Expression modification of uncoupling proteins and MnSOD in retinal endothelial cells and pericytes induced by high glucose: The role of reactive oxygen species in diabetic retinopathy
Expression modification of uncoupling proteins and MnSOD in retinal endothelial cells and pericytes induced by high glucose: The role of reactive oxygen species in diabetic retinopathy
复制标题
DOI：
10.1016/j.exer.2006.03.024
复制
发表时间：
2006-10-01
影响因子：
3.4
通讯作者：
Ho, Patrick C. P.
中科院分区：
文献类型：
--
作者：
Cui, Yan;Xu, Xun;Ho, Patrick C. P.
解偶联蛋白（UCPs）是存在于线粒体内膜的线粒体转运蛋白。它们属于阴离子线粒体载体家族。UCPs可以作为质子载体被代谢物激活，并在呼吸链和ATP合酶的复合物之间产生分流。线粒体内膜对质子的泄漏增加可能是通过限制最大Δ mu（H+）来最小化超氧化物的产生。本研究的目的是检测UCP在视网膜毛细血管细胞中的表达及其在高糖环境下的变化。探讨线粒体活性氧（ROS）和尿连接蛋白（UCPs）在糖尿病视网膜病变发病中的作用。用选择性培养基培养牛视网膜毛细血管内皮细胞和周细胞。将传代细胞在三种不同的葡萄糖浓度（5、23、30 mM）中培养，直到第四代。采用扫描激光共聚焦显微镜（SLCM）检测不同葡萄糖浓度下这些细胞线粒体中活性氧的变化。流式细胞仪检测线粒体膜电位（Deltapsi）、细胞死亡率和凋亡率。免疫细胞化学法检测UCP在视网膜毛细血管细胞中的表达。采用半定量RT-PCR方法检测MnSOD和解偶联蛋白（UCPs）在不同浓度葡萄糖中的表达和修饰。随着培养液中葡萄糖浓度的增加，内皮细胞和周细胞线粒体中的ROS也增加。内皮细胞碱性磷酸酶和细胞死亡率也增加。内皮细胞中ROS与Δ psi和细胞死亡率呈正相关。在周细胞中，不同葡萄糖水平之间的Δ psi和细胞死亡率没有差异。高糖组内皮细胞和周细胞凋亡率高于低糖组。UCP 1和UCP 2在培养的视网膜毛细血管细胞中表达，而UCP 3不表达。在高水平的葡萄糖，UCP 1，UCP 2和MnSOD的表达增加，以适应ROS的生产补偿。当葡萄糖浓度过高（30 mM）时，代偿机制消失。本研究结果表明，高浓度葡萄糖可诱导线粒体活性氧增加。与抗氧化机制有关的蛋白质如MnSOD、UCPs等在一定程度上发挥代偿作用。当葡萄糖浓度太高时，这种补偿作用不充分。(c)2006爱思唯尔有限公司保留所有权利。
Uncoupling proteins (UCPs) are mitochondrial transporters present in the inner membrane of mitochondria. They belong to the family of anion mitochondrial carriers. UCPs could act as proton carriers activated by metabolites and create a shunt between complexes of the respiratory chain and ATP synthase. The increased leakiness of the mitochondrial inner membrane to protons may be to minimize superoxide production by limiting the maximum Delta mu(H+). The purpose of this study was to detect UCP expression in retinal capillary cells and their modification in high levels of glucose. The role of reactive oxygen species (ROS) of mitochondria and UCPs in pathogenesis of diabetic retimipathy was investigated. Bovine retinal capillary endothelial cells and pericytes were cultured with selective culture media, respectively. Passage cells were cultured in three different glucose concentrations (5, 23, 30 mM) until passage four. ROS changes in mitochondria of these cells in different glucose concentrations were detected with scanning laser confocal microscopy (SLCM). The mitochondria membrane potential (Delta psi), cell death rate and apoptosis rate were measured with flowing cytometry. UCP expression in retinal capillary cells was detected by immunocytochemistry. Expression and modification of MnSOD and uncoupling proteins (UCPs) in different concentrations of glucose were detected by means of semi-quantitative RT-PCR. ROS in mitochondria of both endothelial cells and pericytes increased as the glucose concentration of media increased. Alp and cell death rate of endothelial cells increased also. ROS was correlated to Delta psi and cell death rate positively in endothelial cells. No difference in Delta psi and cell death rate among different glucose levels was found in pericytes. Apoptosis rate of endothelial cells and pericytes in high glucose levels was higher than that in lower glucose levels. UCP1 and UCP2 were expressed in cultured retinal capillary cells whereas UCP3 was not. At high levels of glucose, expression of UCP1, UCP2 and MnSOD increased to accommodate ROS production compensatively. The compensative mechanism disappeared when glucose concentration was too high (30 mM). The results of this study showed that increasing mitochondrial ROS could be induced by high glucose concentration. Those proteins related to antioxidation mechanism, such as MnSOD and UCPs, could exert compensative action to a certain extent. This compensative action was insufficient when the glucose concentration was too high. (c) 2006 Elsevier Ltd. All rights reserved.