摘要
目的探讨川芎Chuanxiong Rhizoma调节血脑屏障通透性的作用特点,并从紧密连接蛋白、外排蛋白表达、埃兹蛋白/根蛋白/膜突蛋白(ezrin/radixin/mesin,ERM)磷酸化水平变化的角度阐述影响血脑屏障通透性的机制。方法小鼠单次给予川芎提取物(5.8 g/kg)后iv不同相对分子质量(4×10^(3)、4×10^(4)、7×10^(4)、1.5×10^(5))的异硫氰酸荧光素-葡聚糖(fluorescein isothiocyanate-dextran,FITC-dextran),测定给药后0、0.5、1、2、4、6、8 h脑组织中FITC-dextran的荧光强度,定量描述川芎对体内血脑屏障通透性的影响。建立b End.3细胞单培养血脑屏障模型,给予川芎单体成分洋川芎内酯A、洋川芎内酯H、阿魏酸、洋川芎内酯I、藁本内酯、欧当归内酯A、4-羟基-3-丁基苯酞,比较相同作用时间内血脑屏障模型中荧光素钠(fluorescein sodium,Na-F)的表观渗透系数(apparent permeability coefficient,Papp),确定各成分开放血脑屏障的效应。采用Western blotting检测川芎提取物给药前后小鼠脑组织及洋川芎内酯I给药b End.3细胞中闭锁小带蛋白1(zonula occluden-1,ZO-1)、闭锁蛋白(Occludin)、P-糖蛋白(P-glycoprotein,P-gp)、血管内皮细胞钙黏合素(VE-cadherin)、p-ERM/ERM的表达水平。结果川芎提取物给药0.5 h后可显著增加小鼠脑组织中FITC-dextran(相对分子质量4×103)的透过量(P<0.001),作用强度随给药时间延长减弱,但对相对分子质量4×10^(4)、7×10^(4)、1.5×10^(5)的FITC-dextran透过量无影响。在脑组织中,川芎提取物给药0.5 h时下调ZO-1、Occludin蛋白表达(P<0.05),上调P-gp蛋白表达;给药2 h时上调ZO-1、Occludin蛋白表达(P<0.05、0.01),下调P-gp蛋白表达(P<0.01);给药8 h时,ZO-1、Occludin、P-gp的蛋白表达水平与对照组无明显差异。不同浓度(1、10、100μmol/L)的洋川芎内酯I、洋川芎内酯A、洋川芎内酯H、阿魏酸8 h给药时间内显著增加Na-F透过血脑屏障模型的Papp,其中洋川芎内酯I的促透效应最强。洋川芎内酯I(10μmol/L)给药bEnd.3细胞0.5 h时可下调Occludin、ZO-1、p-ERM/ERM的蛋白表达(P<0.01),给药1 h时上调Occludin、ZO-1、p-ERM/ERM的蛋白表达(P<0.01),给药8 h时Occludin、ZO-1、p-ERM/ERM的蛋白表达水平与对照组无明显差异。结论川芎在一定程度上可增加血脑屏障通透性,且该作用具有时效性,其机制与调节Occludin、ZO-1、P-gp的蛋白表达及ERM磷酸化水平有关。
Objective To explore the effect of Chuanxiong(Chuanxiong Rhizoma)on blood-brain barrier permeability,and explain the mechanism of its effect on blood-brain barrier permeability from the point of view of the changes of tight junction protein,efflux protein expression and ezrin/radixin/mesin(ERM)phosphorylation level.Methods After a single administration of Chuanxiong Rhizoma extract(5.8 g/kg),different molecular weights(4×10^(3),4×10^(4),7×10^(4),1.5×10^(5))fluorescein isothiocyanate-dextran(FITCdextran)were injected intravenously,the fluorescence intensity of FITC-dextran in brain tissue was measured at 0,0.5,1,2,4,6,8 h after administration,and the effect of Chuanxiong Rhizoma on blood-brain barrier permeability in vivo was quantitatively described.The blood-brain barrier model was established by single culture of bEnd.3 cells,monomeric components(senkyunolide A,senkyunolide H,ferulic acid,senkyunolide I,ligustilide,levistilide A,4-hydroxy-3-butylphthalide)in Chuanxiong Rhizoma were given,the apparent permeability coefficient(Papp)of fluorescein sodium(Na-F)in a blood-brain barrier model for the same duration of action were compared,and the effect of each component on opening the blood-brain barrier was determined.Western blotting was used to detect the expressions of zonula occluden-1(ZO-1),Occludin,P-glycoprotein(P-gp),VE-cadherin,p-ERM/ERM protein in brain tissue