Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China.
Department of Pharmacy, The First Affiliated Hospital, Sun Yat-sent University, Guangzhou 510080, China.
Life Sci. 2017 Jan 1;168:28-37. doi: 10.1016/j.lfs.2015.12.054. Epub 2016 Jan 5.
Palmitate, a common saturated free fatty acid, induces endothelial apoptosis in vitro in culture endothelial cells and in vivo in type 2 diabetes mellitus (T2DM) patients. The present study aimed to investigate whether Kv1.5 regulates palmitate-induced endothelial apoptosis and endothelial dysfunction in T2DM.
In vitro experiments were carried out in primary human HUVECs. Apoptosis was analyzed by flow cytometry. Cell viability was determined by Cell Counting Assay Kit-8. The siRNA transfection was employed to knockdown Kv1.5 protein expression. Intracellular and mitochondrial ROS, and mitochondrial membrane potential were detected using fluorescent probes. Male C57BL/6 mice fed with high-sucrose/fat diet were injected with streptozotocin (35mg/kg body weight) to establish T2DM animal model.
We found that palmitate-induced endothelial apoptosis was parallel to a significant increase in endogenous Kv1.5 protein expression in endothelial cells. Silencing of Kv1.5 with siRNA reduced palmitate-induced endothelial apoptosis, intracellular ROS generation, mitochondrial ROS generation and membrane potential (Δψ) alteration and cleaved caspase-3 protein expression; while increased cell viability and ratio of Bcl-2/Bax. Furthermore, we observed that Kv1.5 protein expression increased in endothelial cells of thoracic aorta of T2DM mice. Silencing of Kv1.5 significantly improved the endothelium-dependent vasodilation in thoracic aortic rings of T2DM mice.
These results demonstrate that suppression of Kv1.5 protects endothelial cells against palmitate-induced apoptosis via inhibiting mitochondria-mediated excessive ROS generation and apoptotic signaling pathway, suggesting that Kv1.5 may serve as a therapeutic target of treatment for endothelial dysfunction induced by palmitate and lipid metabolism in T2DM patients.
棕榈酸是一种常见的饱和游离脂肪酸，可在体外培养的内皮细胞中和 2 型糖尿病（T2DM）患者体内诱导内皮细胞凋亡。本研究旨在探讨 Kv1.5 是否调节棕榈酸诱导的 T2DM 内皮细胞凋亡和内皮功能障碍。
在原代人脐静脉内皮细胞（HUVECs）中进行体外实验。通过流式细胞术分析细胞凋亡。通过 Cell Counting Assay Kit-8 测定细胞活力。采用 siRNA 转染敲低 Kv1.5 蛋白表达。使用荧光探针检测细胞内和线粒体 ROS 以及线粒体膜电位。雄性 C57BL/6 小鼠用高蔗糖/脂肪饮食喂养，并注射链脲佐菌素（35mg/kg 体重）建立 T2DM 动物模型。
我们发现棕榈酸诱导的内皮细胞凋亡与内皮细胞中内源性 Kv1.5 蛋白表达的显著增加平行。用 siRNA 沉默 Kv1.5 可减少棕榈酸诱导的内皮细胞凋亡、细胞内 ROS 生成、线粒体 ROS 生成和膜电位（Δψ）改变以及 cleaved caspase-3 蛋白表达；同时增加细胞活力和 Bcl-2/Bax 的比值。此外，我们观察到 T2DM 小鼠胸主动脉内皮细胞中 Kv1.5 蛋白表达增加。沉默 Kv1.5 可显著改善 T2DM 小鼠胸主动脉环内皮依赖性血管舒张功能。
这些结果表明，抑制 Kv1.5 通过抑制线粒体介导的过多 ROS 生成和凋亡信号通路来保护内皮细胞免受棕榈酸诱导的凋亡，表明 Kv1.5 可能成为治疗 T2DM 患者棕榈酸诱导的脂质代谢和内皮功能障碍的治疗靶点。
