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目的探讨胍丁胺(agmatine,AGM)对脂多糖(lipopolysaccharides,LPS)诱导小鼠巨噬细胞系RAW264.7氧化应激的抑制作用及其机制。方法 RAW264.7细胞经LPS(10μg/m L)刺激发生氧化应激,同时AGM(1 mmol/L)对其进行干预。分为对照组、LPS组、LPS+AGM组、AGM组。药物作用24 h,以2,7-二氢二氯荧光素二乙酸酯(2’,7’-dichlorofluoresce in diacetate,DCFH-DA)为荧光探针检测细胞内活性氧(reactive oxygen species,ROS)水平;Griess法检测细胞内一氧化氮(NO)水平;Western blot检测细胞内的iNOS蛋白及细胞质细胞核内Nrf2蛋白表达;RT-PCR检测Nrf2下游抗氧化酶血红素氧合酶(heme oxygenase-1,HO-1)mRNA的表达。结果与对照组相比,LPS可显著增加RAW264.7细胞中ROS、NO以及iNOS蛋白水平,而AGM干预后上述指标含量均明显降低(P<0.05)。AGM干预组中细胞核的Nrf2表达及其下游分子HO-1 mRNA表达水平较LPS单独刺激组显著升高,表明Nrf2信号通路在AGM的作用下被活化。结论 AGM可通过活化转录因子Nrf2促进抗氧化酶HO-1的表达,进而减轻细胞内的氧化应激损伤。
Objective To investigate the inhibitory effect of agmatine (AGM) on the oxidative stress induced by lipopolysaccharides (LPS) in murine macrophage cell line RAW264.7 and its mechanism. Methods RAW264.7 cells were stimulated with LPS (10μg / ml) for 15 days and oxidative stress was induced by AGM (1 mmol / L). Divided into control group, LPS group, LPS + AGM group, AGM group. Drug-induced 24 h, 2,7-dichlorofluorescein diacetate (DCFH-DA) was used as a fluorescent probe to detect intracellular reactive oxygen species (ROS ). The levels of nitric oxide (NO) in cells were detected by Griess method. The expression of iNOS protein and Nrf2 protein in cytoplasm nucleus were detected by Western blot. The levels of heme oxygenase- 1, HO-1) mRNA expression. Results Compared with the control group, LPS increased the levels of ROS, NO and iNOS in RAW264.7 cells significantly (P <0.05). The expression of Nrf2 in nuclei and the expression of HO-1 mRNA in nuclei of AGM intervention group were significantly higher than those of LPS alone stimulation group, indicating that Nrf2 signaling pathway was activated by AGM. Conclusion AGM can promote the expression of antioxidant enzyme HO-1 through activation of transcription factor Nrf2, and then reduce oxidative stress injury in cells.