阳离子-氯离子共转运体KCC2、NKCC1在对氯苯丙氨酸致急性失眠大鼠模型脑干中的表达

Expression of cation-chloride cotransporters KCC2 and NKCC1 in brainstem of parachlorophenylalanine-induced acute insomnic rats

目的探讨阳离子-氯离子共转运体KCC2、NKCC1在大鼠急性失眠发生机制中的作用。方法SD大鼠腹腔注射对氯苯丙氨酸（PCPA）制作急性失眠大鼠模型，同步设立生理盐水对照组和地西泮干预组，每组6只；用逆转录-聚合酶链反应（RT—PCR）、免疫印迹（Western blot）检测各组大鼠脑干KCC2、NKCC1的表达；用氯离子荧光探针（MQAE），结合激光共聚焦显微镜观察脑干组织细胞内氯离子浓度（[C1-]i）。结果（1）模型组和干预组的KCC2 mRNA及蛋白表达均低于对照组（mRNA丰度值：0．196±0．021比0．939±0．109，P〈0．05；0．485±0．026比0．939±0．109，P〈0．05；蛋白相对值：0．363±0．058比0．967±0．155，P〈0．05；0．663±0．106比0．967±0．155，P〈0．05）；干预组高于模型组（mRNA丰度值：0．485±0．026比0．196±0．021，P〈0．05；蛋白相对值：0．663±0．106比0．363±0．058，P〈0．05）。（2）模型组的NKCC1 mRNA及蛋白表达稍高于对照组，但差异无统计学意义（mRNA丰度值：0．344±0．026比0．320±0．019，P〉0．05；蛋白相对值：0．244±0．010比0．230±0．021，P〉0．05）；干预组较对照组和模型组降低（mRNA丰度值：0．066±0．031比0．320±0．019，P〈0．05；0．066±0．031比0．344±0．026，P〈0．05；蛋白相对值：0．131±0．012比0．230±0．021，P〈0．05；0．131±0．012比0．244±0．010，P〈0．05）。（3）模型组的[Cl-]i高于对照组和干预组（相对值：0．0315±0．0039比0．0164±0．0019，P〈0．05；0．0315±0．0039比0．0182±0．0013，P〈0．05）；干预组稍高于对照组，但差异无统计学意义（相对值：0．0182±0．0013比0．0164±0．0019，P〉0．05）。结论（1）脑干KCC2表达下调及[Cl-]i的升高可能参与大鼠急性失眠的病理机制；（2）地西泮可能通过上调KCC2和下调NKCC1的表达，进而降低[Cl-]i而发挥镇静催眠作用。

Objective To explore the possible roles of KCC2 and NKCC1 in the pathological mechanism of acute insomnia in rats. Methods A total of 18 Sprague-Dawley rats were randomly selected into model, interference and normal control groups. The expressions of KCC2 and NKCC1 in brainstem were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. The concentration of intracellular Cl^-（ [Cl^-] i） in brainstem was detected by fluorescence probe MQAE with laser confocal microscopy. Results （ 1 ） Comparing with the control group, both KCC2 mRNA and protein expression were down-regulated in the model and interference groups （mRNA ：0. 196±0. 021 vs 0. 939±0. 109, P 〈0. 05; 0. 485 ± 0. 026 vs 0. 939 ± 0. 109, P 〈 0. 05 ; protein expression ： 0. 363 ± 0. 058 vs 0. 967 ± 0. 155, P 〈0. 05; 0. 663±0. 106 vs 0. 967±0. 155, P 〈0. 05）. However they became up-regulated in the interference group versus the model group （ mRNA：0. 485 ± 0. 026 vs 0. 196 ± 0. 021, P 〈 0. 05 ; protein expression：0. 663 ±0. 106 vs 0. 363 ±0. 058, P 〈0. 05）. （2） Comparing with the control group, both NKCC1 mRNA and protein expression in the model group were slightly up-regulated. But statistical difference was insignificant （mRNA：0. 344 ± 0. 026 vs 0. 320 ± 0. 019, P 〉 0. 05 ; protein expression：0. 244 ± 0. 010 vs 0. 230 ± 0. 021, P 〉 0. 05 ）. There was down-regulation in the interference group versus the model and control groups （mRNA：0.066±0.031 vs 0.320 ±0.019, P〈0.05; 0.066±0.031 vs 0. 344±0.026, P〈0.05;protein expression：0. 131 ± O. 012 vs O. 230 ± O. 021, P 〈 O. 05; O. 131 ± O. 012 vs O. 244 ± O. 010, P 〈 O. 05 ）. （3） Comparing with the control group, [ Cl^-] i became up-regulated in the model group （0. 0315 ±O. 0039 vs O. 0164 ± O. 0019, P 〈 O. 05 ）. It was down-regulated in the interference group versus the model group （0. O182 ± O. 0013 vs O. 0315 ±O. 0039, P 〈 O. 05 ）, but higher than control group without statistical difference （0. 0182±0. 0013 vs 0.0164±0.0019, P 〉 0.05）. Conclusion The down-regulation of KCC2 and rise of [ Cl^- ] i in brainstem may participate in the pathological mechanism of acute insomnia in rats.And the mechanism of sedative-hypnotic diazepam may be operate through an up-regulation of KCC2, a down-regulation of NKCC1 and decreased [ Cl^- ] i.