Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : February 2, 2016
Accepted : July 19, 2016
Published online : September 30, 2016

Original article

Melatonin has no direct effect on inflammatory gene expression in CML-HSA
stimulated RAW264.7 cells
A. N. M. Mamun-Or-Rashid, Wakako Takabe, Yoshikazu Yonei
Anti-Aging Medical Research Center and Glycative Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyoto, Japan

Abstract

Objective: Advanced glycation end products (AGEs), such as N ε- (carboxymethyl)-lysine (CML), modified proteins activate
macrophages and increase the production of inflammatory cytokines via receptors for advanced glycation end products(RAGE) mediated pathway. These compounds ultimately lead to the progress of acute and chronic inflammatory diseases
like rheumatoid arthritis, knee osteoarthritis, locomotive syndrome and so on. Therefore, to suppress the upregulation of
inflammatory gene expression is helpful way in both prevention and cure. Melatonin secretion declines with age, whereas
AGE production and accumulation increases. In this study, we examined the effect of melatonin on CML-HSA (humanserum albumin)-induced inflammatory gene expression in RAW264.7 cells.
Methods: The mouse macrophage cell line RAW264.7 were treated with 10 and 0.1 μg/mL melatonin or 2 μL/mL dimethyl
sulfoxide (DMSO) as the vehicle control for 24 hours, followed by stimulation by 0.5 μg/mL CML-HSA for 3 hours. TNFαprotein content was analyzed by enzyme-linked immunosorbent assay (ELISA) and the inflammatory gene mRNA expression
was measured by real-time polymerase chain reaction (RT-PCR). Cell viability was determined using a cell counting kit-8(CCK-8) and lactate dehydrogenase (LDH) assay.
Results: TNFα secretion was significantly elevated by CML-HSA stimulation for 3 hours (p < 0.001). Pretreatment withmelatonin did not inhibit TNFα production, TNFα, RAGE, inducible nitric oxide synthase (iNOS), interleukin-1 beta (IL-1β),or IL-6 mRNA expression. Cell viability was not affected by the experimental conditions.
Conclusion: These data suggested that melatonin does not have any inhibitory or stimulatory effect on CML-HSA-induced(glycative stress-induced) inflammatory gene expression in RAW264.7 cells.

KEY WORDS: glycative stress, N ε -(carboxymethyl)-lysine (CML), advanced glycation end products (AGEs),		

receptor for AGEs (RAGE), inflammation

Introduction

Advanced glycation end products (AGEs) are implicatedin the pathogenesis of diabetic complications, cancer 1), retinal
disease 2), cardiovascular disease 3), Alzheimer’s disease 4),
respiratory disorders 5), liver disease 6), chronic inflammatory
diseases (atherosclerosis, asthma, arthritis), myocardial
infarction, nephropathy, retinopathy, periodontitis andneuropathy 7) by AGE-RAGE (receptor for advanced glycation
end products) interaction. AGEs also can interact with
SR-A (macrophage scavenger receptor Type I and II ),OST-48 (oligosaccharyl transferase-4) (AGE-R1), 80 K-H
phosphoprotein ( protein kinase C substrate) (AGE-R2),
Galectin-3 (AGE-R3), LOX-1 ( lectin-like oxidized low
density lipoprotein receptor-1), and CD36.
Contact Address: Professor Yoshikazu Yonei, MD, PhD
Anti-Aging Medical Research Center and Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1-3, Tataramiyakodani, Kyotanabe-shi, Kyoto, 610-0394 Japan
Phone/Fax: +81-774- 65- 6394 E-mail: yyonei@mail.doshisha.ac.jp
Co-authors: Mamun-Or-Rashid ANM, mamunbtgeiu@gmail.com ;Takabe W, wtakabe@mail.doshisha.ac.jp

N ε -(carboxymethyl)-lysine (CML) is a major antigenic
AGE which can activate RAGE-mediated reactive oxygen
species (ROS), MAP kinase and nuclear factor-kappa B(NF- K B), ultimately leading to inflammatory cytokine
formation 8). Increased CML levels are observed in the
cerebral blood vessels of both diabetic patients and in thestreptozotocin- treated rat model of diabetes mellitus 9).
These evidences indicate that CML contributes to diabetes
complications. CML has been identified in synovial tissue ofrheumatoid arthritis (RA) patients, and presumably causes
the perpetuation of inflammation in the joint 10). In obese
subjects, CML accumulation in adipose tissue, as evidenced
by the decreased plasma levels of CML, plays an importantrole in adipose tissue inflammation 11).

