melatonin & pi

1. Melatonin inhibits Prevotella intermedia lipopolysaccharide-
induced production of nitric oxide and interleukin-6 in murine
macrophages by suppressing NF-jB and STAT1 activity
Introduction
Periodontal disease is a chronic inflammatory process
accompanied by the destruction of surrounding connective
tissue and alveolar bone, and sometimes loss of teeth [1].
Recent evidence suggests that periodontal disease is a
potential risk factor for several systemic diseases including
cardiovascular disease, diabetes, stroke, and preterm low
birth-weight infants, and hence the treatment of periodon-
tal infection contributes to effective prevention and man-
agement of these systemic disorders [2–4].
The primary causative agents of periodontal disease are
particularly gram-negative anaerobic bacteria that accu-
mulate in the gingival sulcus. Prevotella intermedia is a
major periodontal pathogen that is dominant in the
periodontal pockets of patients with adult periodontitis
[5, 6]. This bacterium has also been frequently recovered
from subgingival flora in patients with acute necrotizing
ulcerative gingivitis [7] and pregnancy gingivitis [8].
Lipopolysaccharide (LPS) is a major component of
the outer membrane of gram-negative bacteria, including
P. intermedia. It has the ability to trigger a number of host
cells, especially mononuclear phagocytes, to produce and
release a wide variety of proinflammatory cytokines,
including tumor necrosis factor alpha (TNF-a),
interleukin-1b (IL-1b), IL-6, and IL-8 [9]. In addition, LPS
can induce significant production of nitric oxide (NO) in a
variety of cell types including macrophages [10, 11]. NO has
recently received considerable attention as a novel type of
mediator [12]; inhibition of nitric oxide synthase (NOS)
activity and NO production frequently limits the progression
and severity of experimental inflammatory diseases includ-
ing osteoarthritis, glomerulonephritis, and colitis [13, 14].
LPS preparations extracted from oral black-pigmented
bacteria including P. intermedia have been reported to
possess unique chemical and immunobiological properties
quite different from those of the classical LPSs from the
family Enterobacteriaceae such as Escherichia coli and
Salmonella species [15]. Kirikae et al. [16] also indicated
that the active molecule(s) and mode of action of P. inter-
media LPS are quite different from those of LPS from
Salmonella. Hashimoto et al. [17] demonstrated the struc-
ture of lipid A from P. intermedia ATCC 25611 LPS to be
composed of a diglucosamine backbone with a phosphate
at the 4-position of the nonreducing side sugar, as well as
five fatty acids containing branched long chains. Moreover,
they also found that the lipid A activates murine cells
through a TLR4-mediated signaling pathway.
Abstract: Although a range of biological and pharmacological activities of
melatonin have been reported, little is known about its potential anti-
inflammatory efficacy in periodontal disease. In this study, we investigated
the effects of melatonin on the production of inflammatory mediators by
murine macrophages stimulated with lipopolysaccharide (LPS) from
Prevotella intermedia, a major cause of inflammatory reactions in the
periodontium, and sought to determine the underlying mechanisms of action.
Melatonin suppressed the production of nitric oxide (NO) and interleukin-6
(IL-6) at both gene transcription and translation levels in P. intermedia LPS-
activated RAW264.7 cells. P. intermedia LPS-induced NF-jB-dependent
luciferase activity was significantly inhibited by melatonin. Melatonin did
not reduce NF-jB transcriptional activity at the level of IjB-a degradation.
Melatonin blocked NF-jB signaling through the inhibition of nuclear
translocation and DNA-binding activity of NF-jB p50 subunit and
suppressed STAT1 signaling. Although further research is required to clarify
the detailed mechanism of action, we conclude that melatonin may
contribute to blockade of the host-destructive processes mediated by these
two proinflammatory mediators and could be a highly efficient modulator of
host response in the treatment of inflammatory periodontal disease.
Eun-Young Choi1
, Ji-Young Jin1
,
Ju-Youn Lee2,3
, Jeom-Il Choi2,3
, In
Soon Choi1
and Sung-Jo Kim2,3
1
Department of Biological Science, College of
Medical and Life Sciences, Silla University,
Busan, Korea; 2
Department of Periodontology,
School of Dentistry, Pusan National University,
Yangsan, Gyeongsangnam-do, Korea;
3
Medical Research Institute, Pusan National
University, Busan, Korea
Key words: interleukin-6, lipopolysaccharide,
melatonin, nitric oxide, periodontal disease,
Prevotella intermedia
Address reprint requests to Sung-Jo Kim,
Department of Periodontology, School of
Dentistry, Pusan National University,
Beomeo-ri, Mulgeum-eup, Yangsan,
Gyeongsangnam-do 626-870, Korea.
