摘要
目的探讨高级氧化蛋白产物(AOPPs)对大鼠胰岛微血管内皮细胞(IMECs)凋亡的影响及可能机制。方法以体外分离培养的大鼠IMECs为研究对象,用不同浓度AOPPs(100、200、300μg/ml)干预不同时间(12、24、48及72h)。荧光染色及流式细胞术观察AOPPs对IMECs细胞凋亡的影响;DHE荧光探针检测细胞内活性氧簇(ROS)水平;Western blot检测凋亡相关蛋白的表达及ELISA检测凋亡蛋白酶(Caspase-3)及Caspase-9的活性。结果 AOPPs在体外可呈剂量、时间依赖性的诱导IMECs凋亡[其中,AOPPs 200μg/ml干预48h凋亡率为(23.48±4.69)%],增加细胞内ROS产生[AOPPs 200μg/ml组ROS荧光强度为(58.43±2.71)],与对照组比较差异有统计学意义(P<0.05);减少抗凋亡蛋白Bcl-2、增加促凋亡蛋白Bax的表达,提高下游Caspase-3及Caspase-9的活性(分别为对照组的2.5倍和3.0倍)(P<0.05)。结论 AOPPs可诱导胰岛微血管内皮细胞凋亡,其机制与还原型辅酶-Ⅱ(NADPH)氧化酶介导的氧化应激(OS)有关。
Objective To explore the effect and mechanism of advanced oxidative protein products （AOPPs） on the apoptosis of islet microvascular endothelial cells （IMECs）. Methods The rat IMECs cultured in vitro were randomly divided into control group and AOPPs‐treated groups（AOPPs 100 ,200 , 300 μg/ml treated for 12 ,24 ,48 ,72 h ,respectively）. The effects of AOPPs on IMECs apoptosis were tested by fluorescent staining and flow cytometry. The ROS level was detected by DHE fluorescent probe. The activity of apoptotic protease caspase‐3 and caspase‐9 were determined by ELISA. The expression of apoptotic related proteins were evaluated by Western blot. Results Apoptosis of IMECs was induced by AOPPs in a dose and time‐dependent manner. The apoptotic rate of 200 μg/ml AOPPs‐treated cells for 48 h was （23.48 ± 4.69 ）% . The DHE‐sensitive ROS generation of the AOPPs 200 μg/ml group was significantly higher than control group（58.43 ± 2.71） （P〈0.05）. Exposure to AOPPs for 48 h caused a significant increase in the expression of the pro‐apoptotic protein bax and a decrease in the expression of anti‐apoptotic protein Bcl‐2（P〈0.05）. Also ,AOPPs‐treatment enhanced the activity of apoptotic protease caspase‐3 and caspase‐9 by 2.5 and 3.0 fold（P〈 0.05） respectively as compared with control group. Conclusion AOPPs can induce the apoptosis of IMECs. The mechanism may be related to NADPH oxidase‐mediated oxidative stress （OS）.
出处
《中国糖尿病杂志》
CAS
CSCD
北大核心
2015年第8期751-755,共5页
Chinese Journal of Diabetes
基金
国家自然科学基金(81270966)
广东省自然科学基金(S2012010009494)
广东省药学会科学研究基金(2013X07)
关键词
高级氧化蛋白产物
胰岛微血管内皮细胞
还原型辅酶-Ⅱ氧化酶
氧化应激
凋亡
Advanced oxidative protein products （AOPPs）
Islet microvascular endothelial cells（IMECs）
NADPH oxidase（NOX）
Oxidative stress（OS）
Apoptosis
