氯沙坦对血管紧张素Ⅱ诱导大鼠肾小管上皮细胞氧化应激反应的作用  

Role of losartan in angiotensin Ⅱ-induced oxidative stress in rat renal tubular epithelial cells

作　　者：彭张哲[1] 陶立坚[1] 王菱[1] 宁旺斌[1] 谢艳云[1] 王纳遂[1] 李冰心[1] 唐怡庭[1] 

机构地区：[1]中南大学湘雅医院肾内科,长沙410078

出　　处：《中华肾脏病杂志》2009年第3期204-209,共6页Chinese Journal of Nephrology

基　　金：基金项目：湖南省自然科学基金（07-JJ3056）;教育部科学技术研究重点项目（107133）;教育部博士点基金（20060533056） 志谢 本实验均在医学遗传学国家重点实验室完成,真挚感谢医学遗传学国家重点实验室提供的技术支持以及仪器设备等硬件支持

摘　　要：目的探讨血管紧张素Ⅰ型受体阻断剂氯沙坦对血管紧张素Ⅱ（AngⅡ）诱导的大鼠肾小管上皮细胞氧化应激反应及转化生长因子B1（TGF-β1）产生的影响。方法以大鼠肾小管上皮细胞株（NRK-52E）为研究对象，分别给予氯沙坦（10^-5mol／L）和（或）NADPH氧化酶抑制剂联二亚苯碘锚（DPI，10^-5mol／L）预处理1h后再加入AngⅡ（10^-7mol／L）刺激24h，同时设立空白培养基作为对照组。实时定量PCR和Western印迹法分别检测4组细胞NADPH氧化酶p22phox、p47phox、Nox-4 mRNA和蛋白水平及TGF-β mRNA水平。用2，7-二氯荧光素双乙酸盐（H2DCF-DA）作为荧光探针，采用荧光分光光度仪检测细胞内活性氧（ROS）的水平。应用比色法测定细胞上清液中超氧化物歧化酶（SOD）水平。结果AngⅡ（10^-7mol／L）刺激细胞15min后开始产生大量的ROS，至60min达平台期。单纯AngⅡ刺激1h后，NRK-52E细胞上清液中SOD水平显著低于对照组（27．31μU／L比48．29μU／L，P〈0．01）；经氯沙坦预处理。1h后再加入AngⅡ刺激1h细胞上清液中SOD水平升高至36．37μU／L，与AngⅡ组差异有统计学意义（P〈0．01）。单纯AngⅡ刺激NRK-52E细胞24h可显著上调NADPH氧化酶p22phox、p47phox和Nox-4 mRNA和蛋白水平，其mRNA水平分别为对照组的5．57倍、5．55倍和9．41倍（均P〈0．01），蛋白水平分别为对照组的4．53倍、4．17倍和6．50倍（均P〈0．01）。经氯沙坦干预后，p22phox、p47phox和Nox-4 mRNA水平分别为对照组的2．71倍、2．18倍和5．23倍（均P〈0．01），蛋白水平分别为对照组的3．20倍、2．30倍和4．30倍（均P〈0．01）。AngⅡ刺激细胞24h后TGF-β1 mRNA表达也显著增多，为对照组的4．36倍（P〈0．01），氯沙坦处理细胞后TGF-β1 mRNA水平为对照组的1．57倍。结论氯沙坦通过抑制NADPH氧化酶的表达和增加SOD的表达减少ROS的产生，并能通过减少ROS产生，�Objective To investigate the effects of losartan on angiotensin （Ang） Ⅱ-induced the generation of oxidative stress and expression of transforming growth factor β1（TGF-β1） in rat proximal tubular epithelial cells and to explore its underlying mechanism. Methods NRK-52E cells, a rat proximal tubular epithelial cell line, were applied to explore the antioxidation and antifibrosis of losartan. The expression of three subunits of nicotinamide-adenine dinucleotide phosphate （NADPH） oxidase, including p47phox, Nox-4, p22phox, and TGF-β1 were determined by real-time RT-PCR and/or Western blot. The generation of reactive oxygen species （ROS） was measured by DCF fluorescence analysis. Superoxide dismutase （SOD） in the supernatant was measured by colorimetric method. Results 10.7 mol/L Ang Ⅱ up-regulated p22prox, p47phox and Nox-4 mRNA and protein expression, and the mRNA increased by 5.57-fold, 5.55-fold and 9.41-fold at 24 h （P〈0.01, respectively） and the protein increased by 4.53-fold, 4.17-fold and 6.50-fold at 24 h （P〈0.01, respectively） as compared with control. Losartan greatly reduced the mRNA elevation of p22prox, p47phox and Nox-4 by 2.71-fold, 2.18-fold and 5.23-fold （P〈0.01, respectively） and reduced the protein elevation by 3.20-fold, 2.30-fold and 4.30-fold （P〈0.01, respectively） as compared with control. Losartan also inhibited ROS generation induced by Ang Ⅱ in rat proximal tubular epithelial cells. SOD level in the supernatant was markedly decreased after Ang Ⅱ stimulation, while losartan could increase SOD levels （P 〈0.01）. Furthermore, losartan signficantly inhibited Ang Ⅱ-induced TGF-β1 mRNA up-regulation by 64% （P 〈0.01）. Conclusions Losartan acts as an anti-oxidative and anti-fibrotic agent via the mechanisms of blocking NADPH oxidase-dependent oxidative stress and inhibiting TGF-β1 expression.

关 键 词：洛沙坦 NADPH氧化酶 活性氧 纤维化 转化生长因子Β1 血管紧张素Ⅱ 

分 类 号：R285.5[医药卫生—中药学]