Research A rticle
Sakuranetin Inhibits Inflammatory Enzyme, Cytokine, and
Costimulatory Molecule Expression in Macrophages through
Modulation of JNK, p38, and STAT1
Ki-Young Kim
1
and Hee Kang
2
1
Departmen t of Genetic Engineering, College of Life Science and Graduate School of Biotechnology,
Kyung Hee U niversity, Yongin, Republic of Korea
2
Graduate School of East-West Medical Science, Kyung Hee University , Yongin, Republic of Kor ea
Correspondence should be addressed to Hee Kang; shehee@khu.ac.kr
Received June ; Revis ed August ; Accepted August
Academic Editor: Michele Navarra
Copyright © K.-Y. Kim and H. Kang. 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.
Sakuranetin is avonoid phytoalexin that serves as a plant antibiotic and exists in Prunus and several other plant species. Recently,
we identied the anti-inammatory eect of Prunus yedoensis and found that there were few studies on the potential anti-
inammatory activity of sakuranetin, one of the main constituents of Prunus yedoensis. Here, we isolated peritoneal macrophages
from thioglycollate-injected mice and examined whether sakuranetin aected the response of the macrophages in response to
lipopolysaccharide (LPS) plus interferon- (IFN-) 𝛾 or LPS only. Sakuranetin suppressed the synthesis of iNOS and COX in
LPS/IFN-𝛾 stimulated cells and the secretion of TNF-𝛼, IL-, and IL- in LPS stimulated cells. e surface expression of the
costimulatory molecules, CD and CD, was also decreased. Among the LPS-induced signaling molecules, STAT, JNK, and
p phosphorylation was attenuated. ese ndings are evidence that sakuranetin acts as anti-inammatory avonoid and further
study is required to evaluate its in vivo ecacy.
1. Introduction
Inammatory responses are protective against further tissue
damage and help to repair wounds. Inammatory stimuli not
only are conned to microbes, but also include endogenously
generated substances, as seen with gout and atherosclerosis
[]. Inammation should be self-limiting, but when this
capacity is impaired, the response will result in continued
tissue destruction. e reasons for the chronicity of inam-
mation include microbes that evade the immune system,
accumulating metabolic or cellular byproducts, and autoim-
mune diseases generated by unknown causes.
Depending on the time required to initially respond, the
site of rst contact with the antigen, and the ability to acquire
memory, the immune system is divided into innate and
adaptive systems. Cells that belong to the innate immune sys-
tem confront the antigens and respond to them immediately
butdonotacquirememory.Ontheotherhand,adaptive
immune cells make rst contact with antigens in secondary
lymphoid tissue such as lymph nodes, which explains why
they take time to respond, and acquire memory, letting
the cells mount a faster response to the next exposure of
the antigen. Macrophages belong to the innate immune
system but present antigens to T cells, acting as a bridge
betweentheinnateandadaptiveimmunesystems.Generally,
macrophages are the rst sensor to detect and react to foreign
microbes and, when necessary, recruit other circulating white
blood cells to the site []. During inammatory responses,
macrophagesrecognizethepresenceofthecausativeagent
through pattern recognition receptors such as toll-like recep-
tor(TLR)andactivatetheNF-𝜅Bpathwayandmitogen-
activated protein kinases (MAPK) pathway, terminating in
the expression of inammatory enzymes such as inducible
nitric oxide synthases (iNOS) and cyclooxygenase- (COX-)
and inammatory cytokines such as tumor necrosis factor-
(TNF-) 𝛼, interleukin- (IL-) , and IL- []. In addition,
Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2016, Article ID 9824203, 8 pages
http://dx.doi.org/10.1155/2016/9824203
Evidence-Based Complementary and Alternative Medicine
macrophages upregulate the surface expression of costimu-
latory molecules such as CD/CD and CD in order to
form stable contacts with T cells. us, the above molecules
are targets of anti-inammatory agents for the control of
chronic inammation.
Sakuranetin is avonoid phytoalexin that serves as a plant
antibiotic [] and exists in the Prunus species, Baccharis
species, Betula species, and rice []. Recently, we identied
the in vitro and in vivo anti-inammatory eects of Prunus
yedoensis bark [, ] and found that reports on the anti-
inammatory mechanism of sakuranetin, one of the main
constituents of Prunus yedoensis bark, were scarce. A litera-
turesearchonsakuranetinshowedthatitinhibitschemically
induced edema in mice [ ] and alleviates the allergen-
induced lung injury model through control of NF-𝜅B [].
