摘要
目的探讨视黄醇类X受体(RXR)激动剂对高糖诱导人急性单核细胞白血病细胞(THP-1)产生的氧化应激的影响及作用机制。方法体外培养THP-1,以25mmol/L葡萄糖干预,模拟糖尿病患者体内环境,通过流式细胞仪和荧光显微镜的方法检测细胞内的活性氧族水平。分别应用实时荧光定量聚合酶链反应(PCR)和免疫印迹的方法检测THP-1烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶亚基NOX2/gp91phox和p22phox的表达水平,用免疫印迹方法检测蛋白激酶C(PKC)蛋白的磷酸化水平。结果在高糖环境下(葡萄糖终浓度为25mmol/L),THP-1内活性氧族生成明显增加(9.54±0.49比2.3±0.58,P<0.05),NADPH氧化酶NOX2/gp91phox和p22phox亚基的mRNA和蛋白表达水平明显上调。RXR激动剂9顺式维甲酸(9-cis-RA)可明显抑制高糖诱导的活性氧族生成及NADPH氧化酶NOX2/gp91phox和p22phox亚基表达水平的增加幅度,且具有浓度依赖性;10-7mol/L浓度的SR11237(RXR的特异性配体)与等浓度9-cis-RA具有相似的抑制效应。PKC抑制剂LY3335(20μmol/L)明显抑制高糖环境下活性氧族的生成及NADPH氧化酶NOX2/gp91phox和p22phox亚基蛋白表达水平的增加幅度。RXR激动剂9-cis-RA和SR11237可抑制高糖诱导的PKC蛋白的磷酸化。结论 PKC的活化参与了高糖诱导THP-1产生的氧化应激反应,RXR激动剂通过抑制PKC蛋白的活化对抗高糖诱导THP-1产生的氧化应激反应。
Objective To investigate the effects of retinoid X receptor （RXR） agonists on high-glucoseqnduced oxidative stress and the underlying mechanisms in human acute monocytic leukemia cell line （THP-1）. Methods THP-1 were cultured in medium 199 with normal concentration of glucose at 5.5 mmol/L. For high glucose treatment, glucose was increased to 25 mmol/L. Reactive oxygen species （ROS） was detected by flow cytometry and confocal micoroscopy. Phosphorylated protein kinase C （PKC） and the expression level of NADPH oxidase subunits NOX2/gp91phox and p22phox were determined by quantitative realtime PCR and immunoblotting. Results Compared to the normal concentration, high glucose significantly increased ROS production （9.54 ± 0.49 vs 2.3 ±0.58, P〈0.05） as well as the expression of NOX2/gp91phox and p22phox subunits. Treatment of THP-1 with 9-cis- RA [RXR natural agonist） resulted in a significant inhibition （in concentration-dependent manner） of high-glucose- induced ROS production and the expression of NOX2/gp91phox and p22phox subunits. SRl1237 （RXR-specific ligand） at 10 7 mol/L showed the same effect as 10-7 mol/L of 9-cis-RA on high-glucose-induced ROS production and the expression of NOX2/gp91phox and p22phoX subunits. PKC inhibitor LY3335 at 20 tzmol/L demonstrated a similar effect as RXR agonists on high-glucose-induced oxidative stress. Furthermore, RXR agonists rapidly inhibited high-glucose-induced activation of protein kinase C. an upstream activator of NADPH oxidase. Conclusion PKC wasinvolved in the high-glucose-induced oxidative stress in THP-1. RXR agonists inhibited high-glucose-induced oxidative stress through depressing PKC activity in THP-1 cells.
出处
《中华高血压杂志》
CAS
CSCD
北大核心
2012年第10期956-961,共6页
Chinese Journal of Hypertension
基金
国家自然科学基金青年基金资助项目(30900586)
