Suppression of inducible nitric oxide synthase by ()-isoeleutherin from the bulbs of

Eleutherine americana through the regulation of NF-B activity

Su-Hyun Song
a
, Hye-Young Min
a
, Ah-Reum Han
a
, Joo-Won Nam
a
, Eun-Kyoung Seo
a
, Seoung Woo Park
b
,
Sang Hyung Lee
c
, Sang Kook Lee
a,

a
College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
b
School of Medicine, Kangwon National University, Chuncheon, Gangwon 200-947, Republic of Korea
c
Boramae Medical Center, Seoul National University College of Medicine, Seoul 156-707, Republic of Korea
a b s t r a c t a r t i c l e i n f o
Article history:
Received 26 August 2008
Received in revised form 29 November 2008
Accepted 2 December 2008
Keywords:
()-Isoeleutherin
Eleutherine americana
Nitric oxide (NO)
Inducible nitric oxide synthase (iNOS)
Nuclear factor kappa beta (NF-B)
Anti-inammatory
Eleutherine americana Merr. et Heyne (Iridaceae) has been used as a folk medicine for the treatment of coronary
disorders andwound-healing. Inour previous phytochemical study, several pyranonaphthoquinoids, including
()-isoeleutherin, were isolated from the bulbs of E. americana. Because inhibitors of inducible nitric oxide
synthase (iNOS) might be useful as anti-inammatory agents, we investigated the potential of pyrano-
naphthoquinoids to inhibit iNOS activity. We found that ()-isoeleutherin inhibits lipopolysaccharide (LPS)-
stimulated induction of nitric oxide (NO) in a dose-dependent manner (IC
50
=7.4 M). Using western blots
and reverse transcription-polymerase chain reaction, we showed that ()-isoeleutherin suppresses the
expression of iNOS protein and mRNA. In addition, ()-isoeleutherin inhibited the expression of various
cytokines such as interleukin-1andinterferon-. Activation of the transcriptional activity of NF-B by LPS was
also inhibited by treatment with ()-isoeleutherin, suggesting that ()-isoeleutherin-mediated suppression
of iNOS expression is associated with the regulation of transcription factor NF-B. These ndings suggest that
()-isoeleutherin might be a novel anti-inammatoryagent, and that it may work by inhibiting NFkB activation
in macrophages.
2008 Elsevier B.V. All rights reserved.
1. Introduction
Nitric oxide (NO) is a signaling molecule synthesized by the
enzyme nitric oxide synthase (NOS) from the oxidation of L-arginine.
There are at least three isoforms of NOS; two of them(cNOS; neuronal
and endothelial NOS) are constitutive forms; the other is inducible
form (iNOS) [1]. Compared to cNOS, which is constitutively expressed
and essential for maintaining tissue homeostasis, iNOS, in contrast, is
induced in response to various proinammatory stimuli including
lipopolysaccharide (LPS), tumor necrosis factor-(TNF-), interferon-
(IFN-), and interleukin-6 (IL-6), and mediates several inammatory
responses [2,3]. Since the overproduction of NO by iNOS is responsible
for inammation, iNOS has become a new target for developing new
substances for the treatment of chronic inammatory diseases [4].
Eleutherine americana Merr. et Heyne (Iridaceae) is a tropical plant
that originated in South America. The plant is nowmainly cultivated in
Southeast Asia including China. The bulb of this plant has been used as
a folk medicine for the treatment of coronary disorders (angina
pectoris) and intestinal infection, and as a diuretic and purgative [57].
Previous phytochemical studies on the bulb of E. americana resulted in
the isolation of several naphthoquinoids [810]. In addition, pharma-
cological studies with some of these isolated compounds were found
to have several biological activities including anti-HIV, antifungal,
antibacterial, and anticancer (by topoisomerase II inhibition) activities
[1013]. Very recently, our co-author reported the isolation of
naphthoquinoids that modulate Th cell-mediated immune responses
[14]. However, to the best of our knowledge, anti-inammatory
constituents from E. americana have not yet been reported. In the
present study, we tried to determine whether ()-isoeleutherin
possesses anti-inammatory effects by modulation of the expression
of inducible nitric oxide synthase (iNOS).
2. Materials and methods
2.1. Chemicals
Dulbecco's modied Eagle medium (DMEM), fetal bovine serum
(FBS), sodium pyruvate, L-glutamine, antibioticsantimycotics solu-
tion, and trypsin-EDTA were purchased from Invitrogen Co. (Grand
Island, NY, USA). Lipopolysaccharide (LPS, E. coli 0111: B4), 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and
other chemicals, unless indicated otherwise, were from Sigma (St.
