Oleuropein Suppresses LPS-Induced Inflammatory Responses in 2 RAW 264.7 Cell and Zebrafish

Abstract

Oleuropein is one of the primary phenolic compounds present in olive leaf. In this study, the anti-inflammatory 11 effect of oleuropein was investigated using lipopolysaccharide (LPS)-stimulated RAW 264.7 and a zebrafish model. The 12 inhibitory effect of oleuropein on LPS-induced NO production in macrophages was supported by the suppression of inducible 13 nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In addition, our enzyme immunoassay showed that oleuropein 14 suppressed the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-6 (IL-6). Oleuropein 15 inhibited the translocation of p65 by suppressing phosphorylation of inhibitory kappa B-α (IκB-α). Oleuropein also decreased 16 activation of ERK1/2 and JNK, which are associated with LPS-induced inflammation, and its downstream gene of AP-1. 17 Furthermore, oleuropein inhibited LPS-stimulated NO generation in a zebrafish model. Taken together, our results 18 demonstrated that oleuropein could reduce inflammatory responses by inhibiting TLR and MAPK signaling, and may be used as 19 an anti-inflammatory agent. 20 KEYWORDS: oleuropein, anti-inflammation, RAW 264.7 cell, zebrafish, NF-κB(p-65)

Full text

1Oleuropein Suppresses LPS-Induced Inflammatory Responses in
2RAW 264.7 Cell and Zebrafish
3Su-Jung Ryu,
†
Hyeon-Son Choi,
‡
Kye-Yoon Yoon,
†
Ok-Hwan Lee,
§
Kui-Jin Kim,
∥
and Boo-Yong Lee*
,⊥
4
†
Department of Biomedical Science, CHA University, Kyonggi 463-836, South Korea
5
‡
Department of Food Science and Technology, Seoul Women’s University, 621 Hwarang-ro, Nowon-gu, Seoul 139-774, South Korea
6
§
Department of Food Science and Biotechnology, Kangwon National University, Chunchenon 200-701, South Korea
7
∥
Laboratory for Lipid Medicine & Technology, Department of Medicine, Harvard Medical SchoolMassachusetts General Hospital,
8149 13th Street, Charlestown, Massachusetts 02129, United States
9
⊥
Department of Food Science and Biotechnology, CHA University, Kyonggi 463-836, South Korea
10 ABSTRACT: Oleuropein is one of the primary phenolic compounds present in olive leaf. In this study, the anti-inflammatory
11 effect of oleuropein was investigated using lipopolysaccharide (LPS)-stimulated RAW 264.7 and a zebrafish model. The
12 inhibitory effect of oleuropein on LPS-induced NO production in macrophages was supported by the suppression of inducible
13 nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In addition, our enzyme immunoassay showed that oleuropein
14 suppressed the release of pro-inflammatory cytokines such as interleukin-1β(IL-1β) and interleukin-6 (IL-6). Oleuropein
15 inhibited the translocation of p65 by suppressing phosphorylation of inhibitory kappa B-α(IκB-α). Oleuropein also decreased
16 activation of ERK1/2 and JNK, which are associated with LPS-induced inflammation, and its downstream gene of AP-1.
17 Furthermore, oleuropein inhibited LPS-stimulated NO generation in a zebrafish model. Taken together, our results
18 demonstrated that oleuropein could reduce inflammatory responses by inhibiting TLR and MAPK signaling, and may be used as
19 an anti-inflammatory agent.
20 KEYWORDS: oleuropein, anti-inflammation, RAW 264.7 cell, zebrafish, NF-κB(p-65)
21 ■INTRODUCTION
22 The olive, Olea europaea, is an evergreen tree that grows in the
23 Mediterranean, Asia, and Africa.
1
Its fruit is commonly used as a
24 source of olive oil, which is important for the Mediterranean
25 diet. Olive leaf is also known to be a natural resource of various
26 beneficial polyphenols. The olive leaf is commonly used as a
27 traditional medicine for malaria and fever in Mediterranean
28 countries.
2
Many studies have been performed to examine the
29 phytochemicals in olive leaf; compounds such as tyrosol,
30 kaempterol, hydroxytyrosol, and oleuropein have been
31 identified.
