2型糖尿病对雄性小鼠听觉功能及内耳Otoferlin、VGLUT3、Myosin Ⅶa蛋白表达的影响Myosin Ⅶa蛋白表达的影响

Effects of type diabetes on auditory function and expression of Otoferlin，VGLUT3 and Myosin Ⅶa proteins in inner ear in male mice

关键词:  听觉功能  2型糖尿病  耳畸蛋白

Key words:  auditory function  type diabetics  Otoferlin

摘要:

目的　观察2型糖尿病对雄性小鼠听觉功能，静纤毛超微结构和耳畸蛋白（Otoferlin）、囊泡谷氨酸转运体3（vesicle glumate transporter，VGLUT3）、肌球蛋白 Ⅶa（MyosinⅦa）蛋白表达的影响。方法　按体重将12只6周龄雄性C57BL/6J小鼠随机分为两组（对照组和糖尿病组，n=6）。对照组小鼠食用饲料含铁量为(8.26±67)mg/kg，糖尿病组小鼠食用饲料含铁量为(8.39±03)g/kg。在实验开始前及实验第4、8、12、16周时测定小鼠血糖，实验第16周时测定葡萄糖耐量及胰岛素耐量，测定听觉诱发电位。分离左侧耳蜗基底膜，用免疫荧光法观察Otoferlin、VGLUT3与Myosin Ⅶa在内外毛细胞的定位与分布。分离右侧半数耳蜗用作western blot分析蛋白表达，另外半数耳蜗分离基底膜，用扫描电镜观察内毛细胞静纤毛形态。结果　实验第8、12、16周，糖尿病组小鼠血糖水平升高，与对照组比较差异有显著性意义(P<0.05）。糖尿病组小鼠糖耐量减弱，胰岛素抵抗指数升高，与对照组比较差异有显著性意义(P<0.05）。糖尿病组小鼠听力诱发电位阈值在刺激频率为2和3 kHz时升高，与对照组比较差异有显著性意义(P<0.05）。糖尿病组小鼠内毛细胞静纤毛排列正常，未见紊乱及数目减少；糖尿病组Otoferlin在内毛细胞底部表达减少，与对照组比较差异有显著性意义(P<0.05）。 结论型糖尿病致雄性小鼠听觉功能损伤，可能与Otoferlin 蛋白在耳蜗毛细胞中表达下调有关。

Abstract:

Objective To observe the effects of type diabetes in auditory function，ultrastructure of stereocilia，expression of Otoferlin，VGLUT3 and Myosin Ⅶa proteins inner ear in male mice.Methods Twelve C57Bl/6 male mice aged week old were randomly assigned evenly to two groups (control group and diabetic group).Control group and diabetic group was fed a diet with iron content (8.26±67) mg/kg and (8.39±03) g/kg，respectively.Blood glucose levels were measured before and，8，12，16 weeks after iron-content diet feeding.Glucose tolerance test and insulin tolerance test，auditory brainstem response (ABR) test were performed at6 weeks after iron-content diet feeding.The basilar membranes of the left cochleae were separated for assessment of the location of Otoferlin，VGLUT3 and MyosinⅦa in inner and outer hair cells by immunofluorescence.One half of right cochleae were used for assessment of protein expressions by western blot，the other half were used for assessment of the morphology of stereocilia by scanning electron microscope.Results Compared with control group，blood glucose levels were significantly higher in diabetic group at 8，12，16 weeks after iron-content diet feeding (P<0.05); and glucose levels of mice from diabetic group were significantly higher at 0，60，120 mins after the end of glucose tolerance test(P<0.05)，and HOMA-IR index of mice from diabetic group were significantly higher (P<0.05).The ABR threshold shifts of mice in diabetic group were significantly higher at，3 kHz frequencies (P<0.05).The location of stereocilia of cochlear inner hair cell was normal in mice from diabetic group.The Otoferlin expression in the diabetic group was down-regulated in basal of inner hair cell compared with control group (P<0.05).Conclusion Abnormal auditory function in male mice of diabetic group might be related with the down-regulated expression of Otoferlin protein in cochlear hair cells.