Yamagata Kazuo, Xie Yajie, Suzuki Sayaka, Tagami Motoki
Department of Food Bioscience and Biotechnology, College of Bioresource Sciences, Nihon University (NUBS), Fujisawa, Japan.
Department of Food Bioscience and Biotechnology, College of Bioresource Sciences, Nihon University (NUBS), Fujisawa, Japan.
Phytomedicine. 2015 Apr 15;22(4):431-7. doi: 10.1016/j.phymed.2015.01.011. Epub 2015 Feb 21.
Tumor necrosis factor alpha (TNF-α) promotes the expression of adhesion molecules and induces endothelial dysfunction, a process that can lead to atherosclerosis. Green tea consumption can inhibit endothelial dysfunction and attenuate the development of arteriosclerosis. The purpose of this study was to examine whether epigallocatechin-3-gallate (EGCG) prevents TNF-α-dependent endothelial dysfunction. Here, we compared the regulatory effects of the green tea components EGCG and L-theanine against TNF-α-induced stimulation of adhesion molecule expression and apoptosis induction, which is associated with autophagy. Monocytic cell adhesion to human endothelial cells was measured using a fluorescently-labeled cell line, U-937. Caspase 3/7 activity was examined with a fluorescent probe and fluorescence microscopy. In addition, we analyzed the expression of several genes by RT-PCR. TNF-α-modulation of LC3 and VCAM1 protein levels were investigated by Western blot (WB). TNF-α induced adhesion of U937 cells to endothelial cells, and gene expression associated with adhesion molecules and apoptosis. On the other hand, EGCG and L-theanine inhibited TNF-α-induced adhesion of U937 cells to endothelial cells and inhibited increases in ICAM1, CCL2 and VCAM1 expression. Furthermore, EGCG and L-theanine inhibited TNF-α-induced apoptosis-related gene expression (e.g., CASP9), and caspase activity while inhibiting TNFα-induced VCAM1, LC3A and LC3B protein expression. Meanwhile, treatment of endothelial cells with autophagy inhibitor 3-methyladenine (3-MA) blocked EGCG-induced expression of CASP9. Together, these results indicate that EGCG can modulate TNF-α-induced monocytic cell adhesion, apoptosis and autophagy. We thus conclude that EGCG might be beneficial for inhibiting TNF-α-mediated human endothelial disorders by affecting LC3 expression-related processes.
肿瘤坏死因子α（TNF-α）可促进黏附分子的表达并诱导内皮功能障碍，这一过程可导致动脉粥样硬化。饮用绿茶可抑制内皮功能障碍并减缓动脉硬化的发展。本研究的目的是检验表没食子儿茶素-3-没食子酸酯（EGCG）是否能预防TNF-α依赖性内皮功能障碍。在此，我们比较了绿茶成分EGCG和L-茶氨酸对TNF-α诱导的黏附分子表达刺激以及与自噬相关的凋亡诱导的调节作用。使用荧光标记的细胞系U-937测量单核细胞对人内皮细胞的黏附。用荧光探针和荧光显微镜检查半胱天冬酶3/7活性。此外，我们通过逆转录聚合酶链反应（RT-PCR）分析了几个基因的表达。通过蛋白质印迹法（WB）研究TNF-α对微管相关蛋白1轻链3（LC3）和血管细胞黏附分子1（VCAM1）蛋白水平的调节。TNF-α诱导U937细胞与内皮细胞黏附以及与黏附分子和凋亡相关的基因表达。另一方面，EGCG和L-茶氨酸抑制TNF-α诱导的U937细胞与内皮细胞黏附，并抑制细胞间黏附分子1（ICAM1）、趋化因子配体2（CCL2）和VCAM1表达的增加。此外，EGCG和L-茶氨酸抑制TNF-α诱导的凋亡相关基因表达（如半胱天冬酶9（CASP9））以及半胱天冬酶活性，同时抑制TNF-α诱导的VCAM1、LC3A和LC3B蛋白表达。同时，用自噬抑制剂3-甲基腺嘌呤（3-MA）处理内皮细胞可阻断EGCG诱导的CASP9表达。综上所述，这些结果表明EGCG可调节TNF-α诱导的单核细胞黏附、凋亡和自噬。因此，我们得出结论，EGCG可能通过影响与LC3表达相关的过程，对抑制TNF-α介导的人类内皮疾病有益。
