Pashevin Denis O, Nagibin Vasyl S, Tumanovska Lesya V, Moibenko Alex A, Dosenko Victor E
Department of General and Molecular Pathophysiology, Bogomoletz Institute of Physiology, National Academy of Science of Ukraine, Kiev, Ukraine.
Pathobiology. 2015;82(6):290-8. doi: 10.1159/000440982. Epub 2015 Nov 12.
Polymorphic mononuclear neutrophils (PMN) are very important cells participating in nonspecific defense of the organism. Among their well-known functions, the formation of neutrophil extracellular traps (NET) is interesting and potentially dangerous for the mechanisms of other cells. Ubiquitin-dependent proteasomal proteolysis is a very important regulator of all cellular activities, but the role of proteasomal proteolysis in NET formation has not been investigated.
We performed experiments with PMN activated to form NET with phorbol 12-myristate 13-acetate (PMA) and the application of a proteasome inhibitor. We also added activated neutrophils to primary culture of isolated rat neonatal cardiomyocytes with or without anoxia-reoxygenation modeling.
The data obtained show that proteasomes participate in NET formation and proteasome inhibitors facilitate the blocking of the NET program. The percentage of NET after PMA application was 70.8 ± 7.2 and the proteasome inhibitor decreased this amount to 4.7 ± 0.9%. In coculture with cardiomyocytes during anoxia-reoxygenation, this effect prevented cardiac cell death induced by activated PMN. The stimulation of NET formation by PMA in coculture with isolated cardiomyocytes led to an increase in the number of necrotic cardiomyocytes of up to 33.1 ± 12.9% and a corresponding decrease in living cardiomyocytes to 66.9 ± 12.9%. The number of living cardiomyocytes in coculture after incubation with both PMA and proteasome inhibitor was 76.6 ± 13.3% (p < 0.05), and the number of necrotic cardiomyocytes was 23.4 ± 13.3% (p < 0.05).
Proteasome inhibition blocks NET formation and prevents cardiomyocyte necrosis in coculture with activated neutrophils.
多形核中性粒细胞（PMN）是参与机体非特异性防御的非常重要的细胞。在其众多已知功能中，中性粒细胞胞外陷阱（NET）的形成很有趣，且对其他细胞机制可能具有危险性。泛素依赖性蛋白酶体蛋白水解是所有细胞活动的非常重要的调节因子，但蛋白酶体蛋白水解在NET形成中的作用尚未得到研究。
我们用佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）激活PMN以形成NET，并应用蛋白酶体抑制剂进行实验。我们还将活化的中性粒细胞添加到分离的大鼠新生心肌细胞的原代培养物中，有无缺氧复氧模型。
所得数据表明蛋白酶体参与NET形成，蛋白酶体抑制剂促进NET程序的阻断。应用PMA后NET的百分比为70.8±7.2，蛋白酶体抑制剂将此量降低至4.7±0.9%。在缺氧复氧期间与心肌细胞共培养时，这种作用可防止活化的PMN诱导的心脏细胞死亡。与分离的心肌细胞共培养时，PMA刺激NET形成导致坏死心肌细胞数量增加至33.1±12.9%，存活心肌细胞相应减少至66.9±12.9%。用PMA和蛋白酶体抑制剂孵育后共培养中存活心肌细胞的数量为76.6±13.3%（p<0.05），坏死心肌细胞的数量为23.4±13.3%（p<0.05）。
蛋白酶体抑制可阻断NET形成，并防止与活化中性粒细胞共培养时的心肌细胞坏死。
