摘要
目的探讨晚期蛋白氧化产物(AOPPs)对大鼠胰岛微血管内皮细胞(IMECs)体外增殖、迁移及再血管化功能的影响及可能机制。方法分为空白对照(NC)组、鼠血清白蛋白(RSA)组、不同浓度AOPP组(100、200、300μg/ml)及AOPPs 200μg/ml+NADPH氧化酶抑制剂(Apocynin)组。CCK8试剂盒检测IMECs增殖活性;Transwell小室、Matrigal胶及显微镜下细胞计数法检测AOPPs对IMECs体外迁移及再血管化的影响;化学发光法检测还原型辅酶-Ⅱ(NADPH)氧化酶活性;免疫印迹检测磷酸化丝-苏氨酸激酶(p-Akt)、磷酸化内皮型一氧化氮合酶(p-eNOS)蛋白的表达及ELISA检测一氧化氮(NO)水平。结果 AOPPs可呈剂量、时间依赖性降低IMECs体外增殖能力、迁移能力及再血管化能力,增强细胞内NADPH氧化酶的活性,降低细胞内p-Akt、p-eNOS的表达水平以及细胞内NO的水平(P<0.05)。结论 AOPPs可导致胰岛微血管内皮细胞增殖、迁移及再血管化功能受损,可能与下调p-Akt及p-eNOS的表达水平以及加强NADPH氧化酶介导的eNOS脱偶联有关。
Objective To explore the effect and mechanism of advanced oxidation protein products （AOPPs） on the proliferation, migration activity and revascularization of islet microvascular endothelial cells （IMECs）. Methods The isolated ceils were treated with 100,200,300 μg/ml AOPPs, 200 μg/ml rat serum albumin （RSA） or 200μg/ml AOPPs＋apocynin. The effects of AOPPs on proliferation, migration and revascularization activity of IMECs were tested respectively by CCK-8 method, transwell assay and Matrigel. The activity of NAPDH oxidase was detected by chemiluminescence. The effect of AOPPs on the expression of p-Akt and p-eNOS were evaluated by western blot. The level of NO was determined by ELISA. Results Proliferation, migration and revascularization of IMECs was decreased by AOPPs in dose and timed-dependent manner （ P ~ 0. 05）. AOPPs enhanced the activity of NADPH oxidase（P〈0. 05）and decreased the expressions of p-Akt and p-eNOS and the level of NO in IMECs（P〈 0.05）. Conclusion AOPPs can induce the injury and dysfunction of IMECs. The mechanism may be related to the down-regulation of p-Akt and p-eNOS and increasing the activity of NADPH oxidase mediated eNOS uncoupling.
出处
《中国糖尿病杂志》
CAS
CSCD
北大核心
2017年第9期836-841,共6页
Chinese Journal of Diabetes
基金
国家自然科学基金青年基金(81500623)
广东省科技计划项目(2014A020212489)
关键词
晚期蛋白氧化产物
胰岛微血管内皮细胞
增殖
迁移
再血管化
还原型辅酶-Ⅱ氧化酶
Advanced oxidation protein products
Islet microvaseular endothelial cells
Proliferation
Migration
Revascularization
Nicotinamide adenine dinucleotide phosphate oxidase
