Department of Physiology, School of Medicine, Ajou University, Suwon, Republic of Korea.
Life Sci. 2012 Feb 27;90(9-10):365-72. doi: 10.1016/j.lfs.2011.12.011. Epub 2011 Dec 29.
Fructose-1,6-bisphosphate (FBP) is a glycolytic intermediate with neuroprotective action in various brain injury models. However, the mechanism underlying the neuroprotection of FBP has not been fully defined. In this study, we investigated whether FBP inhibits endotoxin-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in microglial cells and explored the possible mechanisms of the effects of FBP.
Murine microglial cell line BV2 and primary cultured murine microglial cells were used. NO production and iNOS expression were determined by Griess reaction, RT-PCR and Western blot. Luciferase assay using iNOS promoter-luciferase (iNOS-Luc) construct was adopted for measuring transcriptional activity.
FBP dose-dependently suppressed lipopolysaccharide (LPS)-induced NO production, along with reducing the expression of iNOS at both the protein and mRNA level in primary cultured murine microglia and BV2 cells. FBP significantly inhibited iNOS promoter activity but stabilized iNOS mRNA. Among transcription factors known to be related to iNOS expression, activator protein (AP-1) activation was significantly blocked by FBP. FBP suppressed LPS-induced phosphorylation of three MAPK subtypes-p38 MAPK, JNK and ERK. FBP inhibited LPS-induced production of reactive oxygen species (ROS) and decreased intracellular GSSG/GSH ratio.
Our findings suggest that FBP attenuates the LPS-induced iNOS expression through inhibition of JNK and p38 MAPK, which might be related to ROS downregulation.
1,6-二磷酸果糖（FBP）是一种糖酵解中间产物，在各种脑损伤模型中具有神经保护作用。然而，FBP 神经保护作用的机制尚未完全确定。在本研究中，我们研究了 FBP 是否抑制内毒素诱导的一氧化氮（NO）产生和诱导型一氧化氮合酶（iNOS）在小胶质细胞中的表达，并探讨了 FBP 作用的可能机制。
使用小鼠小胶质细胞系 BV2 和原代培养的小鼠小胶质细胞。通过格里斯反应、RT-PCR 和 Western blot 测定 NO 产生和 iNOS 表达。采用 iNOS 启动子-荧光素酶（iNOS-Luc）构建体进行荧光素酶测定以测量转录活性。
FBP 剂量依赖性地抑制脂多糖（LPS）诱导的 NO 产生，并降低原代培养的小鼠小胶质细胞和 BV2 细胞中 iNOS 的蛋白和 mRNA 表达。FBP 显著抑制 iNOS 启动子活性，但稳定 iNOS mRNA。在已知与 iNOS 表达相关的转录因子中，AP-1 激活被 FBP 显著阻断。FBP 抑制 LPS 诱导的三种 MAPK 亚型（p38 MAPK、JNK 和 ERK）的磷酸化。FBP 抑制 LPS 诱导的活性氧（ROS）产生并降低细胞内 GSSG/GSH 比。
我们的发现表明，FBP 通过抑制 JNK 和 p38 MAPK 来减轻 LPS 诱导的 iNOS 表达，这可能与 ROS 下调有关。
