脱嘌呤/脱嘧啶核酸内切酶1在脂多糖介导肺微血管内皮细胞氧化反应中的调控作用

Modulation of apurinic/apyrimidinic endonuclease on oxidative stress reaction of pulmonary microvascular endothelial cells induced by lipopolysaccharides

目的探讨脱嘌呤/脱嘧啶核酸内切酶1(APE1)在脂多糖介导肺微血管内皮细胞(PMVECs)氧化反应中的调控作用。方法将体外培养人PMVECs分成4组:对照组(未加脂多糖的培养细胞)及各剂量脂多糖组(分别予以0.1、1、10μg/ml剂量脂多糖刺激培养细胞)。分别在脂多糖刺激6、12、24 h检测细胞内活性氧及一氧化氮的水平并进行比较。同时利用携带目的基因慢病毒载体转染PMVECs,将其分成对照组,脂多糖组,GFP组及APE1组。对照组为未加脂多糖的培养细胞,脂多糖组为1μg/ml剂量脂多糖刺激的培养细胞,GFP组为1μg/ml的脂多糖刺激培养慢病毒空载体转染的细胞,APE1组为1μg/ml的脂多糖刺激培养携带APE1基因慢病毒转染的细胞。在12 h检测细胞内活性氧、一氧化氮的水平以及细胞APE1的表达情况。结果与对照组比较,各剂量组脂多糖刺激6、12、24 h后一氧化氮及活性氧分泌明显上升(P均<0.05),且同一剂量组随着时间的推移,一氧化氮及活性氧分泌的比较,差异均具有统计学意义(P均<0.05)。在转染PMVECs12 h后,脂多糖组APE1的表达明显低于对照组,而APE1组细胞APE1的表达较脂多糖组显著提高(P均<0.05)。与对照组相比,脂多糖组能够诱导PMVECs的一氧化氮及活性氧生成显著增加,而APE1组能够一定程度抑制一氧化氮及活性氧的产生(P均<0.05)。结论 APE1可通过抑制一氧化氮及活性氧的分泌从而减轻脂多糖介导PMVECs的氧化应激反应。

objective To investigate the modulation of apurinic/apyrimidinic endonuclease （APEI） on oxidative stress reaction of pulmonary microvascular endothelial cells （PMVECs） induced by lipopolysaceharides （LPS）. Methods PMVECs were cultured in vitro and randomly divided into 4 groups： control group （Not polysaccharide of cultured ceils）, LPS group （polysaceharide of cultured ceils with 0.1, 1, 10 μg/ml, respectively）. The levels of reactive oxygen species （ROS） and NO were measured at 6, 12, 24 h after LPS stimulation. Meanwhile, the PMVECs were transfected with lentiviruses and randomly divided into 4 groups： control group （Not polysaccharide of cultured cells）, LPS group （1 μg/ml polysaccharide of cultured cells）, GFP group （lentivirus vector transfected empty cells with 1 μg/ml polysaccharide） and APE1 group （APE1 gene transfected lentivirus cells with 1 μg/ml polysaccharide）. The levels of ROS, NO and APE1 expression cultured after 12 h were detected and compared. Results Compared with the control group, the levels of ROS and NO in the PMVECs induced by different dose of LPS for 6, 12, 24h respectively were increased obviously （all P〈 0.05）, and at the same dose group, the levels of ROS and NO showed significant differences with the time lapse （all P〈 0.05）. After 12 h of the transfection, the APE1 expression in the LPS group was lower than those in the control and APE1 groups （all P〈0.05）. The levels of ROS, NO in the LPS group after stimulated with 1 μg/ml LPS for 12 h were higher than those in the control and APE1 groups （all P〈 0.05）. Conclusion The APE1 can inhibit the levels of ROS and NO, and thereby relieve the oxidative stress reaction of PMVECs induced by LPS,