Department of Respiratory Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China.
Int J Mol Med. 2013 Jul;32(1):187-94. doi: 10.3892/ijmm.2013.1379. Epub 2013 May 13.
Apoptosis of human pulmonary artery endothelial cells (HPAECs) is the initial step and triggering event for pulmonary hypertension (PH). However, little is known about the actions of nicorandil on HPAECs in vitro. In the present study, we investigated the anti-apoptotic effect of nicorandil on HPAECs exposed to hypoxia, and explored the underlying mechanism(s) of action. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Annexin V and propidium iodide staining, and Hoechst 33342 staining assay were employed to detect apoptosis. In addition, the protein expression of Bax, Bcl-2, caspase-9 and -3, endothelial nitric oxide synthase (eNOS), nuclear factor-κB (NF-κB) and IκBα were determined by western blotting to investigate the possible mechanisms. We found that exposure to hypoxia for 24 h significantly decreased cell viability and increased cell apoptosis. Pretreatment with nicorandil (100 µM) effectively abolished the influence of hypoxia on HPAECs. However, these protective effects of nicorandil were significantly inhibited by an antagonist of mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP) channels, 5-hydroxydecanoate (5-HD, 500 µM), and by an eNOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 300 µM). We further observed that nicorandil could upregulate the decreased protein expression of eNOS and IκBα, and downregulate the increased protein expression of NF-κB, induced by hypoxia. In addition, nicorandil inhibited the enhancement of caspase-3 and -9 expression, and the increase in the Bax/Bcl-2 expression ratio, induced by hypoxia. However, these effects were also abolished by 5-HD and L-NAME. Collectively, these findings suggest that nicorandil inhibits hypoxia-induced apoptosis of HPAECs through activation of mitoKATP channels and increased eNOS expression, which in turn inhibits the NF-κB pathway and the mitochondrial apoptotic pathway.
人肺动脉内皮细胞 (HPAEC) 的凋亡是肺动脉高压 (PH) 的初始步骤和触发事件。然而，关于尼可地尔在体外对 HPAEC 的作用知之甚少。在本研究中，我们研究了尼可地尔对缺氧暴露的 HPAEC 的抗凋亡作用，并探讨了其作用机制。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐 (MTT) 评估细胞活力。使用 Annexin V 和碘化丙啶染色以及 Hoechst 33342 染色法检测细胞凋亡。此外，通过 Western blot 测定 Bax、Bcl-2、caspase-9 和 -3、内皮型一氧化氮合酶 (eNOS)、核因子-κB (NF-κB) 和 IκBα 的蛋白表达，以探讨可能的机制。我们发现，缺氧暴露 24 小时显著降低细胞活力并增加细胞凋亡。尼可地尔 (100 µM) 预处理可有效消除缺氧对 HPAEC 的影响。然而，这些尼可地尔的保护作用被线粒体三磷酸腺苷敏感钾 (mitoKATP) 通道拮抗剂 5-羟基癸酸 (5-HD，500 µM) 和 eNOS 抑制剂 NG-硝基-L-精氨酸甲酯 (L-NAME，300 µM) 显著抑制。我们进一步观察到，尼可地尔可以上调缺氧诱导的 eNOS 和 IκBα 蛋白表达降低，下调 NF-κB 蛋白表达升高。此外，尼可地尔抑制了缺氧诱导的 caspase-3 和 -9 表达增强以及 Bax/Bcl-2 表达比值升高。然而，这些作用也被 5-HD 和 L-NAME 消除。综上所述，这些发现表明，尼可地尔通过激活 mitoKATP 通道和增加 eNOS 表达来抑制缺氧诱导的 HPAEC 凋亡，从而抑制 NF-κB 途径和线粒体凋亡途径。
