摘要
目的探讨银杏内酯B(GB)对TNFα刺激的人脐静脉内皮细胞(HUVECs)中活性氧(ROS)产生的影响及其机制。方法将预先培养好的HUVECs分组:正常对照组;TNFα刺激组;GB预处理组:分别用GB(25 mg/L),GB(50 mg/L),GB(100 mg/L)预处理细胞1 h后再用TNFα刺激24 h;质粒转染组:用转染p47phox的siRNA作用24h后再用TNFα刺激24 h。用小分子RNA干扰(siRNA)技术消除人脐静脉内皮细胞的NADPH氧化酶p47phox亚基;用分子探针2,7-DCF测定各组细胞内ROS的产生量;用RT-PCR、细胞免疫组化及Western blotting等方法检测处理后各组细胞的p47phoxmRNA和蛋白的表达。结果 TNFα刺激使细胞内ROS的产生量较对照组增加了155.4%(P=0.003);GB(25 mg/L),GB(50 mg/L),GB(100 mg/L)分别使TNFα诱导的HUVECs内ROS的水平降低了9.2%(P=0.157),35.4%(P=0.014),48.0%(P=0.005)。TNFα作用细胞24 h后,p47phox的mRNA表达水平较对照组增加了212.8%(P=0.009),蛋白表达增加了156.2%(P=0.001);GB预处理使TNFα诱导的p47phox mRNA表达水平降低了43.6%(P=0.021),蛋白表达水平降低了53.0%(P=0.002)。p47phox的siRNA完全阻断TNFα诱导ROS的产生。结论 GB能够抑制内皮细胞中TNFα诱导的ROS的产生,主要机制是通过抑制NADPH氧化酶p47phox亚基的表达。
Objective To investigate the effect of ginkgo biloba （GB） on generation of ROS and the mechanism involved in the tumor necrosis factor α - stimulated human endothelial ceils. Methods Cuhured HUVECs were divided into normal group; TNFα stimulation group; GB pretreatment group： HUVECs were stimulated 24 h by TNFα after pretreatment 1 h by GB （25 mg/L）, GB（50 mg/L）, GB（ 100 mg/L） ; Plasmid transfection group： HUVECs were stimulated 24 h by TNFα after pretreatment 24 h by p47^phox siRNA. The siRNA expression vector for p47^phox was constructed and used to block the NADPH oxidase in the HUVECs. The intracellular ROS production was detected by using 2,7 - dichlorofluorescin diacetate as probe. The mRNA and protein expression of the p47^phox was determined by using semiquantitative RT -PCR, immunocytochemistery analysis and western blotting analysis in each group after treatment. Results TNFα stimulation caused ROS output increased by 155.4% of control（P = 0. 003 ） ; GB was able to reduced the production of ROS in a dose -dependent manner, GB（25 mg/L）, GB（50 mg/L）, GB （100 mg/L） made the TNFα- induced ROS output decreased 9.2% （P = 0. 157）,35.4 % （P = 0. 014）,48.0% （P = 0. 005）respectively. TNFα stimulation caused p47^phox mRNA increased by 212.8% of control（ P = 0. 009 ） , and also increased p47^phox protein expression by 156.2% of control （ P = 0. 001 ） ; Pretreatment with GB attenuated TNFα - induced p47^phox mRNA by 43.6% （P =0. 021 ） and protein by 53.0% （P =0.002）. Knock down of the p47^phox subunit for NADPH oxidase by siRNA abolished the production of ROS. Conclusion GB has a potent inhibitory effect on the ROS production in the TNFα - stimulated endothelial cells, mainly through the inhibition of NADPH oxidase.
出处
《大连医科大学学报》
CAS
2013年第4期320-324,共5页
Journal of Dalian Medical University
基金
辽宁省教育厅科学研究一般项目(L2011155
L2012321)