1
Oleuropein is one of the major phytochemicals
32 found in olive leaf and is known to have biological effects such
33 as antioxidant, antiobesity, and antimicrobial activity.
3−5
In
34 addition, Drira et al. reported that this olive leaf-derived
35 compound inhibits adipocyte differentiation by suppressing the
36 cell cycle.
4
37 Inflammation is a physiological response against harmful
38 stimuli, such as pathogens, in the body.
6
It exerts protective
39 effects by inducing release of signaling molecules, which
40 neutralize injurious pathogens.
7
However, chronic inflamma-
41 tion has detrimental effects. These inflammatory processes can
42 interfere or destroy healthy cells, even causing cancer or the
43 formation of a plaque on the artery wall.
8
Recent studies have
44 shown that chronic inflammation is also associated with
45 diseases such as diabetes, high blood pressure, and obesity.
9−11
46 The immune system recognizes a variety of pathogens, which
47 trigger production of pro-inflammatory cytokines such as
48 interleukin-6 (IL-6), nitric oxide (NO), inducible nitric oxide
49 synthase (iNOS), and cyclooxygenase-2 (COX-2). The
50
activation of Toll-like receptors (TLRs) is related to the
51
production and activation of these cytokines.
12−14
Nuclear
52
factor kappa-light-chain-enhancer of activated B cells (NF-κB),
53
a signaling molecule in TLR pathways, plays a major role in
54
inflammatory responses by stimulating the expression of pro-
55
inflammatory genes. The activation of NF-κB as a transcription
56
factor requires the degradation of IκBαby phosphorylation.
15,16
57
In addition, activation of MAPK pathways, including p-38, JNK,
58
and ERK, leads to the activation of NF-κB.
17
MAPK pathway
59
also regulated another inflammatory key gene named AP-1 by
60
phosphorylation.
18
The constant activation of these signaling
61pathways can cause excessive inflammatory responses.
62
In the current study, researchers used zebrafish as an in vivo
63
model to assess the anti-inflammatory effect of oleuropein.
64
Zebrafish are a useful vertebrate model in biological research
65
due to their physiological similarity to mammals, availability in
66
large quantities, transparent body, and low cost.
19,20
Recent
67
studies have used zebrafish as a model for drug discovery.
21,22
68
Zebrafish also have innate and acquired immune systems
69
similar to those of mammals,
23
with dynamic and vivid embryo
70
images. In this report, we examined the inhibitory effect of
71
oleuropein on inflammatory responses and signaling in LPS-
72induced RAW 264.7 macrophages and a zebrafish model.
Received: August 5, 2014
Revised: January 22, 2015
Accepted: January 22, 2015
Article
pubs.acs.org/JAFC
© XXXX American Chemical Society ADOI: 10.1021/jf505894b
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
pubsdm_prod |ACSJCA |JCA10.0.1465/W Unicode |research.3f (R3.6.i7:4236 |2.0 alpha 39) 2014/12/19 13:33:00 |PROD-JCAVA |rq_4375856 |2/04/2015 10:39:37 |8|JCA-DEFAULT

73 ■MATERIALS AND METHODS
74 Materials. Dulbecco’s modified Eagle’s medium (DMEM), fetal
75 bovine serum (FBS), penicillin-streptomycin (P/S), and phosphate-
76 buffered saline (PBS) were purchased from Gibco (Gaithersburg,
77 MD). iNOS, COX-2, p65, p-IκB-α,IκB-α, p-ERK, ERK, p-p38, p38, p-
78 JNK, JNK, and GAPDH monoclonal antibodies and secondary
79 antibody were obtained from Cell Signaling Technology (Boston,
80 MA). The enzyme immunoassay (EIA) kits for IL-1βand IL-6 were
81 obtained from BioLegend (San Diego, CA). Maxime PT Premix KIT
82 was purchased from iNtRON (Gyeonggi-do, Korea). iNOS, COX-2,
83 IL-1β, IL-6, and GAPDH oligonucleotide primers were obtained from
84
Bioneer (Seoul, Korea). Compound 2, 3-bis(2-methoxy-4-nitro-5-
85
sulfophenyl)-2H-tetrazolium-5-carbox-anilide (XTT), was purchased
86
from WEL GENE (Daegu, Korea). Oleuropein (>98.0%), TRIzol
87
reagent, diaminofluorophore 4-amino-5-methylanino-2,7-difluorofluor-
88
oescein diacetate (DAF-FM DA), Griess reagent, and lipopolysac-
89
charide (LPS) (Escherichia coli, serotype 0111:04) were obtained from
90Sigma Chemical Co. (St. Louis, MO).
