Division of Nephrology, Department of Internal Medicine, Asan Medical Center, University of Ulsan, Seoul, Republic of Korea.
Cell Biol Int. 2013 Jan;37(1):19-28. doi: 10.1002/cbin.10003. Epub 2012 Dec 4.
Atherosclerosis develops from cascades of inflammatory processes. Spleen tyrosine kinase (Syk) and monocyte chemotatic protein-1 (MCP-1) play important roles in the pathogenesis of atherosclerosis. Mycophenolic acid (MPA) has an anti-inflammatory effect. We have investigated whether MPA regulates Syk to repress tumour necrosis factor-α (TNF-α)-induced MCP-1 production in cultured human aortic endothelial cells. Expression of MCP-1 mRNA and its protein were measured by real time RT-PCR and ELISA, respectively. Reactive oxygen species (ROS) production were measured using 2'7'-dichlorofluorescein diacetate. Activation of AP-1 and NF-κB were assessed by electrophoretic mobility shift assay. Tyrosine phosphorylation of Syk was examined by Western blot analysis. TNF-α increased MCP-1 at both mRNA and protein levels. TNF-α-induced MCP-1 mRNA expression was inhibited by N-acetylcysteine (NAC), Syk inhibitor, Syk-siRNA and MPA. TNF-α-induced MCP-1 protein production was also inhibited by Syk inhibitor and MPA. TNF-α increased DNA binding activity of AP-1 and NF-κB, whereas both AP-1 and NF-κB decoy oligodeoxynucleotides downregulated TNF-α-induced MCP-1 mRNA expression. TNF-α increased ROS generation, which was inhibited by NAC and MPA, but not by Syk inhibitor. TNF-α increased tyrosine phosphorylation of Syk, which was attenuated by NAC and MPA. MPA and Syk inhibitor attenuated TNF-α-induced DNA binding activity of NF-κB and AP-1. TNF-α induced MCP-1 expression via activation of AP-1 and NF-κB. AP-1 and NF-κB were mediated through ROS, followed by Syk. MPA exerts anti-inflammatory effect by inhibiting MCP-1 expression via suppression of ROS and Syk.
动脉粥样硬化是由一系列炎症过程发展而来的。脾酪氨酸激酶（Syk）和单核细胞趋化蛋白-1（MCP-1）在动脉粥样硬化的发病机制中起重要作用。霉酚酸（MPA）具有抗炎作用。我们研究了 MPA 是否通过调节 Syk 来抑制肿瘤坏死因子-α（TNF-α）诱导的培养的人主动脉内皮细胞中 MCP-1 的产生。通过实时 RT-PCR 和 ELISA 分别测量 MCP-1 mRNA 和其蛋白的表达。使用 2'7'-二氯荧光素二乙酸酯测量活性氧（ROS）的产生。通过电泳迁移率变动分析评估 AP-1 和 NF-κB 的激活。通过 Western blot 分析检查 Syk 的酪氨酸磷酸化。TNF-α增加了 MCP-1 的 mRNA 和蛋白水平。N-乙酰半胱氨酸（NAC）、Syk 抑制剂、Syk-siRNA 和 MPA 抑制了 TNF-α诱导的 MCP-1 mRNA 表达。TNF-α诱导的 MCP-1 蛋白产生也被 Syk 抑制剂和 MPA 抑制。TNF-α增加了 AP-1 和 NF-κB 的 DNA 结合活性，而 AP-1 和 NF-κB 诱饵寡脱氧核苷酸下调了 TNF-α诱导的 MCP-1 mRNA 表达。TNF-α增加了 ROS 的产生，NAC 和 MPA 抑制了 ROS 的产生，但 Syk 抑制剂没有抑制 ROS 的产生。TNF-α增加了 Syk 的酪氨酸磷酸化，NAC 和 MPA 减弱了 Syk 的磷酸化。MPA 和 Syk 抑制剂减弱了 TNF-α诱导的 NF-κB 和 AP-1 的 DNA 结合活性。TNF-α通过激活 AP-1 和 NF-κB 诱导 MCP-1 的表达。AP-1 和 NF-κB 通过 ROS 介导，随后是 Syk。MPA 通过抑制 ROS 和 Syk 抑制 MCP-1 的表达发挥抗炎作用。
