血管紧张素Ⅱ通过NADPH氧化酶源性氧化应激促进肾小球系膜细胞单核细胞趋化蛋白1表达

NADPH oxidase-derived reactive oxygen species involved in angiotensin 11-induced monocyte chemoattractant protein-1 expression in mesangial cells

目的探讨氧化应激在血管紧张素（Ang）Ⅱ诱导的系膜细胞单核细胞趋化蛋白1（MCP-1）表达中的作用。方法体外培养人肾小球系膜细胞，应用即时定量PCR检测MCP-1表达；荧光探针2，7-二氯二氢荧光素乙酰乙酸（DCFDA）检测细胞内活性氧的产生；化学发光法检测尼克酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶活性；Western blot检测p47^phox和p67phox膜转位。24只雄性C57BL／6小鼠随机分为三组：正常对照组、模型组和夹竹桃麻素治疗组。采用酶联免疫吸附试验（ELISA）检测尿中8-isoprostane和白蛋白水平。结果AngⅡ呈剂量依赖性诱导MCP-1表达，100nmoL／L AngⅡ诱导的MCP-1表达是对照组的3．56倍。AngⅡ呈时间依赖性和剂量依赖性促进系膜细胞活性氧产生。AngⅡ刺激3min，系膜细胞内活性氧产生明显增加，至60min达到高峰。1、10和100nmol/L AngⅡ刺激60min，活性氧产生分别是对照组的1．82、2．92和4．08倍。AT1R拮抗剂氯沙坦完全阻断AngⅡ诱导的活性氧产生，而AT2R拮抗剂PD123319无抑制作用。NADPH氧化酶抑制剂夹竹桃麻素和二联苯碘几乎完全阻断AngⅡ诱导的活性氧产生，而线粒体复合体Ⅰ抑制剂鱼藤酮、黄嘌呤氧化酶抑制剂别嘌醇、环氧化酶抑制剂吲哚美辛、脂氧化酶抑制剂去甲二氢化愈创木酸、细胞色素P450氧化酶抑制剂酮康唑以及一氧化氮合成酶抑制剂N-硝基-L-精氨酸甲酯（L—NAME）对AngⅡ诱导的活性氧产生均无明显影响。AngⅡ显著刺激NADPH氧化酶活化及p47phox和p67phox膜转位。氯沙坦、乙酰半胱氨酸（NAC）、夹竹桃麻素和二联苯碘阻断AngⅡ诱导的MCP-1表达。AngⅡ灌注后尿中8-isoprostane、白蛋白排泄及肾组织中MCP-1表达分别是对照组的5．45、16．65和2．50倍；肾组织中NADPH氧化酶活性以及p47phox和p67phox膜转位分别是对照组的2．69、2．97和2．67倍。NADPH氧化酶抑制剂夹竹桃麻素显著减轻AngⅡ诱导的蛋白尿和氧化应激，并抑制MCP-1表达。结论NADPH氧化酶来源的活性氧调控AngⅡ诱导的MCP-1表达，抑制NADPH氧化酶活性可减轻AngⅡ诱导的肾脏损害。

Objective To investigate the origin of oxidative stress induced by angiotensin Ⅱ （ Ang Ⅱ ） in human mesangial cells and the role of reactive oxygen species （ ROS ） in Ang Ⅱ -induced monocyte chemoattraetant protein-1 （MCP-1） expression. Methods MCP-1 expression was determined by real time RT-PCR. ROS production was measured by DCFDA fluorescence. Nieotinamide adenine dinucleotide phosphate （NADPH） oxidase activity was examined by lucigenin chemiluminescence, p47phox and p67phox transloeation was assayed by Western blot. Twenty-four male mice were randomly divided into three groups： the control,the Ang Ⅱ infusion [ Ang Ⅱ 400 ng/（ kg . min）], and the apocynin treatment. Ang Ⅱ was infused by subcutaneously osmotic minipump for 14 days. Urinary albumin and 8-isoprostane excretion were measured by ELISA. Results In cultured human mesangial cells, Ang Ⅱ induced the MCP-1 expression in a dose-dependent manner with 3.56 fold increase as compared with the control. Ang Ⅱ increased intracellular ROS production as early as 3 min with the peak at 60min and was in a time and dose-dependent. Incubation with different dosages of Ang Ⅱ （ 1 nmol/L, 10 nmol/L, and 100 nmol/L Ang Ⅱ ） for 60 min, ROS production increased at 1.82, 2. 92, and 4. 08 folds respectively. Ang Ⅱ-induced ROS generation was sensitive to diphenyleneiodorfium sulfate （DPI, 10 μmol/L） and apocynin （500 μmol/L）, two structurally distinct NADPH oxidase inhibitors. In contrast, inhibitors of other oxidantproducing enzymes, including the mitochondrial complex Ⅰ inhibitor 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. Ang Ⅱ-induced ROS generation was inhibited by the AT1 antagonist losartan （10 μmol/L） but not the AT2 antagonist PD123319 （10 μmol/L）. Ang Ⅱ treatment induced translocation of cytosolic of p47phox and p67phox to the membrane. The antioxidants almost abolished Ang Ⅱ-induced MCP-1 expression. Ang Ⅱ infusion increased urinary and p67 translocation by 2. 69-, 2. 97-, and 2. 67-fold, respectively. Conclusions NADPH oxidase-derived ROS is involved in Ang Ⅱ - induced MCP-1 expression. Inhibition of NADPH oxidase alleviates Ang Ⅱ -induced renal injury.