TIGAR在顺铂导致耳蜗螺旋神经节细胞损伤中的作用及机制研究

It has been documented that cisplatin could damage spiral ganglion neuron (SGN) and so contributes to the initiation of sensorineural hearing loss, to date, however, the exact mechanisms responsible for this disorder have not been fully elucidated. Previously, we found that TP53-induced glycolysis and apoptosis regulator (TIGAR) could express in SGN and the exprssion could be changed while the SGN was damaged by cisplatin. We also found that the Wnt signaling pathway could regulate the expression of TIGAR in SGN after being damaged by cisplatin, as well as it protected the SGN from the damage. However, the effects and mechanisms of TIGAR played during the damage process, and how Wnt signaling regulate the expression of TIGAR and protect the SGN from being damaged by cisplatin are still unclear. On the basis of our previous researches, the present project is designed, utilizing the primary cultured SGN and adult mice damaged by cisplatin separately, as the experiment models in vitro or in vivo, to investigate the effects of TIGAR on the SGN damage induced by cisplatin. In particular, this work will focus on clarify the mechanisms of TIGAR on the regulation of ROS and apoptosis in this damage process by using transgenetic mice. Furthermore, we will explore the mechanisms underlying the regulation of Wnt signaling pathway on TIGAR expression and the protection effect of Wnt pathway, mediated by TIGAR, on the SGN from being damaged by cisplatin, through activating and inhibiting Wnt signaling both in vitro and in vivo. Our new research will lends insight into the etiology and mechanisms responsible for the SGN damage induced by cisplatin, which may, in turn, offer novel effective therapeutic targets to sensorineural hearing loss.

顺铂可损伤耳蜗螺旋神经节细胞（SGN）导致感音神经性聋，但其致病分子机制仍不明。我们前期研究发现，TP53诱导的糖酵解和凋亡调节因子（TIGAR）可在SGN内表达并在顺铂损伤SGN后表达出现改变，Wnt信号通路可调控该损伤中TIGAR的表达，且保护了顺铂诱导的SGN损伤。然而TIGAR在该损伤中发挥的作用及机制，Wnt通路与TIGAR的调控关系并未阐明。本项目在前期工作基础上，拟分别通过离体和活体的顺铂损伤SGN模型，研究TIGAR在该损伤中的作用，并通过转基因小鼠明确TIGAR在该损伤中调控SGN内活性氧生成及凋亡发生的作用机制，进一步在离体和活体实验中激活或抑制Wnt信号通路，阐明Wnt通路对TIGAR表达的调控作用，及TIGAR介导下Wnt信号对顺铂损伤SGN发挥保护作用的机制，以期为TIGAR在顺铂损伤SGN中的作用和机制研究提供新的理论依据，为感音神经性聋的治疗提供新的作用靶点。

项目背景：顺铂是临床上广泛应用的广谱抗癌药，但其具有严重的耳毒性不良反应，可造成双侧听力损失。研究表明，顺铂可损伤耳蜗螺旋神经节细胞（SGN）导致感音神经性聋，但是目前为止其致病的分子机制并不明确。本项目前期研究发现，Wnt信号通路在顺铂损伤SGN过程中被激活； TP53诱导的糖酵解和凋亡调节因子（TIGAR）在顺铂损伤SGN后表达改变，Wnt信号通路可调控该损伤中TIGAR的表达。但是在顺铂损伤SGN导致耳聋过程中，Wnt通路及TIGAR发挥的作用及机制，Wnt通路与TIGAR的调控关系均尚未阐明。.主要研究内容：为探讨Wnt信号通路在TIGAR的介导下对于顺铂损伤SGN发挥保护作用及修复听力的机制，本项目分别通过离体和活体的SGN顺铂损伤模型，1）探讨顺铂对于SGN 的损伤作用及作用机制；2）阐明TIGAR在顺铂导致SGN损伤中的作用，及TIGAR在该损伤中调控SGN内ROS生成及抑制凋亡的作用机制；3）进一步在离体和活体实验中分别激活和抑制Wnt信号通路，探讨Wnt信号通过调节TIGAR的表达，并由TIGAR介导发挥保护SGN免受顺铂损伤的作用及机制。.重要结果和关键数据：1）构建了离体和活体的SGN顺铂损伤模型，证明顺铂可以损伤SGN，诱导其凋亡及细胞内ROS水平升高；2）首次在细胞及动物水平上证明了Wnt信号通路对于顺铂导致的SGN损伤的保护作用及作用机制，发现顺铂损伤SGN过程中可以激活Wnt/β-catenin信号通路， 过表达Wnt信号可以减少顺铂导致的SGN损伤； 而相反的，抑制Wnt信号通路可以加剧SGN损伤及加重听力损失。3）TIGAR在在顺铂损伤SGN后表达升高，Wnt/β-catenin信号激活或抑制可以相应的增加或减少TIGAR的表达，使用病毒载体过表达TIGAR后可以减少SGN内的活性氧水平，减少Caspase 3表达以及增加SGN的存活率。 而使用抗氧化剂后可以挽救β-catenin缺陷小鼠中顺铂引起的严重的SGN损失。.科学意义：本研究首次证明了顺铂损伤可以激活SGN中Wnt信号通路，以此调控TIGAR表达从而抑制SGN中的氧化应激以及凋亡情况，最终保护SGN对抗顺铂诱导的损伤，从而为保护SGN免受损伤并修复听力提供了新的作用靶点和实验依据，对于感音神经性聋的防治可发挥重要的作用。