Objective To investigate the role of reactive oxygen species(ROS) in urotensin E (UⅡ)-induced monocyte chemoattractant protein-1 (MCP-1) expression in RAW264. 7 cells. Methods RAW264. 7 cells were stimulated by U Ⅱ (1(10-10 to 10-7 mol/L) for 12 h( MCP-1 mRNA expression) and 18 h(MCP-1 protein secretion) respectively. The cells were pretreated with reduced nicotinamide adenine dinucleotide phosphate(NADPH) oxidase inhibitor diphenyle-neiodonium(DPI 10 μmol/L) or antioxidant N-acetylcysteine(NAC 3 mmol/L) for 0.5 h,then were stimulated by 10"7 mol/L UⅡ for 12 h(MCP-1 mRNA expression) and 18 MMCP-1 protein secretion) respectively. The expression of p47phox,a subunit of NADPH oxidase,was detected by RT-PCR after treated with UⅡ for different periods. ROS levels were assessed by flow cytometer after exposure to U Ⅱ for different hours. Results MCP-1 protein secretion and mRNA expression were increased by 57. 0% and 100. 5% after stimulated by U Ⅱ 10-7 mol/L(P＜0. 01),respectively. The p47phox mRNA expression was increased by U Ⅱ,and UⅡ -induced ROS production by macrophage reached the maximal effect at 6 h. Both DPI and NAC were able to inhibit U Ⅱ -induced MCP-1 secretion and mRNA expression. Conclusion U Ⅱ-induced MCP-1 mRNA expression and protein secretion may be mediated by NADPH oxidase-derived ROS.%目的 观察尾加压素Ⅱ(UⅡ)对小鼠巨噬细胞(RAW264.7细胞)表达和分泌单核细胞趋化蛋白1(MCP-1)的影响.方法 RAW264.7细胞用UⅡ10-10、10-9、10-8和10-7 mol/L刺激后,检测MCP-1 mRNA和蛋白的水平;还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶抑制剂二苯基碘(DPI)或抗氧化剂N-乙酰半胱氨酸(NAC)预处理细胞后,再加入UⅡ10-7 mol/L刺激,检测MCP-1 mRNA表达和蛋白的分泌;用UⅡ刺激细胞不同时间段后,检测NADPH氧化酶亚基的表达水平;用UⅡ刺激细胞后,流式细胞仪检测细胞活性氧的生成.结果 与不加UⅡ时比较,UⅡ10-7 mol/L刺激12h后,巨噬细胞MCP-1mRNA表达和蛋白分泌分别提高了100.5%和57.0%(P＜0.01).UⅡ刺激12 h后,p47phox mRNA表达为不加UⅡ时的1.96倍(P＜0.01).UⅡ刺激6h后,巨噬细胞活性氧为不加UⅡ时的1.8倍(P＜0.01).与单用UⅡ刺激比较,用DPI或NAC预处理后,MCP-1mRNA表达分别下调21.8%和36.2%(P＜0.01),蛋白分泌分别下降22.3%和19.5% (P＜0.05).结论 UⅡ可能通过NADPH氧化酶依赖的活性氧介导了小鼠巨噬细胞MCP-1的表达和分泌上调.
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