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Glycative Stress Research 2016; 3 (3): 141- 151(c) Society for Glycative Stress Research

Melatonin doesn’t Suppress CML-HSA-induced Inflammatory Gene Expression in RAW264.7 Cells
In our previous study, CML-HSA (0.5 to 2 μg/mL) was
found to trigger tumor necrosis factor-alfa (TNFα; a major
inflammatory cytokine) production in RAW264.7 cells;
the TNFα level reached at peak after 3 hour of CML-HSA
stimulation and declined thereafter 12). In another study, we
found that pretreatment with several kinds of plant extracts
can significantly lower TNFα production in CML-HSAinduced RAW264.7 cells 13).
Melatonin (N-acetyl-5-methoxytryptamine) is a natural
mammalian hormone synthesized by methylation of serotoninby SAMe (S-adenosyl methionine); it is mainly found in thepineal gland, but there is also some in retina, bone morrow,
lymphocytes, and even in foods such as oats. Melatonin has
a dual role as a potent anti-inflammatory 14-17) and an antioxidant 18); it also has regulatory roles in sexual activity
development 19), immunomodulation 20), and cardiovascularfunctions 21). Further, it has been suggested to have anti-aging
properties 22, 23). Melatonin secretion declines progressively
with increasing age; menopause is time related with asubstantial, sharp decrease in melatonin secretion 23-26).
Taken together, these facts that melatonin production drops
significantly with age after puberty and that there is an
age-related increase in CML accumulation both suggest a
relation with many of the disturbances seen in the elderly.
Pathological inflammation is closely related to numerous
different diseases, however the current anti-inflammatory
drugs have significant side effects. Thus, it is important to
identify new pharmacological approaches to reduce chronic
inflammation without impairing the physiologic inflammatory
responses. In this study, we have focused on the effectof melatonin on CML-HSA-induced inflammatory gene
expression in RAW264.7 cells.

Materials and Methods
Materials

Melatonin was obtained from Sigma-Aldrich (cat #M5250, St. Louis, MO, USA) and dissolved into dimethyl
sulfoxide (DMSO, Wako, Osaka, Japan). DMSO was also
used as a vehicle control for treatments. CML-HSA
was obtained from (CircuLex, Nagano, Japan). All otherchemicals, of analytical grade, were obtained from SigmaAldrich or Wako.

Cell culture

Murine monocyte/macrophage RAW264.7 (ATCC ®
TIB-71 T M ) cells were purchased from American Type
Culture Collection (ATCC; Manassas, VA, USA). Cells weregrown in Dulbecco’s modified Eagle’s medium (DMEM;
Sigma-Aldrich, St. Louis, MO, USA) supplemented with
10% fetal bovine serum (FBS) (Nichirei Biosciences, Tokyo,
Japan), penicillin 100 units/mL, streptomycin 100 μg/mL
and amphotericin B 25 μg/mL at 37 °C under the condition
of 5% CO 2 12).

Cell viability assessment

assay were used. WST-8 assay was performed using Cell
Counting Kit-8 (CCK-8) according to the manufacturer’s
instructions (Dojindo, Kumamoto, Japan). LDH activity assay
was performed as described by Saito et al. 27). RAW264.7
cells were seeded at 2.5 × 10 4 cells/well in 96-well plates
and incubated for 24 hours, then cells were treated withmelatonin or DMSO for further 24 hours. For WST-8 assay,
after 2 hours treatment of CML-HSA, 10 μL/well CCK-8
was added and incubated for another 1 hour. Absorbance at
450 nm was measured by a spectrophotometer 13).
For LDH assay, cultured media (50 μL) from each well
was collected and used to determine the effect of CMLHSA and melatonin on cell cytotoxicity in RAW264.7 cells.

