Epac1信号介导大鼠噪声暴露后内耳毛细胞损伤作用与机制

Epac1 signaling mediates the injury of inner ear hair cells induced by noise exposure in rats and its mechanism

目的探究环腺苷酸结合蛋白1(Epac1)信号分子介导噪声暴露大鼠内耳毛细胞损伤作用及其机制。方法20只SPF级SD大鼠均分为对照组和噪声暴露组。噪声暴露组大鼠给予频率为4 kHz,声压级为101 dB SPL,暴露8 h。噪声暴露前后检测大鼠听性脑干反应(ABR)。噪声暴露结束后处死大鼠通过耳蜗基底膜铺片染色、免疫荧光和透射电镜,观察噪声暴露后大鼠内耳毛细胞损伤以及Epac1蛋白在耳蜗中的功能定位。免疫蛋白印迹法检测耳蜗组织中Epac1、Rap1、CaMK-Ⅱ、Bax、Bcl-2、cleaved caspase-3(CC3)、cleaved caspase-9(CC9)等蛋白的表达。结果与对照组比较,噪声暴露组大鼠的ABR阈值增加(P<0.05);耳蜗铺片染色结果显示,噪声暴露组大鼠耳蜗三层外毛细胞的缺失高于对照组(P<0.05);透射电镜检测结果显示,噪声暴露组大鼠毛细胞底部溶解,静纤毛丢失或融合增多,细胞器损伤严重,线粒体外膜破裂,线粒体脊断裂或消失,出现空泡化;免疫荧光检测结果显示,噪声暴露组中大鼠外毛细胞中Epac1呈现高表达(P<0.05);Western blot检测结果显示,噪声暴露组大鼠耳蜗组织中Epac1、Rap1和CaMK-Ⅱ蛋白的表达上调(P<0.05),凋亡相关蛋白Bcl-2表达下调,Bax、CC3和CC9蛋白表达上调(P<0.05)。结论Epac1-Rap1信号通路介导了噪声暴露条件下内耳的早期病理性损伤过程,并参与调控噪声暴露诱导的内耳毛细胞凋亡。Epac1-Rap1通路有望成为新的干预噪声性耳聋的潜在靶点。

Objective To investigate the effect of(exchange protein directly activated by cAMP-1)on inner ear hair cell injury with noise-induced hearing loss and its potential mechanism in rats.Methods Twenty Specific pathogen-free(SPF)Sprague-Dawley(SD)rats were randomly divided into normal control group and noise exposure group.The rats of noise exposure were exposed to 4 kHz at 101 dB sound pressure level(SPL)for 8 h.Auditory brainstem responses(ABR)were measured in animals before noise exposure and 24 h after noise exposure.Surface preparation,transmission electron microscopy and immunohistochemistry were performed on cochlea tissuesto elucidate changes in Epac expression in rat after noise exposure.The expression levels of Epac1、Rap1、CaMK-Ⅱ、Bax、Bcl-2、cleaved caspase3(CC3)and cleaved caspase9(CC9)were analyzed using Western blot.Results There was found a stable temporary threshold shift after noise exposure(P<0.05).The missing of outer hair cells occurred after noise exposure(P<0.05).Transmission electron microscopy indicated that the epidermis plate of HCs was partially dissolved,with loss or fusion of stereocilia,some HC organelles showed serious injuries after noise exposure.Epac1 immunostaining intensities were substantially enhanced in OHCs after noise exposure(P<0.05).The expression levels of Epac1,CaMK-Ⅱand Rap1 protein were significantly up-regulated after noise exposure(P<0.05).The expression level of Bcl-2 was significantly down-regulated after noise exposure(P<0.05).The expression levels of Bax,CC3 and CC9 were significantly up-regulated after noise exposure(P<0.05).Conclusion Epac1-Rap1 signaling pathway mediates the early pathological damage in noise-exposed cochlea,and participates in the regulation of inner ear hair cells apoptosis.Epac1-Rap1 pathway is expected to become a new target for intervention in noise-induced hearing loss.