Inhibitory effect of a novel resveratrol derivative on nitric oxide production in lipopolysaccharide-activated microglia

Article abstract of DOI:10.1691/ph.2008.8553

Excessive nitric oxide (NO) production by activated microglial cells has been implicated in various neurodegenerative diseases. In the present study, we found that a new resveratrol derivative, (E)-5-(3-nitrostyryl)benzene-1,3-diol (RV06), has a more potential inhibitory effect on the production of NO in LPS-activated N9 microglial cells, and the result was confirmed on primary rat microglial cells. Further studies showed that RV06 inhibited LPS-induced iNOS expression in N9 microglial cells, with no activity on direct scavenging nitric oxide radical in a cell-free environment. The results suggest that RV06 might be a potential anti-inflammatory agent or leading compound which can inhibit inflammatory responses of microglia.

Full text of DOI:10.1691/ph.2008.8553

ORIGINAL ARTICLES
Department of Pharmacology1, Department of Pharmaceutical Engineering2, Sino-American Laboratory of Neuroimmunophar-
macoly3, Shenyang Pharmaceutical University, Shenyang, P.R. China
Inhibitory effect of a novel resveratrol derivative on nitric oxide production
in lipopolysaccharide-activated microglia
X. L. Meng1, 3, G. L. Chen2, J. Y. Yang1, 3, S. Wang1, 3, C. F. Wu1, 3, J. M. Wang3
Received March 11, 2008, accepted April 11, 2008
Dr. Chun Fu Wu, Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road,
Shenhe District, 110016 Shenyang, P.R. China
wucf@syphu.edu.cn, chunfuw@gmail.com
Pharmazie 63: 671–675 (2008)
doi: 10.1691/ph.2008.8553
Excessive nitric oxide (NO) production by activated microglial cells has been implicated in various
neurodegenerative diseases. In the present study, we found that a new resveratrol derivative, (E)-5-(3-
nitrostyryl)benzene-1,3-diol (RV06), has a more potential inhibitory effect on the production of NO in
LPS-activated N9 microglial cells, and the result was confirmed on primary rat microglial cells. Further
studies showed that RV06 inhibited LPS-induced iNOS expression in N9 microglial cells, with no activ-
ity on direct scavenging nitric oxide radical in a cell-free environment. The results suggest that RV06
might be a potential anti-inflammatory agent or leading compound which can inhibit inflammatory re-
sponses of microglia.
1. Introduction
ities on NO production from LPS-activated microglia. In
this study, we found that a novel resveratrol derivative,
(E)-5-(3-nitrostyryl)benzene-1,3-diol (RV06), performs
fairly better than resveratrol on NO production in N9 mi-
croglia cell line and primary rat microglia. In order to
understand the mechanism of action of RV06, we detected
the NO-scavenging activity of RV06 in non-cellular sys-
tems and the inhibitory effect on iNOS induction in N9
microglia.
Microglia are the resident immune cells of the central ner-
vous system (CNS). Excessive amount of inflammatory
mediators produced by activated microglia, such as nitric
oxide (NO), oxygen radicals, and pro-inflammatory cyto-
kines, are involved in the pathogenesis of a different neu-
rodegenerative diseases such as Alzheimer’s disease (AD),
Parkinson’s disease (PD), and multiple sclerosis (MS)
(Dickson et al. 1993; Gonzalez-Scarano and Baltuch 1999;
Mcgeer et al. 1993; Meda et al. 1995).
B
A
Resveratrol (trans-3,40,5-trihydroxystilbene), a polyphenol
present in relatively large amounts in grapes and red wine,
possesses a variety of biological actions including anti-in-
flammatory, chemopreventive, neuroprotective, and cardio-
protective properties (Anekonda 2006; Bradamante et al.
2004; Jang et al. 1997; Okawara et al. 2007; Wang et al.
2002). The inhibitory effect of resveratrol on microglial ac-
tivation has been previously reported, and resveratrol was
found to suppress the production of tumor necrosis factor-a
(TNF-a) and NO induced by lipopolysaccharide (LPS) in
microglia (Bi et al. 2005). The inhibitory effects on PGE2
and IL-1b production, as well as radical and ROS scaveng-
ing properties of resveratrol in microglia have also been
reported (Bureau et al. 2008; Candelario-Jalil et al. 2007;
Kim et al. 2007; Lorenz et al. 2003). However, due to its
photosensitivity and metabolic instability (Goldber et al.
1995; Jeandet et al. 1995; Trela et al. 1996), as well as the
relatively high concentrations of resveratrol which are re-
quired to modulate microglial activation (Bi et al. 2005;
Lorenz et al. 2003), chemical modifications of resveratrol to
find new and stable derivatives which can more efficiently
modulate microglial activation have been undertaken.
HO
HO
HO
HO
OH
NO2
RV06
resveratrol
2. Investigations, results and discussion
It is well known that excessive pro-inflammatory cytokine
and NO productions by activated microglia play an impor-
tant role in neurodegenerative diseases. This is the first
report for the potent inhibitory activity of RV06, a novel
resveratrol derivative, on LPS-induced iNOS expression
and NO release, reflecting the activation of microglial
cells.
By comparing the chemical structure of RV06 with that of
resveratrol, it is obvious that RV06 resembles resveratrol
in terms of chemical structure but should be more stable
than resveratrol. Thus, RV06 might be more stable to ex-
ert persistent biological effects.
Thus, a series of resveratrol derivatives and analogues
were synthesized and screened for their inhibitory activ-
In order to exclude the possibility that the cytotoxic action
of RV06 caused inhibition of LPS-stimulated NO produc-
Pharmazie 63 (2008) 9
671

