基于Trx-ROS靶因子调控径路的老年性耳蜗神经细胞凋亡机制与中药干预作用的研究

The pathogenesis of presbycusis is very complicated. Previous animal preliminary researchs showed that national patent prescription, healthy ear compound of traditional Chinese medicine(TCM), had the effect on preventing ROS-induced and caspase-mediated apoptosis of cochlear neurons. But theoretically, target factors of regulating ROS were numerous, and the concrete regulatory mechanisms and the relationship each other were still unknown. Latest research suggested that TCM possessed the effect on regulating Trx-1 and Trx-2 in animal models with antagonizing the ototoxicity of gentamicin, accordingly, put forward the hypothesis that pathway of Trx system may play an important role in regulating ROS by target proteins in presbycusis. The healthy ear compound plays a key role in delaying the apoptosis of cochlear neural cells by affecting the Trx system and its peripheral regulatory proteins. In view of this, we will use the animal model of presbycusis，apply proteomics, PCR，multi-overlap immunofluorescence and other molecular biological techniques, and be combined with the methods of morphological research such as cochlear slice and flat surface preparation, and then explore and confirm the influence of Trx-ROS regulating pathway on the apoptosis of cochlear neural cells with age-related hearing loss and the relationship each other in these targeting regulatory molecules so that could further clarify the pathogenesis of presbycusis , and reveal the molecular acting targets by using the TCM and scientific essence on theory of treating deafness by TCM, or provide scientific basis in order to exploit TCM for preventing and treating hearing loss.

老年性聋病机错综复杂，前期动物研究初步表明, 国家专利方复方健耳剂具有阻止 ROS介导caspase启动耳蜗神经细胞凋亡的作用，但理论上调控ROS靶因子众多，具体调控机制及互相之间关系仍未明了，最新研究提示，该方在拮抗庆大霉素耳毒性动物模型中具有调控Trx-1和Trx-2作用，据此提出假说，Trx系统径路可能在老年性聋靶向调控ROS中扮演着重要角色，中药通过影响Trx系统及其外围各靶向调控蛋白质分子起到延缓老年性聋的关键作用，鉴此，我们将利用老年性聋动物模型，采用蛋白质组学、PCR、多重免疫荧光等分子生物学技术，配合耳蜗铺片、耳蜗切片等形态学研究方法，探索和明确Trx-ROS调控径路对老年性耳蜗神经细胞凋亡影响和这些靶向调控分子之间关系，进一步阐明老年性聋发病机制，揭示中药作用分子靶点与机制和中医治聋理论科学实质，为开发防聋治聋中药提供科学依据。

随着我国人口老年化，老年性聋患者急剧增加，因此，开展药物防治老年性聋研究意义重大。本项目主要研究内容为承接上一个国家基金项目，进一步探讨老年性聋动物模型不同月龄变化，利用形态学多重免疫荧光技术、透射电镜技术、蛋白组学技术、免疫组化技术、RT-PCR技术、蛋白免疫印迹技术等探讨老年性耳蜗毛细胞、螺旋神经节神经元凋亡及中医干预作用机制。通过研究，首次揭示C57BL/6J、CBA/CaJ小鼠在历时2年观测察中，不同月龄耳蜗毛细胞损害特征和发展变化规律，C57BL/6J毛细胞损害比CBA/CaJ早。首次从细胞超微结构层面揭示老年性感音神经细胞凋亡特征和发展变化规律。首次揭示中药作用机制主要是围绕ROS为核心，通过调控Trx--ROS- caspase、Trx- ASK1- -caspase、SOD或BDNF-ROS- caspase径路相关因子而产生系列生物学效应。通过蛋白组学研究，发现互相交集蛋白共有351种，其中对中药组与模型组逐一完成具有显著差异的个体蛋白功能分析52种，对中药在调节免疫、增加神经细胞蛋白合成、促进能量代谢、对抗基因损伤和畸变以及肿瘤和遗传病发生、降低活性氧、对抗衰老等方面所起作用获重大发现。这些研究对进一步揭示老年性聋感音神经细胞凋亡特征和发展变化规律，阐明老年性聋病理与中药干预作用机制，开发防治感音神经性聋药物具有重要意义。