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;

• 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
• 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
• 3:上海药品评查中心

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(Authors): 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;

DOI: 10.19367/j.cnki.2096-8388.2020.10.004

参考文献(References)：

• [1]ZHENG X,LI P,LU X. Research advances in cytochrome P450-catalysed pharmaceutical terpenoid biosynthesis in plants[J]. Journal of Experimental Botany,2019,70(18):4619-4630.
• [2]ZHOU L,CUI M,ZHAO L,et al. Potential metabolic drugdrug interaction of Citrus aurantium L.(Rutaceae)evaluating by its effect on 3 CYP450[J]. Frontiers in Pharmacology,2018,9:895.
• [3]曹昌娥．灯盏花素通过PXR及CAR对CYP3A4及CYP2C19的影响及其作用机制研究[D]．大理:大理大学，2017.
• [4]LOLODI O,WANG Y M,WRIGHT W C,et al. Differential regulation of CYP3A4 and CYP3A5 and its implication in drug discovery[J]. Current Drug Metabolism,2017,18(12):1095-1105.
• [5]ZHANG D,WU G D,ZHANG Y D,et al. Effects of digeda-4 decoction on the CYP450 activities in rats using a cocktail method by HPLC[J]. Biomed Research International,2018,2018:1415082.
• [6] WANG J,ZHAI T,CHEN Y. Effects of honokiol on CYP450 activity and transporter mRNA expression in type2 diabetic rats[J]. International Journal of Molecular Sciences,2018,19(3):815.
• [7]RAFFA R B,JR R T,JR J V P. Treating opioid-induced constipation in patients taking other medications:Avoiding CYP450 drug interactions[J]. Journal of Clinical Pharmacy and Therapeutics,2019,44(3):361-371.
• [8]贵州省药品监督管理局．贵州省中药材、民族药材质量标准[M]．贵阳:贵州科学技术出版社，2003.
• [9]巩仔鹏，李梅，熊荻菲菲，等．基于体内外炎症模型研究羊耳菊提取物的抗炎活性[J]．天然产物研究与开发，2017,29(12):2050-2055.
• [10]熊荻菲菲，朱迪，谭丹，等．羊耳菊药材的HPLC指纹图谱研究[J]．中国中药杂志，2015,40(3):480-483.
• [11]巩仔鹏，陈亭亭，侯靖宇，等．基于UHPLC/Q-TOF/MS分析羊耳菊有效组分的入血成分[J]．中国药理学通报，2017,33(11):1605-1610.
• [12]侯靖宇，陆苑，潘洁，等．UPLC-MS法测定羊耳菊中6种成分的含量[J]．天然产物研究与开发，2015,27(11):1917-1921.
• [13]张德骞．鼻康片联合生理海水鼻喷雾剂治疗干燥性鼻炎的临床研究[J]．当代医学，2016,22(36):181-183.
• [14]朱佳佳．针刺联合双羊喉痹通颗粒治疗喉痹30例[J]．中国中医药现代远程教育，2016,14(22):116-117,147.
• [15]刘跃，唐丽，牟景丽，等．菊黄上清含片体外抗病毒及免疫作用研究[J]．中国医院药学杂志，2013,33(20):1673-1677.
• [16]宋菲，杨健，王春，等．Cocktail探针药物法评价羊耳菊对大鼠CYP450酶活性的影响[J]．中国实验方剂学杂志，2019,25(2):60-67.
• [17]LI J,ZHAO J,WANG H,et al. Microrna-140-3p enhances the sensitivity of hepatocellular carcinoma cells to sorafenib by targeting pregnenolone X receptor[J]. Onco Targets and Therapy,2018,11:5885-5894.
• [18]ABE T,SHIZU R,SASAKI T,et al. Functional interaction between pregnane X receptor and yes-associated protein in xenobiotic-dependent liver hypertrophy and drug metabolism[J]. Journal of Pharmacology and Experimental Therapeutics,2019,371(3):590-601.
• [19]TAKESHITA A,TAGUCHI M,KOIBUCHI N,et al. Putative role of the orphan nuclear receptor SXR(steroid and xenobiotic receptor)in the mechanism of CYP3A4 inhibition by xenobiotics[J]. Journal of Biological Chemistry,2002,277(36):32453-32458.
• [20]MOORE L B,GOODWIN B,JONES S A,et al. St. John’s wort induces hepatic drug metabolism through activation of the pregnane X receptor[J]. Proceedings of the National Academy of Sciences of the United States of America,2000,97(13):7500-7502.
• [21]TEBBENS J D,AZAR M,FRIEDMANN E,et al. Mathematical models in the description of pregnane X receptor(PXR)-regulated cytochrome P450 enzyme induction. International Journal of Molecular Sciences,2018,19(6):1785.
• [22]NICOLUSSI S,DREWE J,BUTTERWECK V,et al.Clinical relevance of St. John's wort drug interactions revisited[J]. British Journal of Pharmacology,2020,177(6):1212-1226.
• [23]KAUR P,ROBIN,MEHTA R G,et al. Progression of conventional hepatic cell culture models to bioengineered Hep G2 cells for evaluation of herbal bioactivities[J]. Biotechnology Letters,2018,40(6):881-893.
• [24]GMEZLECHN M J,TOLOSA L,DONATO M T,et al.Upgrading Hep G2 cells with adenoviral vectors that encode drug-metabolizing enzymes:application for drug hepatotoxicity testing[J]. Expert Opinion on Drug Metabolism&Toxicology,2017,13(2):137-148.
• [25]王晓飞，魏璐嫚，王以婷，等．DNA甲基化参与PXR介导的CYP3A4的表达[J]．中国药理学与毒理学杂志，2019,33(10):821-822.
• [26]BARDOWELL S A,STEC D E,PARKER R S. Common variants of cytochrome P450 4F2 exhibit altered vitamin E-{omega}-hydroxylase specific activity[J]. The Journal of Nutrition,2010,140(11):1901-1906.
• [27]KOT M,HADUCH A,PAPP M,et al. The effect of chronic treatment with Lurasidone on rat liver cytochrome P450expression and activity in the chronic mild stress model of depression[J]. Drug Metabolism and Disposition,2017,45(12):1336-1344.

文章评论(Comment)：