Wnt通路调控Lgr5蛋白在乙苯耳毒性中的作用及机制研究

Ethylbenzene can induce ototoxicity with hearing loss in occupational population and rat in vivo. Previous animal research has shown ethylbenzene might induce enhanced hearing threshold and decreased number of hair cells in rat. A previous study investigating relationship between ethylbenzene and hearing loss of petrochemical workers by our group indicated ethylbenzene can cause higher incidence of hearing loss in petrochemical workers exposed to ethylbenzene collaborated with noise than those exposed to noise alone. Ethylbenzene independently contributed to hearing loss of workers. Mitochondria-mediated apoptotic pathway was involved in ethylbenzene-induced toxic effects. However the mechanism underlying its ototoxicity still remains unknown. The Wnt/β-catenin pathway and Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) is known to play crucial roles in development and function of Corti by participating proliferation and differentiation of hair cells. Our hypothesis is Lgr5 protein and both of the Wnt/β-catenin and mitochondria-mediated apoptotic pathways might be involved in ethylbenzene-induced ototoxicity. The two pathways and Lgr5 expression are investigated in rat Corti tissue and hair cell HEI-OC1, respectively. Furthermore, Renovated of cellular apoptosis, hair cell function in Corti will be observed after Wnt pathway was up-regulated by lentiviral vector of β-catenin over-expression. Our study might provide the experimental basis for molecular mechanism of ethylbenzene-induced ototoxicity and hearing loss, and theoretical basis for targeted protein involved with renovation of ethylbenzene-induced hearing loss.

乙苯可导致职业暴露人群和大鼠听力损伤，为耳毒性化学物。动物实验显示乙苯可导致大鼠听力阈值增加，耳蜗毛细胞减少。本课题组在前期乙苯与石化工人听力损伤关系的调查研究中证实乙苯是导致职业人群听力损伤的独立相关因素。线粒体凋亡通路为乙苯毒作用的位点之一，但其在乙苯耳毒性机制中的作用尚未见报道。最新研究表明Wnt/β-catenin通路及富含亮氨酸重复单位的G蛋白偶联受体5（Lgr5）蛋白参与耳蜗毛细胞增殖和分化，在耳蜗组织发育和功能中起关键作用。本研究以Lgr5蛋白为切入点，细胞内Wnt/β-catenin通路和线粒体凋亡通路为轴线，观察两条通路在乙苯耳毒性机制的作用，同时利用慢病毒过表达上调动物的耳蜗组织和HEI-OC1细胞的β-catenin表达，观察耳蜗组织细胞形态、毛细胞功能修复及动物听力改善情况，为阐明乙苯耳毒性作用机制提供实验依据，为寻找改善乙苯所致听力损伤的靶蛋白奠定理论基础。

乙苯可导致职业暴露人群和大鼠听力损伤，为耳毒性化学物。既往动物实验显示乙苯可导致大鼠听力阈值增加，耳蜗毛细胞减少。本课题组在前期乙苯与石化工人听力损伤关系的调查研究中证实乙苯是导致职业人群听力损伤的独立相关因素。最新研究表明Wnt/β-catenin通路及富含亮氨酸重复单位的G蛋白偶联受体5（Lgr5）蛋白参与耳蜗毛细胞增殖和分化，在耳蜗组织发育和功能中起关键作用。本研究以Lgr5蛋白为切入点，利用整体动物和体外耳蜗前体细胞（CPCs）模型，观察Wnt/β-catenin通路在乙苯耳毒性机制的作用，同时利用慢病毒过表达上调大鼠耳蜗组织和CPCs细胞的β-catenin表达，观察毛细胞功能修复及动物听力改善情况。研究结果提示乙苯可导致大鼠听力损伤，可能与Wnt/β-catenin通路相关蛋白及Lgr5蛋白表达受抑有关；β-catenin过表达慢病毒干预后可上调Wnt/β-catenin通路相关蛋白及Lgr5表达，大鼠听力有一定程度恢复；乙苯可导致CPCs细胞损伤和凋亡，可能与Wnt/β-catenin通路相关蛋白和Lgr5蛋白表达受抑有关；β-catenin过表达处理后可上调部分Wnt/β-catenin通路相关蛋白和Lgr5表达，细胞凋亡减少；β-catenin敲减处理后可下调部分Wnt/β-catenin通路相关蛋白和Lgr5表达，抑制CPCs增殖。上述研究结果为乙苯耳毒性和致听力损伤的致病机制提供了研究依据，也为寻找改善乙苯所致听力损伤的靶蛋白提供理论依据。