| 摘要: | 過度活化的巨噬細胞會釋出大量的發炎因子，包括一氧化氮 (NO) 及細胞激素 (TNF-?恁AIL-12，IFN-?珛?)。文獻指出，大量被誘導型一氧化氮合成酶 (iNOS, type II) 生成的NO，會造成許多臨床上的疾病，包括多重硬化症、敗血性休克、阿茲海默症等。本研究發現9-phenoxyacridine之衍生合成藥物 (CYL-418b) 在LPS所活化的巨噬細胞 (RAW 264.7) 中，可以有效的抑制NO的生成。CYL-418b抑制NO的生成，主要是經由抑制iNOS的轉錄及其mRNA穩定性，但不影響iNOS的酵素活性及MAPKs (ERK、JNK與p38) 的磷酸化。此藥物除了可以顯著抑制iNOS的基因表現之外，還可以抑制LPS活化RAW 264.7細胞所釋放的COX-2、IFN-?狺垌NF-?恁A但對IL-10的基因表現並沒有影響。為進一步探討CYL-418b如何抑制基因轉錄活性，本研究分別利用EMSA、Lusiferase assay及Western blotting等方法，檢測會影響這些發炎因子基因表現的轉錄因子活性是否受藥物影響。結果顯示此藥物不會藉由影響AP-1、NF-?羠及ISRE之轉錄活性而抑制LPS活化iNOS基因轉錄。然而，CRE與轉錄因子之間的結合及轉錄活性卻顯著被抑制。由此推測CYL-418b會經由抑制CREB/ATF2之活性，進而影響了LPS刺激巨噬細胞所產生的發炎因子。
Proinflammatory factors, such as nitric oxide (NO) and cytokines (TNF-??, IL-12, IFN-??, …etc.) can be induced by over activated macrophages. Recent studies have shown that large amount of NO produced by inducible NOS (iNOS, type Ⅱ) is associated with the incidence of several diseases, such as multiple sclerosis, septic shock and Alzheimer’s disease. In this study, we have found that a synthetic derivative of 9-phenoxyacridine, CYL-418b, can effectively block LPS-induced NO, COX-2, and TNF-?? production in murine macrophages (RAW 264.7). The inhibition of NO produced by CYL-418b was mediated by significant reduction of iNOS protein level, mRNA synthesis and stability, but not by inactivating iNOS enzyme and the phosphorylation of MAPK (ERK, JNK and p38). Our results demonstrated that CYL-418b not only could reduce iNOS mRNA expression, but also reduce COX-2, TNF-?? and IFN-?? mRNA expression. However, IL-10 mRNA expression was not affected. We performed EMSA, luciferase reporter assay and western blotting to further investigate how CYL-418b suppressed these inflammatory factors at the transcription level. Our results elucidated that the inhibitory effects of CYL-418b were through CRE-mediated regulatory activity, but not through the activities of NF-?羠, AP-1 and ISRE. We suggest that CYL-418b affects the expression of inflammatory factors by decreasing mRNA stability and CREB/ATF2 regulatory activity. |
