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Research article
Received: 17 December 2014, Revised: 4 February 2015, Accepted:
4 February 2015 Published online in Wiley Online Library: 15 April
2015
(wileyonlinelibrary.com) DOI 10.1002/jat.3143
48
PM2.5-induced oxidative stress increasesadhesion molecules
expression in humanendothelial cells through the
ERK/AKT/NF-κB-dependent pathwayWei Ruia, Longfei Guana, Fang
Zhanga*, Wei Zhang and Wenjun Dinga*
ABSTRACT: The aim of this study was to explore the intracellular
mechanisms underlying the cardiovascular toxicity of air
partic-ulate matter (PM) with an aerodynamic diameter of less than
2.5μm (PM2.5) in a human umbilical vein cell line, EA.hy926.
Wefound that PM2.5 exposure triggered reactive oxygen species (ROS)
generation, resulting in a significant decrease in cell
viability.Data fromWestern blots showed that PM2.5 induced
phosphorylation of Jun N-terminal kinase ( JNK), extracellular
signal regula-tory kinase (ERK), p38 mitogen-activated protein
kinase (MAPK) and protein kinase B (AKT), and activation of nuclear
factorkappa B (NF-κB). We further observed a significant increase
in expressions of intercellular adhesion molecule-1 (ICAM-1)
andvascular adhesionmolecule-1 (VCAM-1) in a time- and
dose-dependentmanner.Moreover, the adhesion ofmonocytic THP-1
cellsto EA.hy926 cells was greatly enhanced in the presence of
PM2.5. However, N-acetylcysteine (NAC), a scavenger of ROS,
preventedthe increase of ROS generation, attenuated the
phosphorylation of the above kinases, and decreased the NF-κB
activation aswellas the expression of ICAM-1 and VCAM-1.
Furthermore, ERK inhibitor (U0126), AKT inhibitor (LY294002) and
NF-κB inhibitor(BAY11-7082) significantly down-regulated
PM2.5-induced ICAM-1 and VCAM-1 expression as well as adhesion of
THP-1 cells,but not JNK inhibitor (SP600125) and p38MAPK inhibitor
(SB203580), indicating that ERK/AKT/NF-κB is involved in the
signalingpathway that leads to PM2.5-induced ICAM-1 and VCAM-1
expression. These findings suggest PM2.5-induced ROSmay function
assignalingmolecules triggering ICAM-1 and VCAM-1 expressions
through activating the ERK/AKT/NF-κB-dependent pathway, andfurther
promoting monocyte adhesion to endothelial cells. Copyright © 2015
John Wiley & Sons, Ltd.
Keywords: PM2.5; oxidative stress; adhesion factor;
ERK/AKT/NF-κB; reactive oxygen species; EA.hy926 cells
*Correspondence to: Fang Zhang and Wenjun Ding, Laboratory of
Environmentand Health, College of Life Sciences, University of
Chinese Academy of Sciences.No. 19A Yuquan Road, Beijing 100049,
China.E-mail: [email protected]; [email protected]
aLaboratory of Environment and Health, College of Life Sciences,
University ofChinese Academy of Sciences, No. 19A Yuquan Road,
Beijing100049, China
IntroductionAirborne fine particles with a mean aerodynamic
diameter of lessthan 2.5μm (PM2.5), a component of air pollution,
has been epide-miologically associated with cardiovascular diseases
(Peters et al.,1997; Pope et al., 2004; Alfaro-Moreno et al., 2007;
Laden et al.,2006; Riediker, 2007). Accumulating evidences in vivo
studies havebeen proposed that exposure to environmentally relevant
inhaledparticulate matter enhances atherosclerosis (AS) through
induc-tion of vascular reactive oxygen species (ROS) (Ying et al.,
2009;Hemmingsen et al., 2011; Wauters et al., 2013). In the
cardiovascu-lar system, expression of cell adhesion molecules is
one of the ini-tial events by activated endothelium. Endothelial
activation,characterized by increased inflammation, is an early
event in endo-thelial dysfunction (Horstman et al., 2004). Cell
adhesionmoleculesinduce monocytes adhesion and transmigration to
thesubendothelial space (Wood et al., 1993; Krieglstein and
Granger,2001). Normally, intercellular adhesion molecule-1
(ICAM-1,CD54) and vascular adhesion molecule-1 (VCAM-1, CD106), as
im-portant cell adhesion molecules, are members of the
immuno-globulin superfamily of proteins and crucially mediates
theadhesion of lymphocytes, monocytes, eosinophils and basophilsto
vascular endothelium (Hubbard and Rothlein, 2000; Barreiroet al.,
2002; Yang et al., 2005). The interaction between ICAM-1and VCAM-1
and their specific ligands expressed onmicrovascularendothedlial
cells could be involved in cell adhesion (Collins et al.,2000;
Cybulsky et al., 2001). ICAM-1 and VCAM-1 expressions are
J. Appl. Toxicol. 2016; 36: 48–59 Copyright © 2015 John
the consequence of stimulation by specific molecules
includingtumor necrosis factor-α (TNF), interleukin-1 (IL-1) and
interferon-γ(IFN-γ) (Kume et al., 1992; Springer, 1994). However,
the mecha-nisms responsible for the effects of PM2.5-induced
oxidative stresson ICAM-1 and VCAM-1 expressions at the surface of
endothelialcells have not been fully elucidated.
PM2.5-induced oxidative stress has been considered as a
keymolecular mechanism of PM2.5-mediated toxicity (Deng et
al.,2013). Oxidative stress is caused by an imbalance
betweenproduction of reactive oxygen species (ROS) and activity of
anti-oxidant mechanisms (Limon-Pacheco and Gonsebatt, 2009). ROSare
comprised of oxygen radicals such as the superoxide anion(O2
·-), alkoxyradicals (RO·), peroxy radicals (ROO·) and hydroxyl
radical(·OH) as well as non-radical forms like hydrogen peroxide
(H2O2) orother hydroperoxides (ROOH). A study by Kunsch and
Medford(1999) has implicated ROS as signal transduction agents in
strainedendothelium.
Exposure of cells to ROS or ROS generation systems has longbeen
known to be capable of stimulating various cellular signaling
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PM2.5 effect adhesion molecule expression via the ERK/AKT/NF-κB
pathway
49
pathway (Eckers and Klotz, 2009). ROS appear to be involved in
theactivation of redox-sensitive signal transduction such as
themitogen-activated protein kinases (MAPKs) and PI3K/AKT path-ways
(Harris and Shi, 2003; Huang et al., 2011; Naik and Dixit,2011).
