Study of brain arachidonic acid metabolism using knockou

利用敲除技术研究脑花生四烯酸代谢

• 批准号: 7327031
• 负责人: francesca bosetti
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7327031
• 关键词: Study; brain; arachidonic; acid; metabolism

(1) Cyclooxygenase (COX) enzymes represent the rate-limiting step in the metabolism of arachidonic acid (AA) to prostaglandins (PGs), a pathway activated in neuroinflammation and implicated in several neurodegenerative disorders. To gain a better insight into the specific roles of COX isoforms and characterize the interactions between upstream and downstream enzymes in brain AA cascade, we examined the expression and activity of COX-2 and phospholipase A2 enzymes (cPLA2 and sPLA2), as well as the expression of terminal prostaglandin E synthases (cPGES, mPGES-1, and mPGES- 2) in wild type and COX-1-/- mice. Brain PGE2 concentration was significantly increased, whereas thromboxane B2 (TXB2) concentration was decreased in COX-1-/- compared to wild type mice. There was a compensatory up-regulation of COX-2, accompanied by the activation of the nuclear factor (NF)-kappaB pathway, and also an increase in the upstream cPLA2 and sPLA2 enzymes. Overall, our data suggest that COX-1 and COX-2 play a distinct role in brain PG biosynthesis, with basal PGE2 production being metabolically coupled with COX-2 and TXB2 production being preferentially linked to COX-1. Additionally, COX-1 deficiency can affect the expression of reciprocal and coupled enzymes, COX-2, Ca2+ -dependent PLA2, and terminal mPGES-2. (2) COX-2 is expressed under basal conditions in areas of the brain susceptible to excitotoxicity, a form of toxicity that occurs from over activation of excitatory neurotransmitter systems such as glutamate. While many studies have attempted to determine the role of COX-2 in excitotoxicity through pharmacological inhibition of the enzyme, the results are controversial. We attempted to further study the role of COX in excitotoxicity by testing the susceptibility of mice deficient in either COX-1 or COX-2 to kainic acid (KA) excitotoxicity. COX-1-/-, COX-2-/- and wild type mice were injected intraperitoneally with saline or 10 mg/kg KA, a sublethal dose which induced seizure activity, and video recorded for 2 hours after the injection. Median Racine seizure score (RSS) was significantly elevated in KA-exposed COX-2 -/- mice (RSS=4)compared to wild type (RSS=1) and COX-2+/- mice (RSS=1). COX-1 -/- mice did not differ from COX-1 +/+ mice in the magnitude of KA-induced seizures. Only COX-2 -/- mice exposed to KA exhibited neurons positive for Fluoro-Jade B (FJB), a histochemical stain that detects neuronal degeneration 24 hours after injection. FJB positive neurons were detected in the CA1 and CA3 regions of the hippocampus, amygdala and thalamus. In summary, COX-2 -/- , but not COX-1 -/-, mice exhibit an increased sensitivity to KA-induced seizure activity and neuronal damage, suggesting that COX-2 may be protective against KA-induced excitotoxicity. (3) The specific role of each of COX-1 and COX-2 isoforms in neuroinflammation is still unclear. Therefore, we investigated the role of COX-1 and COX-2 in the neuroinflammatory response induced by intracerebroventricular injection of lipopolysaccharide (LPS) using COX-1-/-, COX-2-/- and wild type mice. In wild type mice LPS increased activated microglia in the cortex, corpus callosum, and cerebral ventricles and resulted in morphological changes of astrocytes in the cortex, hippocampus, and areas surrounding the cerebral ventricles. These changes were accompanied by the up-regulation of the proinflammatory mediators IL-1beta, TNF-alphaand PGE2. Reactive gliosis, expression of proinflammatory cytokines, and brain PGE2 levels, and translocation and activation of NF-kappaB and mitogen-activated protein kinases, important factors for signaling events during an inflammatory response, were significantly decreased in COX-1-/- mice. Protein oxidation, a critical factor contributing to the secondary progression of the inflammatory reaction and oxidative damage was also reduced in the COX-1-/- mice. In contrast, the mRNA expression of IL-1beta, TNF-alpha and of markers of activated microglia and astrocytes (CD11B and GFAP) was significantly increased after LPS injection in the COX-2-/- mice compared to wild-type mice. These observations suggest that COX-1 enhances whereas COX-2 attenuates LPS-induced acute neuroinflammation. (4) To investigate the role of COX-2 in the cerebrovascular coupling, hemodynamic and neuronal responses to forepaw stimulation were measured in alpha-chloralose-anesthetized rats before and after intravenous injection of Meloxicam (MEL), a selective COX-2 inhibitor, and following a bolus of PGE2, a prominent vasodilatatory product of COX-2. Both MEL and PGE2 had a significant effect on the activation-elicited cerebral blood flow (CBF) and blood-oxygenation-level-dependent (BOLD) responses, quantified using continuous arterial spin labeling magnetic resonance imaging, without affecting the baseline perfusion. Meloxicam decreased brain COX enzymatic activity by 57 +/- 14% and decreased the stimulation-induced CBF response to 32 +/- 2% and BOLD to 46 +/- 1% of their respective pre-drug amplitudes. In turn, PGE2 bolus resulted in a partial recovery of functional hyperemia, with the CBF response recovering to 52 +/- 3% and the BOLD response to 56 +/- 2% of their values prior to MEL administration. These findings suggest a modulatory role of COX-2 products in the cerebrovascular coupling and provide evidence for existence of a functional metabolic buffer.

