水杨酸钠通过DA受体影响耳蜗螺旋神经节神经元NMDA受体与GABAa受体亚单位的表达

The sodium salicylate affects the expression of NMDA receptor and GABAa receptor subunits in spiral ganglion neurons of the cochlea through DA receptor

目的:观察水杨酸钠(SS)对大鼠耳蜗螺旋神经节神经元(SGN)的多巴胺受体(DR)亚型表达的影响,以及观察SS作用下,DR对SGN的N-甲基-D-天冬氨酸(NMDA)受体和γ-氨基丁酸(GABA)a受体亚单位的影响,探讨SS介导的受体之间的相互作用。方法:应用免疫荧光技术,检测新生SD大鼠SGN中DR两家族DR1、DR2的表达;应用RT-PCR技术,检测SS处理后的SGN中DR1和DR2亚型的mRNA变化,观察在激活和抑制DR活动时SS对SGN的NMDA受体亚单位NR1、GABAa受体亚单位GABRα2的mRNA表达的影响。结果:免疫荧光显示:在SGN的胞体与突起均有DR1、DR2的免疫荧光显示。RT-PCR结果显示:(1)SGN有DR亚型(DRd1~DRd5)、GABRα2、NR1的表达;(2)5mmol/L SS作用SGN 30min后,除DRd3外,其余DR亚型及GABRα2、NR1的mRNA表达量均明显升高,其中DRd1表达增加34.64%(t=-5.123,P=0.007);DRd2表达增加34.60%(t=-5.206,P=0.006);DRd4表达增加20.87%(t=-3.337,P=0.029);DRd5表达增加26.42%(t=-6.054,P=0.004);GABRα2表达增加30.41%(t=-2.839,P=0.047);NR1表达增加39.22%(t=-6.243,P=0.003);(3)SS分别与多巴胺(100μmol/L)、DR1激动剂(SKF38393,20μmol/L)、DR2激动剂(Quinpirole,20μmol/L)分别作用SGN 30min后,GABRα2、NR1的mRNA表达量均明显升高,其中,GABRα2表达分别增加21.78%、27.45%、33.02%、33.42%(F=12.399,P=0.001),NR1表达分别增加28.70%、26.82%、29.03%、35.05%(F=50.395,P=0.000);(4)SS+DR1拮抗剂(SCH23390,20μmol/L)、SS+DR2拮抗剂(Eticlopride,20μmol/L)分别作用SGN 30min后,与单独SS作用相比,GABRα2的mRNA表达分别减少29.56%、37.10%(F=22.101,P=0.000),NR1的mRNA表达分别减少37.62%、32.83%(F=72.933,P=0.000)。结论:SS可诱导SGN的大部分DR亚型的mRNA表达明显升高;SS可能通过DR影响SGN上GABAaR、NMDAR的mRNA表达。

Objective：To investigate mRNA expression of dopamine receptor subtypes in the rat cochlear spi- ral ganglion neurons （SGN） following exposure to the sodium salicylate. In addition, we observed the effect of so- dium salicylate on N methyl -D- aspartic acid （NMDA） receptor subunit NR1 and gamma aminobutyric acid （GA- BA）a receptor subunit GABRa2 mRNA under the circumstance of DR activation or blocking. Moreover, we also focused on the the interaction between receptors mediated by SS. Method.： Immunofluorescence techniques were ap- plied to detect DR （DR1 and DR2） expression in cultured rat SGN. Moreover, RT-PCR was performed to assess NR1 and GABRα2 subunit mRNA. Result： Immunofluorescence images showed co-localization of DR1/DR2 and β Ⅲ-tubulin protein in SGN bodies and axons. RT-PCR results illustrated that ①DR subtypes of DRdl- 5 were detected in the SGN. @The mRNA expression of all subtypes of DR and GABRa2, NR1 subunits were obviously upregulated except DRd3 after treatment with sodium salicylate. Among them, DRdl expression increased 34.64% （t=-5. 123, P= 0. 007）, DRd2 expression increased 34.60% （t=-5. 206, P= 0. 006）, DRd4 expression increased 20.87 % （t=-3. 337, P=0. 029）, DRd5 expression increased 26.42 % （t= -6. 054, P= 0.004）, GABRa2 expression increased 30.41% （t= -2. 839, P=0. 047）, NR1 expression increased 39.22% （t= -6. 243, P=0. 003）. @After exposure to sodium salicylate （5 mmol/L）, dopamine （100μmol/L）, DR1 agonist （SKF38393, 20μmol/L）, DR2 ago- nist （Quinpirole, 20μmol/L）, GABRa2 expression increased 21.78%, 27.45%, 33.02%, 33.42% respectively （F=12.399, P= 0.001）, and NR1 expression increased 28.70%, 26.82%, 29.03%, 35.05% （F=50. 395, P=0. 000） respectively. @Compared with the group of sodium salicylate treatment alone, both sodium salicylate ＋ DR1 an- tagonist （SCH23390, 20μmol/L） group and sodium salicylate -l- DR2 antagonists （Eticlopride, 20 μmol/L） group had a suppression on GABRa2 and NR1 mRNA expression. GABRa2 mRNA reduced 29.56%, 37.10% （F=22. 101, P=0. 000） and NR1 mRNA expression decreased 37.62%, 32.83% respectively （F=72. 933, P=0. 000）. Conclusion： Most of the DR subtypes mRNA expression in SG-N were increased following exposure to sodium salicylate. DR may be in- volved in the effect of sodium salicylate on GABAaR and NMDAR mRNA expression.