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目的探讨H2O2预处理对核转录因子-κB(NF-κB)的激活作用,并观察NF-κB在H2O2预处理诱导的适应性细胞保护中的作用。方法在PC12细胞建立H2O2预处理对抗高浓度H2O2诱导细胞凋亡的实验模型,分组如下:(1)空白对照组;(2)预处理组;(3)损伤组;(4)预处理+损伤组;(5)TPCK+预处理+损伤组;(6)TPCK组。应用碘化丙啶(PI)染色流式细胞术检测细胞凋亡率,甲氮甲唑蓝(MTT)法检测细胞存活率,免疫印迹法(Westernblot)测定NF-κB的表达水平,电泳迁移实验(EMSA)检测NF-κBDNA结合活性。结果H2O2预处理PC12细胞上调NF-κBp65的表达,增强DNA结合活性,并能明显地增加高浓度H2O2引起的NF-κBp65的表达(与损伤组比较,P<0.01)。H2O2预处理能使PC12细胞对抗高浓度H2O2引起的损伤,提高细胞存活率,降低细胞凋亡率(与损伤组比较,P<0.01)。NF-κB抑制剂甲苯磺酰苯丙氨酰氯甲酮(TPCK)可拮抗H2O2预处理对NF-κBp65的激活作用,并减弱H2O2预处理诱导的适应性细胞保护作用(与预处理+损伤组比较,P<0.01)。结论H2O2预处理对NF-κB的激活作用可能是其引起的适应性细胞保护机制之一。
Objective To investigate the activation of nuclear factor-κB (NF-κB) by H2O2 preconditioning and to explore the role of NF-κB in adaptive cell protection induced by H2O2 preconditioning. Methods The experimental model of H2O2 preconditioning against PC12 cells induced by high concentration H2O2 was established in PC12 cells. The experimental groups were as follows: (1) blank control group; (2) pretreatment group; (3) injury group; (4) pretreatment + injury Group; (5) TPCK + pretreatment + injury group; (6) TPCK group. The apoptotic rate was detected by propidium iodide (PI) staining, the cell viability was determined by MTT assay, the expression of NF-κB was detected by Western blotting, the electrophoretic mobility shift assay (EMSA) to detect NF-κB DNA binding activity. Results H2O2 pretreatment up-regulated the expression of NF-κBp65 in PC12 cells, increased the DNA binding activity, and significantly increased the expression of NF-κBp65 induced by H2O2 (P <0.01 compared with the injury group). Pretreatment with H2O2 could protect PC12 cells against H2O2-induced cell injury, increase cell viability and decrease apoptosis (compared with the injury group, P <0.01). TPCK, an inhibitor of NF-κB, could antagonize the activation of NF-κBp65 by H2O2 pretreatment and attenuate the adaptive cytoprotection induced by H2O2 pretreatment (compared with pretreatment + injury group , P <0.01). Conclusion The activation of NF-κB by H2O2 preconditioning may be one of the adaptive cytoprotection mechanisms.