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

作　　者：杨畅[1] 宋菲[1] 何俊奇 王永林[1] 李勇军[2] 兰燕宇[2] 陈一飞 刘亭[1] 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,Guiyang 550004,Guizhou,China;Engineering Research Center for the Development and Application of Ethnic Medicine and TCM of Ministry of Education,Guizhou Medical University,Guiyang 550004,Guizhou,China;Shanghai Center for Drug Evaluation and Inspection,Shanghai 201203,China)

机构地区：[1]贵州医科大学省部共建药用植物功效与利用国家重点实验室&贵州省药物制剂重点实验室,贵州贵阳550004 [2]贵州医科大学民族药与中药开发应用教育部工程研究中心,贵州贵阳550004 [3]上海药品评审核查中心,上海201203

出　　处：《贵州医科大学学报》2020年第10期1137-1142,共6页Journal of Guizhou Medical University

基　　金：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]。

摘　　要：目的:探讨羊耳菊活性部位(IC-MAC)对孕烷X受体(PXR)高表达HepG2细胞中细胞色素P4503A4酶(CYP3A4)蛋白表达的影响。方法:将质粒pcDNA3.1-PXR转化至DH5α感受态大肠杆菌中扩增并提取质粒,采用FuGENE 6转染试剂,将pcDNA3.1-PXR质粒转染至人肝癌HepG2细胞,以重组PXR为对照,采用Western blot技术测定PXR-HepG2和HepG2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HepG2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HepG2细胞相比,转染pcDNA3.1-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HepG2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HepG2细胞中CYP3A4表达(P<0.05),且PXR-HepG2细胞上调幅度较HepG2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HepG2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 P4503A4 enzyme(CYP3A4)in HepG2 cells with high expression of pregnane X receptor(PXR).Methods:The pcDNA3.1-PXR plasmid was transformed into DH5αescherichia coli for the amplification and extraction,and FuGENE 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),10μmol/L],which were treated for 24,48 and 72 h respectively.Then,Western blot was adopted to detect the expression level of CYP3A4 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 CYP3A4(P<0.05)in PXR-HepG2 and HepG2 cells,and that the expression level of CYP3A4 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 CYP3A4 in PXR-HepG2 cells with high PXR than that in HepG2 cells,indicating that IC-MAC can regulate the expression of CYP3A4 protein through PXR.

关 键 词：催化域 羊耳菊 活性部位 瞬时转染 细胞色素CYP3A4 孕烷X受体 

分 类 号：R969.2[医药卫生—药理学] R285.6[医药卫生—药学]