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Anti-inammatory effect of Momordica grosvenori Swingle

extract through suppressed LPS-induced upregulation of iNOS


and COX-2 in murine macrophages
Min-Hsiung Pan
a,
*
, Ji-Rui Yang
a
, Mei-Ling Tsai
a
, Shengmin Sang
b
, Chi-Tang Ho
c,d,
*
a
Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung 811, Taiwan
b
Nutrition Research Program, University of North Carolina Nutrition Center, North Carolina Research Campus, Julius L. Chambers
Biomedical/Biotechnology Research Institute, North Carolina Central University, 500 Laureate Way, Kannapolis, NC 28081, USA
c
Department of Food Science, Rutgers University, New Brunswick, NJ 08901-8520, USA
d
Graduate Institute of Food Science and Technology, National Taiwan University, Taipei 100, Taiwan
A R T I C L E I N F O
Article history:
Received 22 July 2008
Accepted 24 December 2008
Available online 24 February 2009
Keywords:
Momordica grosvenori Swingle
Inducible NO synthesis (iNOS)
NFjB
RAW 264.7 monocyte/macrophages
Lipopolysaccharide (LPS)
Mitogen-activated protein (MAPK)
Phosphatidylinositol 3-kinase (PI3K)
Cyclooxygenase-2 (COX-2)
A B S T R A C T
Momordica grosvenori Swingle, a traditional medicinal herb, is known to possess anti-
inammatory anti-oxidative, anti-diabetic, and nephroprotective activities. Here, we inves-
tigated the inhibitory effects of M. grosvenori Swingle extract (MSE) on the induction of NO
synthase (iNOS) and cyclooxygenase-2 (COX-2) in murine RAW 264.7 cells activated with
lipopolysaccharide (LPS). Western blotting and reverse transcriptase polymerase chain
reaction (RT-PCR) analyses demonstrated that MSE signicantly blocked protein and mRNA
expression of iNOS and COX-2 in LPS-induced macrophages. Treatment with MSE resulted
in the reduction of LPS-induced nuclear translocation of nuclear factor-jB (NFjB) subunit
and the dependent transcriptional activity of NFjB by blocking phosphorylation of inhibitor
jB (IjB)a and p65 and subsequent degradation of IjBa. Transient transfection experiments
using NFjB reporter constructs indicated that MSE inhibits the transcriptional activity of
NFjB in LPS-stimulated mouse macrophages. MSE also inhibited LPS-induced activation
of PI3K/Akt, extracellular signal-regulated kinase 1/2 and p38 MAPK. Taken together, these
results show that MSE down regulates inammatory iNOS and COX-2 gene expression in
macrophages by inhibiting the activation of NFjB by interfering with the activation PI3K/
Akt/IKK and MAPK. These results have important implication for using MSE towards the
development of effective anti-inammatory agents.
Crown Copyright 2009 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Momordica grosvenori Swingle, a fruit with traditional medici-
nal properties, is known to demonstrate anti-inammatory
antioxidant, anti-diabetic, and nephroprotective properties
(Song et al., 2006, 2007). A recent report has shown that cuc-
urbitane glycosides from M. grosvenori Swingle extract (MSE)
exhibits anti-carcinogenic activity (Takasaki et al., 2003), but
the molecular mechanisms involved was not claried. The
present study was aimed to investigate the effects of MSE
on LPS-induced iNOS and COX-2 expression and to explore
underlying molecular mechanisms in murine macrophage.
1756-4646/$ - see front matter Crown Copyright 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.jff.2009.01.003
Abbreviations: iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NO, nitric oxide; COX-2, cycoloxygenase-2; PGE
2
,
prostaglandin E
2
; IjB, inhibitor jB; NF-jB, nuclear factor-jB; MAPK, mitogen-activated protein kinase.
* Corresponding authors: Tel.: +886 7 361 7141; fax: +886 7 361 1261 (M.-H. Pan).
E-mail address: mhpan@mail.nkmu.edu.tw (M.-H. Pan).