E-mail: sungjokim@pusan.ac.kr
Received August 17, 2010;
accepted October 5, 2010
J. Pineal Res. 2011; 50:197–206
Doi:10.1111/j.1600-079X.2010.00829.x
2010 The Authors
Journal of Pineal Research 2010 John Wiley Sons A/S
Journal of Pineal Research
197
Molecular,
Biological,
Physiological
and
Clinical
Aspects
of
Melatonin

2. With the current understanding of periodontal disease
etiology and pathogenesis, it became apparent that host
responses to the specific causative bacteria and their
metabolic products are a major determinant of disease
pathogenesis. Recent work has demonstrated, in addition
to bacterial control, that modulation of the host inflam-
matory response is a plausible therapeutic strategy for
periodontal disease [18–20]. LPSs from periodontal patho-
gens stimulate secretion of host inflammatory mediators
such as NO and cytokines in immune cells and thereby
initiate the host inflammatory response associated with
periodontal disease [21–25]. Host modulatory agents
directed at inhibiting NO and specific cytokines appear
to be beneficial in terms of attenuating periodontal
disease progression and potentially enhancing therapeutic
responses.
Melatonin (N-acetyl-5-methoxytryptamine) is synthesized
and released from the pineal gland [26], but more recently it
has been identified in many other cells and organs as well [27].
Melatonin is involved in the regulation of circadian and
seasonal rhythms [28]. In addition, melatonin has been
implicated as a remarkable molecule with significantly
broader actions including antioxidant [29, 30], oncostatic
[31], and immunomodulatory properties [32].
Although a range of biological and pharmacological
activities of melatonin have been reported, little is known
about its potential anti-inflammatory efficacy in periodon-
tal disease. Therefore, in this study, we investigated the
effects of melatonin on the production of inflammatory
mediators by macrophages stimulated with LPS from
P. intermedia, a major cause of inflammatory periodontal
disease, and sought to determine the underlying mecha-
nisms of action.
Materials and methods
Reagents
Melatonin, N-p-Tosyl-l-phenylalanine chloromethyl ketone
(TPCK), DNase, RNase, and proteinase K were obtained
from Sigma-Aldrich (St. Louis, MO, USA). SP600125,
SB203580, PD98029, and AG490 were purchased from
Calbiochem (San Diego, CA, USA). Antibodies against
iNOS, ERK, phospho-ERK, JNK, phospho-JNK, p38,
phospho-p38, IjB-a, STAT1, and phospho-STAT1 were
obtained from Cell Signaling Technology (Beverly, MA,
USA), while antibodies against NF-jB p65, NF-jB p50,
b-actin, and PARP-1 were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA).
Bacteria and culture conditions
P. intermedia ATCC 25611 was used throughout. It was
grown anaerobically on the surface of enriched Trypticase
soy agar containing 5% (v/v) sheep blood, or in GAM
broth (Nissui, Tokyo, Japan) supplemented with 1 lg/mL
menadione and 5 lg/mL hemin. Plate-grown cultures were
routinely incubated for 4 days and used as the inoculum for
liquid growth. Liquid-grown cells were incubated for
approximately 24 hr, to late exponential growth phase.
They were collected by centrifugation at 12,000 · g for
20 min at 4C, washed three times with phosphate-buffered
saline (PBS, pH 7.2), and lyophilized. Culture purity
was assessed by gram staining and plating on solid
medium.
LPS isolation
LPS was prepared from lyophilized P. intermedia ATCC
25611 cells by the standard hot phenol–water method [33].
Briefly, 90% phenol was added to bacteria suspended in
pyrogen-free distilled water, and the mixture was extracted
twice at 68C for 20 min. After cooling, the aqueous phase
was separated by centrifugation at 7000 · g for 15 min, and
the pooled aqueous extract was dialyzed extensively against
distilled water at 4C. The dialyzed extract was centrifuged
at 105,000 · g for 3 hr and lyophilized to yield crude
extract. This was treated with DNase (25 lg/mL) and
RNase (25 lg/mL) in 0.1 m Tris (pH 8.0) at 37C overnight
to remove nucleic acids. Any contaminating protein was
then hydrolyzed with proteinase K (50 lg/mL), followed by
heating at 60C for 1 hr, and incubating overnight at 37C.
