To identify the role of RIP3 in ouabain-induced necroptosis and offer clinical implications to prevent spiral ganglion neurons (SGNs) from death, ouabain was applied in SGNs derived from fetal rats and injected into Sprague–Dawley rats to construct injury model in vitro and in vivo, respectively. The necroptosis rate of SGNs was determined by flow cytometry and MTT assays. The protein levels and phosphorylation of RIP3 were evaluated using western blotting and immunofluorescence. SGNs injury was observed using H&E staining and immunofluorescence. The hearing function of rats was evaluated by the auditory brainstem response (ABR) and Distortion Product Otoacoustic Emissions (DPOAE) methods. Ouabain caused dose-dependent necroptosis in SGNs and significant loss of SGNs of the cochlear axis in vivo. RIP3 and pRIP3 were upregulated with SGNs injury promoted, and RIP3 overexpression promoted ouabain-induced necroptosis in SGNs in vitro, which could be suppressed by necrostatin-1. RIP3 knockdown inhibited ouabain-induced necroptosis and reduced the phosphorylation of MLKL but no RIP3-dependent effect on the level of MLKL. RIP3 inhibition in vivo protected rats from ouabain-induced hearing damage with reducing ABR threshold shifts and promoting DPOAE amplitudes, while overexpression of RIP3 enhanced ouabain-induced injury that could be partially reversed by necrostatin-1. A decrease of SGNs density and an upregulation of pRIP3 were observed with RIP3 overexpression, which was in contrast when RIP3 was silenced. Therefore, RIP3 was essential for mediating necroptosis in ouabain-induced SGNs damage. Targeting RIP3 may prevent SGNs from death in clinical practice, and finally help the treatment of sensorineural hearing loss.

为了确定 RIP3 在哇巴因诱导的坏死性凋亡中的作用，并为防止螺旋神经节神经元 (SGN) 死亡提供临床意义，将哇巴因应用于源自胎鼠的 SGN，并注射到 Sprague-Dawley 大鼠体内，构建体外和体内损伤模型。分别是vivo 。通过流式细胞术和MTT测定法测定SGN的坏死率。使用蛋白质印迹和免疫荧光评估 RIP3 的蛋白质水平和磷酸化。使用 H&E 染色和免疫荧光观察 SGN 损伤。采用听觉脑干反应（ABR）和失真产物耳声发射（DPOAE）方法评价大鼠的听力功能。哇巴因在体内引起 SGN 的剂量依赖性坏死性坏死和耳蜗轴 SGN 的显着损失。 RIP3 和 pRIP3 上调，促进 SGN 损伤，RIP3 过表达促进哇巴因诱导的体外 SGN 坏死性凋亡，而 necrostatin-1 可以抑制这种坏死性凋亡。 RIP3 敲低可抑制哇巴因诱导的坏死性凋亡并降低 MLKL 的磷酸化，但对 MLKL 水平没有 RIP3 依赖性影响。体内抑制 RIP3 可保护大鼠免受哇巴因诱导的听力损伤，减少 ABR 阈值变化并促进 DPOAE 振幅，而 RIP3 的过度表达则增强哇巴因诱导的损伤，而 necrostatin-1 可以部分逆转这种损伤。 RIP3 过表达时观察到 SGN 密度降低和 pRIP3 上调，而 RIP3 沉默时则相反。因此，RIP3 对于介导哇巴因诱导的 SGN 损伤中的坏死性凋亡至关重要。 在临床实践中，靶向RIP3可能会阻止SGN死亡，最终有助于感音神经性听力损失的治疗。