treat the macrophages. We believe that results from this study
would not only add our knowledge of mechanisms concerning DM-
associated AS, but also provide new clues for gene-targeted ther-
apies in the future.
2. Material and methods
2.1. AGEs-bovine serum albumin (BSA) preparation
AGEs-BSA was prepared according to the protocol described in
our previous investigation [11]. Briefly, BSA (Hyclone) was incu-
bated with 0.1 mmol/L glyceraldehyde (Sigma) in 0.2 mmol/L
NaPO4 buffer (pH ¼ 7.4) under sterile conditions at 37  C for 7 days.
Then the unincorporated sugars were removed by using chroma-
tography with PD-10 desalting column (GE Healthcare) and dialysis
against PBS. Nonglycated BSA was prepared by the same procedure
without glyceraldehydes as control.
2.2. Peritoneal macrophage isolation
The mouse peritoneal macrophages were prepared as stationary
phenotype (M0) according to the protocol described previously
[12]. Briefly, 8-week-old BALB/c mice (SPF, Animal Experimental
Center, Xi’an Jiaotong University) received 3 mL peritoneal injection
of 2 mL 3% thioglycollate medium (Solarbio). Mice were sacrificed
by CO2 suffocation 3 days and further received peritoneal injection
of 3 mL 0.05% ethylenediaminetetraacetic acid which was dissolved
in phosphate-buffered saline (PBS). Then thioglycollate-elicited
peritoneal macrophages were harvested. Cell pellet were
collected after centrifugation at 1200g for 5 min at 4  C and then
washed by PBS. Then the resulted cells were cultured with RPMI-
1640 medium (Hyclone) supplemented with 10% FBS (Hyclone) at
37  C for 1 h. Then the unattached cells were removed after washed
by PBS three times. All animal experimental protocol were
reviewed and approved by Institutional Animal Care and Use
Committee of Xi’an Jiaotong University.
2.3. RAGE over-expression vector transfections
The cDNA sequence of RAGE was synthesized and inserted into
pcDNA3.1() vector (Invitrogen) to construct a recombinant
pcDNA3.1()/RAGE over-expression vector by GenePharma based
603
on previous publications [13,14]. Vectors were mixed with Lip-
ofectamine 2000 transfection reagent (Invitrogen) at a 1:1 ratio (v/
v) at 25  C for 20 min. Macrophages were then incubated with FBS-
free RPMI1640 medium supplemented with pcDNA3.1()/RAGE
over-expression vector at final concentration of 60 nmol/L for 6 h.
Then the medium was replaced with RPMI1640 medium supple-
mented with FBS.
2.4. siRNA transfection
RNA interfering (RNAi) technique was used to silence TLR4
expression in macrophages. The targeting sequence of siRNA
against tlr4 was 50 -GUCUCAGAUAUCUAGAUCU-30 which has been
proved effective previously [15]. When assistance of HiPerFect
siRNA transfection reagent (Qiagen), the synthesized siRNA (Gen-
ePharma) or scramble control siRNA (GenePharma) were trans-
fected into macrophages at final concentration of 10 mmol/L.
2.5. Cell treatments
For AGEs exposure, the cultured macrophages were respectively
treated with serially diluted AGEs at final concentrations of 2.5, 5
and 10 mmol/L for 48 h. For exogenous ROS stimulation, macro-
phages were treated with hydrogen peroxide (H2O2) at final con-
centration of 10, 20 and 50 mmol/L for 24 h [16]. Several
macrophages were transfected with above siRNA or pcDNA3.1()/
RAGE over-expression vector 12 h prior to AGEs exposure. Several
macrophages were pre-treated with specific TLR4 inhibitor TAK-
242 (Abmole) at final concentration of 10 mmol/L 12 h prior to
AGEs administration.
2.6. In situ ROS detection
2,7- dichlorofluorescein (DCFH-DA, Molecular Probes) was used
to detect the intracellular ROS generation in situ. Harvested mac-
rophages were washed by PBS and then incubated with DCFH-DA at
final concentration of 10 mmol/L in a humidified dark chamber at
37  C for 30 min. Fluorescent images were then captured using an
inverted fluorescence microscope. Fluorescent intensities which
stood for ROS levels were analyzed by using ImagePro Plus (Version
5.0) software.
2.7. Immunofluorescence staining
The immunofluorescence staining was carried out according to
our previous description [17]. Harvested macrophages were pre-
pared as slides and fixed with 4% paraformaldehyde. After per-
meabilized with 0.2% Triton X-100 (Sigma Aldrich) and blocked
with blocking buffer (Abcam), slides were incubated with primary
antibody against inducible nitric oxide synthase (iNOS) at 4  C for
8 h. After washing, slides were then incubated with Alexa Fluor-488
conjugated secondary antibody (Invitrogen) at room temperature
in dark for 1 h. An inverted fluorescence microscope was used to
capture the fluorescent images which were further analyzed by
using ImagePro Plus (Version 5.0) software.
2.8. Enzyme linked immunosorbent assay (ELISA)
Pro-inflammatory cytokines concentrations in cell culture me-
dium supernatant were measured by using interleukin (IL)6 ELISA
kit, IL12 ELISA kit and tumor necrosis factor (TNF)a ELISA kit
(Beyotime). Measurements were carried out according to the pro-
tocols provided by the manufacturer.
604 Z. Liu et al. / Biochemical and Biophysical Research Communications 531 (2020) 602e608

