葛根素对AngⅡ诱导HUVECs细胞组织因子表达的影响及其机制研究  

Influence of puerarin on the expression of tissue factor induced by angiotensin Ⅱ and its mechanism in vascular endothelial cells

作　　者：冉文卓[1] 王宏健[1] 廖晓红[1] 文志斌[1] 胡静[1] 申晓辉[1] 何晓凡[1] 

机构地区：[1]中南大学基础医学院生理学系,湖南长沙410078

出　　处：《中国现代医学杂志》2012年第12期13-18,共6页China Journal of Modern Medicine

基　　金：中南大学硕士学位论文创新项目(No:2011SSXT108)

摘　　要：目的观察葛根素(Pue)对血管紧张素I(IAngII)诱导人脐静脉血管内皮细胞株(HUVECs)组织因子(TF)表达的影响,并探讨其作用机制。方法 HUVECs培养用DMEM完全培养基;一期凝固法测总的细胞促凝活性;TF活性的鉴定用乏Ⅶ血浆和TF单克隆抗体。采用酶联免疫吸附双抗体夹心法(ELISA)测定TF抗原;TFmR NA采用R T-PCR的方法检测。用免疫化学染色观察NF-κB的变化。结果①与对照组相比,不同浓度的AngI(I 10-10～10-6mol/L)促进HUVECs细胞TF活性和mR NA的表达,并具有明显的量效关系(P<0.05);单用Pue(62.5～500 mg/L)不能抑制HUVECs细胞的TF活性(P>0.05);在给予AngⅡ之前30 min用Pue(62.5～500 mg/L)预处理HUVECs细胞,Pue可呈剂量依赖式地抑制AngI(I10-7mol/L)诱导TF PCA,TF抗原和TF mR NA表达增加的作用(P<0.05),在500 mg/L时抑制作用达到最强;500 mg/L Pue抑制AngI(I10-7mol/L)对TF表达的刺激作用也呈时间依赖性,在12 h抑制作用最强。②一氧化氮合酶(NOS)的抑制剂L-NAME可明显地阻抑Pue拮抗AngII促进HUVECs TF PCA和TF mR NA的增加的作用(P<0.05)。③免疫组织化学染色显示HUVECs细胞经AngⅡ处理45 min后,细胞核内NF-κB颗粒明显增多,但经Pue处理HUVECs细胞15 min后,再加入AngII作用45 min,核内棕黄色NF-κB颗粒则明显减少。结论 Pue可抑制AngII诱导HUVECs TF的表达,NO途径参与上述抑制过程。Pue和AngII可能是通过影响NF-κB的活化发挥作用的。【Objective】The purpose of this study was to observe the effects of puerarin on the expression of tissue factor（TF） induced by angiotensin Ⅱ（AngⅡ） in human umbilical vein endothelium derived cell line（HUVECs） and to elucidate the inhibitory mechanism of puerarin on the expression of TF induced by angiotensinII.【Methods】HUVECs were cultured in DMEM.TF activity was determined with one-stage clotting assay measuring total cellular procoagulant activity（PCA）.PCA was recognized as TF activity by FVII deficient plasma and TF monoclonal antibody methods.TF antigen was determined with enzyme linked immunosorbent assay（ELISA）.TF mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction（RT-PCR）.Immunohistochemical analysis was performed to evaluate the activation of NF-κB.【Results】①Compared with the control,a gradual increase in PCA（r=0.9528,P0.05） was observed in HUVECs stimulated with increasing concentration of AngII（10-10～10-6 mol/L）.Puerarin（62.5～500）mg/L alone could not inhibit the expression of TF in HUVECs（P0.05）.When pretreated with puerarin（62.5～500）mg/L,puerarin could inhibit the expression of TF activity,TF antigen and mRNA induced by AngⅡ（P0.05）,and 500 mg/L was the strongest concentration.500 mg/L puerarin decreased TF activity and TF antigen in HUVECs on a time dependent manner,reaching a maximum level after 12 h.②L-NAME alone,which is the inhibitor of NOS,had no marked effects on PCA and TF mRNA in HUVECs,but L-NAME significantly blocked the inhibitory effects of puerarin on TF PCA and mRNA expression induced by AngⅡ（P0.05）.③Immunohistochemical analysis demonstrated that NF-κB translocation from cytoplasm to the nucleus was observed after treatment of endothelial cells with AngII.puerarin inhibited AngII-induced NF-κB translocation.【Conclusion】AngⅡ can induce the expression of TF in vascular endothelial cells and puerarin inhibits this effect at mRNA level.NO pathway participates in the i

关 键 词：葛根素 血管紧张素II 组织因子 血管内皮细胞 细胞内信号转导 

分 类 号：R-33[医药卫生] R-3