GLP-1对隐性听力损失的保护作用及机制研究

Hidden hearing loss (HHL), a recently described auditory disorder, has been proposed to affect auditory neural processing and hearing acuity in subjects with normal audiometric thresholds. Based on recent surveys, HHL exists widely in the population and is closely related to tinnitus and age-related hearing loss, and lacks effective treatment. Glucagon-like peptide-1 (GLP-1) has neuroprotective effects in central neuropathy: blast-induced brain injury, and stroke. Our previous studies have found that GLP-1 receptor (GLP-1R) is expressed in the auditory system; GLP-1 can alleviate the damage of the auditory system caused by low intensity blast exposure and increase the amplitude of ABR wave I, suggesting that GLP-1 may reduce the damage of synapses and neurons. In this project, the relationship between GLP-1 and neurotrophic factors in the cochlea, and the role of PI3K/Akt signaling pathway in the regulation of growth and proliferation of spiral ganglion neurons were investigated by culturing the whole mount or surface preparation of the cochlea. MTT assay and Trypan blue staining were used to investigate the growth and proliferation ability, and the anti-apoptosis and -necrosis effects of GLP-1 on spiral neurons were investigated by Tunel and Prodium Iodide staining. And then, the protective effects of GLP-1 on synapse and spiral ganglion neuron function in animals with hidden hearing loss were investigated by ABR, DPOAE and Electrocochleogram. The protective effects of GLP-1 on synaptic structural integrity were further investigated by CtBP2/Bassoon and GluA2 immunofluorescence staining of presynaptic and posterior membranes, in order to clarify the molecular mechanism of GLP-1's protection against HHL and provide a new targets and treatment strategies for the prevention and treatment of HHL.

隐性听力损失(HHL)是指听力阈值正常但听觉功能仍有损伤的现象。HHL广泛存在，与耳鸣及老年性耳聋有密切关系，缺乏有效的治疗方法。胰高血糖素样肽-1（GLP-1）对中枢神经病变、爆炸性脑损伤等有保护作用。我们发现GLP-1受体表达于听觉系统；GLP-1可缓听觉系统的损伤，提高ABRI波的幅值；提示其可能减少突触及神经元损伤。本项目利用全基底膜培养，考察GLP-1与神经营养因子间的关系和调控方式；应用MTT和台盼蓝实验考察GLP-1对神经元细胞生长、增殖的调控；Tunel和PI实验考察其抗神经元细胞凋亡、坏死的作用；通过ABR、DPOAE、耳蜗电图考察GLP-1对突触及螺旋神经元功能的保护；进一步CtBP2及GluA2荧光染色突触前、后膜，考察GLP-1对突触结构的保护，及PI3K/Akt等信号通路在其中的作用，以期明确GLP-1对HHL保护的分子机制，为HHL的防治提供新的靶点和治疗策略。

耳蜗中的神经节是听觉传导通路中的第一个中继元件，将毛细胞的声音刺激转换成的电信号传递到听觉中枢。要恢复因感音神经性听力损失而降低的听力，预防或减轻耳蜗神经纤维和神经细胞的退化至关重要。胰高血糖素样肽-1受体（Glucagon-likepeptide1receptor，GLP-1R）广泛表达于人和小鼠体内多种组织，已有许多实验表明GLP-1R激活在抑制细胞凋亡、减轻炎症反应、延缓神经退行性变等方面发挥广泛的调控作用。但是，目前GLP-1/GLP-1R在耳科学领域还缺乏研究，本研究旨在探索GLP-1R在小鼠耳蜗中的表达情况，及是否能对螺旋神经元的损伤起到一定的保护作用及其作用机制。. 我们的研究结果显示，GLP-1R表达于螺旋神经元，1mM的卡那霉素可诱导体外培养的螺旋神经元损伤，GLP-1R激动剂艾塞那肽（exenatide）对卡那霉素诱导的螺旋神经元细胞损伤有保护作用，提高了细胞生存率，减轻了神经纤维损伤，提高了SOD活性和GSH-Px水平，降低了MDA、ROS含量，激活了Nrf2/HO-1通路。在动物实验中，卡那霉素加呋塞米处理可以诱导小鼠螺旋神经元损伤，经过艾塞那肽处理可以减轻螺旋神经元损伤；Western blot结果显示，卡那霉素加呋塞米处理组促凋亡蛋白 Cleaved-caspase-3、Bax蛋白水平升高，抗凋亡蛋白 Bcl-2表达水平下降，经过艾塞那肽组 Cleaved-caspase-3、Bax表达较卡那霉素组降低，Bcl-2表达升高。以上结果表明，GLP-1/GLP-1R信号通路可能通过Nrf2/HO-1信号通路在卡那霉素诱导的螺旋神经元损伤中发挥了减轻氧化损伤、减轻凋亡和神经保护作用。GLP-1R激动剂有望成为听神经保护的药物，可以为耳聋的防治提供新的方向。