2.9. Real-time polymerase chain reaction (PCR)
Real-time PCR was used to determine the expression level of
TLR4 at transcription level. TRIzol (TaKaRa) was used to extract total
RNA from macrophages, which were then reversely transcribed
into cDNA with PrimeScript RT Master Mix (TaKaRa). SYBR Premix
Ex Taq™ II (TaKaRa) was used to perform real-time PCR. GAPDH
was used as internal control. Gene expression levels were
expressed as fold changes which were calculated using 2-△△ct
method. The primers were listed below: RAGE forward: 50 -
GGACCCTTAGCTGGCACTTAGA-3’; reverse: 50 - GAGTCCCGTCT-
CAGGGTGTCT-3’. TLR4 forward: 50 -AGACATCCAAAGGAA-
TACTGCAA-3’; reverse: 50 -GCCTTCATGTCTATAGGTGATGC-3’.
GAPDH forward: 50 -CTCCATTCCTCCTCCAGACACT-3’; reverse: 50 -
AAGGAGAGGCAGTTTGGCTTC-3’.
2.10. Western blotting
Harvested macrophages were lysed with RIPA lysis buffer (Santa
Cruz) supplement with PMSF (Santa Cruz). Cytoplasmic protein and
nuclear protein were extracted by using Cytoplasmic Protein
Extraction Kit (Beyotime) and Nuclear Protein Extraction Kit
(Beyotime). A BCA kit (Invitrogen) was used to determine the
protein concentrations. Protein samples were then subjected to 10%
SDS-PAGE then transferred to PVDF membranes. After blocked with
blocking buffer (Abcam) for 1 h, the membranes were then incu-
bated with antibodies against RAGE (Abcam, 1:500), TLR4 (Abcam,
1:1000), phosphor-STAT1 (CST, 1:500), STAT1 (CST, 1:500), GAPDH
(Abcam, 1:5000) and Histone H3 (CST, 1:2000) at 4  C for 8 h. After
washed by TBST, the membranes were then incubated with HRP-
conjugated secondary antibodies (Abcam) at room temperature for
1 h. Then the membranes were developed by Western Blotting
Luminal Reagent (Santa Cruz) and visualized on X-ray films. Soft-
ware ImagePro Plus (Version 5.0) was used to analyze the optic
densities of the immunoblots.
2.11. Statistics
All data were presented in a (mean ± SD) manner in this study
which were then analyzed by using SPSS (Version 17.0) software.
One-way analysis of variance (ANOVA) was used to analyze the
differences between groups. Difference were considered statisti-
cally significant when P < 0.05.
3. Results
3.1. AGEs exposure induced macrophage M1 polarization which
was suppressed by both TLR4 silencing and inhibitor
As demonstrated in Fig. 1A and Fig. 1B, the tlr4-siRNA trans-
fection significantly down-regulated the expression of TLR4 in
macrophages. The expression of iNOS which is the marker of M1
macrophage phenotype was detected by immunofluorescence
staining. As shown in Fig. 1C, AGEs treatment increased iNOS