J O U R N A L O F F U N C T I O N A L F O O D S 1 ( 2 0 0 9 ) 1 4 5 1 5 2
avai l abl e at www. sci encedi r ect . com
j our nal homepage: www. el sevi er . com/ l ocat e/ j f f
Lipopolysaccharide (LPS), a component of the cell walls of
gram-negative bacteria, induces the activation of monocytes
and macrophages and involves the production of proinam-
matorycytokines (Nicholas et al., 2007). Nitricoxide(NO) is pro-
duced endogenously by a family of nitric oxide synthases
(NOSs) with a wide range of physiological and pathophysiolog-
ical actions (Moncada et al., 1991; Nathan&Xie, 1994). NOS en-
zymes are classied into two groups. One group, (cNOS), is
constitutively present in several cell types (e.g. neurons and
endothelial cells) (Liu & Hotchkiss, 1995). The other group, the
inducible form(iNOS), which is expressed invarious cell types,
including macrophages, is induced in response to proinam-
matory cytokines and bacterial LPS (Geller et al., 1993; Rockey
et al., 1998). ExcessiveandprolongediNOS-mediatedNOgener-
ation has been linked with inammation and tumorigenesis.
Cyclooxygenase-2(COX-2) isaninducibleenzymecatalyzing
the conversion of arachidonic acid to prostaglandins. Recent
studies have suggested that increased levels of prostaglandins
and cyclooxygenase activity may play important roles in multi-
ple epithelial cancers such as colon carcinoma (Dubois et al.,
1998; Levy, 1997). COX-2-derivedbioactivelipids, includingpros-
taglandin E
2
, are potent inammatory mediators that promote
tumor growth and metastasis by stimulating cell proliferation,
invasion, and angiogenesis (Mann et al., 2005). Therefore, high
levels of prostaglandins may promote the development of
malignancy (Yoshimura et al., 2005).
Previous reports have showna potential role for tyrosine ki-
nase in LPS promoter that contain 24 transcriptional factor
binding sites, including those for NFjB family, which appear
to be essential for the enhanced iNOS and COX-2 gene expres-
sion seen in macrophages exposed to LPS (Pan et al., 2006; Xie
et al., 1994). The p65 NFjB also seems to be responsible for
inducing iNOS in astrocytes (Feinstein et al., 1996). Activation
of NFjB by LPS is induced by a cascade of events leading to
the activation of inhibitor jB (IjB) kinases (IKKs), whichin turn
phosphorylates IjB and leads to the degradation of NFjB and
its translocation to the nucleus (Griscavage et al., 1996). In this
pathway, NFjB transcriptional activity is independent of IjBa
degradation and is regulated by phosphorylation of NFjB.
Phosphorylation at Ser 536 on the p65 subunits is mediated
by IKK during LPS stimulation (Yang et al., 2003). Ser 536 phos-
phorylation is responsible for the recruitment of coactivators
as p300, promoting the transcriptional activation of NFjB and
subsequent production of inammatory cytokines (Chen
et al., 2004). These IKKs canbe activated through phosphoryla-
tion by upstream kinases, including NFjB-inducing kinase,
mitogen-activate protein kinase, and protein kinase C (Chio
et al., 2004; Kim et al., 2005). In addition, many studies imply
cytokine in the induction of transcription activity of NFjB
through extracellular signal-regulated 1/2 (ERK)1/2 (p42/44),
p38 MAPK, and PI3K/AKT pathways (Agarwal et al., 2005; Bozi-
novski et al., 2002; Chandrasekar et al., 2004; Je et al., 2004).
Importantly, NO has been shown to be involved in regulating
cycoloxygenase-2 (COX-2), which plays a pivotal role in colon
tumorigenesis (Landino et al., 1996). Collectively, suppression
of enzyme induction and the activities of iNOS/COX-2 is an
important approachtopreventing carcinogenesis inseveral or-
gans, including the stomachand colon(Nosho et al., 2005). The
recent emphasis on the role of NO in pathological conditions
suchas atherosclerosis include hypercholesterolaemia, hyper-
tension, diabetes, smoking, and cardiovascular disease has led
to the discovery of newtherapeutic agents. Here we found that
MSE was able to protect against LPS-induced inammation by
blocking the activation of NFjB, p44/42 MAPK and PI3K/Akt,
thereby inhibiting the iNOS and COX-2 expression.
