Inhibition of LPS-Induced Cellular Inflammation by Phytotoxin Aristolochic Acid

• Main Authors: I-Tzu, 陳怡孜
• Other Authors: Biing-Hui Liu
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/09225779948415073428

碩士 === 中山醫學大學 === 生物醫學科學學系碩士班 === 96 === Aristolochic acid (AA) is a group of natural compounds widely found in Artistolochia species and is mainly composed of aristolochic acid I (AAI) and aristolochic acid II (AAII). Recently AA is found to be nephrotoxic and carcinogenic to humans and rodents. However, the extract of Artistolochia serving as Chinese herbal medicine has been commonly used to treat arthritis and rheumatism for hundreds of years. In addition, AA compound has the ability to increase phagocytic activity of phagocytes and has potential anti-inflammatory properties. In the present study, we focused on the anti-inflammatory effect of AA on the rat cell line (RAW 264.7) and further studied its reaction mechanism. Lipopolysaccharide (LPS) was used to stimulate RAW 264.7 macrophages to generate a cellular inflammation model. When RAW 264.7 macrophages were treated with LPS, the levels of nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) mRNA and protein expression were all increased. Co-treatment of RAW 264.7 macrophages with LPS and various concentrations of AA led to the down-regulation of LPS-induced NO, iNOS protein and mRNA levels in a concentration-dependent manner. Since AA above 100 μM showed no significant effect on the cellular viability of RAW 264.7, the AA concentration we used can efficiently inhibit LPS-induced cellular inflammation without showing cellular toxicity. The induction of iNOS gene expression is known to be correlated with the regulation of various transcription factors, so both in vitro and in vivo assays were conducted to investigate if AA can affect the DNA binding activity of these transcription factors. By the in vitro Electrophoretic Mobility Shift Assays (EMSA), we found that exposure of LPS-treated cells to AA did not block the NF-κB DNA binding activity mediated by LPS. By using Luciferase reporter assay in the cellular experiment, we observed that the AA treatment did not alter the luciferase activities of reporter plasmids harboring NF-κB, AP-1, ISRE or GAS DNA binding sequences in front of luciferase gene. Besides, the presence of AA showed no influence on the status of LPS-induced STAT-1 phosphorylation and did not significantly inhibit the translocation of NF-κB from cytoplasm to nucleus. However, high concentrations of AA could obviously suppress the phosphorylation of I-κB induced by LPS; I-κB protein is known to be an inhibitor of NF-κB. To further confirm that AA is able to decrease iNOS gene transcription, three luciferase reporter plasmids containing 1588 bp, 1008 bp or 360 bp iNOS promoter sequence from the starting site were constructed. The results demonstrated that AA could apparently decrease the LPS-induced luciferase activities of all the constructed plasmids, suggesting that the inhibitory effect of AA may be associated with an unknown DNA sequence within the 360 bp iNOS promoter.