反式激活蛋白-NEMO结合域抑制核因子-κB活化在胆红素诱导大鼠海马神经元凋亡中的作用

Role of nuclear factor-κB activation in bilirubin-induced rat hippocampal neuronal apoptosis and the effect of TAT-NBD intervention

目的明确核因子-κB(NF-κB)活化在胆红素诱导大鼠海马神经元凋亡中的作用,及反式激活蛋白-NEMO结合域(TAT-NBD)对胆红素神经毒性的干预作用。方法原代培养海马神经元分为对照组、胆红素组、TAT-NBD早期干预组、持续干预组及晚期干预组。免疫细胞化学法检测NF-κB p65蛋白表达,改良MTT法、Annexin V-FITC/PI双染法及TUNEL法检测细胞相对存活率及凋亡率,ELISA法检测原代培养基上清IL-1β水平。结果胆红素组海马神经元NF-κB p65蛋白平均光密度值(MOD)明显高于对照组(P<0.01),6、24 h达高峰。TAT-NBD早期干预组细胞相对存活率为(80.784±9.767)%低于对照组(P<0.01)、高于胆红素组(P<0.01),细胞凋亡率为(14.100±2.252)%(Annexin V-FITC/PI双染法)、(12.883±1.629)%(TUNEL法)高于对照组(P<0.01)、低于胆红素组(P<0.01),IL-1β水平为15.348±0.812 pg/ml低于胆红素组(P<0.05)。TAT-NBD持续干预及晚期干预组细胞存活率、凋亡率、IL-1β水平与胆红素组无显著性差异(P>0.05)。结论 NF-κB双向调控胆红素诱导的海马神经元凋亡。TAT-NBD抑制NF-κB早期高峰有神经保护作用,有可能用于胆红素脑损伤的预防。

Objective To investigate the role of nuclear factor-κB（NF-κB） activation in bilirubin-induced apoptosis of rat hippocampal neurons and the effect of TAT-NBD intervention on bilirubin neurotoxicity. Methods Primary-cultured rat hippocampal neurons were treated with TAT-NBD in the initial 6 or 24 h or in the latter 6 h during a 24-h bilirubin exposure of the cells （early, continuous and late intervention groups, respectively）. Immunocytochemistry was performed to detect NF-κB p65 protein expression, and the cell survival and apoptosis were assessed with a modified MTT assay, Armexin V-FITC/PI and TUNEL assay. IL-1β concentration in the supematant was determined with ELISA. Results Compared with the control cells, bilirurin-treated cells showed a significantly increased NF-KB p6S protein expression （P〈0.01）, which reached the peak level at 6 and 24 h （P〈0.01）. The cell survival rate in early TAT-NBD intervention group was （80.784±9.767）%, significantly lower than that of the control group （P〈0.01） but higher than that of bilirubin group （P〈0.01）; the apoptotic rate in early TAT-NBD intervention group was significantly higher than that of control group （P〈0.01） but lower than that of bilirubin group （P〈0.01）. IL-1β concentration was significantly lower in early TAT-NBD intervention group （15.348±0.812 pg/ml） than in bilirubin group （P〈0.05）. The continuous and late TAT-NBD intervention groups showed comparable cell survival rate, apoptotic rate and IL-1β concentration with bilirubin group （P〉0.05）. Conclusion NF-κB bidirectionally regulates bilirubin-induced apoptosis of rat hippocampal neurons. Selective inhibition of the early peak of NF-κB by TAT-NBD offers neuroprotective effect. TAT-NBD can be potentially used for prophylaxis of bilirubin-induced brain injury.