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Life Sciences 79 (2006) 2022 2031

www.elsevier.com/locate/lifescie

Inhibitory effect of curcumin on nitric oxide production from


lipopolysaccharide-activated primary microglia
Ki Kyung Jung a,b , Hae Sung Lee a,b , Jae Youl Cho c,, Won Cheol Shin c , Man Hee Rhee d ,
Tae Gyun Kim a , Ju Hye Kang a , Seung Hee Kim a , Sungyoul Hong b , Seog Youn Kang a,1
a

Pharmacology Department, National Institute of Toxicological Research, KFDA, Seoul 122-704, Republic of Korea
b
Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea
c
School of Biotechnology and Bioengineering, Kangwon National University, Chuncheon 200-701, Republic of Korea
Laboratory of Physiology and Signaling, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Republic of Korea
Received 16 November 2005; accepted 26 June 2006

Abstract
Curcumin has been shown to exhibit anti-inflammatory, antimutagenic, and anticarcinogenic activities. However, the modulatory effect of
curcumin on the functional activation of primary microglial cells, brain mononuclear phagocytes causing the neuronal damage, largely remains
unknown. The current study examined whether curcumin influenced NO production in rat primary microglia and investigated its underlying
signaling pathways. Curcumin decreased NO production in LPS-stimulated microglial cells in a dose-dependent manner, with an IC50 value of
3.7 M. It also suppressed both mRNA and protein levels of inducible nitric oxide synthase (iNOS), indicating that this drug may affect iNOS
gene expression process. Indeed, curcumin altered biochemical patterns induced by LPS such as phosphorylation of all mitogen-activated protein
kinases (MAPKs), and DNA binding activities of nuclear factor-B (NF-B) and activator protein (AP)-1, assessed by reporter gene assay. By
analysis of inhibitory features of specific MAPK inhibitors, a series of signaling cascades including c-Jun N-terminal kinase (JNK), p38 and NFB was found to play a critical role in curcumin-mediated NO inhibition in microglial cells. The current results suggest that curcumin is a
promising agent for the prevention and treatment of both NO and microglial cell-mediated neurodegenerative disorders.
2006 Elsevier Inc. All rights reserved.
Keywords: Curcumin; Primary microglia; NO; iNOS; p38; JNK; NF-B; AP-1

Introduction
Microglial cells are a class of brain mononuclear phagocytes
that carry out phagocytosis, antigen presentation, the secretion of
cytokine and the production of inflammatory mediators such as
eicosanoids, reactive oxygen species and nitric oxide (NO)
(Simmons and Murphy, 1992; Wang et al., 2002). Like other
tissue macrophage's role in pathophysiology, these cells par Corresponding author. Cho is to be contacted at the School of Biotechnology
and Bioengineering, Kangwon National University, 192-1, Hyoja-2-dong,
Chuncheon 200-701, Republic of Korea. Tel.: +82 33 250 6562; fax: +82 33 253
6560.
E-mail address: jaecho@kangwon.ac.kr (J.Y. Cho).
1
Pharmacology Department, National Institute of Toxicological Research,
KFDA, 5 Nokbun-Dong, Eunpyung-Ku, Seoul 122-704, Republic of Korea.
Tel.: +82 2 380 1812; fax: +82 2 901 8386.
0024-3205/$ - see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2006.06.048

ticipate in certain brain inflammatory conditions via excessive


production of the inflammatory mediators. Indeed, over-activated
microglial cells are found in many neurodegenerative diseases
such as Alzheimer's disease, multiple sclerosis and human
immunodeficiency virus-associated dementia (Aloisi, 1999). For
this, the effective control of microglial cell function in numerous
neuronal diseases is regarded as an important therapeutic target.
NO released from microglia is known to induce neurotoxicity
(Dawson et al., 1991; Joe and Lokesh, 1994; Kim and Ko, 1998).
This pathological situation is mediated by the formation of the
potent oxidizing agent, peroxynitrite (ONOO), by the chemical
reaction between NO and superoxide (Beckman et al., 1994;
Lipton et al., 1993) or the formation of toxic dopamine oxidative
products (Cook et al., 1996). Therefore, NO production is a
critical step in modulation of NO-mediated diseases. The
generation of NO following an inflammatory stimulation is

