中性粒细胞胞外陷阱网通过促进STAT3磷酸化 诱导内皮细胞凋亡参与下肢动脉再狭窄

neutrophil extracellular traps; endothelial cell; apoptosis; neointima formation; restenosis of lower extremity artery; mice

关键词:  中性粒细胞陷阱网  内皮细胞  凋亡  内膜增生  下肢动脉再狭窄  小鼠

Key words:  neutrophil extracellular traps  endothelial cell  apoptosis  neointima formation  restenosis of lower extremity artery  mice

摘要:

目的通过体内体外实验探索中性粒细胞陷阱网（neutrophil extracellular traps, NETs）参与下肢动脉再狭窄发生、发展的分子机制。 方法建立导丝损伤血管内膜增生的ApoE-/- C57BL/6小鼠为再狭窄组，同时建立相同入路但不使用导丝扩张血管者为假手术组，每组4只小鼠。观察两组小鼠NETs相关蛋白表达情况，使用DNase I 10mL/（kg·d-1）进行干预，观察其对内膜增生及STAT3激活的影响，并进行统计学分析。体外刺激中性粒细胞提取NETs，刺激内皮细胞（endothelial cells, ECs），观察ECs的凋亡水平以及STAT3的磷酸化水平；最后在体外敲减ECs的STAT3，观察NETs下游STAT3磷酸化及其对ECs凋亡的影响。 结果与假手术组相比，再狭窄造模组NETs相关蛋白和STAT3的磷酸化水平显著上调；DNase Ⅰ治疗后可以缓解内膜增生，且下调STAT3磷酸化的水平。在体外NETs能上调ECs中STAT3的磷酸化水平同时促进ECs的凋亡，而敲减STAT3可以缓解NETs诱导的ECs的凋亡。 结论NETs可以通过促进STAT3的磷酸化促进ECs凋亡，促进血管损伤后内膜增生，参与下肢动脉再狭窄的发生、发展。

Abstract:

ObjectiveTo explore the molecular mechanism of neutrophil extracellular traps(NETs) involving in the occurrence and development of arterial restenosis in lower limbs through in vitro and in vivo experiments. MethodsThe restenosis model was established by guide-wire injury on ApoE-/- C57BL/6 mice, and a sham operation was performed in sham group. The expression of NETs-related proteins and p-STAT3 was detected in two groups. The effects of DNase Ⅰ[10mL/(kg·d-1)] on neointima formation and STAT3 activation were observed. At the same time, neutrophils were stimulated in vitro and NETs were extracted. The endothelial cells(ECs) were stimulated with the extracted NETs, and the apoptosis of ECs was observed and the expression of p-STAT3 was detected. The level of STAT3 in ECs was knocked down in vitro to observe the effect on the activation of STAT3 downstream and the NETs-induced apoptosis of ECs. ResultsCompared with sham group, NETs-related proteins and phosphorylation level of STAT3 in restenosis model group were significantly up-regulated. DNase Ⅰ treatment alleviated neointima formation and down-regulate the level of STAT3 phosphorylation. NETs promoted the apoptosis of ECs and up-regulated the phosphorylation level of STAT3 in ECs, while knocking down STAT3 inhibited the phosphorylation of STAT3 and apoptosis of ECs induced by NETs. ConclusionNETs can promote apoptosis of ECs through STAT3 pathway, promote neointima formation and participate in the occurrence and development of restenosis of lower extremity arteryafter vascular injury.