γ-Mangostin inhibits inhibitor-κB kinase activity and decreases lipopolysaccharide-induced cyclooxygenase-2 gene expression in C6 rat glioma cells
γ-Mangostin inhibits inhibitor-κB kinase activity and decreases lipopolysaccharide-induced cyclooxygenase-2 gene expression in C6 rat glioma cells
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DOI：
10.1124/mol.104.002626
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发表时间：
2004-09-01
影响因子：
3.6
通讯作者：
Ohizumi, Y
中科院分区：
文献类型：
--
作者：
Nakatani, K;Yamakuni, T;Ohizumi, Y
我们研究了从药用植物山竹果的果实船体中纯化的γ-mangostin对C6大鼠胶质瘤细胞自发性前列腺素E-2（PGE（2））释放和诱导型环氧合酶2（考克斯-2）基因表达的影响。用γ-mangostin处理18小时，以浓度依赖性方式有效抑制PGE（2）的自发释放，IC 50值约为2 μ M，即使在30 μ M时也不影响细胞活力。通过免疫印迹和逆转录聚合酶链反应，我们发现，γ-mangostin浓度依赖性抑制脂多糖（LPS）诱导的考克斯-2蛋白及其mRNA的表达，但不是那些组成性考克斯-1环氧合酶。因为已知LPS刺激抑制剂kappaB（IkappaB）激酶（IKK）介导的IkappaB磷酸化，随后其降解，这又诱导核因子（NF）-kappaB核转位，导致考克斯-2基因的转录激活，所以检查了γ-芒果毒素对控制NF-kappaB激活的IKK/IkappaB级联的影响。使用从C6细胞提取物免疫沉淀的IKK蛋白的体外IKK测定显示，该化合物以浓度依赖性方式抑制IKK活性，IC 50值约为10 μ M。通过免疫印迹法测定，还观察到一致性γ-倒捻子素以浓度依赖性方式降低LPS诱导的IkappaB降解和磷酸化。此外，荧光素酶报告基因分析表明，γ-mangostin减少了LPS诱导的NF-κ B和人考克斯-2基因启动子区域依赖性转录的激活。γ-Mangostin还抑制大鼠角叉菜胶诱导的足水肿。这些结果表明，γ-mangostin直接抑制IKK活性，从而阻止NF-κ B靶基因考克斯-2基因的转录，可能减少体内炎症因子刺激的PGE（2）产生，是一种新的有用的抗炎药物开发的先导化合物。
We investigated the effect of gamma-mangostin purified from the fruit hull of the medicinal plant Garcinia mangostana on spontaneous prostaglandin E-2 (PGE(2)) release and inducible cyclooxygenase 2 (COX-2) gene expression in C6 rat glioma cells. An 18-h treatment with gamma-mangostin potently inhibited spontaneous PGE(2) release in a concentration-dependent manner with the IC50 value of approximately 2 muM, without affecting the cell viability even at 30 muM. By immunoblotting and reverse-transcription polymerase chain reaction, we showed that gamma-mangostin concentration-dependently inhibited lipopolysaccharide (LPS)-induced expression of COX-2 protein and its mRNA, but not those of constitutive COX-1 cyclooxygenase. Because LPS is known to stimulate inhibitor kappaB (IkappaB) kinase (IKK)-mediated phosphorylation of IkappaB followed by its degradation, which in turn induces nuclear factor (NF)-kappaB nuclear translocation leading to transcriptional activation of COX-2 gene, the effect of gamma-mangostin on the IKK/IkappaB cascade controlling the NF-kappaB activation was examined. An in vitro IKK assay using IKK protein immunoprecipitated from C6 cell extract showed that this compound inhibited IKK activity in a concentration-dependent manner, with the IC50 value of approximately 10 muM. Consistently gamma-mangostin was also observed to decrease the LPS-induced IkappaB degradation and phosphorylation in a concentration-dependent manner, as assayed by immunoblotting. Furthermore, luciferase reporter assays showed that gamma-mangostin reduced the LPS-inducible activation of NF-kappaB - and human COX-2 gene promoter region-dependent transcription. gamma-Mangostin also inhibited rat carrageenan-induced paw edema. These results suggest that gamma-mangostin directly inhibits IKK activity and thereby prevents COX-2 gene transcription, an NF-kappaB target gene, probably to decrease the inflammatory agent-stimulated PGE(2) production in vivo, and is a new useful lead compound for anti-inflammatory drug development.