活性氧介导脂多糖联合三磷酸腺苷诱导的人肺动脉内皮细胞炎症小体活化
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江苏省呼吸病临床医学研究中心


Lipopolysaccharide combinied with adenosine triphosphate induces inflammasome activation in human pulmonary artery endothelial cells by reactive oxygen species
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    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 domain-containing 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-

    Abstract:

    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 DCFH-DA 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 N-acetylcysteine. Conclusion: High level ROS plays an important role in LPS combined with ATP-induced NLRP3 inflammasome activation as well as apoptosis of HPAECs.

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王再亮,孔 辉,曾晓宁,刘汶睿,闫晓培,王艳丽,解卫平,王 虹.活性氧介导脂多糖联合三磷酸腺苷诱导的人肺动脉内皮细胞炎症小体活化[J].南京医科大学学报(自然科学版),2015,(7):968-974

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  • 收稿日期:2015-02-17
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  • 在线发布日期: 2015-07-12
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