2. Materials and methods
2.1. Reagents
Lipopolysaccharide (LPS) (Escherichia coli 0127: E8), sulfanil-
amide, naphthylethylenediamine dihydrochloride, and dithi-
othreitol (DTT) were purchased from Sigma Chemical Co.
(St. Louis, MO, USA).
2.2. Momordica grosvenori Swingle extract
M. grosvenori Swingle extract (MSE) was a gift from Ibis Amer-
ica, LLC (Branchburg, NJ, USA). The level of mogroside V in
MSE used in this study was quantied using the following
HPLC method by comparing the peak area of the test sample
with the standard curve. Any information on the molecular
weight of mogroside V was puried from the extracts and
its structure was conrmed by comparing its NMR data with
those reported in the literature (Si et al., 2005). The nal per-
centage of the mogroside V in the tested MSE was 25.9%.
TheHPLCsystemconsistedof aWaters717refrigeratedauto-
sampler, a HITACHI L-6200A intelligent pump, and a UVVIS
detector (Waters 490E programmable multiwavelength detec-
tor). ASupelcosil C18reversed-phasecolumn(150 mm 4.6 mm
inner diameter; Supelco Co., Bellefonte, PA, USA) was used. The
HPLC was performed under isocratic elution with 22% acetoni-
trile and78%water. The solvent was lteredwith0.2 lmNylao
membrane lter and degassed. The owrate was maintained at
1 mL/min. The running time for each sample was 30 min. The
injectionvolume was 50 lL for each sample solution. The wave-
length of UV detector was set at 190, 195, 210, and 254 nm for
channels 14, respectively. The limit of detection was 100 ng/
mL and the limit of quantication was 1 lg/mL.
2.3. Cell culture
RAW 264.7 cells, derived from murine macrophages, were ob-
tained from the American Type Culture Collection (Rockville,
MD, USA). RAW 264.7 cells were cultured in Roswell Park
Memorial Institute (RPMI) 1640 media (without phenol red)
supplemented with 10% endotoxin-free, heat-inactivated fe-
tal calf serum (GIBCO, Grand Island, NY, USA), 100 units/mL
penicillin, and 100 lg/mL streptomycin. When the cells
reached a density of 23 10
6
cells/mL, they were activated
by incubation in medium containing E. coli LPS (100 ng/mL).
Various concentrations of test compounds dissolved in di-
methyl sulphoxide (DMSO) were added together with LPS.
Cells were either treated with 0.05% DMSO as vehicle control.
2.4. Determination of prostaglandin E2 (PGE2)
The culture medium of control and treated cells was col-
lected, centrifuged and stored at 70 C until tested. The level
146 J O U R N A L O F F U N C T I O N A L F O O D S 1 ( 2 0 0 9 ) 1 4 5 1 5 2
of PGE
2
released into the culture medium was quantied
using a specic enzyme immunoassay (EIA) according to the
manufacturers instructions (Amersham, Arlington Heights,
IL, USA; Suh et al., 1998).
2.5. Nitrite assay
The nitrite concentration in the culture medium was mea-
sured as an indicator of NO production, according to the Gri-
ess reaction (Kim et al., 1995). One hundred microlitres of
each supernatant were mixed with the same volume of Griess
reagent (1% sulphanilamide in 5% phosphoric acid and 0.1%
naphthylethylenediamine dihydrochloride in water). Absor-
bance of the mixture at 550 nm was measured with an en-
zyme-linked immunosorbent assay plate reader (Dynatech
MR-7000; Dynatech Labs, Chantilly, VA, USA).
2.6. Cytotoxicity assay
The RAW 264.7 cells were cultivated at a density of 2 10
5
cells in a six-well plate. The MSE studied were added to the
medium 18 h after inoculation. The cells were harvested after
18 h. Viability was determined by trypan blue exclusion and
microscopy examination.
