ENDOGENOUS MODULATION OF COCHLEA INJURY

耳蜗损伤的内源性调节

• 批准号: 6873746
• 负责人: LEONARD P RYBAK
• 金额: $ 32.53万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY SCH OF MED
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6873746
• 关键词: NAD(P)H dehydrogenase; acoustic nerve; biological signal transduction; chemoprevention; chinchilla; cisplatin; cochlea; ear hair cell; ear pharmacology; enzyme activity; flavonoids; flow cytometry; free radicals; gel mobility shift assay; immunocytochemistry; injury; laboratory rat; nitric oxide synthase; ototoxin; oxidative stress; polymerase chain reaction; purinergic receptor; terpenes; western blottings; xanthine oxidase

DESCRIPTION (provided by applicant): Cisplatin is a potent chemotherapeutic agent widely used to treat patients with a variety of malignant neoplasms. Severe side effects including nephrotoxicity, neurotoxicity and ototoxicity limit doses that can be used. Although progress has been made to limit nephrotoxicity, ototoxicity continues to compromise the quality of life of cancer survivors. Experiments outlined in this application seek to continue to define the mechanisms of cisplatin ototoxicity in order to find rational therapeutic approaches to maximizing efficacy and minimizing toxicity. Studies proposed will utilize hair cell lines developed from the Immortomouse cochlea in combination with live animal experiments. These investigations will systematically characterize the role of a key enzyme in the cochlea, NADPH oxidize. It generates super oxide and other free radicals that can activate downstream effectors in the apoptotic pathway, leading to hair cell death and hearing loss. Data from the current period of support demonstrate the presence of NADPH oxidizes in the chinchilla cochlea, and that this enzyme is dramatically activated by acoustic trauma. Experiments with hair cells of the OC-k3 cell line show that the enzyme is present in these cells and is strongly activated by cisplatin exposure. The proposed research will elucidate the modes of cisplatin-induced activation of NADPH oxidize, downstream signaling which lead to apoptosis and the interaction of endogenous and exogenous compounds that protect the cochlea. We will also explore the roles of two other enzymes that could generate free radicals in the cochlea following cisplatin exposure: inducible nitric oxide synthase and xanthine oxidize. These investigations will address four specific aims: 1A) to examine the characteristics of NADPH oxidize in cochlear tissues; 1B) to elucidate the mechanisms of activation of cochlear NADPH oxidize by cisplatin; 2) to examine the cytoprotective effect of adenosine A1receptor (A1AR) activation in the cochlea; 3) to examine the mechanisms of protection against cisplatin-induced ototoxicity by the standardized extract of the natural product, Gingko biloba (Egb 761) and its components, the terrenes and falconoid; 4) to elucidate the role of p53 activation by cisplatin in causing hair cell death using the p53 inhibitor pifithrin. Experiments will be carried out in hair cell lines initially, and then confirmed by in vivo experiments using local and systemic administration of cisplatin and protective agents. Hair cells will be tested for free radical generating enzymes using immunocytochemistry and Western blotting for INOS and protein kinas C, RT-PCR for the subunits of NADPH oxidize, and Lucien assay for xanthenes oxides activity. Assays for NF-kappaB will be performed using the electrophoretic mobility shift assay, lmmunocytochemical staining for A1 AR, t-BID, Bax, Bcl-2, cytochrome C, caspase- 3, and Annexin V on tissue sections and using flow cytometry with the hair cell lines. The results of these experiments should provide novel insights into the mechanisms of cisplatin-induced ototoxicity and new methods for chemoprevention.

描述（由申请人提供）：顺铂是一种有效的化疗药物，广泛用于治疗各种恶性肿瘤患者。严重的副作用包括肾毒性、神经毒性和耳毒性，限制了可以使用的剂量。虽然在限制肾毒性方面取得了进展，但耳毒性继续损害癌症幸存者的生活质量。本申请中概述的实验试图继续定义顺铂耳毒性的机制，以找到合理的治疗方法来最大化功效和最小化毒性。拟议的研究将利用从永生鼠耳蜗开发的毛细胞系与活体动物实验相结合。这些研究将系统地描述耳蜗中关键酶NADPH氧化的作用。它产生超氧化物和其他自由基，可以激活凋亡途径中的下游效应器，导致毛细胞死亡和听力损失。从目前的支持期间的数据表明，NADPH氧化的存在下，在灰鼠耳蜗，这种酶是显着激活声创伤。用OC-k3细胞系的毛细胞进行的实验表明，该酶存在于这些细胞中，并且通过顺铂暴露而被强烈激活。拟议的研究将阐明顺铂诱导的NADPH氧化激活的模式，导致细胞凋亡的下游信号传导以及保护耳蜗的内源性和外源性化合物的相互作用。我们还将探讨其他两种酶的作用，可以产生自由基在耳蜗顺铂暴露后：诱导型一氧化氮合酶和黄嘌呤氧化。本研究的主要目的是：（1）研究耳蜗组织NADPH氧化的特点，（2）研究顺铂激活耳蜗NADPH氧化的机制，（3）研究腺苷A1受体（A1 AR）激活对耳蜗细胞的保护作用，（4）研究顺铂对耳蜗NADPH氧化的影响。3）研究银杏叶标准提取物（Egb 761）及其组分对顺铂耳毒性的保护机制，terrenes和falconoid; 4）阐明顺铂激活p53在使用p53抑制剂pifithrin引起毛细胞死亡中的作用。实验最初将在毛细胞系中进行，然后通过使用顺铂和保护剂的局部和全身给药的体内实验进行确认。将使用免疫细胞化学和蛋白质印迹法检测毛细胞中的自由基生成酶（INOS和蛋白激酶C），使用RT-PCR检测NADPH氧化亚基，使用Lucien试验检测氧杂蒽活性。将使用电泳迁移率变动试验、组织切片上的A1 AR、t-BID、Bax、Bcl-2、细胞色素C、半胱天冬酶-3和膜联蛋白V的免疫细胞化学染色以及使用毛细胞系的流式细胞术进行NF-κ B的试验。这些实验的结果应该提供新的见解顺铂诱导的耳毒性和化学预防的新方法的机制。