91
Cell Culture. RAW 264.7 macrophage cells (American Type
92
Culture Collection, CL-173, and passage 5−7) were cultured in
93
DMEM with 1.5 g/L sodium bicarbonate, 1% P/S, and 10% FBS at 37
94°C and in a humidified chamber with a 5% CO2atmosphere. Cells
Figure 1. Chemical structure of oleuropein (A) and its effect on cell viability (B). RAW 264.7 cells (1 ×104/well) were treated with oleuropein
(100−400 μg/mL) for 12, 24, 48, and 72 h. Cell viability was determined using the XTT assay (B). Data are presented as means with standard
deviations of three replicates. Results were analyzed by ANOVA and Duncan’s multiple range test (p< 0.05).
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/jf505894b
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
B

95 were incubated with 100, 200, and 300 μM oleuropein, and then
96 stimulated with LPS at 1 μg/mL for the indicated times.
97 XTT Assay. RAW 264.7 cells (∼1×104per well) were seeded in a
98 96-well plate and incubated in a CO2incubator at 37 °C for 12, 24, 48,
99 and 72 h. Cells were treated with various concentrations (100−400
100 μg/mL) of oleuropein and incubated for 12, 24, 48, and 72 h, after
101 which 2, 3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-
102 carbox-anilide (XTT) was added to the culture medium. The
103 cytotoxicity of oleuropein was determined on the basis of the
104 absorbance at 450 and 690 nm measured using an ELISA plate reader.
105 Nitrite Determination in RAW 264.7. RAW 264.7 cells (∼1×
106 104per well) were plated in 96-well plates, treated with various
107 concentrations of oleuropein and then incubated with or without LPS
108 (1 μg/mL) for 24 h. Nitrite levels, which reflect NO levels, in culture
109 media were determined using the Griess reaction. Cell culture medium
110 (100 μL) was mixed with 100 μL of Griess reagent and incubated at
111 room temperature for 15 min. Absorbance was then measured at 540
112 nm using an ELISA reader. Nitrite levels in samples were determined
113 using a standard sodium nitrite curve.
114 Enzyme-Linked Immunosorbent Assay (ELISA). RAW 264.7
115 cells were pretreated with various oleuropein concentrations for 1 h
116 and then further stimulated with LPS (1 μg/mL) for 24 h. The
117 supernatants were collected and stored at −80 °C until cytokine
118 analysis. IL-1βand IL-6 levels in supernatants were determined using
119 ELISA MAX Kits (BioLegend, San Diego, CA), according to the
120 manufacturer’s instructions.
121 RNA Isolation and Reverse Transcription Polymerase Chain
122 Reaction. Total RNA was extracted using TRIzol (Invitrogen,
123 Carlsbad, CA), and 1 μg of the total RNA was used to produce
124 cDNA using an RT-PCR system. Amplification of the target genes was
125
performed using specific oligonucleotide primers by PCR. The primers
126
used were as follows: iNOS, forward (5′-CCCTTCCGA-
127
AGTTTCTGGCAGCAG-3′) and reverse (5′-GGCTGTCAGAGC-
128
CTCGTGGCTTTG-3′); COX-2, forward (5′-ATGCTCCTG-
129
CTTGAGTATGT-3′) and reverse (5′-CACTACATCCTG-
130
ACCCACTT-3′); IL-6, forward (5′-
131
CCATCTCTCCGTCTCTCACC-3′) and reverse (5′-
132
AGACCGCTGCCTGTCTAAAA-3′); IL-1β,forward(5′-
133
CAGGATGAGGACATGAGCACC-3′) and reverse (5′-
134
CTCTGCACACTCAAACTCCAC-3′); GAPDH, forward (5′-
135
AACTTTGGCATTGTGGAAGG-3′) and reverse (5′-
136
ACACATTGGGGGTAGGAACA-3′). The PCR products were
137
separated on 1.0% agarose gels, stained with ethidium bromide, and
138
photographed. The expression levels were quantified by scanning using
139a gel documentation and analysis system (ImageJ, SAS).
