Department of Life Sciences, National Chung-Hsing University, Taichung 402, Taiwan.
J Biomed Sci. 2013 Nov 19;20(1):86. doi: 10.1186/1423-0127-20-86.
Epigallocatechin-3-gallate (EGCg) with its potent anti-oxidative capabilities is known for its beneficial effects ameliorating oxidative injury to cardiac cells. Although studies have provided convincing evidence to support the cardioprotective effects of EGCg, it remains unclear whether EGCg affect trans-membrane signalling in cardiac cells. Here, we have demonstrated the potential mechanism for cardioprotection of EGCg against H2O2-induced oxidative stress in H9c2 cardiomyoblasts.
Exposing H9c2 cells to H2O2 suppressed cell viability and altered the expression of adherens and gap junction proteins with increased levels of intracellular reactive oxygen species and cytosolic Ca2+. These detrimental effects were attenuated by pre-treating cells with EGCg for 30 min. EGCg also attenuated H2O2-mediated cell cycle arrest at the G1-S phase through the glycogen synthase kinase-3β (GSK-3β)/β-catenin/cyclin D1 signalling pathway. To determine how EGCg targets H9c2 cells, enhanced green fluorescence protein (EGFP) was ectopically expressed in these cells. EGFP-emission fluorescence spectroscopy revealed that EGCg induced dose-dependent fluorescence changes in EGFP expressing cells, suggesting that EGCg signalling events might trigger proximity changes of EGFP expressed in these cells. Proteomics studies showed that EGFP formed complexes with the 67 kD laminin receptor, caveolin-1 and -3, β-actin, myosin 9, vimentin in EGFP expressing cells. Using in vitro oxidative stress and in vivo myocardial ischemia models, we also demonstrated the involvement of caveolin in EGCg-mediated cardioprotection. In addition, EGCg-mediated caveolin-1 activation was found to be modulated by Akt/GSK-3β signalling in H2O2-induced H9c2 cell injury.
Our data suggest that caveolin serves as a membrane raft that may help mediate cardioprotective EGCg transmembrane signalling.
表没食子儿茶素没食子酸酯（EGCg）具有强大的抗氧化能力，其有益作用可改善心肌细胞的氧化损伤。虽然已有研究为 EGCg 的心脏保护作用提供了令人信服的证据，但 EGCg 是否影响心肌细胞的跨膜信号传递仍不清楚。在这里，我们已经证明了 EGCg 对 H9c2 心肌细胞中 H2O2 诱导的氧化应激的心脏保护作用的潜在机制。
将 H9c2 细胞暴露于 H2O2 会抑制细胞活力，并改变黏附连接和缝隙连接蛋白的表达，同时增加细胞内活性氧和细胞质 Ca2+的水平。用 EGCg 预处理细胞 30 分钟可减轻这些有害作用。EGCg 还通过糖原合酶激酶-3β（GSK-3β）/β-连环蛋白/细胞周期蛋白 D1 信号通路减弱 H2O2 介导的细胞周期阻滞在 G1-S 期。为了确定 EGCg 如何靶向 H9c2 细胞，将增强型绿色荧光蛋白（EGFP）异位表达在这些细胞中。EGFP 发射荧光光谱显示，EGCg 在表达 EGFP 的细胞中诱导剂量依赖性荧光变化，表明 EGCg 信号事件可能触发这些细胞中表达的 EGFP 的近距离变化。蛋白质组学研究表明，EGFP 与 67 kD 层粘连蛋白受体、窖蛋白-1 和 -3、β-肌动蛋白、肌球蛋白 9、波形蛋白在表达 EGFP 的细胞中形成复合物。使用体外氧化应激和体内心肌缺血模型，我们还证明了窖蛋白在 EGCg 介导的心脏保护中的作用。此外，发现 EGCg 介导的窖蛋白-1 激活受 Akt/GSK-3β 信号通路调节，可减轻 H2O2 诱导的 H9c2 细胞损伤。
我们的数据表明，窖蛋白作为质膜筏，可能有助于介导 EGCg 的跨膜信号传递。
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