贵州医科大学学报

2020, v.45;No.241(10) 1137-1142

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羊耳菊活性部位对PXR高表达HepG2细胞中CYP3A4蛋白表达的影响
Effect of Active Constituents from Inula Cappa on the Protein Expression of CYP3A4 in HepG2 Cells with High PXR Expression

杨畅;宋菲;何俊奇;王永林;李勇军;兰燕宇;陈一飞;刘亭;
YANG Chang;SONG Fei;HE Junqi;WANG Yonglin;LI Yongjun;LAN Yanyu;CHEN Yifei;LIU Ting;State Key Laboratory of Functions and Applications of Medicinal Plants & Guizhou Provincial Key Laboratory of Pharmaceutics,Guizhou Medical University;Engineering Research Center for the Development and Application of Ethnic Medicine and TCM of Ministry of Education,Guizhou Medical University;Shanghai Center for Drug Evaluation and Inspection;

摘要(Abstract):

目的:探讨羊耳菊活性部位(IC-MAC)对孕烷X受体(PXR)高表达Hep G2细胞中细胞色素P4503A4酶(CYP3A4)蛋白表达的影响。方法:将质粒pc DNA3.1-PXR转化至DH5α感受态大肠杆菌中扩增并提取质粒,采用Fu GENE6转染试剂,将pc DNA3.1-PXR质粒转染至人肝癌Hep G2细胞,以重组PXR为对照,采用Western blot技术测定PXR-HepG2和Hep G2细胞中PXR的表达,并筛选得到高表达PXR的PXR-HepG2细胞模型;将PXR-HepG2细胞和Hep G2细胞分别分为阴性对照组(DMSO组,0.1%)、药物处理组(25、50、100 mg/L IC-MAC)和阳性药物组[PXR激动剂利福平(RIF)组,10μmol/L],分别处理24、48及72 h后,采用Western blot技术测定细胞中CYP3A4蛋白的表达。结果:Western blot结果显示,与Hep G2细胞相比,转染pc DNA3.1-PXR质粒的PXR-HepG2细胞中PXR表达明显上调(P <0.01),证明PXR-HepG2细胞模型构建成功; 25、50及100 mg/L IC-MAC分别作用于PXR-HepG2和Hep G2细胞24、48和72 h后,与DMSO组相比,25、50及100 mg/L IC-MAC均可上调PXR-HepG2和Hep G2细胞中CYP3A4表达(P <0.05),且PXR-HepG2细胞上调幅度较Hep G2细胞更大。结论:PXR高表达的PXR-HepG2细胞中IC-MAC上调CYP3A4蛋白表达的幅度大于Hep G2细胞,提示IC-MAC可能通过PXR来调控CYP3A4蛋白的表达。
Objective: To investigate the effect of main active constituents from Inula Cappa( ICMAC) on the protein expression of cytochrome P450 3 A4 enzyme( CYP3 A4) in HepG2 cells with high expression of pregnane X receptor( PXR). Methods: The pcDNA3. 1-PXR plasmid was transformed into DH5α escherichia coli for the amplification and extraction,and Fu GENE  6 transfection reagent was used to transfect pcDNA3. 1-PXR plasmid into human hepatocarcinoma HepG2 cells. By comparing the recombined PXR,Western blot technology was adopted to detect the expression of PXR in HepG2 and PXR-HepG2,and to screen and obtain the PXR-HepG2 cell model with high PXR expression. The PXR-HepG2 and HepG2 cells were divided into negative control group( DMSO group,0. 1%),drug groups of different IC-MAC concentrations( 25,50,and 100 mg/L IC-MAC) and positive drug group[PXR agonist rifampicin group( RIF group),10 μmol/L],which were treated for 24,48 and 72 h respectively. Then,Western blot was adopted to detect the expression level of CYP3 A4 protein.Results: Western blot experiment results showed that,compared with HepG2 cells,PXR expression was significantly up-regulated in PXR-HepG2 cells transfected with pcDNA3. 1-PXR plasmid( P <0. 01),indicating that the PXR-HepG2 cell model was successfully constructed. After PXR-HepG2 and HepG2 cells were treated with 25,50,and 100 mg/L IC-MAC for 24,48,and 72 h,the results showed that,compared with the negative control group,25,50,and 100 mg/L IC-MAC could all significantly up-regulate the expression of CYP3 A4( P < 0. 05) in PXR-HepG2 and HepG2 cells,and that the expression level of CYP3 A4 protein in PXR-HepG2 cells was significantly higher than that in HepG2 cells. Conclusion: IC-MAC has more effect on up-regulating the expression of CYP3 A4 in PXR-HepG2 cells with high PXR than that in HepG2 cells,indicating that IC-MAC can regulate the expression of CYP3 A4 protein through PXR.

关键词(KeyWords): 催化域;羊耳菊;活性部位;瞬时转染;细胞色素CYP3A4;孕烷X受体
catalytic domain;Inula cappa;active constituents;transient transfection;cytochrome CYP3A4;pregnane X receptor(PXR)

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(U1812403);; 贵州省科学技术厅人才团队项目[黔科合平台人才(2016)5613,5677]

作者(Author): 杨畅;宋菲;何俊奇;王永林;李勇军;兰燕宇;陈一飞;刘亭;
YANG Chang;SONG Fei;HE Junqi;WANG Yonglin;LI Yongjun;LAN Yanyu;CHEN Yifei;LIU Ting;State Key Laboratory of Functions and Applications of Medicinal Plants & Guizhou Provincial Key Laboratory of Pharmaceutics,Guizhou Medical University;Engineering Research Center for the Development and Application of Ethnic Medicine and TCM of Ministry of Education,Guizhou Medical University;Shanghai Center for Drug Evaluation and Inspection;

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DOI: 10.19367/j.cnki.2096-8388.2020.10.004

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