The yield of LPS was about 0.26%. The protein content of
the purified LPS, determined by the method of Markwell
et al. [34], was less than 0.1%. Coomassie blue staining of
overloaded sodium dodecyl sulfate (SDS)-polyacrylamide
gels did not reveal any visible protein bands in the purified
LPS, confirming the purity of the preparation (data not
shown).
Cell cultures
The murine macrophage cell line RAW264.7 (American
Type Culture Collection, Rockville, MD) was grown in
Nunc flasks in Dulbeccos modified Eagles medium
(DMEM) supplemented with 100 U/mL of penicillin,
100 lg/mL streptomycin, 10 mm HEPES, 2 mm l-gluta-
mine, 0.2% NaHCO3, 1 mm sodium pyruvate, and 10%
[v/v] heat-inactivated FBS in a humidified chamber with
5% CO2/95% air at 37C. At confluence, the medium and
nonadherent cells were removed and replaced with fresh
culture medium. After an additional 24 hr of culture, the
cells were harvested by gentle scraping with a rubber
policeman, washed three times, and viable cells counted.
The cells were seeded into 24-well culture plates at a density
of 5 · 105
cells/well and incubated for at least 12 hr to
allow them to adhere to the plates. After washing three
times with medium, various concentrations of P. intermedia
LPS and melatonin were added and the cells were cultured
for 24 hr, after which culture supernatants were collected
and assayed for NO and IL-6.
Cytotoxicity assay
The cellular toxicity of melatonin was assessed by the MTT
assay, which is based on the conversion of 3-(4,5-dim-
ethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
to formazan by mitochondrial dehydrogenases [35]. Cells
were incubated with various concentrations of P. interme-
dia LPS and melatonin for 24 hr, and MTT was added to
the cultures to a final concentration of 0.5 mg/mL. After
incubation at 37C in 5% CO2 for 2 hr, the supernatant
Choi et al.
198

3. was removed and the cells were solubilized in dimethyl
sulfoxide (DMSO). The extent of reduction of MTT to
formazan within the cells was quantified by measuring
absorbance at 570 nm with a Spectra Max 250 ELISA
Reader (Molecular Devices, Sunnyvale, CA, USA).
Cell viability is expressed as a percentage of the control
value.
Measurement of NO production
NO production was assayed by measuring the accumula-
tion of the stable oxidative metabolite, nitrite (NO2
)
), in
culture supernatants [36]. Briefly, 5 · 105
cells/well were
stimulated in 24-well tissue culture plates for 24 hr, and
100 lL of Griess reagent (1% sulfanilamide, 0.1% naph-
thylethylene diamine dihydrochloride, and 2.5% phospho-
ric acid) (Sigma) was added to equal volumes of culture
supernatants in a 96-well flat-bottomed microtiter plate and
left at room temperature for 10 min. Optical densities at
540 nm were read with a Spectra Max 250 ELISA Reader
(Molecular Devices), and nitrite concentrations were cal-
culated from a standard curve established with serial
dilutions of NaNO2 (Sigma) in culture medium.
Measurement of IL-6 production
The amount of IL-6 secreted into the culture medium was
determined by enzyme-linked immunosorbent assay (ELI-
SA) using a commercially available kit (OptEIA; BD
Pharmingen, San Diego, CA, USA) according to protocols
recommended by the manufacturer.
RNA extraction and real-time polymerase chain
reaction for iNOS and IL-6 mRNA
Cells were plated in 100-mm tissue culture dishes at a density
of 1 · 107
cells/dish and treated with various concentrations
of P. intermedia LPS and melatonin for 24 hr. Following
incubation, they were washed twice with PBS and collected
by centrifugation. Total RNA was isolated with an RNeasy
Mini Kit (Qiagen, Valencia, CA, USA) according to the
manufacturers instructions. cDNA was prepared from 1 lg
of the total RNA using iScript cDNA Synthesis Kit (Bio-
Rad, Hercules, CA, USA). A real-time PCR was performed
using the CFX96 real-time PCR detection system (Bio-Rad)
with specific primers for mouse iNOS and IL-6. As an
endogenous control, b-actin primer was used. PCR was
conducted with SsoFast EvaGreen Supermix (Bio-Rad)
according to the manufacturers instruction. Thermal cycler
conditions were as follows: After denaturing at 98C for
30 s, PCR was performed for 45 cycles, each of which
consisted of denaturing at 95C for 1 s, annealing/extending
at 60C for 5 s. The following PCR primers for iNOS
(130 bp), IL-6 (162 bp), and b-actin (149 bp) were used:
iNOS sense, 5¢-GCACCACCCTCCTCGTTCAG-3¢ and
antisense, 5¢-TCCACAACTCGCTCCAAGATTCC-3¢; IL-6
sense, 5¢-GCCAGAGTCCTTCAGAGAGATACAG-3¢ and
antisense, 5¢-GAATTGGATGGTCTTGGTCCTTAGC-3¢;
b-actin sense, 5¢-TGAGAGGGAAATCGTGCGTGAC-3¢
and antisense, 5¢-GCTCGTTGCCAATAGTGATGA-
CC-3¢. Each assay was normalized to b-actin mRNA.