K.K. Jung et al. / Life Sciences 79 (2006) 20222031

catalyzed by inducible NO synthase (iNOS) and its regulation


depends on the formation of a multiple intracellular signaling
complex composed of Janus kinases, protein tyrosine kinases,
protein kinase C, and mitogen-activated protein kinases (MAPKs)
as well as transcription system such as nuclear factor-B (NF-B)
and activator protein (AP)-1 (Nick et al., 1999; Kim et al., 2003a;
Kang et al., 2004).
There is increasing interest in the role of food components in
health and disease. One such component is curcumin. Curcumin
is a major chemical component of turmeric (Curcuma longa)
which is used as a spice to give a specific flavor and yellow
color in curry. The form of herbal powder turmeric has been
used for centuries as an anti-inflammatory remedy in Asian
medicine. Curcumin has been shown to display anti-inflammatory, antioxidant, and anticarcinogenic properties (Huang et al.,
1997; Kumar et al., 1998). The anti-inflammatory effects of
curcumin are most likely mediated through its ability to inhibit
expression of pro-inflammatory genes such as cyclooxygenase2, adhesion molecules, chemokines, cytokines, metalloproteinases (MMP), lipoxygenase, and iNOS (Huang et al., 1991;
Surh et al., 2001; Woo et al., 2005) via down-regulation of
transcription factors such as NF-B, AP-1 and Egr-1 (Aggarwal
et al., 2003; Kang et al., 2004) and intracellular signaling
pathways such as Janus kinase (JAK)-STAT signaling (Kim
et al., 2003a). A recent study has shown that curcumin reduces
oxidative damage and amyloid pathology in an Alzheimer
transgenic mouse (Lim et al., 2001).
Although the anti-inflammatory properties of curcumin are
known, its molecular basis in neuronal system is not fully
understood yet. In particular, how curcumin can modulate NO
production in primary microglial cells has not been fully elucidated yet in terms of its molecular aspects, although it has been
reported that curcumin blocked iNOS expression via suppressing JAK-STAT inflammatory signaling (Kim et al., 2003a,b).
Therefore, in the present study, the NO inhibitory effect of
curcumin and its molecular mechanism in primary microglia
were carefully investigated. From this study, our results suggest
that curcumin may effectively modulate the pathophysiological
function of microglia via regulating iNOS gene expression
mediated by a series of signaling cascades including c-Jun Nterminal kinase (JNK), p38, and NF-B.
Materials and methods
Reagents
Curcumin (1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione), lipopolysaccharide (LPS; from Salmonella
enteriditis), interferon-, aminoguanidine (AG), a selective
iNOS inhibitor, and MTT were purchased from Sigma-Aldrich
Chemical Co. (St. Louis, MO). PD98059 (a extracellular signalregulated kinase [ERK] inhibitor), SB203580 [a p38 inhibitor],
and SP600125 (a c-Jun N-terminal kinase [JNK] inhibitor) were
obtained from Calbiochem. (La Jolla, CA). Minimum essential
medium (MEM), Dulbecco's modified Eagle's medium
(DMEM), neurobasal A medium, fetal bovine serum (FBS),
Penicillinstreptomycin antibiotics and TrypsinEDTA were

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purchased from GIBCO BRL Life Technologies (Grand Island,


NY). The BV2 cell line was obtained from American Tissue
Culture Center (Manassas, VA). [-32P]-ATP (250 Ci), Western blotting detection system, and ECL Plus were from
Amersham-Pharmacia Biotech. Ltd. (Little Chalfont, Buckinghamshire, UK). Antibodies to OX-42 (CD11b), glial fibrillary
acidic protein (GFAP), and iNOS were purchased from
Chemicon International Inc. (Temecula, CA). Antibodies to
p65 (Rel A) of NF-B, -actin, IB- and IB- were from
Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies to
JNK, phospho-JNK (p-JNK), ERK, p-ERK, p38, and p-p38
were obtained from cell signaling Technology (Beverley, MA).
Protein assay kit and gelatin were obtained from Bio-Rad Lab
(Hercules, CA). Other common chemicals all came from SigmaAldrich Chemical Co. (St. Louis, MO).
Primary microglial cell culture
Experimental protocols were approved by the Animal Care
and Use Committees, National Institute of Toxicology Center,
and conformed to regulations detailed in the National Institutes
of Health publication Guide for the Care and Use of Laboratory
Animals (revised in 1996). All efforts were made to minimize
the suffering and the number of animals used according to the
abovementioned guidelines and derived guidelines on the ethical
use of animals. Harlan SpragueDawley rats were purchased
from the Dae-Han/Biolink Experimental Animal Center (Daejeon, Korea). Microglial cells were cultured from the cerebral
cortices of 1 to 3 day old rats by a modification of Park's method
(Park et al., 1999). In brief, the meninges were removed from the
forebrains, and tissues collected from forebrains were triturated
into single cells using fire-polished long Pasteur pipettes in
serum-free media. Cells were plated onto T-75-cm2 flask in
MEM containing 10% heat-inactivated FBS and incubated at
37 C in a humidified incubator containing 5% CO2 and 95% air.
The following week, the cells were replaced with new media and
incubated for 12 weeks. Microglial cells were then purified
from the initial mixed culture by sequential shaking at 180 rpm
for 30 min at room temperature. The resultant supernatant was
collected and centrifuged at 150 g for 5 min. The pellet was
suspended in new neurobasal A media containing 10% FBS,
applied to a nylon mesh to remove astrocytes and cell clumps,
and then plated on a culture slide or in 24-well culture plates. The
plate was under the culture hood, allowing to settle it for 1 h.
After 1 h, microglial cells were further purified by washing twice
with serum-free media and grown in new neurobasal A media.
To determine the purity of the microglial cells, immunocytochemical analysis was carried out using microglial-specific OX42 and astrocyte-specific GFAP antibodies. These cultures were
N95% OX-42 positive and GFAP negative as determined by
immunocytochemistry indicating that they were composed of
microglial cells.
Measurement of nitrite
NO production in cell culture supernatants was spectrophotometrically evaluated by measuring the nitrite, a stable end product

