Abstract
BackgroundPost-ischemic microglial activation may contribute to neuronal damage through the release of large amounts of pro-inflammatory cytokines and neurotoxic factors. The involvement of microRNAs (miRNAs) in the pathogenesis of disorders related to the brain and central nervous system has been previously studied, but it remains unknown whether the production of pro-inflammatory cytokines is regulated by miRNAs.MethodsBV-2 and primary rat microglial cells were activated by exposure to oxygen-glucose deprivation (OGD). Global cerebral ischemia was induced using the four-vessel occlusion (4-VO) model in rats. Induction of pro-inflammatory and neurotoxic factors, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and nitric oxide (NO), were assessed by ELISA, immunofluorescence, and the Griess assay, respectively. The miRNA expression profiles of OGD-activated BV-2 cells were subsequently compared with the profiles of resting cells in a miRNA microarray. BV-2 and primary rat microglial cells were transfected with miR-181c to evaluate its effects on TNF-α production after OGD. In addition, a luciferase reporter assay was conducted to confirm whether TNF-α is a direct target of miR-181c.ResultsOGD induced BV-2 microglial activation in vitro, as indicated by the overproduction of TNF-α, IL-1β, and NO. Global cerebral ischemia/reperfusion injury induced microglial activation and the release of pro-inflammatory cytokines in the hippocampus. OGD also downregulated miR-181c expression and upregulated TNF-α expression. Overproduction of TNF-α after OGD-induced microglial activation provoked neuronal apoptosis, whereas the ectopic expression of miR-181c partially protected neurons from cell death caused by OGD-activated microglia. RNAinterference-mediated knockdown of TNF-α phenocopied the effect of miR-181c-mediated neuronal protection, whereas overexpression of TNF-α blocked the miR-181c-dependent suppression of apoptosis. Further studies showed that miR-181c could directly target the 3′-untranslated region of TNF-α mRNA, suppressing its mRNA and protein expression.ConclusionsOur data suggest a potential role for miR-181c in the regulation of TNF-α expression after ischemia/hypoxia and microglia-mediated neuronal injury.
Highlights
Post-ischemic microglial activation may contribute to neuronal damage through the release of large amounts of pro-inflammatory cytokines and neurotoxic factors
Hypoxia/ischemia induces microglial activation and the release of pro-inflammatory cytokines and neurotoxic factors Microglia activated by ischemic conditions have been shown to release pro-inflammatory and neurotoxic factors, such as IL-1, tumor necrosis factor (TNF)-α and nitric oxide (NO), which may be responsible for severe brain-tissue damage [2,3]
Real-time PCR and enzyme-linked immunosorbent assay (ELISA) were used to assess the induction of IL-1β and TNF-α in BV-2 microglia exposed to oxygen-glucose deprivation (OGD)
Summary
Post-ischemic microglial activation may contribute to neuronal damage through the release of large amounts of pro-inflammatory cytokines and neurotoxic factors. Microglia display a quiescent phenotype in the healthy brain, but they become highly activated after brain insult, when they produce reactive oxygen and nitrogen species and pro-inflammatory cytokines [1,2,3,4,5]. The available evidence has indicated that microglial activation results in the overproduction of pro-inflammatory cytokines, which may contribute to the development and progression of neurodegenerative disorders [7]. Activated microglia, by releasing pro-inflammatory cytokines, participate in the inflammatory response associated with cerebral ischemia[1]. Overproduction of inflammatory factors from ischemia-activated microglia is thought to mediate post-ischemic neuronal damage [14]
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