Immunoblotting analysis
Cells were plated in 60-mm tissue culture dishes, at a
density of 4 · 106
cells per dish, and treated with various
concentrations of P. intermedia LPS and melatonin for the
indicated periods of time. To prepare cell lysates, cells were
washed three times with ice-cold PBS and lysed by
incubating for 30 min on ice with 200 lL of lysis buffer
(50 mm TrisÆCl [pH 8.0], 150 mm NaCl, 0.002% sodium
azide, 0.1% SDS and 1% Nonidet P-40) containing
protease inhibitors (1 mm phenylmethanesulfonyl fluoride,
5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 5 mg/mL
leupeptin). The cell lysates were centrifuged at 10,000 · g
for 10 min to remove insoluble material. The nuclear
fraction was prepared from cells using the ActiveMotif
nuclear extract kit (Active Motif, Carlsbad, CA, USA)
according to the manufacturers instructions. Briefly, the
cells were washed with ice-cold PBS/phosphatase inhibi-
tors, collected with a cell scraper, and harvested by
centrifugation. The cell pellet was then resuspended in
hypotonic buffer and then kept on ice for 15 min. The
suspension was then mixed with detergent and centrifuged
for 30 s at 14,000 · g. The nuclear pellet obtained was
resuspended in complete lysis buffer in the presence of the
protease inhibitor cocktail, incubated for 30 min on ice,
and centrifuged for 10 min at 14,000 · g. The resulting
supernatant, corresponding to nuclear fraction, was col-
lected and stored at )80C until use. Protein concentrations
were determined with the bicinchoninic acid (BCA) protein
assay reagents (Pierce, Rockford, IL, USA) according to
the manufacturers instructions. The same amount of
protein (30 lg) was then subjected to SDS–polyacrylamide
gel electrophoresis (SDS–PAGE) on 10–12% acrylamide
gels with 3% stacking gels. The resolved proteins were
transferred to a nitrocellulose membrane by electroblotting,
and the blots were blocked for 1 hr in PBST (PBS with
0.1% Tween-20) containing 3% nonfat dry milk, followed
by incubation with specific primary antibodies. They were
then washed three times for 10 min each with PBST,
incubated with horseradish peroxidase-conjugated second-
ary antibodies at room temperature for 1 hr, and visualized
by enhanced chemiluminescence (Cell Signaling Technol-
ogy) as recommended. The intensity of each protein-specific
band was quantified by densitometer with densitometric
software.
Transfection and luciferase assay
RAW 264.7 cells were plated at 1.5 · 105
cells per well in
24-well plates the day before transfection and grown to
80–90% of confluence. Cells were transiently cotransfected
with the plasmids pNF-jB-Luc (Sratagene, Santa Clara,
CA, USA) and pRL-TK (Promega, Madison, WI, USA)
using FuGENE HD Transfection Reagent (Roche Applied
Science, Indianapolis, IN, USA) according to the manu-
facturers protocol. Briefly, the transfection mixture con-
taining 0.35 lg of pNF-jB-Luc and 0.15 lg of pRL-TK
was mixed with the Fugene HD reagent and added to the
cells. After 24 hr of transfection, cells were incubated
with melatonin (1000 lm) in the absence or presence of
P. intermedia LPS (10 lg/mL). After 12 hr of incubation,
Effects of melatonin on periodontal disease
199

4. luciferase activity in the cell lysate was determined by using
the Dual-Luciferase Reporter Assay System (Promega)
with a SpectraMax L microplate luminometer (Molecular
Devices). The firefly luciferase activity was normalized
to the Renilla luciferase activity. The level of luciferase
activity was determined as a ratio in comparison with cells
with no stimulation. All transfections were performed in
triplicate.
