复方聪耳胶囊及其拆方干预离体和在体AmAn中毒性耳蜗神经细胞凋亡形态学及相关因子影响机制研究

Aminoglycoside antibiotics ( AmAn ) ototoxic mechanism is very complex, and many aspects of them is not yet clear, so prevention on the ototoxicity is very difficult. It was common reason of deafness and then disability induced by AmAn. Cong-er preparation of Chinese herbal compound based on the treating rules of Bushen-Jianpi-Huoxuetonqiao of TCM is our clinical effective prescription in treating sensorineural tinnitus and deafness. Previous animal experiments showed it could delay the apoptosis of age-related cochlear hair cells and spiral ganglion neuron, some herbal of them had also antagonism on gentamicin ototoxicity. By said above, we put forward the hypothesis: treating rule with Bushen-Jianpi-Huoxuetonqiao from the original prescription in resisting AmAn ototoxicity may be better than treating rule with Bushen- Huoxue, Jianpi- Huoxue from its disassembled prescriptions, at the same time, by the research in intervention of the dominant treating rules, ascertain influence of related factors regulation. To test and verify this hypothesis, through take animal AmAn ototoxic model in vitro based on the cochlear organic culture or in vivo, use molecular biology technology in immunohistochemistry, multiple immunofluorescence ,PCR, combine with the methods of cochlea preparation and cochlea section, the treating effect on the compound of TCM and its disassembled prescriptions will be compared according to TCM different treating rule, meanwhile, explore related factors regulating effects, reveal TCM effects and mechanism from cell level to molecular level. It is of great significance for verifying the meaning of TCM theoretical guidance, optimizing prescription, guiding clinical drugs use and developing new drugs.

氨基糖甙类抗生素(AmAn)耳毒性聋病机复杂，迄今尚未明了，防治甚为棘手,是因聋致残常见原因。以补肾健脾、活血通窍为治法的复方聪耳剂是我们临床治疗后天性感音神经性耳鸣耳聋有效验方，前期动物研究表明，它具有延缓老年性耳蜗毛细胞、螺旋神经节神经元凋亡的作用，其中部分药物具有拮抗庆大霉素耳毒性聋的作用，但原方及拆方对比作用与机制仍不清楚。据此提出假说：原方从脾肾瘀论治对抗AmAn耳毒性优于拆方从肾或从脾论治，同时通过优势治法干预研究，探明相关因子的调控影响。鉴此，我们将利用耳蜗器官体外培养建立的AmAn耳毒性动物模型、配合在体模型，采用免疫组化、多重免疫荧光、PCR等为主的分子生物学技术，结合耳蜗铺片、耳蜗切片等方法，按照中医不同治则，对复方聪耳剂及其拆方进行比较，同时探讨相关因子调控影响，从分子、细胞等水平揭示其作用和机制，这对于验证中医理论，优化组方，指导临床用药，开发新药均具有重大意义。

伴随着氨基甙类抗生素抗生素（AmAn）的广泛应用，由其导致的耳毒性聋患者逐年大量增加。因其病因病机复杂，迄今尚无临床专门防治药物，一旦致聋不可逆转，是因聋致残的主要原因，因此，开发临床防治药物意义重大。复方建耳剂是项目负责人临床验方，本项目旨在主要通过常用的AmAn-庆大霉素耳毒性聋动物模型，主要利用耳蜗铺片技术、免疫组化技术、RT-PCR技术、蛋白免疫印迹技术、荧光免疫探针技术等，探索庆大霉素耳毒性聋病理，复方建耳剂及其拆方干预作用与药效机制，结果表明，一定剂量GM连续使用可造成豚鼠耳蜗外毛细胞和听力严重损害，而且以外毛细胞为主要损害靶细胞，同时肾小球、肾小管以及肾功能破坏明显。复方健耳剂具有显著防护GM耳、肾毒性损害作用，复方健耳剂拆方之一的健脾补气药组方也有一定的防护GM耳、肾毒性损害作用，他们的作用机制与其通过Trx-ROS径路，或SOD-ROS径路，抑制氧化应激反应，减轻氧化应激损伤，干预ROS引起caspase介导的细胞程序性死亡密切相关，其中通过护肾，促进GM排泄，是其间接护耳的另一重要机制。此外，通过同属于感音神经性聋的老年性聋动物模型研究，还证明了复方健耳剂通过清除细胞内氧自由基，阻止ROS产生，保护线粒体，阻止细胞色素C引起caspase介导的细胞程序性死亡这一重要作用机制。研究提示，古代中医耳聋从脾、肾论治具有科学依据。对于病机复杂的难治性感音神经性聋，多种治法优于某个单纯治法，体现了中医整体观理论的科学意义，这对临床耳聋防治具有十分重要的现实意义。