Wei Guoqian, Zhu Dayong, Sun Yongtao, Zhang Lan, Liu Xian, Li Ming, Gu Jinxia
Department of Cardiology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China.
Department of General Surgery, Heilongjiang Provincial Hospital, Harbin, Heilongjiang Province, China.
J Biochem Mol Toxicol. 2021 Jun;35(6):1-8. doi: 10.1002/jbt.22766. Epub 2021 Apr 1.
Atherosclerosis is a common cardiovascular disease with high morbidity and mortality. It is reported to be related to oscillatory shear stress (OSS)-induced endothelial dysfunction and excessive production of inflammatory factors. Azilsartan, a specific antagonist of the angiotensin II receptor, has been approved for the management of hypertensive subjects with diabetes mellitus type II (DMII). The present study will investigate the effects of azilsartan against OSS-induced endothelial dysfunction and inflammation, as well as the underlying mechanism.
Cell viability was detected using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay were used to determine the expression levels of IL-6, TNF-α, IL-1β, VCAM-1, and ICAM-1 in human aortic endothelial cells (HAECs). Generation of reactive oxygen species (ROS) was measured using 2'-7'dichlorofluorescin diacetate (DCFH-DA) staining, and the level of reduced glutathione (GSH) was evaluated using a commercial kit. The adhesion of THP-1 monocytes to HAECs was evaluated using calcein-AM staining. The expression level of KLF6 was determined using qRT-PCR and Western blot analysis.
According to the result of the MTT assay, 5 and 10 μM azilsartan were considered as the optimized concentrations applied in the present study. The elevated production of IL-6, TNF-α, and IL-1β, increased levels of ROS, decreased levels of reduced GSH, upregulated VCAM-1, ICAM-1, and E-selectin, and the aggravated adhesion of THP-1 cells to HAECs induced by OSS were all reversed by the introduction of azilsartan. The downregulation of KLF6 induced by OSS was significantly reversed by azilsartan. By knocking down the expression of KLF6, the suppressed adhesion of THP-1 cells to the HAECs, and the downregulation of VCAM-1 and ICAM-1 induced by azilsartan in OSS-stimulated HAECs were greatly reversed.
The protective effects of azilsartan against OSS-induced endothelial dysfunction and inflammation might be mediated by KLF6.
动脉粥样硬化是一种常见的心血管疾病，发病率和死亡率都很高。据报道，它与振荡剪切应力（OSS）诱导的内皮功能障碍和炎症因子的过度产生有关。阿齐沙坦是一种血管紧张素II受体特异性拮抗剂，已被批准用于治疗II型糖尿病（DMII）合并高血压的患者。本研究将探讨阿齐沙坦对OSS诱导的内皮功能障碍和炎症的影响及其潜在机制。
采用MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐）法检测细胞活力。采用定量逆转录聚合酶链反应（qRT-PCR）和酶联免疫吸附测定法测定人主动脉内皮细胞（HAECs）中白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、血管细胞黏附分子-1（VCAM-1）和细胞间黏附分子-1（ICAM-1）的表达水平。使用2'-7'-二氯荧光素二乙酸酯（DCFH-DA）染色测定活性氧（ROS）的产生，并使用商用试剂盒评估还原型谷胱甘肽（GSH）的水平。采用钙黄绿素-AM染色评估THP-1单核细胞与HAECs的黏附情况。使用qRT-PCR和蛋白质印迹分析测定KLF6的表达水平。
根据MTT测定结果，5和10μM阿齐沙坦被认为是本研究中应用的最佳浓度。阿齐沙坦的引入逆转了OSS诱导的IL-6、TNF-α和IL-1β产生增加、ROS水平升高、还原型GSH水平降低、VCAM-1、ICAM-1和E-选择素上调以及THP-1细胞与HAECs黏附加剧的情况。阿齐沙坦显著逆转了OSS诱导的KLF6下调。通过敲低KLF6的表达，阿齐沙坦在OSS刺激的HAECs中诱导的THP-1细胞与HAECs黏附受抑制以及VCAM-1和ICAM-1下调的情况被极大地逆转。
阿齐沙坦对OSS诱导的内皮功能障碍和炎症的保护作用可能由KLF6介导。