Fig. 1. A: columns indicated relative expression levels of tlr4 mRNA in negative control macrophages or macrophages transfected with scramble siRNA or tlr4-siRNA. B: left panel
demonstrated immunoblots of TLR4 and GAPDH in macrophages. Columns on the right panel indicated relative expression levels of TLR4 in negative control macrophages or
macrophages transfected with scramble siRNA or tlr4-siRNA respectively. C: captured immunofluorescent staining images of iNOS, DAPI and their merged results were demon-
strated on the left side. Columns on the right side indicated mean fluorescence intensities of Alexa Fluor-488 in AGEs- exposed macrophages which pre-treated with tlr4-siRNA or
TLR4 specific inhibitor TAK-242 respectively. (n ¼ 6, [*P < 0.05]).
 Z. Liu et al. / Biochemical and Biophysical Research Communications 531 (2020) 602e608 605

expression on macrophages in a concentration-dependent manner. which was reduced by both tlr4-siRNA and TAK-242 pre-
Both of the tlr4-siRNA and TLR4 inhibitor pre-treatment signifi- treatments.
cantly reduced iNOS expression on AGEs-exposed macrophages.

3.2. TLR4 silencing and inhibition failed affecting RAGE/ROS 3.4. AGEs exposure increased macrophage M1 phenotypic cytokines
signaling activation in macrophages exposed to AGEs secretion which were reduced by TLR4 silencing and inhibition

Macrophage RAGE expression levels were assessed by Western
blotting which were demonstrated in Fig. 2A. DCFH-DA assay was
used to determine the intracellular ROS generation in situ which
was demonstrated in Fig. 2B. AGEs exposure significantly increased
RAGE expression in macrophages in a concentration-dependent
manner. The tlr-siRNA and TAK-242 pre-treatment did not show
any significant effects on RAGE expression and intracellular ROS in
AGEs-exposed macrophages.
The results from ELSIA which was used to measure the M1
phenotypic pro-inflammatory cytokines concentrations in cell
culture medium supernatant were demonstrated in Fig. 4A. AGEs
exposure significantly elevated IL6, IL12 and TNFa concentrations
in culture medium supernatant of macrophages in a concentration-
dependent manner. The pre-treatment of both tlr4-siRNA and TLR4
inhibitor TAK-242 reduced IL6, IL12 and TNFa concentrations in
culture medium supernatant from AGEs-exposed macrophages.

3.3. TLR4 depletion and inhibition reduced STAT1 signaling

activation in AGEs- incubated macrophages
The activation of STAT1 signaling in macrophages was elevated
by Western blotting of extracted cytoplasmic and nuclear protein.
As demonstrated in Fig. 3A and Fig. 3B, AGEs exposure significantly
up-regulated expression levels of TLR4 as well as phosphorylation
levels of STAT1 in cytoplasm, which were dramatically inhibited by
tlr4-siRNA pre-treatment. The pre-treatment of TLR4 specific in-
hibitor TAK-242 down-regulated STAT1 phosphorylation level
without affecting TLR4 expression level in cytoplasm. As demon-
strated in Fig. 3C, AGEs exposure significantly increased STAT1
expression levels in nuclei in a concentration-dependent manner
3.5. RAGE/ROS was identified as the up-stream signaling of TLR4 in
macrophages
In order to testify ROS/TLR4 was the down-stream effecter of
RAGE signaling, a recombinant pcDNA3.1()/RAGE over-expression
vector was transfected into macrophages. As demonstrated in
Fig. 4B, RAGE over-expression significantly increased intracellular
ROS generation as well as TLR4 expression in macrophages.
Moreover, in order to testify TLR4 was the down-stream effecter of
ROS, macrophages were exposed to exogenous ROS originating
from H2O2. As shown in Fig. 4C, H2O2 exposure significantly up-
regulated TLR4 expression in a concentration-dependent manner.