Isolation of total RNA and RT-PCR

RAW264.7 cells were seeded into 24-well plates at adensity of 1 × 10 5 cells/well and incubated for 24 hours,
and then, cells were pre-treated with melatonin or DMSO
for further 24 hours. Then, cells were stimulated using 0.5
μg/mL CML-HSA for 3 hours.
Total RNA was extracted using Isogen II reagent (Nippon
Gene, Toyama, Japan) according to the manufacturer’s
protocol. RNase-free DNase-treated total RNA (500 ng)
was reverse-transcribed with PrimeScript T M RT Master Mix(Takara Bio Inc., Japan) using Applied Biosystems 2720
Thermal cycler.
RT-PCR was performed with a Thunderbird T M SYBRqPCR mix (Toyobo Co., Ltd., Osaka, Japan) according to
manufacturer’s protocol with gene-specific primers as listed
in Table 1. Briefly, the amplification reactions were carriedout on a AB Applied Biosystems StepOnePlus Real-Time
PCR system with an initial hold step (95°C for 1 min) and40 cycles of PCR (95°C for 15 seconds, 60°C for 60 seconds),
followed by dissociation curve. The comparative CT method
was used to determine the amount of target, normalized
to an endogenous reference (GAPDH; glyceraldehyde-3phosphate dehydrogenase).

TNFα measurement by ELISA

Cells were pre-treated with melatonin or DMSO for 24hours, and then, medium was replaced with DMEM containing
0.5 μg/mL CML-HSA for 3 hours. TNFα concentration in
the cell culture medium was measured by ELISA (mouse
TNFα ELISA kit, ab100747; Abcam, Cambridge, United
Kingdom) as described by the manufacturer. Absorbancewas measured at 450 nm by a spectrophotometer. TNFαconcentrations were calculated using the standard curve
prepared by the recombinant mouse TNFα with known
concentrations (93.75 to 6,000 pg/mL).

Statistical analysis

Data were expressed as mean ± standard deviation (SD).
All statistical analysis were performed using Tukey-Kramer
test for intergroup comparison in all of the experiments.
Differences were considered significant at a significancelevel of 5%.

For determination of cell viability of RAW264.7
cells, WST-8 assay and lactate dehydrogenase (LDH)_ 14 2 _Glycative Stress Research

Table 1. Primers for RT-PCR

GeneSequence (5 → 3 )GAPDH
ForwardTGAAGGTCGGTGTGAACGGATTGGC

ReverseCATGTAGGCCATGAGGTCCACCAC

Forward

ACCCTCACACTCAGATCATCTTC

ReverseTGGTGGTTTGCTACGACGT

ForwardACAACCACGGCCTTCCCTACTT

ReverseCACGATTTCCCAGAGAACATGTG

ForwardCCAAGCCCTCACCTACTTCC

ReverseCTCTGAGGGCTGACACAAGG

ForwardTGTAATGAAAGACGGCACACC

ReverseTCTTCTTTGGGTATTGCTTGG

Forward

ACTACCGAGTCCGAGTCTACC

ReverseGTAGCTTCCCTCAGACACACA

28)

TNFα
29)

IL-6
29)

iNOS
29)

IL -1β
30)

RAGE
31)

RT-PCR, real-time polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TNF, tumor necrosis factor; IL,interleukin; iNOS, inducible nitric oxide synthase; RAGE, receptor for AGEs; AGEs, advanced glycation end products.

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Melatonin doesn’t Suppress CML-HSA-induced Inflammatory Gene Expression in RAW264.7 Cells

Results

Cell viability assessment

First, we analyzed the effect of melatonin and CMLHSA on cell viability in RAW264.7 cells. WST-8 assay
was performed to measure the percentage of viable cells
in response to melatonin and CML-HSA treatment for 24hours ( Fig. 1A). Also conditioned medium was collected tomeasure LDH released into the media from damaged cells( Fig. 1B ). Either 10 μg/mL melatonin or 0.5 μg/mL CMLHSA did not affect cell viability in RAW264.7 cells.