MAPK has been implicated in cardiovascular disease (Wadaand
Penninger, 2004; Panka et al., 2006). MAPK is a group of pro-tein
Serine/threonine kinases that are activated in response to avariety
of extracellular stimuli and mediate signal transductionfrom the
cell surface to the nucleus, including c-Jun NH2-terminalkinases (
JNK), extracellular signal-regulated kinases (ERK) and p38MAPK
(Davis, 2000). ERKs, AKT kinases, stress-activated proteinkinases
and nuclear factor-kappa B (NF-κB) are the potentialtargets of ROS
in endothelial cells (Singh et al., 2002). Among them,ERKs are
important mediators of cell proliferation. JNK and p38MAPK as
stress-activated protein kinases are involved in apoptosis.p38 MAPK
has been implicated in the upregulation of ICAM-1resulting in
endothelial dysfunction. NF-κB are involved in endothe-lial
dysfunction and vascular inflammation (Gilmore, 2006), whereasAKT
kinases has been associated with antiapoptotic signaling andare
regulated by ROS in the angiotensin II-stimulated smoothmusclecells
(Irani, 2000). Therefore, ROS serve as important cellular
signalingmechanisms that may be responsible for the induction of
vascularlesions and subsequent atherosclerosis (Singh et al.,
2002).
Using 99mtechnetium-labeled carbon particles, which are
verysimilar to the ultrafine fraction of actual pollutant
particles,Nemmar et al. (2002) demonstrated that inhaled particles
pass rap-idly into the systemic circulation in healthy volunteers,
suggestingdirect impacts on the heart and vessels of particulate
air pollution.In the present study, using a human endothelial cell
line, EA.hy926,as an in vitromodel, we addressed the question of
whether PM2.5-induced ROS generation affects ICAM-1 and VCAM-1
expressionsand the adhesion of human monocytic THP-1 cells to the
EA.hy926 monolayer. It is known that EA.hy926 cells normally
expressICAM-1 and VCAM-1, so this cell line may be useful model to
studythe interactions of cardiovascular endothelial cells with
stressfulstimuli (Yu et al., 2010; Wei et al., 2011; Yi et al.,
2012; Napierskaet al., 2013). The involvement of ROS-dependent JNK,
ERK1/2,p38 MAPK, AKT phosphorylation and NF-κB activation were
alsoevaluated using selective kinase inhibitors.
Materials and Methods
Materials
A human umbilical vein cell line (EA.hy926) cells were
obtainedfrom the China Center for Type Culture Collection
(Shanghai,China). Human monocytic leukaemia cell line THP-1 cells
wereobtained from the Cell Bank of Peking Union Medical
College(Beijing, China). Dulbecco’s Modified Eagle’s Medium
(DMEM),Roswell Park Memorial Institute (RPMI) 1640 medium
andpenicillin/streptomycin were purchased from Gibco (Grand island,
NY,USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide(MTT), dimethylsulfoxide (DMSO),
2’,7’-dichlorodihydrofluoresceindiacetate (DCFH-DA) were purchased
from Sigma (St Louis, MO,USA). SP600125, SB203580, BAY11-7082 and
LY294002 wereobtained from Beyotime (Shanghai, China). U0126 was
obtainedfrom Promega (Madison, WI, USA). Fetal bovine serum was
pur-chased from PAA (Linz, AUS). Twenty-four well, 6-wells,
96-wellsplates and cell culture dishes were obtained from
CostarCambridge (Tewksbury, MA, USA). FITC-anti-ICAM-1 and
PE-anti-VCAM-1 antibody were purchased from Biolegend (San
Diego,CA, USA). JNK, ERK1/2, p38 MAPK, AKT, p65, phospho-JNK,
J. Appl. Toxicol. 2016; 36: 48–59 Copyright © 2015 John
phospho-ERK1/2, phospho-p38, phospho-AKT antibodies
werepurchased from Bioworld (Louis Park, MN, USA).
Particle Collection and Preparation
PM2.5 samples were collected continuously at Yuquan
Road,Beijing, China in September to October 2012. The sampling
sitewas located in University Campus, about 20m from a
streetcharacterized by moderate traffic and commercial activities.
Theresidential area is characterized by low traffic density. In
thesurrounding areas, large industrial and thermoelectric plants
wereabsent. Sampling inlets were installed at a height of 10m
abovethe ground. PM2.5 samples were collected on Teflon
filters(diameter=47mm; Whatman, Piscataway, NJ, USA) for
biologicalassay using a low volume samplers (42 lmin–1, URG, Chapel
Hill,NC, USA) for 12h (08:00 to 20:00 hours). Before or after
sampleswere collected, the Teflon filters were equilibrated in a
conditionof 30% relative humidity and 25 °C room temperature for
over48h and then weighted on a high-precision microbalance(AG258;
Mettler Toledo, Columbus, OH, USA) to measure the massof collected
PM2.5. All sampled filters were stored in the darkness at–20 °C
before further chemical and physical characterization.Blanks
(unexposed filters) were prepared using the same methodas for the
samples except for sampling, which were used as acontrol in all
experiments.PM2.5 samples on Teflon filters were extracted
according to the
method of Imrich et al. (2000). Briefly, Teflon filters were put
into a50-ml centrifuge tube, probe-sonicated for 1min in
ultra-pure(18.2 MΩ/cm) water, the were then dried filters by drying
oven,equilibrated for 48 h and weighed on a microbalance.
Accordingthe weight decrease of filters, we qualified the mass of
particleswhich suspend in water. The extract particles were put
together,andmade the concentration at 5mgml–1, then stored at –80
°C untilphysical and chemical characterization analysis or cell
exposure.
PM2.5 Physical and Chemical Characterization
The size distribution of PM2.5 was performed by scanning
electronmicroscopy (SEM, JSM-6700F, JEOL, JAPAN) at a magnification
of30 000× and accelerating voltage of 5 kV. After being dried
over-night, the samples were fixed onto stubs by double-sided
tapeand sputter coated with gold for scanning electron
microscope(SEM) observation. The size distribution of PM2.5 in
suspensionwas analyzed using a Nano-Zetasizer (1000 HS; Malvern
Instru-ment Ltd., Worcestershire, UK) based on a dynamic light
scatteringmeasurement technique. Before measurement, PM2.5 was
firstlysuspended in serum-free culture medium, and sonicated for 30
sat 40W in a bath to disperse the PM2.5 using an ultrasonic
processor(VCX130, Sonics, Newtown, CT, USA). The particle Z-Average
wasreported.Inorganic elements, organic and elemental carbon (OC
and EC),
water soluble inorganic ions and polycyclic aromatic
hydrocarbons(PAHs) were detected in our extract PM2.5 samples.
