Al Mamun Bhuyan A, Nüßle Sigrid, Cao Hang, Zhang Shaqiu, Lang Florian
Department of Internal Medicine III, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany.
Department of Molecular Medicine II, Medical Faculty, Heinrich Heine University, Duesseldorf, Germany.
Cell Physiol Biochem. 2017;43(2):492-506. doi: 10.1159/000480476. Epub 2017 Sep 20.
BACKGROUND/AIMS: The 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase inhibitor simvastatin has been shown to trigger apoptosis of several cell types. The substance has thus been proposed as an additional treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the extracellular face of the erythrocyte cell membrane. Signaling contributing to stimulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), induction of oxidative stress, increase of ceramide abundance, and activation of SB203580-sensitive p38 kinase. The present study explored, whether simvastatin induces eryptosis and aimed to shed light on cellular mechanisms involved.
Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant.
A 48 h exposure of human erythrocytes to simvastatin (1 µg/ml) significantly decreased the forward scatter, significantly augmented the percentage of annexin-V-binding cells, significantly increased Fluo3-fluorescence, and significantly enhanced DCFDA fluorescence. Simvastatin tended to increase ceramide abundance, an effect, however, escaping statistical significance. The effect of simvastatin on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of SB203580 (2 µM).
Simvastatin stimulates eryptosis, an effect at least in part due to Ca2+ entry, oxidative stress, and p38 kinase.
背景/目的：3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶抑制剂辛伐他汀已被证明可引发多种细胞类型的凋亡。因此，该物质被提议作为恶性肿瘤的一种辅助治疗手段。与有核细胞的凋亡类似，红细胞可能会发生红细胞程序性死亡，即自杀性红细胞死亡。红细胞程序性死亡的特征包括细胞皱缩和细胞膜磷脂酰丝氨酸外翻至红细胞膜的细胞外表面。导致红细胞程序性死亡刺激的信号包括胞质Ca2+活性（[Ca2+]i）增加、氧化应激诱导、神经酰胺丰度增加以及SB203580敏感的p38激酶激活。本研究探讨了辛伐他汀是否诱导红细胞程序性死亡，并旨在阐明其中涉及的细胞机制。
采用流式细胞术通过膜联蛋白-V结合来定量细胞表面磷脂酰丝氨酸的暴露情况，通过前向散射来定量细胞体积，通过Fluo3荧光来定量[Ca2+]i，通过DCFDA依赖性荧光来定量活性氧（ROS）丰度，并利用特异性抗体来定量神经酰胺丰度。通过上清液中的血红蛋白浓度来估计溶血情况。
将人红细胞暴露于辛伐他汀（1μg/ml）48小时后，前向散射显著降低，膜联蛋白-V结合细胞的百分比显著增加，Fluo3荧光显著增加，DCFDA荧光显著增强。辛伐他汀倾向于增加神经酰胺丰度，然而，该效应未达到统计学显著性。去除细胞外Ca2+和添加SB203580（2μM）可显著减弱辛伐他汀对膜联蛋白-V结合的影响。
辛伐他汀刺激红细胞程序性死亡，该效应至少部分归因于Ca2+内流、氧化应激和p38激酶。