Here,wesoughttoinvestigatetheanti-inammatoryactivity
of sakuranetin and its mechanism using lipopolysaccharide
(LPS) plus interferon- (IFN-) 𝛾 or LPS stimulated macro-
phage model.
2. Materials and Methods
2.1. Animals. Seven-week-old male BALB/c mice (Samtaco,
Osan, Korea) were purchased and kept in a temperature- and
humidity-controlled, pathogen-free animal facility at Kyung
Hee University. e mice were provided with standard mouse
chow and water ad libitum in accordance with the Guide for
the Care and U se of Laboratory Animals issued by the United
States National Research Council (), and the protocol
(KHUSASP(GC)--) was approved by the Kyung Hee
Universi ty Institutional Animal Care and Use Committee.
2.2. Cell Culture. Mice were injected intraperitoneally with
mL o f .% sterile thioglycollate solution (BD, Sparks, MD,
USA). ree days later, mice were sacriced by cervical
dislocation and macrophages were isolated by peritoneal
lavage with cold DMEM. Aer centrifugation, cells were
resuspended in DMEM with % fetal bovine s erum (FBS;
Hyclone, Utah, USA) and % penicillin-streptomycin and
incubated overnight in a humidied atmosphere of % CO
2
at
∘
C. Aer nonadherent cells were removed, cells were
seeded for subsequent assays.
2.3. Viability Assay. Cells were seeded in quadruplicate in -
well plates and stimulated for h at increasing concentra-
tions of sa kuranetin (Sigma, St. Louis, MO, USA). Cell
viability was determined using the MTS (-(,-dimethylthi-
azol--yl)--(carboxymethoxyphenyl)--(-sulfophenyl)-H-
tetrazolium) reduction method (CellTiter One Solution
Cell Proliferation Assay Kit, Promega, Madison, WI, USA),
based on the measurement of mitochondrial respiration in
living cells. Optical density was measured at nm with
a microplate reader (Molecular Devices, Sunnyvale, CA,
USA).
2.4. Measurement of Nitrites. Cells were stimulated with
ng/mL of recombinant IFN-𝛾 (BD Pharmingen, San Diego,
CA, USA) and ng/mL LPS (Sigma) in the presence
of sakuranetin or 𝜇M dexamethasone (Sigma) for h.
Supernatant was obtained for the evaluation of nitrite levels
using the Griess Reagent System (Promega). e absorbance
at nm was measured with the microplate reader.
2.5. Cytokine Measurement. Cells were cultured with ng/
mL LPS and sakuranetin for h or h. e cytokine
levels from appropriately diluted supernatants were measured
by ELISA according to the manufacturer’s protocol (BD
Pharmingen).
2.6. Western Blotting. To detect iNOS and COX-, cells were
stimulated with LPS/IFN-𝛾 inthepresenceofsakuranetin
for h. To detect phospho-STAT, cells were pretreated with
sakuranetin for h and then stimulated with LPS for h. To
detect I𝜅B𝛼 a nd phospho-MAPK, cells were pretreated with
sakuranetin for h and then LPS was added for min. Tota l
cell extracts were prepared by resuspending the cells in lysis
buer ( mM Tris-HCl, pH .; mM NaCl; mM EDTA;
mM NaF; .% NP-; and % Triton X-) containing a
phosphatase inhibitor cocktail (Sigma) and an X pert protease
inhibitor cocktail (GenDEPOT, TX, USA). Protein concen-
tration was determined using the Bradford assay. Cell extracts
were separated on an % or % sodium dodecyl sulfate-
polyacr ylamide gel and were transferred to polyvinylidene
uoride membrane. e membranes were blocked with %
skim milk in Tris-buered saline with .% Tween (TBST)
for h and then incubated overnight at
∘
CwithiNOS,I𝜅B𝛼,
tubulin, or GAPDH (Santa Cruz Biotechnology, Santa Cruz,
CA, USA), phospho-STAT, STAT, phospho-JNK, JNK,
phospho-p, p, phospho-ERK, or ERK (Cell Signaling
Technology, CA, USA) diluted at / in % skim milk in
TBST. e blots were washed with TBST and incubated for h
with anti-rabbit horseradish peroxidase-conjugated antibody
(diluted at : in % skim milk in TBST). Protein
bands were detec ted with EzWestLumi plus (ATTO, Japan)
andanalyzedusinganEZ-CaptureMG(ATTO).eband
density of each protein was quantied using ImageJ soware
and normalized with internal control.