2.7. Western blotting
Afterward, the stimulated murine macrophage cell line RAW
264.7 cells were washed with PBS and lysed in an ice-cold
RIPA buffer (25 mM TrisHCl pH 7.2; 0.1%, SDS; 1%, Triton
X-100; 1%, sodium deoxycholate; 0.15 M, NaCl; EDTA (ethyl-
enediaminetetra acetic acid) 1 mM) containing 1 mM of phe-
nylmethylsulphonyl uoride (PMSF), 10 lg/mL of aprotinin,
1 mM of sodium orthovanadate and 5 lg/mL of leupeptin. Pro-
tein concentrations were determined using the bicinchoninic
acid assay (BCA) method (Pierce, Rockford, IL, USA). The sam-
ples (50 lg of protein) were mixed with vefold sample buffer
containing 0.3 M TrisHCl (pH 6.8), 25% 2-mercaptoethanol,
12% sodium dodecyl sulphate (SDS), 25 mM EDTA, 20% glyc-
erol, and 0.1% bromophenol blue. The mixtures were boiled
at 100 C for 5 min and were pre-run on a stacking gel and
then resolved by 12% SDS-polyacrylamide minigels at a con-
stant current of 20 mA. For electrophoresis, proteins on the
gel were electro-transferred onto a 45 lm immobile mem-
brane (PVDF; Millipore Corp., Bedford, MA, USA) with transfer
buffer composed of 25 mM TrisHCl (pH 8.9), 192 mM glycine,
and 20% methanol as described previously (Pan et al., 2000).
The membrane was then incubated with the primary anti-
body of COX-2 or iNOS (Transduction Laboratories, Lexington,
KY, USA) and cytosolic fraction (for IjBa, p65). The membrane
was blocked overnight at room temperature with blocking
solution (20 mM TrisHCl pH 7.4, 125 mM NaCl, 0.2% Tween
20, 1% bovine serum albumin, and 0.1% sodium azide), anti-
phospho (Ser 32)-specic IjBa (New England Biolabs, Ipwich,
MA, USA), or anti-b-actin monoclonal antibodies (Oncogene
Science Inc., Uniondale, NJ, USA) at room temperature for
1 h. The anti-phospho-Akt (Ser473), anti-phospho-p65
(Ser536), anti-phospho-p38 (Thr180/Tyr182), anti-phospho-
JNK, anti-phospho-ERK1/2 (Thr202/Tyr204), ERK, JNK, p38,
and Akt antibodies obtained from Cell Signaling Technology
(Beverly, MA, USA) were used to determine the level of phos-
phorylated proteins. The membranes were subsequently
probed with anti-mouse or anti-rabbit IgG antibody conju-
gated to horseradish peroxidase (Transduction Laboratories,
Lexington, KY, USA) and visualized using enhanced chemilu-
minescence (ECL, Amersham). The densities of the
bands were quantitated with a computer densitometer
(AlphaImager
TM
2200 System). All the membranes were
stripped and reprobed for b-actin (Sigma Chemical Co., St.
Louis, MO, USA) as loading control.
2.8. Reverse transcription polymerase chain reaction (RT-
PCR)
Total RNA was isolated from mouse macrophage RAW 264.7
cell using Trizol Reagent according to the manufacturers
instructions (Invitrogen, Life Technologies, Carlsbad, CA,
USA). Changes in the steady-state concentration of mRNA
in iNOS, COX-2 and G3PDH were assessed by RT-PCR. Total
RNA (2 lg) was converted to cDNA in a series of standard
10-lL reverse transcription reactions. DNA amplication was
carried out in Ready To Go PCR Beads (Amersham Pharma-
cia Biotech, Piscataway, NJ, USA). The initial conditions were
42 C for 60 min. Amplication 30 cycles of iNOS were 95 C
for 40 s, 65 C for 60 s, and 72 C for 2 min, followed by a
10 min extension at 72 C. The thermal cycle conditions of
COX-2 were initiated at 42 C for 60 min, then 30 cycles of
amplication (94 C for 45 s, 55 C for 60 s, and 72 C for
2 min) were performed followed by 10 min extension at
72 C. The PCR products were separated by electrophoresis
on a 1% agarose gel and visualized by ethidium bromide
staining. Amplication of G3PDH served as a control for sam-
ple loading and integrity. PCR was performed on the cDNA
using the following sense and antisence primer: iNOS, for-
ward primer 5
0
-CCCTTCCGAAGTTTCTGGCAGCAGC-3
0
(2944
2968), reverse primer 5
0
-GGCTGTCAGAGAGCCTCGTGGCTTT-
GG-3
0
(34163440); COX-2, forward primer 5
0
-GGAGAGACTA-
TCAAGATAGTGATC-3
0
(10941117), reverse primer 5
0
-ATGGT-
CAGTAGACTTTTACAGCTC-3
0
(19311954); b-actin, forward
primer 5
0
-AAGAGAGGCATCCTCACCCT-3
0
, reverse primer 5
0
-
TACATGGCTGGGGTGTTGAA-3
0
. Conrmation of the correct
amplicons was obtained by direct DNA sequencing of the
PCR products.
