Evaluation of Endogenous TRP Agonists in Human Burns

描述（由申请人提供）：烧伤通常会导致持续性疼痛，可用的镇痛药无法很好地控制。烧伤疼痛的机制还不完全清楚。然而，TRPV 1（瞬时受体电位V1）受体似乎是枢转参与烧伤后疼痛。在我们的初步数据中，~67%的烧伤后热痛觉过敏被外周注射TRPV 1受体拮抗剂阻断。此外，烧伤的人皮肤提取物通过TRPV 1受体机制注射到大鼠体内时产生热痛觉过敏。这些新的数据表明，TRPV 1显着有助于热烧伤后损伤。有趣的是，TRPV 1激活的确切机制仍然未知。汇聚的证据表明，来自亚油酸或花生四烯酸的氧化脂质在组织损伤期间释放并激活TRPV 1和/或TRPA 1，导致伤害感受器去极化和疼痛。这些脂质中的许多是通过酶如细胞色素P450（CYP）或脂氧合酶（LOX）的作用形成的。这是特别重要的，因为对GLUE Grant的分析表明，编码这些酶的转录本早在烧伤后0-3天就升高了。因此，我们提出了一个中心假设，即氧化脂质在人体烧伤后酶促形成，并通过激活TRPV 1和/或TRPA 1促进烧伤后疼痛。具体目标1：鉴定氧化亚油酸形成激活TRPV 1或TRPA 1的代谢物的酶。我们已经分析了NIGMS支持的GLUE Grant微阵列数据库中烧伤皮肤与对照人类皮肤的转录物，发现CYP和LOX水平显著升高，持续长达12个月。本目标将使用COS表达系统来评估这些酶是否能够使用膜片钳电生理学将亚油酸氧化成TRPV 1或TRPA 1激动剂，并在临床前研究中确定这些化合物的功能活性。进一步的研究将评估这些脂质之间可能的环境相互作用，以激活TRP通道。具体目标2：鉴定氧化花生四烯酸形成激活TRPV 1或TRPA 1的代谢物的酶。目标2将使用与目标1相同的方法，但将侧重于花生四烯酸代谢物。这一新的假设具有很强的科学和医学意义。从科学的角度来看，它将TRPV 1的重点从瞬时热量的检测器扩展到热诱导损伤后很长时间内从烧伤皮肤释放的氧化脂质的检测器。从医学的角度来看，这一假设是创新的，因为它导致了一种全新的治疗烧伤疼痛的药理学方法-通过阻断烧伤后组织释放的TRPV 1和/或TRPA 1活性化合物的酶促合成。本提案旨在全面测试这一潜在的突破性假设的持续烧伤后疼痛。

DESCRIPTION (provided by applicant): Burns often lead to persistent pain not well managed by available analgesics. The mechanisms of burn pain are incompletely understood. However, the TRPV1 (transient receptor potential V1) receptor appears to be pivotally involved in post-burn pain. In our preliminary data, ~67% of post-burn thermal hyperalgesia is blocked by peripheral injection of a TRPV1 receptor antagonist. In addition, extracts of burned human skin produce thermal hyperalgesia when injected into rats via a TRPV1 receptor mechanism. These novel data suggest that TRPV1 significantly contributes to post-thermal burn injuries. Interestingly, the precise mechanism(s) for the activation of TRPV1 remains unknown. Converging evidence indicates that oxidized lipids derived from linoleic or arachidonic acid are released during tissue injury and activate TRPV1 and/or TRPA1, resulting in nociceptor depolarization and pain. Many of these lipids are formed by the actions of enzymes such as cytochrome P450s (CYP) or lipoxygenase (LOX). This is of particular significance since analysis of the GLUE Grant indicates that transcripts encoding these enzymes are elevated as early as 0-3 days after burns. Therefore, we propose the central hypothesis that oxidized lipids are enzymatically formed after human burn injuries and contribute to post-burn pain by activation of TRPV1 and/or TRPA1. The present Aims will: Specific Aim 1: Identify enzymes that oxidize linoleic acid to form metabolites that activate TRPV1 or TRPA1. We have analyzed the NIGMS-supported GLUE Grant microarray database of transcripts from skin with burn injury vs. control human skin and found significantly elevated levels of CYPs and LOXs that persist for up to 12 months. This Aim will use a COS expression system to evaluate whether these enzymes are capable of oxidizing linoleic acid into TRPV1 or TRPA1 agonists using patch clamp electrophysiology and determine the functional activity of these compounds in preclinical studies. Additional studies will evaluate possible entourage interactions among these lipids for activating TRP channels. Specific Aim 2: Identify enzymes that oxidize arachidonic acid to form metabolites that activate TRPV1 or TRPA1. Aim 2 will use the same approach as Aim 1, but will focus on arachidonic acid metabolites. This novel hypothesis has strong scientific and medical implications. From a scientific perspective, it expands the focus of TRPV1 from being a detector of transient heat, to a detector of oxidized lipids released from burned skin long after the heat-induced injury. From a medical perspective, this hypothesis is innovative since it leads to a fundamentally new pharmacological approach for treating burn pain - by blocking the enzymatic synthesis of TRPV1- and/or TRPA1-active compounds released from tissues after burns. The present proposal is designed to comprehensively test this potentially ground-breaking hypothesis of persistent post-burn pain.