Department of Neurology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China.
Laboratory Research Center, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China.
J Neuroinflammation. 2018 Mar 15;15(1):83. doi: 10.1186/s12974-018-1117-5.
BACKGROUND: Ischemic stroke-induced neuroinflammation is mainly mediated by microglial cells. The nuclear factor kappa B (NF-κB) pathway is the key transcriptional pathway that initiates inflammatory responses following cerebral ischemia. OTULIN, a critical negative regulator of the NF-κΒ signaling pathway, exerts robust effects on peripheral immune cell-mediated inflammation and is regarded as an essential mediator for repressing inflammation in vivo. The effect of OTULIN on inflammatory responses in the central nervous system (CNS) was previously unstudied. This current study investigated the anti-inflammatory effect of OTULIN both in vitro and in vivo in ischemic stroke models. METHODS: Sprague-Dawley (SD) rats were subjected to transient middle cerebral artery occlusion (tMCAO) or an intraperitoneal injection of lipopolysaccharide (LPS). Overexpression of the OTULIN gene was utilized to observe the effect of OTULIN on ischemic stroke outcomes. The effect of OTULIN overexpression on microglia-mediated neuroinflammation was examined in rat primary microglia (PM) and in the microglial cell line N9 after induction by oxygen-glucose deprivation (OGD)-treated neuronal medium. The activation and inflammatory responses of microglia were detected using immunofluorescence, ELISA, and qRT-PCR. The details of molecular mechanism were assessed using Western blotting. RESULTS: In the tMCAO rats, the focal cerebral ischemia/reperfusion injury induced a continuous increase in OTULIN expression within 72 h, and OTULIN expression was increased in activated microglial cells. OTULIN overexpression obviously decreased the cerebral infarct volume, improved the neurological function deficits, and reduced neuronal loss at 72 h after reperfusion, and it also inhibited the activation of microglia and attenuated the release of TNF-α, IL-1β, and IL-6 by suppressing the NF-κB pathway at 24 h after tMCAO. In vitro, OTULIN overexpression inhibited the microglia-mediated neuroinflammation by reducing the production of TNF-α, IL-1β, and IL-6 via depressing the NF-κB pathway in both PM and N9 cells. CONCLUSIONS: OTULIN provides a potential therapeutic target for ischemic brain injury by ameliorating the excessive activation of microglial cells and neuroinflammation through repressing the NF-κB signaling pathway.
背景：缺血性脑卒中引起的神经炎症主要由小胶质细胞介导。核因子 kappa B（NF-κB）途径是脑缺血后引发炎症反应的关键转录途径。OTULIN 是 NF-κB 信号通路的关键负调控因子，对外周免疫细胞介导的炎症具有强大的作用，被认为是体内抑制炎症的重要介质。OTULIN 对中枢神经系统（CNS）炎症反应的影响此前尚未研究过。本研究在缺血性脑卒中模型中体内和体外研究了 OTULIN 的抗炎作用。 方法：Sprague-Dawley（SD）大鼠接受短暂性大脑中动脉闭塞（tMCAO）或腹腔注射脂多糖（LPS）。过表达 OTULIN 基因，观察 OTULIN 对缺血性脑卒中结局的影响。在氧葡萄糖剥夺（OGD）处理的神经元培养基诱导后，在大鼠原代小胶质细胞（PM）和小胶质细胞系 N9 中观察 OTULIN 过表达对小胶质细胞介导的神经炎症的影响。使用免疫荧光、ELISA 和 qRT-PCR 检测小胶质细胞的激活和炎症反应。通过 Western blot 评估分子机制的详细信息。 结果：在 tMCAO 大鼠中，局灶性脑缺血再灌注损伤诱导 OTULIN 表达在 72 h 内持续增加，并且 OTULIN 表达增加在激活的小胶质细胞中。OTULIN 过表达明显减少脑梗死体积，改善再灌注后 72 h 的神经功能缺损，并减少神经元丢失，同时在 tMCAO 后 24 h 还通过抑制 NF-κB 通路抑制小胶质细胞的激活并减轻 TNF-α、IL-1β 和 IL-6 的释放。体外，OTULIN 过表达通过在 PM 和 N9 细胞中通过抑制 NF-κB 通路减少 TNF-α、IL-1β 和 IL-6 的产生来抑制小胶质细胞介导的神经炎症。 结论：OTULIN 通过抑制 NF-κB 信号通路改善小胶质细胞过度激活和神经炎症，为缺血性脑损伤提供了一种潜在的治疗靶点。
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