200μl (1mg)PM2.5 samples were used for inorganic elements detection
by aciddigestion (HNO3:HF=7:3), followed by measurement using
induc-tively coupledplasma-mass spectrometry (ICP-MS, Thermo,
ElementalX7, Waltham, MA, USA) for concentrations of Ti, V, Cr, Mn,
Co, Ni, Cu,As, Sr, Mo, Cd, Cs, Ba, Pb, Zn, Fe, Ca, K, Mg and Na.
The water solubleinorganic ion composition (SO4
2-, NO3- , NH4
+ and Cl-) was determinedwith ion chromatography (Dionex-600,
Sunnyvale, CA, USA).PM2.5 samples were frozen drying to powder to
detect OC/EC
and PAHs. PM2.5 powder were put on the quartz filters which
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W. Rui et al.
50
preheated at 450 °C for 4 h, then EC and OC in PM2.5
weremeasured on-filter using a thermal-optical analyzer
(SunsetLaboratories, Hillsborough, NC, USA) according to the
ACE-Asiabase case protocol as described in the methods of Zhanget
al. (2008). PAHs was measured by a thermal desorption at300 °C
coupled with cold trapping, and followed by gaschromatography-mass
spectrometry (GC/MS) analyzes (Model6890N; Aglient, Santa Clara,
CA, USA) equipped with a 60-mHP-5MS column as described by Zhang et
al. (2009). FifteenPAHs were analyzed in the PM2.5 samples
including acenaphthylene,acenaphthene, fluorene, phenanthrene,
anthracene, fluoranthene,pyrene, benz[a]anthracene, chrysene,
benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene,
indeno[123-cd]pyrene, dibenz[a,h]anthracene and coronene.
Cell Culture and PM2.5 Treatment
Human umbilical vein EA.hy926 cells were cultured in high
glucoseDMEM supplemented with 10% (v/v) FBS, 100 IUml–1
penicillinand 100μgml–1 streptomycin at 37 °C in a 5% CO2
humidifiedatmosphere. All cell exposure experiments were performed
at80–90% of cell confluence with viability≥ 90% determined bythe
trypan blue staining. The cells were harvested using 0.25%trypsin
and sub-cultured into 6-well plates, 24-well plates, or96-well
plates according to the selection of experiments. Cellswere allowed
to attach the surface in DMEM supplemented 10%FBS for 24 h prior to
treatment. Then, the culture medium wasreplaced with serum-free
medium and the cells were incubatedwith freshly dispersed PM2.5
preparations at a final concentrationof 25, 50, 100 or 200μgml–1
for 1, 3, 6, 12 or 24h at 37 °C with orwithout NAC (5mM)
pretreatment, respectively. For the inhibitoryeffect experiments,
the cells were pretreated with or without inhi-bitors of JNK
(SP600125, 25μm), ERK (U0126, 10μm), p38 MAPK(SB203580, 25μm) and
PI3K/AKT (LY294002, 25μm) or NF-κB(BAY11-7082, 5μm) and then
treated with PM2.5 for the indicatedconcentration and duration,
respectively.
Human monocytic leukaemia cell line THP-1 cells were culti-vated
in suspension in RPMI-1640 medium containing 10% FBS,100 IUml–1
penicillin and 100μgml–1 streptomycin at 37 °C in 5%CO2 and 95% air
humidified atmosphere.
MTT Assay
Cell viability was assessed using the MTT assay (Wei et al.,
2011).The EA.hy926 cells were plated into 96-well plates at a
density of1.0 × 104 cells per well in 100μl of medium and cultured
for 24 h.After incubation, EA.hy926 cells were treated with 0, 25,
50, 100and 200μgml–1 PM2.5 for 1, 3, 6, 12 and 24h, respectively.
Afterthe exposure, 20μl of MTT (0.5mgml–1 in PBS) was added to
eachwell and incubated for 1 h at 37 °C. Cells were then treated
with100μl of dimethyl sulfoxide (DMSO). The absorbance was
quanti-fied at 492nm by a microplate spectrophotometer (Thermo
MK3,Waltham, MA, USA). The viability of the treated cells was
expressedas a percentage of untreated cells, whichwas assumed to be
100%.
Reactive Oxygen Species Assay
The level of intracellular ROS generation was determined
bymeasuring the oxidative conversion of DCFH-DA to
fluorescentcompound dichlorofluorescin (DCF). Thus, the
intracellular ROSgeneration of cells can be investigated using the
DCFH-DA as anindicator to detect and quantify intracellular
produced reactive
Copyright © 2015 Johnwileyonlinelibrary.com/journal/jat
oxygen species (Wan et al., 1993; Deng et al., 2013). Briefly,
EA.hy926 cells (2× 105 cells) were treated with 25, 50, 100
and200μgml–1 PM2.5 for 1, 3, 6, 12 and 24h, respectively. The
cellswere lysed with 400mM of NaOH. The fluorescence intensity
wasdetected in a fluorescencemulti-well plate reader (Berthold,
TriStarLB 941, Baden-Württemberg, Germany) with excitation
andemission wavelengths of 488 nm and 530nm. Results weremeasured
as the fluorescence intensity change of untreated cells.
Western blotting analisis
After exposure to PM2.5, EA.hy926 cells were separated from
theculture medium and lysed in RIPA buffer (50mM Tris-HCl, pH
8.0,150mMNaCl, 1% Triton X-100, 0.5% sodium deoxycholate
supple-mented with protease inhibitor cocktail) and phosphotase
inhi-bitor cocktail at 4 °C for 30min. Whole cell lysates were
collectedusing centrifugation at 12 000 g for 5min at 4 °C. Nuclear
proteinswere purified using a nuclear and cytoplasmic protein
extractionkit (Beyotime, Beijing, China) according to the
manufacturer’sinstruction. Then proteins were subjected to 10%
sodium dodecylsulfate-polyacrylaminde gel electrophoresis
(SDS-PAGE). The gelswere transferred to a PVDF membrane by semidry
electrophoretictransfer at 1mA/cm2 for 60min. The PVDF membranes
were thenblocked in 5% non-fat milk at room temperature for 1 h and
thenincubated with the primary antibodies diluted (1:1000)
inTris/buffered saline/Tween-20 (TBST) containing 5% bovine
serumalbumin for overnight in 4 °C, and then incubated with the
secon-dary antibody diluted (1:1000) at room temperature for 1 h.
