ATP调控组蛋白乙酰化在噪声性听力损伤的机制研究

Apoptosis of outer hair cells (OHC) in basal part of cochlea is a typical pathology of noise-induced hearing loss (NIHL). However, the mechanism of apoptosis of OHC remains unclear. Our preliminary results show that: 1) in noise-induced permanent threshold shift (PTS) mice model (Broad band noise, 2-20 KHz, 106 dB SPL, 120 min), intracellular adenosine 5'-triphosphate (ATP) of OHC decreases after 30 min noise exposure; 2) in the mean time, the acetylation of Histone H3K9 in OHC decreases significantly; 3) Histone deacetylase (HDAC) inhibitor sodium butyrate (SB) significantly attenuates NIHL when applied before noise exposure as determined by auditory brainstem response (ABR) test.. In cell nucleus, ATP is required for ATP-citrate lyase (ACL)-dependent production of Acetyl Coenzyme A, which is a necessary substrate for Histone acetylation in nucleus by enzyme Histone acetyltransferase (HAT). HAT transfers acetyl group from Acetyl Coenzyme A to Histone promoting gene expression. Another type of enzyme, Histone deacetylase (HDAC), removes acetyl from Histone inhibiting gene expression. Histone acetylation is an important epigenetic modification modulating gene expression. Histone deacetylation, caused by decreasing of HAT, increasing of HDAC, or insufficient pool of Acetyl Coenzyme A, play an important role in neuronal cell death and aminoglycoside-induced cochlea hair cell apoptosis.. Base on our preliminary results, we therefor propose that in the early stage of noise exposure, decreased intracellular ATP causes insufficient pool of Acetyl Coenzyme A in nucleus, induces Histone deacetylation, down-regulates the expression of cell survival genes, and results in apoptosis of OHC. To examine our hypothesis we propose three specific aims by using mouse model of NIHL and organ of Corti organotypic culture model of ATP inhibition. In specific aim 1, we will examine how intracellular ATP decrease afftects Histone acetylation in OHC. In specific aim 2, we will examine how Histone deacetylation, by using HDAC inhibitor, affects apoptosis and survival of OHC. In specific aim 3, we will screen cell survival genes affected by Histone H3K9 deacetylation in OHC. Results from these experiments promise to significantly contribute to our understanding of how noise exposure induces apoptosis of OHC via ATP decrease-induced Histone deacetylation, and will aid in the design of new and effective prophylactic intervention in NIHL.

耳蜗外毛细胞凋亡是噪声性听力损伤的典型病理改变，但其机制尚未阐明。我们前期研究发现，噪声暴露早期耳蜗底转外毛细胞内ATP下降、组蛋白H3K9的乙酰化水平显著降低；组蛋白去乙酰化酶抑制剂可以预防噪声性听力损伤。由于细胞核内以ATP为底物合成的乙酰辅酶A为组蛋白乙酰化修饰提供乙酰基，我们假设，噪声暴露早期耳蜗外毛细胞内ATP下降导致细胞核内乙酰辅酶A生成受阻，组蛋白乙酰化水平降低，关键基因表达下调，触发细胞凋亡。本课题拟通过小鼠噪声性听力损伤和体外Corti器ATP抑制模型，研究ATP下降对耳蜗外毛细胞组蛋白乙酰化水平的影响；应用组蛋白去乙酰化酶抑制剂、高通量基因分析方法，研究组蛋白去乙酰化对耳蜗外毛细胞凋亡的影响，筛选获得受组蛋白去乙酰化影响显著下调的基因。本课题旨在阐明ATP下降-组蛋白去乙酰化-外毛细胞凋亡这一机制在噪声性听力损伤中的作用，为噪声性耳聋临床干预新方法的建立奠定理论基础。

耳蜗外毛细胞凋亡是噪声性听力损伤的主要病理改变，但其损伤机制尚未阐明。本项目通过永久性阈移噪声性听力损伤小鼠动物模型和体外培养耳蜗Corti器基底膜腺嘌呤核苷三磷酸（ATP）剥夺模型，研究噪声暴露早期耳蜗外毛细胞内ATP下调导致组蛋白的乙酰化水平下降，组蛋白去乙酰化酶（HDAC）抑制剂通过恢复组蛋白乙酰化水平预防噪声引起的耳蜗外毛细胞损伤。分光光度法检测发现在噪声暴露条件下耳蜗外毛细胞内ATP水平下降，106 dB SPL广谱噪声（2-20 KHz）条件下噪声暴露0、30、60和120 分钟后耳蜗组织ATP值分别为15.5×10-7 M，8.5×10-7 M，3.1×10-7 M和3.5×10-7 M。间接免疫荧光染色法同时发现耳蜗外毛细胞内AMPKα蛋白磷酸化水平升高。蛋白免疫印迹法发现同样条件噪声暴露30分钟开始耳蜗组织细胞内核心组蛋白H2A，H3，H4乙酰化水平开始下降，噪声暴露后60分钟开始耳蜗外毛细胞内核心组蛋白H2B乙酰化水平开始下降，并以组蛋白H3为例行间接免疫荧光染色法检测细胞特异性，发现噪声暴露30分钟后耳蜗外毛细胞内组蛋白H3乙酰化水平开始下降。同时，蛋白免疫印迹法发现噪声暴露30分钟后耳蜗组织内活化型Caspase3水平上升。采用蛋白印迹法和间接免疫荧光染色法意外发现噪声暴露后耳蜗组织内HDAC1和HDAC4水平上调，噪声暴露30分钟后耳蜗外毛细胞内HDAC4水平上升。分光光度法检测体外培养耳蜗Corti器基底膜在1 μM寡霉素作用下2小时开始细胞内ATP水平下降，蛋白免疫印迹法发现AMPKα蛋白磷酸化水平升高，蛋白免疫印迹法和间接免疫荧光染色法发现耳蜗外毛细胞内核心组蛋白H3乙酰化水平同时下调。荧光染色发现在这个体外培养条件下8小时外毛细胞出现纤毛明显紊乱和毛细胞缺失。荧光染色和扫描电镜发现HDAC抑制剂环庚烷异羟肟酸（SAHA）可减少强噪声暴露引起的耳蜗毛细胞丢失。SAHA可减轻耳蜗外毛细胞损伤程度及各个频率上减轻强噪声引起脑干诱发电位（ABR）反应阈移。本项目阐明一个新的噪声性听力损伤信号转导机制：噪声暴露后ATP降低—组蛋白乙酰化水平下降—耳蜗毛细胞凋亡。本项目具有潜在的临床应用前景：研究发现HDAC抑制剂可以预防噪声性听力损伤，已知效力温和的HDAC抑制剂丁酸钠可以通过膳食纤维消化获得，有望通过提高膳食中纤维的含量预防噪声性听力损伤。