沉默囊性纤维化跨膜传导调节因子对大鼠海马神经元细胞线粒体功能及氧化应激的影响

目的探讨沉默囊性纤维化跨膜传导调节因子(cystic fibrosis transmembrane conductance regulator,CFTR)在大鼠海马神经元细胞线粒体功能及氧化应激中的作用。方法18只SD大鼠随机分为CFTR小干扰RNA(small interfering RNA,siRNA)组、阴性对照组、空白对照组各6只。分离3组海马神经元细胞,取对数生长期细胞进行实验。CFTR siRNA组神经元细胞转染CFTR siRNA,阴性对照组转染阴性对照siRNA,空白对照组不转染。转染后培养48 h,采用Western blot法检测3组神经元细胞线粒体CFTR蛋白相对表达量,JC-1荧光探针检测线粒体膜电位,荧光分光光度法检测线粒体活性氧(reactive oxygen species,ROS)和过氧化氢(hydrogen peroxide,H2O2)表达,氧电极法测定线粒体呼吸控制率(respiratory control rate,RCR),荧光素酶发光法检测线粒体三磷酸腺苷(adenosine triphosphate,ATP)浓度,高效液相色谱法检测线粒体谷胱甘肽(glutathione,GSH)和氧化型谷胱甘肽(oxidized glutathione,GSSG)表达。结果转染后培养48 h,CFTR siRNA组神经元细胞线粒体CFTR蛋白相对表达量(0.09±0.01)、线粒体膜电位(247.16±34.28)mV]、ATP(11.71±2.54)mol/g]、RCR(4.69±0.82)、GSH(0.71±0.12)nmol/μg]、GSH/GSSG值(0.31±0.08)低于空白对照组0.42±0.07、(432.18±56.04)mV、(24.18±3.64)mol/g、7.94±1.21、(1.62±0.23)nmol/μg、1.65±0.27]和阴性对照组0.38±0.06、(434.07±52.13)mV、(22.97±3.43)mol/g、7.83±1.18、(1.57±0.21)nmol/μg、1.63±0.24](P<0.05),线粒体H2O2水平(1.73±0.11)高于空白对照组(1.00±0.02)和阴性对照组(1.08±0.06)(P<0.05);空白对照组神经元细胞线粒体CFTR蛋白相对表达量及线粒体膜电位、ATP、RCR、H2O2、GSH、GSH/GSSG值与阴性对照组比较差异均无统计学意义(P>0.05)。结论沉默大鼠神经元细胞线粒体CFTR可引起海马神经元细胞线粒体功能障碍,增强氧化应激反应。

Influence of silencing CFTR on mitochondrial function and oxidative stress of rat hippocampal neurons

Objective To investigate the influences of cystic fibrosis transmembrane conductance regulator(CFTR)on mitochondrial function and oxidative stress of rat hippocampal neurons.Methods Eighteen SD rats were randomly divided into CFTR siRNA group,negative control group and blank control group,with 6 rats in each group.The hippocampal neuron cells were isolated in three groups and the cells in logarithmic growth phase were obtained for experiment.CFTR siRNA group was transfected with CFTR siRNA,negative control group was transfected with negative control siRNA,and blank control group was no transfected.After culture for 48 h,the relative expression of CFTR was detected by Western blot,the mitochondrial membrane potential was measured by JC-1 fluorescence intensity method,the levels of reactive oxygen species(ROS)and mitochondrial hydrogen peroxide(H2O2)were determined by fluorescence intensity method,the mitochondrial respiratory control rate(RCR)was measured by oxygen electrode method,the mitochondrial adenosine triphosphate(ATP)concentration was measured by luminescence spectrophotometry,and the levels of mitochondrial glutathione(GSH)and oxidized glutathione(GSSG)were determined by high performance liquid chromatography.Results The relative expression of CFTR protein,mitochondrial membrane potential,ATP,RCR,GSH and the GSH/GSSG value were lower in CFTR siRNA group(0.09±0.01,(247.16±34.28)mV,(11.71±2.54)mol/g,4.69±0.82,(0.71±0.12)nmol/μg,0.31±0.08)than those in blank control group(0.42±0.07,(432.18±56.04)mV,(24.18±3.64)mol/g,7.94±1.21,(1.62±0.23)nmol/μg,1.65±0.27)and negative control group(0.38±0.06,(434.07±52.13)mV,(22.97±3.43)mol/g,7.83±1.18,(1.57±0.21)nmol/μg,1.63±0.24)(P<0.05).The mitochondrial H2 O2 was higher in CFTR siRNA group(1.73±0.11)than that in blank control group(1.00±0.02)and negative control group(1.08±0.06)(P<0.05).The relative expression of CFTR protein,mitochondrial membrane potential,ATP,RCR,H2 O2,GSH and GSH/GSSG showed no significant differences between blank control group and negative control group(P>0.05).Conclusion Silenced CFTR in mitochondria of rat hippocampal neurons would cause mitochondrial dysfunction and enhance oxidative stress.