NADPH氧化酶在转化生长因子β1诱导大鼠肾小管上皮细胞转分化中的作用

目的探讨NADPH氧化酶在转化生长因子β1（TGF-β1）诱导大鼠肾小管上皮细胞（NRK-52E）转分化中的作用。方法用TGF-β1（10μg/L）刺激NRK-52E细胞不同时间,观察α-平滑肌肌动蛋白（α-SMA）、E-钙黏蛋白（E-cadherin）、纤溶酶原激活物抑制剂1（PAI- 1）及Ⅰ型胶原（Col-Ⅰ）的表达。部分实验中细胞在TGF-β1刺激前用NADPH氧化酶抑制剂DPI预处理1 h。用激光共聚焦显微镜观察细胞内活性氧（ROS）的产生。用RT-PCR方法检测NADPH氧化酶p22phox、gp91phox、p47phox和p67phox亚单位mRNA的表达。α-SMA、E-cadherin、PAI-1及Col-ⅠmRNA及蛋白的表达分别采用RT-PCR、Western印迹和细胞免疫化学检测。结果TGF-β1可显著上调NADPH氧化酶p67phox亚单位mRNA的表达,8 h及24 h时分别为对照组的2.43倍及3.59倍（P〈0.01）。TGF-β1可显著促进细胞ROS的产生,5 min时已是对照组的2.5倍（P〈0.05）。DPI预处理同时可显著逆转TGF-β1诱导NRK-52E细胞ROS的产生（P〈0.05）、α-SMA的表达上调、E-cadherin的表达下调以及PAI-1和Col-Ⅰ的表达上调。结论TGF-β1可促进NRK-52E细胞增加ROS的产生。ROS介导了TGF-β1诱导NRK-52E细胞的转分化,促进肾脏纤维化。

Role of NADPH oxidase in TGF-β1-induced epithelial mesenchymal transition in rat renal tubular epithelial cells

Objective To investigate the role of NADPH oxidase in tubular epithelial-myofibroblast transition(EMT) induced by TGF-β1 in rat renal tubular epithelial cells. Methods Growth arrested and synchronized rat renal tubular epithelial cells (NRK-52E) were stimulated by 10 μg/L TGF-β1 for different time. Part of experimental cells were pretreated with DPI, an inhibitor of NADPH oxidase, for 1 h. The expression of NADPH oxidase subunits in cultured NRK-52E cells was messured by semi-quantitive RT-PCR. Intracellular ROS generation was visualized using H2DCF-DA by confocal microscope. The expression levels of α-SMA and E-cadherin were measured by RT-PCR, Western blot and immunocytochemistry, respectively. The mRNA and protein expression of PAI-1 and Col-Ⅰwere assessed by RT-PCR and Western blot respectively. Results TGF-β1 significantly upregulated the p67phox subunits mRNA expression, which was 2.43 folds and 3.59 folds of control group in 8 h and 24 h (P<0.01). TGF-β1 promoted the synthesis of cellular ROS, which was 2.5 folds of control group after 5 min stimulation (P<0.05). DPI could decrease the generation of ROS induced by TGF-β1(P<0.05). DPI effectively reversed TGF-β1-induced expression of α-SMA, E-cadherin, Col-Ⅰ, and PAI-1 in rat renal tubular epithelial cells. Conclusion TGF-β1 can significantly increase intracellular ROS and NADPH oxidase-derived ROS mediates TGF-β1-induced EMT in rat renal tubular epithelial cells.