and bEnd.3 cells before and after administration of Chuanxiong Rhizoma and its components.Results After 0.5 h of administration of Chuanxiong Rhizoma extract,it significantly increased the transmittance of FITC-dextran(molecular weight 4×10^(3))in brain tissue of mice(P<0.001),and the intensity of action was weaken with prolonged administration time,but had no effect on the transmittance of FITC-dextran(molecular weight of 4×10^(4),7×10^(4),1.5×10^(5)).In brain tissue,after 0.5 h of administration of Chuanxiong Rhizoma extract,the expressions of ZO-1 and Occludin protein were downregulated(P<0.05),and the expression of Pgp protein was upregulated;After 2 h of administration,ZO-1 and Occludin protein expressions were upregulated(P<0.05,0.01),and P-gp protein expression was downregulation(P<0.01);At 8 h of administration,there was no significant difference in the protein expression levels of ZO-1,Occludin,and P-gp compared with control group.Within 8 h administration time of different concentrations(1,10,100μmol/L)of senkyunolide I,senkyunolide A,senkyunolide H and ferulic acid,Papp of Na-F penetration through the bloodbrain barrier model was significantly increased,with senkyunolide I had the strongest promoting effect.bEnd.3 cells were treated with senkyunolide I(10μmol/L)for 0.5 h,the protein expressions of Occludin,ZO-1 and p-ERM/ERM were downregulated(P<0.01),and the protein expressions of Occludin,ZO-1 and p-ERM/ERM were upregulated at 1 h of treatment(P<0.01).At 8 h of treatment,there was no significant difference in the protein expression levels of Occludin,ZO-1 and p-ERM/ERM compared with control group.Conclusion Chuanxiong Rhizoma can increase the permeability of blood-brain barrier to a limited extent,and the effect is timedependent,and its mechanism is related to the change of protein expression level of Occludin,ZO-1,P-gp and ERM phosphorylation level.
作者
刘明妍
刘力榕
冯梦晗
朱美霞
张银环
闫晓宁
方聪
屈碧琼
贾志鑫
刘洁
肖红斌
LIU Mingyan;LIU Lirong;FENG Menghan;ZHU Meixia;ZHANG Yinhuan;YAN Xiaoning;FANG Cong;QU Biqiong;JIA Zhixin;LIU Jie;XIAO Hongbin(School of Chinese Medicine,Beijing University of Chinese Medicine,Beijing 102400,China;Research Center for Chinese Medicine Analysis and Transformation,Beijing University of Chinese Medicine,Beijing 102400,China;Beijing Research Institute of Chinese Medicine,Beijing University of Chinese Medicine,Beijing 102400,China)
出处
《中草药》
CAS
CSCD
北大核心
2024年第1期138-147,共10页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(81774155)
国家自然科学基金资助项目(82173957)。
作者简介
刘明妍(1996—),女,硕士,研究方向为中药药效物质基础研究。Tel:18810627605 E-mail:mingyan_0415@163.com;通信作者:肖红斌,男,博士,教授,研究方向为中药作用物质及机理解析、中药新药开发。Tel:(010)53911883 E-mail:hbxiao69@163.com。