Effect of melatonin on CML-HSA-induced
inflammatory gene expression

Cells were pre-treated with melatonin as 10 & 0.1 μg/mL
or 2 μL/mL DMSO (vehicle control) for 24 hours, followed
by stimulation by 0.5 μg/mL CML-HSA for 3 hours. RNA
was extracted and performed RT-PCR analysis to measuremRNA expression of TNFα and condition medium wereused for ELISA to determine secreted TNFα. CML-HSA
induced both mRNA levels ( Fig. 2A) and secreted protein
levels into culture medium (Fig. 2B ). In this condition, pretreatment of 10 μg/mL melatonin lowered CML-HSAinduced TNFα mRNA expression compared to the vehicle
control (Fig. 2A), however, the secreted protein levels of
TNFα did not altered by melatonin ( Fig. 2B ). To further
investigation of inflammation responses, we performed RTPCR analysis against inflammatory genes such as iNOS, IL1β, IL-6. There was a trend of inducing inflammatory genes
by CML-HSA, however, these are not significant and notaltered by melatonin ( Fig. 3 ).

Inhibition of basal expression of inflammatory
gene by melatonin

In Figs. 2 and 3, melatonin did not show significant
inhibition of inflammatory genes in response to CML-HSA,
we further investigated that the effect of melatonin on basal
expression levels of inflammatory genes in RAW264.7 cells.
In case of without CML-HSA treatment, melatonin showeda trend to decrease basal mRNA levels of TNFα and IL-6
expression ( Figs. 4A and 4B ) and significantly inhibited
iNOS and IL-1β ( Figs. 4C and 4D ).

Neither CML-HSA nor melatonin did alter
expression of RAGE mRNA in RAW264.7 cells

RAGE is a well known receptor for AGEs. In this study,
we examined that the effect of CML-HSA and melatoninon RAGE expression. As shown in Figs. 5A and 5B, neither
CML-HSA nor melatonin did affect RAGE expression.

Discussion

AGEs can disrupt or change cellular functions by (a) theformation of cross-links between key molecules, permanently
altering cellular structure; and (b) activation of AGEs receptors
which causes upregulation of the transcription factor NF-K B

and its target genes 32). The engagement of AGEs with RAGE
triggers NADPH-oxidase, increasing intracellular oxidative
stress, evokes signaling pathways such as protein kinase C,
mitogen-activated protein kinase (MAPK) and the extracellularsignal-regulated kinase; it activates proinflammatorytranscription factor NF-K B and activator protein 1 (AP-1),
and subsequently induces thrombogenic, fibrogenic, adhesive,
chemoattractive and proinflammatory [i.e. TNFα, IL-1, IL6, matrix metalloproteinase (MMP), vascular endothelial
growth factor (VEGF)] gene expression 12, 33-36). IL-1 (α and β),IL-6 and TNF (α and β) induce macrophage differentiation
into osteoclast-like multinucleated cells and bone resorption
by increasing the bone-resorbing activity of formed osteoclasts
in vitro and in vivo 37-42).
TNFα was also reported to enhance the receptor activatorof nuclear factor kappa-B ligand (RANKL) signaling by
inducing the formation of osteoclastic cells in the presenceof low concentrations of RANKL from bone marrow
macrophages in vitro 40, 42), indicating that TNFα is a crucial
differentiation factor for osteoclasts. TNFα and M-CSF(macrophage colony-stimulating factor) play an importantrole in local osteolysis in chronic inflammatory diseases.
TNFα strongly induces differentiation of M-CSF-dependentbone marrow macrophage (MDBM) cells into mature
osteoclasts, TRAP-positive multinucleated cells (MNCs)
in the presence of M-CSF, and in the presence or absenceof RANKL 42). Diseases like rheumatoid arthritis, knee
osteoarthritis, locomotive syndrome, aseptic loosening,and periodontal diseases have been associated with theaccumulation of pro-inflammatory cytokines, TNFαand/or
IL-1 and IL-6, which likely facilitates local bone destruction
by stimulating osteoclast differentiation and activation 43-45).
Therefore, proinflammatory cytokine production is closely
related to bone diseases.
Proteins are usually glycated through their lysine residues
and in humans, histones in the cell nucleus are the richestin lysine, and therefore the formation and accumulation ofglycated protein CML is very common 46). Elevated levels of
CML and pentosidine in serum may be a cause of bone loss
due to disturbed bone remodeling in osteoporosis patients 47).
RAGE, when binding with AGEs, contributes to age- anddiabetes-related chronic inflammatory diseases suchas atherosclerosis, asthma, rheumatoid arthritis, knee
osteoarthritis, locomotive syndrome, osteoporosis, myocardial
infarction, nephropathy, retinopathy, periodontitis andneuropathy 7). Thus AGEs induce inflammatory gene
expression and these inflammatory cytokines subsequently
lead to destructive bone diseases. Thus, the inhibition of
inflammatory gene expression is of great importance inchronic inflammatory and destructive bone diseases.
Inducible nitric oxide synthase (iNOS) is a key enzyme
for generating NO (a free radical with an unpaired electron)as an immune defense mechanism by activated macrophages.
It is the proximate cause of septic shock and may function in
autoimmune disease. IRF1 and NF-K B-dependent activationof the inducible NOS promoter supports an inflammationmediated stimulation of this transcript. iNOS produces large
quantities of NO upon stimulation, such as by proinflammatory
cytokines (e.g. TNFα, IL -1 and interferon gamma) 48).
Pathologic generation of nitric oxide through increased iNOS
production may decrease tubal ciliary beats and smoothmuscle contractions and thus affect embryo transport, which
may consequently result in ectopic pregnancy 49, 50). Therefore,_ 14 4 _Glycative Stress Research