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K.K. Jung et al. / Life Sciences 79 (2006) 20222031

of NO. Nitrite was determined by the Griess reaction (Ding et al.,


1988; Cho et al., 2000). Fifty microliters of the culture supernatant
was mixed with an equal volume of Griess reagent (0.1%
naphthyl-ethylenediamine, 1% sulfanylamide, and 2.5% phosphoric acid). Absorbance was measured in a microplate reader at
540 nm, using a calibration curve with sodium nitrite standards.

iNOS, ACA ACG TGG AGA AAA CCC CAG GTG, and ACA
GCT CCG GGC ATC GAA GAC C were used as a sense and
anti-sense primer, respectively. For the GAPDH, CTG CCA CTC
AGA AGA CTG TGG and CTT GAT GTC ATC ATA CTT GGC
were used as a sense and anti-sense primer, respectively (Lee
et al., 2003a).

Reverse transcription-polymerase chain reaction (RT-PCR)

Western blot analysis

Total RNAwas prepared from rat primary microglial cells with


TRIzol reagent (Roche Molecular Biochemicals, Mannheim,
Germany), according to the manufacturer's instructions. Two
micrograms of total RNA were reverse-transcribed and then used
for PCR as a template as previously described (Hong et al., 2003).
The primers (Bioneer, Daejeon, Korea) used for amplifying iNOS
and GAPDH, a housekeeping gene, were as follows: for the

For Western blot analysis, cells were pelleted, washed with


PBS, pH 7.5, and resuspended in lysis buffer consisting of Tris
HCl (pH 6.8) buffer containing 2% SDS, 1 mM EDTA, 100 mM
DTT, and protease inhibitor cocktail. After 30 min on ice, cell
lysates were cleared by centrifugation for 5 min at 10,000 g.
10 g of total proteins in Tris-glycine SDS electrophoresis buffer
was loaded on 10% acrylamide gels and subjected to SDS-PAGE

Fig. 1. Effects of curcumin on NO production and iNOS expression in rat primary microglial cells stimulated with LPS. (A) Primary microglial cells (1.5 x106 cells/
ml) (upper panel) or BV2 cells (5 x106 cells/ml) (lower panel) pretreated with the indicated concentrations of curcumin or AG (0.2 mM) were stimulated with 1 g/ml
of LPS. Cultured medium was then collected after 24 h, and nitrite level was determined by Griess reaction. Results are the mean SEM of three independent
experiments performed with triplicate. Asterisk indicates significant difference from LPS-stimulated group (: P b 0.05, : P b 0.01). (B) Transcriptional expression
of iNOS was assessed by RT-PCR using LPS-activated microglial cells pre-treated with curcumin (4 and 8 M). (C) Whole cell lysates were prepared from 5 106 cells
of microglia treated with LPS and/or curcumin. iNOS and -actin expression was analyzed by immunoblotting with antibody to iNOS. (D) Immunocytochemical
analysis was carried out using Vectastain ABC kit as described in Materials and methods. The data are representative of three different experiments with similar results.

