核因子-κB圈套寡核苷酸对核因子-κB活性及创伤炎症反应大鼠肝脏功能损害的影响

Effect of nuclear factor -κB (NF-κB) decoy oligodeoxynucleotide on NF-κB activity and liver function impairment in rats following traumatic inflammation

目的 探讨核因子 (NF) -κB圈套寡核苷酸 (ODN)对NF -κB活性及创伤炎症反应大鼠肝脏功能损害的影响。 方法 利用电泳迁移率改变实验 (EMSA) ,测定合成的环状哑铃形圈套ODN对NF -κB的DNA结合能力的竞争抑制作用 ;利用NF -κB反应性报告细胞株HEK -不稳定增强型绿色荧光蛋白 (d2EGFP) ,观察圈套ODN瞬时转染对报告基因表达的影响。Wistar大鼠 96只 ,随机分为对照组、创伤性炎症组、圈套ODN组和无关ODN组。利用EMSA检测各组肝脏组织NF -κB的DNA结合活性 ,用逆转录 -聚合酶链反应 (RT -PCR)检测大鼠肝脏组织TNF -α、白细胞介素 - 6 (IL - 6 )mRNA水平 ,酶联免疫吸附实验 (ELISA)法检测大鼠肝脏组织肿瘤坏死因子 -α(TNF -α)、IL - 6的蛋白水平。 结果 设计的环状哑铃形圈套ODN对NF -κB的DNA结合能力具有竞争抑制作用 ,1 μg ml和 2 μg ml圈套ODN瞬时转染HEK -d2EGFP ,可明显抑制p6 5蛋白诱导的d2EGFP表达。大鼠创伤性炎症后 3h ,肝脏NF -κB活性开始升高 ,伤后 1 2h达高峰。肝脏组织TNF -α、IL - 6mRNA水平和蛋白水平也明显上升 ,与NF -κB的活性改变一致。圈套ODN治疗后 ,大鼠肝脏组织TNF -α、IL - 6的表达明显下降 ,肝脏功能明显好转 ,而无关ODN则没有治疗效果。 结论 设计的靶向NF

Objective To study the effect of nuclear factor-κB (NF-κB) decoy oligodeoxynucleotide (ODN) on NF-κB activity and liver function impairment in rats following traumatic inflammation. Methods Ninety-six Wistar rats were randomly divided into four groups: control group, traumatic inflammation group, traumatic inflammation group, decoy ODN group and mutant decoy ODN group. All rats were sacrificed 3, 6,12,24,48 and 72 hours, respectively after traumatic inflammation. Electrophoretic mobility shift assay (EMSA) was employed to determine the competitive inhibitory effect of circular dumbbell decoy ODN on DNA binding activity of NF-κB and measure the DNA binding activity of NF-κB in the liver tissues. Meanwhile, NF-κB-responsive destabilized enhanced green fluorescent protein (d2EGFP) reporter system, ie, HEK-d2EGFP, was used to observe the effect of decoy ODN on reporter gene expression driven by NF-κB, RT-PCR to measure gene expressions of TNF-α and interleukin-6 (IL-6) in liver tissues and ELISA to determine protein level of TNF-α and IL-6. Results Circular dumbbell decoy ODN could competitively inhibit the DNA binding activity of NF-κB in vitro. The transient co-transfection of HEK-d2EGFP by decoy ODN at the concentrations of 1 μg/ml and 2 μg/ml significantly suppressed the d2EGFP expression induced by p65 protein. After traumatic inflammation, DNA binding activity of NF-κB in the liver tissues increased gradually at the 3rd hour and reached the peak at the 12th hour. So were the TNF-α, IL-6 mRNA and protein levels. After in vivo administration of decoy ODN, the expressions of TNF-α and IL-6 gene in the liver tissues significantly decreased with recovery of the liver function. While the mutant ODN had no curative effect. Conclusions Circular dumbbell decoy ODN targeting to NF-κB can effectively inhibit the release of mediators including TNF-α and IL-6 in rat liver tissues and thereby improve the liver function through specifically antagonizing the activity of NF-κB.