140
Western Blot Analysis. RAW 264.7 cells were washed with PBS
141
buffer, lysed with lysis buffer, and then centrifuged to remove cell
142
debris. The total protein content of the supernatant was determined
143
using the Bradford assay. Protein extracts (50 μg) were separated using
144
SDS-PAGE and transferred to polyvinylidene fluoride membranes.
145
The membranes were immunoblotted with primary antibodies specific
146
for iNOS, COX-2, p65, p-IκB-α,IκB-α, p-ERK, ERK, p-p38, p38, p-
147
JNK, JNK, and GAPDH overnight. Secondary antibodies conjugated
148
to horseradish peroxidase (1:1000) were then applied for 1 h. The
149
bands were visualized using enhanced chemiluminescence (ECL) and
150detected using the LAS imaging software (Fuji, New York, NY).
151
Nitrite Determination in Zebrafish. Synchronized zebrafish
152
embryos were collected and rearranged using a pipet at 20 embryos/
153well in six-well plates containing 2 mL of egg water. After 7−9h
Figure 2. Inhibition by oleuropein of NO production and iNOS and COX-2 expression in LPS-stimulated RAW 264.7 cells. LPS-stimulated RAW
264.7 cells were treated with oleuropein (100, 200, or 300 μM) for 4 h, followed by oleuropein (100, 200, or 300 μM) and/or LPS for 24 h. Nitric
oxide concentrations (A) in the culture media were determined by Griess assay. mRNA (B) and protein (C) levels were determined by RT-PCR and
Western blot, respectively. The results were quantified using the ImageJ software (D). Data are presented as means with standard deviations of three
replicates. Results were analyzed by ANOVA and Duncan’s multiple range test (p< 0.05).
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/jf505894b
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
C

154 postfertilization (hpf), embryos were incubated with or without
155 various concentrations of oleuropein for 1 h. Zebrafish were stimulated
156 by LPS (5 μg/mL) for 24 h at 28.5 °C. The zebrafish embryos were
157 then transferred into fresh embryo medium. NO levels in the
158 inflammatory zebrafish model were measured using a fluorescent
159 probe dye, diaminofluorophore 4-amino-5-methylanino-2,7-difluoro-
160 fluoroescein diacetate (DAF-FM DA). Transformation of DAF-FM
161 DA by NO generates highly fluorescent triazole derivatives. Following
162 stimulation by LPS, the zebrafish larvae were transferred into 96-well
163 plates and treated with DAF-FM DA solution (1 μM) for 1 h in the
164 dark at 28.5 °C. After incubation, the zebrafish larvae were rinsed in
165 fresh zebrafish embryo medium and anesthetized with tricaine
166 methanesulfonate solution before observation. The fluorescence
167 intensity of individual zebrafish larvae was quantified using an
168 ECLIPSE E600 (Nikon, Tokyo, Japan).
169 Statistical Analysis. All experiments were performed in triplicate.
170 The results were analyzed statistically using an analysis of variance
171 (ANOVA) and Duncan’s multiple range test. A pvalue < 0.05 was
172 considered to indicate statistical significance (SAS Instititue, NC).
173
■RESULTS AND DISCUSSION
174
Effect of Oleuropein on RAW 264.7 Cell Viability.
175 f1
There is the chemical structure of oleuropein in Figure 1A.
176
Oleuropein was nontoxic to the RAW 264.7 cells at the
177
indicated range of concentrations (Figure 1B) and also has no
178
toxicity at serial time −12, 24, 48, and 72 h. No morphological
179
changes in the cells were observed on the basis of microscopic
180
analysis (data not shown). This result shows that oleuropein
181
has no effect on cell apoptosis. Accordingly, all of the following
182
experiments were performed using oleuropein concentrations
183of 100, 200, and 300 μM.
