摘要
目的 :探讨脂多糖(lipopolysaccharide,LPS)联合三磷酸腺苷(adenosine triphosphate,ATP)对人肺动脉内皮细胞(human pulmonary artery endothelial cells,HPAECs)NOD样受体热蛋白结构域相关蛋白3(Nod-like receptor pyrin domaincontaining protein 3,NLRP3)炎症小体活化的影响及其相关机制。方法 :LPS联合或不联合ATP建立HAPECs炎症损伤模型;CCK-8法测定细胞活力;ELISA法检测细胞上清白细胞介素(interleukin,IL)-1β及IL-18含量;Western blot法评价半胱氨酸天冬氨酸蛋白酶-1(caspase-1)、p-p38及p-p65水平;DCFH-DA荧光探针法观察细胞内外活性氧(reactive oxygen species,ROS)变化;Annexin V/PI标记流式细胞术检测细胞凋亡。结果:LPS单独作用对细胞活力无显著影响,联合ATP后细胞活力显著下降,细胞上清IL-1β及IL-18含量增加,caspase-1、p-p38及p-p65表达水平上调,细胞内外ROS升高,细胞凋亡率升高;ROS清除剂乙酰半胱氨酸可抑制上述效应。结论:LPS联合ATP激活HPAECs内NLRP3炎症小体、介导细胞凋亡与ROS水平升高密切相关。
Objective:To investigate whether lipopolysaccharide(LPS) combined with adenosine triphosphate(ATP) activates Nod-like receptor pyrin domain-containing protein 3(NLRP3) inflammasome in human pulmonary artery endothelial cells(HPAECs), and the underlying mechanism. Methods: HAPECs were stimulated by LPS with or without ATP to establish inflammation damage model.Cell vitality was assessed by cell counting kit-8. The levels of IL-1β and IL-18 in supernatant were analyzed by ELISA. The expressions of caspase-1, p-p38 and p-p65 were determined by Western blot. Reactive oxygen species(ROS) was detected by DCFHDA fluorescent probe. Cell apoptosis was evaluated by annexin V and PI staining assay. Results: LPS alone had no effects on HPAECs. By combined with ATP, LPS significantly inhibited cell viability. The levels of IL-1β and IL-18 in supernatant, the expressions of caspase-1, p-p38 and p-p65 in cytoplasm, the concentrations of intracellular and extracellular ROS as well as cell apoptosis were up-regulated in HPAECs activated by LPS combined with ATP. These effects were inhibited by ROS scavenger Nacetylcysteine. Conclusion: High level ROS plays an important role in LPS combined with ATP-induced NLRP3 inflammasome activation as well as apoptosis of HPAECs.
出处
《南京医科大学学报(自然科学版)》
CAS
CSCD
北大核心
2015年第7期968-974,共7页
Journal of Nanjing Medical University(Natural Sciences)
基金
江苏省呼吸病临床医学研究中心