中文摘要：

目的：观察RSC-364细胞外源性亚硝基化对cAMP反应元件结合蛋白（CREB）活性的影响。方法：提取RSC-364细胞总蛋白，与100μmol／L还原型谷胱甘肽（GSH）、100μmol／LNO供体亚硝基化谷胱甘肽（GSNO）、10mmol／L亚硝基化抑制剂二硫苏糖醇（DTT）及溶剂单独或联合应用孵育15min完成外源性亚硝基化，采用生物素转化法与Western blotting技术检测亚硝基化蛋白表达水平；分别用溶剂或重组大鼠白细胞介素-1β（rIL-1β）10μg／L孵育RSC-364细胞1h，提取细胞核蛋白，经外源性亚硝基化后用电泳迁移率改变分析（elec-trophoresis mobility shift assay，EMSA）观察亚硝基化作用对CREB活性的影响。结果：RSC-364细胞总蛋白经Gs-NO孵育后亚硝基化蛋白表达水平明显增高，而应用DTT可抑制亚硝基化水平；GSNO与RSC-364细胞核蛋白孵育后，明显抑制了rIL-1β诱导的CREB活性（P〈0．01），GSNO的作用可被DTr所逆转（P〈0．01）。结论：NO可通过亚硝基化作用抑制RSC-364细胞CREB活性。

英文摘要：

AIM ： To investigate the effect of protein S-nitrosylation on cAMP response element-binding protein（CREB） activity in RSC- 364 cells. METHODS：①RSC -364 cells cytoplasmic extracts were incubated with 100μmol/L GSH （control chemical） , 100μmol/L GSNO （a donor of NO） in the presence or absence of 10 mmol/L DTT（inhibitor of S-nitrosylation） for 15 min at room temperature. The S-nitrosylated proteins were determined by biotin switch assay. The expression of S-nitrosylated proteins was assayed by Western blotting. ②RSC - 364 cell nuclear extracts were subjected to S -nitrosylation and electrophoretic mobility shift assay （EMSA） to analyze the CREB DNA binding activity after 1 h stimulation of rIL-1β（ 10μg/L）. RESULTS： GSNO obviously increased the expression of nitrosy-lated proteins and inhibited the CREB activity, which was reversed by DTT. CONCLUSION ： S-nitrosylation may inhibit the CREB activity in RSC-364 cells.