麝香酮对斑马鱼血脑屏障超微结构及Claudins基因表达的影响  

The effect of muscone on blood brain barrier ultrastructure and expression of Claudins in zebrafish

作　　者：陈怡君[1] 桑洁[2] 赵宝全 刘萍[4] CHEN Yi-jun;SANG Jie;ZHAO Bao-quan;LIU Ping(Department of Pharmacy,Hainan Hospital of Chinese PLA General Hospital,San'ya 572013,China;Tianjin University of Traditional Chinese Medicine,Tianjin 300073,China;State Key Laboratory of Toxicology and Medical Countermeasures,Institute of Toxicology and Pharmacology,Academy of Military Medical Sciences,Academy of Medical Sciences,Beijing 100850,China;Section of Health Economic Management,Hainan Hospital of Chinese PLA General Hospital,San'ya 572013,China)

机构地区：[1]解放军总医院海南医院药剂科,三亚572013 [2]天津中医药大学中药系,天津300073 [3]军事科学院军事医学研究院毒物药物研究所,抗毒药物与毒理学国家重点实验室,北京100850 [4]解放军总医院海南医院卫生经济科,三亚572013

出　　处：《国际药学研究杂志》2020年第12期1121-1128,共8页Journal of International Pharmaceutical Research

基　　金：国家自然科学基金资助项目(81173572)。

摘　　要：目的研究麝香酮对斑马鱼(担尼鱼)血脑屏障(BBB)超微结构及其紧密连接蛋白Claudins基因表达的影响,以阐明麝香酮开启BBB的机制。方法给成年斑马鱼腹腔注射麝香酮溶液,计算半数致死剂量(LD50),利用LD50的1/5剂量进行后续实验;利用透射电子显微镜观察给予麝香酮30 min、1 h、4 h、8 h后,BBB超微结构的变化;选取与BBB密切相关的Clau⁃dins,利用实时荧光定量PCR(RT-qPCR)检测麝香酮作用1 h时的mRNA表达的变化,从中选取对麝香酮较为敏感的基因,进一步对敏感基因在不同时间点(0 min、15 min、30 min、1 h、2 h、4 h)的表达进行检测,验证其时效性。结果斑马鱼腹腔注射麝香酮溶液24 h的LD50是65.20μmol/g体质量,后续实验采用13.04μmol/g体质量的剂量给斑马鱼腹腔注射给药;给予麝香酮后,随时间推移BBB的基膜出现断裂、膨大的现象,紧密连接结构疏松,内皮细胞出现皱缩,给药1 h时最为严重,之后随时间延长BBB超微结构逐渐恢复,给药4 h后BBB的结构逐渐恢复正常;RT-qPCR检测结果表明,给药1 h时BBB相关的Claudins大部分基因mRNA表达下调,其中Claudin-2、-5a、-7b下调最明显(P值分别为0.005、0.002、0.002)。对Claudin-2、-5a、-7b基因不同时间点的表达情况进行检测,结果表明,给药15 min时,各基因均出现了表达的上调,30 min时表达回落,1 h时其表达下调到最低点,然后逐渐上升恢复。其中Claudin-2在4 h时表达略高于正常水平,差异不显著(P>0.05),Claudin-5a在4 h时表达基本恢复至正常水平(P>0.05),Claudin-7b在4 h时表达未恢复至正常水平,有极显著性差异(P<0.01)。结论麝香酮能使Claudins基因表达下调,致使紧密连接结构疏松,加之基膜结构破坏,内皮细胞皱缩,使得BBB的屏障作用减弱,从而开启BBB。基因可逆性的下调以及BBB结构可逆性的改变说明麝香酮对BBB的开启是生理性的,对脑部不会带来病理性损伤。Objective To investigate the effect of muscone on the blood brain barrier(BBB)ultrastructure and the expres⁃sion of the tight junction protein Claudins in BBB tight junctions,in order to elucidate the mechanism of muscone on BBB.Methods Muscone was injected intraperitoneally into adult zebrafish(Danio rerio)for calculating LD50 and 1/5 of LD50 was used for subsequent experiments.Transmission electron microscopy was used to observe the changes in BBB ultrastructure after 30 min,1 h,4 h and 8 h of muscone treatment.After selecting the members of Claudins closely related to BBB,reverse transcription quantitative polymerase chain reaction(RT-qPCR)was performed to examine the changes in the Claudins mRNA expression after 1 h of muscone treatment.The Claudins sensitive to muscone were selected to further examine their mRNA expression at different time points(0 min,15 min,30 min,1 h,2 h,4 h)after drug administration to investigate their time course.Results The LD50 of muscone solution injected intraperi⁃toneally into adult zebrafish for 24 h was 65.20μmol/g body weight,and the dose of 13.04μmol/g body weight for injecting intraperi⁃toneally into adult zebrafish was used in subsequent experiments.After muscone treatment,basement membrane fracture and swell⁃ing,tight junction deformity and endothelial cell shrinkage were all observed in the BBB ultrastructure over time.These changes were most serious at 1 h,and then gradually recovered with time.Four hours after muscone treatment,the BBB ultrastructure gradually returned to its normal state;The results of RT-PCR showed that the mRNA expression for most of the Claudins related to BBB were down-regulated after 1 h of muscone treatment,among which the Claudin-2,-5a and-7b mRNA expressions were most significantly down-regulated(P=0.005,0.002,0.002,respectively).The expression of Claudin-2,-5a,-7b mRNA was detected at different time points.The expression of Claudin-2,-5a and-7b mRNA was up-regulated 15 minutes after muscone administration,decreased from 30 min,reachi

关 键 词：麝香酮 血脑屏障 超微结构 CLAUDINS 

分 类 号：R915[医药卫生—微生物与生化药学] R966[医药卫生—药学]