ORIGINAL ARTICLES
A
LPS
Con
A
120
LPS
Con
120
100
80
60
40
20
0
###
100
80
60
40
20
0
*
***
***
***
Con
0
.3
1
3
10
30 Res
Con
0
.3
1
3
10
30
Res
RV06 (mM)
RV06 (mM)
B
120
100
80
LPS
Con
LPS
Con
B
###
120
100
80
60
40
20
0
**
**
**
***
60
***
40
20
0
Con
0
.3
1
3
10
30
Res
Con
0
.3
1
3
10
30 Res
RV06 (mM)
RV06 (mM)
Fig. 1: Effect of RV06 on microglial cell viability. N9 cells (A) or primary
microglial cells (B) were treated with RV06 (0.3–30 mM) or resver-
atrol (10 mM) in the presence of LPS (1 mg/ml) for 48 h. Cell viabil-
ity was examined by MTT reduction assays and the results were
expressed as percentage of surviving cells over control cells (cul-
tured in medium alone). Data were represented as mean ꢀ SEM of
three separate experiments
Fig. 2: Effect of RV06 on NO production induced by LPS in microglial
cells. N9 cells (A) or primary microglial cells (B) were treated with
RV06 (0.3–30 mM) or resveratrol (10 mM) in the presence of LPS
(1 mg/ml). The results were expressed as the percentage values tak-
ing LPS treatment group as 100%. Data were represented as mean
ꢀ SEM of three separate experiments. ### P < 0.001 as compared
with the control group (cultured in medium alone), * P < 0.05,
** P < 0.01, *** P < 0.001 as compared with only LPS groups
tion, we investigated the effect of RV06 on the cell viabi-
lity. Treatment of N9 or primary rat microglial cells with
RV06 (0.3–30 mM) in the presence of LPS for 48 h did
not provoke any cytotoxicity (Fig. 1A and B). LPS alone
(1 mg/ml) did not cause significant loss of cell viability
(data not shown). These data indicated that the effect of
RV06 on NO production from LPS-activated microglia
was not due to its cytotoxic action.
NO is synthesized from l-arginine by NOS in various
cells and tissues. NOS is broadly classified into two sub-
types: the constitutive “neuronal” and “endothelial” NOS
(cNOS) and inducible NOS (iNOS) (Schroeder and Kuo
1995). iNOS is expressed only when the cells are exposed
to LPS or cytokines such as interferon-g and TNF-a. NO
produced by iNOS is a key mediator and its production is
a crucial step in microglia activation (Possel et al. 2000).
Thus, we detected the effect of RV06 on LPS-induced NO
production in microglial cells. The result showed that ni-
trite, a NO product, was accumulated in the medium after
48 h incubation with LPS. RV06 inhibited LPS-induced
NO production in N9 and primary rat microglial cells in a
concentration-dependent manner, with the IC50 being 15.1
and 9.0 mM, respectively. In the parallel experiments, res-
veratrol (10 mM) significantly reduced NO production
(Fig. 2A and B). Most important is that RV06 has more
potential inhibitory effect on NO production in N9 and
primary rat microglial cells (IC50 ¼ 15.1 and 9.0 mM, re-
spectively) than that of resveratrol (IC50 ¼ 20.8 and
15.5 mM, respectively). These results suggest that RV06
may act as a more efficiently NO inhibitor in microglial
cells than resveratrol.
It is demonstrated that resveratrol has a strong NO-scaveng-
ing effect (Chan et al. 2000). To investigate the NO
scavenging capacity of RV06, we used sodium nitroprus-
side (SNP) as a donor for NO in a cell-free assay. SNP is
an unstable molecule that breaks down when exposed to
light and NO is found as NOþ. SNP was used to test the
NO-scavenging ability of various substances (Mirkov et al.
2004; Yen et al. 2001). In this study, resveratrol (10 mM)
significantly reduced NO levels of SNP solution. How-
ever, no change was observed when RV06 (0.3–30 mM)
was added to the SNP solution (Fig. 3). This result indi-
cated that, unlike resveratrol, RV06 has no NO-scavenging
activity. Therefore, this is not likely to be the main me-
chanism of action of RV06 to inhibit NO levels in med-
ium of microglial cells.
In our previous studies, we evaluated the inhibition of res-
veratrol on iNOS expression in LPS-activated microglia
672
Pharmazie 63 (2008) 9

