银杏叶提取物(EGb761)通过调控AMPK/KLF4途径抑制血管平滑肌细胞表型转化  

Inhibition Effect of Ginkgo biloba Extract(EGb761)on the Phenotypic Switch of Vascular Smooth Muscle Cells through Regulation of APMK/KLF4 Signaling Pathway

作　　者：周静 徐慧敏[3] 黄鑫涛 孔永红 张文才[4] 王永辉 ZHOU Jing;XU Hui-min;HUANG Xin-tao;KONG Yong-hong;ZHANG Wen-cai;WANG Yong-hui(Department of Phamacy,Zhumadian Central Hospital,Zhumadian 463000,China;Department of Cardiovascular Medicine,Zhumadian Central Hospital,Zhumadian 463000,China;Department of Phamacy,The Second Affiliated Hospital of Zhejiang University School of Medicine,Hangzhou 310009,China;Department of Cardiology,First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China)

机构地区：[1]驻马店市中心医院药学部,河南驻马店463000 [2]驻马店市中心医院心内科,河南驻马店463000 [3]浙江大学医学院附属第二医院药剂科,杭州310009 [4]郑州大学第一附属医院心内科,郑州450000

出　　处：《中国药学杂志》2020年第8期604-610,共7页Chinese Pharmaceutical Journal

基　　金：国家自然科学基金项目资助(81500362)。

摘　　要：目的探讨银杏叶提取物(EGb761)对血小板源生长因子(PDGF)诱导的血管平滑肌细胞(VSMCs)表型转化的影响及其机制。方法体外培养原代VSMCs,使用20 ng·mL^-1的PDGF诱导VSMCs表型转化,采用MTT法和划痕实验检测不同浓度(1、10、100μg·mL^-1)EGb761对VSMCs增殖和迁移的作用;采用免疫荧光和Western blot分别检测VSMCs肌丝排列情况和表型转化相关蛋白α平滑肌肌动蛋白(α-SMA)、calopnin、骨桥蛋白(OPN)以及AMPK/KLF4通路蛋白表达的改变。结果与对照组相比,PDGF诱导VSMCs增殖和迁移明显增强,而EGb761呈浓度依赖性抑制PDGF诱导的VSMCs增殖和迁移;与对照组相比,PDGF组细胞肌丝F-actin排列紊乱、荧光减弱;α-SMA、calponin表达减弱,而OPN表达增强;PDGF+EGb761组细胞肌丝F-actin排列较整齐、荧光较强;α-SMA、calponin表达增强,OPN表达减弱,且呈浓度依赖性。此外,与对照组相比,PDGF能明显诱导AMPK磷酸化水平增加和KLF4表达上调,EGb761呈浓度依赖性抑制AMPK/KLF4通路的激活。使用AMPK通路抑制剂compound C抑制AMPK/KLF4通路后,EGb761对PDGF介导的VSMCs表型转化的抑制作用得到加强;反之,使用AMPK通路激活剂AICAR激活AMPK/KLF4通路后,EGb761对介导的VSMCs表型转化的抑制作用得到逆转。结论EGb761通过抑制APMK/KLF4通路拮抗PDGF诱导的VSMCs表型转化。OBJECTIVE To explore the effects of Ginkgo biloba extract(EGb761)on platelet derived growth factor(PDGF)-induced phenotypic switch of vascular smooth muscle cells(VSMCs)and its potential mechanisms.METHODS VSMCs were cultured in vitro,and 20 ng·mL^-1 PDGF was used to induce the phenotypic switch of VSMCs.MTT assay and wound healing assay were performed to determine the effects of various concentration(1,10,100μg·mL-1)of EGb761 on cell proliferation and migration,respectively;immunofluorescence and Western blot assay were used to detect the arrangement of myofilament,the expression of phenotypic proteins includingα-SMA,calopnin and OPN,as well as the protein expression of AMPK/KLF4 signaling pathway.RESULTS Compared with the control group,PDGF significantly promoted the proliferation and migration of VSMCs.However,EGb761 treatment inhibited PDGF-induced cell proliferation and migration in a concentration-dependent manner.Compared with the control group,PDGF treatment induced disordered arrangement of myofilament and reduced the fluorescence intensity of F-actin.In addition,PDGF significantly decreased the expression ofα-SMA and calponin,whrease increased the expression of OPN in VSMCs,when compared with the control group.VSMCs in PDGF+EGb761 group showed well-aligned myofilament and enhanced the fluorescence intensity of F-actin;the expressions ofα-SMA and calponin were increased and OPN was decreased in the PDGF+EGb761 group when compared with the PDGF group.Meanwhile,as compared with the control group,PDGF increased the level of phosphorylated AMPK and the expression levels of KLF4,which was inhibited by the addition of EGb761 in a concentration dependent manner.After inhibition of AMPK/KLF4 signaling pathway with the use of specific AMPK pathway inhibitor compound C,the inhibitory effect of EGb761 on PDGF-mediated phenotypic switch of VSMCs was enhanced;vice versa,the activation of AMPK/KLF4 signaling pathway with AMPK pathway activator AICAR and the inhibitory effect of EGb761 on PDGF-mediated phenotypic

关 键 词：动脉粥样硬化 血管平滑肌细胞 EGB761 表型转化 

分 类 号：R725.6[医药卫生—儿科]