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目的观察去甲肾上腺素(norepinephrine,NE)预处理人巨噬细胞基质金属蛋白酶-9(MMP-9)的表达及其致动脉粥样硬化机制。方法体外培养的U937巨噬细胞,以10-10、10-9、10-8、10-7mol/L NE分别刺激细胞0、1、3、6、12、24 h。RT-PCR法测细胞MMP-9 mRNA水平,Western blot法检测细胞MMP-9蛋白表达,ELISA法测细胞培养液上清中MMP-9蛋白水平的表达。预先加入抗氧化剂NAC(10 mmol/L)和NAD(P)H氧化酶抑制剂DPI(10-5mol/L)孵育1 h后再用不同浓度的NE分别刺激细胞24 h,用荧光探针法检测细胞内ROS的生成;用RT-PCR及ELISA法检测细胞MMP-9mRNA和蛋白的表达。结果 NE浓度时间依赖性地增加巨噬细胞MMP-9 mRNA(10-7mol/L NE为对照组的4.65倍,P<0.01)及蛋白(10-7mol/L NE为对照组的4.14倍,P<0.01)表达;随着NE浓度的升高,ROS的生成分别为对照组的2.18、3.22、3.64、4.50倍(P<0.01)。NAC和DPI都一定程度地抑制了MMP-9 mRNA[DPI组为NE组的0.752倍(P<0.05),NAC组为NE组的0.500倍(P<0.01)]和蛋白(DPI组为NE组的0.698倍,NAC组为NE组的0.671倍,均P<0.05)的表达。结论 NE浓度和时间依赖性地诱导人巨噬细胞U937 MMP-9的表达,通过NAD(P)H氧化酶介导的ROS通路而促进炎症反应,促进AS的发生、发展。
Objective To investigate the expression of matrix metalloproteinase-9 (MMP-9) and its mechanism of atherosclerosis in human macrophages pretreated with norepinephrine (NE). Methods U937 macrophages cultured in vitro were stimulated with 10-10,10-9,10-8,10-7 mol / L NE for 0,1,3,6,12,24 h. The level of MMP-9 mRNA was detected by RT-PCR, the expression of MMP-9 protein was detected by Western blot and the level of MMP-9 protein in the culture supernatant by ELISA. The cells were stimulated with NAC (10 mmol / L) and NAD (P) H oxidase inhibitor DPI (10-5 mol / L) for 1 h, then stimulated with different concentrations of NE for 24 h, The generation of intracellular ROS was detected. The expression of MMP-9 mRNA and protein was detected by RT-PCR and ELISA. Results NE concentration increased the mRNA expression of MMP-9 in macrophages in a time-dependent manner (4.65 folds of 10-7 mol / L NE, P <0.01) and 4.14 folds of 10-7 mol / L NE <0.01). With the increase of NE concentration, ROS production was 2.18, 3.22, 3.64 and 4.50 times higher than that of the control group (P <0.01). Both NAC and DPI inhibited MMP-9 mRNA to a certain degree [DPI group: 0.752 times as much as NE group (P <0.05), NAC group: 0.500 times as much as NE group (P <0.01) 0.698 times of NAC group, 0.671 times of NAC group, all P <0.05). Conclusion NE concentration and time-dependent induction of U937 MMP-9 expression in human macrophages can promote the inflammatory response and promote the development and progression of AS through NAD (P) H oxidase-mediated ROS pathway.