银杏叶提取物对活化血小板诱导HCAECs氧化应激的作用及其机制  

Effects of Ginkgo biloba extract on production of ROS in human coronary artery endothelial cells induced by activated platelets

作　　者：苟学蕊 李忠东[2] 王兆浩 史尔兰 王建昌[4] 

机构地区：[1]空军总医院老年病科,北京100142 [2]空军总医院药学部,北京100142 [3]北大医疗鲁中医院神经外科,山东淄博255400 [4]空军总医院

出　　处：《空军医学杂志》2016年第4期246-251,共6页Medical Journal of Air Force

基　　金：首都医学发展科研基金(SF200JII06)

摘　　要：目的利用活化血小板诱导血小板刺激人冠状动脉内皮细胞(human coronary artery endothelial cells,HCAECs),建立离体氧化应激模型。探讨不同剂量的银杏叶提取物(Ginkgo biloba extract,EGb)对细胞内NADPH氧化酶4(NOX4)及Rho A的表达及对细胞内活性氧(reactive oxygen species,ROS)的产生所形成的影响。方法用内皮细胞培养基特供胎牛血清(加入内皮细胞生长添加剂)于37℃、5%CO2细胞培养箱中培养细胞,用第6代细胞进行实验。将HCAECs随机分为5组:生理盐水对照组,活化血小板组,4、40、400μg/ml EGb实验组。除对照组外,其他4组细胞用2×108/ml的活化血小板刺激12 h后,实验组细胞再分别用不同剂量的EGb干预12 h。用DCFH-DA探针检测细胞内ROS的产生,用荧光显微镜观察;用Western Blot技术检测细胞内NOX4和Rho A的表达。结果荧光显微镜观察结果显示,活化血小板刺激HCAECs后,与生理盐水对照组相比,ROS产生明显增加(P<0.05);与活化血小板组相比,EGb对ROS产生的抑制作用呈剂量相关(P<0.05)。Western Blot结果显示,与生理盐水对照组相比,活化血小板刺激HCAECs后,NOX4和Rho A的表达量增加(P<0.05);与活化血小板组相比,EGb对NOX4表达的抑制作用呈剂量相关性(P<0.05),对Rho A表达有抑制作用(P<0.05),但不呈剂量相关性。结论 EGb能抑制体外培养的活化血小板诱导的HCAECs内ROS、NOX4和Rho A的表达,EGb治疗冠状动脉粥样硬化性心脏病的机制可能是通过协同抑制NOX4和Rho A的表达,从而使ROS表达量减少,抑制细胞内的氧化应激反应。ObjectiveTo establish a model of oxidative stress invitro via HCAECs induced by activated-platelets in order to explore whether different dosages of Ginkgo biloba extract （EGb） might synergistically suppress reactive oxygen species （ROS） produced in HCAECs by regulating the expression of NOX4 and RhoA in HCAECs.MethodsHCAECs stressed with activated platelets 2＆#215;108/ml, were divided into 4 groups： activated platelets group, 4, 40, 400μg /ml EGb groups. In addition, HCAECs served as the control group. EGb （4, 40 or 400μg/ml） was treated for 12 h. ROS in HCAECs was measured with a DCFH-DA probe and observed by fluorescence microscopy. The expression of NOX4 and RhoA was examined by Western Blot.ResultsThe results of fluorescence microscopy showed that in HCAECs, intracellular ROS was markedly increased （P〈0.05）, after stimulation of activated platelets, compared with HCAECs control group. Compared with activated platelets group, EGb inhibited ROS in HCAECs in a dosage dependent manner （P〈0.05）. By Western blot analysis, we found that cowpared with HCAECS control group, the expression of NOX4 and RhoA in HCAECs stresscd by activated platelets was markedly enhanced （P〈0.05 ）. Compared with activated platelet group, EGb reduced the expression of NOX4 in a dosage dependent manner （P〈0.05）.ConclusionEGb can attenuate the expression of ROS, NOX4 and RhoA in HCAECs induced by activated platelets in vitro. That the mechanism of EGb in the treatment of coronary atherosclerotic heart disease may be correlated with the inhibition of ROS production by suppressing NOX4 and RhoA expression.

关 键 词：动脉粥样硬化 活化血小板 活性氧 NADPH氧化酶4 RHOA 

分 类 号：R714.252[医药卫生—妇产科学]