Wang Xi, Wang Ye, Ding Zhong-jia, Yue Bo, Zhang Peng-zhi, Chen Xiao-dong, Chen Xin, Chen Jun, Chen Fu-quan, Chen Yang, Wang Ren-feng, Mi Wen-juan, Lin Ying, Wang Jie, Qiu Jian-hua
Department of Otolaryngology-Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, 17 Changle Western Road, Xi'an 710032, China.
Department of Otolaryngology-Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University, 17 Changle Western Road, Xi'an 710032, China; Ji-guan Hospital, Lanzhou Military Region, Air Force of PLA, Lanzhou, Gansu 730000, China.
Neurosci Lett. 2014 Aug 22;578:111-6. doi: 10.1016/j.neulet.2014.06.042. Epub 2014 Jun 30.
Spiral ganglion neuron (SGN) injury is a generally accepted precursor of auditory neuropathy. Receptor-interacting protein 3 (RIP3) has been reported as an important necroptosis pathway mediator that can be blocked by necrostatin-1 (Nec-1). In our study, we sought to identify whether necroptosis participated in SGN injury. Ouabain was applied to establish an SGN injury model. We measured the auditory brain-stem response (ABR) threshold shift as an indicator of the auditory conditions. Positive β3-tubulin immunofluorescence staining indicated the surviving SGNs. RIP3 expression was evaluated using immunofluorescence, quantitative real-time polymerase chain reaction and western blot. SGN injury promoted an increase in RIP3 expression that could be suppressed by application of the necroptosis inhibitor Nec-1. A decreased ABR threshold shift and increased SGN density were observed when Nec-1 was administered with apoptosis inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD). These results demonstrated that necroptosis is an indispensable pathway separately from apoptosis leading to SGN death pathway, in which RIP3 plays an important role.
螺旋神经节神经元（SGN）损伤是听觉神经病变公认的先兆。据报道，受体相互作用蛋白3（RIP3）是一种重要的坏死性凋亡途径介质，可被坏死性凋亡抑制剂-1（Nec-1）阻断。在我们的研究中，我们试图确定坏死性凋亡是否参与SGN损伤。应用哇巴因建立SGN损伤模型。我们测量听觉脑干反应（ABR）阈值变化作为听觉状况的指标。β3微管蛋白免疫荧光染色阳性表明存活的SGN。使用免疫荧光、定量实时聚合酶链反应和蛋白质印迹法评估RIP3表达。SGN损伤促进RIP3表达增加，应用坏死性凋亡抑制剂Nec-1可抑制这种增加。当Nec-1与凋亡抑制剂N-苄氧羰基-Val-Ala-Asp-氟甲基酮（Z-VAD）联合使用时，观察到ABR阈值变化降低和SGN密度增加。这些结果表明，坏死性凋亡是一条独立于凋亡导致SGN死亡途径的不可或缺的途径，其中RIP3起重要作用。
