曲美他嗪抑制自噬减少中性粒细胞胞外核酸网形成

Trimetazidine reduces formation of neutrophil extracellular traps via inhibiting autophagy

目的研究曲美他嗪(trimetazidine TMZ)对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)体外诱导中性粒细胞胞外核酸网(neutrophil extracellular traps,NETs)形成的影响及其与自噬的关系。方法密度梯度离心法提取小鼠骨髓中性粒细胞,ox-LDL体外干预建立NETs诱导模型;采用TMZ、LY294002、Rapamycin干预ox-LDL对NETs的诱导;免疫荧光法观察NETs标志物MPO-DNA的释放;PicoGreen试剂盒定量检测cfDNA/nets含量;Western blot检测髓过氧化物酶(MPO)、自噬相关蛋白Beclin-1、LC3b的表达。结果ox-LDL以浓度-时间依赖的方式刺激中性粒细胞释放MPO-DNA复合物,使上清cfDNA/nets含量明显增高;细胞自噬相关蛋白LC3b、Beclin-1及MPO的蛋白水平亦随ox-LDL浓度增加上调;TMZ预处理能抑制NETs释放,降低LC3b、Beclin-1及MPO的蛋白表达,该结果可被PI3K通路阻断剂LY294002模拟,而用mTOR抑制剂Rapamycin预处理则结果相反。结论ox-LDL以浓度-时间依赖的方式诱导中性粒细胞释放NETs;TMZ可下调中性粒细胞自噬水平抑制ox-LDL对NETs的诱导作用,减少NETs产生。

Aim To study the effects of trimetazidine(TMZ)on theformationof neutrophil extracellular traps(NETs)induced by oxidized low-density lipoprotein(ox-LDL)in vitro and its relationship associated with cell autophagy.Methods The bone marrow neutrophils of mice were extracted by density gradient centrifugation,and NETs induction model was established using ox-LDL.Furthermore,TMZ,autophagy inhibitor LY294002 and autophagy inducer Rapamycin were added to disturb the induction of NETs induced by oxLDL.The production of NETs marker MPO-DNA and the content of cfDNA/nets in the supernatantwere observed.Meanwhile,Western blot was employed to determine the protein expression of myeloperoxidase(MPO),beclin-1 and LC3b in neutrophils.Results Treatment of ox-LDL to adhered neutrophils could lead to the formation of extracellular MPO-DNA and cfDNA/nets generation in a time-and concentration-dependent manner.The protein expression of LC3b,beclin-1 and MPO in neutrophils was up-regulated aftertreatedwith ox-LDL.TMZ pretreatment significantly inhibited NETs release and reduced the protein expression of LC3b,beclin-1 and MPO in neutrophils,which could be simulated by PI3K/AKT pathway blocker LY294002,while AKT/mTOR inhibitor rapamycin preconditioning did the opposite.Conclusions Ox-LDLinduces the formation of NETs in a time-and concentration-dependent manner.TMZ could down-regulate the level of autophagy and neutrophils,inhibit the induction of ox-LDL on NETs and reduce the release of NETs.