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.

可溶性环氧化物水解酶和环氧化物脂肪酸参与氨暴露后角膜损伤：损伤机制和使用 sEH 抑制剂和生物稳定 EpFA 模拟物的潜在治疗方法。

• 批准号: 10708436
• 负责人: BRUCE D HAMMOCK
• 金额: $ 48.42万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10708436
• 关键词: Acids; Acute; Adverse effects; Alkalies; Ammonia; Anti-Inflammatory Agents; Blindness; Cell Culture Techniques; Cell Death Induction; Cells; Cicatrix; Cornea; Corneal Injury; Data; Defect; Development; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Epithelium; Epoxide hydrolase; Epoxy Compounds; Exposure to; Eye; Fatty Acids; Fibrosis; Fluorescein; Flushing; Goals; Human; Hydration status; Immune; Immunohistochemistry; In Vitro; Inflammation; Inflammatory; Injury; Liquid substance; Location; Mass Spectrum Analysis; Measures; Mixed Function Oxygenases; Modeling; Mus; Pathologic; Pathology; Pathway interactions; Phase Ia Trial; Polyunsaturated Fatty Acids; Prevention; Proteins; Reporting; Role; Signal Pathway; Stains; Testing; Therapeutic; Therapeutic Uses; Time; Topical application; Ulcer; Wound models; analog; burn model; burn wound; chemical stability; clinical translation; corneal epithelial wound healing; corneal epithelium; design; epithelial wound; healing; histopathological examination; human tissue; improved; in vitro Model; in vivo; inhibitor; interest; liquid chromatography mass spectroscopy; model development; neovascularization; novel; response; response to injury; tissue culture; wound; wound healing

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.

氨溶液（20%）（AMM）具有高毒性，是CCRP感兴趣的物质。急性氨中毒的主要目标 暴露的是角膜。角膜氨暴露导致碱性伤口，这可能导致大量的 潜在的致盲病理特征和经常的眼睛损失。很少有研究描述AMM 角膜损伤和导致这些损伤的机制，没有建立模型进行这样的研究。 我们假设，导致AMM诱导的角膜病变的重要途径是 碱烧伤伤口是环氧脂肪酸（EpFA）通过可溶性环氧化物羟化酶（sEH）的快速水合。 EpFA是长链CYP450的抗炎、抗炎和抗纤维化代谢产物， 多不饱和脂肪酸因此，AMM损伤诱导的EpFAs的sEH水合破坏了细胞的天然代谢。 角膜中存在的炎症消退和抗纤维化途径。此外，我们假设AMM 使用局部应用的sEH抑制剂（sEHI）和/或天然的sEH抑制剂， EpFA如Escherichia coli，包括我们实验室开发和合成的新型sEHI和EpFA模拟物。 该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暴露造成损伤的机制，并开发潜在的 用于治疗AMM诱导的伤口的治疗剂。鉴于AMM伤口至少部分是由于暴露于 这些治疗剂可能具有广泛的应用。该项目的具体目标是： 目标1.开发新的、可靠的体外和体内小鼠角膜AMM损伤模型来验证这一假设 sEH活性降低小鼠角膜AMM暴露后EpFA浓度，并确定 对照角膜和AMM损伤后角膜中sEH的细胞定位。 目标2.检验AMM诱导的角膜损伤可以通过局部使用 应用sEHI和/或EpFA。 目标3.定量人角膜中的sEH和EpFA水平并形成可重复的人角膜AMM损伤 在培养的原代人角膜细胞中检查AMM暴露对sEH和EpFA的影响的模型 和离体供体角膜边缘。 体内小鼠伤口愈合研究和细胞培养模型将在Watsky博士的实验室中进行， 模型目前正在开发中，沿着免疫组织化学研究。sEH和EpFA定量 将在Hammock博士的实验室通过ELISA和质谱进行分析。待测治疗药物 局部应用于AMM损伤的角膜将包括在Hammock博士的 Hammock和维克博士实验室设计和合成的生物稳定的EpFA模拟物。人体组织 和细胞培养AMM暴露模型的开发将在Watsky博士的实验室使用供体角膜进行 轮辋和从这些轮辋中培养的细胞，这些轮辋通常在实验室中获得和使用。