K.K. Jung et al. / Life Sciences 79 (2006) 20222031

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Fig. 2. Role of MAPKs in curcumin-mediated NO production in LPS-stimulated primary microglial cells. (A) Whole cell lysates were prepared from 5106 cells of microglia
that were stimulated with LPS (1 g/ml) for 30 min after a 2-h pretreatment with curcumin (4 or 8 M). The total or phosphorylation forms of ERK, p-ERK, p38, p-p38, JNK
and p-JNK were analyzed by immunoblotting, as described in Materials and methods. The data are representative of three different experiments with similar results. (B) Effect of
MAPK inhibitors (SP600125 [15 M], SB203580 [25 M], and PD98059 [50 M]) on NO production in LPS (1 g/ml)-activated primary microglial cells (1.5 106 cells/ml)
(upper panel) or BV2 cells (5106 cells/ml) (lower panel) was assessed. Cultured medium was collected after 24 h, and nitrite level was determined by Griess reaction. Results
are the meanSEM of three independent experiments performed with triplicate. Asterisk indicates significant difference from LPS-stimulated group (: P b 0.05, : P b 0.01).

before transfer to polyvinylidene difluoride membranes. Membranes were blocked with Tris-buffered saline and, 5% nonfat dry
milk before being probed with primary antibodies to iNOS, p65,
IB-, IB-, JNK, p-JNK, p38, p-p38, ERK, and p-ERK. All
blots were visualized with enhanced chemiluminescence (Amersham-Pharmacia Biotech Inc.).
Immunocytochemistry
Microglial cells were seeded onto culture slides. After 24 h,
media were removed and slides were air-dried. The cells were
then fixed with 50% methanol and 50% acetone for 30 min at
room temperature. Following three rinses in phosphate buffed
saline (PBS, pH 7.6), sections were blocked with 1% goat serum
in PBS for 30 min, and incubated with primary antibodies against
OX-42, GFAP, and iNOS dissolved in PBS containing 0.3%
Triton X-100. Incubations were performed for 1618 h at 4 C.
After several washes in PBS, sections were incubated with
biotinylated anti-mouse or anti-rabbit secondary antibodies
(1:200, Vector Laboratories) for 1 h at room temperature. Staining
was visualized by the biotin-avidin-peroxidase method (ABC
Elite Kit, Vector Laboratories) using diaminobenzidine as
chromogen. Negative control data were obtained with the isotype
antibodies of each tested antibody (data not shown).

BV2 cell culture and transient transfection assay


Mouse microglial cell line BV2 cells were grown in DMEM,
supplemented with 10% FBS, streptomycin, and penicillin, as
described previously. Transfection was performed by a standard
calcium phosphate method. BV2 cells were transfected with 4 g
of the reporter construct (NF-B-luc or AP-1-luc) and 1 g pRSV
Renillar-Luc. Plasmids used for transient transfection assays were
prepared by Qiagen (Santa Clarita, CA) columns. After 48 h, cells
were harvested and luciferase assays were performed as previously
described (Woo et al., 2005). To correct for differences in transfection efficiencies among different DNA precipitates, luciferase
activity was normalized to that of Renilla luciferase activity. All
transfection assays were carried out at least three times in triplicate.
Electrophoretic mobility shift assay
Nuclear extraction
Nuclear extracts were prepared by the method previously
reported (Kim et al., 2005). Microglial cells (3 106 cells/well)
were seeded into 6-well plates and treated with or without
curcumin for 2 h, followed by exposure to 1 g of LPS. The cells
then were washed twice with ice-cold PBS followed by
incubation on ice for 15 min with 0.2 ml of ice-cold lysis buffer

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Fig. 3. Effects of curcumin and MAPK inhibitors on transcriptional activation of NF-B and AP-1. (A) Effect of curcumin and MAPK inhibitors (SP600125 [15 M], SB203580
[25 M], and PD98059 [50 M]) on LPS-induced NF-B and AP-1 reporter gene activity was examined as described in Materials and methods. Results are the meanSEM of
three independent experiments performed with triplicate. Asterisk indicates significant difference from LPS-stimulated group (: Pb 0.05, : Pb 0.01). (B) Nuclear extracts were
prepared from 3106 cells of microglia that were stimulated with LPS for 24 h after a 2-h pretreatment with curcumin. Nuclear translocation of NF-B was evaluated with EMSA,
as described in Material and methods. (C) Whole cell lysates were prepared from 5106 cells of microglia that were stimulated with LPS for 24 h after a 2-h pretreatment with
curcumin. Expression of p65, I-B ( and ) and -actin was analyzed by immunoblotting as described in Materials and methods. (D) Nuclear extracts were prepared from 3106
cells of microglia that were stimulated with LPS for 24 h after a 2-h pretreatment with MAPK inhibitors (SP600125 [15 M], SB203580 [25 M], and PD98059 [50 M]).
Nuclear translocation of NF-B was evaluated with EMSA, as described in Materials and methods. The data are representative of three different experiments with similar results.

