噪声刺激致豚鼠外毛细胞凋亡时半胱氨酸天冬氨酸蛋白酶3激活和凋亡诱导因子转移

目的探讨噪声损伤后豚鼠外毛细胞发生凋亡的通路及其机制。方法分离解剖对照组（12只）和噪声暴露组（12只）豚鼠耳蜗，利用免疫荧光抗体和免疫荧光染料，分别染色细胞核、线粒体、凋亡诱导因子（apoptosis inducing factor，AIF）和半胱氨酸天冬氨酸蛋白酶3（Caspase 3），按表面制备法行耳蜗铺片，观察凋亡和坏死毛细胞内的荧光信号。结果对照组正常耳蜗毛细胞内没有激活的Caspase 3，AIF分布在毛细胞的线粒体内。实验组动物暴露于声压级120dB的白噪声环境中每天4h，连续2d后，耳蜗外毛细胞出现死亡，表现为坏死和凋亡两种方式，但以凋亡为主。在凋亡的外毛细胞中，Caspase 3被激活，AIF从线粒体转移到细胞核中；而在坏死的外毛细胞中，没有Caspase 3的激活，只有AIF自线粒体向细胞核的转移。结论噪声损伤后耳蜗外毛细胞的凋亡以Caspase依赖型的通路为主；线粒体释放的AIF在外毛细胞死亡通路中起重要作用。

Caspase 3 activation and apoptosis inducing factor translocation in noise exposure induced out hair cells apoptosis

OBJECTIVE: To investigate the pathway and mechanism of noise exposure induced out hair cells (OHC) apoptosis. METHODS: The cochleae of control and noise exposure group were dissected. The activity of caspase 3, an important mediator of apoptosis, in OHC, was examined with carboxyfluorescein-labeled fluoromethyl ketone (FMK)-peptide inhibitors. The apoptosis inducing factor (AIF) translocation from mitochondria in OHC were further examined by immunohistology method. The nuclei were labeled with PI and the mitochondrion was labeled with Mito-tracker. Whole mount organ of Corti was prepared. Morphological and fluorescent change was observed use confocal microscope. RESULTS: In the normal OHC, AIF is distributed where the mitochondria were located and no activated caspase 3 was observed. After the animals exposed to broadband noise at 122 dB in 4 h/day for 2 days, both apoptosis and necrosis were appeared in OHC. AIF translocated from mitochondrion to nuclei in apoptotic and necrotic OHC following noise exposure. The noise exposure triggered activation of caspase 3 in apoptic hair cells. But no caspase 3 activation appeared in necrotic OHC. CONCLUSIONS: These findings indicated that the caspase-dependent pathway is an important pathway in noise exposure induced apoptosis. And AIF also involves OHC death pathway following noise exposure.