欧前胡素通过抑制TLR4/NF-κB信号通路抗脑缺血再灌注损伤

Imperatorin is a type of coumarin compound isolated from the fruit of Cnidium monnieri (L) Cusson or the roots of Glehnia littoralis, or Angelica dahurica. It has been reported that imperatorin possesses several biological activities, including anti-inflammatory, antioxidant, calcium antagonistic, and vasodilatory properties, and so on. We previously found that imperatorin exerts protective effects in a rat model of cerebral ischemia/reperfusion injury. However, the mechanism underlying imperatorin neuroprotection has not been fully elucidated. Therefore, the present study was designed to determine the mechanisms of action of neuroprotective properties of imperatorin in a rat model of focal cerebral ischemia (2 h)/reperfusion (24 h) injury in vivo and in an oxygen-glucose deprivation (OGD) injury model of primary cultured astroglial and microglial cells from neonatal rats in vitro. We intend to use enzyme-linked immunosorbent assays (ELISA) and Real-time PCR, and Western blot as well as immunofluorescence staining assay to assess the effects of imperatorin on the production of the pro-inflammatory mediators, the expression of Toll-like receptor-4 (TLR4) and myeloid differentiation factor 88 (MyD88), and NF-κB p65 translocation to nucleus following focal cerebral ischemia-reperfusion injury, respectively. Furthermore, we also will examine the effect of imperatorin on the OGD induced activation of astroglial and microglial cells in vitro and the activation of TLR4/MyD88/NF-κB signaling pathway. The results will reveal that imperatorin has the ability to inhibit inflammatory response and neuronal cell death through alleviation of TLR4/NF-κB signaling pathway activation, and provide evidence that imperatorin might be a potential neuroprotective agent for ischemic cerebrovascular diseases.

欧前胡素是一种从蛇床子果实、北沙参根或白芷根中提取的香豆素类化合物，具有抗炎、抗氧化、钙拮抗和舒张血管等作用。我们前期研究发现，欧前胡素对脑缺血再灌注损伤有保护作用，但是其作用的机制还未阐明，本课题利用大鼠局灶性脑缺血2h/再灌注24h和离体原代培养的乳鼠大脑皮层星形胶质细胞和小胶质细胞氧糖剥夺模型，采用ELISA、Real-time PCR、Western blot 和免疫荧光染色等技术，测定欧前胡素对大鼠脑缺血再灌注损伤后炎症介质的产生、TLR4和MyD88表达及NF-κB p65核转位的影响；我们还将观察欧前胡素对氧糖剥夺致星形胶质细胞和小胶质细胞活化及对TLR4/ MyD88/NF-κB通路激活的影响，揭示欧前胡素抗脑缺血损伤的机制是通过抑制TLR4/NF-κB信号通路，减轻炎症反应，抑制神经细胞死亡。通过本课题研究，为欧前胡素用于缺血性脑血管病提供理论依据。

欧前胡素是一种从蛇床子果实、北沙参根或白芷根中提取的呋喃香豆素类化合物，具有抗炎、抗氧化、钙拮抗和舒张血管等作用。我们前期研究发现，欧前胡素对脑缺血再灌注损伤有保护作用，但是其作用的机制还未阐明，本课题利用大鼠局灶性脑缺血2h/再灌注24h模型，采用ELISA、Western blot 和免疫荧光染色等技术，检测了欧前胡素对大鼠局灶性脑缺血/再灌注后大脑皮层中活性氧簇（reactive oxygen species，ROS）的堆积和氧化应激损伤以及神经细胞凋亡的影响，测定欧前胡素对大鼠脑缺血再灌注损伤后大脑皮层中炎症介质的产生的影响，检测诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）、环氧化酶-2（cyclooxygenase-2，COX-2）、Toll样受体4（Toll-like receptors，TLR4）、接头蛋白髓样分化因子88（myeloid differentiation primary response protein 88，MyD88）、TNF受体相关因子6（TNF receptor associated factor-6，TRAF6）和磷酸化IκBα（p-IκBα）及核内NF-κB p65的蛋白表达的水平，同时还观察了欧前胡素对星形胶质细胞活化及对TLR4表达的影响。研究结果显示，欧前胡素能改善大鼠脑缺血再灌注损伤后神经功能行为缺陷评分、减轻脑水肿和降低脑梗死范围，改善缺血区局部脑血流量，抑制大脑皮层中ROS的堆积和减轻氧化应激损伤以及抑制神经细胞细胞凋亡；进一步研究显示，欧前胡素能抑制大鼠脑缺血再灌注后脑组织中炎症介质的产生，抑制iNOS、COX-2 、TLR4、MyD88、TRAF6和p-IκBα及细胞核内NF-кB p65的蛋白表达，抑制脑缺血再灌注后星形胶质细胞激活。我们的研究结果提示，欧前胡素可能通过抑制氧化应激反应、减轻TLR4/NF-κB介导的炎症反应和抑制神经细胞死亡，从而发挥对脑缺血再灌注损伤的保护作用。本项目的研究结果可为欧前胡素开发用于防治缺血性脑血管病提供理论依据。