Otolaryngology-Head and Neck Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China; Department of Pathology and Pathophysiology, School of Medicine, Shandong University, Jinan, China.
Otolaryngology-Head and Neck Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China; Shandong Provincial Key Laboratory of Otology, Jinan, China.
Free Radic Biol Med. 2018 May 20;120:342-355. doi: 10.1016/j.freeradbiomed.2018.02.025. Epub 2018 Feb 17.
Phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1) gene encodes a serine/threonine kinase, which acts as a molecular sensor of mitochondrial health necessary for mitochondrial quality control. The present study was designed to examine whether PINK1 expressed in C57BL/6 murine cochlea and HEI-OC1 cells and, if so, to investigate the possible mechanisms underlying the action of PINK1 in cisplatin-induced death of sensory hair cells (HCs) and spiral ganglion neurons (SGNs) in vitro. The expression pattern of PINK1, formation of parkin particles, and autophagy were determined by immunofluorescent staining. The expressions of PINK1, LC3B, cleaved-caspase 3 and p-JNK were measured by Western blotting. The levels of reactive oxygen species (ROS) were evaluated by DCFH-DA and Mito-Sox Red staining. The mitochondrial membrane potential was detected by Tetramethylrhodamine methyl ester perchlorate (TMRM) and Rhodamine 123. Cell viability and apoptosis were examined by CCK8 assay, TUNEL staining and Annexin V Apoptosis Detection Kit, respectively. We found that PINK1 was widely expressed in the cytoplasm in HCs, SGNs, stria vascularis of C57BL/6 cochlea and HEI-OC1 cells and, notably, the expression level in cochlear HCs and SGNs of postnatal day 4 (P4) mice was higher than that in adult mice. Moreover, treatment with 30 μM cisplatin elicited the formation of ROS, which, in turn, led to PINK1 activation, parkin recruitment, autophagy formation and JNK pathway relevant to apoptosis in HEI-OC1 cells, HCs, and SGNs. Meanwhile, co-treatment with ROS scavenger N-acetyl-L-cysteine (NAC) or HO consumer catalase-polyethylene glycol (PEG-catalase) inhibited parkin recruitment, alleviated autophagy formation, and mitigated JNK pathway related apoptosis. In addition, PINK1 silencing resulted in a lower level of autophagy, but, a higher mortality in HEI-OC1 cells treated with cisplatin. Taken together, data from this work reveal that PINK1 possesses the protective effect via induction of autophagy and resistance of apoptosis under cisplatin stimulus in sensory HCs and SGNs, implying that PINK1 might serve as an important regulator of cisplatin-elicited ototoxicity.
磷酸酶和张力蛋白同源物（PTEN）诱导的假定激酶 1（PINK1）基因编码一种丝氨酸/苏氨酸激酶，作为线粒体健康的分子传感器，对于线粒体质量控制是必要的。本研究旨在探讨 PINK1 是否在 C57BL/6 鼠耳蜗和 HEI-OC1 细胞中表达，如果是这样，还探讨 PINK1 在顺铂诱导的感觉毛细胞（HCs）和螺旋神经节神经元（SGNs）体外死亡中的可能作用机制。通过免疫荧光染色确定 PINK1 的表达模式、parkin 颗粒的形成和自噬。通过 Western blot 测定 PINK1、LC3B、cleaved-caspase 3 和 p-JNK 的表达。通过 DCFH-DA 和 Mito-Sox Red 染色评估活性氧（ROS）水平。通过四甲基罗丹明甲酯过氯酸盐（TMRM）和 Rhodamine 123 检测线粒体膜电位。通过 CCK8 测定、TUNEL 染色和 Annexin V Apoptosis Detection Kit 分别检测细胞活力和细胞凋亡。我们发现 PINK1 在 C57BL/6 耳蜗和 HEI-OC1 细胞中的 HCs 和 SGNs 细胞质中广泛表达，特别是在出生后 4 天（P4）小鼠耳蜗 HCs 和 SGNs 中的表达水平高于成年小鼠。此外，用 30μM 顺铂处理会引发 ROS 的形成，进而导致 PINK1 激活、parkin 募集、自噬形成以及与凋亡相关的 JNK 通路在 HEI-OC1 细胞、HCs 和 SGNs 中。同时，用 ROS 清除剂 N-乙酰-L-半胱氨酸（NAC）或 HO 消耗剂过氧化氢酶聚乙二醇（PEG-catalase）共处理抑制了 parkin 募集，减轻了自噬形成，并减轻了 JNK 通路相关的凋亡。此外，PINK1 沉默导致自噬水平降低，但在顺铂处理的 HEI-OC1 细胞中死亡率更高。总之，这项工作的数据表明，PINK1 通过诱导自噬和对顺铂刺激的抗凋亡作用，在感觉 HCs 和 SGNs 中具有保护作用，这表明 PINK1 可能作为顺铂诱导耳毒性的重要调节剂。
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