DNA-binding activity of NF-jB
Cells were plated in 60-mm tissue culture dishes, at a
density of 4 · 106
cells per dish, and treated with various
concentrations of P. intermedia LPS and melatonin for the
indicated periods of time. After extracting the nuclear
protein as described earlier, the DNA-binding activity of
NF-jB in nuclear extract was assayed by using a TransAM
NF-jB p65/NF-jB p50 transcription factor assay kits
(Active Motif) according to the manufacturers recom-
mended procedures. Oligonucleotide with the NF-jB con-
sensus binding site (5¢-GGGACTTTCC-3¢), to which the
activated NF-jB contained in nuclear extracts specifically
binds, has been immobilized on a 96-strip well plate. The
activated NF-jB p65 and p50 specifically bound to this
oligonucleotide was detected using specific antibodies to
NF-jB p65 and p50, respectively. A Spectra Max 250
ELISA Reader (Molecular Devices) was used to read the
sample absorbance, with results expressed as optical density
(OD) emitted at 450 nm.
Statistical analysis
Data are expressed as means ± S.D., and statistical
analysis was performed using Students t-test with
P 0.05 considered statistically significant.
Results
To assess the effects of melatonin on the P. intermedia LPS-
induced NO and IL-6 production, RAW264.7 cells were
challenged with different doses of melatonin (0, 10, 100, and
1000 lm) in the absence or presence of P. intermedia LPS
(10 lg/mL) for 24 hr, and the levels of NO and IL-6 in the
culture supernatants were measured. As shown in Fig. 1,
P. intermedia LPS stimulation led to marked increases of
NO and IL-6 levels. Melatonin effectively suppressed the
P. intermedia LPS-induced production of NO and IL-6,
and these effects of melatonin were concentration depen-
dent. Of note, melatonin nearly completely blocked the
IL-6 secretion at the concentration of 1 mm. Immunoblot
analysis showed that melatonin also reduced P. intermedia
LPS-induced iNOS protein expression (Fig. 2). This result
indicated that melatonin inhibited the production of NO by
reducing iNOS protein expression in LPS-stimulated
RAW264.7 cells. Real-time PCR analysis showed that
melatonin also reduced P. intermedia LPS-induced iNOS
and IL-6 mRNA expression in a dose-dependent manner
(Fig. 3). No notable effects on cell viability were observed
when the cells were exposed up to 1 mm of melatonin for
24 hr as determined by MTT assay (data not shown),
indicating that the suppression of NO and IL-6 production
could not be attributable to a direct cytotoxic effect by
melatonin.
To elucidate which signaling pathways lead to the effects
of melatonin on NO and IL-6 production induced by
P. intermedia LPS, we first tested the roles of MAPKs,
NF-jB, and JAK-2/STAT1 in the P. intermedia LPS-
induced production of NO and IL-6. P. intermedia LPS-
induced production of NO and IL-6 was significantly
inhibited by the specific JNK inhibitor SP600125 and p38
inhibitor SB203580, whereas ERK inhibitor PD98029 had
no effect (Fig. 4). In addition, treatment with either NF-jB
inhibitor TPCK or JAK2/STAT1 inhibitor AG490 signif-
(A)
(B)
Fig. 1. Effects of melatonin on Prevotella intermedia lipopolysac-
charide (LPS)-induced production of NO (A) and IL-6 (B) in
RAW264.7 cells. Cells were incubated with different doses of
melatonin (0, 10, 100, and 1000 lm) in the absence or presence of
P. intermedia LPS (10 lg/mL). Supernatants were removed after
24 hr and assayed for NO and IL-6. The results are means ± S.D.
of three independent experiments. **P 0.01 versus P. intermedia
LPS alone.
Fig. 2. Effects of melatonin on Prevotella intermedia lipopolysac-
charide (LPS)-induced expression of iNOS protein in RAW264.7
cells. Cells were incubated with different doses of melatonin (0, 10,
100, and 1000 lm) in the absence or presence of P. intermedia LPS
(10 lg/mL) for 24 hr. iNOS protein synthesis was measured by
immunoblot analysis of cell lysates using iNOS-specific antibody. A
representative immunoblot from two separate experiments with
similar results is shown.
Choi et al.
200

5. icantly attenuated LPS-induced production of NO and IL-6
(Fig. 4), suggesting that the JNK, p38, NF-jB, and JAK2/
STAT1 pathways are involved in NO and IL-6 production
induced by P. intermedia LPS.
We then determined whether melatonin inhibited
P. intermedia LPS-induced NO and IL-6 production by
the regulation of JNK or p38 pathways activated by LPS.
As anticipated, stimulation with P. intermedia LPS resulted
in the phosphorylation of JNK and p38 (Fig. 5). However,
melatonin failed to prevent LPS from activating either JNK
or p38 (Fig. 5). These findings suggest that the JNK
and p38 pathways are not involved in the inhibition of
P. intermedia LPS-induced NO and IL-6 release by
melatonin.
