C-peptide reduces high-glucose-induced apoptosis of endothelial cells and decreases NAD(P)H-oxidase reactive oxygen species generation in human aortic endothelial cells
C-peptide reduces high-glucose-induced apoptosis of endothelial cells and decreases NAD(P)H-oxidase reactive oxygen species generation in human aortic endothelial cells
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DOI：
10.1007/s00125-011-2251-0
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发表时间：
2011-10-01
期刊：
影响因子：
8.2
通讯作者：
Luppi, P.
中科院分区：
文献类型：
--
作者：
Cifarelli, V.;Geng, X.;Luppi, P.
在高血糖期间产生的活性氧（ROS）与糖尿病血管并发症的发展有关。高糖增加内皮细胞的氧化应激并诱导细胞凋亡。暴露于葡萄糖的内皮细胞中活性氧的主要来源是NAD（P）H氧化酶。几项研究表明，C肽，胰腺β细胞内胰岛素原裂解的产物，在某些血管功能障碍模型中显示抗炎作用。然而，这种效应背后的分子机制尚不清楚。我们假设C肽通过降低NAD（P）H氧化酶的活化来减少葡萄糖诱导的ROS产生，并防止凋亡。在存在或不存在C肽的情况下，将人主动脉内皮细胞（HAEC）暴露于25 mmol/l葡萄糖，并通过ELISA、TUNEL和免疫印迹法检测蛋白质量和caspase-3和其他凋亡标志物的活性。使用ROS敏感染料氯甲基-2 '，7'-二氯二氢荧光素二乙酸酯（CM-H-2-DCDFA）通过流式细胞术测量细胞内ROS。光泽精测定NAD（P）H氧化酶活性。采用免疫印迹和ELISA法研究了NAD（P）H亚基ras相关的C3肉毒毒素底物1（rho家族，小GTP结合蛋白Rac 1）（RAC-1）的膜和胞浆水平及其GTP酶活性。用实时荧光定量PCR检测RAC-1（也称为RAC 1）基因表达，C肽显著降低caspase-3水平和活性，上调抗凋亡因子B细胞CLL/淋巴瘤2（BCL-2）的产生。C肽可抑制葡萄糖诱导的ROS产生，这与NAD（P）H氧化酶活性降低、RAC-1膜产生和GT3活性降低有关，C肽可通过减少RAC-1向膜的转运和NAD（P）H氧化酶的激活而发挥内源性抗氧化分子的作用。通过防止氧化应激，C肽保护内皮细胞免受葡萄糖诱导的凋亡。
Reactive oxygen species (ROS) generated during hyperglycaemia are implicated in the development of diabetic vascular complications. High glucose increases oxidative stress in endothelial cells and induces apoptosis. A major source of ROS in endothelial cells exposed to glucose is the NAD(P)H oxidase enzyme. Several studies demonstrated that C-peptide, the product of proinsulin cleavage within the pancreatic beta cells, displays anti-inflammatory effects in certain models of vascular dysfunction. However, the molecular mechanism underlying this effect is unclear. We hypothesised that C-peptide reduces glucose-induced ROS generation by decreasing NAD(P)H oxidase activation and prevents apoptosisHuman aortic endothelial cells (HAEC) were exposed to 25 mmol/l glucose in the presence or absence of C-peptide and tested for protein quantity and activity of caspase-3 and other apoptosis markers by ELISA, TUNEL and immunoblotting. Intracellular ROS were measured by flow cytometry using the ROS sensitive dye chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H-2-DCDFA). NAD(P)H oxidase activation was assayed by lucigenin. Membrane and cytoplasmic levels of the NAD(P)H subunit ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) (RAC-1) and its GTPase activity were studied by immunoblotting and ELISA. RAC-1 (also known as RAC1) gene expression was investigated by quantitative real-time PCR.C-peptide significantly decreased caspase-3 levels and activity and upregulated production of the anti-apoptotic factor B cell CLL/lymphoma 2 (BCL-2). Glucose-induced ROS production was quenched by C-peptide and this was associated with a decreased NAD(P)H oxidase activity and reduced RAC-1 membrane production and GTPase activity.In glucose-exposed endothelial cells, C-peptide acts as an endogenous antioxidant molecule by reducing RAC-1 translocation to membrane and NAD(P)H oxidase activation. By preventing oxidative stress, C-peptide protects endothelial cells from glucose-induced apoptosis.