摘要
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.
出处
《神经解剖学杂志》
CAS
CSCD
北大核心
2016年第6期698-704,共7页
Chinese Journal of Neuroanatomy