We then investigated whether melatonin inhibited
P. intermedia LPS-induced production of NO and IL-6
via regulation of NF-jB pathway. We performed luciferase
reporter assay to determine whether melatonin is able to
inhibit NF-jB transcriptional activity. Incubation of trans-
fected RAW264.7 cells with P. intermedia LPS for 12 hr
increased NF-jB-dependent luciferase activity about
5-fold, and this activation was significantly attenuated by
melatonin (Fig. 6A). To determine whether the inhibitory
action of melatonin was because of its effect on P. inter-
media LPS-induced degradation of IjB-a, upstream signal-
ing pathway of NF-jB, the cytoplasmic levels of IjB-a
protein were examined by immunoblotting. As shown in
Fig. 6B, the degradation of IjB-a induced by P. intermedia
LPS was not inhibited when cells were cotreated with
melatonin. Because melatonin inhibited P. intermedia LPS-
induced NF-jB transcriptional activity without a diminu-
tion of IjB-a degradation, we next examined whether
melatonin prevents the nuclear translocation of the subun-
its of NF-jB, i.e., p65 and p50, which is immediately
occurred downstream IjB-a degradation. Nuclear fractions
were isolated and immunoblotted with antibodies against
NF-jB p65 and p50. Whereas nuclear translocation of p50
subunit induced with P. intermedia LPS was dose-depen-
dently hampered in the presence of melatonin, melatonin
did not affect p65 nuclear translocation (Fig. 6C). Finally,
we determined whether melatonin could affect NF-jB-
dependent transcription by inhibiting the binding of NF-jB
to DNA. The DNA-binding activity of NF-jB in nuclear
extract was analyzed by using the ELISA-based NF-jB
p65/NF-jB p50 transcription factor assay kits (Active
(A)
(B)
Fig. 3. Effects of melatonin on Prevotella intermedia lipopolysac-
charide (LPS)-induced iNOS (A) and IL-6 (B) mRNA expression in
RAW264.7 cells. Cells were incubated with different doses of
melatonin (0, 10, 100, and 1000 lm) in the absence or presence of
P. intermedia LPS (10 lg/mL) for 24 hr. Real-time PCR was per-
formed with EvaGreen Supermix, b-actin being used as an
endogenous control. Data are presented as percentage of P. inter-
media LPS alone. The results are means ± S.D. of three inde-
pendent experiments. **P 0.01 versus P. intermedia LPS alone.
(A)
(B)
Fig. 4. Involvement of MAPKs, NF-jB, and JAK2/STAT1 path-
ways in Prevotella intermedia lipopolysaccharide (LPS)-induced
production of NO (A) and IL-6 (B) in RAW264.7 cells. Cells were
pretreated with various kinase inhibitors for 1 hr or 30 min and
then stimulated with P. intermedia LPS (10 lg/mL) for 24 hr.
Supernatants were removed and assayed for NO and IL-6. Data
are presented as percentage of P. intermedia LPS alone. The results
are means ± S.D. of three independent experiments. **P 0.01
versus P. intermedia LPS alone.
Effects of melatonin on periodontal disease
201

6. Motif). DNA-binding activities of NF-jB p65 and
p50 subunits were markedly increased upon exposure to
P. intermedia LPS (Fig. 6D). Whereas the increased NF-jB
p50-binding activity induced by P. intermedia LPS was
dose-dependently attenuated by treatment with melatonin,
melatonin did not affect p65-binding activity (Fig. 6D).
In addition, we examined whether melatonin regulates
P. intermedia LPS-induced NO and IL-6 production
through inhibiting the STAT1 pathway. STAT1 phosphor-
ylation induced by P. intermedia LPS was significantly
inhibited by melatonin (Fig. 7). This result suggests that
melatonin exerts its effects on P. intermedia LPS-induced
NO and IL-6 production via regulating the STAT1
pathway.
Discussion
Because production of NO and IL-6 has been recognized as
a marker in a variety of human diseases associated with
inflammation [37–39], we investigated whether melatonin
could downregulate the production of these inflammatory
mediators in macrophages stimulated with LPS from
P. intermedia, the causative agent of inflammatory peri-
odontal disease, and attempted to elucidate possible mech-
anisms of action. Macrophages are known to be the main
producer of NO and IL-6 and a dense infiltration of
inflammatory cells, including macrophages, occurs in the
gingival connective tissues of patients with periodontal
disease [40].