Themembranes were washed with TBST four times for 5min
each.Immunoreactive bands were detected with ECL
reagents(Millipore, Billerica, MA, USA) according to the
manufacturer’sinstructions. β-Actinwas used as loading controls for
the total proteincontent and showed no differences between
groups.
Analysis for Adhesion Molecule Expression
The levels of ICAM-1 and VCAM-1 expression in EA.hy926 cellswere
detected using flow cytometry analysis as previouslydescribed (Ali
et al., 2004; Huang et al., 2014). After EA.hy926cells were exposed
to 0, 25, 50 or 100μgml–1 PM2.5 for 24h or100μgml–1 PM2.5 for 1, 3,
6, 12 and 24h, respectively. Cellswere harvested from plates with
trypsin and centrifuged at 800 gfor 5min. The pellet was labeled
with anti-ICAM-1 antibody (1:50dilution) or anti-VCAM-1 antibody
(1:50 dilution) in PBS supple-mented with 0.3% of bovine serum
albumin (PBSA) at 4 °C for30min in the dark. Subsequently, the
labeled cells were rinsedtwice with cold phosphate-buffered saline
(PBS) in microfugetubes and centrifuged at 800 g for 5min to remove
anyunattached antibody. ICAM-1 and VCAM-1 expression weremeasured
by a flow cytometer (Beckman FC500, Brea, CA, USA)and expressed as
the mean fluorescence intensity.
Preparation of Fluorescent THP-1 Cells
To visualize the THP-1 cells adherent to the EA.hy926 cells,
fluores-cent THP-1 cells were prepared according to the method of
Kinardet al. (2001). In brief, the THP-1 cells were labeledwith a
fluorescentdye, calcein-AM, by incubating 1.0× 107 cells in 4ml of
RPMI-1640containing 10% FBS and 40μl of calcein-AM to make a
finalcalcein-AM concentration of 10μM at 37 °C for 1 h. Dye
loadingwas stopped by adding 4ml of cold RPMI-1640 containing
10%FBS to the suspension and then centrifuging.
Fluorescence-labeled
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PM2.5 effect adhesion molecule expression via the ERK/AKT/NF-κB
pathway
cells were resuspended in 4ml of RPMI-1640 containing 10% FBSand
prepared for use in adhesion experiments.
Adhesion Experiment of THP-1 Cells
To investigate the effect of the concentration of PM2.5 added to
acell culture medium on the adhesion of THP-1 cells to
EA.hy926cells, cells were exposed to 25, 50 or 100μgml–1 PM2.5 for
24h or100μgml–1 PM2.5 for 1, 3, 6, 12 and 24h, respectively. For
theinhibitory effect experiments, the cells were pretreated with
orwithout inhibitors of JNK (SP600125, 25μm), ERK (U0126, 10μm),p38
MAPK (SB203580, 25μm) and PI3K/AKT (LY294002, 25μm) orNF-κB
(BAY11-7082, 5μm) and then treated with 100μgml–1 PM2.5for 24 h,
respectively. The cells in the EA.hy926 culture wererinsed three
times with 1ml of PBS (pH 7.3) to wash out thePM2.5. Then 500μl of
new culture medium containing antibioticswas put in each well. Then
20-30μl of suspension of calcein-AM-labeled THP-1 cells (5.0 × 105
cells) was added to each culturewell. They were incubated for 1 h
at 37 °C in 5% CO2 and 95%air humidified atmosphere. After that,
the suspension of THP-1 cellsin each well was discarded, and the
EA.hy926 culture was gentlywashed three times with 1ml of PBS
(pH7.3) in order to removenon-adherent THP-1 cells. Adherent
fluorescence-labeled THP-1 cellswere observed under a fluorescence
microscope (Nikon, Eclipse Ti,Japan) (Kim et al., 2007). The
fluorescence intensity of the adherentTHP-1 cells was determined
using a Multi-well Plate Reader withexcitation and emission
wavelengths of 488 and 530nm.
Figure 1. PM2.5 size distribution. (A) Scanning electron
microscope (SEM)images of PM2.5 (Bar, 0.1 μm;magnification 30
000×). (B) PM2.5 size distribu-tion in the Dulbecco’s Modified
Eagle’s Medium (DMEM) culture mediumwas detected by dynamic light
scattering.
Table 1. Metals detected in PM2.5
Metals Concentration (ngm–3)
Ti 56.58± 2.04V 2.66± 0.05Cr 8.61± 0.18Mn 51.71± 1.02Co 0.55±
0.01Ni 3.93± 0.06Cu 26.62± 0.51As 14.31± 0.38Sr 11.10± 0.16Mo
1.39±0.03Cd 2.87± 0.05Cs 0.86± 0.01
Statistical Analysis
Data are expressed as the means ± SD. The differences betweenthe
mean values of two groups were determined by Student’st-test.
Associations between the different variables were exam-ined by
one-way ANOVA, followed by post hoc comparisonsusing the Tukey’s
multiple paired comparison test. Statisticalsignificance was set at
P< 0.05.
Results
Physichemical Characteristics of PM2.5
The morphology of PM2.5 by SEM was shown in Fig. 1A.
Dynamiclight scattering measurement of PM2.5 showed a size range
of0.04–5μm with a mean of 0.28μm (Fig. 1B).
Among the total of 20 metal elements measured, Fe, Pb, Zn, Tiand
Mn were the most abundant elements in the PM2.5 samples(Table 1).
The analytical results of water soluble inorganic ions inPM2.5
showed SO4
2- (12.49± 0.97μgm–3), NO3- (7.18± 0.69μgm–3),
NH4+ (4.78± 0.29μgm–3) and Cl- (4.09 ± 0.35μgm–3),
respectively
(Table 2). The average concentrations of OC and EC in thePM2.5
were 18.51± 3.25μgm
–3 and 2.90± 0.07μgm–3, and theOC/EC ratio was 6.39 (Table 3).
Acenaphthylene, acenaphthene,benzo[b]fluoranthene, benzo[a]pyrene
and chrysene were thedominant PAHs in PM2.5 (Table 4).
