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目的:观察重组白细胞介素-1β(rIL-1β)和重组干扰素-γ(rIFN-γ)联合诱导的亚硝基化反应对cAMP反应元件结合蛋白(CREB)的DNA结合活性的影响。方法:(1)分别用细胞因子(rIL-1β和rIFN-γ的混合物)、诱导型一氧化氮合酶抑制剂氨基胍(AG)及溶剂单独或联合孵育大鼠滑膜细胞12h,收集培养液上清进行一氧化氮(NO)含量测定;并提取各组总蛋白,其中细胞因子组的蛋白与巯基氧化修饰还原剂二硫苏糖醇(DTT)反应15min,采用生物素转化法与免疫印迹技术检测各组蛋白质的亚硝基化修饰水平。(2)分别用rIL-1β、rIL-1β和rIFN-γ的混合物、AG及溶剂单独或联合孵育滑膜细胞12h,提取各组核蛋白,其中rIL-1β+rIFN-γ组核蛋白与DTT反应15min,采用电泳迁移率改变分析技术观察各组CREB的DNA结合活性。结果:(1)细胞经rIL-1β和rIFN-γ孵育后,NO生成增多(P<0.01),蛋白质的亚硝基化修饰水平升高,而AG降低了NO的水平(P<0.01)并抑制了亚硝基化反应,DTT与总蛋白直接作用也可抑制亚硝基化反应。(2)rIL-1β和rIFN-γ共孵育明显抑制rIL-1β单独诱导的CREB的DNA结合活性(P<0.01),而AG抑制了rIL-1β和rIFN-γ的作用,DTT与核蛋白直接反应也可逆转细胞因子共孵育的作用,使CREB的DNA结合活性增高(P<0.01)。结论:IL-1β和IFN-γ共同孵育滑膜细胞可诱导细胞适度产生NO,使细胞内蛋白质的亚硝基化修饰水平升高,导致CREB发生可逆性巯基氧化修饰,抑制其DNA结合活性。
OBJECTIVE: To observe the effects of nitrosylation induced by recombinant interleukin-1β (rIL-1β) and recombinant interferon-γ (rIFN-γ) on the DNA binding activity of cAMP response element binding protein (CREB). Methods: (1) Synoviocytes were incubated with cytokines (a mixture of rIL-1β and rIFN-γ) and AGN (inhibitor of nitric oxide synthase) and solvent alone or in combination for 12 hours respectively. The content of nitric oxide (NO) in the supernatant of the supernatant was measured, and the total protein of each group was extracted. The protein of the cytokines was reacted with the dithiothreitol (DTT) Imprinting technique was used to detect the nitrosylation level of each group of proteins. (2) Synoviocytes were incubated with mixtures of rIL-1β, rIL-1β and rIFN-γ, AG and solvent separately or in combination for 12h, and the nuclear proteins of each group were extracted. The nuclear proteins of rIL-1β + rIFN- Reaction 15min, using electrophoretic mobility shift assay to observe the CREB DNA binding activity of each group. Results: (1) NO production was increased (P <0.01) and nitrosylation level of protein was increased in the cells incubated with rIL-1β and rIFN-γ, while AG decreased NO level (P <0.01) Inhibit the nitrosylation reaction, DTT and total protein can also inhibit the direct nitrosylation reaction. (2) The co-incubation of rIL-1β and rIFN-γ significantly inhibited the DNA-binding activity of CREB induced by rIL-1β (P <0.01), while AG inhibited the effect of rIL-1β and rIFN- Reactions also reversed cytokine co-incubation and increased CREB DNA binding activity (P <0.01). CONCLUSION: Co-incubation of IL-1β and IFN-γ by synovial cells can induce moderate NO production in cells and increase the nitrosylation of intracellular proteins, resulting in reversible sulfhydryl oxidation of CREB and inhibition of its DNA-binding activity.