Role of Epoxygenated Fatty Acids in Modulating Pain

描述(由申请人提供)：对于慢性疼痛状态，对新的疼痛疗法的需求很大。花生四烯酸(ARA)级联通路是炎症性疼痛的关键通路，许多研究涉及该级联通路的环氧合酶(COX)和脂氧合酶(LOX)分支。然而，第三个分支-细胞色素P450途径的重要性正在显现，它为痛觉调制提供了新的替代方案。与炎症性COX和LOX代谢物不同，细胞色素P450产生的ARA代谢物环氧二十碳三烯酸(EETs)是一种有效的生理性抗炎/抗痛敏分子。这些生物活性脂质的半衰期很短，通过可溶性环氧化物水解酶(sEH，EC 3.3.2.3)抑制其降解可产生抗伤害作用。SEH抑制剂在炎症性和神经病理性疼痛模型中是有效的，但sEH抑制如何减轻疼痛尚不清楚，值得进一步研究这一新途径。SEH在外周和中枢神经系统、神经元和神经胶质细胞中都有很好的表达，并具有空间选择性 在大脑区域之间的表达。SEH抑制剂(SEHIs)可阻断伤害性行为，同时保持正常的伤害性感觉和运动功能。因此，在这项研究中，我们将检验环氧化脂肪酸和sEHI在疼痛病理生理学中具有调节作用的假设。为了解决这一假设，将监测两种不同疼痛模型中皮肤和腰椎脊髓中EpFAs及其降解产物的水平。然后通过不同的途径在两种不同的疼痛模型中调节EPFAs的水平：a)通过外源性EPFAs，b)通过抑制sEH，c)通过抑制磷酸二酯酶和d)通过新型合成的抗sEH介导的降解的EPFAs类似物。我们将利用角叉菜胶诱导的炎性疼痛模型来鉴定抗痛敏的EpFAs。抗伤害性EPFAs的生物活性将在一个临床相关的模型--切口疼痛模型中得到验证。我们将结合行为分析、生化和代谢分析来了解EpFAs与伤害性通路的相互作用。我们将建立一个特征/诊断LC/MS/MS生成的大鼠足底角叉菜胶和足底切口痛模型的代谢谱。关于生物活性脂类代谢产物的改变，尤其是对切口处疼痛的研究较少。目标是 代谢产物(80种生物活性脂质)来自AA级联的所有三个分支，包括主要的COX、LOX、细胞色素P450和sEH产物，包括EPA和DHA的脂质代谢产物，使我们能够研究代谢产物通量和生物级联之间的串扰。这些研究将促进对EpFAs和sEH抑制剂抗伤害性作用机制的深入理解，并为疼痛反应中脂类和生化变化的潜在机制提供新的见解。这些实验产生的知识可能会导致开发出新型的药物，这些药物针对以前没有被识别的途径。

DESCRIPTION (provided by applicant): Great demand for novel pain therapies exists for chronic pain states. The arachidonic acid (ARA) cascade is a pivotal inflammatory pain pathway, and many studies addressed the cyclooxygenase (cox) and lipoxygenase (lox) branches of this cascade. However, the emerging importance of a third branch, the cytochrome P450 pathway, offers novel alternatives for pain modulation. In contrast to the inflammatory cox and lox metabolites, the cytochrome P450-generated ARA metabolites, epoxyeicosatrienoic acids (EETs), are potent physiological anti-inflammatory/antihyperalgesic molecules. These bioactive lipids have very short half lives and inhibiting their degradation mediated by soluble epoxide hydrolase (sEH, EC 3.3.2.3) leads to antinociception. Inhibitors of sEH are effective in models of inflammatory and neuropathic pain, but it is unclear how sEH inhibition attenuates pain, warranting further investigation of this novel pathway. The sEH is well expressed in the peripheral and central nervous system, in the neurons and the glial cells, with spatial selectivity of expression among brain regions. Inhibitors of sEH (sEHIs) block nocifensive behavior, while leaving intact normal nociceptive and motor function. Thus in this study we will test the hypothesis that epoxygenated fatty acids and sEHI have a modulatory role in pain pathophysiology. To address this hypothesis, the levels of the EpFAs and their degradation products in the skin and the lumbar spinal cord in two distinct models of pain will be monitored. Levels of EpFAs will then be modulated in two distinct models of pain by different approaches; a) by exogenous EpFAs, b) by inhibiting the sEH, c)by inhibiting phosphodiesterases and d) by novel synthetic analogues of EpFAs that are resistant to sEH mediated degradation. We will utilize the carrageenan induced inflammatory pain model to identify anti- hyperalgesic EpFAs. The bioactivity of anti-nociceptive EpFAs will be validated in a clinically relevant model, the incisional pain model. We will use a combination of behavioral assays, biochemical and metabolomic analysis to understand the interactions of EpFAs with nociceptive pathways. We will produce a characteristic/diagnostic LC/MS/MS generated metabolomic profile of the intraplantar carrageenan and the plantar incisional pain models in rats. The incisional pain in particular is less well studied in regard to alterations in bioactive lipid metabolites. The target metabolites (80+ bioactive lipids) are from all three branches of the AA cascade including major cox, lox, cytochrome P450 and the sEH products including lipid metabolites of EPA and DHA, allowing us to investigate metabolite flux and crosstalk between biological cascades. The proposed studies will result in an advanced understanding of the antinociceptive mechanism of action of EpFAs and sEH inhibitors, and provide novel insights to the underlying mechanisms of lipidomic and biochemical alterations in response to pain. The knowledge generated by these experiments is likely to lead to the development of novel classes of agents that target pathways that have not been recognized previously.