Aristolochic Acid Inhibits iNOS Gene Expression Induced by LPS or IFN-gamma in RAW 264.7 Macrophages

• Main Authors: Ming-Chao, 劉銘超
• Other Authors: 劉秉慧 教授
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/72786039528335604811

碩士 === 中山醫學大學 === 生物醫學科學學系碩士班 === 98 === Aristolochic Acid (AA), a group of natural compound widely found in Artistolochia species, is composed of AAI and AAII. AA I in the herbal medicine is found to be nephrotoxic and carcinogenic to human. To study the immunosuppressive ability of AAI, lipopolysaccharide (LPS) or Interferon gamma (IFN-γ) -stimulated RAW 264.7 macrophage cells were used as a model to examine the effects of AAI on the expression of the inducible nitric oxide synthase (iNOS) gene. When RAW 264.7 macrophages were treated with LPS (50 ng/ml) or IFN-γ (10ng/ml) for 18 h, the levels of nitric oxide (NO), iNOS protein and iNOS mRNA expression were all significantly increased. However, the presence of AAI (30-100μM) down-regulated the expression of LPS/IFN-γ-induced NO, iNOS protein and mRNA in a dose-dependent manner. To confirm whether AAI was able to decrease iNOS gene expression at the transcription level, we constructed a series of luciferase reporter plasmids which contained various promoter regions (-1588 to +121) of iNOS gene. AAI was found to inhibit the LPS/IFN-γ-induced iNOS expression by modulating the nuclear factor-κB (NF-κB) binding element located at nucleotides −86 to −76. The results of electrophoretic gel mobility shift assay (EMSA) also supported the inhibitory effect of AAI on the DNA binding activity of NF-κB. In addition, Western blotting demonstrated LPS-induced I-κB phosphorylation was significantly suppressed by AAI. Treatment of RAW 264.7 with AAI also down-regulated the LPS-induction of TNF-α and IL-6, two NF-κB regulated genes. Furthermore, the exposure of transient transfectant to AAI did not affect the luciferase activities of reporter construct that contained iNOS mRNA 3''-UTR, indicating that AAI does not inhibit iNOS gene expression at the post transcriptional level. Taken together, the data herein suggest that in activated RAW 264.7 macrophages, AAI regulates iNOS gene expression at the transcriptional level, and inhibition of NF-κB activation may be associated with the immunomodulatory effect of AAI.