ORIGINAL ARTICLES
RV06 may result from the inhibition of the enzyme ex-
140
120
100
80
pression. Moreover, we previously demonstrated that, by
blocking IjBa degradation and p38 mitogen-activated pro-
tein kinases (MAPKs) phosphorylation, resveratrol acts to
reduced LPS-induced NO production in N9 microglial
cells (Bi et al. 2005). Whether these mechanisms contrib-
ute to the inhibition of NO production of RV06 in LPS-
activated microglia needs to be further investigated. More-
over, further studies are required to understand whether it
might be a leading compound for future drug develop-
ment. These include assessment of bioavailability, efficacy,
and safety in vivo.
SNP
Con
###
**
60
40
20
In summary, the data presented here demonstrated for the
first time that RV06, a novel resveratrol derivative, exerted
a more potential anti-inflammatory effect by inhibiting
LPS-induced production of NO in microglial cells.
Furthermore, RV06 reduced LPS-induced protein expres-
sion of iNOS, but did not display direct NO-scavenging
activity up to 30 mM in cell-free system. Though the exact
mechanism of this suppression is not clearly understood at
present stage, it can be assumed that RV06 might be a
potential anti-inflammatory agent or leading compound
which can inhibit inflammatory responses of microglia.
0
Con
0
.3
1
3
10
30
Res
RV06 (mM)
Fig. 3: Effect of RV06 on NO levels in SNP solution. SNP solution
(2.5 mM) in PBS was incubated alone or with RV06 (0.3–30 mM)
or resveratrol (10 mM) in light at room temperature for 60 min.
Data were represented as mean ꢀ SEM of three separate experi-
ments. ### P < 0.001 compared to the control group (PBS alone),
**P < 0.01 as compared with the SNP group
3. Experimental
3.1. Materials
Fetal bovine serum (FBS) and Iscove’s modified Dulbecco’s medium
(IMDM) were purchased from Gibco BRL (Grand Island, NY, USA). LPS
(E5: 055) was purchased from Sigma Chemical Co. (St. Louis, MO,
USA). Monoclonal antibody MRC OX-42 to rat CD11b/c was from Immu-
noTools, Friesoythe, Germany. Thiazolyl blue (MTT) was from Sino-
American Biotechnology (Beijing, China). iNOS antibody was from BD
Biosciences (San Diego, CA).
Fig. 4: Effect of RV06 on iNOS expression induced by LPS in N9 micro-
glial cells. N9 cells were treated with RV06 (3–30 mM) in the pre-
sence of LPS (1 mg/ml) for 24 h. The expression of iNOS protein
was detected by Western blot. The experiments were repeated three
times and similar results were obtained
3.2. Synthesis of resveratrol and RV06
All chemical reagents for synthesizing the compounds were purchased
from a local supplier and were analytical grade. Melting points were deter-
mined with a Yanaco micro melting point apparatus and were not cor-
rected. NMR spectra were recorded at 300 MHz using Bruker ARX-300
instrument with TMS as internal reference. Mass spectra (ESI-MS) were
measured on Agilent 1100 Series MSD Trap (SL).
Resveratrol and RV06 were synthesized as described previously (Chen
et al. 2005). The route of the synthesis of RV06 is shown in the Scheme.
Starting from 3,5-dihydroxybenzoic acid, methyl 3,5-dimethoxybenzate
was prepared in good yield and followed by reduction with LiAlH4. Subse-
quent treatment with SOCl2 afforded 3,5-dimethoxylbenzyl chloride with a
yield of about 94%. Using the Wittig-Horner reaction, only the trans iso-
mer was obtained. BBr3 was used for deprotection of methyl groups in
CH2Cl2 to afforded RV06, which is yellow solid, m.p. 196–197 ꢁC.
(Bi et al. 2005). Further experiments were carried out to
examine whether the inhibitory effect of RV06 on NO
production was associated with decreased expression of
iNOS in LPS-stimulated microglia. The result of Western
blot analysis showed that unstimulated cells expressed
very low levels of iNOS protein whereas treatment of N9
microglia with LPS (1 mg/ml) for 24 h led to significant
increases in iNOS protein. LPS-stimulated increase of
iNOS was suppressed in the presence of RV06 (3–30 mM)
(Fig. 4). Thus, the inhibitory activity of NO production by
Scheme Reagents and conditions: i. Me2SO4, K2CO3, CH3COCH3, reflux 3 h, 93.6%; ii. LiAlH4, EtOEt, reflux 4 h, 92.3%; iii. SOCl2, EtOEt, rt 3 h,
94.4%; iv. P(OEt)3, reflux 3 h; v. NaOEt, RCHO, DMF, rt 3 h, 67% (for iv, v steps); vi. BBr3, CH2Cl2, ꢂ10 ꢁC 2 h, 90%
H3CO
HO
H3CO
iii
ii
i
CH2OH
COOH
COOCH3
H3CO
HO
H3CO
H3CO
O2N
H3CO
H3CO
O
CHO
iv
OEt
CH2P
CH2Cl
v
OEt
NO2
H3CO
H3CO
H3CO
vi
HO
HO
NO2
Pharmazie 63 (2008) 9
673

