Microarray Analysis of Gene Expression Profiles in Response to Treatment with Melatonin in Lipopolysaccharide Activated RAW 264.7 Cells

Ju Yeon Ban; Bum Sik Kim; Soo Cheol Kim; Dong Hwan Kim; Joo-Ho Chung

doi:10.4196/kjpp.2011.15.1.23

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Ban, Kim, Kim, Kim, and Chung: Microarray Analysis of Gene Expression Profiles in Response to Treatment with Melatonin in Lipopolysaccharide Activated RAW 264.7 Cells

Original Article

Korean J Physiol Pharmacol 2011;15(1):23-29.

Published online: 18 February 2011

DOI: https://doi.org/10.4196/kjpp.2011.15.1.23

Microarray Analysis of Gene Expression Profiles in Response to Treatment with Melatonin in Lipopolysaccharide Activated RAW 264.7 Cells

Ju Yeon Ban^1,^∗, Bum Sik Kim^2,^∗, Soo Cheol Kim², Dong Hwan Kim³, Joo-Ho Chung^4,

¹Department of Pharmacology and Institute of Tissue Regeneration Engineering (ITREN), College of Dentistry, Dankook University, Cheonan 330-714, Korea

²Department of Thoracic and Cardiovascular Surgery, Kyung Hee University, Seoul 130-701, Korea

³Department of Physical Medicine and Rehabilitation, Kyung Hee University, Seoul 130-701, Korea

⁴Pharmacology 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.

Received 3 January 2011 Revised 8 February 2011 Accepted 8 February 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.

Keywords: Macrophages, Melatonin, Microarray, Lipopolysaccharide

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Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Table 1.

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′

Table 2.

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

Table 3.

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

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