噪声损伤引起耳蜗外毛细胞凋亡时细胞核内单链DNA的产生及EndoG的转移

ssDNA production and EndoG translocation in noise exposure induced hair cell death

目的观察噪声损伤后耳蜗外毛细胞内单链DNA和EndoG的变化,探讨耳蜗外毛细胞的死亡机制。方法豚鼠随机分为噪声暴露组、MNNG耳蜗灌流组和对照组(每组各12只);小鼠随机分为噪声暴露组和对照组(每组12只)。分离解剖耳蜗后,用碘化丙啶(PI)染色细胞核、Pholloidin染色F-actin,免疫荧光抗体分别染色单链DNA(ssDNA)、核酸内切酶G(Endonuclease G EndoG)和凋亡诱导因子(Apoptosis inducing factors,AIF),制备耳蜗铺片,激光共聚焦显微镜下观察凋亡和坏死毛细胞内的荧光信号变化。结果(1)暴露于120dBSPL的白噪声环境中每天4小时,连续2天后引起豚鼠和小鼠耳蜗外毛细胞凋亡时,其细胞核内产生ssDNA,而在正常细胞内没有三ssDNA;(2)在正常情况下,EndoG分布于耳蜗毛细胞的细胞核外,在暴露于上述噪声后发生凋亡和坏死的豚鼠耳蜗外毛细胞中,EndoG从细胞核外转移到细胞核内,细胞核中的EndoG显著增加;(3)豚鼠耳蜗外淋巴灌流烷化剂MNNG后发生耳蜗外毛细胞凋亡和坏死,在凋亡和坏死的耳蜗外毛细胞中,AIF自线粒体转移到细胞核,其变化与噪声损伤引起耳蜗外毛细胞凋亡和坏死时一致。结论噪声刺激或烷化剂MNNG灌流后,造成耳蜗外毛细胞DNA损伤,产生ssDNA,引起AIF和EndoG自线粒体释放,激活Caspase-3,AIF和EndoG进一步向细胞核转移,最终使细胞核内的DNA降解,导致耳蜗毛细胞的死亡。

Objective The changes of single strand DNA and EndoG were assessed to investigate the mechanism of noise exposure induced death of out hair cells （OHCs）. Methods The guinea pigs were divided into control, MNNG cochlear perfusion and noise exposure groups of 12 animals each . The mice were divided into control and noise exposure groups of 12 animals each. The cochleae of animals were dissected. Propidium iodide （PI）, a DNA intercalating fluorescent probe, was used to trace the morphological changes in OHC nuclei. F-actin staining was used to determine missing OHCs. The single strand DNA （ssDNA） in apoptotic OHCs of guinea pigs and mice, and apoptosis inducing factor （AIF） and Endonuclease G （EndoG） in OHCs of guinea pigs were examined by immunofluorescence method. Morphological and flourescent changes were assessed using confocal microscopy in whole-mount organ of Corti preparations. Results （1） After the animals were exposed to broadband noise at 122 dB SPL in 4 h/day for 2 days, both apoptosis and necrosis appeared in OHCs. The single strand DNA in apoptotic OHCs were observed both in guinea pigs and mice. （2） In normal OHCs, EndoG was distributed outside of nuclei. EndoG translocated from outside to inside of the nuclei in both apoptotic and necrotic OHCs following noise exposure. （3） The MNNG cochlear perfusion and noise exposure both caused the transloctation of AIF from the mitochondria to the nuclei. The translocation of AIF took place in both apoptotic and necrotic OHCs. Conclusion These findings indicate that both noise exposure and cochlear MNNG perfusion injure the nuclear DNA of OHCs, then the action of Caspase-3 is triggered, AIF and EndoG are translocated to nuclei, and finally, DNA is broken and death of OHCs induced.