14-3-3ε激活NF-κB通路参与氧糖剥夺/再灌注诱导的原代培养大鼠脊髓星形胶质细胞活化

14-3-3ε participates in the activation of primarily cultured astrocytes from rats spinal cord induced by oxygen-glucose deprivation reoxygenation through activating NF-κB pathway

目的:研究14-3-3ε蛋白对氧糖剥夺/再灌注(oxygen-glucose deprivation reperfusion,OGDR)诱导的脊髓星形胶质细胞活化的影响及其机制。方法:原代培养新生SD大鼠脊髓星形胶质细胞建立OGDR模型,用CCK-8试剂盒检测细胞活力,乳酸脱氢酶(lactate dehydrogenase,LDH)活性检测试剂盒检测LDH活性,用ELISA法检测谷氨酸浓度、TNF-α和IL-1β的表达,Real-Time PCR和Western Blot分别检测14-3-3εmRNA和蛋白表达,用Western Blot检测核转录因子-B(Nuclear transcription factor kappa B,NF-κB)、星形胶质细胞活化标志物胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、胶质瘢痕标志物neurocan和phosphacan蛋白的表达;转染14-3-3εsiRNA沉默14-3-3ε表达,将细胞分为对照组、OGDR组、非特异性转染(Scramble)组和14-3-3εsiRNA组,检测14-3-3εsiRNA预处理对OGDR星形胶质细胞GFAP、neurocan和phosphacan以及NF-κB蛋白表达的影响。用NF-κB特异性抑制剂PDTC预处理细胞,观察NF-κB通路是否参与14-3-3ε对星形细胞的活化。结果:OGDR引起细胞活力降低,LDH活性、谷氨酸浓度、TNF-α和IL-1β分泌升高;OGDR处理后,细胞14-3-3εmRNA和蛋白表达均显著升高,同时NF-κB、GFAP、neurocan和phosphacan蛋白的表达均升高,与对照组相比差异均具有统计学意义(P<0.05);14-3-3ε沉默后,OGDR细胞GFAP、neurocan、phosphacan和NF-κB蛋白的表达水平均显著下调(P<0.05)。PDTC预处理后,14-3-3εsiRNA对星形胶质细胞活化的抑制作用增强。结论:14-3-3ε可能是通过激活NF-κB通路参与OGDR诱导的脊髓星形细胞活化。

Objective： To investigate the effect and mechanism of 14-3-3ε on the activation of spinal cord astrocytes induced by oxygen-glucose deprivation reoxygenation （OGDR）. Methods： Primary spinal cord astrocytes from SD rats were used to build OGDR model. Then cell viability, LDH activity, the concentration of glutamate and the level of inflammatory cytokines （TNF-α and IL-1β） were detected by CCK-8 assay, LDH activity assay kit and ELISA, respectively. The mRNA and protein expression of 14-3-3ε was detected by Real-Time PCR and Western Blot. The level of NF-KB and glial scar associated proteins （ GFAP, neurocan and phosphacan） were determined by Western Blot. After silenced the expression of 14-3-3ε by 14-3-3ε siRNA transfection, cells were divided into control group, OGDR group, nonspecific transfeeted （Scramble） group and 14-3-3ε siRNA transfection group. The effect of 14-3-3ε siRNA on the protein expres-sion of NF-κB, GFAP, neurocan and phosphacan in spinal cord astrocytes with OGDR was detected. Pyrrolidine dithiocarbamate （ PDTC）, an inhibitor of NF-κB, was used to analyze the influence of NF-κB pathway on the effect of 14-3-3ε siRNA on the activation of astrocyte. Results： OGDR induced the decrease of cell viability and the increase of LDH activ- ity, glutamate concentration, the levels of TNF-α and IL-1β. The mRNA and protein expression of 14-3-3ε were upregulated after OGDR treatment compared with contral group （P 〈 0. 05）. The protein levels of GFAP, neurocan, phosphacan and NF-κB was significantly upregulated after OGDR, but downregulated after 14-3-3ε knockdown （P 〈 0. 05 ）. After PDTC preconditioning, the inhibitory effect of 14-3-3ε siRNA on the activation of astrocyte was increased. Conclusion： 14-3-3ε involved in the activation of spinal cord astrocyte which may be associated with NF-κB signaling pathway.