that consisted of 10 mM HEPES (pH 7.9) containing 1.5 mM


MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.2 mM PMSF. Cells
were then scraped and collected in 1.5 ml Eppendorf tubes and
placed on ice for an additional 30 min. The homogenates then
underwent centrifugation at 4 C at 1200 g for 10 min. The
resulting pellets were washed once with 0.5 ml of ice ice-cold
lysis buffer and incubated on ice for 1 h with 80 l of nuclear
extraction buffer composed of 20 mM HEPES (pH 7.9) buffer
containing 20% (v/v) glycerol, 420 mM NaCl, 1.5 mM MgCl2,
0.2 mM EDTA, 0.5 mM DTT and 0.2 mM PMSF. The resulting
homogenates were centrifuged at 4 C at 110,000 g for 15 min.
The supernatants were collected and stored at 70 C until use.
Electrophoretic mobility shift assay for NF-B
NF-B, which binds to B enhancer elements on DNA, was
detected in nuclear extracts by an electrophoretic mobility shift
assay (EMSA), according to our previous report (Kim et al.,

2005). For EMSA, 40 g of nuclear proteins were incubated for


30 min at room temperature with approximately 100,000 cpm of
an oligonucleotide containing NF-B consensus sequence (5AGT TGA GGG GAC TTT CCC AGG C-3) that had 5-end
labeled with [-32P] ATP using T4 polynucleotide kinase
(Promega). Supershift and competitive binding assay for NF-B
was carried out using a p65 antibody and non-labeled cold
probe (data not shown). Samples were then electrophoresed in a
6% nondenaturing polyacrylamide gel. Autoradiographs were
obtained from radioactive signals.
Statistical analysis
Unless otherwise stated, all measurements were made from
four or ten observations and at least three separate experiments.
Data are expressed as mean SEM. For statistical comparison,
results were analyzed using ANOVA/Scheffe's post-hoc test and
KruskalWallis/MannWhitney test. P value b 0.05 was

K.K. Jung et al. / Life Sciences 79 (2006) 20222031

considered a statistically significant difference. All statistical tests


were carried out using the computer program STATISTICA,
version 4.5 (StatSoft Inc, Microsoft corporation, Oklahoma,
USA).
Results
Inhibition of NO production by curcumin in primary microglia
First, we investigated whether curcumin can inhibit LPSmediated induction of NO production in rat primary microglial
cells. Cells were treated with curcumin and AG, a selective iNOS
inhibitor, in the culture medium for 2 h, followed by stimulation
with LPS for 20 h. LPS caused greater than 10-fold increases in
NO production compared to the control. LPS-induced NO
production was suppressed by AG, and also inhibited by
curcumin (1 to 8 M) in a dose-dependent manner with an IC50
value of 3.7 M (Fig. 1A, left panel). Furthermore, the inhibitory
activity of curcumin was also shown in the experiments with
BV2 cells, a microglial cell line. Thus, NO production in LPSactivated BV2 cells was concentration-dependently suppressed
by curcumin (Fig. 1A, lower panel), suggesting that it may act as
a potent NO inhibitor in microglial cells.
Cell viability was also measured by MTT assay to determine
whether the inhibitory effects of curcumin on NO production
were attributable to non-specific cytotoxicity. In the presence of
up to 8 M curcumin, a concentration at which NO synthesis
was almost inhibited, cell viability was not different from that of
LPS-stimulated cells (data not shown). Next, to determine
whether the inhibitory effect of curcumin on NO production is
due to inhibition of iNOS protein, RT-PCR, Western blotting
and immunohistochemical analyses were carried out. Fig. 1B,
C, and D showed that curcumin dose-dependently diminished
iNOS expression at the transcriptional and translational levels.
Effect of curcumin on MAPK activation
To understand the inhibitory mechanism by which curcumin
strongly regulates NO production in primary microglial cells, the
effect of curcumin on LPS-induced signaling activation was
examined. To do this, MAPKs (Erk1/2, p38 and JNK) were first
chosen, based on previous reports (Nick et al., 1999; Bodles and
Barger, 2005; Woo et al., 2005). As shown in Fig. 2A, curcumin
down-regulated the activation of all MAPKs, as indicated by their
phosphorylation patterns, a hallmark for activation, without
alteration of normal protein levels. To determine which MAPK is
critical for NO production in primary microglia, specific
inhibitors of MAPKs were employed in a NO assay under the
same conditions. As shown in Fig. 2B (upper panel), SP600125, a
JNK inhibitor, and SB203580, a p38 inhibitor, but not PD98059,
an ERK inhibitor, strongly suppressed NO release in rat primary
microglial cells stimulated by LPS. Furthermore, these inhibitory
patterns were also similarly demonstrated in the case of BV2 cells
(Fig. 2B, lower panel), suggesting that NO release from
microglial cells is dependent on the activation of p38 and JNK
and that their inhibition by curcumin may lead to suppressive
conditions.