International Immunopharmacology 9 (2009) 298302
Corresponding author. College of Pharmacy, Ewha Womans University, 11-1
Daehyun-Dong, Seodaemun-Ku, Seoul 120-750, Republic of Korea. Tel.: +82 2 3277
3023; fax: +82 2 3277 2851.
E-mail address: sklee@ewha.ac.kr (Sang Kook Lee).
1567-5769/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.intimp.2008.12.003
Contents lists available at ScienceDirect
International Immunopharmacology
j our nal homepage: www. el sevi er. com/ l ocat e/ i nt i mp
Louis, MO, USA). ()-Isoeleutherin (Fig. 1) was isolated from the bulbs
of E. americana (900 g) as described previously [14].
2.2. Cell culture
Mouse macrophage RAW 264.7 cells, obtained from the American
Type Culture Collection (ATCC, Rockville, MD, USA), were cultured in
DMEM supplemented with 10% heat-inactivated FBS, 100 units/ml
penicillin, 100 g/ml streptomycin, and 0.25 g/ml amphotericin B.
RAW 264.7 cells stably transfected with a pNF-kB-SEAP-NPT reporter
plasmid (SEAP-RAWcells) were kindly provided by Dr. Yeong Shik Kim
(Seoul National University, Korea). SEAP-RAW cells were maintained
in DMEM containing 500 g/ml G418. All cells were incubated at 37 C
under 5% CO
2
in a humidied atmosphere.
2.3. Nitrite assay
To evaluate the inhibitory activity of test materials on LPS-induced
NOproduction, RAW264.7 cells in 10% FBS-DMEMwithout phenol red
were plated in 24 well plates (510
5
cells/ml), and incubated for 24 h.
Cells were washed with PBS, replaced with fresh media, and then
incubated with 1 g/ml LPS in the presence or absence of test com-
pounds. After an additional 20 h incubation, media were collected and
analyzed (by the Griess reaction) for nitrite accumulation as an
indicator of NO production. Briey, 180 l of Griess reagents (0.1%
N-(1-naphthyl)ethylenediamine dihydrochloride in H
2
O and 1% sul-
fanilamide in 5% H
3
PO
4
) were added to 100 l of each supernatant
from LPS or sample-treated cells in 96 well plates. Absorbance was
measured at 540 nm and nitrite concentration was determined by
comparison with a sodium nitrite standard curve. Percent inhibition
was expressed as [1-(NO level of test samples / NO levels of vehicle-
treated control)] 100. The IC
50
value, the sample concentration
resulting in 50% inhibition of NO production, was determined using
non-linear regression analysis (% inhibition versus concentration).
2.4. Cytotoxicity assay (MTT assay)
After the Griess reaction, MTT solution (nal concentration=
500 g/ml) was added to each well and further incubated for 4 h at
37 C. Media were discarded, and dimethyl sulfoxide (DMSO) was
added to each well to dissolve generated formazan. The absorbance
was measured at 570 nm and % survival was determined by com-
parison with the control group.
2.5. Preparation of total cell lysates
RAW 264.7 cells (510
5
cells/ml in 60 mm dish) were incubated
with or without various concentrations of ()-isoeleutherin and LPS
(1 g/ml) for 16 h. To obtain total cell lysates, cells were washed with
ice-cold PBS and lysed in boiling 2 sample loading buffer (250 mM
TrisHCl (pH 6.8), 4% SDS, 10% glycerol, 0.006% bromophenol blue,
50 mM sodium uoride, 5 mM sodium orthovanadate, and 2%
-mercaptoethanol). Cell lysates were boiled for an additional
20 min and stored at 20 C. The protein content of cell lysates was
determined by the BCA method.
2.6. Western blots
Equal amounts of cell lysates (4050 g) were subjected to 8% and
10% SDS-PAGE and electro-transferred onto polyvinylidene diuoride
(PVDF) membranes (Millipore, CITY?, MA, USA). Membranes were
blocked in PBST (PBS with 0.1% Tween-20) containing 5% non-fat dry
milk for 1 h at room temperature. After washing three times with
PBST, membranes were incubated with primary antibodies against
iNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA), -actin (Sigma)
Fig. 1. Chemical structure of ()-isoeleutherin.
