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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
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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).
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.
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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).
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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.
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