白藜芦醇对高糖诱导的肾小管上皮细胞转分化的抑制作用  

Inhibitory role of resveratrol in high glucose-induced renal tubular epitheliamesenchymal transition and its underlying mechanism

作　　者：何婷[1] 管旭[1] 孙蕾[1] 杨可[1] 肖堂利[1] 徐新丽[1] 杨琴[1] 赵景宏[1] 

机构地区：[1]第三军医大学新桥医院肾内科,重庆400037

出　　处：《第三军医大学学报》2014年第15期1572-1576,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81270290)~~

摘　　要：目的探讨白藜芦醇对高糖诱导人肾小管上皮细胞(HK-2)发生上皮-间充质细胞转分化(epitheliamesenchymal transition,EMT)的抑制作用及机制。方法体外培养HK-2细胞,分为正常对照组(NG,5.5 mmol/L)、高糖组(HG,30 mmol/L)、白藜芦醇组(RES,10μmol/L)、高糖(30 mmol/L)+白藜芦醇(10μmol/L)组(HG+RES)、二苯基碘(diphenyleneiodonium)组(DPI,10μmol/L)、高糖(30 mmol/L)+DPI(10μmol/L)组(HG+DPI)。倒置显微镜下观察高糖对细胞形态的影响。免疫荧光检测上皮细胞标志物波形蛋白(cytokeratin)及间充质细胞标志物波形蛋白(vimentin)的表达变化,Western blot检测α平滑肌肌动蛋白(α-SMA)、E钙黏蛋白(E-cadherin)、NADPH氧化酶亚基(NOXs)的表达变化,DCFH-DA法检测细胞内ROS水平。结果与正常对照组相比,高糖处理后的HK-2细胞失去原有形态,呈长梭形改变,上皮细胞标志物cytokeratin及E-cadherin表达明显减少,间充质细胞标志物vimentin及α-SMA表达明显升高(P<0.01),同时NADPH氧化酶亚基NOX1、NOX4表达明显升高(P<0.01),NOX2无明显变化,ROS产生明显增多[(1 723.82±303.97)vs(2 579.36±353.76),P<0.01],而加入白藜芦醇或NADPH氧化酶抑制剂DPI预孵育后,均能抑制高糖诱导的HK-2细胞EMT(P<0.01)和ROS[(1 803.36±199.54)或(1 837.45±266.83)vs(2 579.36±353.76),P<0.01]产生增多,并且白藜芦醇可以明显抑制高糖诱导的NOX1以及NOX4表达升高(P<0.01)。结论白藜芦醇可能通过抑制NADPH氧化酶降低肾小管上皮细胞ROS生成,进而抑制高糖状态下HK-2细胞EMT的发生。Objective To determine the effect of resveratrol on high glucose-induced epithelia- mesenehymal transition （EMT） of renal tubular epithelial cell line HK-2 and its underlying mechanism. Methods Cultured HK-2 cells were divided into 6 groups, that is, normal glucose （ NG, 5.5 mmoL/L） , high glucose （ HG, 30 mmoL/L）, resveratrol （ RES, 10 μmoL/L） , high glucose and resveratrol （ HG ＋ RES, 30 mmol/L ＋ 10 μmol/L）, diphenyleneiodonium （DPI, NADPH oxdiase inhibitor, 10 μ mol/L）, and high glucose and DPI group （HG ＋ DPI, 30 mmol/L ＋ 10 μmol/L）. Inverted microscopy was used to observe the morphological changes in high glucose-induced HK-2 cells. Expression of cytokeratin and vimentin was detected by immunofluorescence assay, and that of of a-smooth muscle actin （α-SMA） , E-cadherin, NADPH oxdiase subunits （NOXs） were measured by Western blotting. Intraeellular reactive oxide species （ROS） were detected by DCFH-DA fluorescence probe. Results After incubated with high glucose, HK-2 cells turned into long spindle-shape compared with NG group. Protein levels of epithelial markers E-cadherin and cytokeratin were significantly decreased, while those of the mesenchymal markers α-SMA and vimentin were increased （P 〈 O. 01 ）. Protein levels of NADPH oxidase subunits NOX1 and NOX4 were significantly decreased （P 〈0.01 ）, but that of NOX2 had no change, and intracellular ROS production was significantly increased （ 1 723.82 ± 303.97 vs 2 579.36±353.76, P 〈 0.01 ）. The pre-incubation of resveratrol or DPI obviously inhibited EMT （ P 〈 O. 01 ） and ROS production （ 1 803.36 ± 199.54 and 1 837.45± 266.83 vs 2 579.36± 353.76, P 〈0. 01 ） in HK-2cells induced by high glucose, while resveratrol markedly inhibited NOX1 and NOX4 expression （P 〈0.01 ）. Conclusion Resveratrol may reduce ROS production through suppressing NADPH oxidase in renal epithelial cells, and thus inhibit the EMT of the cells induced by high glucose.

关 键 词：白藜芦醇 高糖 上皮-间充质转分化 NADPH氧化酶 

分 类 号：R282.71[医药卫生—中药学] R322.61[医药卫生—中医学]