2.7. Flow Cytometry. Cells were washed t wice in cold phos-
phate buered saline (PBS) and resuspended at ×
6
cells/mL in FACS buer (PBS/.% NaN
3
/% FBS). Cells
were blocked with rat anti-mouse CD/CD (BD Pharmin-
gen) at
∘
C for min and then stained for min with
FITC-conjugated anti-mouse-CD and PE-conjugated anti-
mouse CD (BD Pharmingen) on ice in the dark. For isotype
controls, FITC-conjugated rat IgGa 𝜅 or PE-conjugated rat
IgGa 𝜅 (BD Pharmingen) was used. e cells were washed
twice and resuspended in FACS buer. Ten thousand cells
werecollectedforeachsampleandanalyzedonaNaviosFlow
Cytometer (Beckman Coulter, Brea, CA, USA). e data were
analyzed with Kaluza soware.
2.8. Statistical Analysis. Statistical analysis was performed
using Student’s 𝑡-test or ANOVA followed by the SNK test
using IBM Statistics SPSS version . 𝑃 values less than .
were considered signicant.
Evidence-Based Complementary and Alternative Medicine
0
20
40
60
80
100
120
0 2 10 50 100 200 400
% control
S (𝜇M)
∗∗∗
F : Eects of sakuranetin on cell viability. Mouse peritoneal macrophages were c ultured with s akuranetin for h and cell viability was
determined using the MTS assay. Data are represented as a percentage of control cells ( 𝜇g/mL) (𝑛=4).
∗∗∗
𝑃 < 0.005 versus control.
0
10
20
30
40
50
0
0 10 50 100
Dex
S (𝜇M)
∗∗∗
∗∗∗
∗∗∗
∗∗∗
Nitrite (𝜇M)
LPS/IFN-𝛾
∗
(a)
Tub ul in
iNOS
COX2
0 0 10 50 100 DexS (𝜇M)
LPS/IFN-𝛾
(b)
F : Eects of sakuranetin on the release of nitric oxide and the synthesis of inducible NO synthase (iNOS) and COX in LPS/interferon-
(IFN-) 𝛾 stimulated cells. Mouse peritoneal macrophages were stimulated with IFN-𝛾 and LPS in the presence of sakuranetin for h. (a)
NO in the supernatant was detected by the Griess reaction. Dexamethasone ( 𝜇M) was treated as a reference drug. Data are expressed as
mean ± SD (𝑛=3),
∗
𝑃 < 0.05 and
∗∗∗
𝑃 < 0.005 versus controls (LPS/IFN-𝛾 treated cells). (b) e expression of iNOS and COX protein was
analyzed by Western blotting using tubulin as an internal control. One of the three independent experiments is shown.
3. Results
3.1. Eect of Sakuranetin on Cytotoxicity. First, we sought to
determine t he noncytotoxic range of sakuranetin using the
MTS assay. A culture of peritoneal macrophages incubated
with 𝜇Mforhresultedinnoeectoncellviability,
but cells incubated with 𝜇M sakuranetin showed a rapid
decrease in number (Figure ). Based on these results,
subsequentassayswereperformedatnohigherthan𝜇M.
3.2.EectofSakuranetinontheNOProductionandthe
Expression of iNOS and COX-2 in LPS/IFN-𝛾Stimulated Cells.
When LPS and IFN-𝛾 are coadministered to macrophages,
full production of NO occurs []. In an eort to explore
the anti-inammatory potential of sakuranetin, we rst
measured the level of NO in the supernatant from acti-
vated macrophages. Sakuranetin was added to the cells
simultaneously with those inammatory stimuli. Dexam-
ethasone ( 𝜇M)wasusedasareferencechemical.SinceNO
has a short half-life [], the level of nitrite, another product
obtained during NO synthesis, was measured using the col-
orimetric method. A reduction in NO release by sakuranetin
occurred in a dose-dependent manner (Figure (a)). en we
examined whether this reduction was due to iNOS protein
inhibition. e suppressive eect of sakuranetin on iNOS
protein was dose-dependent, as measured by Western blot-
ting (Figure (b)). We also measured the level of COX- pro-
tein from the same cells. A higher concentration ( 𝜇M) of
sakuranetin was required to inhibit COX- protein than that
used in iNOS protein synthesis.
