National Research Institute of Chinese Medicine, Taipei, Taiwan.
Eur J Pharmacol. 2011 Jan 10;650(1):102-9. doi: 10.1016/j.ejphar.2010.10.004. Epub 2010 Oct 14.
Psoralidin has been reported to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production, but the mechanisms of the action remain unclear. Thus, the impact of psoralidin on signaling pathways known to be implicated in NO synthesis was explored in LPS-activated RAW264.7 macrophages by using RT-PCR and Western blotting. Consistent with NO inhibition, psoralidin suppressed LPS-induced expression of inducible NO synthase (iNOS) by abolishing IκB kinase (IKK) phosphorylation, IκB degradation and nuclear factor κB (NF-κB) nuclear translocation without effecting mitogen-activated protein kinases (MAPKs) phosphorylation. Exposure to wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression, suggesting activation of such a signal pathway might also be phosphoinositide-3-kinase (PI3K) dependent. By using Src inhibitor PP2, Janus kinase 2 (JAK-2) inhibitor AG490, Bruton's tyrosine kinase (Btk) inhibitor LFM-A13 and spleen tyrosine kinase (Syk) inhibitor piceatannol, the results showed that piceatannol clearly repressed NO production more potently than the other inhibitors. Furthermore, piceatannol significantly repressed LPS-induced PI3K/Akt phosphorylation and the downstream IKK/IκB activation, suggesting that Syk is an upstream key regulator in the activation of PI3K/Akt-mediated signaling. In fact, transfection with siRNA targeting Syk obviously reduced iNOS expression. Interestingly, LPS-induced phosphorylations of Syk and PI3K-p85 were both significantly blunted by psoralidin treatment. The present results show that interfering with Syk-mediated PI3K phosphorylation might contribute to the NO inhibitory effect of psoralidin via blocking IKK/IκB signaling propagation in LPS-stimulated RAW 264.7 macrophages.
补骨脂素已被报道可抑制脂多糖（LPS）诱导的一氧化氮（NO）产生，但作用机制尚不清楚。因此，本研究在 LPS 激活的 RAW264.7 巨噬细胞中通过 RT-PCR 和 Western blot 技术探索了补骨脂素对已知参与 NO 合成的信号通路的影响。与抑制 NO 生成一致，补骨脂素通过消除 IκB 激酶（IKK）磷酸化、IκB 降解和核因子 κB（NF-κB）核易位来抑制 LPS 诱导的诱导型一氧化氮合酶（iNOS）的表达，而不影响丝裂原活化蛋白激酶（MAPKs）磷酸化。wortmannin 的暴露消除了 IKK/IκB/NF-κB 介导的 iNOS 表达，表明这种信号通路的激活也可能依赖于磷酸肌醇 3-激酶（PI3K）。使用Src 抑制剂 PP2、Janus 激酶 2（JAK-2）抑制剂 AG490、Btk 抑制剂 LFM-A13 和脾酪氨酸激酶（Syk）抑制剂白藜芦醇，结果表明白藜芦醇比其他抑制剂更明显地抑制 NO 生成。此外，白藜芦醇显著抑制 LPS 诱导的 PI3K/Akt 磷酸化和下游 IKK/IκB 激活，表明 Syk 是激活 PI3K/Akt 介导信号的上游关键调节因子。事实上，用靶向 Syk 的 siRNA 转染明显降低了 iNOS 的表达。有趣的是，补骨脂素处理明显减弱了 LPS 诱导的 Syk 和 PI3K-p85 的磷酸化。本研究结果表明，干扰 Syk 介导的 PI3K 磷酸化可能通过阻断 LPS 刺激的 RAW264.7 巨噬细胞中 IKK/IκB 信号转导，导致补骨脂素的 NO 抑制作用。