Table 1
Sequences of gene specic primers used in PCR
Genes Sequences
Mouse iNOS Sense 5-ATGTCCGAAGCAAACATCAC-3
Antisense 5-TAATGTCCAGGAAGTAGGTG-3
Mouse -actin Sense 5-TGTGATGGTGGGAATGGGTCAG-3
Antisense 5-TTTGATGTCACGCACGATTTCC-3
Mouse IL-1 Sense 5-TGCAGAGTTCCCCAACTGGTACATC-3
Antisense 5-GTGCTGCCTAATGTCCCCTTGAATC -3
Mouse IFN- Sense 5-GAGTTACACTGCCTTTGCC-3
Antisense 5-GATTCACTACCAGTCCCAGA-3
Fig. 2. ()-Isoeleutherin dose-dependently inhibited LPS-induced NOproduction in LPS-
stimulated macrophage cells. (A) RAW 264.7 cells were stimulated with 1 g/ml LPS in
the presence or absence of ()-isoeleutherin. After 20 h, cultured media were collected
and analyzed for nitrite using the Griess reaction. Data are expressed as meanS.D. of
triplicate tests. pb0.05 was considered statistically signicant. (B) Cell viability was
measured by MTT assay as described in Materials and methods.
299 S.-H. Song et al. / International Immunopharmacology 9 (2009) 298302
for 3 h at room temperature or overnight at 4 C. Membranes were
washed three times with PBST and incubated with corresponding
secondary antibodies (Santa Cruz) for 90 min at room temperature.
The blots were washed three times with PBST, and visualized using an
enhanced chemiluminescence (ECL) western blotting detection
system (Lab Frontier, Suwon, Korea).
2.7. Reverse transcriptase-polymerase chain reaction (RT-PCR)
RAW 264.7 cells were stimulated with 1 g/ml LPS in a presence or
absence of ()-isoeleutherin for 4 h. Total cellular RNA was extracted
with TRI reagent (Sigma) according to the manufacturer's recom-
mended procedure. One microgram of total RNA was reverse-
transcribed using oligo-(dT)
15
primers and avian myeloblastosis virus
(AMV) reverse transcriptase (Promega, Madison, WI, USA). PCR was
done in a reaction mixture containing the obtained cDNA, 0.2 mM
dNTP mixture (Promega), 10 pmol of target gene-specic primers, and
0.25 unit of Taq DNA polymerase (Promega) using a GeneAmp PCR
system 2400 (Applied Biosystems, Foster, CA, USA). Each of the PCR
steps was done as follows: initial denaturation step for 4 min at 94 C;
2530 cycles of the amplication step consisting of denaturation for
30 s at 94 C, annealing for 30 s at 55 C, andelongationfor 30 s at 72 C;
a nal extension step for 5 min at 72 C. PCR products were separated
by 2% agarose gel electrophoresis, stained with SYBR-Gold (Molecular
Probes, Eugene, Oregon, USA), and visualized by UV transillumination.
Forward and reverse primers used for this study are detailed inTable 1.
2.8. Report gene assay for NF-B transcriptional activity in
LPS-stimulated RAW cells
To determine the effect of ()-isoeleutherin on the activation of NF-
B, a reporter gene assay was done as described previously with some
modications [15]. SEAP-RAW cells were plated at a density of 510
5
cells/well in a 12-well plate and incubated for 24 h. The cells were
treated with ()-isoeleutherin for 2 h and then treated with LPS (1 g/
ml) for an additional 18 h. Cell culture supernatants were heated at
65 C for 5 min, and then reacted with SEAP assay buffer (2 M die-
thanolamine, 1 mM MgCl
2
, 500 M 4-methylumbelliferyl phosphate
(MUP)] in darkness at 37 C for 1 h. Fluorescence from the product
of the SEAP/MUP reaction was measured in relative uorescence units
(RFU) using a 96-well plate uorometer with excitation at 360 nmand
emission at 449 nm and normalized by protein concentration. Data
are expressed as a proportion of isoeleutherin-treated to vehicle-
treated control cells without LPS.
2.9. Statistics
All experiments were repeated at least three times. Data are
presented as meansSE for the indicated number of independently
performed experiments. Student's t-test was used for the determina-
tion of statistical signicance. pb0.05 was considered signicant.
