肽聚糖通过 TLR2 受体、MyD88、磷脂酰肌醇 3-激酶/AKT 和 NF-κB 途径增强 BV-2 小胶质细胞中的促炎细胞因子表达。
Peptidoglycan enhances proinflammatory cytokine expression through the TLR2 receptor, MyD88, phosphatidylinositol 3-kinase/AKT and NF-kappaB pathways in BV-2 microglia.
机构信息
Institute of Medical Science, China Medical University, Taichung, Taiwan.
出版信息
Int Immunopharmacol. 2010 Aug;10(8):883-91. doi: 10.1016/j.intimp.2010.04.026. Epub 2010 May 5.
In this study, we investigated the signaling pathways involved in inflammatory production caused by peptidoglycan (PGN), a cell wall component of the gram-positive bacterium, in BV-2 microglia. PGN caused a concentration- and time-dependent increase in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein levels. In addition, PGN also induced IL-1 beta, TNF-alpha and IL-6 mRNA up-regulation in a concentration-dependent manner. Moreover, PGN also increased Toll-like receptor 2 (TLR2) expression in BV-2 microglia. Administration of TLR2 neutralizing antibody effectively inhibited PGN-induced iNOS and COX-2 expression. On the other hand, PGN-induced iNOS and COX-2 up-regulation were attenuated by PI3-kinase inhibitors (LY294002 and wortmannin), and an AKT inhibitor. Treatment of BV-2 microglia with PGN caused a time-dependent activation of PI3-kinase (p85) and AKT. PGN-induced PI3-kinase/AKT activation, iNOS and COX-2 expression were also inhibited by MyD88 inhibitory peptide. Treatment of cells with NF-kappaB inhibitor (pyrrolidine dithiocarbamate), I kappaB alpha phosphorylation inhibitor (Bay 117082), or I kappaB protease inhibitor (l-1-tosylamido-2-phenylethyl chloromethyl ketone) inhibited PGN-induced iNOS and COX-2 expression. Furthermore, stimulation of cells with PGN also activated IKK alpha/beta, I kappaB alpha phosphorylation, I kappaB alpha degradation, p65 phosphorylation at Ser(536), and increased kappaB-luciferase activity. PGN-induced IKK alpha/beta phosphorylation, I kappaB alpha phosphorylation, and I kappaB alpha degradation were further inhibited by pre-treatment with PI3-kinase inhibitors. Moreover, PGN-mediated increase of kappaB-luciferase activity was also inhibited by pre-transfection with dominant-negative mutants of p85, AKT, IKK alpha or IKK beta. Our data demonstrate that PGN-induced iNOS, COX-2 and proinflammatory cytokine expression was mediated through the TLR2/MyD88/PI3-kinase/AKT pathway, which in turn initiates IKK alpha/beta and NF-kappaB activation in BV-2 microglia.
在这项研究中，我们研究了肽聚糖（PGN）引起的炎症产生的信号通路，PGN 是革兰氏阳性菌细胞壁的组成部分，在 BV-2 小胶质细胞中。PGN 导致诱导型一氧化氮合酶（iNOS）和环氧化酶-2（COX-2）mRNA 和蛋白水平的浓度和时间依赖性增加。此外，PGN 还以浓度依赖性方式诱导 IL-1β、TNF-α和 IL-6 mRNA 的上调。此外，PGN 还增加了 BV-2 小胶质细胞中 Toll 样受体 2（TLR2）的表达。TLR2 中和抗体的给药有效抑制了 PGN 诱导的 iNOS 和 COX-2 的表达。另一方面，PI3-激酶抑制剂（LY294002 和wortmannin）和 AKT 抑制剂减弱了 PGN 诱导的 iNOS 和 COX-2 的上调。用 PGN 处理 BV-2 小胶质细胞引起 PI3-激酶（p85）和 AKT 的时间依赖性激活。PGN 诱导的 PI3-激酶/AKT 激活、iNOS 和 COX-2 的表达也被 MyD88 抑制肽抑制。用 NF-κB 抑制剂（吡咯烷二硫代氨基甲酸酯）、IκBα磷酸化抑制剂（Bay 117082）或 IκB 蛋白酶抑制剂（l-1-甲苯磺酰基-2-苯乙基氯甲基酮）处理细胞可抑制 PGN 诱导的 iNOS 和 COX-2 的表达。此外，用 PGN 刺激细胞还激活了 IKKα/β、IκBα磷酸化、IκBα降解、p65 在 Ser（536）的磷酸化和增强的κB-荧光素酶活性。用 PI3-激酶抑制剂预处理进一步抑制了 PGN 诱导的 IKKα/β磷酸化、IκBα磷酸化和 IκBα降解。此外，PGN 介导的κB-荧光素酶活性的增加也被 p85、AKT、IKKα或 IKKβ的显性负突变体的预转染抑制。我们的数据表明，PGN 诱导的 iNOS、COX-2 和促炎细胞因子的表达是通过 TLR2/MyD88/PI3-激酶/AKT 途径介导的，该途径继而在 BV-2 小胶质细胞中启动 IKKα/β和 NF-κB 的激活。
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