（1）环氧酶（COX）酶代表蛛网膜酸（AA）对前列腺素（PGS）代谢的限制步骤，这是一种在神经炎症中激活并与几种神经退行性疾病有关的途径。为了更好地了解Cox同工型的特定作用，并表征了脑AA AA级联体中上游和下游酶之间的相互作用，我们检查了Cox-2和磷脂酶A2酶的表达和活性（CPLA2和SPLA2和SPLA2），以及末端ProstaglandE e andpers eynthase eNspers andp andp andp andp andp andp andp andp andp andp andp andp andp andp andp andp andp andp and。 COX-1 - / - 小鼠。与野生型小鼠相比，COX-1 - / - 的脑PGE2浓度显着升高，而COX-1 - / - 中血栓烷B2（TXB2）浓度降低。 COX-2的补偿性上调伴随着核因子（NF）-Kappab途径的激活，以及上游CPLA2和SPLA2酶的增加。总体而言，我们的数据表明，COX-1和COX-2在脑PG生物合成中起着独特的作用，基础PGE2的产生与COX-2和TXB2的产生相结合，优先与COX-1相关。另外，COX-1缺乏会影响倒数和耦合酶，Cox-2，Ca2+依赖性PLA2和末端MPGES-2的表达。 （2）COX-2在脑部易感兴奋性毒性的区域的基础条件下表达，这种毒性形式来自兴奋性神经递质系统（如谷氨酸）的过度激活。尽管许多研究试图通过药理抑制酶来确定COX-2在兴奋性毒性中的作用，但结果是有争议的。我们试图通过测试小鼠在COX-1或COX-2对Kainic Acid（Ka）兴奋性兴奋性毒性毒性中缺乏小鼠的敏感性来进一步研究Cox在兴奋性毒性中的作用。将COX-1 - / - ，COX-2 - / - 和野生型小鼠腹膜内注射盐水或10 mg/kg ka，这是一种诱发癫痫发作活性的余量剂量，注射后记录了2小时的视频。与野生型（RSS = 1）和COX-2 +/-小鼠相比，在KA暴露的COX-2 - / - 小鼠中，RACINE癫痫发作评分（RSS）显着升高（RSS = 4）（RSS = 1）。在Ka诱导的癫痫发作的大小中，Cox-1 - / - 小鼠与Cox-1 +/ +小鼠没有差异。仅暴露于KA的COX-2 - / - 小鼠对氟-jade B（FJB）的神经元表现出阳性，这是一种组织化学染色，在注射后24小时检测神经元变性。在海马，杏仁核和丘脑的CA1和CA3区域中检测到FJB阳性神经元。总而言之，COX-2 - / - 但不可以COX-1 - / - 对KA诱导的癫痫发作活性和神经元损伤的敏感性增加，这表明COX-2可以保护Ka诱导的兴奋性毒素。 （3）COX-1和COX-2同工型在神经炎症中的特定作用尚不清楚。因此，我们使用COX-1 - / - ，COX-2 - / - 和野生型小鼠研究了Cox-1和Cox-2在脂多糖（LPS）中诱导的神经炎症反应中的作用。在野生型小鼠中，LPS增加了皮质，call体和脑室的活化小胶质细胞，并导致皮质，海马和脑心室区域中星形胶质细胞的形态变化。这些变化伴随着促炎性介质IL-1Beta，TNF-Alphaand PGE2的上调。反应性神经胶质性，促炎细胞因子的表达以及脑PGE2水平以及NF-kappab和有丝分裂原激活的蛋白激酶的易位和激活，炎性反应期间信号事件的重要因素在COX-1 - / - 小鼠中显着降低。在COX-1 - / - 小鼠中，蛋白质氧化也是有助于炎症反应和氧化损伤的关键因素。相反，与野生型小鼠相比，LPS注射LPS后，LPS注射LPS后，IL-1BETA，TNF-α的mRNA表达以及活化的小胶质细胞和星形胶质细胞（CD11b和GFAP）的mRNA表达显着增加。这些观察结果表明，COX-1增强了，而COX-2减弱了LPS诱导的急性神经炎症。 (4) To investigate the role of COX-2 in the cerebrovascular coupling, hemodynamic and neuronal responses to forepaw stimulation were measured in alpha-chloralose-anesthetized rats before and after intravenous injection of Meloxicam (MEL), a selective COX-2 inhibitor, and following a bolus of PGE2, a prominent vasodilatatory product of COX-2. MEL和PGE2都对激活引起的脑血流（CBF）和血氧水平依赖性（BOLD）反应都有显着影响，并使用连续的动脉自旋标记磁共振成像对其进行了量化，而不会影响基层灌注。美洛昔康将脑Cox酶活性降低了57 +/- 14％，并降低了刺激引起的CBF对32 +/- 2％的反应，并将其BOLD降低至46 +/- 1％，为其各自的前药物振幅。反过来，PGE2推注会导致功能性充血的部分回收，而CBF响应恢复到52 +/- 3％，并且在MEL给药之前对其值的56 +/- 2％的粗体响应。这些发现表明COX-2产物在脑血管耦合中的调节作用，并为存在功能代谢缓冲液的存在提供了证据。