李小艳;王小花;黄小强;吴婷婷;许文;吴水生;
LI Xiao-yan;WANG Xiao-hua;HUANG Xiao-qiang;WU Ting-ting;XU Wen;WU Shui-sheng;School of Pharmacy,Fujian University of Traditional Chinese Medicine;Centre of Biomedical Research&Development,Fujian University of Traditional Chinese Medicine;

• 1:福建中医药大学药学院
• 2:福建中医药大学生物医药研发中心

摘要(Abstract)：

利用酵母GAL4 BD-UAS反式激活分析(transactivation assay)系统在哺乳动物细胞中建立以过氧化物酶体增殖物受体的配体结合结构域(PPARs-LBD,包括PPARα,PPARβ和PPARγ)为靶点的体外筛选模型,并应用该模型对泽泻化学成分进行筛选,研究泽泻14种化学成分对PPARs的激活作用。首先构建GAL4 BD-PPARs LBD融合表达质粒p Bind-PPARs-LBD,采用Lipofectamine将表达质粒p Bind-PPARs-LBD与含有UAS:luciferase报告质粒p GL4.35共转染293T细胞。以PPARs的激动剂(PPARα:非诺贝特;PPARβ:L165041;PPARγ:罗格列酮)为阳性对照,通过测定萤火虫荧光素酶活性来验证系统是否构建成功,并用该系统来考察泽泻中14种化学成分对PPARs靶点激活的影响。PPARs的阳性激动剂结果显示系统构建成功,用该系统分析泽泻化学成分结果显示泽泻单体化合物5,6,7,8,13,14对PPARα有激活作用,化合物5,7对PPARβ有激活作用,而化合物6,7,8,12对PPARγ有激活作用,其中化合物12对PPARγ有显著激活能力。该研究在哺乳动物293T细胞中成功构建以PPARα/β/γ配体结合结构域为靶点的体外筛选细胞模型,并成功应用于泽泻PPARα/β/γ的天然激动剂的筛选。
Peroxisome proliferators activated receptors( PPARs) are closely related to human chronic disease,such as diabetes mellitus and the other metabolic diseases. In this study,a cell-based PPARs( PPAR α/β/γ) model was developed for the screening of PPARs agonists from Alismatis Rhizoma( AR). Firstly,293 T cells were transfected with the reconstructed plasmid p Bind-PPAR( α,β,or γ)-LBD and reporter gene p GL4. 35,and the known PPARs agonists were used as the positive control( fenofibrate for PPARα,L165041 for PPARβ,and rosiglitazone for PPARγ). The ability of activation for PPARs was evaluated by analyzing the expression value of luciferase. Afterward,the 14 pure triterpenoids isolate from AR were analyzed on the developed PPARα,PPARβ and PPARγscreening assay method. The results showed that the compounds 5,6,7,8,13 and 14 from AR have the ability of activation for PPARα. The compounds 5 and 7 from AR have the ability of activation for PPARβ. The compounds 6,7,8 and 12 from RA have the ability of activation for PPARγ. In this study,the compound 12 from AR were found to display significant activation on PPARγ for the first time. AR triterpenoids extracts had the ability of activation for PPARα,PPARβ and PPARγ. The results suggested that triterpenoids extracts from AR were PPARα,PPARβ and PPARγ agonists. The results will help to provide reference for clinical application of AR,and establish a model for PPARs on 293 T cell,which can be used to screen and evaluate PPARs natural agonists.

关键词(KeyWords)： 过氧化物酶体增殖物激活受体(PPARs);激动剂;泽泻;三萜成分
peroxisome proliferator-activated receptors;agonist;Alismatis Rhizoma;active triterpenoids ingredients

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(U1205022);; 福建省自然科学基金项目(2014J01352);; 福建省卫生厅青年科研课题(2013-2-55);; 福建省教育厅A类科技课题(JA15242);; 福建中医药大学校管课题(X2014107-学科,X2015011-平台);; 大学生创新创业训练项目(201610393059)

作者(Authors): 李小艳;王小花;黄小强;吴婷婷;许文;吴水生;
LI Xiao-yan;WANG Xiao-hua;HUANG Xiao-qiang;WU Ting-ting;XU Wen;WU Shui-sheng;School of Pharmacy,Fujian University of Traditional Chinese Medicine;Centre of Biomedical Research&Development,Fujian University of Traditional Chinese Medicine;

参考文献(References)：

• [1]Francisco A Monsalve,Radha D Pyarasani,Fernando DelgadoLopez,et al.Peroxisome proliferator-activatedreceptor targets for the treatment of metabolic diseases[J].Mediators Inflamm,2013,2013(4):287.
• [2]Bogna Grygiel-Górniak.Peroxisome proliferator-activated receptors and their ligands:nutritional and clinical implications——a review[J].Nutr J,2014,13:17.
• [3]Daisuke Usuda,Tsugiyasu Kanda.Peroxisome proliferator-activated receptors for hypertension[J].World J Cardiol,2014,6(8):744.
• [4]Gao D,Zhang Y,Yang F,et al.In vitro screening and evaluation of 37 traditional Chinese medicines for their potential to activate peroxisome proliferator-activated receptors-γ[J].Pharmacogn Mag,2016,12(46):120.
• [5]姜慧洁,张晓静,张慧,等.基于PPARs靶标改善胰岛素抵抗的中药活性成分研究进展[J].中国中药杂志,2015,40(22):4355.
• [6]Tian T,Chen H,Zhao Y Y.Traditional uses,phytochemistry,pharmacology,toxicology and quality control of Alisma orientale(Sam.)Juzep:a review[J].J Ethnopharmacol,2014,158:373.
• [7]Rau O,Wurglics M,Dingermann T,et al.Screening of herbal extracts for activation of the human peroxisome proliferator-activated receptor[J].Pharmazie,2006,61:952.
• [8]Xu W,Li T,Qiu J F,et al.Anti-proliferative activities of terpenoids isolated from Alisma orientalis and their structure-activity relationships[J].Anticancer Agents Med Chem,2015,15(2):228.
• [9]许文,罗奋熔,赵万里,等.泽泻降糖活性提取物化学成分研究[J].中草药,2014,45(22):3238.
• [10]许文,徐明涛,赵万里,等.泽泻提取物三萜类成分含量测定研究[J].药物分析杂志,2015,35(2):210.
• [11]Zhao W L,Huang X Q,Li X Y,et al.Qualitative and quantitative analysis of major triterpenoids in Alismatis Rhizoma by high performance liquid chromatography/diode-array detector/quadrupole-time-of-flight mass spectrometry and ultra-performance liquid chromatography/triple quadrupole mass spectrometry[J].Molecules,2015,20:13958.
• [12]罗奋熔,丘建芳,许文,等.泽泻总三萜提取纯化工艺的实验室试验及中试验证[J].中国医药工业杂志,2014,45(8):729.
• [13]Xia Zhining,Lin Yexin,Guo Lixia,et al.Development of a cellbased high-throughput peroxisome proliferator-activated receptors(PPARs)screening model and its application for evaluation of the extracts from Rhizoma Coptis[J].J Asian Nat Prod Res,2013,15(3):225.
• [14]环奕,彭军,王悦,等.非激动剂PPARγ配体的筛选方法建立和应用[J].药学学报,2014,49(12):1658.
• [15]Vázquez-Carrera M.Unraveling the effects of PPARβ/δon insulin resistance and cardiovascular disease[J].Trends Endocrinol Metab,2016,27(5):319.