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

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

• Ban JY
• Kim BS
• Kim SC
• Kim DH
• Chung JH

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.

Keyword

Macrophages; Melatonin; Microarray; Lipopolysaccharide

MeSH Terms

Down-Regulation
Gene Expression
Macrophages
Melatonin
Microarray Analysis
Pineal Gland
Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Reverse Transcription
Transcriptome
Up-Regulation
Melatonin

Figure

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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.

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  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.

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  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.

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  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.

Reference

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