Department of Pharmacology, School of Pharmaceutical Sciences, Shandong University, PR China.
Biol Pharm Bull. 2010;33(3):432-8. doi: 10.1248/bpb.33.432.
A tetramethylpyrazine analogue, CXC195, was synthesized by the Boekelheide reaction, in which the second methyl group of tetramethylpyrazine (TMP) was replaced with (4,4'-fluorine) diphenyl-methyl-1-piperazidine, the active group of flunarizine. We have observed protective effects of CXC195 on vascular endothelial cell survival under oxidative stress in previous study. The aim of the present study was to investigate the effects of CXC195 against apoptosis induced by hydrogen peroxide in human umbilical vein endothelial cells (HUVECs). Accordingly, a biochemical approach to elucidate the apoptotic signal pathways was attempted. HUVECs were exposed to 150 muM H(2)O(2) for 12 h, resulting in an increase of apoptotic cells assessed by the nuclear staining assay and flow cytometry. Mitochondrial membrane potential was detected by retention of rhodamine123. The concentration of free intracellular calcium was determined by fura-2/AM fluorometry. Co-incubation with CXC195 reduced the percentage of apoptotic cells and inhibited the loss of mitochondrial membrane potential and intracellular calcium overload induced by H(2)O(2). Induction of p53, the activation of caspase-3 by H(2)O(2) which accompanying downregulation of bcl-2, was blocked by CXC195. In addition, CXC195 clearly improved phosphorylation levels of the antiapoptotic extracellular signal-regulated kinase-1/2 (ERK1/2) in cells undergoing oxidative damage. Moreover, CXC195 showed stronger effects on inhibition of apoptotic cells and loss of mitochondrial membrane potential and activation of phosphorylated ERK1/2 than TMP. These results suggest that CXC195 prevents reactive oxygen species-induced apoptosis through inhibition of the mitochondria-dependent caspase-3 pathway and ERK pathway to show a better beneficial effect in protecting endothelial cells than TMP.
一种四甲基吡嗪类似物 CXC195 通过 Boekelheide 反应合成，其中四甲基吡嗪（TMP）的第二个甲基被（4,4'-氟）二苯甲基-1-哌嗪取代，这是氟桂利嗪的活性基团。在之前的研究中，我们观察到 CXC195 在氧化应激下对血管内皮细胞存活的保护作用。本研究旨在探讨 CXC195 对人脐静脉内皮细胞（HUVEC）中过氧化氢诱导的细胞凋亡的影响。因此，尝试了一种阐明凋亡信号通路的生化方法。将 HUVEC 暴露于 150μM H2O2 12 小时，通过核染色法和流式细胞术评估凋亡细胞的增加。通过保留 rhodamine123 检测线粒体膜电位。通过 fura-2/AM 荧光法测定细胞内游离钙浓度。CXC195 共孵育可降低凋亡细胞的百分比，并抑制 H2O2 诱导的线粒体膜电位丧失和细胞内钙超载。H2O2 诱导的 p53 以及伴随 bcl-2 下调的 caspase-3 激活被 CXC195 阻断。此外，CXC195 可明显改善氧化应激细胞中抗凋亡细胞外信号调节激酶-1/2（ERK1/2）的磷酸化水平。此外，CXC195 在抑制凋亡细胞、线粒体膜电位丧失和磷酸化 ERK1/2 激活方面的作用强于 TMP。这些结果表明，CXC195 通过抑制线粒体依赖性 caspase-3 途径和 ERK 途径来防止活性氧诱导的细胞凋亡，从而在保护内皮细胞方面比 TMP 表现出更好的有益效果。