A

B
5

LDH release [fold change]

Cell viability [% control]

150

100

50

4

3

2

1

0

0
DMSO

0.1 µg/ml 10 µg/ml

DMSO

melatonin

0.1 µg/ml 10 µg/ml
melatonin

CML-HSACML-HSA

Fig. 1. Effect of CML-HSA and melatonin on cell viability in RAW264.7 cells.

Cells were pre-incubated with melatonin or DMSO for 24 hours, then stimulated with CML-HSA for further 3 hours. A. WST-8 assay was
performed to determine cell viability. B. Condition medium was used for LDH assay to assess dead cells. All data were shown as the mean
±standard deviation (n = 3) of the ratios against no addition. CML, carboxymethyl-lysine; HSA, human serum albumin; DMSO, dimethyl
sulfoxide; LDH, lactate dehydrogenase.

A

B
3500

＊＊

＊＊

＊＊

3000

Secreted TNFα [pg/ml]

Expression levels of TNFα
[Fold change]

5

4

3

2

1

2500
2000
1500
1000
500
0

0
DMSO

0.1 µg/ml 10 µg/ml

DMSO

0.1 µg/ml 10 µg/ml
melatonin

melatonin

CML-HSACML-HSA

Fig. 2. Effect of melatonin on CML-HSA induced TNFα gene expression.

Cells were pre-incubated with melatonin or DMSO for 24 hours, then stimulated with CML-HSA for further 3 hours. A. TNFα mRNAlevel was examined by RT-PCR. B. Secreted TNFα was determined using condition medium by ELISA. All data obtained were normalized
by GAPDH value and were shown as the mean ± standard deviation (n = 3) of the ratios against no addition. ** p < 0.01 vs. no addition by
Tukey-Kramer test. CML, carboxymethyl-lysine; HSA, human serum albumin; TNF, tumor necrosis factor; DMSO, dimethyl sulfoxide;GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

_ 145 _

Melatonin doesn’t Suppress CML-HSA-induced Inflammatory Gene Expression in RAW264.7 Cells

B
3

3

2.5

2.5

Expression levels of iNOS[Fold change]

Expression levels of IL-6
[Fold change]

A

2
1.5
1
0.5
0

2
1.5
1
0.5
0

DMSO

0.1 µg/ml

10 µg/ml

DMSO

0.1 µg/ml

melatonin

10 µg/ml

melatonin

CML-HSACML-HSAC

Expression levels of IL-1β[Fold change]

3
2.5
2
1.5
1
0.5
0
DMSO

0.1 µg/ml

10 µg/ml

melatonin

CML-HSA

Fig. 3. Effect of melatonin on CML-HSA-induced inflammatory genes expression in RAW264.7 cells.

