α2-肾上腺素能受体在水杨酸钠诱导大鼠耳蜗螺旋神经节神经元兴奋毒性中的表达变化

Expression Changes of α2 -Adrenergic Receptor in Sodium Salicylate-induced Excitotoxicity of Rat Cochlear Spiral Ganglion Neurons

目的 观察α2-肾上腺素能受体(α2-adrenergic receptor, α2-AR)在大鼠耳蜗螺旋神经节神经元(spiral ganglion neuron, SGN)中的分布,以及水杨酸钠(sodium salicylate, SS)作用SGN不同时间点后α2-AR各亚型的表达改变,以探讨其在SGN兴奋毒性中的作用。方法 将新生SD大鼠随机分成:对照组、SS处理6 h组、SS处理12 h组和SS处理24 h组,每组2只。急性分离各组SGN后进行原代培养48 h,对照组用新的培养基培养,不做处理,余三组分别用5 mM水杨酸钠处理6 h、12 h、24 h,用免疫荧光染色技术检测SGN中α2-AR各亚型的表达情况;采用实时荧光定量PCR(RT-qPCR)和Western blot方法检测SS作用不同时间后α2-AR各亚型的mRNA和总蛋白表达改变情况。结果 (1)免疫荧光染色表明SGN上存在α2A-AR、α2B-AR、α2C-AR三种亚型的表达;(2)RT-qPCR结果显示在SGN中α2-AR各亚型mRNA相对表达量α2A-AR>α2B-AR(P<0.01),α2A-AR>α2C-AR(P<0.01),α2B-AR和α2C-AR之间差异无统计学意义;与对照组相比,5 mM SS作用SGN 6 h、12 h、24 h后α2A-AR表达量均明显增高(P<0.05);SS作用12 h后α2C-AR表达量开始增高(P<0.05),而α2B-AR呈升高趋势,但差异无统计学意义(P>0.05);(3)Western blot结果显示SGN中α2-AR各亚型蛋白表达量α2A-AR>α2B-AR(P<0.01),α2A-AR>α2C-AR(P<0.05),α2B-AR和α2C-AR之间差异无统计学意义,与mRNA表达一致。与对照组相比,5 mM SS作用SGN 12 h后α2A-AR总蛋白开始下降(P<0.05),作用24 h后α2C-AR总蛋白下降(P<0.05),α2B-AR总蛋白不变。结论 α2-AR三种亚型在SGN中均有表达,其中α2A-AR、α2C-AR可能介导了SS诱导的SGN兴奋毒性且α2A-AR比α2C-AR反应更快速。

Objective To observe the distribution of α2-adrenergic receptor(α2-AR) in rat cochlear spiral ganglion neuron(SGN) and the expression changes of α2-AR subtypes in SGN treated with sodium salicylate(SS) at different time points to explore its possible role in SGN excitotoxicity. Methods Newborn SD rats were randomly divided into 4 groups: control group, SS treatment for 6-hour group, SS treatment for 12-hour group and SS treatment for 24-hour group, with 2 rats in each group. After acute isolation of SGN from each group, primary culture was carried out for 48 hours. The expression of α2-AR subtypes in SGN was detected by immunofluorescence staining, and the changes of mRNA and total protein expression of α2-AR subtypes after SS treatment were detected by real-time fluorescence quantitative PCR(RT-qPCR) and Western blot. Results(1)Immunofluorescence staining showed that there were three subtypes of α2A-AR, α2B-AR and α2C-AR on SGN.(2)The results of RT-qPCR showed that the relative expression of mRNA of α2-AR subtypes in SGN was α2A-AR > α2B-AR, α2A-AR > α2C-AR, but there was no significant difference between α2B-AR and α2C-AR. Compared with the control group, the expression of α2A-AR increased significantly after treatment with 5 mM SS for 6 hours, 12 hours and 24 hours. After treatment with SS for 12 hours, the expression of α2C-AR began to increase, while the expression of α2B-AR showed an increasing trend, but the difference was not statistically significant.(3)Western blot results showed that the protein expression of α2-AR subtypes in SGN was α2A-AR > α2B-AR, α2A-AR > α2C-AR. There was no significant difference between α2B-AR and α2C-AR, which was consistent with mRNA expression. Compared with the control group, the total protein of α2A-AR began to decrease after SGN was treated with 5 mM SS for 12 hours, and the total protein of α2C-AR decreased after 24 hours of treatment, while the total protein of α2B-AR remained unchanged. Conclusion Three subtypes of α2-AR were all expressed in SGN. α2A-AR and α2C-AR mediated SGN excitotoxicity induced by SS, and the response of α2A-AR was faster than α2C-AR.