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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/L NO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DTT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。
Objective: To observe the effect of exogenous nitrosylation of RSC-364 on the activity of cAMP response element binding protein (CREB). Methods: The total protein of RSC-364 cells was extracted and incubated with 100μmol / L reduced glutathione (GSH), 100μmol / L NO donor nitrosylated glutathione (GSNO) and 10mmol / L nitrosylation Dithiothreitol (DTT) and solvent alone or in combination for 15 min to complete the exogenous nitrosylation. The nitrosylated protein expression level was detected by biotin transformation method and Western blotting technique. Soluble or recombinant RSC-364 cells were incubated with rIL-1|Â (10|Ìg / L) for 1h, then the nuclear protein was extracted and exogenous nitrosated. The changes of electrophoretic mobility shift assay (EMSA) The effect of nitrosylation on the activity of CREB was observed. Results: The expression level of nitrosylated protein in RSC-364 cells incubated with GS-NO significantly increased, while the application of DTT inhibited the nitrosylation level. GSNO and RSC-364 nuclear protein incubated significantly inhibited the expression of rIL- 1β-induced CREB activity (P <0.01), and the effect of GSNO reversed by DTT (P <0.01). Conclusion: NO inhibits the CREB activity of RSC-364 cells through nitrosylation.