Yang Lijuan, Kan Enci Mary, Lu Jia, Wu Chunyun, Ling Eng-Ang
Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore.
J Neuroinflammation. 2014 Aug 23;11:148. doi: 10.1186/s12974-014-0148-9.
We reported previously that amoeboid microglial cells in the postnatal rat brain expressed 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) both in vivo and in vitro; however, the functional role of CNPase in microglia has remained uncertain. This study extended the investigation to determine CNPase expression in activated microglia derived from cell culture and animal models of brain injury with the objective to clarify its putative functions.
Three-day-old Wistar rats were given an intraperitoneal injection of lipopolysaccharide to induce microglial activation, and the rats were killed at different time points. Along with this, primary cultured microglial cells were subjected to lipopolysaccharide treatment, and expression of CNPase was analyzed by real-time reverse transcription PCR and immunofluorescence. Additionally, siRNA transfection was employed to downregulate CNPase in BV-2 cells. Following this, inducible nitric oxide synthase, IL-1β and TNF-α were determined at mRNA and protein levels. Reactive oxygen species and nitric oxide were also assessed by flow cytometry and colorimetric assay, respectively. In parallel to this, CNPase expression in activated microglia was also investigated in adult rats subjected to fluid percussion injury as well as middle cerebral artery occlusion.
In vivo, CNPase immunofluorescence in activated microglia was markedly enhanced after lipopolysaccharide treatment. A similar feature was observed in the rat brain after fluid percussion injury and middle cerebral artery occlusion. In vitro, CNPase protein and mRNA expression was increased in primary microglia with lipopolysaccharide stimulation. Remarkably, inducible nitric oxide synthase, IL-1β, TNF-α, reactive oxygen species and nitric oxide were significantly upregulated in activated BV-2 cells with CNPase knockdown. siRNA knockdown of CNPase increased microglia migration; on the other hand, microglial cells appeared to be arrested at G1 phase.
The present results have provided the first morphological and molecular evidence that CNPase expression is increased in activated microglia. CNPase knockdown resulted in increased expression of various inflammatory mediators. It is concluded that CNPase may play an important role as a putative anti-inflammatory gene both in normal and injured brain.
我们之前报道过，新生大鼠脑中的阿米巴样小胶质细胞在体内和体外均表达2',3'-环核苷酸3'-磷酸二酯酶（CNPase）；然而，CNPase在小胶质细胞中的功能作用仍不明确。本研究进一步展开调查，以确定源自细胞培养和脑损伤动物模型的活化小胶质细胞中CNPase的表达情况，目的是阐明其假定功能。
给3日龄的Wistar大鼠腹腔注射脂多糖以诱导小胶质细胞活化，并在不同时间点处死大鼠。与此同时，对原代培养的小胶质细胞进行脂多糖处理，通过实时逆转录PCR和免疫荧光分析CNPase的表达。此外，采用小干扰RNA（siRNA）转染来下调BV-2细胞中的CNPase。在此之后，在mRNA和蛋白质水平测定诱导型一氧化氮合酶、白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）。还分别通过流式细胞术和比色法评估活性氧和一氧化氮。与此同时，在遭受液压冲击损伤以及大脑中动脉闭塞的成年大鼠中，也对活化小胶质细胞中的CNPase表达进行了研究。
在体内，脂多糖处理后活化小胶质细胞中的CNPase免疫荧光明显增强。在液压冲击损伤和大脑中动脉闭塞后的大鼠脑中也观察到类似特征。在体外，脂多糖刺激后原代小胶质细胞中CNPase蛋白和mRNA表达增加。值得注意的是，在CNPase敲低的活化BV-2细胞中，诱导型一氧化氮合酶、IL-1β、TNF-α、活性氧和一氧化氮均显著上调。CNPase的siRNA敲低增加了小胶质细胞的迁移；另一方面，小胶质细胞似乎停滞在G1期。
目前的结果提供了首个形态学和分子证据，表明活化小胶质细胞中CNPase表达增加。CNPase敲低导致各种炎症介质的表达增加。得出的结论是，CNPase在正常和受损大脑中可能作为一种假定的抗炎基因发挥重要作用。
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