184
Effect of Oleuropein on LPS-Induced NO Production
185
and Expression of iNOS and COX-2. Overproduction of
186
iNOS-mediated nitric oxide is a representative inflammatory
187
reaction, and may be involved in other negative cellular
188
physiologies such as mutagenesis, DNA damage, and the
189formation of N-nitrosoamine.
24−26
Cyclooxygenase-2 (COX-
Figure 3. Effect of oleuropein on IL-6 and IL-1βmRNA and protein levels in LPS-stimulated RAW 264.7 cells. Cells were treated with oleuropein
(100, 200, or 300 μM), followed by LPS. Cells were then incubated for a further 4 or 24 h. IL-6 and IL-1βmRNA levels were determined by RT-
PCR and visualized on a gel. (A) Cytokine levels in culture media were determined using an enzyme immunoassay kit. (B) Data are presented as
means with standard deviations of three replicates. Results were analyzed by ANOVA and Duncan’s multiple range test (p< 0.05).
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/jf505894b
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
D

190 2), another inflammatory marker, is also associated with
191 production of proinflammatory substances such as prostaglan-
192 dins, and is upregulated during inflammation. In particular,
193 COX-2-activated pathways, which are responsible for the
194 conversion of arachidonic acid to prostaglandin and other
195 eicosanoids, are of clinical importance as major targets for
196 nonsteroid anti-inflammatory drugs such as aspirin, which is
197 commonly used for inflammation and pain.
27
However, these
198 drugs have several side effects such as gastrointestinal bleeding,
199 swelling of skin tissue, and allergy. We examined the effect of
200 oleuropein on LPS-induced NO production, a mediator of the
f2 201 inflammatory response. As shown in Figure 2A, the NO level in
202 culture medium was reduced by oleuropein treatment in a
203 dose-dependent manner. This decrease in NO production was
204 due to the downregulation of iNOS, a major pro-inflammatory
205 enzyme that produces NO, at the mRNA and protein levels
206 (Figure 2B,C). In addition, COX-2 (also an inflammatory
207 marker) mRNA and protein levels were decreased by
208 oleuropein treatment in a dose-dependent manner (Figure
209 2B,C). Expression of iNOS and COX-2 in mRNA was reduced
210 by 42% and 43%, respectively, and in protein levels was reduced
211 by 72% and 45%, respectively, by 300 μM oleuropein (Figure
212 2D). Our data showed that oleuropein, an olive compound,
213 decreased LPS-induced NO production dose-dependently by
214 downregulating iNOS (Figure 2A,B), which is closely
215
associated with the synthesis of NO and COX-2 expression.
216
This result indicated that oleuropein could be a potential anti-
217
inflammatory phytochemical. Recent studies identified various
218
phytochemicals with anti-inflammatory effects. Resveratrol,
219
EGCG, and tyrosol are also known to downregulate COX-2
220and iNOS.
28−30
221
Effect of Oleuropein on LPS-Induced IL-1βand IL-6
222
Release and Their mRNA Expressions. Inflammation
223
generally involves the abnormal regulation of cytokines.
224
Cytokines such as IL-6 and IL-1βare pro-inflammatory in
225
vitro and in vivo.
31
Inflammation generally involves the
226
abnormal regulation of cytokines. Cytokines such as IL-6 and
227
IL-1βare pro-inflammatory in vitro and in vivo, and play
228
important roles in the extent of inflammation and recruit other
229
immune cells implicated in the pathogenesis of diverse
230
inflammatory conditions, such as rheumatoid arthritis and
231
septic shock.
32
We further examined the anti-inflammatory
232
effect of oleuropein by assaying levels of pro-inflammatory
233
cytokines. RT-PCR analysis showed that expression of IL-1β
234
and IL-6 (inflammatory cytokines) was reduced by oleuropein
235 f3
treatment (Figure 3A). Levels of these cytokines in culture
236
medium were decreased by oleuropein treatment in a dose-
237
dependent manner. IL-6 and IL-1βlevels were decreased,
238
respectively, compared with the LPS-induced group at 300 μM
239oleuropein (Figure 3B). In particular, IL-1βin the presence of
Figure 4. Effect of oleuropein on the phosphorylation of IκB-αand the nuclear translocation of NF-κB. Cells were pretreated with oleuropein (100,
200, or 300 μM) for 1 h and then with LPS (1 μg/mL) for 15 min. Protein levels were determined by Western blotting. NF-κb p65 levels in the
cytosol and nucleus were quantified using the ImageJ software and normalized to Lamin B and β-actin, respectively. (A) The results were quantified
using the ImageJ software and normalized to IκBα(B). Data are presented as means with standard deviations of three replicates. Results were
analyzed by ANOVA and Duncan’s multiple range test (p< 0.05).