Melatonin may be beneficial for the treatment of
oxidative stress-related pathologies of oral cavity including
periodontal disease [41]. Scavenging of free radicals by
melatonin in the inflamed gingival tissue would be poten-
tially valuable in reducing the degree of periodontal tissue
destruction. Moreover, melatonin stimulates alveolar bone
regeneration by promoting osteoblast differentiation [42]
and favoring the synthesis of type I collagen fibers [43].
Both salivary and gingival crevicular fluid melatonin levels
decreased in subjects with periodontitis compared to
clinically healthy subjects, indicating that melatonin may
play a protective role against periodontal disease [44, 45].
Thus, melatonin may have potential use in the treatment of
periodontal disease, although further studies are encour-
aged to validate this hypothesis.
The results of the present study indicate that melatonin
suppresses the production of NO and IL-6 at both gene
transcription and translation levels in P. intermedia LPS-
activated RAW264.7 cells. NO is thought to have an
important role in the pathogenesis of inflammatory peri-
odontal disease as it does in other inflammatory diseases.
Enhanced production of NO has been demonstrated in
periodontal disease [46], and gingival tissues from patients
with chronic periodontitis have higher levels of iNOS
protein and mRNA than healthy tissue [47–50]. Macro-
phages, polymorphonuclear cells, and fibroblasts are the
sources of iNOS in periodontal tissues, with endothelial
cells also contributing [47–50]. Moreover, LPS from Acti-
nobacillus actinomycetemcomitans, a major pathogen of
early-onset periodontitis, induced significant production of
NO in macrophages [21, 22], and LPSs from P. intermedia
and P. nigrescens, the causative agents of inflammatory
periodontal disease, fully induced iNOS expression and NO
production in the murine macrophage cell line, RAW264.7,
in the absence of other stimuli [23, 24]. Additionally, IL-6 is
also important in the pathogenesis of periodontal disease.
Clinically, IL-6 levels in sites with periodontal disease are
higher than those in healthy sites and closely related to the
severity of periodontal disease [51, 52]. Moreover, it has
been well demonstrated that IL-6 is a potent bone resorp-
tive agent, induces osteoclastogenesis, and hence plays
important roles in alveolar bone resorption in periodontal
disease [53, 54]. Blockade of NO and IL-6, therefore, could
be a highly efficient tool for blocking the development and
progression of inflammatory periodontal disease.
It is generally accepted that multiple signal transduction
pathways participate in LPS-induced activation of macro-
phages and resultant production of proinflammatory
mediators, and MAPK and NF-jB pathways play critical
roles. However, the results of this study suggest that
MAPK pathways are not involved in the inhibition of
P. intermedia LPS-induced NO and IL-6 release by mela-
tonin. NF-jB is a transcription factor that plays a critical
role in the expressions of proinflammatory cytokines and
other mediators [55–57]. NF-jB is comprised of homo- or
heterodimers of five different Rel proteins, p65 (RelA), p50
(NF-jB1), p52 (NF-jB2), c-Rel, and RelB. In unstimulated
cells, NF-jB is present in the cytoplasm in an inactive form
bound to the inhibitory jB (IjB) proteins. IjB becomes
phosphorylated, ubiquitinated, and then degraded upon
stimulation with a broad range of stimuli, including LPS.
Then, the activated NF-jB dimers are translocated into the
nucleus, bind to jB-binding sites in the promoter regions of
target genes, and induce the transcription of various
proinflammatory mediators including iNOS and IL-6 [58,
59]. In this study, luciferase reporter gene analysis indicated
that P. intermedia LPS-induced NF-jB transcriptional
activity was significantly inhibited by melatonin. Melatonin
did not inhibit NF-jB transcriptional activity at the level of
IjB-a degradation. As shown previously [60, 61], melatonin
blocked NF-jB signaling through the inhibition of nuclear
translocation and DNA-binding activity of NF-jB p50
subunit induced with P. intermedia LPS. Both NF-jB p65
Fig. 5. Effects of melatonin on Prevotella intermedia lipopolysac-
charide (LPS)-induced phosphorylation of JNK and p38 in
RAW264.7 cells. Cells were incubated with different doses of
melatonin (0, 10, 100, and 1000 lm) in the absence or presence of
P. intermedia LPS (10 lg/mL) for 30 min (for JNK) or 15 min (for
p38). Cells lysates were subjected to immunoblot analysis using
specific antibodies. A representative immunoblot from two sepa-
rate experiments with similar results is shown.
Choi et al.