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Inhibition of NF-B activation by curcumin


To investigate whether the expression of iNOS is mainly
dependent on the activation of NF-B and AP-1, we analyzed
whether curcumin modulated NF-B and AP-1 activities using
transient transfection assays with the reporter constructs. As
indicated in Fig. 3A, curcumin blocked both NF-B-luc and
AP-1-luc activities induced by LPS stimulation. In the case of
MAPK inhibitors, two inhibitors (SP600125 and SB203580)
were very effective in NF-B-luc activity assay, but all diminished AP-1 activity. Since curcumin more strongly suppressed
NF-B-luc, we further explored the inhibitory process in NF-B
activation pathway. As shown in Fig. 3B and C, curcumin dose
dependently inhibited NF-B binding assessed by EMSA without alteration of p65, a component of NF-B dimmer, and also
suppressed degradation of I-Bs ( and ), a critical process for
translocation of NF-B to the nuclei, shown in LPS stimulation.
Similarly, exposure of microglial cells to SP600125 and
SB203580 but not PD98059 also down-regulated DNA binding
activity of NF-B (Fig. 3D), suggesting that p38 and JNK play a
central role in NF-B activation process and that these are the
major inhibitory pathways managed by curcumin treatment.
Specific NF-B binding was confirmed by supershift and competitive assay with antibody to p65 and an unlabeled probe (data
not shown), as reported previously (Kim et al., 2005).
Discussion
Microglial cells play a central role in pathological events
leading to neuronal cell death occurring in ischemia and neurodegenerative diseases such as Alzheimer's disease, Parkinson's
disease and multiple sclerosis. Nonetheless, not many papers
have reported on primary microglial cells due to cultural difficulties. In particular, curcumin's regulating effect on primary
microglial cell function has not been reported yet, even though
there are numerous studies on its molecular pharmacological
features. In the current study, therefore, the potential immunomodulatory effects of curcumin on NO production in microglial
cells were carefully evaluated using primary microglial cells
activated by inflammatory stimuli such as LPS (Szabo and
Thiemermann, 1995; Macmicking et al., 1997).
As reported previously using other macrophage cell lines
(Chan et al., 1998; Chan, 1995; Brouet and Oshima, 1995), in
our study, curcumin clearly prevented microglia from neurotoxic circumstance caused by excessive NO production. Thus,
as indicated in Fig. 1, curcumin strongly blocked NO generation
from both primary and cell lined microglial cells mediated by
LPS and other pathological stimuli such as IFN- and A(2535) (Bodles and Barger, 2005) in a dose-dependent manner
(data not shown). Curcumin-mediated inhibition was due to
suppression of iNOS expression at the transcriptional and
translational levels, according to a line of evidence that 1) AG, a
selective iNOS inhibitor (Corbett and McDaniel, 1996),
diminished NO production under the same conditions and 2)
a down-regulated level of iNOS but not eNOS or constitutive
NOS (data not shown) was observed by RT-PCR, immunoblotting and immunohistochemical analyses (Fig. 1). The inhibitory

