PINK1在外源性胁迫条件致感音神经性聋中的作用机制研究

It has been documented that the abnormity of hair cells is considered as a pivotal determinant attributable to sensory hearing loss. The phosphatase and tensin homologue (PTEN)-induced putative kinase-1 (PINK1) gene is closely related to mitochondrial respiration regulation, ROS generation, mitophagy, intracellular transport, and nuclear signaling. Previously, we found that PINK1 was highly expressed in hair cell line HEI-OC1 and cochlear hair cells of murine inner ear and, also, that the exposure of cis-platinum to HEI-OC1 led to the Parkin accumulation on mitochondria and the occurrence of mitophagy. However, the precise mechanisms of PINK1 in the process of hair cell damage are largely undeciphered to date. On the basis of the previous study, the present project was designed, using the wild type and PINK1-knock out mice as well as HEI-OC1 as experimental materials, to further investigate that: 1)whether PINK1 is closely related to the development of inner ear and the formation of hearing; 2) whether PINK1 can mediate the repair of the damaged mitochondria via the activation of two pathways of MDV and mitophagy, whereby exerting the protective role against the hearing impairment; and 3) whether the up-regulation of PINK1 expression can influence the p-JNK and P53 pathways as well as the endoplasmic reticulum stress, thereby enhancing resistance of hair cells to exogenous stresses. This will be helpful for elucidating the mechanism underlying the action of PINK1 in the pathogenesis of hair cell damage, which might, in turn, provide a promising therapeutic target for the sensory hearing loss.

毛细胞功能障碍是感音性耳聋病理发生的的关键因素。研究表明，同源性磷酸酶-张力蛋白诱导的激酶1（PINK 1）与线粒体呼吸、ROS生成、线粒体自噬、等密切相关。 我们前期发现，PINK1在小鼠耳蜗毛细胞及HEI-OC1毛细胞系中高表达，且顺铂处理HEI-OC1细胞引起PARKIN在线粒体聚集和自噬。但PINK1在毛细胞损伤时的作用机制尚不清楚。本项目拟在前期基础上，应用野生型小鼠、PINK1基因敲除小鼠和HEI-OC1毛细胞系，进一步研究：1）PINK1是否与小鼠的内耳发育及听力形成相关；2）PINK1是否通过MDV和线粒体自噬两条途径介导的损伤线粒体修复，在听力损伤中起到重要的保护作用；3）上调PINK1的表达是否可影响p-JNK、P53信号通路及内质网应激，增加毛细胞对外源性胁迫的抵抗。本研究将有助于揭示PINK1在毛细胞损伤中的病理机制，进而为感音性耳聋的治疗提供新的作用靶点。

听觉损失是主要以内耳毛细胞损害为病理基础的最常见的感官缺陷疾病之一, 已经成为全球当前重大公共卫生问题。基于前期实验数据，我们提出同源性磷酸酶-张力蛋白诱导的激酶1（phosphatase and tensin homologue (PTEN)-induced putative kinase 1, PINK 1）可能参与外源性胁迫因素对耳蜗听觉细胞的损伤过程。.本课题的主要内容和研究结果如下：(1) 我们首次发现，PINK1在C57BL/6小鼠耳蜗HCs、SGNs、血管纹和HEI-OC1细胞的细胞质中广泛点状表达。 (2) 顺铂处理HEI-OC1细胞能够引起时间依赖性活性氧（ROS）的形成，线粒体膜电位的损伤；同时，顺铂可引起PINK1的表达变化及其下游parkin分子的聚集、线粒体自噬的形成和p-JNK相关的细胞凋亡。 (3)干扰PINK1表达，能减轻顺铂所致的PINK1信号通路的激活; 抑制JNK信号通路能够提高细胞存活率。(4)顺铂处理小鼠耳蜗基底膜，能引起毛细胞内ROS 水平增加、PINK1/parkin信号通路的激活、自噬及凋亡水平的增加；顺铂处理小鼠耳蜗窝轴，能引起SGNs内ROS 水平增加、线粒体膜电位的受损、PINK1/parkin信号通路的激活、自噬及凋亡水平的增加。 (5)庆大霉素处理HEI-OC1细胞能够引起时间依赖性ROS的形成和线粒体膜电位的损伤；庆大霉素处理HEI-OC1细胞能够引起PINK1的表达呈波动性变化。(6)庆大霉素处理小鼠耳蜗基底膜，能引起毛细胞内ROS 水平增加、线粒体膜电位的受损、PINK1/parkin信号通路的激活、自噬及凋亡水平的增加。本研究表明PINK1信号通路可以通过激活线粒体自噬及抑制p53信号通路，起到对抗顺铂及庆大霉素引起的细胞损伤作用。推断PINK1在抵抗耳毒性药物引起的损伤方面可能具有普遍性，为药物性聋病的治疗和预防提供了新的调控靶点。