Fig. 2. A: immunoblots of RAGE and GAPDH in macrophages were demonstrated on the left side. Columns indicated the relative expression levels of RAGE in AGEs- exposed
macrophages which pre-treated with tlr4-siRNA or TLR4 specific inhibitor TAK-242 respectively. B: captured fluorescent images of DCFH-DA, bright images of light microscope and
their merged results were demonstrated. Columns indicated mean fluorescence intensities of DCFH-DA in AGEs- exposed macrophages which pre-treated with tlr4-siRNA or TLR4
specific inhibitor TAK-242 respectively. (n ¼ 6, [*P < 0.05]).
606 Z. Liu et al. / Biochemical and Biophysical Research Communications 531 (2020) 602e608

Fig. 3. A: immunoblots of TLR4 and GAPDH in macrophage cytoplasm were demonstrated. Columns indicated the relative expression levels of cytoplasmic TLR4 in AGEs- exposed
macrophages which pre-treated with tlr4-siRNA or TLR4 specific inhibitor TAK-242 respectively. B: left panel demonstrated immunoblots of phosphorylated STAT1 (S727) and
STAT1 in macrophage cytoplasm. Columns indicated the relative phosphorylation levels of cytoplasmic STAT1 in AGEs- exposed macrophages which pre-treated with tlr4-siRNA or
TLR4 specific inhibitor TAK-242 respectively. C: immunoblots of STAT1 and Histone H3 in macrophage nuclei were demonstrated. Columns indicated relative expression levels of
nuclear STAT1 in AGEs- exposed macrophages which pre-treated with tlr4-siRNA or TLR4 specific inhibitor TAK-242 respectively. (n ¼ 6, [*P < 0.05]).

4. Discussion experienced higher risks of developing adverse cardiovascular

Macrophages are recruited and accumulated, contributing to the
development and progress of AS plaques. AS macrophages exhibit
heterogeneous phenotypes presenting distinct functions in
response to different external stimuli. There are mainly two
macrophage phenotypes which are known as the classically acti-
vated pro-inflammatory macrophages (M1) and alternatively acti-
vated anti-inflammatory macrophages (M2) [18,19]. Functionally,
M1 macrophages take part in exacerbating AS plaque progression
and instability by producing pro-inflammatory cytokines and
inducing oxidative stress. Macrophages located in the plaque
shoulder which is believed as the most unstable region of AS plaque
mainly present M1 phenotype [20].
Multiple clinical trials have revealed the fact that DM patients
events such as myocardial infarction and stroke [21]. These events
were pathologically characterized by AS. In other words, the AS is
more aggressive under circumstance of DM. AGEs are largely
fostered in DM and have been considered as typical pathological
diabetic metabolite. Content of AGEs was supposed to be associated
with the major adverse cardiovascular events (MACE) of patients
with DM [22]. Thus, the aggravating role of AGEs in AS progression
could be proposed. In the present study, we found that AGEs
exposure significantly polarized quiescent macrophages toward M1
phenotype which was evidenced by increase of M1 macrophage
hall marker iNOS. This could be one of the underlying mechanisms
explaining AS lesions exacerbation in DM.
AGEs act as oxidative stress inducer in multiple diabetic organs
by activating RAGE/NADPH oxidase [23]. M1 macrophages are
 Z. Liu et al. / Biochemical and Biophysical Research Communications 531 (2020) 602e608 607