3.3. Eect of Sakuranetin on Soluble Inamma tory Cytokine
Expression. We stimulated macrophages with LPS in the
Evidence-Based Complementary and Alternative Medicine
###
##
###
###
0
500
1000
1500
2000
2500
3000
3500
0 0 10 50 100 Dex
LPS
S (𝜇M)
∗∗∗
∗∗∗
∗∗∗
∗∗∗
TNF-𝛼 (pg/ml)
6 h
24 h
(a)
###
#
#
###
###
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
IL-6 (pg/ml)
0 0 10 50 100 Dex
LPS
S (𝜇M)
6 h
24 h
∗∗∗
∗∗
∗∗
∗
∗
(b)
###
#
###
###
0
500
1000
1500
2000
2500
IL-12 (pg/ml)
0 0 10 50 100 Dex
LPS
S (𝜇M)
∗∗∗
∗∗∗
∗∗∗∗∗
6 h
24 h
(c)
F : Sakuranetin decreases the secretion of tumor necrosis factor- (TNF-) 𝛼, interleukin- (IL-) , and IL-. Mouse peritoneal
macrophages were stimulated with LPS in the presence of sakuranetin or dexamethasone ( 𝜇M)forhorh,andthelevelsofTNF-𝛼
(a), IL- (b), and IL- (c) in the supernatant were analyzed by ELISA. Data are expressed as mean ± SD (𝑛=3).
∗
𝑃 < 0.05,
∗∗
𝑃 < 0.01,and
∗∗∗
𝑃 < 0.005 versus contro ls (cells treated with LPS for h);
#
𝑃 < 0.05,
##
𝑃 < 0.01,and
###
𝑃 < 0.005 versus controls (cells treated with LPS
for h).
presence of sakuranetin for h or h, and the levels of
TNF-𝛼, IL-, and IL- were measured by ELISA. From a gap
in cytokine level at h and h, it was clear that the peak
of TNF-𝛼 secretion was earlier than those of IL- and IL-
(Figure ). Sakuranetin at and 𝜇M and dexamethasone
decreased the levels of all the cytokines tested at each time
point.
3.4. Eect of Sakuranetin on Surface Costimulatory Molecules.
We analyzed the inuence of sakuranetin on the surface
expression of costimulatory molecules CD and CD
using ow c ytometr y. Treatment of macrophages with LPS
increased the mean uorescence intensity (MFI) of CD
from . to . and that of CD from . to .
(Figure ). e MFI value of CD was decreased in
Evidence-Based Complementary and Alternative Medicine
CD86
Fluorescence intensity
CD40
Isotype
Counts
LPS (−)
LPS (+)
10
0
10
1
10
2
10
3
H
HHH
A
A
A
A
35
33 23 22
32 27
21
22
CD86
Fluorescence intensity
Counts
10
0
10
1
10
2
10
3
CD86
Fluorescence intensity
Counts
10
0
10
1
10
2
10
3
CD86
Fluorescence intensity
Counts
10
0
10
1
10
2
10
3
Fluorescence intensity
Isotype
Counts
LPS (−)
LPS (+)
10
0
10
1
10
2
10
3
CD40
Fluorescence intensity
Counts
10
0
10
1
10
2
10
3
CD40
Fluorescence intensity
Counts
10
0
10
1
10
2
10
3
CD40
Fluorescence intensity
Counts
10
0
10
1
10
2
10
3
LPS (+)/S 100 𝜇M
LPS (+)/S 100 𝜇M
(a)
0
2
4
6
8
10
12
14
0 0 10 50 100
Surface molecules (MFI)
CD86
CD40
LPS
∗∗∗
∗∗∗
∗∗∗
∗∗∗
S (𝜇M)
∗
(b)
F : Sakuranetin decreases the expression of costimulatory molecules. Mouse peritoneal macrophages were stimulated with LPS in the
presence of sakuranetin for h. e cells were stained for FITC-conjugated CD antibody or PE-conjugated CD antibody and analyzed
using ow cytometry. (a) Representative histograms are shown. (b) e value of mean uorescence intensity (MFI) was analyzed and data
are expressed as mean ± SD (𝑛=4).
∗
𝑃 < 0.05 and
∗∗∗
𝑃 < 0.005 versus controls (cells treated with LPS only).
a dose-dep endent manner with % and % of control
cells at and 𝜇M, respectively. CD expression was
decreased by % at 𝜇M compared with controls.
3.5. Eects of Sakuranetin on I𝜅B𝛼 Degradation and MAPK
Activation. e inammatory gene expression initiated by
LPS/TLR signaling depends on the NF-𝜅BandMAPK
signaling pathways. I𝜅B𝛼 plays a critical role in the control
of NF-𝜅B signaling by preventing it from migrating to the
nucleus []. Sakuranetin had no eect on I𝜅B𝛼 degradation
at min (Figure ). We examined the inuence of saku-
ranetin on the activation of MAPK (p, JNK, and ERK).
e expression of phosphorylated JNK was attenuated in a