Kono Mari, Saigo Katsuyasu, Yamamoto Shiori, Shirai Kohei, Iwamoto Shuta, Uematsu Tomoko, Takahashi Takayuki, Imoto Shion, Hashimoto Makoto, Minami Yosuke, Wada Atsushi, Takenokuchi Mariko, Kawano Seiji
Scientific Research, Scientific Affairs, Sysmex Corporation, Kobe, Japan.
Faculty of Pharmacological Sciences, Himeji Dokkyo University, Himeji, Japan.
Clin Exp Pharmacol Physiol. 2016 Oct;43(10):915-20. doi: 10.1111/1440-1681.12612.
Iron-chelating agents, which are frequently prescribed to transfusion-dependent patients, have various useful biological effects in addition to chelation. Reactive oxygen species (ROS) produced by neutrophils can cause pulmonary endothelial cell damage, which can lead to acute lung injury (ALI). We previously reported that deferasirox (DFS), an iron-chelating agent, inhibits phorbol myristate acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP)-induced ROS production in neutrophils, in vitro. Here, we investigate whether DFS inhibits vacuolization in neutrophils and neutrophil extracellular trap (NET) formation. Human neutrophils were incubated with DFS and stimulated with PMA or fMLP. Human neutrophils were separated from heparinized peripheral blood using density gradient centrifugation, and subsequently incubated with DFS. After 10 minutes, neutrophils were stimulated by PMA or fMLP. Vacuole formation was observed by electron microscopy. For observing NET formations using microscopes, immunohistological analyses using citrullinated histone H3 and myeloperoxidase antibodies, and SYTOX Green (an impermeable DNA detection dye) staining, were conducted. NET formation was measured as the quantity of double-stranded DNA (dsDNA), using the AccuBlue Broad Range dsDNA Quantitation Kit. DFS (50 μmol/L) inhibited vacuole formation in the cytoplasm and NET formation. Additionally, 5-100 μmol/L concentration of DFS inhibited the release of dsDNA in a dose-independent manner. We demonstrate that DFS inhibits not only ROS production but also vacuolization and NET formation in neutrophils. These results suggest the possibility of protective effects of DFS against NET-related adverse effects, including ALI and thrombosis.
经常给依赖输血的患者开铁螯合剂，除了螯合作用外，铁螯合剂还有各种有益的生物学效应。中性粒细胞产生的活性氧（ROS）可导致肺内皮细胞损伤，进而导致急性肺损伤（ALI）。我们之前报道过，铁螯合剂地拉罗司（DFS）在体外可抑制佛波酯肉豆蔻酸酯乙酸酯（PMA）或甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）诱导的中性粒细胞ROS生成。在此，我们研究DFS是否抑制中性粒细胞的空泡化和中性粒细胞胞外陷阱（NET）形成。将人中性粒细胞与DFS孵育，并用PMA或fMLP刺激。通过密度梯度离心从肝素化外周血中分离出人中性粒细胞，随后与DFS孵育。10分钟后，用PMA或fMLP刺激中性粒细胞。通过电子显微镜观察空泡形成。为了使用显微镜观察NET形成，进行了使用瓜氨酸化组蛋白H3和髓过氧化物酶抗体的免疫组织学分析以及SYTOX Green（一种不可渗透的DNA检测染料）染色。使用AccuBlue宽范围双链DNA定量试剂盒将NET形成测量为双链DNA（dsDNA）的量。DFS（50μmol/L）抑制细胞质中的空泡形成和NET形成。此外，5 - 100μmol/L浓度的DFS以剂量非依赖性方式抑制dsDNA的释放。我们证明DFS不仅抑制ROS生成，还抑制中性粒细胞的空泡化和NET形成。这些结果表明DFS可能对包括ALI和血栓形成在内的NET相关不良反应具有保护作用。
