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剂，广泛用于治疗多种恶性肿瘤患者。包括肾毒性、神经毒性和耳毒性在内的严重副作用限制了可使用的剂量。尽管在限制肾毒性方面已取得进展，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旨在继续确定顺铂耳毒性的机制，以便找到最大化疗效和最小化毒性的合理治疗方法。拟议的研究将利用从永生鼠耳蜗中开发的毛细胞系并结合活体动物实验。这些研究将系统地表征耳蜗中关键酶 NADPH 氧化的作用。它产生超氧化物和其他自由基，可以激活细胞凋亡途径中的下游效应器，导致毛细胞死亡和听力损失。当前支持期间的数据表明，龙猫耳蜗中存在 NADPH 氧化，并且这种酶会因声损伤而显着激活。对 OC-k3 细胞系的毛细胞进行的实验表明，这种酶存在于这些细胞中，并且通过暴露于顺铂而被强烈激活。拟议的研究将阐明顺铂诱导的 NADPH 氧化激活模式、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。我们还将探讨顺铂暴露后在耳蜗中产生自由基的另外两种酶的作用：诱导型一氧化氮合酶和黄嘌呤氧化酶。这些研究将解决四个具体目标：1A）检查耳蜗组织中 NADPH 氧化的特征； 1B) 阐明顺铂激活耳蜗NADPH氧化的机制； 2）检查耳蜗中腺苷A1受体（A1AR）激活的细胞保护作用； 3) 研究天然产物银杏叶（Egb 761）及其成分土烯类和黄酮类化合物的标准化提取物对顺铂引起的耳毒性的保护机制； 4) 使用p53抑制剂pifithrin阐明顺铂激活p53在导致毛细胞死亡中的作用。实验首先在毛细胞系中进行，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证实。将使用免疫细胞化学和蛋白质印迹法检测 INOS 和蛋白激酶 C、RT-PCR 检测 NADPH 氧化亚基，并使用 Lucien 测定检测呫吨氧化物活性，检测毛细胞中自由基生成酶的情况。将使用电泳迁移率变动测定、组织切片上的 A1 AR、t-BID、Bax、Bcl-2、细胞色素 C、caspase-3 和膜联蛋白 V 免疫细胞化学染色以及对毛细胞系使用流式细胞术进行 NF-kappaB 测定。这些实验的结果应该为顺铂诱导的耳毒性机制和化学预防的新方法提供新的见解。