Soluble epoxide hydrolase and epoxide fatty acid involvement in corneal injury after ammonia exposure: Mechanisms of injury and potential therapeutics using sEH inhibitors and biostable EpFA mimics.

氨水(20%)(AMM)是剧毒的，与CCRP有关。急性氨中毒的主要目标 暴露在眼底的就是角膜。角膜氨气暴露会导致碱创伤，这可能会导致大量 潜在致盲的病理特征，经常失去眼睛。很少有研究描述AMM 关于角膜损伤和导致这些损伤的机制，目前还没有确定的模型来进行这类研究。 我们假设导致AMM诱导的角膜病变的一个重要途径 碱烧伤创面是由可溶性环氧化物羟基酶(SEH)催化环氧脂肪酸(EpFA)快速水合而成。 EpFAs是一种抗炎、消炎、抗肝纤维化的长链代谢产物 多不饱和脂肪酸。因此，AMM损伤诱导的EPFAs的sEH水化破坏了自然的 角膜中存在消炎和抗纤维化的途径。此外，我们假设AMM 使用局部应用sEH抑制剂(SEHI)和/或天然药物可以最大限度地减少或解决角膜损伤 EPFA，如EETs，包括新的sEHI和EpFA模拟物，由我们的实验室开发和合成。 这个项目的长期目标有几个方面，总体目标是获得更好的 了解角膜AMM暴露造成的损伤背后的机制，并开发潜力 治疗AMM所致伤口的治疗方法。鉴于AMM的伤口至少部分是由于接触了 这些疗法都是极碱性药物，具有广阔的应用前景。该项目的具体目标是： 目的1.建立新颖、可靠的体外和体内小鼠角膜AMM损伤模型，以验证这一假说 SEH活性降低了小鼠角膜AMM暴露后的EpFA浓度，并确定了 角膜基质损伤后和对照角膜中sEH的细胞定位。 目的2.检验AMM引起的角膜损伤可以通过局部用药减轻或解决的假设 应用sEHI和/或EPFA。 目的3.定量测定人角膜中sEH和EpFA的水平并建立可重复的人角膜AMM损伤 AMM暴露对原代培养人角膜上皮细胞sEH和EpFAs影响的模型研究 和体外供体角膜缘。 体内小鼠伤口愈合研究和细胞培养模型将在沃茨基博士的实验室进行，使用 目前正在开发的模型，以及免疫组织化学研究。SEH和EpFA定量 分析将在Hammock博士的实验室通过ELISA法和质谱仪进行。治疗药物将接受测试 局部应用于AMM损伤的角膜将包括由Hammock博士合成的sEHIs和EpFA Lab，以及在Hammock和Vic博士的实验室设计和合成的生物稳定的EpFA模拟物。人体组织 细胞培养AMM暴露模型的开发将在Watsky博士的实验室进行，使用捐献的角膜 轮缘和从这些轮缘培养出来的细胞，通常在实验室中获得和使用。

Ammonia solution (20%) (AMM) is highly toxic and of interest to the CCRP. A major target for acute ammonia exposure is the cornea. Corneal ammonia exposure results in an alkali wound, which can lead to a host of potentially blinding pathologic features and often times loss of the eye. There are few studies describing AMM cornea wounds and the mechanisms leading to those injures, with no established model to carry out such studies. We hypothesize that a significant pathway leading to pathologies associated with AMM-induced corneal alkali burn wounds is the rapid hydration of epoxy fatty acids (EpFA) by soluble epoxide hydroxylase (sEH). EpFAs are anti-inflammatory, inflammation-resolving and anti-fibrotic CYP450 metabolites of long chain polyunsaturated fatty acids. Consequently, AMM injury-induced sEH hydration of EpFAs disrupts the natural inflammation resolving and anti-fibrotic pathways present in the cornea. Moreover, we hypothesize that AMM corneal injuries can be minimized or resolved using topically applied sEH inhibitors (sEHI) and/or natural EpFAs such as EETs, including novel sEHI and EpFA mimics developed and synthesized in our labs. The long-term objectives of this project are several fold, with the overall goals being to gain a better understanding of the mechanisms behind injuries created by corneal AMM exposure, and to develop potential therapeutics to treat AMM-induced wounds. Given that AMM wounds are at least in part due to exposure to an extremely alkali agent, these therapeutics could have broad applications. The project Specific Aims are: Aim 1. Develop novel, reliable in vitro and in vivo mouse cornea AMM-injury models to test the hypothesis that sEH activity reduces EpFA concentrations following mouse cornea AMM exposure, and determine the cellular location of sEH in control corneas and in corneas following AMM injury. Aim 2. Test the hypothesis that AMM-induced corneal injuries can be diminished or resolved using topically applied sEHI and/or EpFAs. Aim 3. Quantify sEH and EpFA levels in human corneas and develop repeatable human cornea AMM-injury models to examine the effects of AMM exposure on sEH and EpFAs in cultured primary human corneal cells and ex vivo donor cornea rims. The in vivo mouse wound healing studies and cell culture models will be carried out in Dr. Watsky's lab using models currently in development, along with immunohistochemistry studies. sEH and EpFA quantitative analysis will be performed by ELISA and mass spectroscopy in Dr. Hammock's lab. Therapeutics to be tested and topically applied to AMM-wounded corneas will include sEHIs and EpFAs synthesized in Dr. Hammock's lab, and biostable EpFA mimics designed and synthesized in the labs of Drs. Hammock and Vic. Human tissue and cell culture AMM exposure model development will be carried out in Dr. Watsky's lab using donor cornea rims and cells cultured from those rims, which are routinely obtained and utilized in the lab.