202

7. and 50 accumulate in the nucleus in a variety of cell types in
response to LPS. While NF-jB p65, RelB, and c-Rel have
transactivation domains and directly promote gene tran-
scription, p50 lacks such a domain and does not directly
stimulate gene transcription [62]. p50, therefore, usually
forms a heterodimer with other NF-jB subunits and
participates in target gene transcription [59, 63]. NF-jB
has been considered as a potential target molecule for the
treatment of inflammatory diseases, and its inhibition by
melatonin would be useful in the treatment of periodontal
disease.
The signal transducer and activator of transcription
(STAT) signaling pathway plays an essential role in the
regulation of inflammatory responses [64]. The seven STAT
(A)
(C)
(D)
(B)
Fig. 6. Effects of melatonin on Prevotella
intermedia lipopolysaccharide (LPS)-in-
duced NF-jB activation in RAW264.7
cells. (A) Cells were transiently cotrans-
fected with pNF-jB-Luc and pRL-TK for
24 hr. The transfected cells were then
incubated with different doses of melato-
nin (0 and 1000 lm) in the absence or
presence of P. intermedia LPS (10 lg/mL).
After 12 hr of incubation, luciferase
activity in the cell lysate was determined
using the dual-luciferase assay. The firefly
luciferase activity was normalized to the
Renilla luciferase activity. The level of
luciferase activity was determined as a
ratio in comparison with cells with no
stimulation. All transfections were per-
formed in triplicate. **P 0.01 versus
P. intermedia LPS alone. (B, C, D) Cells
were incubated with different doses of
melatonin (0, 10, 100, and 1000 lm) in the
absence or presence of P. intermedia LPS
(10 lg/mL). (B) After 30 min of incuba-
tion, IjB-a degradation was determined
by immunoblot analysis of cell lysates
using antibody against IjB-a. A repre-
sentative immunoblot from two separate
experiments with similar results is shown.
(C, D) After 30 min (for NF-jB p65) or
8 hr (for NF-jB p50) of incubation, the
nuclear fraction was isolated from cells.
(C) Nuclear translocation of NF-jB
subunits was assessed by immunoblot
analysis using antibodies against NF-jB
p65 and p50. A representative immuno-
blot from two separate experiments with
similar results is shown. (D) DNA-binding
activity of NF-jB in nuclear extracts was
assessed by using the ELISA-based NF-
jB p65/NF-jB p50 transcription factor
assay kits. The results are means ± S.D.
of two independent experiments.
*P 0.05 versus P. intermedia LPS
alone.
Fig. 7. Effects of melatonin on Prevotella intermedia lipopolysac-
charide (LPS)-induced phosphorylation of STAT1 in RAW264.7
cells. Cells were incubated with different doses of melatonin (0, 10,
100, and 1000 lm) in the absence or presence of P. intermedia LPS
(10 lg/mL) for 4 hr. Expression of phospho-STAT1 was measured
by immunoblot analysis of cell lysates. Total STAT1 was used as
an internal control. A representative immunoblot from two sepa-
rate experiments with similar results is shown.
Effects of melatonin on periodontal disease
203

8. family members are identified in mammals, and each one
binds to a different DNA sequence [65]. Different STATs
form homo- or heterodimers. The STAT family of tran-
scription factors is activated by Janus kinases (JAKs) [66,
67]. Activated STAT dimers translocate into the nucleus
and induce the transcription of their target genes. STAT1,
downstream of JAK2, appears to be an important tran-
scription factor for LPS-induced gene expressions in
macrophages [68]. In this study, melatonin exerted its
effects on the inhibition of P. intermedia LPS-induced NO
and IL-6 production via regulating the STAT1 pathway.
Thus, the STAT1 signaling pathway could be an attractive
molecular target for treating inflammatory periodontal
disease.
In conclusion, the present study shows for the first time
that melatonin strongly suppresses NO and IL-6 produc-
tion induced by LPS from P. intermedia, a major cause of
inflammatory periodontal disease, in macrophages. The
underlying mechanisms of melatonin involve the inhibition
of NF-jB and STAT1 pathways in LPS-stimulated macro-
phages. Although further research is required to clarify the
detailed mechanism of action, we conclude that melatonin
may contribute to blockade of the host-destructive pro-
cesses mediated by these two proinflammatory mediators
and could be a highly efficient modulator of host response
in the treatment of inflammatory periodontal disease.
Further in vivo studies are required to better evaluate the
potential of melatonin as a therapeutic agent to treat
periodontal disease.
Acknowledgements
This study was supported by Medical Research Institute
Grant (2009-3), Pusan National University.
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