缺氧/复氧后肾小管上皮细胞ICAM-1表达及Apocynin对其干预作用的研究

Study on expression of ICAM-1 and interventional effect of apocynin on renal tubular epithelial cells after hypoxia reoxygenation

目的:研究缺氧/复氧后肾小管上皮细胞氧化应激的变化细胞间黏附分子-1(ICAM-1)蛋白和mRNA的表达及NADPH氧化酶抑制剂Apocynin对其的影响。方法:采用肾小管上皮细胞(HK2)建立缺氧(细胞置于37℃、95%N_2、5%CO_2环境中)/复氧(细胞置于37℃、95%O_2、5%CO_2环境中)细胞模型,设正常对照组,缺氧0.5 h、1 h、2 h/复氧24 h组,及不同浓度(0.05 mmol/L、0.25 mmol/L、0.5 mmol/L)Apocynin干预组。流式细胞术检测细胞内氧化应激水平,逆转录-聚合酶链反应(RT-PCR)法测定细胞ICAM-1 mRNA表达,流式细胞术检测ICAM-1蛋白表达。结果:缺氧/复氧后HK2细胞内氧化应激水平提高,ICAM-1 mRNA和蛋白表达增强,随缺氧时间的延长,细胞内氧化应激水平逐渐升高,ICAM-1mRNA和蛋白表达也逐渐增强;Apocynin呈剂量依赖关系抑制细胞内氧化应激和ICAM-1 mRNA、蛋白表达的上调。结论:缺氧/复氧诱导细胞内氧化应激的产生,诱导ICAM-1表达上调;Apocynin可以通过抑制细胞内氧化应激抑制ICAM-1的上调。

Objective：To investigate the mechanism of expression of intracellular adhesion molecular-1 （ICAM- 1） on renal tubular epithelial ceils after hypoxia/reoxygenation （H/R） exposure, and to evaluate the effects of apocynin on H/R injury. Methods：The renal epithelial ceils （HK2） in culture were divided into following groups.. normal control group,0.5,1,2 hours of H/R group, 0.05, 0.25, 0.5 mmol/L apocynin＋2 hours of hypoxia/ reoxygenation group. In all the groups, the reoxygenation time was 24 hours. The flow cytometry was used to detect the oxidative stress as well as expression of ICAM-1 in HK2. Real time quantitative reverse transcription-polymerase chain reaction（RT-PCR） was used to detect expression of mRNA of ICAM-1. Results： The oxidative stress to HK2 was induced after H/R. The expressions of ICAM-1 protein and mRNA were increasd after H/R. Oxidative stress in HK2 was inhibited by apocynin with a dose-dependent manner. The expressions of ICAM-1 protein and mRNA were inhibited by apocynin in a dose-dependent manner. Conclusions： Hypoxia/reoxygenation induces oxidative stress of HK2 ceils with an increase in the expression of ICAM-1. Apocynin protects HK2 cells against H/R injury by inhibiting oxidation and the up-regulation of ICAM 1.