异氟醚/丙泊酚不同配伍对大鼠海马神经元缺氧损伤时GABAA受体α1亚基稳态的影响

Effects of different ratios of medicine dosage for isoflurane and propofol on GABAA receptor a1 sub-unit proteostasis during hypoxia injury to hippocampa｜ neurons of rats

目的评价异氟醚/丙泊酚不同配伍对大鼠海马神经元缺氧损伤时GABAA受体（GABAAR）α1亚基稳态的影响。 方法原代培养Wistar大鼠胎鼠海马神经元，采用随机数字表法将神经元分为6组（n＝60）：正常对照组（C组）、缺氧组（H组）、异氟醚组（I组）、丙泊酚组（P组）、异氟醚/丙泊酚不同配伍组（IP1组和IP2组）。H组缺氧6 h；I组和P组缺氧6 h后分别经1.9 %异氟醚和22.4 μmol/L丙泊酚孵育3 h；IP1组和IP2组缺氧6 h后分别经1.0%异氟醚＋6.7 μmol/L丙泊酚、1.4 %异氟醚＋3.4 μmol/L丙泊酚孵育3 h；随后更换正常培养基培养。培养24 h时采用CCK-8法检测细胞活力，采用qPCR法检测GABAAR α1亚基mRNA的表达，采用Western blot法检测胞膜GABAAR α1亚基的表达，采用免疫沉淀和Western blot法检测GABAAR α1亚基内质网相关降解（ERAD）水平，采用免疫荧光检测CCAAT/增强子结合蛋白同源蛋白（CHOP）的表达。 结果与C组比较，其余5组神经元活力降低，GABAAR α1亚基mRNA和胞膜GABAAR α1亚基表达下调，CHOP表达上调，GABAAR α1亚基ERAD水平升高（P〈0.05）。与H组比较，I组、P组和IP2组神经元活力降低，GABAAR α1亚基mRNA和胞膜GABAAR α1亚基表达下调，CHOP表达上调，GABAAR α1亚基ERAD水平升高（P〈0.05），IP1组上述指标差异无统计学意义（P〉0.05）；与I组或P组比较，IP1组和IP2组神经元活力升高，GABAAR α1亚基mRNA和胞膜GABAAR α1亚基表达上调，CHOP表达下调，GABAAR α1亚基ERAD水平降低（P〈0.05）。与IP1组比较，IP2组神经元活力降低，GABAAR α1亚基mRNA和胞膜GABAAR α1亚基表达下调，CHOP表达上调，GABAAR α1亚基ERAD水平升高（P〈0.05）。 结论1.0%异氟醚＋6.7 μmol/L丙泊酚配伍时不加重缺氧诱导的大鼠海马神经元GABAAR α1亚基稳态破坏。

Objective To evaluate the effects of different ratios of medicine dosage for isoflurane and propofol on GABAA receptor （GABAAR） txj subunit proteostasis during hypoxia injury to hippocampal neurons of rats. Methods The hippoeampal neurons isolated from fetal rats obtained from Wistar rats wereprimarily cultured and divided into 6 groups （ n = 60 each） using a random number table： control group （group C）, hypoxia group （group H）, isoflurane group （group I）, propofol group （group P） and dif- ferent ratios of medicine dosage for isoflurane and propofol groups （ group IPt and group IP2 ）. The cells were subjected to hypoxia for 6 h in group H. Cells were incubated for 3 h with 1.9 % isoflurane and with 22.4 μmol/L propofol after being subjected to hypoxia for 6 h in I and P groups, respectively. Ceils were incubated for 3 h with 1.0% isoflurane and 6.7 μmol/L propofol and with 1.4 % isoflurane and 3.4 μmol/L propofol after being subjected to hypoxia for 6 h in IP1 and IP2 groups, respectively. Then the culture medi- um was replaced with plain culture medium. At 24 h of incubation, the cells were collected for measure- ment of cell viability by CCK-8 assay, GABAA R cq mRNA expression （by quantitative polymerase chain reaction） , GABAAR expression in the cytomembrane （by Western blot） , level of GABAAR c subunit endoplasmic reticulum-associated degradation （ERAD） （by immunoprecipitation and Western blot） and CCAAT/enhancer-binding protein homologous protein （CHOP） expression （by immunofluorescence）. Re- suits Compared with group C, the cell viability was significantly decreased, the expression of GABAAR al mRNA and GABAAR cq in cytomembrane was down-regulated, the expression of CHOP was up-regula- ted, and the level of GABAAR cq subunit ERAD was increased in the other five groups （P〈0. 05）. Com- pared with group H, the cell viability was significantly decreased, the expression of GABAA R cq mRNA and GABAa R a1 in cytomembrane was down-regulated, the expression of CHOP was up-regulated, and the level of GABAAR cq subunit ERAD was increased in I, P and IP2 groups （P 〈0.05） , and no significant change was found in the parameters mentioned above in group IP1 （P〈0.05）. Compared with group I or group P, the cell viability was significantly increased, the expression of GABAAR a1 mRNA and GABAAR al in cytomembrane was up-regulated, the expression of CHOP was down-regulated, and the level of GABAAR cq subunit ERAD was decreased in IPl and IP2 groups （P〈0. 05）. Compared with group IP1, the cell viability was significantly decreased, the expression of GABAAR cq mRNA and GABAAR cq in cy- tomembrane was down-regulated, the expression of CHOP was up-regulated, and the level of GABAAR cq subunit ERAD was increased in group IP2 （P〈0.05）. Conclusion Combination of 1.0% isoflurane and 6.7 μmol/L propofol does not aggravate hypoxia-induced destruction of GABAA R cq subunit proteostasis in hippocampal neurons of rats.