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背景还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶是在高糖环境下内皮细胞活性氧离子(ROS)产生的重要来源,视黄醇类X受体(RXR)能够抑制高糖诱导下内皮细胞ROS的生成,但对NADPH氧化酶的表达水平的影响尚不清楚。目的探讨高糖干预下血管内皮细胞NADPH氧化酶NOX4、gp91phox和p22phox亚基表达水平的变化及RXR激动剂的影响。方法体外培养人脐静脉血管内皮细胞(HUVECs),以33mmol/L葡萄糖干预,模拟糖尿病患者体内环境,通过流式细胞学方法检测细胞内的ROS水平,采用干扰RNA(siRNA)技术检测NADPH氧化酶NOX4、gp91phox和p22phox亚基在高糖诱导下内皮细胞产生氧化应激反应中的作用,分别应用实时荧光定量PCR和免疫印迹杂交的方法检测NADPH氧化酶各亚基的表达水平。结果高糖处理后HUVECs内ROS的水平显著增加(6.58±0.24对2.47±0.12,P<0.05)。siRNA检测结果显示NOX4、gp91phox和p22phox亚基参与了高糖诱导下HUVECs内ROS的生成过程。高糖处理显著上调NADPH氧化酶NOX4、gp91phox和p22phox亚基的mRNA水平(NOX4:0.390±0.020比0.110±0.006,P<0.05;gp91phox:0.480±0.040比0.090±0.005,P<0.05;p22phox:0.025±0.002比0.005±0.001,P<0.05)和蛋白水平。RXR激动剂9-cis-RA和SR11237抑制高糖诱导下ROS生成,以及NADPH氧化酶NOX4、gp91phox和p22phox各亚基mRNA和蛋白水平的表达。结论RXR激动剂通过下调高糖诱导下内皮细胞NADPH氧化酶的表达,对抗高糖诱导内皮细胞产生的氧化应激反应。
Background Nicotinic acid adenine dinucleotide phosphate (NADPH) oxidase is an important source of reactive oxygen species (ROS) production in endothelial cells under high glucose conditions. Retinol X receptor (RXR) is able to inhibit high glucose Induced endothelial cell ROS production, but the impact of NADPH oxidase expression levels is unclear. Objective To investigate the changes of expression of NADPH oxidase NOX4, gp91phox and p22phox subunits in vascular endothelial cells under high glucose and the effect of RXR agonist. Methods Human umbilical vein endothelial cells (HUVECs) were cultured in vitro. The intramuscular environment of diabetic patients was induced by 33 mmol / L glucose intervention. The level of ROS was measured by flow cytometry. The expression of NADPH oxidase NOX4, gp91phox and p22phox subunits in endothelial cells under high glucose induced oxidative stress response were detected by real-time fluorescent quantitative PCR and Western blot hybridization method to detect the NADPH oxidase subunit expression levels. Results The levels of ROS in HUVECs were significantly increased after high glucose treatment (6.58 ± 0.24 vs. 2.47 ± 0.12, P <0.05). The results of siRNA showed that NOX4, gp91phox and p22phox subunits were involved in the ROS production in HUVECs induced by high glucose. High glucose treatment significantly up-regulated the mRNA levels of NADPH oxidase NOX4, gp91phox and p22phox subunits (NOX4: 0.390 ± 0.020 vs. 0.110 ± 0.006, P <0.05; gp91phox: 0.480 ± 0.040 vs. 0.090 ± 0.005, P <0.05; p22phox: 0.025 ± 0.002 vs 0.005 ± 0.001, P <0.05) and protein levels. RXR agonists 9-cis-RA and SR11237 inhibited the ROS production induced by high glucose and the mRNA and protein levels of NADPH oxidase NOX4, gp91phox and p22phox. Conclusion RXR agonists can prevent oxidative stress induced by high glucose in endothelial cells by down-regulating the expression of NADPH oxidase in endothelial cells.