Steroid Responsive Mechanisms in the Ear

项目摘要 糖皮质激素（泼尼松、甲泼尼龙、地塞米松）已被用于控制数十年 听力损失然而，很少有人知道的细胞和分子过程的耳朵，是在他们的 控制了解这些激素反应机制对于我们理解正常耳蜗的 功能，以及设计适当的临床治疗。因此，本研究的长期目标是 以充分表征耳朵的类固醇驱动的细胞和分子机制。这项研究的进展 表明MRL/MpJ-Faslpr自身免疫小鼠的听力损失对糖皮质激素和 泼尼松龙和盐皮质激素醛固酮。此外，研究表明，糖皮质激素与 对糖皮质激素受体和盐皮质激素受体的亲和力相等，从而调节耳蜗基因 分别通过免疫抑制和离子稳态表达。因此，我们的工作假设是， 耳中存在两种类固醇反应机制：钠和钾的直接转运（离子稳态） 盐皮质激素受体介导的基因表达，以及间接的炎症基因抑制 糖皮质激素受体介导的机制。计划的研究将描述这些类固醇驱动的 用类固醇治疗耳中的细胞和分子过程，其将在功能上分离受体， 测量它们控制的体内平衡和炎症基因表达。调查的具体目的 耳朵的这些类固醇驱动机制是： 目的1：确定内耳离子稳态和炎症基因表达的剂量依赖性控制， 盐皮质激素醛固酮和糖皮质激素泼尼松龙。 目的2：确定内耳离子稳态和炎症基因表达的最有效控制 两种类固醇的联合剂量。 目的3：确定耳蜗的细胞和分子功能是由每个类固醇受体介导的。 目的4：确定是否有效的内耳稳态和抗炎基因表达可以诱导 中耳类固醇递送。 在所有研究中，1）内耳功能将通过听性脑干反应测听术进行评估， 耳蜗内电位; 2）将通过光学和电子显微镜评估内耳形态; 3）全身 自身免疫性疾病将通过血清免疫复合物、血细胞比容和抗核抗体进行评估; 4） 耳蜗特异性自身抗体将用ELISA进行评估，5）类固醇介导的耳蜗基因产物将 用ELISA、Western印迹、细胞因子RNA表达和定量RT-PCR进行评估。的结果 这些研究将提供关于耳朵的细胞和分子机制的新发现， 类固醇的控制这也将为类固醇治疗的潜在发展奠定重要基础 比目前使用的更有效。项目叙述 本研究旨在评估类固醇控制维持内分泌的机制， 耳内稳态研究旨在更好地了解糖皮质激素的作用， 盐皮质激素在正常内耳基因表达中的作用，以及类固醇治疗如何影响内耳基因表达。 可以逆转听力损失具有自身免疫性内耳疾病的小鼠模型（MRL/lpr）， 类固醇反应性疾病，将被用来区分离子稳态和免疫 类固醇的抑制功能和两种类固醇组合的功效。最后 将比较中耳注射与全身递送的功能影响，以确定 每种方法控制糖皮质激素和盐皮质激素受体介导基因的能力 在耳蜗中的表达。特别感兴趣的是炎症细胞因子的免疫抑制 和相关的转录因子相比，控制离子转运途径的调节 体液平衡

Project Summary Glucocorticoids (prednisone, methlyprednisolone, dexamethasone) have been employed for decades for control of hearing loss. However, little is known of the cellular and molecular processes of the ear that are under their control. Knowledge of these steroid-responsive mechanisms is critical for our understanding of normal cochlear function, as well as the design of appropriate clinical therapies. Therefore, the long term goal of this research is to fully characterize the steroid-driven cellular and molecular mechanisms of the ear. Progress on this study has shown that hearing loss in the MRL/MpJ-Faslpr autoimmune mouse responds to both the glucocorticoid prednisolone and the mineralocorticoid aldosterone. Furthermore, studies have shown glucocorticoids bind with equal affinity to both the glucocorticoid receptor and the mineralocorticoid receptor, thus regulating cochlear gene expression via immune suppression and ion homeostasis, respectively. Therefore, our working hypothesis is that two steroid-responsive mechanisms exist in the ear: a direct sodium and potassium transport (ion homeostatic) gene expression mediated by the mineralocorticoid receptor, and an indirect inflammatory gene suppression mechanism mediated by the glucocorticoid receptor. The planned studies will characterize these steroid driven cellular and molecular processes in the ear with steroid treatments that will functionally isolate the receptors and measuretheionhomeostaticand inflammatory gene expression they control. The specific aims to investigate these steroid driven mechanisms of the ear are: Aim 1: Determine the dose-dependent control of inner ear ion homeostatic and inflammatory gene expression by the mineralocorticoid aldosterone and the glucocorticoid prednisolone. Aim 2: Determine the most effective control of both inner ear ion homeostatic and inflammatory gene expression processes by combination doses of the two steroids. Aim 3: Determine which cochlear cellular and molecular functions are mediated by each steroid receptor. Aim 4: Determine if effective inner ear homeostatic and anti-inflammatory gene expression can be induced by middle ear steroid delivery. In all studies, 1) inner ear function will be assessed by auditory brainstem response audiometry and endocochlear potential; 2) inner ear morphology will be assessed by light and electron microscopy; 3) systemic autoimmune disease will be assessed by serum immune complexes, hematocrits, and antinuclear antibodies; 4) cochlearspecificautoantibodies willbeassessedwithELISA,and5)steroid-mediatedcochleargeneproducts will be assessed with ELISA, Western blot, cytokine RNA expression, and quantitative RT-PCR. The results from these studies will provide new findings regarding the cellular and molecular mechanisms of the ear that are under the control of steroids. This also will lay important groundwork for the potential development of steroid therapies more effective than those currently employed. Project Narrative A study is proposed to evaluate the mechanisms by which steroids control the maintenance of inner ear homeostasis. Studies are designed to better understand the role of glucocorticoids and mineralocorticoids in normal inner ear gene expression, and how steroid treatments may be operating to reverse hearing loss. Mouse models (MRL/lpr) with autoimmune inner ear disease, a steroid responsive disorder, will be employed to differentiate the ion homeostasis and immune suppressive functions of steroids and the efficacy of the two steroid classes combined. Finally, the functional impact of middle ear injections versus systemic delivery will be compared to determine the ability of each method to control glucocorticoid and mineralocorticoid receptor-mediated gene expression in the cochlea. Of particular interest are immune suppression of inflammatory cytokines and related transcription factors compared to the regulation of ion transport pathways that control fluid homeostasis.