H_2O_2预处理对NF-κB的激活作用特征及其细胞保护作用

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。

Characteristics of effect of H2O2 preconditioning on NF-κB activation and its role in the cytoprotection in PC12 cells

Aim To explore the effect of H2O2 precon-ditioning on nuclear factor-kappa B(NF-κB)activa-tion and the role NF-κB in cytoprotection induced by H2O2 preconditioning in PC12 cells.Methods The experimental model of H2O2 preconditioning against apoptosis induced by H2O2 at a high concentration was established,and experimental groups were divided as follows:(1)control;(2)precondition;(3)H2O2;(4)precondition+H2O2;(5)TPCK+ precondition+H2O2;(6)TPCK.The percentage of apoptotic cells was measured by flow cytometry(FCM)with propidium iodide stain.The viability of PC12 cells was assessed by MTT assay.The level of NF-κB expression was detected by Western blot.The NF-κB DNA binding activity was detected by electrophoretic mobility shift assay(EMSA).Results H2O2 preconditioning significantly up-regulated NF-κB p65 expression and promoted its DNA binding activity.Furthermore,H2O2 preconditioning obviously enhanced the expression of NF-κB p65 induced by H2O2 at a high dose(P<0.01,compared with H2O2 group).H2O2 preconditioning markedly protected PC12 cells against injury induced by H2O2 at a high dose,increasing the viability of cells and decreasing the percentage of apoptotic cells(P<0.01,compared with H2O2 group).N-tosyl-L-phenylalanine chloromethyl ketone(TPCK),an inhibitor of NF-κB antagonized H2O2 preconditioning-induced NF-κB activation and the adaptive cytoprotection(P<0.01,compared with PC+H2O2 group).Conclusion NF-κB was activated by H2O2 preconditioning,which was one of the underlying mechanisms underlying adaptive cytoprotection induced by H2O2 preconditioning.