Aslan Mutay, Canatan Duran
Akdeniz University School of Medicine. Department of Biochemistry, Antalya, Turkey.
Exp Hematol. 2008 Nov;36(11):1535-44. doi: 10.1016/j.exphem.2008.07.004. Epub 2008 Sep 21.
Interaction of nitric oxide (NO) with enzymatic sources of reactive species exerts modulatory actions on inflammatory signaling mechanisms.
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.
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-phenylalanine 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-phenylalanine stimulation revealed a significant decrease in SCD neutrophils compared to controls.
Depletion of GSH in SCD neutrophils may impact on rates of NO consumption and reflects increased oxidative stress associated with neutrophil activation.
一氧化氮（NO）与活性物质的酶源相互作用，对炎症信号传导机制发挥调节作用。
测定了镰状细胞病（SCD）患者和健康对照者分离出的中性粒细胞中的NADPH氧化酶、总过氧化物酶、环氧化酶（COX）活性以及NO消耗。还分析了谷胱甘肽（GSH）水平和诱导型NO合酶（NOS-2）的表达，分别以评估细胞内氧化还原状态和NO生成。
通过测量超氧化物释放来进行NADPH氧化酶的功能测定，在基础条件下以及对N-甲酰甲硫氨酰亮氨酰苯丙氨酸刺激的反应中，对照组和SCD组的超氧化物释放相似。通过分光光度法评估的过氧化物酶活性，SCD中性粒细胞与对照组相比无显著差异。通过试剂盒测定的总COX活性，在SCD中性粒细胞中显著增加。在SCD中观察到的总COX活性增加是由于COX-2活性增强，使用同工型特异性抑制剂DuP-697和SC-560进行区分。对SCD和对照中性粒细胞中COX-2蛋白的蛋白质印迹分析证实了患病组中酶活性增加。对SCD患者中性粒细胞裂解物的蛋白质印迹分析显示，与对照组相比，NOS-2蛋白含量显著增加。对GSH以及硝酸盐/亚硝酸盐水平的分光光度测量显示，SCD中性粒细胞中GSH减少，硝酸盐/亚硝酸盐含量增加。在基础条件下以及N-甲酰甲硫氨酰亮氨酰苯丙氨酸刺激后对NO消耗的电化学测量显示，与对照组相比，SCD中性粒细胞中NO消耗显著减少。
SCD中性粒细胞中GSH的消耗可能影响NO消耗速率，并反映出与中性粒细胞活化相关的氧化应激增加。
