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学治疗剂，广泛用于治疗各种恶性肿瘤的患者。严重的副作用，包括可以使用的肾毒性，神经毒性和耳毒性极限剂量。尽管已经取得了进展来限制肾毒性，但耳毒性继续损害癌症幸存者的生活质量。该应用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，以最大程度地提高疗效和最大程度地减少毒性。提出的研究将利用由不朽的耳蜗开发的毛线细胞系与现场动物实验结合使用。这些研究将系统地表征关键酶在耳蜗中的作用，即NADPH氧化。它产生超级氧化物和其他自由基，可以激活凋亡途径中的下游效应子，从而导致毛细胞死亡和听力损失。当前支持时期的数据表明，NADPH在龙猫耳蜗中氧化，并且这种酶被声学创伤大大激活。对OC-K3细胞系的毛细胞进行的实验表明，这些酶存在于这些细胞中，并通过顺铂暴露强烈激活。拟议的研究将阐明顺铂诱导的NADPH氧化，下游信号传导的激活模式，从而导致细胞凋亡以及保护耳蜗的内源性和外源性化合物的相互作用。我们还将探索另外两种酶的作用，这些酶在顺铂暴露后可能会在耳蜗中产生自由基：诱导型一氧化氮合酶和黄嘌呤氧化。这些研究将针对四个具体目的：1a）检查在耳蜗组织中NADPH氧化的特征； 1b）阐明了通过顺铂氧化的耳蜗纳德剂的激活机制； 2）检查耳蜗中腺苷A1受体（A1AR）活化的细胞保护作用； 3）检查自然产物Gingko Biloba（EGB 761）及其成分，Terrenes和Terrenes和Falconoid的标准化提取物，对顺铂诱导的耳毒性的保护机制； 4）为了阐明顺铂使用p53抑制剂pifithrin引起毛细胞死亡的p53激活的作用。实验最初将在毛细胞系中进行，然后通过体内实验使用局部和全身给药，并使用顺铂和保护剂进行了实验。毛细胞将使用免疫细胞化学的自由基生成酶进行测试，并为iNOS和蛋白质kinas C，rt-PCR，NADPH氧化亚基的RT-PCR进行蛋白质印迹，以及用于氧化氧化氧化物的Lucien分析。 NF-kappab的测定将使用电泳迁移率转移测定法，对A1 AR，T-BID，BAX，BCL-2，细胞色素C，Caspase-3和Annexin v的LMMUNOCYTO染色以及在组织区上的ANCEPASE-3和ANNEXIN V，以及使用毛细胞系的流式囊肿仪。这些实验的结果应提供有关顺铂诱导的耳毒性机制和化学预防的新方法的新见解。