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) 具有剧毒，CCRP 对此很感兴趣。急性氨的主要目标 暴露的是角膜。角膜氨暴露会导致碱伤，从而导致许多 可能导致致盲的病理特征，并且常常导致失明。描述 AMM 的研究很少 角膜损伤以及导致这些损伤的机制，目前还没有建立模型来进行此类研究。 我们假设，导致与 AMM 诱导的角膜相关的病理的重要途径 碱烧伤伤口是通过可溶性环氧化物羟化酶（sEH）快速水合环氧脂肪酸（EpFA）。 EpFA 是长链 CYP450 代谢物，具有抗炎、消炎和抗纤维化作用 多不饱和脂肪酸。因此，AMM 损伤诱导的 EpFAs sEH 水合破坏了天然的 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 sEH 抑制剂 (sEHI) 和/或天然药物可以最大程度地减少或解决角膜损伤 EpFA，例如 EET，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多个，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 引起的伤口的疗法。鉴于 AMM 伤口至少部分是由于暴露于 由于强碱剂，这些疗法可能具有广泛的应用。该项目的具体目标是： 目标 1. 开发新颖、可靠的体外和体内小鼠角膜 AMM 损伤模型来检验假设 小鼠角膜 AMM 暴露后 sEH 活性降低了 EpFA 浓度，并确定 对照角膜和 AMM 损伤后角膜中 sEH 的细胞位置。 目标 2. 检验以下假设：局部使用可减少或解决 AMM 引起的角膜损伤 应用 sEHI 和/或 EpFA。 目标 3. 量化人角膜中的 sEH 和 EpFA 水平并开发可重复的人角膜 AMM 损伤 用于检查 AMM 暴露对培养的原代人角膜细胞中 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 博士的实验室中使用 目前正在开发模型以及免疫组织化学研究。 sEH 和 EpFA 定量 分析将在 Hammock 博士的实验室中通过 ELISA 和质谱法进行。待测试的疗法 局部应用于 AMM 损伤的角膜将包括 Hammock 博士合成的 sEHI 和 EpFA 实验室，以及在博士实验室设计和合成的生物稳定 EpFA 模拟物。吊床和维克。人体组织 细胞培养 AMM 暴露模型开发将在 Watsky 博士的实验室中使用供体角膜进行 边缘和从这些边缘培养的细胞，这些细胞通常在实验室中获得和使用。