ERK及AP-1对二氧化硅上调人肺上皮细胞纤溶酶原激活物抑制因子-1表达的作用

摘要

目的 探讨细胞外信号调控激酶(ERK)/激活蛋白(AP-1)信号通路在二氧化硅诱导人肺上皮细胞(A549)纤溶酶原激活物抑制因子-1(PAI-1)表达中的作用.方法 以人肺上皮细胞A549为研究对象,200 μg/ml二氧化硅刺激0-24h.干预实验采用姜黄素预处理以及c-Jun显性负性突变体(TAM67)瞬时转染阻断AP-1活性;PD98059预处理阻断ERK活性.以Western印迹检测ERK激酶活力、PAI-1蛋白表达,EMSA检测AP-I DNA结合活性.结果 (1)SiO2作用A549细胞4、8、16 h,AP-1DNA结合活性分别为对照的1.3、1.3、2.1倍,差异有统计学意义(P＜0.05);10、25、50μmol/L浓度姜黄素对二氧化硅诱导的PAI.1蛋白表达抑制率分别为20%、63%、65%,差异有统计学意义(P＜0.05);TAM-67对二氧化硅诱导的PAI.1蛋白表达的抑制率为59%,与二氧化硅刺激组比,差异有统计学意义(P＜0.05).(2)二氧化硅作用A549细胞诱导ERK激酶活化;PD98059对二氧化硅诱导的PAI-1蛋白表达的抑制率为51%,与二氧化硅刺激组比较,差异有统计学意义(P＜0.05).(3)PD98059对二氧化硅诱导的AP-1 DNA结合活性的抑制率为73%,与二氧化硅刺激组比较,差异有统计学意义(P＜0.05).结论 ERK/AP-1信号通路参与调控SiO2诱导的人肺上皮细胞PAl-1蛋白表达.%Objective To investigate the role of extracellular signal-regulated kinase (ERK)/activator protein-1 (AP-1) signaling pathway in SiO2-induced plasminogen activators inhibitor-1 (PAI-1) protein expression in human lung epithelial cells A549. Methods A549 cells were cultured and then stimulated with 200 μg/ml SiO2 for 0-24 h. To prevent AP-1 activity,Curcumin was added into culture medium before incubated with SiO2 and transient TAM-67 tranfection was performed. In addition, PD98059 was pretreated with cells to prevent ERK activity. The PAI-1 protein expression and ERK activity were evaluated by Western blot. The AP1 DNA binding activity was tested by EMSA. Results (1)At 4,8 and 16 h after exposure to SiO2, the fold change of AP-1 DNA binding activity (relative to the control group) were1.3,1.3,and 2.1 ,respectively (P＜0.05 ). 10,25,50 μmol/L Curcumin inhibited SiO2-induced PAI-1 protein expression (inhibition ratio: 20%,63%, 65%; P＜0.05). TAM-67 downregulated SiO2-induced PAI-1 protein expression(inhibition ratio: 59%, P＜0.05). (2)SiO2 activated ERK and PD98059 downregulated SiO2-induced PAI-1 protein expression (inhibition ratio:51% ,P＜0.05). (3)PD98059 downrngulated SiO2-indueed AP-1 DNA binding activity (inhibition ratio:73%,P ＜0.05). Conclusion ERK/AP-1 signaling pathway is responsible for SiO2-induced PAI-1 protein expression.