Acute blood glucose fluctuation enhances rat aorta endothelial cell apoptosis, oxidative stress and pro-inflammatory cytokine expression in vivo.
Acute blood glucose fluctuation enhances rat aorta endothelial cell apoptosis, oxidative stress and pro-inflammatory cytokine expression in vivo.
复制标题
急性血糖波动促进大鼠主动脉内皮细胞凋亡、氧化应激和促炎细胞因子表达。
DOI：
10.1186/s12933-016-0427-0
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发表时间：
2016-08-05
影响因子：
9.3
通讯作者：
Han P
中科院分区：
文献类型：
--
作者：
Wu N;Shen H;Liu H;Wang Y;Bai Y;Han P
糖尿病（DM）的并发症不仅与血糖升高有关，而且与血糖波动有关。然而，血糖波动在糖尿病并发症发病机制中的具体作用机制尚不清楚。本研究在体内研究了急性波动型高血糖和持续性高血糖对血管内皮细胞凋亡、功能、氧化应激和炎症的影响。将大鼠分为3组（n = 10/组），分别给予48 h生理盐水（SAL组）、连续50%葡萄糖（恒定高糖组[CHG]）和间歇50%葡萄糖（急性血糖波动组[AFG]）。采用酶联免疫吸附试验（ELISA）试剂盒定量测定血浆8-异前列腺素、白细胞介素-6 （IL-6）、肿瘤坏死因子-α (TNF-α)和细胞间粘附分子-1 （ICAM-1）水平。血浆胰岛素水平采用放射免疫测定（RIAs）试剂盒测定。收集主动脉段。用比色试剂盒检测主动脉内皮细胞制备的内皮匀浆中丙二醛（MDA）水平和谷胱甘肽过氧化物酶（GSH-PX）活性。采用末端脱氧核苷酸转移酶dUTP缺口末端标记法（TUNEL）检测血管内皮细胞凋亡情况。采用等长张力记录法评估内皮功能障碍。Western blot检测大鼠主动脉内皮细胞中B细胞淋巴瘤-2 （Bcl-2）、Bcl-2相关X蛋白（Bax）、caspase-3、caspase-3 p17、3-硝基酪氨酸（3-NT）和p47phox蛋白的表达。采用双氢乙二酸依赖荧光显微形貌法测定主动脉冷冻切片内皮细胞活性氧（ROS）的形成。实时定量PCR检测血管内皮细胞中IL-6、TNF-α和ICAM-1 mrna的表达。AFG组主动脉内皮细胞凋亡及功能障碍显著（P < 0.05）。与CHG组相比，AFG降低了Bcl-2和pro caspase-3水平，提高了Bax线粒体易位和caspase-3 p17蛋白水平（P < 0.05）。AFG和CHG均可诱导β细胞功能障碍和胰岛素抵抗（P < 0.05）。AFG升高血浆MDA和8-异前列腺素水平，血管内皮细胞氧化应激，血浆和血管内皮细胞炎症因子水平（P < 0.05）。急性血糖波动可引起内皮细胞明显的氧化应激和炎症反应，增加单核细胞对内皮细胞的粘附，促进内皮细胞凋亡，导致严重的心血管损伤。
Complications of diabetes mellitus (DM) are related not only to elevated plasma glucose, but also plasma glucose fluctuations. However, the specific mechanism underlying the role of plasma glucose fluctuation in the pathogenesis of DM complications remains poorly understood. In the present study, the influence of acute fluctuant hyperglycemia and persistent hyperglycemia on vascular endothelial cell apoptosis, function, oxidative stress and inflammation was examined in vivo. Rats were assigned to three different groups (n = 10/group) that received 48-h infusions of saline (SAL group), continuous 50 % glucose (constant high glucose group [CHG]), or intermittent 50 % glucose (acute blood glucose fluctuation group [AFG]). Plasma 8-isoprostaglandin, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) levels were quantified by using enzyme-linked immunosorbent assay (ELISA) commercial kits. Plasma insulin levels were measured by radioimmunoassays (RIAs) using kits. The aortic segment was collected. The levels of malondialdehyde (MDA) and activity of glutathione peroxidase (GSH-PX) were measured in endothelial homogenates prepared from endothelial cells harvested from the aorta using colorimetric kits. Apoptosis of vascular endothelial cells was determined with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Endothelial dysfunction was assessed by isometric tension recording to evaluate the endothelial function. The expression of B cell lymphoma-2 (Bcl-2), Bcl-2 Associated X protein (Bax), pro caspase-3, caspase-3 p17, 3-nitrotyrosine (3-NT) and p47phox protein in rat aortic endothelial cells were tested with Western blot analysis. Endothelial cells reactive oxygen species (ROS) formation was determined using dihydroethidium-dependent fluorescence microtopography in aortic cryo-sections. Expression of IL-6, TNF-α and ICAM-1 mRNAs in vascular endothelial cells were determined by real-time quantitative PCR. Endothelial cells apoptosis and dysfunction were observed significantly in the aortas of the AFG group (P < 0.05). The AFG had reduced Bcl-2 and pro caspase-3 levels and enhanced Bax mitochondrial translocation and caspase-3 p17 protein levels in comparison with the CHG group (P < 0.05). Both AFG and CHG induced β-cell dysfunction and insulin resistance (P < 0.05). AFG increased MDA and 8-isoprostaglandin levels in plasma, oxidative stress in vascular endothelial cells, and inflammatory cytokines in plasma and vascular endothelial cells (P < 0.05). Acute glucose fluctuation may cause significant oxidative stress and inflammation in endothelial cells, increase the adhesion of monocytes to endothelial cells, and elevate endothelial cell apoptosis, resulting in severe cardiovascular injury.