REACTIVE OXYGEN SPECIES FROM MITOCHONDRIA INDUCE CYCLOOXYGENASE-2 GENE EXPRESSION IN HUMAN MESANGIAL CELLS : POTENTIAL ROLE IN DIABETIC NEPHROPATHY

高血压增加了牛内皮细胞中来自线粒体电子传递链的活性氧物质（ROS）的产生（Nishikawa等人，2000 ; Nature 404：787-90）。由于一些研究已经假设了在早期糖尿病中观察到的肾小球高滤过中，前列腺素（PGs）的作用，我们评估了线粒体ROS对培养的人肾小球系膜细胞（HMC）中诱导型环氧合酶（考克斯-2）表达的影响。我们首先证实，与5.6 mM葡萄糖相比，用30 mM葡萄糖孵育HMC显著增加COX-2 mRNA，但不增加考克斯-1 mRNA。同样，30 mM葡萄糖孵育HMC显著增加线粒体膜电位、细胞内ROS产生、考克斯-2蛋白表达和PGE_2合成，这些事件被线粒体代谢抑制剂TTFA或CCCP或解偶联蛋白-1或锰超氧化物歧化酶过表达完全抑制。体内实验证实糖尿病小鼠肾小球考克斯-2 mRNA和蛋白表达增加。此外，高血糖诱导考克斯-2基因启动子激活，启动子区域的两个NF-κB（核因子κB）结合位点突变使其完全失效。结果提示，高血糖可增加线粒体活性氧的产生，进而激活NF-κB，诱导考克斯-2 mRNA的表达，增加考克斯-2蛋白的表达，促进PGE_2的合成。这一系列事件可能参与了糖尿病肾病的发病机制。

Hyperglycemia increases the production of reactive oxygen species (ROS) from the mitochondrial electron transport chain in bovine endothelial cells (Nishikawa et al. 2000 ; Nature 404:787-90). Because several studies have postulated a role for prostaglandins (PGs) in the glomerular hyperfiltration seen in early diabetes, we evaluated the effect of mitochondrial ROS on expression of the inducible isoform of cyclooxygenase (COX-2) in cultured human mesangial cells (HMC). We first confirmed that incubation of HMC with 30 mM glucose significantly increased C0X-2 mRNA, but not COX-1 mRNA, compared with 5.6 mM glucose. Similarly, incubation of HMC with 30 mM glucose significantly increased mitochondrial membrane potential, intracellular ROS production, COX-2 protein expression and PGE_2 synthesis, and these events were completely suppressed by TTFA or CCCP, inhibitors of mitochondrial metabolism, or by overexpression of uncoupling protein-1 or manganese superoxide dismutase. In vivo study, it was confirmed that expression of COX-2 mRNA and protein was increased in glomeruli of diabetic mice. In addition, hyperglycemia induced activation of the COX-2 gene promoter, which was completely abrogated by mutation of two NF-κB (nuclear factor κB) binding sites in the promoter region. Our results suggest that hyperglycemia increases mitochondrial ROS production, resulting in NF-κB activation, COX-2 mRNA induction, COX-2 protein production and PGE_2 synthesis. This chain of events might contribute to the pathogenesis of diabetic nephropathy.