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/jf505894b
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
E

240300 μM oleuropein was reduced to the level of the control
241(Figure 3B). Therefore, oleuropein suppressed the LPS-
242induced release of pro-inflammatory cytokines. Our results
243showed that LPS induced release of cytokines (Figure 3B) was
244effectively reduced by oleuropein treatment at the mRNA
245(Figure 3A). However, we did not explore whether other
246cytokines, such as TNF-αand IL-10, were also regulated by
247oleuropein. Analysis of these cytokines would provide more
248information on the anti-inflammatory mechanism of oleur-
249opein.
250Effect of Oleuropein on LPS-Induced IκB Phosphor-
251ylation and NF-κB Translocation. Regulation of inflamma-
252tory cytokines and inflammatory responses is transcriptionally
Figure 5. Inhibitory effect of oleuropein on LPS-induced activation of
MAP kinases and AP-1 in RAW 264.7 cells. RAW 264.7 were
pretreated with oleuropein (100, 200, or 300 μM) for 1 h and then
with LPS (1 μg/mL) for 15 min. Total proteins (50 μg) were
subjected to Western blotting (A and C). The protein levels were
quantified using the ImageJ software, and those of the phosphorylated
forms were normalized to total protein levels (B). The data of AP-1
were quantified using the ImageJ software and normalized to GAPDH
(D). Data are presented as means with standard deviations of three
replicates. Results were analyzed by ANOVA and Duncan’s multiple
range test (p< 0.05).
Figure 6. Effect of oleuropein on LPS-induced NO production in
zebrafish embryo. Zebrafish embryos were pretreated with oleuropein
for 1 h and then exposed to LPS (5 μg/mL) for 24 h (A). The NO
level was measured after staining with DAF-FM-DA (A). The results
were quantified using the ImageJ software. (B) Data are presented as
means with standard deviations of three replicates. Results were
analyzed by ANOVA and Duncan’s multiple range test (p< 0.05).
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/jf505894b
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
F

253 governed by NF-κb transcription factors. These transcription
254 factors regulate inflammatory related genes in the nucleus,
255 where they bind to the DNA of pro-inflammatory mediators to
256 induce their transcription.
33,28
In addition, transcriptional
257 activity of NF-κB is dependent on IκBαphosphorylation,
34
258 by which p65, a subunit of NF-κB, translocates into the nucleus
259 to promote the expression of inflammatory genes including
260 iNOS, COX-2, and cytokines such as IL-6 and IL-1β.
35,36
Our
261 results showed that oleuropein-mediated inhibition of IκBα
f4 262 phosphorylation (Figure 4B) blocks the translocation of p65
263 from the cytosol to the nucleus (Figure 4A), suggesting that
264 oleuropein exerts its anti-inflammatory effects by suppressing
265 NK-κB, a major component of the TLR pathway. The nuclear
266 NF-κB level increased significantly after LPS treatment
267 compared with the normal control (Figure 4A). Oleuropein
268 (300 μM) inhibited the translocation of p65, a subunit of NF-
269 κB, into the nucleus by 40% compared with the LPS-induced
270 control. However, lower concentrations of oleuropein exerted
271 no significant effects on the translocation of p65 into the
272 nucleus. Cytosolic NF-κB levels decreased with LPS treatment,
273 but increased in the oleuropein-treated group (300 μM). This
274 result was correlated with the nuclear NF-κB pattern, i.e., a
275 reduction in the presence of oleuropein (300 μM) (Figure 4A).
276 Translocation of NF-κB into the nucleus is associated with
277 phosphorylation of IκB-αin the TLR4 pathway, a major
278 inflammation pathway. Accordingly, we examined the phos-
279 phorylation of IκB-α, a mediator of NF-κB activation.
