Identification of anti-inflammatory target genes of Rhizoma coptidis extract in lipopolysaccharide-stimulated RAW264.7 murine macrophage-like cells.
OBJECTIVE
Rhizoma coptidis is used widely in traditional Oriental medicine to treat inflammatory diseases. The aim of this study was to identify the anti-inflammatory target genes of Rhizoma coptidis extract (CEX) in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophage-like cells.


METHODS
RAW264.7 cells were treated with CEX in the absence or presence of LPS for 6h, and changes in gene expression profiles were analyzed using oligonucleotide DNA microarrays. The results of microarray analysis were validated by semiquantitative reverse transcription-polymerase chain reaction. To confirm the anti-inflammatory activity of CEX, the concentrations of cytokines released into the media were measured by sandwich ELISA, NO production was assessed using the Griess reagent, and iNOS expression levels were determined using immunoblot analysis.


RESULTS
Microarray analysis revealed that activation of RAW264.7 cells with LPS elicited marked changes in mRNA expression of numerous genes known to be associated with inflammatory responses. Treatment of the cells with CEX suppressed the expression of various cytokines/chemokines, cell surface molecules, adhesion molecules, and growth factors. An ELISA also showed a decrease in the secretion of IL-1alpha, GM-CSF, and IL-6 but not of TNF-alpha. iNOS protein expression and NO production were also reduced by CEX treatment.


CONCLUSIONS
The data obtained in this study demonstrate that CEX exerts its anti-inflammatory effect by inhibiting the expression of various proinflammatory cytokines and cell surface molecules involved in inflammatory responses at the transcriptional level. These data support the traditional use of CEX as an anti-inflammatory agent and should provide useful information for the understanding of the pharmacological effects of CEX.
Kim
Jong Min
JM
Department of Bioscience & Biotechnology, Sejong University, Kwangjin-gu, Seoul, Republic of Korea.
Jung
Hyun Ah
HA
Choi
Jae Sue
JS
Lee
Na Gyong
NG
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology* / Q000633:toxicity; D015153:Blotting, Western; D002460:Cell Line; D039323:Coptis; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects; D018836:Inflammation Mediators / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010936:Plant Extracts / Q000494:pharmacology* / Q000633:toxicity; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027343:Rhizome; D013997:Time Factors
D000893:Anti-Inflammatory Agents; D016207:Cytokines; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D010936:Plant Extracts; D012333:RNA, Messenger; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse