Study of brain arachidonic acid metabolism knockout mice

脑花生四烯酸代谢敲除小鼠的研究

• 批准号: 7132267
• 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/7132267
• 关键词: DNA binding protein; I kappa B beta; arachidonate; brain; eicosanoid metabolism; enzyme activity; enzyme biosynthesis; enzyme deficiency; fatty acid biosynthesis; genetically modified animals; histochemistry /cytochemistry; kainate; laboratory mouse; neurophysiology; neurotoxins; nuclear factor kappa beta; phospholipase A2; phosphorylation; prostaglandin endoperoxide synthase; prostaglandins; protein isoforms; synaptotagmin; thromboxanes

(1) To investigate the interaction between upstream and downstream enzymes involved in brain prostaglandin (PG) synthesis, we examined the expression and activity of cyclooxygenase (COX)-1, of different phospholipase A2 (PLA2) enzymes, and of prostaglandin E2 synthase (PGES) enzymes in COX-2 deficient mice. The PGE2 level was decreased by 52% in the COX-2 knockout mice brain, indicating a significant role of COX-2 in formation of PGE2. However, when we added exogenous arachidonic acid (AA) to brain homogenates, COX activity was increased in the COX-2 deficient mice, suggesting a compensatory increase in COX-1 expression and an intracellular compartmentalization of the COX isozymes. Activity and expression of cytosolic cPLA2 and secretory sPLA2 enzymes, supplying AA to COX, were significantly increased. Our results indicate that compensatory mechanisms exist in COX-2 deficient mice and that microsomal PGES-2 is functionally coupled with COX-2. Thus, this pathway might represent a novel target for anti-inflammatory and neuroprotective drugs. Then, we further examined how COX-2 deficiency was affecting the NF-kappaB pathway, which controls COX-2 expression. We found a decrease in NF-kappaB DNA-protein binding activity, which was accompanied by a reduction of the phosphorylation state of both I-kappaBalpha and p65 proteins in the COX-2 deficient mice. The mRNA and protein levels of p65 were also reduced in COX-2 deficient mice, whereas total cytoplasmic I-kappaB protein level was not significantly changed. Taken together, these changes may be responsible for the observed decrease in NF-kappaB DNA binding activity. NF-kappaB DNA binding activity was selectively affected in the COX-2 deficient mice compared to the wild type as there was no significant change in NFATc DNA binding activity. Overall, our data indicate that constitutive brain NF-kappaB activity is decreased in COX-2 deficient mice and suggest a reciprocal coupling between NF-kappaB and COX-2. (2) To determine the specific role of COX-1 in brain AA cascade, we examined the expression and activity of COX-2 as well as the expression and activity of the PLA2 enzymes (group IV cPLA2 and group V sPLA2), which generate AA, and of known downstream PGES isoforms, which generate biologically active PGE2, in brain from COX-1 deficient mice. The expression and activity of brain cPLA2 and sPLA2 were significantly increased in COX-1-deficient mice compared to the wild-type. We also find a compensatory increase in COX-2 expression, accompanied by an up-regulation of the NF-kappaB pathway. Downstream enzymes also were affected, as the protein levels of mPGES-1 and -2, but not cPGES, were decreased in COX-1-deficient mice. Brain PGE2 level was increased and thromboxane B2 level was decreased in COX-1-deficient mice, suggesting that these end-products are specifically derived from COX-1 and COX-2 metabolism of AA, respectively. Taken together, these data are consistent with our hypothesis that the COX-1 deficiency results in the altered expression of the remaining enzyme that regulate mobilization and conversion of AA to prostaglandins. (3) 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. Mice deficient in either COX-1 or COX-2 and their wild-types 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 deficient mice compared to wild type and heterozygous mice. COX-1 deficient mice did not differ from wild type mice in the magnitude of KA-induced seizures. Only COX-2 deficient mice exhibited neurons positive for Fluoro-Jade B (FJB), a histochemical stain that detects neuronal degeneration, 24 hours after KA injection. FJB positive neurons were detected in areas known to be affected by KA such as the CA1 and CA3 regions of the hippocampus, amygdala and thalamus. In summary, COX-2 deficient, but not COX-1 deficient, mice exhibit an increased sensitivity to KA-induced seizure activity and are more susceptible than wild type mice to excitotoxic neuronal damage, suggesting that COX-2 may be protective against excitotoxicity.

（1）研究参与脑前列腺素（PG）合成的上游和下游酶之间的相互作用，我们检查了不同磷脂酶A2（PLA2）酶的环氧合酶（COX）-1的表达和活性，以及​​前磷酸酶A2（PLA2）酶以及前磷酸素E2合酶（PGES）的表达和活性。在COX-2基因敲除小鼠大脑中，PGE2水平降低了52％，表明COX-2在PGE2形成中起着重要作用。但是，当我们在脑匀浆中添加外源性蛛网膜酸（AA）时，COX-2缺乏小鼠的Cox活性增加，这表明COX-1表达的补偿性增加和Cox同工酶的细胞内分室化。向COX供应AA的胞质CPLA2和分泌SPLA2酶的活性和表达显着增加。我们的结果表明，COX-2缺陷小鼠中存在补偿机制，并且微粒体PGES-2在功能上与COX-2耦合。因此，该途径可能代表了抗炎和神经保护药物的新靶标。然后，我们进一步研究了COX-2缺乏症如何影响控制COX-2表达的NF-kappab途径。我们发现NF-kappab DNA-蛋白结合活性的降低，伴随着COX-2缺陷小鼠中I-kappabalpha和p65蛋白的磷酸化态降低。在COX-2缺乏小鼠中，p65的mRNA和蛋白质水平也降低，而总细胞质I-kappab蛋白水平没有显着改变。综上所述，这些变化可能导致观察到的NF-kappab DNA结合活性的降低。与野生型相比，NF-kappab DNA结合活性在COX-2缺陷小鼠中有选择地影响，因为NFATC DNA结合活性没有显着变化。总体而言，我们的数据表明，COX-2缺陷小鼠的组成型脑NF-kappab活性降低，并提出NF-kappab和Cox-2之间的相互耦合。 （2）确定COX-1在脑AA级联反应中的具体作用，我们检查了Cox-2的表达和活性以及PLA2酶的表达和活性（IV组CPLA2和V组V SPLA2），从而在生物学上产生了生物学上活跃的PGE2 PGE2，从而在Cox-1中产生了生物学上活跃的PGE2，从而产生了AA和已知的下游PGES PGES PGES同种型。与野生型相比，在COX-1缺陷型小鼠中，脑CPLA2和SPLA2的表达和活性显着增加。我们还发现COX-2表达的补偿性增加，并伴随着NF-kappab途径的上调。下游酶也受到影响，因为在COX-1缺陷型小鼠中，MPGES-1和-2而不是CPGE的蛋白质水平降低。在COX-1缺陷型小鼠中，脑PGE2水平升高，血栓烷B2水平降低，这表明这些终产物分别是源自AA的COX-1和COX-2代谢。综上所述，这些数据与我们的假设一致，即COX-1缺乏会导致其余酶的表达改变，从而调节AA的动员和转化为前列腺素。 （3）COX-2在脑部易感兴奋性毒性区域的基础条件下表达，这种毒性形式是从兴奋性神经递质系统（如谷氨酸）的过度激活中发生的。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。将缺乏COX-1或COX-2及其野生型的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剂量，并在注射后记录了2小时的视频。与野生型和杂合小鼠相比，在KA暴露的COX-2缺乏小鼠中，RACINE癫痫发作评分（RSS）显着升高。在Ka诱导的癫痫发作的大小中，COX-1缺乏小鼠与野生型小鼠没有差异。仅COX-2缺乏小鼠在KA注射后24小时后，氟-jade B（FJB）表现出神经元阳性的组织化学染色，这是一种检测神经元变性的组织化学染色。在已知受KA影响的区域（例如海马，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缺乏但不足Cox-1的小鼠对KA诱导的癫痫发作活性的敏感性增加，并且比野生型小鼠对兴奋性神经元损伤更容易受到敏感，这表明COX-2可能具有保护性抗兴奋性。