Whan Han J, Gon Lee B, Kee Kim Y, Woo Yoon J, Kyoung Jin H, Hong S, Young Lee H, Ro Lee K, Woo Lee H
College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea.
Br J Pharmacol. 2001 Jun;133(4):503-12. doi: 10.1038/sj.bjp.0704099.
We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) by ergolide, sesquiterpene lactone from Inula britannica. iNOS activity in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS. Ergolide markedly decreased the production of prostaglandin E(2) (PGE(2)) in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner, without alteration of the catalytic activity of COX-2 itself. Ergolide decreased the level of iNOS and COX-2 protein, and iNOS mRNA caused by stimulation of LPS/IFN-gamma in a concentration-dependent manner, as measured by Western blot and Northern blot analysis, respectively. Ergolide inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF-kappaB as well as IkappaB-alpha degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF-kappaB itself or an upstream molecule of NF-kappaB. Ergolide also directly inhibited the DNA-binding activity of active NF-kappaB in LPS/IFN-gamma-pretreated RAW 264.7 macrophages. These results demonstrate that the suppression of NF-kappaB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF-kappaB resulted from blockade of the degradation of IkappaB and the direct modification of active NF-kappaB, leading to the suppression of the expression of iNOS and COX-2, which play important roles in inflammatory signalling pathway.
我们研究了旋覆花中的倍半萜内酯麦角灵抑制诱导型一氧化氮合酶（iNOS）和环氧化酶-2（COX-2）的机制。用麦角灵处理可显著减弱脂多糖（LPS）/γ干扰素（IFN-γ）刺激的RAW 264.7巨噬细胞无细胞提取物中的iNOS活性。其对iNOS的抑制作用与LPS/IFN-γ刺激的RAW 264.7巨噬细胞培养基中亚硝酸盐积累的减少呈浓度依赖性平行关系。然而，其抑制作用并非源于对一氧化氮合酶催化活性的直接抑制。麦角灵以浓度依赖性方式显著降低LPS/IFN-γ刺激的RAW 264.7巨噬细胞无细胞提取物中前列腺素E2（PGE2）的产生，而不改变COX-2自身的催化活性。通过蛋白质免疫印迹法（Western blot）和Northern印迹法分别检测发现，麦角灵以浓度依赖性方式降低LPS/IFN-γ刺激引起的iNOS和COX-2蛋白水平以及iNOS mRNA水平。麦角灵抑制核因子-κB（NF-κB）激活，NF-κB是响应LPS/IFN-γ时iNOS和COX-2表达所必需的转录因子。这种作用伴随着NF-κB亚基p65核转位的平行减少以及IκB-α降解。此外，用半胱氨酸处理可完全阻断这些作用，表明麦角灵的这种抑制作用可能是通过NF-κB自身或NF-κB上游分子的烷基化介导的。麦角灵还直接抑制LPS/IFN-γ预处理的RAW 264.7巨噬细胞中活性NF-κB的DNA结合活性。这些结果表明，麦角灵对NF-κB激活的抑制作用可能归因于对IκB降解的阻断导致NF-κB核转位的抑制以及对活性NF-κB的直接修饰，从而导致在炎症信号通路中起重要作用的iNOS和COX-2表达受到抑制。