3. Results
3.1. Inhibition of NO production by ()-isoeleutherin in LPS-stimulated
RAW 264.7 cells
In order to evaluate whether ()-isoeleutherin affects NO produc-
tion, the production of nitrite, the stable metabolite of NO was used as
an indicator of NO production; NO was monitored in cultured LPS-
stimulated RAW264.7 macrophage cells. Treatment with LPS (1 g/ml)
markedly increased the production of NO from a basal level of 1.9
0.9 M to 25.51.5 M over 20 h of incubation. In this assay system,
L-NMMA, a positive control for a non-selective inhibitor of NOS [16],
Fig. 3. Effect of ()-isoeleutherinoniNOSproteinandmRNAexpressioninLPS-stimulatedRAW264.7cells. (A) Cells weretreatedwithLPS(1g/ml) and()-isoeleutherin(030M) for 16h.
After incubation, cell extracts were obtained and subjected to western blot analysis as described in Materials and methods. Data are representative of three separate experiments. -actin
was usedas aninternal standard (left panel). Relative density was expressed by the ratio of iNOS/-actinat eachband(right panel). (B) RAW264.7 cells were treatedwith LPS (1 g/ml) and
()-isoeleutherin(030M) for 4 h. Total RNAwas isolatedand1 g of RNAwas usedfor reverse-transcription. The cDNAobtainedwas usedfor the polymerase chainreaction. PCRproducts
were separated by 2% agarose gel electrophoresis, stained with SYBR-Gold, and visualized under a UV transilluminator. Data are representative of three separate experiments. -actinwas
used as an internal standard (left panel). Relative density was expressed by the ratio of iNOS/-actin at each band (right panel).
300 S.-H. Song et al. / International Immunopharmacology 9 (2009) 298302
exhibited an IC
50
of 6.5 M. When the cells were simultaneously
treated with various concentrations of ()-isoeleutherin (030 M)
and LPS, NO production was signicantly inhibited in a concentra-
tion-dependent manner with an IC
50
value for ()-isoeleutherin of
7.4 M (Fig. 2(A)). Over 98% inhibition of NO production was shown
at 30 M. No signicant effect on cell viability was observed at test
concentrations up to 30 M ()-isoeleutherin as determined by MTT
assay (N95% cell survival), indicating that the inhibition of NO pro-
duction by ()-isoeleutherin was not mediated by a cytotoxic effect
(Fig. 2(B)).
3.2. Suppression of iNOS protein and mRNA expression
To elucidate the mechanism of action of ()-isoeleutherin on the
inhibition of NO production, the effects of ()-isoeleutherin on iNOS
protein and gene expression were determined. As shown in Fig. 3(A),
treatment with LPS (1 g/ml) for 16 h markedly increased the ex-
pression of iNOS protein. This effect was suppressed in a concentra-
tion-dependent manner by co-treatment with ()-isoeleutherin,
indicating that ()-isoeleutherin inhibited iNOS protein expression.
In addition, to further investigate the effect of ()-isoeleutherin on
LPS-induced enhancement of iNOS mRNA expression, steady-state
levels of iNOS mRNA were determined using RT-PCR analysis. As
illustrated in Fig. 3(B), ()-isoeleutherin signicantly inhibited, in a
concentration-dependent manner, the increase in iNOS mRNA levels
that had been induced by LPS, suggesting that inhibition of NO
production by ()-isoeleutherin might be correlated with the sup-
pression of iNOS expression at translational and transcriptional levels.
3.3. Suppression of expression of proinammatory cytokines by
()-isoeleutherin
To determine the effect of ()-isoeleutherin on the expression of
pro-inammatory cytokines, the levels of steady-state transcripts of
interleukin-1 (IL-1) and interferon- (IFN-) were analyzed by
RT-PCR. As shown in Fig. 4, treatment with LPS (1 g/ml) for 4 h
markedly increased the expression of IL-1 and IFN- mRNA, but
co-treatment with ()-isoeleutherin (040 M) dose-dependently
suppressed the expression of these pro-inammatory cytokines.
3.4. Effect of ()-isoeleutherin on LPS-induced NF-B transcriptional
activity
It is well known that NF-B is a major transcription factor for the
induction of the iNOS gene by LPS [17]. To further investigate whether
NF-B is an important target of the action of ()-isoeleutherin in
LPS-stimulated RAW 264.7 cells, a reporter gene assay for NF-B
transcriptional activity (SEAP) was used. Treatment with LPS for
18 h elicited a 4.3-fold increase in NF-B transcriptional activity, but
treatment with ()-isoeleutherin (040 M) markedly and dose-
dependently inhibited LPS-induced increases in NF-B transcrip-
tional activity (Fig. 5).
