Objective To investigate the role of activation of ROS/ALOX5/NLRP3 signaling pathway in hypoxia-induced pro-liferation of pulmonary artery endothelial cells .Methods The human pulmonary arterial endothelial cells (HPAEC) were cultured in normoxia (21％O2 ) and hypoxia (1％O2 ) for 24,48,72 and 96 hours,respectively.At the same time,subsets of reactive oxygen spe-cies (ROS) scavenger,Trolox and ALOX5 inhibitor,Zileuton were set up.The CCK-8 method was used to detect the cell proliferation rate.The change of ROS level in HPAEC under hypoxia was detected by probe H 2-DCFA.Western blot was used to detect the expres-sion of ALOX5,NLRP3 and caspase-1 protein after intervention of hypoxia ,Trolox and Zileuton,respectively.Results HPAEC were significantly proliferated at 48,72 and 96 hours after hypoxia,reaching (135.6±29.4)％,(185.3±21.7)％ and (212.3±39.0)％, respectively.At the same time,intracellular ROS at 24-,48-and 72-hour was increased significantly and return to normal at 96 hour.The protein expressions of ALOX5,NLRP3 and caspase-1 were increased with the prolonged hypoxia time .Pretreatment with Trolox and Zileuton inhibited the expressions of NLRP 3 and caspase-1 in the inflammasome ,and the cell proliferation rate was decreased signifi-cantly.Conclusion ROS/ALOX5/NLRP3 signaling pathway is significantly activated in hypoxia-induced proliferation of HPAEC ,and inhibition of this pathway can significantly reduce hypoxia-induced endothelial cell proliferation .%目的 考察ROS/ALOX5/NLRP3信号通路相关蛋白激活在低氧所致肺动脉内皮细胞增殖中的作用.方法 将人肺动脉内皮细胞分别在常氧状态(21％O2)和低氧状态(1％O2)下培养24、48、72及96小时,同时低氧状态下分别设立活性氧(ROS)清除剂Trolox组和ALOX5抑制剂齐留通组.采用CCK-8法检测细胞增殖率的改变,活性氧探针H2-DCFA检测低氧状态下肺动脉内皮细胞内ROS的变化,Western blot法分别检测低氧状态及加入Trolox或齐留通干预后ALOX5、NLRP3和Caspase-1蛋白表达的变化.结果 肺动脉内皮细胞在低氧作用48、72及96 h后可见增殖显著,分别达(135.6±29.4)％、(185.3±21.7)％及(212.3±39.0)％.同时,细胞内ROS在24、48及72 h可检测到显著增加,96小时后基本恢复正常.ALOX5、NLRP3和Caspase-1蛋白随低氧时间增加而表达增多.预先加入ROS清除剂Trolox和ALOX5抑制剂齐留通均可抑制NLRP3炎性小体相关蛋白NLRP3和Caspase-1的表达,且细胞增殖率也显著降低.结论 ROS/ALOX5/NLRP3信号通路在低氧所致的肺动脉内皮细胞增殖中显著激活,抑制这一通路可显著减少低氧所致的内皮细胞增殖.
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