280 Oleuropein inhibited IκB-αphosphorylation, suggesting that
281 NF-κB translocation into the nucleus was inhibited by
282 suppression of IκB-αphosphorylation (Figure 4B). Therefore,
283 the inhibitory effect of oleuropein on LPS-induced inflamma-
284 tory responses was due to the deactivation of NF-κb and IκB-α,
285 major components in the TLR4 pathway.
286 Effect of Oleuropein on LPS-Induced Activation of
287 Mitogen-Activated Protein (MAP) Kinase. MAP kinases
288 are another signaling pathway that plays a critical role in
289 inflammation through activation of NFκB.
17
This kinase family
290 is composed of several subgroups, such as JNK, ERK, and p38.
291 The activation of these kinase groups mediates various
292 inflammatory responses in vitro and in vivo.
37−39
The
293 inflammatory response can be activated through the MAP
294 kinase pathway. Thus, we determined whether MAP kinase
295 signaling is involved in oleuropein-mediated inhibition of LPS-
296 induced inflammatory responses. MAP kinase plays a critical
297 role in LPS-induced inflammation signaling at the transcrip-
298 tional level. Since the MAP kinase pathway is phosphorylation-
299 dependent, we examined the phosphorylation status of
300 components of this pathway. Oleuropein significantly decreased
301 LPS activated ERK and JNK by inhibiting their phosphor-
f5 302 ylation (Figure 5A). Phosphorylation of ERK and JNK induced
303 by LPS was reduced by 20% and 62%, respectively, by 300 μM
304 oleuropein (Figure 5B). However, phosphorylation of P38
305 induced by LPS, also a component of the MAP kinase pathway,
306 was not decreased in the presence of oleuropein. Our data also
307 showed that oleuropein decreased AP-1, downstream gene
308 related with MAP kinase (Figure 5C). AP-1 is another
309 inflammatory key gene by regulation NO.
18
Our results
310 indicated that inhibition of ERK and JNK rather than p38
311 contributes to the anti-inflammatory effect of oleuropein in the
312 LPS-induced inflammatory response. This result suggested that
313 the anti-inflammatory effect of oleuropein might be due at least
314 in part to the inhibition of ERK and JNK.
315Effect of Oleuropein on LPS-Induced NO Production
316in Zebrafish Model. We used a zebrafish model to investigate
317the anti-inflammatory effect of oleuropein in vivo. Nitric oxide
318(NO) production in zebrafish was determined using a
319 f6fluorescent probe dye. As shown in Figure 6A, the nitric
320oxide level in zebrafish was elevated after LPS treatment
321compared with the positive control. Therefore, LPS increased
322nitric oxide production in zebrafish, similar to that in RAW
323264.7 cells. LPS-induced increases in NO production were
324significantly suppressed in the presence of oleuropein (Figure
3256A). In particular, 300 μM oleuropein inhibited LPS-induced
326NO production in zebrafish by 62% compared with the LPS
327control group (Figure 6B). Our data showed that oleuropein
328effectively reduced LPS-induced NO production in zebrafish.
329This result was correlated with the in vitro cell culture data,
330suggesting that oleuropein exerts anti-inflammatory effects. To
331our knowledge, this is the first report of an anti-inflammatory
332effect of oleuropein in zebrafish. However, we did not explore
333the genetic regulation of inflammatory factors at the gene and
334protein levels. Thus, further studies on the inflammatory
335responses of zebrafish in the presence of oleuropein are
336required. Oleuropein is a relatively abundant phenolic
337compound in olive leaf compared with other components,
338such as tyrosol and hydroxytyrosol, which have been suggested
339to possess various biological activities; also, our data showed
340that oleuropein inhibited inflammatory responses in RAW
341264.7 cells and zebrafish by suppressing NF-κB translocation
342and the MAP kinase pathway. Doses of oleuropein in the range
343100−300 uM are very high so we think that concentrations
344were supraphysiological. Our study also provides information
345important for the development of anti-inflammatory agents
346containing oleuropein.
347
■AUTHOR INFORMATION
348Corresponding Author
349*Phone: +82-31-725-8371. Fax: +82-31-725-8282. E-mail:
350bylee@cha.ac.kr.
351Notes
352The authors declare no competing financial interest.
353
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