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K.K. Jung et al. / Life Sciences 79 (2006) 20222031

potency (IC50 = 3.7 M) of curcumin in microglial cells was two


to ten-fold higher than that of macrophages including J774.1
(IC50 = 12.3 M), RAW264.7 (IC50 = 6 M), peritoneal macrophages (IC50 = approximately 25 M), and rat mammary glands
(IC50 = approximately 35 M) (Meselhy, 2003; Bhaumik et al.,
2000; Onoda and Inano, 2000; Brouet and Oshima, 1995).
Although it is not clear how microglial cells display higher
sensitivity to curcumin, the potency might be decided by both
direct NO scavenging activity and indirect blockade effects of
NO producing pathways. With reactive oxygen species, neuropathologically, reactive NO has been known to generate
peroxynitrite, a stable toxic component, causing neuro-degenerative diseases (Huie and Padmaja, 1993). Curcumin itself has
been known to be a potent radical scavenger against reactivity
of peroxynitrite, superoxide and nitric oxide (Biswas et al.,
2005; Vajragupta et al., 2004; Kim et al., 2003a,b; Sreejayan
and Rao, 1997). However, under our DPPH [1,1-diphenyl-2picrylhydrazyl] assay condition, we obtained that the direct
scavenging effect of curcumin was not strong with IC50 values
of 91.3 M (data not shown), suggesting that indirect way may
give curcumin a strong NO inhibitory potential in primary
microglial cells. Consequently, our present and previous data
strongly suggest that curcumin may more effectively act on
microglial cells from various oxidative damages than other
target cells, consequently protecting against neuronal cell
degeneration (Hewett et al., 1994).
To produce inflammatory mediators in immune cells, intracellular signaling pathways linked to the activation of transcription
factors should be followed. These include multiple activation of
signaling enzymes such as protein tyrosine kinase (Lyn, Fgr and
Hck) (Khadaroo et al., 2003), JAKs (Kim et al., 2003a), protein
kinase C (, and isoforms) (Cuschieri et al., 2004; Asehnoune
et al., 2005), phosphoinositide 3-kinase (Jhun et al., 2004), MAPKs
(Erk1/2, p38 and SAPK/JNK) (Fiebich et al., 2002), and
transcription factors (such as NF-B, AP-1 and CCAAT
enhancer-binding protein ) (Kim et al., 2005). Therefore, we
next explored the potential inhibitory mechanism of LPS-mediated
NO production by curcumin, since there have been no reports on
the LPS-induced signaling aspects of curcumin in primary
microglial cells, although similar data obtained from IFN-
signaling study were previously published (Kim et al., 2003a). To
do this, MAPK and transcription factors were primarily selected,
based on previous reports (Guzik et al., 2003; Otani, 2004). Fig. 2
indicated that curcumin is able to modulate all MAPK activation, as
judged by analyzing their phosphorylation levels, suggesting that
this compound may act as a broad-spectrum MAPK inhibitor.
Although the mechanism whereby the compound is capable of
down-regulating all MAPK molecules at the same time has not
been fully elucidated yet, it is hypothesized that the inhibitory effect
is due to indirect action. Thus, radical scavenging effect of this
compound is regarded as one of potential drug's action mechanisms
(Vajragupta et al., 2004; Kim et al., 2003a,b; Sreejayan and Rao,
1997), for MAPKs are reported to be activated by cellular redox
system (Torres, 2003; Bundy et al., 2005). Since curcumin was not
strongly scavenged the reactivity of the radicals under our
condition, it is thought that the indirect effect may be not critical
in inhibiting all MAPKs. Considering that protein kinase C,

