Chen Ying, Zhang Ai-hua, Huang Song-ming, Ding Gui-xia, Zhang Wei-zhen, Bao Hua-ying, Wu Hong-mei, Chen Rong-hua
Department of Nephrology, Nanjing Children's Hospital, Affiliated to Nanjing Medical University, Nanjing 210008, China.
Zhonghua Bing Li Xue Za Zhi. 2009 Jul;38(7):456-61.
To investigate the origin of oxidative stress induced by angiotensin II (AngII) in human mesangial cells and the role of reactive oxygen species (ROS) in AngII-induced monocyte chemoattractant protein-1 (MCP-1) expression.
MCP-1 expression was determined by real time RT-PCR. ROS production was measured by DCFDA fluorescence. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was examined by lucigenin chemiluminescence. p47phox and p67phox translocation was assayed by Western blot. Twenty-four male mice were randomly divided into three groups: the control, the AngIIinfusion [AngII 400 ng/(kg.min)], and the apocynin treatment. AngII was infused by subcutaneously osmotic minipump for 14 days. Urinary albumin and 8-isoprostane excretion were measured by ELISA.
In cultured human mesangial cells, AngII induced the MCP-1 expression in a dose-dependent manner with 3.56 fold increase as compared with the control. AngII increased intracellular ROS production as early as 3 min with the peak at 60 min and was in a time and dose-dependent. Incubation with different dosages of AngII (1 nmol/L, 10 nmol/L, and 100 nmol/L AngII) for 60 min, ROS production increased at 1.82, 2.92, and 4.08 folds respectively. AngII-induced ROS generation was sensitive to diphenyleneiodonium sulfate (DPI, 10 micromol/L) and apocynin (500 micromol/L), two structurally distinct NADPH oxidase inhibitors. In contrast, inhibitors of other oxidant-producing enzymes, including the mitochondrial complex Iinhibitor rotenone, the xanthine oxidase inhibitor allopurinol, the cyclooxygenase inhibitor indomethacin, the lipoxygenase inhibitor nordihydroguiaretic acid, the cytochrome P450 oxygenase inhibitor ketoconazole and the nitric oxide synthase inhibitor G-nitro-L-arginine methyl ester were without an effect. AngII-induced ROS generation was inhibited by the AT1 antagonist losartan (10 micromol/L) but not the AT2 antagonist PD123319 (10 micromol/L). AngII treatment induced translocation of cytosolic of p47phox and p67phox to the membrane. The antioxidants almost abolished AngII-induced MCP-1 expression. AngII infusion increased urinary and p67 translocation by 2.69-, 2.97-, and 2.67-fold, respectively.
NADPH oxidase-derived ROS is involved in AngII-induced MCP-1 expression. Inhibition of NADPH oxidase alleviates AngII-induced renal injury.
研究血管紧张素II（AngII）诱导人系膜细胞氧化应激的来源以及活性氧（ROS）在AngII诱导单核细胞趋化蛋白-1（MCP-1）表达中的作用。
通过实时逆转录聚合酶链反应（RT-PCR）测定MCP-1表达。用2′,7′-二氯二氢荧光素二乙酸酯（DCFDA）荧光法检测ROS生成。用光泽精化学发光法检测烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶活性。用蛋白质免疫印迹法检测p47phox和p67phox转位。24只雄性小鼠随机分为三组：对照组、AngII输注组[AngII 400 ng/(kg·min)]和载脂蛋白处理组。通过皮下渗透微型泵输注AngII 14天。用酶联免疫吸附测定（ELISA）法检测尿白蛋白和8-异前列腺素排泄量。
在培养的人系膜细胞中，AngII以剂量依赖性方式诱导MCP-1表达，与对照组相比增加了3.56倍。AngII早在3分钟时就增加细胞内ROS生成，60分钟时达到峰值，且呈时间和剂量依赖性。用不同剂量的AngII（1 nmol/L、10 nmol/L和100 nmol/L AngII）孵育60分钟，ROS生成分别增加1.82倍、2.92倍和4.08倍。AngII诱导的ROS生成对两种结构不同的NADPH氧化酶抑制剂硫酸二苯撑碘鎓（DPI，10 μmol/L）和载脂蛋白（500 μmol/L）敏感。相比之下，其他产生活性氧酶的抑制剂，包括线粒体复合物I抑制剂鱼藤酮、黄嘌呤氧化酶抑制剂别嘌呤醇、环氧化酶抑制剂吲哚美辛、脂氧化酶抑制剂去甲二氢愈创木酸、细胞色素P450加氧酶抑制剂酮康唑和一氧化氮合酶抑制剂G-硝基-L-精氨酸甲酯均无作用。AngII诱导的ROS生成被血管紧张素II 1型受体（AT1）拮抗剂氯沙坦（10 μmol/L）抑制，但不被血管紧张素II 2型受体（AT2）拮抗剂PD123319（10 μmol/L）抑制。AngII处理诱导胞质中的p47phox和p67phox转位至细胞膜。抗氧化剂几乎完全消除了AngII诱导的MCP-1表达。输注AngII使尿中8-异前列腺素和p67转位分别增加2.69倍、2.97倍和2.67倍。
NADPH氧化酶衍生的ROS参与AngII诱导的MCP-1表达。抑制NADPH氧化酶可减轻AngII诱导的肾损伤。