2.9. Transient transfection and luciferase assay
The luciferase assay was performed as described by George
et al. (1997) with some modications. RAW 264.7 cells were
seeded in a 60-mm dish. When the cells reached conuence,
the medium was replaced with serum-free Opti-MEM (Gibco).
The cells were then transfected with the pNFjB-Luc plasmid
reporter gene (Stratagene, Jalla, CA, USA) using Lipofect-
AMINE
TM
reagent (Gibo, NRL, Life Technologies, Inc. Grand Is-
land, NY). After 24 h of incubation, the medium was replaced
with complete medium. After another 24 h, the cells were
trypsinized and equal numbers of cells were plated in 12-well
tissue culture plates for 18 h. The cells were then incubated
with 100 ng/mL LPS and MSE for 3 h. Each well was then
washed twice with cold PBS and harvested in 150 lL of lysis
buffer (0.5 M HEPES pH 7.8, 1% Triton N-101, 1 mM CaCl
2
J O U R N A L O F F U N C T I O N A L F O O D S 1 ( 2 0 0 9 ) 1 4 5 1 5 2 147
and 1 mM MgCl
2
). Luciferase activity was assayed by means of
the LucLite
TM
luciferase reporter gene kit (Packard BioScience
Company, Meriden, CT, USA), with 100 lL of cell lysate used in
each assay. Luminescence was measured on a Top Counter
Microplate Scintillation and Luminescence Counter (Packard
9912 V) in single photon counting mode for 0.1 min/well, fol-
lowing a 5 min adaptation in the dark. Luciferase activities
were determined and normalized to protein concentrations.
2.10. Statistical analysis
Data are presented as means SE of at least three indepen-
dent experiments. One way Students t-test was used to as-
sess the statistical signicance between the LPS- and MSE
plus LPS-treated cells. A P-value <0.05 was considered statis-
tically signicant.
3. Results
3.1. Inhibition of LPS-induced nitrite and PGE2 production
by MSE in RAW 264.7 macrophages
To investigate the anti-inammatory effect of MSE, we tested
their effect on nitrite and prostaglandin production in LPS-
activated macrophages. MSE at 800 lg/mL did not interfere
with the reaction between nitrite and Griess reagents (data
not shown). As shown in Fig. 1, MSE inhibited nitrite produc-
tion by >50% at 500 lg/mL. MSE, at a concentration range of
300800 lg/mL, markedly and concentration-dependently
suppressed nitrite production. After treatment with LPS for
24 h, the medium concentration of PGE
2
had elevated signi-
cantly to 3.3 ng/mL. This increase was markedly inhibited by
different concentrations of MSE (Fig. 2). Inhibition of nitrite
and PGE
2
production was not toxic, as determined by the try-
pan blue exclusion assay (data not shown).
3.2. MSE inhibition of LPS-induced iNOS and COX gene
expression
We next investigated whether MSE might affect levels of iNOS
and COX-2 proteins. As shown inFig. 3, MSE strongly and con-
centration-dependently suppressed the protein levels of both
iNOS and COX-2. These data suggest that translational events
are involved in MSEs inhibition of LPS-induced expression of
iNOS and COX-2. Changes in amounts of iNOS and COX-2 en-
zymes could reect altered protein synthesis or degradation.