Ba 31.09± 0.39
Pb 146±3Zn 253±3Fe 1071±7Ca 1676±16K 1088±6Mg 396±2Na 803±13
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Effect of PM2.5 on Cell Viability in EA.hy926 Cells
EA.hy926 cells were exposed 0, 25, 50, 100 or 200μgml–1 PM2.5
for1, 3, 6, 12, or 24 h, respectively (Fig. 2). After 1-, 3- and
6-h exposureof cells to 25, 50, 100 and 200μgml–1 PM2.5, no
statistically signifi-cant impacts of PM2.5 were detected compared
with the unex-posed control cells. However, a dose-dependent
decrease of cell
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& Sons, Ltd. wileyonlinelibrary.com/journal/jat
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Table 2. Water soluble inorganic ions detected inPM2.5
Water soluble ions Concentration (μgm–3)
SO42- 12.49± 0.97
NO3- 7.18± 0.69
NH4+ 4.78± 0.29
Cl- 4.09 ± 0.35
Table 3. Carbonaceous fractions detected in PM2.5
OC/EC Concentration (μgm–3)
OC 18.51± 3.25EC 2.90± 0.07OC/EC 6.39± 1.27
Table 4. Organic compositions detected in PM2.5
PAHs Concentration (ngm–3)
Acenaphthene 20.64± 1.12Acenaphthylene 20.40± 0.71Anthracene
3.33± 0.15Benz[a]anthracene 5.23± 0.34Benzo[a]pyrene 8.14±
0.53Benzo[b]fluoranthene 9.33± 0.65Benzo[k]fluoranthene 4.89±
0.15Chrysene 6.78± 0.18Coronene 0.55± 0.02Dibenz[a,h]anthracene
0.61± 0.02Fluoranthene 3.29± 0.27Fluorene 0.73±
0.06Indeno[123-cd]pyrene 5.56± 0.24Phenanthrene 0.07± 0.01Pyrene
3.54± 0.18
Figure 2. Effect of PM2.5 exposure on cell viability in EA.hy926
cells. EA.hy926 cells were treated with 0, 25, 50, 100 or 200
μgml–1 PM2.5 for 1, 3,6, 12 or 24 h, respectively. The cell
viability was assayed by the
3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Valuesare the mean± SD of three independent experiments.
*P< 0.05,**P< 0.01 vs. the untreated control cells.
W. Rui et al.
52
viability was observed after 12- and 24-h exposure to 50, 100
and200μgml–1 PM2.5.
Figure 3. Effect of PM2.5 on the reactive oxygen species (ROS)
generationin EA.hy926 cells. EA.hy926 cells were exposed to 0, 25,
50, 100 or200 μgml–1 PM2.5 for 1, 3, 6, 12 or 24 h, respectively.
The ROS levels weredetermined by measuring the oxidative conversion
of DCFH-DA to DCF.To investigate if the PM2.5-induced changes were
related to the decreasein the antioxidant defense, EA.hy926 cells
were pretreated with5 mM N-acetylcysteine (NAC) for 1 h and then
treated with 100 μgml–1 PM2.5 forthe indicated time. Results were
measured as mean fluorescence (arbitraryunits, AU). Values are the
mean± SD of three independent experiments.*P< 0.05, **P<
0.01, ***P< 0.001 vs. the untreated control cells.#P< 0.05,
##P< 0.01 vs. 100μgml–1 PM2.5.
Effect of PM2.5 on Intracellular ROS Generation in
EA.hy926Cells
The potential of PM2.5 to induce intracellular ROS generation in
EA.hy926 cells was evaluated by DCFH-DA intensity. The cells
weretreated with 0, 25, 50, 100 or 200μgml–1 PM2.5 for 1, 3, 6, 12
or24h, respectively. After 1 h of exposure to the indicated
concentra-tion of PM2.5, ROS generation was evident in relation to
the variousconcentrations used (Fig. 3). In addition, Fig. 3 shows
that the highestROS generation was observed after 12h with a
decline thereafter,indicating that ROS generation is dose
dependent. However,increased ROS induced by PM2.5 exposure
(100μgml
–1) was signifi-cantly attenuated by pretreatment with NAC.
Effect of PM2.5 on ICAM-1 and VCAM-1 Expressions on EA.hy926
Cells
In order to establish the role of PM2.5-induced oxidative
stress, wemeasured ICAM-1 and VCAM-1 cell surface expressions in
responseto PM2.5. As shown in Fig. 4, both ICAM-1 and VCAM-1 cell
surfaceexpressions in EA.hy926 cells were significantly increased
in a
Copyright © 2015 Johnwileyonlinelibrary.com/journal/jat
dose- and time-dependent manner (Fig. 4A and B). However,
theincreases in ICAM-1 and VCAM-1 cell surface expressions
inresponse to PM2.5 were attenuated by NAC (Fig. 4A). These
resultsindicated that PM2.5-induced ROS are capable of stimulating
CAMexpression at the cell surface in EA.hy926.
Effect of PM2.5 on THP-1 Cell Adhesion to EA.hy926 Cells
To investigate the effect of PM2.5-induced oxidative stress on
theadhesion of THP-1 cells to the surface of EA.hy926 cells, we
J. Appl. Toxicol. 2016; 36: 48–59Wiley & Sons, Ltd.
-
Figure 4. Effect of PM2.5 on intercellular adhesion molecule-1
(ICAM-1)and vascular adhesion molecule-1 (VCAM-1) expressions in
EA.hy926 cells.EA.hy926 cells were exposed to 0, 25, 50 or 100
μgml–1 PM2.5 for 24 h (A),or 100 μgml–1 PM2.5 for 1, 3, 6, 12 or 24
h (B). The ICAM-1 and VCAM-1 cellsurface expressions were detected
by flow cytometer as described in theMethods section. To
investigate if the PM2.5-induced reactive oxygen spe-cies (ROS)
generation was related to the increase expression of cell adhe-sion
molecule (ICAM-1 and VCAM-1) in the cell surface, EA.hy926
cellswere pretreated with 5mMN-acetylcysteine (NAC) for 1 h and
then treatedwith 100μgml–1 PM2.5 for the indicated time. Values are
the mean ± SD ofthree independent experiments. *P< 0.05, **P<
0.01 vs. the untreatedcontrol cells. ##P< 0.01 vs. 100 μgml–1
PM2.5.
PM2.5 effect adhesion molecule expression via the ERK/AKT/NF-κB
pathway
measured the fluorescence intensity of adherent THP-1 cells in
theEA.hy926 culture after incubation with THP-1 cells in a culture
me-dium containing PM2.5 at 0, 25, 50 or 100μgml
–1 PM2.5 for 24 h or100μgml–1 PM2.5 for 1, 3, 6, 12 and 24h.