commonly found during various mitogenic exposure, was directly


inhibited by curcumin (Woo et al., 2005), there is a possibility that
the compound may have an inhibitory capacity on the broadspectrum kinase activity, which remains to be verified.
It is known that IFN- produced from T helper type I cells acts
as a critical factor of LPS-mediated NO production (Matsuura et al.,
2003). Indeed, anti-IFN- antibody strikingly affected NO
production from primary splenocyte- or bone marrow-derived
macrophages activated by LPS. Normally, the production of IFN-
from T helper cells also requires macrophage- or dendritic cellderived IL-12 and IL-18 (Matsuura et al., 2003), suggesting that
LPS-induced NO production is also IL-12-, IL-18- and IFN-dependent. However, various macrophage-like cells showed
different NO production pattern in which IFN- induced additive
NO release only, not completely inhibited by anti-IFN- antibody,
indicating that there may be another signaling pathways induced by
LPS itself. Considering these, therefore, IFN--induced NO
production pathway may participate in late signaling phase in
primary cell condition or may be involved as another completely
independent event in macrophage cell lines. By the reasons, what
types of macrophages are chosen and what signaling steps are
selected to be tested should be carefully considered to evaluate a
drug's efficacy and its pharmacological mechanism. The fact that
curcumin suppressed IFN--mediated NO production pathway via
suppressing the activity of JAK and STAT machinery (Kim et al.,
2003a,b), therefore, seems thought to explain curcumin's NO
modulation by blockade of relatively late phase activation under
LPS stimulation. In contrast, MAPK chosen in our study are known
to be key regulatory signaling molecules in LPS-mediated early
signaling as well as IL-12/IL-18-and IFN--mediated iNOS
expression. Our paper, therefore, showed that curcumin is able to
regulate early signaling phase raised by LPS stimulation, which is
clearly distinct from previous findings reported by Kim et al.
(2003a,b).
Despite the critical involvement of MAPK in activated
macrophages, which kinds of MAPKs (ERK, p39 and JNK) are
involved in iNOS gene expression seem to conflict yet depending
on cell types [chondrocytes, murine macrophage cell lines (J771
and RAW264.7), rat macrophages, rat primary microglia and C6
glioma cells] and stimulators. Therefore, which MAPKs are truly
involved in NO production from microglial cells were carefully reevaluated using their specific inhibitors in this study. Significantly,
as shown in Fig. 2B, SP600125 and SB203580 interrupted NO
release process in primary microglial cells, as similarly shown in
microglial cell line BV2 cells. More interestingly, these three
inhibitors also showed different inhibitory patterns to NF-B and
AP-1-mediated luciferase activities. According to reporter gene
assays, SP600125 and SB203589 but not PD98059 strongly
blocked NF-B activation, whereas all were effective in AP-1mediated luciferase activity. Hence, these results suggest that NFB activation triggered by JNK and p38 but not ERK play dominant roles in iNOS expression and NO production in microglial
cells rather than AP-1-mediated gene expression. Indeed, these two
inhibitors (SP600125 and SB203580) and curcumin also strongly
blocked DNA binding of NF-B, as evaluated by EMSA (Fig. 3B
and D). Furthermore, curcumin blocked degradation of I-B (
and ), induced by the phosphorylation of IB and the conjunction

K.K. Jung et al. / Life Sciences 79 (2006) 20222031

of I-B to ubiquitin (Baldwin, 1996), without alteration of one of


heterodimeric components, p65 (Fig. 3C), unlike a report that this
compound is able to down-regulate the heterodimeric components
(p50 and p65) upon IFN- treatment (Lee et al., 2005). Therefore,
these results suggest that curcumin-mediated inhibition of JNK,
p38 and NF-B is a critical mechanism explaining its pharmacology in microglial cells. Numerous researchers have reported that
strong activation of p38 and NF-B was observed in brain diseases
such as Parkinson's disease (Wilms et al., 2003; Saccani et al.,
2002; Pyo et al., 1998; Walton et al., 1998; Jeng et al., 2005; Li
et al., 2001) and that JNK and p38 are required for induction of
apoptosis in Parkinson's disease cybrids (Onyango et al., 2005),
suggesting neuro-pathophysiological roles of these molecular
components. Some of evidence that p38 acted as a major kinase
involved in phosphorylation of p65 to enhance its binding property
(Schwenger et al., 1998; Nick et al., 1999; Waetzig et al., 2002),
and that JNK and p38 indirectly up-regulate NF-B binding
activity via enhancing rapid degradation of I-B components
(Spiegelman et al., 2001; Jijon et al., 2004) also indicates their
important roles in NF-B activation. However, our results are still
distinct from others findings that ERK and AP-1 have been
critically involved in the production of NO and even other
inflammatory molecules such as MMP-9 in different cells
including macrophages, C6 cells and astroglioma (Lee et al.,
2003b; Kristof et al., 2001; Woo et al., 2005). However, inhibition
of ERK and AP-1 by curcumin may be involved in expression of
other inflammatory genes, such as cytokines (IL-1, IL-6 and TNF) (Christman et al., 1998). This discrepancy seems to be due to
different cells and stimuli used, but further studies should be
conducted to exactly understand pathological features of each
inflammatory cell.
In conclusion, we found that curcumin strongly regulated
NO production in LPS-activated primary microglial cells via
inhibiting the activation of JNK, p38 and NF-B. The inhibitory
effect by curcumin was higher than with other tissue macrophages and cell lines, indicating that brain microglial cells are
the most predominant cells in curcumin pharmacology. Considering that 1) curcumin is one of dietary phenolic compounds
with low toxicity, found in spice turmeric and 2) pharmacologically effective level of curcumin in brain was obtainable when
treated with liver metabolism regulators such as piperine (Shoba
et al., 1998), our data obtained in primary microglial model
suggest that curcumin is a promising agent for both the prevention and treatment of both NO and microglial cell-mediated
neurodegenerative disorders.
Acknowledgments
This work was supported by grant from the Korea Food and
Drug Administration, Korea.
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