Zhang Haiyan, Jiang Zongpei, Chang Jie, Li Xiaoyan, Zhu Hengmei, Lan Hui Y, Zhou Shu-Feng, Yu Xueqing
Department of Nephrology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.
Nephrology (Carlton). 2009 Apr;14(3):302-10. doi: 10.1111/j.1440-1797.2008.01072.x. Epub 2009 Feb 3.
This study investigated the role of NAD(P)H oxidase in transforming growth factor-beta1 (TGF-beta1)-induced reactive oxygen species (ROS) generation, monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) expression in rat renal tubular epithelial NRK-52E cells.
The cells were treated with 10 ng/mL TGF-beta1, either in the presence or absence of the NAD(P)H oxidase inhibitor, diphenyleneiodonium (DPI), or short hairpin RNA (shRNA) suppressing p67phox expression. Expression of NAD(P)H oxidase subunits, MCP-1, and IL-6 at the mRNA levels was detected by reverse transcription polymerase chain reaction, while expression of NAD(P)H oxidase subunit p67phox protein was analyzed by western blot and MCP-1 by enzyme-linked immunosorbent assay. The cellular ROS generation was visualized using 2',7'-dichlorodihydrofluorescein diacetate by confocal microscopy.
Compared to control, TGF-beta1 upregulated NAD(P)H oxidase subunit p67phox mRNA by 3.59-fold (P < 0.01), but had no effect on p22phox, gp91phox and p47phox NAD(P)H subunits. TGF-beta1 was also able to significantly increase intracellular ROS (P < 0.05), MCP-1 (P < 0.01) and IL-6 (P < 0.05) expression in NRK-52E cells. Further studies showed that generation of ROS and upregulation of MCP-1 and IL-6 by TGF-beta1 were significantly blocked by addition of DPI or shRNA-p67phox (P < 0.01), suggesting that these effects were NAD(P)H oxidase-dependent.
TGF-beta1 differentially regulates the expression of NAD(P)H oxidase subunits and mediates MCP-1 and IL-6 expression in rat renal tubular cells via the NAD(P)H oxidase/p67phox-dependent mechanism.
本研究探讨烟酰胺腺嘌呤二核苷酸磷酸（NAD(P)H）氧化酶在转化生长因子-β1（TGF-β1）诱导大鼠肾小管上皮NRK-52E细胞活性氧（ROS）生成、单核细胞趋化蛋白-1（MCP-1）及白细胞介素-6（IL-6）表达中的作用。
细胞分别在存在或不存在NAD(P)H氧化酶抑制剂二苯碘鎓（DPI）或抑制p67phox表达的短发夹RNA（shRNA）的情况下，用10 ng/mL TGF-β1处理。通过逆转录聚合酶链反应检测NAD(P)H氧化酶亚基、MCP-1和IL-6在mRNA水平的表达，同时通过蛋白质印迹分析NAD(P)H氧化酶亚基p67phox蛋白的表达，并用酶联免疫吸附测定法检测MCP-1。使用二氯二氢荧光素二乙酸酯通过共聚焦显微镜观察细胞内ROS的生成。
与对照组相比，TGF-β1使NAD(P)H氧化酶亚基p67phox mRNA上调3.59倍（P < 0.01），但对p22phox、gp91phox和p47phox NAD(P)H亚基无影响。TGF-β1还能够显著增加NRK-52E细胞内ROS（P < 0.05）、MCP-1（P < 0.01）和IL-6（P < 0.05）的表达。进一步研究表明，添加DPI或shRNA-p67phox可显著阻断TGF-β1诱导的ROS生成以及MCP-1和IL-6的上调（P < 0.01），表明这些作用依赖于NAD(P)H氧化酶。
TGF-β1差异性调节NAD(P)H氧化酶亚基的表达，并通过NAD(P)H氧化酶/p6�phox依赖性机制介导大鼠肾小管细胞中MCP-1和IL-6的表达。
