Abstract

In this study, we investigated the effects of Angiotensin II (Ang II) on insulin-resistant endothelial cells. High glucose and insulin at series of concentrations were used to induce IR in Human Umbilical Vein Endothelial Cells (HUVECs). Successful IR induction was confirmed according to glucose consumption and glycogen content levels. Cell morphology was observed under a microscope. Expression levels of Ang II and Calreticulin (CRT) were measured by ELISA, qRT-PCR and Western blot as appropriate. Cell viability and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. HUVECs with IR were exposed to Ang II at series of concentrations, and then the cell viability, apoptosis and CRT were detected. Rescue assays were performed by transfection of siCRT or overexpression of CRT with or without Ang II stimulation into the HUVECs with IR. Expressions of cell apoptosis-related proteins Bcl-2 and Bax were measured by qRT-PCR and Western blot. Glucose (33.3 mmol/L) and insulin (4 µmol/L) induced significantly strong IR to the HUVECs, with a pathological appearance. Levels of Agn II and CRT were both up-regulated by IR. Cell viability of HUVECs was slightly reduced after IR induction for 2 h, and cell apoptosis rate was increased. In addition, Ang II (10⁻⁷ mol/l) suppressed cell viability and glucose uptake, promoted cell apoptosis and increased CRT, and these effects could be weakened by silencing CRT. Thus, we preliminarily proved that Ang II up-regulates CRT, and CRT knockdown can relieve Ang II-induced injury of HUVECs with IR.

摘要

在这项研究中，我们研究了血管紧张素 II (Ang II) 对胰岛素抵抗内皮细胞的影响。使用一系列浓度的高葡萄糖和胰岛素来诱导人脐静脉内皮细胞 (HUVEC) 中的 IR。根据葡萄糖消耗和糖原含量水平确认成功的 IR 诱导。在显微镜下观察细胞形态。Ang II 和钙网蛋白 (CRT) 的表达水平通过 ELISA、qRT-PCR 和蛋白质印迹酌情测量。分别通过CCK-8测定和流式细胞术测量细胞活力和凋亡。将具有IR的HUVECs暴露于一系列浓度的Ang II，然后检测细胞活力、凋亡和CRT。通过将 siCRT 转染或在有或没有 Ang II 刺激的情况下过表达 CRT 到具有 IR 的 HUVEC 中来进行拯救试验。通过qRT-PCR和Western blot检测细胞凋亡相关蛋白Bcl-2和Bax的表达。葡萄糖 (33.3 mmol/L) 和胰岛素 (4 µmol/L) 对 HUVEC 产生显着强烈的 IR，具有病理表现。Agn II 和 CRT 的水平都被 IR 上调。IR诱导2小时后HUVECs的细胞活力略有降低，细胞凋亡率增加。此外，Ang II (10 IR诱导2小时后HUVECs的细胞活力略有降低，细胞凋亡率增加。此外，Ang II (10 IR诱导2小时后HUVECs的细胞活力略有降低，细胞凋亡率增加。此外，Ang II (10^{− 7} mol/l) 抑制细胞活力和葡萄糖摄取，促进细胞凋亡和增加 CRT，这些作用可以通过沉默 CRT 减弱。因此，我们初步证明Ang II上调CRT，CRT敲低可以减轻Ang II诱导的IR损伤HUVECs。