RT-PCR analysis was done to investigate whether MSE sup-
pressed LPS-mediated induction of iNOS and COX-2 via a pre-
translational mechanism. From the result of this experiment,
we found that MSE marked suppression of iNOS and COX-2 (b-
actin as control gene) gene expression in a dose-dependent
manner in LPS-activated macrophages ( Fig. 4). These data
suggest that MSE may inhibit the expression of iNOS and
COX-2 at the transcription levels. The inhibitory effect of
Fig. 1 Effects of MSE on LPS-induced nitrite production in
RAW 264.7 macrophage. The cells were treated with 100 ng/
mL of LPS only or with different concentrations of MSE for
24 h. At the end of incubation time, 100 lL of the culture
medium was collected for nitrite assay. The values are
expressed as means standard error of triplicate tests.
*
P < 0.05 and
**
P < 0.01 indicate statistically signicant
differences from the LPS-treated group.
Fig. 2 Effects of MSE on LPS-induced PGE
2
production in
RAW 264.7 macrophage. The cells were treated with 100 ng/
mL of LPS only or with different concentrations and MSE for
24 h. At the end of incubation time, 100 lL of the culture
medium was collected for PGE
2
assay. The values are
expressed as means standard error of triplicate tests.
*
P < 0.05,
**
P < 0.01 indicate statistically signicant
differences from the LPS-treated group.
Fig. 3 Effects of MSE on LPS-induced iNOS and COX-2
protein expression in RAW 264.7 cells. The cells were
treated with different concentrations (500, 600, 700, and
800 lg/mL) of MSE for 24 h. Equal amounts of total proteins
(50 lg) were subjected to 10% SDS-PAGE. The expression of
iNOS, COX-2 and b-actin protein was detected by Western
Blot using specic antibodies. This experiment was
repeated three times with similar results.
148 J O U R N A L O F F U N C T I O N A L F O O D S 1 ( 2 0 0 9 ) 1 4 5 1 5 2
MSE on iNOS and COX-2 activities in LPS-activated macro-
phages was further studied in details.
3.3. Reduction of nuclear NFjB level and NFjB activation
by MSE treatment in LPS-stimulated macrophages
Because activation of NFjB is critical for induction of both
iNOS and COX-2 by LPS or other inammatory cytokines, we
used nuclear accumulation to test whether MSE would per-
turb the distribution of NFjB subunits. Nucleus and cytosolic
extracts were prepared and subjected to immunoblot analy-
sis. As shown in Fig. 5A, co-incubation with LPS plus MSE de-
creased NFjB proteins in the nucleus. Lamin B, a nuclear
protein, and b-actin, a cytosolic protein, were used as controls
to conrm that there was no contamination during extraction
of each fraction. In an additional study, transient transfection
with a NFjB-dependent luciferase reporter plasmid was done
to conrm whether MSE inhibited NFjB binding activity in
LPS-induced macrophages. As shown inFig. 5B, MSE inhibited
LPS-induced NFjB transcriptional activity in a dose-depen-
dent manner.
3.4. Inhibitory effects of MSE on LPS-induced
phosphorylation and degradation of IjBa
Because the LPS-mediated translocation of NFjB to the nu-
cleus is preceded by the phosphorylation and proteolytic
degradation of IjBa, we examined the phosphorylated and
un-phosphorylated protein levels of IjBa by immunoblot
analysis. It was found that the treatment with LPS caused ser-
ine-phosphorylation of IjBa protein, as evidenced by the pres-
ence of anti-Ser32-phospho-specic IjBa antibody after 15
60 min and the degradation of IjBa after 30 min. Levels of IjBa
gradually recovered after 4560 min ( Fig. 6A). As shown in
Fig. 6B, treatment with MSE effectively sustained the IjBa pro-
tein content. The pattern of inhibition on IjBa phosphoryla-
tion by MSE was paralleled to the pattern of inhibition on
its degradation. These results suggest that blocking the phos-
phorylation and the degradation of IjBa protein, MSE can in-
hibit the production of NO and PGE
2
, thus preventing the
translocation and activation of NFjB in the nucleus (Fig. 5).
