李小艳+王小花+黄小强+吴婷婷+许文+吴水生

[摘要]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-PPARsLBD融合表达质粒pBind-PPARs-LBD，采用Lipofectamine将表达质粒pBind-PPARs-LBD与含有UAS：luciferase报告质粒pGL4.35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。

[关键词]过氧化物酶体增殖物激活受体（PPARs）； 激动剂； 泽泻； 三萜成分

[Abstract]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 （PPARα/β/γ） model was developed for the screening of PPARs agonists from Alismatis Rhizoma （AR）. Firstly， 293T cells were transfected with the reconstructed plasmid pBind-PPAR （α，β， orγ）-LBD and reporter gene pGL4.35， 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

PPARγ screening 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 293T cell， which can be used to screen and evaluate PPARs natural agonists.

[Key words]peroxisome proliferator-activated receptors； agonist； Alismatis Rhizoma； active triterpenoids ingredients

doi：10.4268/cjcmm20162121

过氧化物酶体增殖物激活受体（PPARs）是核激素受体超家族中的配体激活转录因子，参与许多與慢性疾病密切相关的临床生理和病理过程，成为代谢疾病（2型糖尿病、高血脂、心血管疾病等）治疗的重要靶点^[1]。其作用机制是PPAR与类维甲酸受体（RXR）形成异源二聚体，再与靶基因启动子上的PPRE序列结合，之后通过配体的激活诱导辅阻遏物蛋白的分离和辅激活物的募集，形成PPAR活化复合物，导致糖脂代谢、脂质平衡、脂肪细胞分化的基因表达发生改变^[2]。……