Figure 5 summarizes theresults. As evident from the figure, the
adhesion of THP-1 cells wasgreatly enhanced by the presence of
PM2.5 under a monolayer ofEA.hy926 cells. At any PM2.5
concentration and any time, thenumber of adherent cells in the
EA.hy926 culture was significantlyincreased. However, PM2.5-induced
increases in the number ofadherent cells were significantly
attenuated when EA.hy926 cellswere pretreated with NAC (Fig. 5A).
These results suggested thatPM2.5-induced ROS was involved in
promoting the adhesion ofmonocytes to the endothelial cells.
53
Effect of PM2.5 on ROS-Dependent Signaling Pathways in EA.hy926
Cells
To assess whether JNK, ERK1/2, p38 MAPK, AKT and NF-κB
signal-ing pathways were involved in PM2.5-induced ROS generation,
wefirstly detected the phosphorylation of JNK, ERK1/2, p38 MAPK
and
J. Appl. Toxicol. 2016; 36: 48–59 Copyright © 2015 John
AKT as well as the activation of NF-κB after exposure of
EA.hy926cells to 0, 25, 50 or 100μgml–1 PM2.5 for 24h. As shown in
Fig. 6A,PM2.5 markedly triggered an increase in ERK1/2 and p38
MAPKphosphorylation from 25 to 100μgml–1 PM2.5 after
exposure.Phosphorylations of AKTwere significantly increased after
exposureto 100μgml–1 PM2.5. Moreover, there was a significant
increase inNF-κB activation in a concentration-dependentmanner.
These datasuggested that PM2.5 exposure induces phosphorylation of
JNK,ERK1/2, p38 MAPK, AKT phosphorylation and activation of
NF-κB.To confirm whether PM2.5-induced ROS generation was
involved in the JNK, ERK1/2, p38 MAPK, AKT and NF-κB
signalingpathways in EA.hy926 cells, we further detected the
phosphoryla-tion of JNK, ERK1/2, p38 MAPK, AKT and activation of
NF-κB afterexposure of cells to 100μgml–1 PM2.5 in the presence of
NAC(5mM) for the indicated time. As shown in Fig. 6A,
phosphoryla-tion of JNK, ERK1/2, p38 MAPK, AKT and activation of
NF-κB weresignificantly attenuated after pretreatment with NAC as
comparedwith themarked increase in AKT phosphorylation in the
absence ofNAC (Fig. 6A). These findings indicated that JNK, ERK1/2,
p38MAPK, AKT and NF-κB pathways are involved in ROS generationin
response to PM2.5. Moreover, we also found that treatment withPM2.5
increased JNK, ERK1/2, p38 MAPK, AKT phosphorylation andNF-κB
activation in a time-dependent manner (Fig. 6B).
Effect of ERK1/2, JNK, p38 MAPK, AKT and NF-κB Inhibitors
onPM2.5-Induced ICAM-1 and VCAM-1 Expressions on EA.hy926Cells
To explore the underlying mechanism of JNK, ERK1/2, p38 MAPK,AKT
and NF-κB signaling pathways involved in PM2.5-inducedCAM cell
surface expression and adhesion of monocytes to the en-dothelium,
EA.hy926 cells were next utilized JNK, ERK1/2, p38MAPK, AKT and
NF-κB inhibitors after exposure of EA.hy926 cellsto 100μgml–1 PM2.5
for 1 h. Phosphorylations of JNK, ERK1/2, p38MAPK, AKT and N-p65
were significantly inhibited by the indicatedinhibitors,
respectively (data not shown). As shown in Fig. 7, pre-treatment
with ERK1/2 inhibitor (U0126), AKT inhibitor(LY294002) and NF-κB
inhibitor (BAY11-7082) inhibited ICAM-1and VCAM-1 cell surface
expressions after exposure to PM2.5(P< 0.001). However, JNK
inhibitor (SP600125) and p38 MAPKinhibitor (SB203580) did not
affect on ICAM-1 and VCAM-1 cell sur-face expressions. These data
suggested that ERK/AKT and NF-κBpathways mediate PM2.5-induced
ICAM-1 and VCAM-1 cell surfaceexpressions in EA.hy926 cells.
Effect of ERK1/2, JNK, p38 MAPK, AKT and NF-κB Inhibitors
onPM2.5-Induced THP-1 Cell Adhesion to EA.hy926 Cells
ICAM-1 and VCAM-1 are involved in the adhesion mechanismsthat
underlie interactions between endothelial cells with other
celltypes. We previously showed that PM2.5 increases the
expressionof these adhesion molecules on EA.hy926 cells, and thus
to evalu-ate the functional significance of the observation, we
assessed theadhesion of THP-1 cells to EA.hy926 cells after
pretreatment withJNK inhibitor (SP600125), ERK1/2 inhibitor
(U0126), p38 MAPKinhibitor (SB203580), AKT inhibitor (LY294002) and
NF-κB inhibitor(BAY11-7082). As shown in Fig. 8, the adhesion of
THP-1 cells wasnot affected by pretreatment with JNK inhibitor
(SP600125) andp38 MAPK inhibitor (SB203580) in terms of
fluorescence intensityas compared to PM2.5-treated cells. However,
the fluorescenceintensity markedly appeared to decrease after
pretreatment withERK1/2 inhibitor (U0126), AKT inhibitor (LY294002)
and NF-κB
Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat
-
Figure 5. Effect of PM2.5 on THP-1 cell adhesion to EA.hy926
cells. (A and B) Representative fluorescence microscopic
photographs of Ca-AM-labeled THP-1cell adherent to EA.hy926 cells.
(C andD) Quantitative data from the three independent experiments
are shown in A and B. EA.hy926 cells were exposed to 0,25, 50 or
100 μgml–1 PM2.5 for 24 h (A), or 100μgml
–1 PM2.5 for 1, 3, 6, 12 or 24 h (B). EA.hy926 cells were
incubated with Ca-AM-labeled THP-1 cells for 1 h,and then washed
three times with cold PBS to remove the non-adherent THP-1 cells.
The intensity of adherent fluorescence-labeled THP-1 cells was
ob-served using a fluorescence microscope as described in the
Methods section. The fluorescence intensity was detected using a
multi-well plate reader withexcitation and emission wavelengths of
488 nm and 530 nm. To investigate if the PM2.5-induced reactive
oxygen species (ROS) generation were related tothe increase THP-1
cells adhesion to EA.hy926 cells, EA.hy926 cells were pretreated
with 5mM of N-acetylcysteine (NAC) for 1 h and then treated withμg
ml–1 PM2.5 for the indicated time. Values are the mean ± SD of
three independent experiments. *P< 0.05, **P< 0.01 vs. the
untreated control cells.##P< 0.01 vs. 100 μgml–1 PM2.5.