ORIGINAL ARTICLES
1H NMR (300 MHz, DMSO-d6): d (ppm) 6.01 (1 H, s), 6.50 (2 H, s), 7.20
(5% skimmed milk) and then incubated overnight with iNOS antibody,
followed by horseradish peroxidase conjugated secondary antibody. The
protein of interest was detected by enhanced chemiluminescence reagents.
(1 H, d, J ¼ 16.4 Hz, ––CH¼CH––), 7.30 (1 H, d, J ¼ 16.2 Hz,
ꢂCH¼CH––), 7.65 (1 H, t), 8.08 (1 H, t), 8.42 (1 H, s), 9.33 (2 H, s,
––OH). ESI-MS: 257.9 [M þ H], 279.9 [M þ Na]þ, 255.7 [M ꢂ H]––,
291.7 [M þ Cl]––.
3.9. Statistical analysis
Results were expressed as mean ꢀ SEM of three experiments performed in
triplicate. One-way ANOVA followed by Dunnett’s t-test was used for sta-
tistical analysis (SPSS 13.0 software, SPSS, USA).
3.3. Cell culture
The murine microglial cell line N9 was a kind gift from Dr. P. Ricciardi-
Castagnoli (Universita Degli Studi di Milano-Bicocca, Milan, Italy). The
cells were grown in IMDM supplemented with 5% FBS, 2 mM glutamine,
100 U/ml penicillin, 100 mg/ml streptomycin, and 5ꢃ10ꢂ5 M 2-mercap-
toethanol.
Acknowledgements: This study is supported by the Nature Science Foun-
dation of China (No. 30572248 and No. 39800185) and by the project of
Key Laboratory of New Drug Screen of Liaoning province, China.
Primary rat microglia were prepared from the cortex of newborn SD rats
(1 day) (Department of Laboratory Animal Science, Shenyang Pharmaceu-
tical University) (Barger and Harmon 1997). Briefly, the meninges and
blood vessels of the rat cortex were removed. Tissues were dissociated
with 0.25% trypsin at 37 ꢁC for 10 min, and then the cell suspension was
filtered through a 50-mm diameter nylon mesh. The cells were collected by
centrifugation at 1000 rpm/min for 10 min, re-suspended in IMDM and
then plated in culture flasks. Cells were incubated at 37 ꢁC in a humidified
atmosphere (5% CO2). After 9–11 days, the flasks were shaken on a ro-
tary shaker at 240 rev/min for 1 h. The resulting cell suspension, rich in
microglia, was placed in culture dishes in which the cells adhered after
30 min at 37 ꢁC. The purity of cells obtained was > 95% as verified by
immunocytochemistry using the monoclonal antibody MRC OX-42 to rat
CD11b/c (Bi et al. 2005).
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were washed with ice-cold PBS and lysed for 10 min with RIPA lysis
buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% NP-40, 1%
sodium-deoxycholate, 1 mM phenylmethyl-sulfonylfluoride, 1 mM EDTA,
1 mg/ml pepstatin, 1 mg/ml leupeptin, and 1 mg/ml aprotinin). The protein
concentration in the supernatant fluid of the lysate was measured by the
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