摘要
目的研究补阳还五汤有效组分生物碱、苷对凝血酶刺激的血管内皮细胞株黏附分子表达的影响及其信号转导机制。方法(1)体外培养脐静脉内皮细胞株ECV-304细胞,以凝血酶(Thr)刺激,加入原方、生物碱、苷后培养,免疫细胞化学法检测黏附分子PECAM-1(CD31)、ICAM-1(CD54)、E-selectin和PKCα的表达;(2)培养的ECV-304细胞,分别给予Thr、Thr+H7[1-(5-异喹啉磺酰基)-2-甲基哌嗪]、PMA(佛波脂)、PMA+H7、Thr+CATG(组织蛋白酶-G)、Thr+原方、Thr+生物碱、Thr+苷,免疫细胞化学法检测细胞蛋白激酶Cα(protein kinase Cα,PKCα)、磷酸化p38丝裂原活化蛋白激酶(phospho-p38 mitogen-activated protein kinase,p-p38MAPK)、磷酸化细胞外信号调节激酶(phospho-extracellular signal regula-ted kinase,p-ERK)和核因子-κB(nuclear factor-κB,NF-κB)表达的变化。结果(1)凝血酶刺激后黏附分子PECAM-1、ICAM-1和E-selectin增强(P<0.05)。与凝血酶组比较,原方组这3种黏附分子表达降低(P<0.05);生物碱组和苷组PECAM-1、ICAM-1表达降低(P<0.05),E-selectin表达无显著变化;(2)凝血酶可促进ECV-304细胞PKCα的表达(P<0.01)。PKC激活剂PMA刺激细胞后,PKCα表达增强(P<0.01);PKC抑制剂H7作用于PMA刺激的ECV-304细胞后,PKCα表达被抑制;PAR-1抑制剂CATG作用于凝血酶刺激的细胞后,PKCα表达被显著抑制(P<0.05)。原方(50、100 mg/mL)、生物碱(2 mg/mL)和苷(1.25、2.5、5 mg/mL)均可显著抑制凝血酶诱导的PKCα表达(P<0.05);(3)凝血酶刺激后细胞p-p38MAPK及p-ERK表达无变化,加入PMA、H7、CATG后,p-p38MAPK及p-ERK表达亦无变化。原方及有效组分生物碱和苷对p-p38MAPK和p-ERK表达无显著影响;(4)各组NF-κB的表达均无显著变化。结论补阳还五汤可下调凝血酶诱导的ECV-304细胞黏附分子的表达,生物碱和苷可能是方中产生该作用的有效物质。凝血酶激活ECV-304细胞的作用主要是通过PKC活化而介导的,p38MAPK、ERK、NF-κB信号通路并不是凝血酶活化ECV-304细胞的主要信号途径,补阳还五汤及其有效组分生物碱、苷可能通过抑制凝血酶激活PKC这一信号途径而发挥其抗凝血酶活化血管内皮细胞的作用。
Objective To explore the effect and possible cell signal transduction mechanism of active components (alkaloids and glucosides) of Buyang Huanwu Decoction (BHD) on the vascular adhesion molecule (VAM) expression induced by thrombin (Thr). Methods (1) Endothelial cells (ECV-304) were cultured in vitro and stimulated by Thr, then cultured in media containing BHD whole recipe ( BHDw), its alkaloids (BHDa) or glycosides (BHDg), respectively. The expressions of E-selectin, CD31, CD54, and protein Kinase Ca ( PKCα) were detected by immunocytochemical method. (2) Cultured ECV-304 cells were treated with Thr, Thr + 1-(5-isoquinolinesulfonyl)-2-mythylpiperazine dihydrochloride (H7), phorbol myristate acetate (PMA), PMA + H7, Thr +cathepsin-6 (CATG) as well as Thr plus BHDw, BHDa, and BHDg, respectively after then, changes of PKCα, phospho-p38 mitogen-activated protein kinase (p-p38MAPK), phospho-extracellular signal regulated
出处
《中国中西医结合杂志》
CAS
CSCD
北大核心
2009年第5期430-434,共5页
Chinese Journal of Integrated Traditional and Western Medicine
基金
国家自然科学基金资助项目(No.30572301)
教育部科学研究重点项目(No.204100)
湖南省教育厅重点资助项目(No.03A033)
关键词
补阳还五汤
血管内皮细胞
黏附分子
信号转导
Buyang Huanwu Decoction
vascular endothelial cells
adhesion molecule
signal transduction