W. Rui et al.
J. Appl. Toxicol. 2016; 36: 48–59Copyright © 2015 John Wiley
& Sons, Ltd.wileyonlinelibrary.com/journal/jat
54
-
Figure 6. Effect of PM2.5 on reactive oxygen species
(ROS)-dependent ERK1/2, JNK, p38 MAPK, AKT phosphorylation and
NF-κB activation in EA.hy926 cells.EA.hy926 cells were exposed to
0, 25, 50 or 100μgml–1 PM2.5 for 24 h (A), or 100μgml
–1 PM2.5 for 1, 3, 6, 12 or 24 h (B). Cell lysates were blotted
for phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, phospho-p38 MAPK, p38
MAPK, phospho-AKT, AKT, and nuclear and cytoplasmic protein
extraction of N-p65 and C-p65were blotted, respectively. Density of
phosphorylated-JNK/ERK1/2/P38 MAPK/AKT and N-p65 was relative to
the level of JNK/ERK1/2/P38 MAPK/AKTand C-p65, respectively. To
investigate if the PM2.5-induced ROS generation were related to
JNK, ERK1/2, p38 MAPK, AKT and NF-κB signaling pathways,EA.hy926
cells were pretreated with5 mM N-acetylcysteine (NAC) for 1 h and
then treated with 100 μgml–1 PM2.5 for the indicated time. Values
are themean ± SD of three independent experiments. *P< 0.05,
**P< 0.01 vs. the untreated control cells. #P< 0.01 vs. 100
μgml–1.
PM2.5 effect adhesion molecule expression via the ERK/AKT/NF-κB
pathway
55
inhibitor (BAY11-7082). These findings suggested that ERK/AKTand
NF-κB pathways are involved in the adhesion of THP-1 cellsto
EA.hy926 cells in response to PM2.5.
DiscussionThe present study demonstrates that PM2.5 induced an
increase inintracellular ROS generation, followed by upregulation
JNK,ERK1/2, p38 MAPK and AKT phosphorylation, and nuclear
translo-cation of NF-κB in human EA.hy926 cells in a dose- and
time-dependent manner. The cell surface expressions of
adhesionmolecules, ICAM-1 and VCAM-1, and the adhesion of THP-1
cellto EA.hy926 cells were also significantly increased after the
PM2.5exposure. In contrast, scavenging ROS with NAC
significantlyattenuated the cell surface expression of adhesion
molecules,decreased cell adhesion and markedly reduced
ROS-dependentkinases phosphorylation. Moreover, pretreatment with
ERK1/2 in-hibitor (U0126), AKT inhibitor (LY294002) and NF-κB
inhibitor
J. Appl. Toxicol. 2016; 36: 48–59 Copyright © 2015 John
(BAY11-7082), but not a JNK inhibitor (SP600125) or a p38
MAPKinhibitor (SB203580), significantly inhibited PM2.5-induced
ICAM-1and VCAM-1 cell surface expressions on EA.hy926 cells and
adhe-sion of THP-1 cells to EA.hy926 cells. Taken together, these
resultssuggest that PM2.5-induced ROS may contribute to ICAM-1
andVCAM-1 expressions by activating the ERK/AKT/NF-κB
signalingpathway in the endothelial cells.Many previous studies
have demonstrated that chemical com-
position of PM2.5 is involved in toxicological effects,
including bothorganic and inorganic fractions (Araujo et al., 2008;
Wei et al., 2011;Deng et al., 2013) . Global population-weighted
PM2.5 compositioncontains organic mass (32%), mineral mass (30%),
sulfate (17%),black carbon (7%), ammonium (7%), nitrate (6%) and
sea salt(1%) (Philip et al., 2014). Consistent with our previous
study (Denget al., 2013), the study demonstrates that the high
levels of Fe, Pb,Zn, Ti, Mn, acenaphthylene, acenaphthene,
benzo[b]fluoranthene,benzo[a]pyrene and chrysene are major
constituents of PM2.5. Inaddition to metals, PAHs, OC and EC, PM2.5
also appeared to have
Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jat
-
Figure 7. Effect of ERK1/2, JNK, p38 MAPK, AKT and NF-κB
inhibitors onPM2.5-induced intercellular adhesion molecule-1
(ICAM-1) and vascular ad-hesion molecule-1 (VCAM-1) expressions on
EA.hy926 cells. EA.hy926 cellswere pretreated with U0126 (10 μM),
SP600125 (25 μM), SB203580(25 μM), LY294002 (25μM) and BAY11-7082
(5μM) for 1 h and then treatedwith 100 μgml–1 PM2.5 for 24 h,
respectively. Cells were processed for flowcytometer analysis as
described in the Methods section. Values are themean± SD of three
independent experiments. *P< 0.05, **P< 0.01 vs.the untreated
control cells. #P< 0.01, ##P< 0.001 vs. PM2.5.
W. Rui et al.
56
higher constitutes of water soluble inorganic ions such as SO42-
and
NO3- . All these results obtained from the chemical
characterization
reflect that PM2.5 samples in Beijing are a complex mixture
ofchemicals and a significant contribution from
anthropogenicsource. These constituents can play an important role
in mediatingcardiovascular effects of PM air pollution (Peters et
al., 1997; Popeet al., 2004; Laden et al., 2006; Alfaro-Moreno et
al., 2007; Riediker,2007; Araujo et al., 2008).
The components of PM2.5, including transitionmetals, water
sol-uble inorganic ions, carbonaceous fractions and polycyclic
aro-matic hydrocarbons, caused oxidative stress which have
beendescribed as a common pathway for PM2.5-indcued oxidativedamage
(Gilmore, 2006; Eckers and Klotz, 2009; Ying et al., 2009;Wei et
al., 2011; Feng et al., 2013). In the present study, we foundthat
different concentrations of PM2.5 significantly caused a de-crease
in cell viability (Fig. 2). It has been reported that PM2.5
anddiesel exhaust particles (DEP) induces intracellular ROS
generationin cultured human umbilical vein endothelial cells (Wei
et al., 2011)and the microvascular endothelium (Frikke-Schmidt et
al., 2011;Wauters et al., 2013). Similarly, we also clearly
observed thatPM2.5 induced ROS generation in EA.hy926 cells in a
time- anddose-dependent manner. Moreover, NAC, a ROS scavenger,
signif-icantly attenuated PM2.5-induced ROS generation (Fig. 3),
indicat-ing that PM2.5 is capable of inducing the cytotoxic effect
ofoxidative stress induced by ROS.
