Department of Geriatrics, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China.
Metabolism. 2013 May;62(5):743-52. doi: 10.1016/j.metabol.2012.09.014. Epub 2012 Dec 4.
The purpose of this research was to investigate the effects of ghrelin on circulating endothelial progenitor cells (EPC) directional migration and its underlying molecular mechanisms involved in this process.
MATERIALS/METHODS: EPC were isolated from bone marrow of SD rats by using Percoll density gradient centrifugation, and characterized by double positive for acLDL-Dil uptake and FITC-UEA-1 binding and immunocytochemistry for CD34, CD133, vWF and Flk-1. EPC were treated with different concentrations of ghrelin (10(-9)~10(-6)M) with or without GHSR1a inhibitor [D-Lys3]-GHRP-6, PI3K inhibitor LY294002 and endothelial nitric oxide synthase (eNOS) inhibitor L-NAME, migration of EPC was detected by transwell assay, levels of phosphorylated and total Akt and eNOS were determined by Western-blot analysis and Nitric Oxide (NO) production was measured by Griess assay, respectively.
EPC were successfully obtained by Percoll density gradient centrifugation and ghrelin at 10(-8)M~10(-7)M promoted EPC migration. Ghrelin-induced EPC migration was accompanied by phosphorylation of Akt and eNOS, as well as an increase in NO production. These biochemical events and EPC directional migration induced by ghrelin were completely inhibited by GHSR-1a blocker [D-Lys3]-GHRP-6. PI3K inhibitor LY294002 attenuated ghrelin-induced EPC migration, phosphorylation of Akt and eNOS, and NO production. eNOS inhibitor L-NAME blocked ghrelin-induced EPC migration, phosphorylation of eNOS, and NO production, but had no effect on Akt phosphorylation.
These findings suggest that ghrelin stimulates EPC directional migration via GHSR1a-mediated PI3K/Akt/eNOS/NO signal pathway. It indicates that ghrelin may be used as a therapeutic strategy to treat ischemic diseases by promoting EPC directional migration.
本研究旨在探讨 ghrelin 对循环内皮祖细胞（EPC）定向迁移的影响及其在这一过程中涉及的潜在分子机制。
材料/方法：通过 Percoll 密度梯度离心法从 SD 大鼠骨髓中分离 EPC，通过 acLDL-Dil 摄取和 FITC-UEA-1 结合的双阳性以及 CD34、CD133、vWF 和 Flk-1 的免疫细胞化学来鉴定 EPC。用不同浓度的 ghrelin（10(-9)~10(-6)M）处理 EPC，并用 GHSR1a 抑制剂[D-Lys3]-GHRP-6、PI3K 抑制剂 LY294002 和内皮型一氧化氮合酶（eNOS）抑制剂 L-NAME 预处理，用 Transwell 检测 EPC 迁移，Western blot 分析测定磷酸化和总 Akt 和 eNOS 的水平，Griess 测定法测定一氧化氮（NO）的产生。
通过 Percoll 密度梯度离心成功获得 EPC，ghrelin 在 10(-8)M~10(-7)M 促进 EPC 迁移。Ghrelin 诱导的 EPC 迁移伴随着 Akt 和 eNOS 的磷酸化以及 NO 产生的增加。这些生化事件和 ghrelin 诱导的 EPC 定向迁移完全被 GHSR-1a 阻断剂[D-Lys3]-GHRP-6 抑制。PI3K 抑制剂 LY294002 减弱了 ghrelin 诱导的 EPC 迁移、Akt 和 eNOS 的磷酸化以及 NO 的产生。eNOS 抑制剂 L-NAME 阻断了 ghrelin 诱导的 EPC 迁移、eNOS 的磷酸化和 NO 的产生，但对 Akt 磷酸化没有影响。
这些发现表明，ghrelin 通过 GHSR1a 介导的 PI3K/Akt/eNOS/NO 信号通路刺激 EPC 定向迁移。这表明 ghrelin 可以通过促进 EPC 定向迁移来作为治疗缺血性疾病的治疗策略。
