Jiao Hui, Zhang Zhi, Ma Qinghua, Fu Wei, Liu Zidong
Department of Cardiology, The Third Affilated Hospital of Liaoning Medical University, Jinzhou 121001, P.R. China.
Exp Ther Med. 2013 Mar;5(3):797-802. doi: 10.3892/etm.2013.902. Epub 2013 Jan 17.
The aim of the present study was to investigate the effect of Apelin-13 on cardiomyocyte autophagy and to determine the underlying mechanism of this effect. To establish an autophagic model system, the cardiomyocytes of Sprague Dawley rats (postnatal day 3) were cultured and divided into five groups: normal control (Co), glucose deprivation (GD), GD+Apelin-13, GD+Apelin-13 treated with the Akt-specific inhibitor triciribine (GD+Apelin-13+Triciribine) and triciribine alone (Triciribine). The intracellular autophagosomes were then observed using transmission electron microscopy (TEM) and the expression levels of cellular autophagy-related protein microtubule-associated protein 1 light chain 3 (LC3), phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) protein were measured using western blotting. Compared with the Co group, the ratio of LC3-II/LC3-I increased significantly in all treatment groups, with the exception of the Triciribine group (P<0.05). Compared with the GD group, the ratio of LC3-II/LC3-I was significantly decreased, and the PI3K and mTOR expression was significantly enhanced in the GD+Apelin-13 and GD+Apelin-13+Triciribine groups (P<0.05). Compared with the GD+Apelin-13 group, the ratio of LC3-II/LC3-I increased significantly (P<0.05) and the PI3K expression remained unchanged in the GD+Apelin-13+Triciribine group (P>0.05), but mTOR expression was significantly reduced (P<0.05). GD led to increased numbers of autophagosomes and augmented the LC3-II/LC3-I ratio (P<0.05). Apelin-13 pretreatment attenuated GD-induced cardiomyocte injury, decreased the autophagosome number and the ratio of LC3-II/LC3-I (P<0.05), enhanced PI3K activity (P<0.05) and upregulated the phosphorylation levels of the Akt and mTOR proteins (P<0.05). The Akt-specific inhibitor triciribine weakened the protective role of Apelin-13 (P<0.05). To a certain extent, Apelin-13 inhibited GD-induced cardiomyocyte autophagy, which may be related in part to the activation of the PI3K/Akt/mTOR signaling pathway.
本研究旨在探讨Apelin-13对心肌细胞自噬的影响，并确定其作用的潜在机制。为建立自噬模型系统，培养出生3天的Sprague Dawley大鼠的心肌细胞，并分为五组：正常对照组（Co）、葡萄糖剥夺组（GD）、GD+Apelin-13组、用Akt特异性抑制剂三环己基锡处理的GD+Apelin-13组（GD+Apelin-13+三环己基锡）和单独的三环己基锡组（三环己基锡）。然后使用透射电子显微镜（TEM）观察细胞内自噬体，并使用蛋白质印迹法测量细胞自噬相关蛋白微管相关蛋白1轻链3（LC3）、磷脂酰肌醇-3激酶（PI3K）和雷帕霉素靶蛋白（mTOR）的表达水平。与Co组相比，除三环己基锡组外，所有治疗组中LC3-II/LC3-I的比率均显著增加（P<0.05）。与GD组相比，GD+Apelin-13组和GD+Apelin-13+三环己基锡组中LC3-II/LC3-I的比率显著降低，PI3K和mTOR表达显著增强（P<0.05）。与GD+Apelin-13组相比，GD+Apelin-13+三环己基锡组中LC3-II/LC3-I的比率显著增加（P<0.05），PI3K表达保持不变（P>0.05），但mTOR表达显著降低（P<0.05）。GD导致自噬体数量增加和LC3-II/LC3-I比率升高（P<0.05）。Apelin-13预处理减轻了GD诱导的心肌细胞损伤，减少了自噬体数量和LC3-II/LC3-I的比率（P<0.05），增强了PI3K活性（P<0.05），并上调了Akt和mTOR蛋白的磷酸化水平（P<0.05）。Akt特异性抑制剂三环己基锡削弱了Apelin-13的保护作用（P<0.05）。在一定程度上，Apelin-13抑制了GD诱导的心肌细胞自噬，这可能部分与PI3K/Akt/mTOR信号通路的激活有关。
