Both high level of nitric oxide (NO) and its generating enzyme, inducible NO synthase (iNOS), play important roles in pathophysiological conditions such as inflammatory processes. We previously found that 1,3,5-trihydroxy-4-prenylxanthone (TH-4-PX) isolated from Cudrania cochinchinensis repressed lipopolysaccharide (LPS)-induced NO production in RAW264.7 macrophages. Here we further examined the underlying mechanisms using RT-PCR and Western blot analyses. Consistent with NO inhibition, suppression of LPS-induced iNOS expression by TH-4-PX through abolishing IkappaB kinase (IKK) phosphorylation, IkappaB degradation and nuclear factor-kappaB (NF-kappaB) nuclear translocation was observed. After LPS stimulation, the increased nuclear level of c-Fos and c-Jun (major components of activator protein-1, AP-1) and the phosphorylated level of upstream signal molecules, such as c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase, (ERK) were all significantly suppressed by TH-4-PX, while p38 remained unaffected. A further experiment revealed that TH-4-PX inhibited the phosphorylation of transforming growth factor-beta (TGF-beta)-activated kinase 1 (TAK1), an upstream signaling molecule required for IKK and mitogen-activated protein kinases (MAPKs) activation. Stimulation with LPS also triggered the modification (phosphorylation and ubiquitination) and eventually the proteasomal degradation of membrane-associated interleukin (IL)-1 receptor-associated serine/threonine kinase 1 (IRAK-1), an essential signaling component to toll-like receptor (TLR)-mediated TAK-1 activation. Interestingly, the modified pattern of IRAK-1 in the presence LPS was significantly attenuated by TH-4-PX treatment. In conclusion, TH-4-PX inhibited LPS-induced NF-kappaB and AP-1 activations by interfering with the posttranslational modification (phosphorylation and/or ubiquitinylation) of IRAK-1 in the cell membrane to impede TAK1-mediated activation of IKK and MAPKs signal transduction.

中文翻译：

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1,3,5-三羟基-4-异戊烯基蒽酮通过阻止IRAK-1的翻译后修饰来抑制脂多糖诱导的iNOS表达。

高水平的一氧化氮（NO）及其生成酶（可诱导型NO合酶（iNOS））在病理生理状况（例如炎症过程）中起着重要作用。我们先前发现，从中华dra中分离到的1,3,5-三羟基-4-异戊基蒽酮（TH-4-PX）抑制了脂多糖（LPS）诱导的RAW264.7巨噬细胞中NO的产生。在这里，我们使用RT-PCR和Western blot分析进一步研究了潜在的机制。与NO抑制一致，通过消除IkappaB激酶（IKK）磷酸化，IkappaB降解和核因子-kappaB（NF-kappaB）核易位，抑制了TH-4-PX对LPS诱导的iNOS表达的抑制。LPS刺激后，c-Fos和c-Jun（激活蛋白1的主要成分，AP-1）和上游信号分子（例如c-Jun NH2末端激酶（JNK）和细胞外信号调节激酶（ERK））的磷酸化水平均被TH-4-PX显着抑制，而p38则不受影响。进一步的实验表明，TH-4-PX抑制了转化生长因子-β（TGF-β）激活的激酶1（TAK1）的磷酸化，这是IKK和有丝分裂原激活的蛋白激酶（MAPKs）激活所需的上游信号分子。用LPS刺激还会触发修饰（磷酸化和泛素化），并最终引起膜相关白介素（IL）-1受体相关丝氨酸/苏氨酸激酶1（IRAK-1）的蛋白酶体降解，IRAK-1是通行费样受体的重要信号成分。 （TLR）介导的TAK-1激活。有趣的是，TH-4-PX处理可显着减弱存在LPS时IRAK-1的修饰模式。总之，TH-4-PX通过干扰细胞膜中IRAK-1的翻译后修饰（磷酸化和/或泛素化）来抑制LPS诱导的NF-κB和AP-1活化，从而阻止TAK1介导的IKK和AP-1活化。 MAPKs信号转导。