*Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xian, Shaanxi 710032, Peoples Republic of China.
Cell Biol Int. 2011 Feb;35(2):135-40. doi: 10.1042/CBI20100139.
Ghrelin is thought to directly exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function. Our study demonstrates the ability of ghrelin to promote rat CMEC (cardiac microvascular endothelial cell) proliferation, migration and NO (nitric oxide) secretion. CMECs were isolated from left ventricle of adult male Sprague-Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil-ac-LDL (1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine-labelled acetylated low-density lipoprotein) intake assays were used to identify CMECs. Cells were split into five groups and treated with varying concentrations of ghrelin as follows: one control non-treated group; three ghrelin dosage groups (1×10-9, 1×10-8, 1×10-7 mol/l) and one ghrelin+PI3K inhibitor group (1×10-7 mol/l ghrelin+20 μmol/l LY294002). After 24 h treatment, cell proliferation capability was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and Western blot for PCNA (proliferating cell nuclear antigen) protein expression. Migration of CMECs was detected by transwell assays, and NO secretion of CMECs was measured via nitrate reduction. Protein expression of AKT and phosphorylated AKT in CMECs was measured by Western blot after exposure to various concentrations of ghrelin and the PI3K inhibitor LY294002. Our results indicate that ghrelin significantly enhanced cell growth at concentrations of 10-8 mol/l (0.271±0.041 compared with 0.199±0.021, P = 0.03) and 10-7 mol/l (0.296±0.039 compared with 0.199±0.021, P<0.01). However, addition of the PI3K/AKT inhibitor LY294002 inhibited the ghrelin-mediated enhancement in cell proliferation (0.227±0.042 compared with 0.199±0.021, P = 0.15). At a concentration between 10-8 and 10-7 mol/l, ghrelin caused a significant increase in the number of migrated cells compared with the control group (126±9 compared with 98±7, P = 0.02; 142±6 compared with 98±7, P<0.01), whereas no such change could be observed in the presence of 20 μmol/l of the PI3K/Akt inhibitor LY294002 (103±7 compared with 98±7, P = 0.32). Ghrelin treatment significantly enhanced NO production in a dose-dependent fashion compared with the untreated control group [(39.93±2.12) μmol/l compared with (30.27±2.71) μmol/l, P = 0.02; (56.80±1.98) μmol/l compared with (30.27±2.71) μmol/l, P<0.01]. However, pretreatment with 20 μmol/l LY294002 inhibited the ghrelin-stimulated increase in NO secretion [(28.97±1.64) μmol/l compared with (30.27±2.71) μmol/l, P = 0.37]. In summary, we have found that ghrelin treatment promotes the proliferation, migration and NO secretion of CMECs through activation of PI3K/AKT signalling pathway.
生长激素释放肽被认为直接对心血管系统发挥保护作用，特别是通过促进血管内皮细胞功能。我们的研究表明，ghrelin 能够促进大鼠 CMEC（心脏微血管内皮细胞）的增殖、迁移和 NO（一氧化氮）分泌。CMEC 从成年雄性 Sprague-Dawley 大鼠的左心室通过酶消化分离，并在内皮细胞培养基中维持。通过 Dil-ac-LDL（1,1'-二辛基-3,3,3',3'-四甲基吲哚碳酰化乙酰化低密度脂蛋白）摄取测定来鉴定 CMEC。将细胞分为五组，并分别用不同浓度的 ghrelin 处理如下：一个对照非处理组；三个 ghrelin 剂量组（1×10-9、1×10-8、1×10-7mol/l）和一个 ghrelin+PI3K 抑制剂组（1×10-7mol/l ghrelin+20μmol/l LY294002）。处理 24 小时后，通过 MTT[3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴盐]测定法和增殖细胞核抗原（PCNA）蛋白表达的 Western blot 测定细胞增殖能力。通过 Transwell 测定法检测 CMEC 的迁移，通过硝酸盐还原测定 CMEC 的 NO 分泌。用不同浓度的 ghrelin 和 PI3K 抑制剂 LY294002 处理 CMEC 后，通过 Western blot 测定 AKT 和磷酸化 AKT 的蛋白表达。我们的结果表明，ghrelin 在 10-8mol/l（0.271±0.041 与 0.199±0.021，P=0.03）和 10-7mol/l（0.296±0.039 与 0.199±0.021，P<0.01）浓度下显著增强细胞生长。然而，加入 PI3K/AKT 抑制剂 LY294002 抑制了 ghrelin 介导的细胞增殖增强（0.227±0.042 与 0.199±0.021，P=0.15）。在 10-8 和 10-7mol/l 之间的浓度下，ghrelin 与对照组相比，迁移细胞的数量显著增加（126±9 与 98±7，P=0.02；142±6 与 98±7，P<0.01），而在存在 20μmol/l 的 PI3K/Akt 抑制剂 LY294002 时，没有观察到这种变化（103±7 与 98±7，P=0.32）。与未处理的对照组相比，ghrelin 处理以剂量依赖性方式显著增强 NO 产生[（39.93±2.12）μmol/l 与（30.27±2.71）μmol/l，P=0.02；（56.80±1.98）μmol/l 与（30.27±2.71）μmol/l，P<0.01]。然而，用 20μmol/l LY294002 预处理抑制了 ghrelin 刺激的 NO 分泌增加[（28.97±1.64）μmol/l 与（30.27±2.71）μmol/l，P=0.37]。总之，我们发现 ghrelin 处理通过激活 PI3K/AKT 信号通路促进 CMEC 的增殖、迁移和 NO 分泌。
