地塞米松对烟草烟雾刺激的人巨噬细胞沉默信息调节因子1表达影响的实验研究

Experimental study of dexamethasone on SIRT1 in human macrophage cells stimulated by cigarette smoke

目的:探讨地塞米松(DEX)对烟草烟雾提取物(CSE)刺激的人巨噬细胞中沉默信息调节因子1(SIRT1)表达的影响及其机制。方法:体外培养人类单核细胞系U937细胞,用佛波酯将其诱导分化为人巨噬细胞,将细胞分为5组:对照组(Control)、CSE组(CSE)、地塞米松组(DEX)、烟酰胺组(NAM)、吡咯烷二硫氨基甲酸组(PDTC)。CSE组用1%浓度烟草烟雾提取物刺激24 h;地塞米松组用10-6mol/L地塞米松预处理24 h后,用1%浓度CSE刺激24 h;烟酰胺组20 mmol/L浓度烟酰胺孵育24 h;吡咯烷二硫氨基甲酸组用20 nmol/L浓度吡咯烷二硫氨基甲酸孵育24 h。处理后分别用荧光酶标仪检测各组细胞ROS释放水平,ELISA试剂盒检测细胞上清液IL-6的浓度;蛋白印迹实验(Western blot)检测SIRT1和NF-κB蛋白表达。结果:烟草烟雾引起人巨噬细胞释放ROS增加(P <0. 01),促进IL-6释放(P <0. 01),抑制细胞中SIRT1表达以及促进转录因子NF-κB蛋白表达(P <0. 01)。DEX可抑制烟草烟雾诱导的人巨噬细胞上清中IL-6的含量(P <0. 01),降低烟草烟雾暴露引起的人巨噬细胞内ROS释放(P <0. 01);下调烟草烟雾诱导的人巨噬细胞NF-κB的表达(P <0. 01),增强烟草烟雾抑制的人巨噬细胞SIRT1蛋白表达(P <0. 01)。结论:DEX通过抑制CSE引起的ROS释放从而降低CSE对SIRT1的损害,进而抑制NF-κB表达,最终减少炎症介质IL-6释放。

Objective: To investigate the effect and mechanism of dexamethasone(DEX) on the expression of sirtuin 1(SIRT1 in human macrophage cells stimulated by cigarette smoke extract(CSE).Methods: The human monocytic cell line U937 cells were cultured in vitro,and differentiate to human macrophages by Phorbol esters(PMA).The cells were divided into 5 groups: control group(Control),CSE group(CSE),dexamethasone group(DEX),nicotinamide group(NAM),pyrrolidine dithiocarbamate group(PDTC).CSE group treated with 1% cigarette smoke extract for 24 hours;dexamethasone group pretreatment with 10-6 mol/L dexamethasone 24 h before stimulate by CSE(1%,24 h);nicotinamide group treated with 20 mmol/L nicotinamide for 24 h;pyrrolidine dithiocarbamate group treated with 20 nmol/L pyrrolidine dithiocarbamate for 24 h.Fluorescent microplate reader detected the release of ROS,IL-6 of cell supernatant detected by ELISA;Western blot to detect the expression of SIRT1 and NF-κB protein.Results: Fluorescent microplate reader showed that CSE promotes the release of ROS in human macrophages(P < 0.01),ELISA showed that CSE promote IL-6 release(P < 0.01),Western blot analysis suggested that CSE inhibite the expression of SIRT1 in cells and promote the expression of NF-κB protein(P < 0.01).DEX inhibited release of ROS induced by CSE(P < 0.01),and down-regulated the release of IL-6 in supernatant(P < 0.01),enhanced the expression of SIRT1 protein and down-regulated the expression of NF-κB protein(P < 0.01).Conclusion:DEX protect SIRT1 protein from CSE by suppressing CSE-induced ROS,and inhibiting NF-κB activity,suppressed the release of IL-6.