3.5. Effects of MSE on activation of ERK1/2 (p44/42), p38
MAP kinase, PI3K, and Akt
ERK1/2 (p44/42) and p38 MAPK have been shown to be in-
volved in the LPS-mediated induction of iNOS and COX-2 in
mouse macrophages (Ho et al., 2004; Nick et al., 2000) and
cytokine activation of PI3K/Akt pathway leads to the phos-
phorylation and activation of the NFjB (Sizemore et al.,
1999). Therefore, we investigated the effects of MSE on the
activation of PI3K/Akt, JNK1/2, ERK1/2, and p38 MAPK in
LPS-stimulated macrophages. Activation of MAPK requires
phosphorylation of threonine and tyrosine residues (Rain-
geaud et al., 1995). We used immunoblot analysis with anti-
p85 antibody to investigate whether PI3K pathway was
involved in the MSEs inhibition of LPS-induced RAW 264.7
Fig. 5 Effects of MSE on LPS-induced p65 and p50
translocation and NF-jB activation in RAW 264.7 cells. (A)
Cells were treated with LPS (100 ng/mL) with or without MSE
(600 or 800 lg/mL) for 45 min. Then cytosolic and nuclear
fractions were prepared and analyzed by Western Blotting.
The values below the gure represent change in protein
expression of the bands normalized to Lamin B or b-actin.
(B) The cells were transiently transfected with 2 lg of pNF-
jB-Luc reporter gene, and then treated with LPS (100 ng/mL)
with or without MSE for 12 h. Cells were harvested and the
levels of luciferase activities were determined as described
in Materials and Methods. Results show the
means standard error of triplicate tests.
*
P < 0.05 vs. LPS
treatment.
Fig. 4 RT-PCR analysis of the expression of iNOS and COX-2
mRNA. Cells were treated with LPS (100 ng/mL) and MSE
(500, 600, 700, and 800 lg/mL) for 5 h, and total RNA was
subjected to RT-PCR with the primers iNOS or COX-2 with b-
actin as internal control. The PCR product was resolved in
1.5% agarose gel. Quantication of iNOS and COX-2 RNA
expression were performed by densitometric analysis of the
agarose gel. This experiment was repeated three times with
similar results.
J O U R N A L O F F U N C T I O N A L F O O D S 1 ( 2 0 0 9 ) 1 4 5 1 5 2 149
macrophages. The LPS-stimulated activation of PI3K was
attenuated by MSE (Fig. 7A). To further evaluate the involve-
ment of Akt, a downstream target of PI3K, in LPS-induced
responses, we used immunoblot analysis with anti-phos-
pho-Akt antibody to examine Akt activity in macrophage
cells. When the cells were co-treated with MSE and LPS for
30 min, the LPS-stimulated activation of Akt was attenuated
by MSE in a dose-dependent manner (Fig. 7A). When the cells
were co-treated both MSE and LPS for 30 min, MSE was found
to attenuate the LPS-stimulated activation of JNK1/2, ERK1/2,
and p38 MAPK (Fig. 7B). The results of our immunoblot anal-
yses suggest that MSEs inhibition of iNOS and COX-2 expres-
sion might block LPS-induced NFjB activation by inhibiting
ERK1/2, p38, and PI3K/Akt/IKK pathway, which interrupts
the degradation of IjBa.
4. Discussion
Epidemiological studies have linked the consumption of fruits
and vegetables to reduced risk of several types of human can-
cer (Surh, 2003). Laboratory animal studies have provided evi-
dences that MSE signicantly suppressed 7,12-
dimethylbenz[a] anthracene (DMBA)-initiated and 12-O-tetra-
decanoylphorbol-13-acetate (TPA)-promoted skin carcinogen-
esis (Takasaki et al., 2003). Moreover, MSE has been reported
to exhibit many biological effects including anti-oxidative
and anti-tumor promoter effects (Song et al., 2007; Ukiya
et al., 2002), but their anti-inammatory mechanism is still
not clear. In present study we found that MSE was strong
inhibitor of iNOS and COX-2 expression in LPS-activated
macrophages.
Because there is a causal relationship between inamma-
tion and cancer, iNOS and COX-2 are considered potential
molecular targets for chemoprevention (Chun et al., 2004).
