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）有剧毒，CCRP感兴趣。急性氨的主要目标 暴露是角膜。角膜氨的暴露会导致碱性伤口，这可能导致大量 潜在地具有盲目的病理特征，并且通常会流失眼睛。描述AMM的研究很少 角膜伤口和导致这些伤害的机制，没有建立的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学的重要途径 碱烧伤是可溶性环氧羟化酶（SEH）迅速水合环氧脂肪酸（EPFA）。 EPFA是抗炎，解决炎症和抗纤维化CYP450长链的抗纤维化的CYP450代谢物 多不饱和脂肪酸。因此，AMM损伤引起的EPFA的SEH水合破坏了自然 角膜中存在炎症和抗纤维化途径。而且，我们假设AMM 可以使用局部施加的SEH抑制剂（SEHI）和/或天然来最大程度地减少角膜损伤 EPFA，例如EET，包括在我们的实验室中开发和合成的新型SEHI和EPFA模拟物。 该项目的长期目标是几折，总体目标是获得更好的 了解角膜AMM暴露造成的伤害背后的机制，并发展潜力 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。该项目的特定目的是： 目标1。开发新颖，可靠的体外和体内小鼠角膜AMM伤害模型以检验假设 SEH活性会降低小鼠角膜AMM暴露后EPFA浓度，并确定 AMM损伤后，SEH在对照角膜和角膜中的细胞位置。 AIM 2。检验以下假设，即可以使用局部减少或解决AMM引起的角膜损伤 应用的SEHI和/或EPFA。 AIM 3。量化人角膜中的SEH和EPFA水平并发展可重复的人角膜AMM伤害 模型检查AMM暴露对SEH和EPFA的影响在培养的原代人角膜细胞中 和离体捐赠者角膜边缘。 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 目前正在开发的模型以及免疫组织化学研究。 SEH和EPFA定量 分析将由ELISA和Hammock博士实验室中的质谱进行。要测试的治疗剂 并局部应用于AMM链角膜，包括Hammock博士中合成的SEHIS和EPFA 实验室，以及在DRS实验室中设计和合成的可生物可生物的EPFA模拟物。吊床和维克。人体组织 和细胞培养AMM暴露模型开发将在Watsky博士的实验室使用供体角膜进行 从那些轮辋中培养的边缘和细胞通常在实验室中获得和使用。