摘要
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 effect of norepinephrine（NE） on the expression of matrix metalloproteinase-9（MMP-9） in human macrophages,and to explore its pro-inflammatory and pro-atherosclerotic mechanisms.Methods Murine U937 macrophages were cultured and pretreated with antioxidant NAC（10 mmol/L） and NAD（P）H oxidase inhibitor DPI（10-5 mol/L） for 1 h.Then the cells were treated with different concentrations（10-10,10-9,10-8 and 10-7 mol/L） of NE at different time points（0,1,3,6,12 and 24 h）.MMP-9 mRNA level was analyzed by RT-PCR.MMP-9 protein expression was measured by Western blotting.MMP-9 protein level in cell supernatant was detected by ELISA.Intracellular reactive oxygen species（ROS） generation was observed with fluorescence probes,and intracellular MMP-9 mRNA and protein expressions were detected by RT-PCR and ELISA.Results The mRNA expression level（4.65 times that of the control group in the 10-7 mol/L NE group,P0.01） and protein expression level（4.14 times that of the control group in the 10-7 mol/L NE group,P0.01） of MMP-9 in macrophages were increased by NE in a dose-and time-dependent manner.The levels of ROS were 2.18,3.22,3.64 and 4.50 times that of the control group in the 10-10,10-9,10-8 and 10-7 mol/L NE groups,respectively（P0.01）.NAC and DPI significantly antagonized the effect of NE on MMP-9 mRNA（DPI group was 75.2% that of the control group,P0.05;NAC group was 50% that of the control group,P0.01） and protein expressions（DPI group was 69.8% that of the control group,P0.05;NAC group was 67.1% that of the control group,P0.05）.Conclusion NE upregulates MMP-9 expression in U937 cells in a dose-and time-dependent manner,and induces ROS secretion via NAD（P）H oxidase,which mediates the pro-inflammatory effect of NE to promote the formation and development of atherosclerosis.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2012年第17期1758-1761,共4页
Journal of Third Military Medical University