Cells were pre-incubated with melatonin or DMSO for 24 hours, then stimulated with CML-HSA for further 3 hours. RT-PCR analyses were
performed for A. IL-6, B. iNOS and C. IL-1β. All data obtained were normalized by GAPDH value and were shown as the mean ± standard
deviation (n = 3) of the ratios against no addition. CML, carboxymethyl-lysine; HSA, human serum albumin; DMSO, dimethyl sulfoxide; IL,interleukin ; iNOS, inducible nitric oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

_ 14 6 _Glycative Stress Research

A

B
2

Expression levels of IL-6
[Fold change]

Expression levels of TNFα
[Fold change]

2

1.5

1

0.5

1.5

1

0.5

0

0

DMSO

0.1 µg/ml

10 µg/ml

DMSO

melatoninC

10 µg/ml

melatonin

D
2

Expression levels of IL-1β[Fold change]

2

Expression levels of iNOS[Fold change]

0.1 µg/ml

1.5

1

0.5

＊

1.5

1

0.5

＊

0

0
DMSO

0.1 µg/ml

10 µg/ml

DMSO

melatonin

0.1 µg/ml

10 µg/ml

melatonin

Fig. 4. Effect of melatonin on inflammatory genes expression in RAW264.7 cells.

Cells were incubated with melatonin or DMSO for 24 hours, then RNA was extracted and performed RT-PCR. A. TNFα, B. IL-6, C. iNOS
and D. IL-1β. All data obtained were normalized by GAPDH value and were shown as the mean ± standard deviation (n = 3) of the ratios
against no addition. * p < 0.05 vs. no addition by Tukey-Kramer test. DMSO, dimethyl sulfoxide; TNF, tumor necrosis factor; IL, interleukin; iNOS, inducible nitric oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase .

_ 147 _

Melatonin doesn’t Suppress CML-HSA-induced Inflammatory Gene Expression in RAW264.7 Cells

B
Expression levels of RAGE
[Fold change]

Expression levels of RAGE
[Fold change]

A
2

1.5

1

0.5

2

1.5

1

0.5

0

0
DMSO

0.1 µg/ml 10 µg/ml

DMSO

0.1 µg/ml 10 µg/ml
melatonin

melatonin

CML-HSA

Fig. 5. Effect of melatonin and CML-HSA on RAGE expression.

Cells were incubated with melatonin or DMSO for 24 hours (B), then stimulated CML-HSA for further 3 hours (A). RNA was extracted andperformed RT-PCR against RAGE. All data obtained were normalized by GAPDH value and were shown as the mean ± standard deviation (n= 3) of the ratios against no addition. CML, carboxymethyl-lysine; HSA, human serum albumin; RAGE, receptor for advanced glycation end
products; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase .

inhibition of TNFα, IL -1β, IL- 6, iNOS, and RAGE
expression pathway is of great importance to control chronic
inflammatory diseases. Anti-inflammatory medications
such as steroids have strong anti-inflammatory effects but
can cause undesirable side effects like adrenal suppression,
cataracts, and negative effects on bone metabolism.
Nonsteroidal anti-inflammatory drugs (NSAIDs) can cause
alterations in renal function, hepatic injury, and induce
gastric ulcer 51,52). Development of anti-inflammatory drugs
with a high efficacy and few side effects is necessary forthe treatment of acute and chronic inflammatory diseases
including rheumatoid arthritis, atherosclerosis, Alzheimer’s
disease, knee osteoarthritis, and locomotive syndrome 53).
Melatonin (N-acetyl-5-methoxytriptamine), a hormone
secreted by the pineal gland that is involved in the mediationof circadian rhythms, has anti-inflammatory effects produced
via decreased levels of prostaglandins, NO, and TNFα bothin vitro and in vivo 23, 54-58). Melatonin can also reduce theexpression of COX-2 and iNOS genes 56) and has minimal side
effects in humans 59). Melatonin and its derivatives, benzoylmelatonin and acetyl-melatonin, inhibited nitric oxide NO
and prostaglandin E2 production in lipopolysaccharide (LPS)stimulated RAW264.7 cells in a dose-dependent manner withhalf maximum inhibition concentration (IC 50) values lower
than those of melatonin 60).
In the present study, we investigated that the effect of
melatonin on inflammatory genes in RAW264.7 cells with orwithout glycated protein such as CML-HSA. CML-HSA
significantly stimulated RAW264.7 cells to produce TNFα

