A Helminth Protease Inhibitor Modulates the Lipopolysaccharide-Induced Proinflammatory Phenotype of Microglia in vitro
A Helminth Protease Inhibitor Modulates the Lipopolysaccharide-Induced Proinflammatory Phenotype of Microglia in vitro
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DOI：
10.1159/000444756
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发表时间：
2016-01-01
影响因子：
2.4
通讯作者：
Lucius, Ralph
中科院分区：
文献类型：
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作者：
Behrendt, Peter;Arnold, Philipp;Lucius, Ralph
目的：本研究的目的是检查是否天然蛋白酶抑制剂Av-cystatin（rAv 17）的寄生线虫Acanthocheilonema viteae发挥抗炎作用，在体外模型的脂多糖（LPS）激活的小胶质细胞。方法：从2日龄Wistar大鼠脑中收获原代小胶质细胞，并在有或无rAv 17（250 nM）的情况下培养。在6和24小时后，在上清液中测量一氧化氮（Griess试剂）和TNF-α（ELISA）的释放。在培养2、6和24 h后进行实时PCR以测量IL-1 β、IL-6、TNF-α、考克斯-2、iNOS和IL-10的mRNA表达。为了解决所涉及的信号传导途径，通过免疫细胞化学观察核NF-κ B易位。考马斯亮蓝染色观察小胶质细胞形态学变化。使用单因素ANOVA和Bonferroni事后检验计算组间差异。结果：形态学分析表明，LPS诱导的小胶质细胞向变形虫形态的转化被rAv 17抑制。Av-半胱氨酸蛋白酶抑制剂分别引起促炎细胞因子iNOS和考克斯-2 mRNA表达的时间依赖性下调。这是由IL-10在静息以及LPS刺激的小胶质细胞中的上调表达所证实的。Av-cystatin可减少培养上清液中NO和TNF-α的释放。免疫细胞化学染色显示，NF-κ B B的Av-cystatin在LPS刺激下转位减弱。此外，蛋白质印迹分析揭示了LPS诱导的ERK 1/2通路活化的rAv 17依赖性降低。结论：寄生虫来源的分泌产物Av-cystatin抑制LPS诱导的小胶质细胞的促炎机制与IL-10，一个潜在的关键介质。(C)2016 S. Karger AG，巴塞尔
Objective: The aim of this study was to examine whether the natural protease inhibitor Av-cystatin (rAv17) of the parasitic nematode Acanthocheilonema viteae exerts anti-inflammatory effects in an in vitro model of lipopolysaccharide (LPS)-activated microglia. Methods: Primary microglia were harvested from the brains of 2-day-old Wistar rats and cultured with or without rAv17 (250 n M). After 6 and 24 h the release of nitric oxide (Griess reagent) and TNF-alpha (ELISA) was measured in the supernatant. Real-time PCR was performed after 2, 6 and 24 h of culture to measure the mRNA expression of IL-1 beta, IL-6, TNF-alpha, COX-2, iNOS and IL-10. To address the involved signaling pathways, nuclear NF-kappa B translocation was visualized by immunocytochemistry. Morphological changes of microglia were analyzed by Coomassie blue staining. Differences between groups were calculated using one-way ANOVA with Bonferroni's post hoc test. Results: Morphological analysis indicated that LPS-induced microglial transformation towards an amoeboid morphology is inhibited by rAv17. Av-cystatin caused a time-dependent downregulation of proinflammatory cytokines, iNOS and COX-2 mRNA expression, respectively. This was paralleled by an upregulated expression of IL-10 in resting as well as in LPS-stimulated microglia. Av-cystatin reduced the release of NO and TNF-alpha in the culture supernatant. Immunocytochemical staining demonstrated an attenuated translocation of NF-kappa B by Av-cystatin in response to LPS. In addition, Western blot analysis revealed a rAv17-dependent reduction of the LPS-induced ERK1/2-pathway activation. Conclusion: The parasite-derived secretion product Av-cystatin inhibits proinflammatory mechanisms of LPS-induced microglia with IL-10, a potential key mediator. (C) 2016 S. Karger AG, Basel