依帕司他诱导低氧大鼠肺动脉平滑肌细胞线粒体依赖性凋亡。
Iptakalim induces mitochondria-dependent apoptosis in hypoxic rat pulmonary arterial smooth muscle cells.
机构信息
Department of Respiratory Medicine, The first Affiliated Hospital of Nanjing Medical University Nanjing 210029, China.
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China.
出版信息
Biomed Pharmacother. 2016 Dec;84:773-779. doi: 10.1016/j.biopha.2016.09.031. Epub 2016 Oct 5.
OBJECTIVES
Pulmonary vascular medial hypertrophy in hypoxic pulmonary arterial hypertension (HPH) is caused in part by decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Iptakalim (Ipt), an ATP sensitive potassium channel opener, ameliorates HPH in animal models. Here we investigated the effects of Ipt on proliferation and apoptosis of hypoxic rat PASMCs, and to determine the possible underlying mechanisms.
METHODS
Primary rat PASMCs were isolated and cultured. PASMCs were cultured for 24h in normoxia or hypoxia (5% O) conditions with and without Ipt. Cell proliferation and cycle were determined by MTT assay and flow cytometry, respectively. Mitochondrial membrane potential (Δym) was detected by fluorescence microscope Western blot assays were used to examine the expression of cyclin D, CDK4, endothelin-1 (ET-1), hypoxia-inducible factor-1 (HIF-1), platelet-derived growth factor-BB (PDGF-BB), Bax, Bcl-2, cytochrome c (Cyt c), caspase-9, and caspase-3 in PASMCs.
RESULTS
We found that hypoxia significantly stimulated proliferation and rendered resistance to apoptosis in PASMCs. Ipt suppressed proliferation and induced cell cycle arrest in hypoxia PASMCs. Ipt decreased the expression of cyclin D, CDK4, HIF-1, ET-1, and PDGF-BB in hypoxia PASMCs. It reversed the depolarization of Δψm in hypoxia PASMCs too. Ipt significantly upregulated Bax expression and downregulated Bcl-2 expression, and promoted the release of Cyt c from mitochondria to cytoplasm in hypoxia PASMCs. Furthermore, Ipt significantly activated the caspase cascades evidenced by increased expression of caspase-9 and caspase-3 in hypoxia PASMCs.
CONCLUSIONS
Ipt could inhibit cell proliferation and induce apoptosis associated with cell cycle arrest, decreased ET-1, HIF-1, cyclin D, CDK4, PDGF-BB and Δψm, increased Bax/Bcl-2 ratio, enhanced Cyt c release, and activation of caspases in PASMCs under hypoxia status. Our data indicated that Ipt could be a therapeutic candidate for treatment of HPH.
目的
低氧性肺动脉高压（HPH）中的肺血管中层肥厚部分是由于肺动脉平滑肌细胞（PASMC）凋亡减少引起的。伊帕立林（Ipt）是一种三磷酸腺苷敏感钾通道开放剂，可改善动物模型中的 HPH。在这里，我们研究了 Ipt 对低氧大鼠 PASMC 增殖和凋亡的影响，并确定了可能的潜在机制。
方法
分离并培养原代大鼠 PASMC。将 PASMC 在常氧或低氧（5% O）条件下培养 24 小时，同时加入和不加入 Ipt。通过 MTT 测定和流式细胞术分别测定细胞增殖和细胞周期。用荧光显微镜检测线粒体膜电位（Δym），Western blot 检测 PASMC 中环化蛋白 D、CDK4、内皮素-1（ET-1）、缺氧诱导因子-1（HIF-1）、血小板衍生生长因子-BB（PDGF-BB）、Bax、Bcl-2、细胞色素 c（Cyt c）、caspase-9 和 caspase-3 的表达。
结果
我们发现低氧显著刺激 PASMC 增殖，并使其对凋亡产生抗性。Ipt 抑制低氧 PASMC 的增殖并诱导细胞周期停滞。Ipt 降低低氧 PASMC 中环化蛋白 D、CDK4、HIF-1、ET-1 和 PDGF-BB 的表达。它还逆转了低氧 PASMC 中Δψm 的去极化。Ipt 显著上调 Bax 表达，下调 Bcl-2 表达，并促进 Cyt c 从线粒体向细胞质释放。此外，Ipt 显著激活了 caspase 级联反应，表现为低氧 PASMC 中 caspase-9 和 caspase-3 的表达增加。
结论
Ipt 可抑制细胞增殖并诱导与细胞周期停滞相关的凋亡，降低 ET-1、HIF-1、cyclin D、CDK4、PDGF-BB 和Δψm，增加 Bax/Bcl-2 比值，增强 Cyt c 释放，并激活低氧状态下 PASMC 中的 caspases。我们的数据表明，Ipt 可能是治疗 HPH 的候选药物。
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