Fig. 5. Effect of ()-isoeleutherin on LPS-induced NF-B transcriptional activity. RAW
264.7 cells stably transfected with an NF-kB-SEAP-NPT reporter plasmid were pre-
treated with various concentrations of ()-isoeleutherin for 2 h, and then stimulated
with LPS (1 g/ml) for an additional 18 h. The supernatants were analyzed for secreted
alkaline phosphase activity (SEAP) as described in Materials and methods. Values are
expressed as mean+/S.D. of three independent experiments. pb0.05 was considered
statistically signicant.
Fig. 4. Effect of ()-isoeleutherin on LPS-induced pro-inammatory cytokine expression in macrophage cells. RAW 264.7 cells (510
5
cells/ml) were incubated for 24 h, and then
treated with LPS (1 g/ml) and ()-isoeleutherin (040 M) for an additional 4 h. Total RNA was isolated and 1 g of RNA was used for reverse-transcription. The cDNA obtained was
used in the polymerase chain reaction using Taq DNA polymerase and IL-1 and IFN--specic primers. PCR products were separated by 2% agarose gel electrophoresis, stained with
SYBR-Gold, and visualized under a UV transilluminator. -actin was used as an internal standard (left panel). Relative density was expressed by the ratio of IL-1/-actin or INF- /-
actin at each band (right panel).
301 S.-H. Song et al. / International Immunopharmacology 9 (2009) 298302
4. Discussion
Although the production of NO by macrophages has been viewed
as a host defense mechanism against pathogens, the overproduction
of NOis highly associated with inammation [4], and the expression of
inducible nitric oxide synthase (iNOS) plays a major role in the
production of NO in activated macrophages. Therefore, the regulation
of NO production or iNOS expression levels might be a useful target in
the treatment of inammation. In the course of searching for NO
inhibitors or suppressors of iNOS as potential anti-inammatory
agents from natural products, we investigated ()-isoeleutherin, an
active principle isolated from E. americana, for its ability to modulate
NO production and iNOS expression in activated macrophage cells.
The present results demonstrate that ()-isoeleutherin inhibits the
production of NO in LPS-activated RAW 264.7 macrophage cells in a
dose-dependent manner (Fig. 2(A)), and this effect was shown not to
be due to cytotoxicity (Fig. 2(B)). Subsequent studies revealed that the
inhibitory effect of ()-isoeleutherin on NO production was highly
related to inhibition of the expression of iNOS protein and gene
expression as assessed by western blot and RT-PCR analysis (Fig. 3(A)
and (B)). In addition, pro-inammatory cytokines are also considered
triggering factors for inammatory disorders [18]. The stimulation of
macrophages by LPS enhances the expression of pro-inammatory
cytokines including IL-1 and IFN-. In the present study, treatment
with ()-isoeleutherin inhibited induction of the expression of these
pro-inammatory cytokines, which was concomitant with the sup-
pression of NO production and iNOS expression (Fig. 4). NF-B is a
crucial factor for the expression of pro-inammatory cytokines and
iNOS in LPS-stimulated macrophages [17]. To further investigate the
molecular mechanisms underlying the effects of ()-isoeleutherin
on the expression of iNOS, IL-1 and IFN-, transcriptional activity of
NF-B was determined using a reporter gene assay. When macro-
phages were treated with LPS (1 g/ml) for 18 h, the transcriptional
activity of NF-B was enhanced and treatment with ()-isoeleutherin
inhibited this effect. This suggests that NF-B is a major contributing
transcription factor to the regulation of iNOS gene expression by
()-isoeleutherin (Fig. 5).
In summary, this study suggests that ()-isoeleutherin from
E. americana inhibits NO production in LPS-stimulated RAW 264.7
macrophage cells through the suppression of iNOS protein and mRNA
expression. Furthermore, this suppression of iNOS is, at least in part,
associated with regulation of NF-B activation. Thus, ()-isoeleutherin
might have a clinically useful anti-inammatory effect because it
inhibits iNOS expression and NO overproduction.
Acknowledgments
This work was supported by grant no. R15-2006-020 from the
National Core Research Center (NCRC) program of the Ministry of
Education, Science & Technology (MEST) and the Korea Science and
Engineering Foundation (KOSEF) through the Center for Cell Signaling
& Drug Discovery Research at Ewha Womans University.
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