银杏内酯B对TNFα诱导的人内皮细胞活性氧产生的影响及其机制

1. Department of Cardiology, the First Affiliated Hospital of Dalian Medical University，Dalian 116011，China; 2. Department of Pathophysiology，Basic Medical Academy，Peking Union Medical College, Beijing 100005, China

关键词:  银杏内酯B  肿瘤坏死因子α  内皮细胞  活性氧

Key words:  ginkgo biloba  TNFα  endothelial cells  reactive oxygen species

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p47phox的siRNA作用24 h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 p47phox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p47phox mRNA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， p47phox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p47phox 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）。p47phox 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p47phox亚基的表达。

Abstract:

Objective 〖WTBZ〗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 cells. 〖WTHZ〗Methods 〖WTBZ〗Cultured 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. 〖WTHZ〗Results 〖WTBZ〗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. 〖WTHZ〗Conclusion 〖WTBZ〗GB has a potent inhibitory effect on the ROS production in the TNFα- stimulated endothelial cells, mainly through the inhibition of NADPH oxidase.