Fig. 4. A: Columns indicated the concentrations of IL6 (A), IL12 (B) and TNFa (C) in supernatant from culture medium of AGEs- exposed macrophages which pre-treated with tlr4-
siRNA or TLR4 specific inhibitor TAK-242 respectively. B: immunoblots of RAGE, TLR4 and RAGE were demonstrated. Columns indicated the relative expression levels of tlr4 mRNA,
TLR4, rage mRNA and RAGE in macrophages not transfected or transfected with pcDNA3.1()/RAGE over-expression vector respectively. Captured fluorescent images of DCFH-DA,
bright images of light microscope and their merged results were demonstrated. Columns indicated the mean fluorescence intensities of DCFH-DA in macrophages not transfected or
transfected with pcDNA3.1()/RAGE over-expression vector respectively. C: immunoblots of TLR4 and GAPDH were demonstrated. Columns indicated relative expression levels of
tlr4 mRNA and TLR4 in macrophages treated with H2O2 at concentrations of 0, 10, 20 and 50 mmol/L respectively. (n ¼ 6, [*P < 0.05]).

potent of producing excessive intracellular ROS [24]. Results from
our current study showed that AGEs exposure dramatically
increased RAGE expression and further caused elevation of intra-
cellular ROS contents. Our previous investigation indicated that
accumulation of intracellular ROS caused up-regulation of TLR4
expression in experimental diabetes animal model [6]. TLR4 is a key
member of TLR family which participates in immune responses in
mammals. Notably, elevated TLR4 expression was noted in many
cell types under circumstance of DM [25,26]. In the current study,
we found that TLR4 expression was increased in AGEs- exposed
macrophages followed by activation of RAGE/ROS signaling. Ac-
cording to previous reports, inhibition of TLR4 suppressed macro-
phages polarization toward M1 [27]. Indeed, M1 polarization was
significantly blocked by both TLR4 silencing and inhibitor treat-
ment in macrophages exposed to AGEs. Notably, TLR4 silencing and
inhibition did not alter RAGE and ROS levels, indicating TLR4 was
the down-stream pathway of RAGE/ROS signaling. Our observation
that over-expression of RAGE induced ROS and TLR4 expression and
exogenous ROS stimulated TLR4 expression testified the composi-
tion of RAGE/ROS/TLR4 signaling in macrophages. Thus, it is
reasonable for us to speculate that RAGE/ROS/TLR4 signaling take
responsibility in AGEs- induced macrophage M1 polarization.
It is believed that STAT1 plays a role modulating the crosstalk
between inflammatory responses and TLRs. In a previous study,
TLR4 was proved to phosphorylate STAT1 at residue 727 (S727)
which further translocated to nucleus [9]. Indeed, in this study,
TLR4 silencing and inhibitor treatment dramatically inhibited
STAT1 phosphorylation and nuclear translocation in AGEs- exposed
macrophages. STAT1 was reported to initiate transcription of its
down-stream genes such as iNOS and to interact with nuclear
factor kB (NFkB) to trigger production of pro-inflammatory cyto-
kines [28,29]. In this study, both TLR4 silencing and inhibitor
treatment were found effective in suppressing production of M1
phenotypic cytokines including IL6, IL12 and TNFa in macrophages
exposed to AGEs.
In summary, we can conclude that AGEs induced macrophages
polarization toward M1 by activating RAGE/ROS/TLR4/STAT1
signaling pathway. TLR4 played a key role in this process by linking
oxidative stress and biological effects of nuclear factor STAT1. These
results would enrich our current interpretation on DM associated
rapid AS plaque progression. More importantly, TLR4 of AS macro-
phages could be potentially considered as an ideal therapeutic target.
608 Z. Liu et al. / Biochemical and Biophysical Research Communications 531 (2020) 602e608
Funding sources
This study was supported by Fundamental Scientific Research
Foundation of Xi’an Jiaotong University, Health Scientific Founda-
tion of Shaanxi Province, Innovative Talents Promotion Project of
Shaanxi Province, and Natural Science Basic Research Foundation of
Shaanxi Province.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2020.08.014.
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