Previous studies have shown that ROS act as chemical messen-gers
in the signal transduction process to activate cell
adhesionmolecule expression in endothelial cells (Ying et al.,
2009; Frikke-Schmidt et al., 2011; Hemmingsen et al., 2011; Wauters
et al.,2013; Wei et al., 2011). There is growing evidence that PM
increasesthe adhesion factors expression in endothelium
(Montiel-Davaloset al., 2007; Yatera et al., 2008; Li et al., 2010;
Lee et al., 2012) . In
Copyright © 2015 Johnwileyonlinelibrary.com/journal/jat
the present study, our results showed that PM2.5
significantlyincreased ICAM-1 and VCAM-1 cell surface expressions
and theadhesion of THP-1 cells to EA.hy926 cells in a time-
andconcentration-dependent manner (Figs. 4 and 5). However,
pre-treatment with NAC significantly reduced PM2.5-induced
adhesionfactors cell surface expressions and the adhesion of THP-1
cells toEA.hy926 cells, indicating that PM2.5-induced ROS was
involved instimulating the expression of adhesionmolecules and
recruitmentof inflammatory cells.
MAPK or AKT is an important regulator of cell growth,
survival,proliferation, inflammatory and immune reaction in
response tooxidative stress. (Cowan and Storey, 2003; Wan et al.,
2010). JNK,ERK and p38 MAPK are involved in stimulation of adhesion
factorsexpression in different kinds of cells by different stress
stimuli(Chen et al., 2011; Feng et al., 2013; Olejarz et al.,
2014). In thepresent study, we evaluated if the JNK, ERK, p38 MAPK
and AKTpathways involved in regulating the expression of adhesion
mole-cules and recruitment of inflammatory cells in response to
PM2.5-induce oxidative stress. Here we found that JNK, ERK1/2,
p38MAPKand AKT pathways were activated after exposure to PM2.5 for
theindicated times. However, treatment with U0126 and
LY294002significantly inhibited ICAM-1 and VCAM-1 cell surface
expressionsand the adhesion of THP-1 cells to EA.hy926 cells after
exposure toPM2.5, whereas SP600125 or SB203580 had no significant
effects.Thus, we propose that ERK/AKT pathway is required for the
expres-sion of adhesion molecules and recruitment of inflammatory
cells.In addition, we also observed that NAC markedly suppressed
thephosphorylations of ERK and AKT after PM2.5 exposure, even
whenthe oxidative stress is decreased. The complementary
evidenceindicated that ROS generation is associated with the
activation ofthe ERK/AKT pathway, which contributes to adhesion
moleculesexpression after PM2.5 exposure. These observations are
consistentwith the previous reports that ERK/AKT signaling pathway
isimplicated in ICAM-1 and VCAM-1 expressions in human
aorticendothelial cells (HAECs) (Chen et al., 2011; Feng et al.,
2013;Olejarz et al., 2014).
The transcription factor NF-κB regulates a wide variety of
biolog-ical effects on stress and adaptive responses in various
cell typesand organs ( Jones et al., 2005). Multiple signaling
pathways includ-ing MAPK, PKC, PKA, and AKT pathways act directly
to phosphory-late NF-κB subunits to affect the ability of NF-κB to
bind DNA andincrease the transactivation of NF-κB-dependent genes (
Joneset al., 2005). Thus, the activation of NF-κB and the
subsequentincrease in cell adhesion molecule expression have been
linkedto the intracellular redox state (Khan et al., 1996; Kunsch
andMedford, 1999). Here, our data also showed that NF-κB is
activatedand p65 translocated into nucleus after exposure to
PM2.5.However, activation of NF-κB was significantly attenuated
afterpretreatment with NAC. Moreover, BAY11-7082 inhibited
ICAM-1and VCAM-1 cell surface expressions and the adhesion of
THP-1to EA.hy926 cells after exposure to PM2.5. These observations
areconsistent with the previous reports that NF-κB is responsible
forthe ICAM-1 and VCAM-1 expression (Verna et al., 2006; Chenet
al., 2011; Mehla et al., 2011; Yi et al., 2012; Feng et al.,
2013;Olejarz et al., 2014) .
In conclusion, PM2.5-induced oxidative stress induces the
cellsurface expression of ICMA-1 and VCAM-1 through activating
theERK/AKT/NF-κB signaling pathway, promotingmonocyte adhesionto
endothelial cells. Understanding the signaling pathways impli-cated
in PM2.5-mediated oxidative stress provides new insightsinto the
pathophysiology of cardiovascular diseases associatedwith airborne
pollution.
J. Appl. Toxicol. 2016; 36: 48–59Wiley & Sons, Ltd.
-
Figure 8. Effect of ERK1/2, JNK, p38MAPK, AKT and NF-κB
inhibitors on PM2.5-induced THP-1 cell adhesion to EA.hy926 cells.
EA.hy926 cells were pretreatedwith U0126 (10 μM), SP600125 (25 μM),
SB203580 (25μM), LY294002 (25μM) and BAY11-7082 (5μM) for 1 h and
then treated with 100 μgml–1 PM2.5 for24 h, respectively. The
fluorescence intensity was detected in a fluorescence multi-well
plate reader with excitation and emission wavelengths of 488 and530
nm. The intensity of adherent fluorescence-labeled THP-1 cells was
observed under a fluorescence microscope. The cells were incubated
withCa-AM-labeled THP-1 cells for 1 h, and then washed three times
with phosphate-buffered saline (PBS) to remove the unadherent
cells. Fluorescing mono-cytes that adhered on the endothelial cell
surface were observed under a fluorescencemicroscope as described
in theMethods section (A). The fluorescenceintensity was detected
using a multi-well plate reader with excitation and emission
wavelengths of 488 nm and 530 nm (B). Values are the mean ± SD
ofthree independent experiments. *P< 0.05, **P< 0.01 vs. the
untreated control cells. ##P< 0.01 vs. PM2.5.
PM2.5 effect adhesion molecule expression via the ERK/AKT/NF-κB
pathway
J. Appl. Toxicol. 2016; 36: 48–59 Copyright © 2015 John Wiley
& Sons, Ltd. wileyonlinelibrary.com/journal/jat
57
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W. Rui et al.
58
Acknowledgments
This work was financially supported by grants from
NationalNatural Science Foundation of China (No. 21377127,
11275264,U1432245), and the CAS/SAFEA International Partnership
Programfor Creative Research Teams.
Conflict of interestAll authors declare no competing financial
interest.
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