The present results suggest that MSEs inhibition of the
ERK1/2, p38 MAPK, JNK1/2, and PI3K/Akt signaling pathway
may partially explain how it reduces the induction of iNOS
and COX-2 protein. The possible mechanism is that MSE
down regulates inammatory iNOS and COX-2 gene expres-
sion in macrophages by inhibiting the activation NFjB by
interfering with the activation of PI3K/Akt and MAPK.
Activation of NFjB is necessary for LPS induction of the
iNOS and COX-2 promoter (Xie et al., 1994). NFjB is composed
mainly of two proteins, p50 and p65. In resting cells, the NFjB
heterodimer is held in the cytosol through interaction with
IjB inhibitory proteins (Baeuerle & Henkel, 1994). With expo-
sure to proinammatory stimuli, IjB becomes phosphory-
lated, ubiquitinated, and then degraded. Thus, the liberated
NFjB dimers are translocated to the nucleus, where the tran-
scription of target gene is induced. Results of this work show
that MSE reduces iNOS and COX-2 expression by blocking
transcription of its gene, a conclusion supported by the obser-
vation that it reduced the steady state of iNOS and COX-2
mRNA levels, and promoter activity (as assessed by leucifer-
ase activity assay). Phosphorylation plays an important role
in activating protein tyrosine kinase. Many signaling path-
Fig. 6 Effects of MSE on LPS-induced phosphorylation and
degradation of IjBa. (A) RAW 264.7 cells were treated with
LPS (100 ng/mL) for different times. Total cellular lysates
were prepared for Western Blot analysis. (B) Cells were
treated with LPS (100 ng/mL) and MSE (800 lg/mL) for
different time, and the cellular lysates were prepared and
analyzed for content of IjBa, p-IjBa and b-actin by Western
Blot. These experiments were repeated three times with
similar results.
Fig. 7 Inhibition of ERK1/2, p38 MAPK and PI3K/Akt by MSE
in LPS-activated RAW macrophages. RAW 264.7 cells were
treated with LPS (100 ng/mL) with or without MSE (600 or
800 lg/mL) for 30 min. Cells extracts were then prepared and
analyzed for PI3K and p-PI3K, Akt, p-Akt, JNK and p-JNK,
ERK1/2 and pERK1/2, or p38 and p-p38 by Western Blot.
These experiments were repeated three times with similar
results.
150 J O U R N A L O F F U N C T I O N A L F O O D S 1 ( 2 0 0 9 ) 1 4 5 1 5 2
ways, including PI3K/Akt and mitogen-activated protein ki-
nase, have been proposed to respond to LPS stimulation
(Chen& Wang, 1999). PI3K activation leads to phosphorylation
of phosphatidylinositides, which then activate the down-
stream main target, Akt, which appears to play various
important roles in regulating cellular growth, differentiation,
adhesion, and the inammatory reaction (Carpenter & Cant-
ley, 1996). Activation of PI3K/Akt plays an important role in
the expression of iNOS and COX-2 in vascular smooth muscle
cells, peritoneal macrophages, and mesangial cells (Hattori
et al., 2003; Sheu et al., 2005). Our ndings demonstrate that
the PI3K/Akt pathway is involved in MSEs inhibition of
expression of NFjB-related iNOS and COX-2. In this study,
incubation of RAW 264.7 cells with LPS also brought about
the activation of p38 and ERK1/2. Thus, co-treatment of MSE
only blocked the activation of ERK1/2 and p38MAPK. These re-
sults suggest that MSE suppresses LPS-induced NFjB translo-
cation by inhibiting the activation of MAPK and subsequently
decreasing the protein levels of iNOS and COX-2 (Fig. 8).
Based on the above ndings it is proposed that MSE is a po-
tential and novel chemopreventive agent and may be used in
the future to treat inammation-associated tumorigenesis.
The possible relationship between the bioactive elements
and the structureeffect relationship responsible of MSE on
anti-inammatory activities deserves further investigation.
Acknowledgments
This study was supported by the National Science Council un-
der the Grant numbers, NSC 96-2321-B-022-001 and NSC 95-
2313-B-022-003-MY3.
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