NETosis调控心肌细胞自噬促进小鼠急性心肌梗死的作用机制研究

Mechanism of NETosis Regulating Cardiomyocyte Autophagy to Promote Acute Myocardial Infarction in Mice

目的探究小鼠急性心肌梗死(acute myocardial infarction,AMI)与中性粒细胞外陷阱相关死亡(neutrophil extracellular traps-osis,NETosis)调控心肌细胞自噬的相关性。方法C57BL/6小鼠通过冠状动脉左前降支结扎法构建小鼠AMI模型。小鼠原代心肌细胞通过氧糖剥夺(oxygen glucose deprivation,OGD)处理构建心肌细胞损伤体外模型。PMA(NETosis诱导剂)/DNasⅠ(NETosis抑制剂)处理中性粒细胞,取上清液处理OGD诱导的心肌细胞,雷帕霉素(rapamycin,Rap)处理OGD诱导的心肌细胞。TTC染色检测心肌梗死面积;酶联免疫吸附试验(enzyme-linked immunoadsordent assay,ELISA)检测血清cTnI水平;TUNEL染色检测细胞凋亡;ELISA检测中性粒细胞上清液中NETosis标志物水平,Western blot法检测相关蛋白表达水平。结果TTC染色显示,与假手术组比较,模型组小鼠的心肌梗死面积显著增加,血清中cTnI水平显著升高(P<0.05),心肌细胞凋亡增多,Beclin-1和LC3-Ⅱ/LC3-Ⅰ水平显著升高,p62蛋白表达水平显著降低。相较于对照组,OGD条件下细胞凋亡和cleaved caspase-3的表达水平显著升高(P<0.05)。与对照组比较,PMA组中性粒细胞上清液中NETosis标志物MPO-DNA、MPO、NE水平显著升高;PMA+DNasⅠ组相较于单纯PMA处理组,心肌细胞凋亡水平显著下降;Beclin-1、LC3-Ⅱ/LC3-Ⅰ水平显著降低,p62蛋白表达水平显著升高;相较于PMA+DNasⅠ组,Rap处理能够增强Beclin-1和LC3-Ⅱ/LC3-Ⅰ水平,抑制p62水平(P<0.05),同时也显著逆转了由PMA+DNasⅠ诱导的细胞凋亡降低。结论中性粒细胞NETosis可通过调控心肌细胞自噬促进小鼠AMI,可为AMI治疗药物的研发提供新的方向和理论依据。

Objective To explore the correlation between acute myocardial infarction(AMI)and neutrophil extracellular traps-osis(NETosis)in the regulation of cardiomyocyte autophagy in mice.Methods The mouse model of AMI was established in C57BL/6mice by ligation of the left anterior descending coronary artery.Mouse primary cardiomyocytes were treated with oxygen glucose deprivation(OGD)to build an in vitro model of cardiomyocyte injury.Neutrophils were treated with PMA(NETosis inducer)/DNasⅠ(NETosis inhibitor),the supernatant was taken to treat OGD-induced cardiomyocytes,and rapamycin(Rap)was used to treat OGD-induced cardiomyocytes.The myocardial infarction area was detected by TTC staining;serum cTnI level was detected by enzyme-linked immunoadsordent assay(ELISA);cardiomyocytes apoptosis was detected by TUNEL staining;NETosis marker levels in neutrophil supernatant were detected by ELISA,and related protein expression levels were detected by Western blot.Results TTC staining showed that compared with the sham-operated group,the myocardial infarction area of the mice in the model group was significantly increased,the level of cTnI in serum was significantly increased(P<0.05),the apoptosis of cardiomyocytes was increased,and the levels of Beclin-1 and LC3-Ⅱ/LC3-Ⅰwere significantly increased,the p62 protein expression level was significantly decreased.Compared with the control group,the expression levels of apoptosis and cleaved caspase-3 were significantly increased under OGD conditions(P<0.05).Compared with the control group,the levels of NETosis markers MPO-DNA,MPO,and NE in the neutrophil supernatant in the PMA group were significantly increased;compared with the PMA-treated group the apoptosis level of cardiomyocytes in the PMA+DNasⅠgroup was significantly decreased;the levels of Beclin-1 and LC3-Ⅱ/LC3-Ⅰwere significantly decreased,and the protein expression level of p62 was significantly increased;compared with the PMA+DNasⅠgroup,Rap treatment could enhance the levels of Beclin-1 and LC3-Ⅱ/LC3-Ⅰ,and inhibit the level of p62(P<0.05).It also significantly reversed the decrease in apoptosis induced by PMA+DNasⅠ.Conclusion Neutrophil NETosis can promote AMI in mice by regulating cardiomyocyte autophagy,which can provide a new direction and theoretical basis for the research and development of therapeutic drugs for AMI.