kjpp The Korean Journal of Physiology & Pharmacology Korean J Physiol Pharmacol 1226-4512 2093-3827 Korean J Physiol Pharmacol 10.4196/kjpp.2011.15.1.23 kjpp-15-23 Original Article Microarray Analysis of Gene Expression Profiles in Response to Treatment with Melatonin in Lipopolysaccharide Activated RAW 264.7 Cells BanJu Yeon 1 ∗ KimBum Sik 2 ∗ KimSoo Cheol 2 KimDong Hwan 3 ChungJoo-Ho 4 Department of Pharmacology and Institute of Tissue Regeneration Engineering (ITREN), College of Dentistry, Dankook University, Cheonan 330-714, Korea Department of Thoracic and Cardiovascular Surgery, Kyung Hee University, Seoul 130-701, Korea Department of Physical Medicine and Rehabilitation, Kyung Hee University, Seoul 130-701, Korea Pharmacology and Kohwang Medical Research Institute, School of Medicine, Kyung Hee University, Seoul 130-701, Korea Corresponding to: Joo-Ho Chung, Department of Pharmacology, School of Medicine, Kyung Hee University, Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Korea. (Tel) 82-2-961-0303, (Fax) 82-2-968-0560, (E-mail) jhchung@khu.ac.kr

Equally contributed to first author.

 022011 18022011 15 1 23 29 03012011 08022011 08022011 Copyright © 2011 Korean J Physiol Pharmacol 2011 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract

Melatonin, which is the main product of the pineal gland, has well documented antioxidant and immune-modulatory effects. Macrophages produce molecules that are known to play roles in inflammatory responses. We conducted microarray analysis to evaluate the global gene expression profiles in response to treatment with melatonin in lipopolysaccharide (LPS) activated RAW 264.7 macrophage cells. In addition, eight genes were subjected to real-time reverse transcription polymerase chain reaction (RT-PCR) to confirm the results of the microarray. The cells were treated with LPS or melatonin plus LPS for 24 hr. LPS induced the up-regulation of 1073 genes and the down-regulation of 1144 genes when compared to the control group. Melatonin pretreatment of LPS-stimulated RAW 264.7 cells resulted in the down regulation of 241 genes and up regulation of 164 genes. Interestingly, among genes related to macrophage-mediated immunity, LPS increased the expression of seven genes (Adora2b, Fcgr2b, Cish, Cxcl10, Clec4n, Il1a, and Il1b) and decreased the expression of one gene (Clec4a3). These changes in expression were attenuated by melatonin. Furthermore, the results of real-time PCR were similar to those of the microarray. Taken together, these results suggest that melatonin may have a suppressive effect on LPS-induced expression of genes involved in the regulation of immunity and defense in RAW 264.7 macrophage cells. Moreover, these results may explain beneficial effects of melatonin in the treatment of various inflammatory conditions.

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Cytotoxicity of melatonin was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RAW 264.7 macrophage cells were exposed to melatonin at 0, 50, 100, and 500 μM in the absence or presence of LPS for 24 h. The results are presented as the mean±S.E.M. LPS, lipopolysaccharide.

The effects of melatonin on nitrite production. RAW 264.7 cells were pretreated with 50, 100, and 500 μM of melatonin and then treated with lipopolysaccharides (1.0 μg/ml). The media were harvested 24 h later and assayed for nitrite production. Data are the means±S.E.M. LPS, lipopolysaccharide; Mel, melatonin. ##p< 0.01 compared to control, ∗∗p<0.01 compared to LPS.

Functional analysis of genes selected using the Panther database. (A) Functional categories of genes regulated by LPS treatment in RAW 264.7 macrophage cells. (B) Functional categories of genes regulated by melatonin pretreatment in LPS-stimulated RAW 264.7 macrophage cells.

Validation of microarray results using real-time RT-PCR. Relative expression of each gene in the control was designated as 1, values are mean±S.E.M. LPS, lipopolysaccharide; Mel, melatonin. #p<0.05, ##p<0.01 compared to control, ∗p<0.05, ∗∗p<0.01 compared to LPS.

