依达拉奉干预LPS介导原代小胶质细胞的激活实验

目的 探索依达拉奉 (edaravone, EDA) 对脂多糖 (lipopolysaccharide, LPS) 介导原代小胶质细胞 (Microglia) 激活后的一氧化氮 (nitric oxide, NO) 调控作用.方法 麻醉SD大鼠新生鼠断头取脑, 消化离心等实验方法完成混合胶质细胞的原代培养, 通过生物摇床方法分离、纯化小胶质细胞.利用Griess法检测一氧化氮释放量.结果 通过荧光方法鉴定小胶质细胞的纯化可达到95%以上, 与对照组 (Control) 相比, LPS介导小胶质细胞后激活可见NO浓度显著上升 (P<0.05) , 建模成功;用EDA预处理原代小胶质细胞24 h后, LPS介导的NO下调, 与LPS单独组比较, 差异有统计学意义 (P<0.05) .同时, EDA先预处理24 h, MAPK inhibitor (丝裂原活化蛋白激酶抑制剂) 预处理30 min, 10 ng/m L LPS介导小胶质细胞16 h, NO释放量显著降低, 差异有统计学意义 (P<0.05) .结论 EDA单独应用可抑制LPS介导原代小胶质细胞激活后NO的释放量;EDA同时加用MAPK信号传递通路抑制剂, 加强了EDA减轻LPS介导小胶质细胞激活NO产生.EDA可能与p38MAPK、JNK、ERK信号传导通路抑制剂协同抑制LPS介导的原代小胶质细胞激活有关.

The Experiment of Edaravone on Primary Microglia Activation by Lipopolysaccharide

Objective To explore the effect of Edaravone (EDA) on oxidative stress such as release of nitric oxide (NO) induced by Lipopolysaccharide (LPS) in activation of primary microglia. Me thods There were four groups including control, LPS, LPS + EDA and MAPK inhibitors + EDA + LPS in this study. The primary mixed glial cells were derived from the cortex of Sprague-Dawley rats which were 2 to 3-day old neonatal pups. The primary microglia were separated and purified by Biological Shaker after growing for about ten days. Griess Assay was used to detect NO secreted by activated microglia in four groups. Re s ults The shake-off microglial cells were confirmed as purity of 95% or up via fluorescence staining of CD68. Compared with the control group, NO product of microglial activation mediated by LPS significantly increased while NO value decreased in the group of EDA which was pretreatedt of EDA ahead of LPS for 24 hrs (P<0.05) . After addition of LPS, the incubation was remained for additional 24 h. In addition to EDA pretreatment for 24 h, the addition of three Mitogen-activated protein kinase inhibitors of MAPK signaling pathways were ahead of addition of EDA for 30 min, and then 10 ng/m Lof LPS was added to microglia. All of them incubated with the primary microglia for 24 h after the last addition. NO release was significantly reduced, the difference was statistically significant (P <0.05) .Conclus ion EDA treatment alone could inhibit the release of NO induced by LPS in primary microglia while EDA plus MAPK signaling pathway inhibitor obviously reduced release of NO. EDA may take effect with p38 MAPK, JNK and ERK inhibitors of sub-pathways in some way (s) to co-operate with EDA in modulating oxidative stress in activated microglia induced by LPS. As to the working mechanisms how EDA co-operates with MAPK inhibitors is still remain unknown.