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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 investigate the inhibitory effect and mechanism of resveratrol on epithelial mesenchymal transition (EMT) induced by high glucose in human renal tubular epithelial cells (HK-2). Methods HK-2 cells were cultured in vitro and divided into normal control group (NG, 5.5 mmol / L), high glucose group (HG, 30 mmol / L), resveratrol group (RES, 10μmol / L) 30 mmol / L) + resveratrol (10 μmol / L), diphenyleneiodonium (DPI 10 μmol / L), high glucose / L) group (HG + DPI). Inverted microscope to observe the effect of high glucose on cell morphology. The expression of cytokeratin and vimentin were detected by immunofluorescence. The expressions of α-SMA, E-cadherin, ), NADPH oxidase subunits (NOXs) expression changes, DCFH-DA detection of intracellular ROS levels. Results Compared with the normal control group, the HK-2 cells lost their original morphology and had a long fusiform shape with significantly decreased cytokeratin and E-cadherin expression. The mesenchymal cell markers vimentin and α (P <0.01). The expression of NOX1 and NOX4 in NADPH oxidase subunit were significantly increased (P <0.01), NOX2 was not significantly changed, and ROS production was significantly increased (1 723.82 ± 303.97 vs 579.36 ± 353.76), P <0.01]. However, pretreatment with resveratrol or NADPH oxidase inhibitor DPI could inhibit both EMT (P <0.01) and ROS [(1 803.36 ± 199.54) or (1837.45 ± 266.83) vs (5779.36 ± 353.76), P <0.01], and resveratrol significantly inhibited the high glucose-induced NOX1 and NOX4 expression (P <0.01). Conclusion Resveratrol may reduce ROS production in renal tubular epithelial cells by inhibiting NADPH oxidase, thereby inhibiting EMT in HK-2 cells under high glucose condition.