Primer sequences of genes applied in real-time RT-PCR

Gene Primers
Adora2b Sense: 5′- CTATGCCTACAGGAACCGAGACT –3′
  Antisense: 5′- GTCAGCCAGACTTGTGTAACTCC-3′
Cish Sense: 5′- GTACAGGGATCTTGTCCTTTGC-3′
  Antisense: 5′- GGCTGTAATAGAACCCCAGTACC-3′
Clec4a3 Sense: 5′- ACTCCTCAGACATCGACACAGAC-3′
  Antisense: 5′- ACAGCTCCAGACTTTGTCTTCC-3′
Clec4n Sense: 5′- GTCCCTGAGTCGTATTTTGGAG-3′
  Antisense: 5′-CTGACACCATAGTCCCTTCACTG-3′
Cxcl10 Sense: 5′-CTCTCTCCATCACTCCCCTTTAC –3′
  Antisense: 5′-ACTTAGAACTGACGAGCCTGAGC –3′
Fcgr2b Sense: 5′-GAACTCTTCTACCCAGTGGTTCC –3′
  Antisense: 5′-GCAGTAGTAGTCCCCACTGTGAC –3′
Il1a Sense: 5′- CCAGATCAGCACCTTACACCTAC-3′
  Antisense: 5′-AGGTCGGTCTCACTACCTGTGAT –3′
Il1b Sense: 5′-CCTAAAGTATGGGCTGGACTGT –3′
  Antisense: 5′-CTAGAGAGTGCTGCCTAATGTCC-3′

List of “macrophage-mediated immunity” genes regulated by lipopolysaccharide treatment in RAW 264.7 macrophage cells

Gene symbol Gene name Fold change
S100a4 S100 calcium binding protein A4 -8.88
Ltc4s Leukotriene C4 synthase -6.83
Clec4a3 C-type lectin domain family 4, member a3 -2.83
Clec10a C-type lectin domain family 10, member A -2.38
Alox5ap Arachidonate 5-lipoxygenase activating protein -2.23
Lgals3bp Lectin, galactoside-binding, soluble, 3 binding protein -2.1
Adora2b Adenosine A2b receptor 2.17
Ddt D-dopachrome tautomerase 2.75
Clec4d C-type lectin domain family 4, member d 3
Gbp1 Guanylate binding protein 1 3.07
Il10 Interleukin 10 3.24
Fcgr2b Fc receptor, IgG, low affinity IIb 3.54
Gbp2 Guanylate binding protein 2 4.27
Gbp3 Guanylate binding protein 3 4.4
Clec4e C-type lectin domain family 4, member e 5.9
Cish Cytokine inducible SH2-containing protein 8.19
Cxcl10 Chemokine (C-X-C motif) ligand 10 10.5
Clec4n C-type lectin domain family 4, member n 12.03
S100a8 S100 calcium binding protein A8 (calgranulin A) 41.32
Il1a Interleukin 1 alpha 76.72
Il1b Interleukin 1 beta 148.59
Cxcl2 Chemokine (C-X-C motif) ligand 2 328.7

List of “macrophage-mediated immunity” genes regulated by melatonin pretreatment in lipopolysaccharide-stimulated RAW 264.7 macrophage cells

Gene symbol Gene name Fold change
Cxcl10 Chemokine (C-X-C motif) ligand 10 -7.54
Il1b Interleukin 1 beta -3.87
Adora2b Adenosine A2b receptor -3.13
Fcgr2b Fc receptor, IgG, low affinity IIb -3
Il1a Interleukin 1 alpha -2.73
Clec4n C-type lectin domain family 4, member n -2.61
Cish Cytokine inducible SH2-containing protein -2.46
Colec12 Collectin sub-family member 12 2.21
Cxcl4 Chemokine (C-X-C motif) ligand 4 2.22
Clec4a3 C-type lectin domain family 4, member a3 2.5