基于人孕烷X受体调控CYP3A4转录表达的麦冬皂苷D和麦冬皂苷D'的比较研究  

Comparative study on the effects of ophiopogonin D and ophiopogonin D' on human pregnane X receptor-regulated CYP3A4 transcription

作　　者：黄小燕[1,2] 王宇光[2] 李晗[1,2] 王远[2] 马增春[2] 汤响林[2] 肖成荣[2] 谭洪玲[2] 王怡[1] 高月[2] 

机构地区：[1]天津中医药大学,天津330007 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《中华中医药杂志》2017年第5期2302-2306,共5页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金："十二五"国家科技重大专项(No.2014ZX09304307001-003;No.2015ZX09501004-003-003);国家重点基础研究发展计划(973计划)项目(No.2012CB518402)~~

摘　　要：目的:比较麦冬皂苷D(OP-D)和麦冬皂苷D'(OP-D')量、时、毒、效之间的差异性,明确孕烷X受体(PXR)是否参与OP-D和OP-D'对CYP3A4的转录调控,为中药毒性物质基础及机制研究提供参考。方法:不同浓度的OP-D和OP-D'处理HepG2细胞24h、48h,MTT法检测细胞存活率;瞬时共转染报告基因实验将hPXR表达质粒、CYP3A4报告基因质粒共同转染至HepG2细胞中,比较OP-D(5、10、20、40μmol/L)和OP-D'(0.1、0.25、0.5、1.0μmol/L)对PXR介导的CYP3A4转录调控作用;RT-PCR法比较OP-D和OP-D'对CYP3A4 mRNA的诱导效应差异。结果:OP-D 120μmol/L和OP-D'2μmol/L及以上浓度对细胞存活率产生抑制作用(P<0.01,P<0.001);当共转染体系hPXR表达质粒、CYP3A4报告基因质粒、p LR-TK内参质粒比例为10∶5∶1时,40μmol/L的OP-D和0.5μmol/L的OP-D'诱导CYP3A4报告基因荧光素酶活性至2.17、2.45倍(P<0.01),用空白载体质粒pcDNA3.1代替hPXR表达质粒进行检测时,OP-D和OP-D'对CYP3A4的激活效应消失,阳性对照药利福平对CYP3A4的激活倍数也由原来的4.20倍降至1.27倍(P<0.01);40μmol/L的OP-D和0.5μmol/L的OP-D'对CYP3A4 mRNA诱导效应是对照组的5.12倍和5.48倍(P<0.01)。结论:OP-D和OP-D'的量、时、毒、效差异性较大,PXR参与了OP-D和OP-D'对CYP3A4的转录调控。Objective: To compare the difference of quantity, time, effect and toxic of ophiopogonin D(OP-D) and ophiopogonin D'(OP-D') and investigate whether the pregnane X receptor(PXR) participated in the process of OP-D and OPD' regulating the transcription of CYP3A4 or not in order to provide a reference for basic and mechanism research of Chinese medicine toxic substance. Methods: HepG2 cells were treated by different concentrations of OP-D and OP-D' for 24 hours and 48 hours respectively. Cell survival rate was detected by MTT method. The hPXR gene expression plasmid and CYP3A4 reporter gene plasmid were transfected into HepG2 cells by transient co-transfection of reporter gene experiment. The effects of OP-D(5, 10, 40μmol/L) and OP-D'(0.1, 0.25, 0.5, 1μmol/L) on PXR-mediated transcriptional regulation of CYP3A4 were compared. RT-PCR method was used to compare the difference between OP-D and OP-D' in the inducing effect on CYP3A4 mRNA. Results: According to the results of MTT, OP-D at 120μmol/L and OP-D' at 2μmol/L concentration had inhibitory effect on cell survival rate(P<0.01). Reporter gene screening showed that when the proportion of hPXR, CYP3A4, p RL-TK was 10∶5∶1, OP-D at 40μmol/L increased the CYP3A4 luciferase report gene activity significantly to 2.17-fold compared to the treated group(P<0.01). OP-D' at 0.5μmol/L increased CYP3A4 transcription significantly to 2.45-fold(P<0.01). The effect of OP-D and OP-D' on the activation of CYP3A4 was disappeared when the blank plasmid pcDNA3.1 was used to replace hPXR expression plasmid. The activation of positive control drug rifampicin on CYP3A4 also dropped from 4.20-fold to 1.27-fold(P<0.01). The CYP3A4 mRNA expression was markedly increased 5.12-fold and 2.42-fold by OP-D at 40μmol/L and OP-D' at 0.5μmol/L respectively. Conclusion: The difference of quantity, time, effect and toxic of OP-D and OP-D' were obvious. PXR participated in the process of OP-D and OP-D' regulating the transcription of CYP3A4.

关 键 词：麦冬皂苷D 麦冬皂苷D’ 量-时-毒-效 孕烷X受体 中药安全性评价 

分 类 号：R285[医药卫生—中药学]