Modulation of redox pathways in neutrophils from sickle cell disease patients
Modulation of redox pathways in neutrophils from sickle cell disease patients
复制标题
DOI：
10.1016/j.exphem.2008.07.004
复制
发表时间：
2008-11-01
影响因子：
2.6
通讯作者：
Canatan, Duran
中科院分区：
文献类型：
--
作者：
Aslan, Mutay;Canatan, Duran
Objective.一氧化氮（NO）与活性物质的酶源的相互作用对炎症信号传导机制发挥调节作用。测量了从镰状细胞病（SCD）患者和健康对照中分离的中性粒细胞的NADPH氧化酶、总过氧化物酶、环氧合酶（考克斯）活性和NO消耗。同时检测细胞内谷胱甘肽（GSH）水平和诱导型一氧化氮合酶（NOS-2）的表达，以评估细胞内氧化还原状态和NO的产生。通过测量超氧化物释放来进行NADPH氧化酶的功能测定，这在对照和SCD中是相似的，在基础条件下和响应于N-甲酰基-甲硫氨酰基-亮氨酰基-苯丙氨酸刺激。过氧化物酶的活性，评估荧光定量法，SCD中性粒细胞与对照组相比，没有显着差异。SCD中性粒细胞的总考克斯活性（通过检测试剂盒测定）显著增加。在SCD中观察到的总考克斯活性的增加是由于考克斯-2的活性增强，通过使用亚型特异性抑制剂DuP-697和SC-560进行区分。SCD和对照中性粒细胞中考克斯-2蛋白的Western印迹分析证实了患病组中酶活性增加。SCD患者中性粒细胞裂解物的Western印迹分析显示，与对照组相比，NOS-2蛋白含量显著增加。GSH和硝酸盐/亚硝酸盐水平的分光光度法测量结果显示，减少GSH和SCD中性粒细胞中的硝酸盐/亚硝酸盐含量的增加。在基础条件下和N-甲酰-甲硫氨酰-亮氨酰-苯丙氨酸刺激后NO消耗的电化学测量显示SCD中性粒细胞与对照组相比显著减少。SCD中性粒细胞中GSH的消耗可能影响NO消耗速率，并反映与中性粒细胞活化相关的氧化应激增加。(c)2008 ISEH-血液学和干细胞学会。爱思唯尔公司出版
Objective. Interaction of nitric oxide (NO) with enzymatic sources of reactive species exerts modulatory actions on inflammatory signaling mechanisms.Materials and Methods. NADPH oxidase, total peroxidase, cyclooxygenase (COX) activity, and NO consumption were measured in neutrophils isolated from sickle cell disease (SCD) patients and healthy controls. Glutathione (GSH) levels and expression of inducible NO synthase (NOS-2) were also analyzed to assess intracellular redox state and NO production, respectively.Results. Functional assay of NADPH oxidase was performed by measuring superoxide release, which was similar in control and SCD, both at basal conditions and in response to N-formyl-methionyl-leucyl-phenylaianine stimulation. Peroxidase activity, assessed spectrophotometrically, was not significantly different in SCD neutrophils compared to controls. Total COX activity, measured via an assay kit, was significantly increased in SCD neutrophils. The increase in total COX activity observed in SCD was due to enhanced activity of COX-2, differentiated by using the isoform-specific inhibitors DuP-697 and SC-560. Western blot analysis of COX-2 protein in SCD and control neutrophils confirmed increased enzyme activity in the diseased group. Western blot analysis of neutrophil lysates from SCD patients showed significantly increased NOS-2 protein content, compared to controls. Spectrophotometric measurement of GSH and nitrate/nitrite levels showed a decrease in GSH and an increase in nitrate/nitrite content in SCD neutrophils. Electrochemical measurement of NO consumption both under basal conditions and after N-formyl-methionyl-leucyl-phenylaianine stimulation revealed a significant decrease in SCD neutrophils compared to controls.Conclusions. Depletion of GSH in SCD neutrophils may impact on rates of NO consumption and reflects increased oxidative stress associated with neutrophil activation. (c) 2008 ISEH-Society for Hematology and Stem Cells. Published by Elsevier Inc.