ABCG1在肿瘤坏死因子α诱导的氧化应激中的机制研究

ABCG1 inhibits TNF-α-induced oxidative stress by regulating NADPH oxidase and superoxide dismutase

摘要:

目的  探讨三磷酸腺苷结合盒转运体G1（ABCG1）在肿瘤坏死因子α（TNF-α）诱导的氧化应激中的作用及可能的机制。方法  人脐静脉内皮细胞被特异性ABCG1 siRNA或ABCG1过表达质粒转染或使用LXR（肝X受体）激活剂T0901317预处理，随后给予肿瘤坏死因子（TNF-α）干预12 h。采用DCFHDA-
AM（2’7’-二氯荧光双乙酸盐）荧光探针检测细胞内活性氧簇（ROS）的水平，分光光度仪测量还原型烟酰胺腺嘌呤二核苷酸磷（NADPH）氧化酶活性，实时荧光定量聚合酶链反应法（qRT-PCR）和Western blot检测内皮细胞NADPH氧化酶亚型非吞噬细胞氧化酶4（NOX4）表达及超氧化物歧化酶（SOD）的表达。结果  ABCG1表达上调抑制TNF-α诱导的氧化应激，同时抑制促氧化应激的NADPH氧化酶的活性和NOX4的表达，促进抗氧化的SOD表达。相反，ABCG1表达下调进一步诱导ROS的产生，诱导NADPH氧化酶的活性和NOX4的表达，抑制SOD1表达。结论  ABCG1通过调节NADPH氧化酶/SOD抑制TNF-α诱导的氧化应激。

关键词:

三磷酸腺苷结合盒转运体G1;氧化应激;还原型烟酰胺腺嘌呤二核苷酸磷氧化酶;超氧化物歧化酶

Abstract:

Objective To explore the role of ATP-binding cassette sub-family G member 1 (ABCG1) in oxidative stress production induced by tumor necrosis factor α (TNF-α) and its possible mechanisms. Methods Human umbilical vein endothelial cells (HUVECs) were transfected with specific ABCG1 siRNA or ABCG1 overexpression plasmid or pretreated with liver X receptor agonist T0901317, then were cultured with TNF-α for 12 hours. Intracellular reactive oxygen species (ROS) levels were measured using 6-carboxy-2, 7-dichlorodihydrofluorescein diacetate, diacetoxymethyl ester (CDCFHDA-AM) fluorescence and nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) activity was measured by spectrophotometer. Real time PCR and Western blot were employed to measure the expression of Nox4, one of NADPH oxidase subunit and the expression of superoxide dismutase (SOD). Results ABCG1 upregulation inhibited TNF-α-induced oxidative stress. Furthermore, the activity of NADPH oxidase and the expression of Nox4 were also suppressed by ABCG1 overexpression, but the expression of antioxidant SOD was promoted. Conversely, downregulation of ABCG1 by ABCG1 siRNA both increased the ROS production and promoted the NADPH oxidase activity and Nox4 expression. However, the expression of SOD1 was inhibited. Conclusions The results suggest that ABCG1 attenuates TNF-α-induced oxidative stress by regulating NADPH oxidase and SOD.

Key words:

adenosine triphosphate binding cassette transporter G; oxidative stress; reduced nicotinamide adenine dinucleotide phosphate oxidase; superoxide dismutase