in all of our experimental conditions, but melatonin did notchange the TNFα mRNA level or protein content significantly
compared to the vehicle control group (DMSO 2 μL/mL) in
0.5 μg/mL CML-HSA stimulated RAW264.7 cells. Thoughmelatonin at 10 μg/mL lowered TNFα mRNA expression
slightly compared to the vehicle control group, the secreted
protein levels were the same ( Fig. 2 ). As shown in Fig. 3,
melatonin did not change the iNOS, IL-1β, or IL-6 mRNAlevel significantly compared to the vehicle control group(DMSO 2 μL/mL) in response to CML-HSA. As while,melatonin itself decreased basal mRNA expression levels of
iNOS and IL-1β ( Figs. 4C and 4D). None of the experimental
conditions significantly changed cell viability ( Figs. 1 and 2 )and expression of RAGE ( Fig. 5 ) in RAW264.7 cells.
Melatonin prevented inflammatory nitric oxide NO andprostaglandin E2 production in LPS-stimulated RAW264.7
cells in a dose-dependent manner with IC 50 values 1,500 μM,
and 840 μM, respectively 60) which is much higher than thepeak value of melatonin secretion in humans 0.516~0.775
nM 61, 62). In our experimental conditions we did not findany significant inhibition on inflammatory TNFα, RAGE,iNOS, IL-1β, IL-6 mRNA or TNFα protein expression byusing 0.1~10 μg/mL melatonin (approximately 0.43~43.05μM), 55,548 times higher concentration than the peak value
in humans. As melatonin is a sleep causing hormone andsuch a high concentration cannot reduce tested inflammatory
gene expression by CML-HSA induced RAW264.7 cells
likely indicates that melatonin has no direct effect on CMLmediated inflammatory gene expression by macrophage_ 14 8 _Glycative Stress Researchcells. Therefore, prevention and blockage of CML-modified
protein production, that is glycation, is of great importancefor the prevention of AGEs-mediated health complications.
Melatonin derivatives, benzoyl-melatonin and acetylmelatonin have much higher anti-inflammatory effects 60).
However, further studies will be necessary on the effects ofmelatonin derivatives on CML-HSA-induced inflammatory
gene expression. Also animal studies are necessary as
the immune system consists of many types of cellularinteractions.
In our previous study demonstrated that LPS stimulates
much higher inflammatory gene expression than CML in thesame RAW264.7 cell culture 12). Phiphatwatcharaded C et
al. reported that melatonin can prevent inflammatory gene
expression induced by LPS 60), but not by CML. These data
indicate that CML-HSA might activate different pathway
from LPS activates. Therefore, further studies are necessary
to elucidate the pathways in order to determine effective
blockers of CML-induced inflammatory gene expression.
As another study showed that several foods have potential
anti-glycative and anti-inflammatory activity, some plantmaterials can be used in the future to prevent CML productionin the human body and thereby lessen inflammatory gene
expression.

Conclusion

The present study showed that melatonin pre-treatment
does not have any inhibitory or stimulatory effect on CMLHSA-induced TNFα mRNA expression and protein secretionin RAW264.7 cells.

Acknowledgements

This work was supported by JSPS KAKENHI Grant
Number 26350917 and by the Japanese Council for Science,
Technology and Innovation, SIP (Project ID 14533567 ),“Technologies for creating next-generation agriculture,
forestry and fisheries” (funding agency: Bio-oriented
Technology Research Advancement Institution, NARO).

Conflict of Interest Statement

The authors state that the performance of this study
entailed no issues representing a conflict of interest.

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