Establishment Of Cell-Based Screening Systems For Human PPARs Ligand And Agonist Discovery

Objectives:Peroxisome proliferator-activated receptors (PPARs) can mediate the metabolic pathways of a variety of substances and drug treatment effects. In this study, recombinant plasmid phPPARs-IRES2-EGFP and report plasmid ptk-PPRE×3-luc were co-transfected into 293T cells to imitate PPAR signaling pathway and the feasibility of the molecular platform for drug screening based on PPARs were evaluated by reporter gene expression through positive drug interference. This study aims to provide a reliable cell model for screening candidates of active PPARs ligands.Methods:1)Cell culture and plasmids co-transfection:293T cells were cultured in HG-DMEM containing 10% FBS in a humidified atmosphere of 95% air and 5% CO2 at 37℃. The subcultured cells were incubated in 24-well plate at a density of 8×104 cells per well.293T cells were divided into three groups including phPPARa-IRES2-EGFP transfection group, phPPARy-IRES2-EGFP transfection group and pIRES2-EGFP transfection control group. Recombinant plasmid phPPARs-IRES2-EGFP, firefly luciferase-containing report plasmid ptk-PPRE×3-luc and renilla luciferase-containing inner control plasmid pRL-CMV were co-transfected into each group of 293T cells using lipofectamine 2000 when cells were 90-95% confluented.2)Transfection efficiency:36 h after transfection, GFP expression was observed under a fluorescence microscope and the transfection efficiency was measured by flow cytometry (FCM).3)Specific positive drugs intervention of 293T cells co-transfected system:14h after transfection, each transfection group were treated with 1‰DMSO,10-8 mol/L,10-7 mol/L,10-6 mol/L,10-5 mol/L,10-4 mol/L concentration gradient of a positive drug (phPPARα-IRES2-EGFP transfection group was treated with WY14643; phPPARy-IRES2-EGFP transfection group was treated with rosiglitazone), respectively. Dual-Luciferase Reporter Assay System (DLR) was used to detect the samples luciferase activity at 24 h after positive drug intervention, and the ratio of firefly to renilla luciferase luminescence (F/R values) refered to as luciferase expression activity was caculated; moreover,10-5 mol/L of the positive drugs were used to analysis time dependent relationship at the different time points including 8 h,16 h,24 h, 36 h, and 48 h post-intervention.4)Identification of the cell-based drug screening models specificity:the positive drugs were exchanged between phPPARα-IRES2-EGFP transfection group and phPPARy-IRES2-EGFP transfection group in order to verify the specificity of the cell models mentioned above. Furthermore, the positive drugs were replaced by all-trans-retinoic acid (ATRA, a RXRa specific agonist) with the purpose of clarifying whether the cell models could be activated by ATRA.5)RT-PCR detection:PPARs and RXRa mRNA expression levels of co-transfected 293T cells after being intervened by the positive drugs were assessed by real-time fluorescence quantitative PCR.6)Evaluation of utilization ability of the PPARαdrug screening cell-based model: different fibrates (bezafibrate, ciprofibrate and clofibrate) which were potent hypolipidemic agents in clinical application were used to verify the utilization ability of the PPARa drug screening cell model.Results:1)FCM analysis revealed that the transfection efficiency of phPPARα-IRES2-EGFP and phPPARy-IRES2-EGFP transfection group were 68.30% and 67.56%, repectively. GFP were highly expressed in each transfected 293T cells groups under fluorescent microscope.2)F/R values appeared to have an ideal dose-and time-dependent relationship with the positive drugs intervention in co-transfected 293T cells. Dose-dependent analysis results:In the two kinds of hPPARs transfection system, luciferase reporter gene expression level (F/R values) increased with positive drugs concentration, however, higher drug concentration would inhibit the expression (P<0.01); Time-dependent analysis results:In the two kinds of hPPARs transfection system, luciferase reporter gene expression level increased as prolonged time with the 10-5 mol/L of the positive drugs intervention (P<0.01).3)PPARa agonist WY14643 was unable to activate phPPARγ-IRES2-EGFP transfected 293T cells reaction system, whereas rosiglitazone could partly activate phPPARa-IRES2-EGFP transfected 293T cells with response intensity lower than WY14643(P<0.05). In addition, both of hPPARs transfection systems could not be activated by ATRA.4)RT-PCR results:In phPPARα-IRES2-EGFP transfected 293T cells group, hPPARαmRNA expression levels were 4 orders of magnitude higher than control group while hPPARγmRNA expression levels were 2～3 orders of magnitude higher than control group in phPPARγ-IRES2-EGFP transfected group, which demonstrated that the recombinant plasmids were efficiently expressed at mRNA level. RXRαmRNA expression levels were the same and low in three groups.5)As expected, bezafibrate, ciprofibrate and clofibrate responded ideally in the PPARαdrug screening 293T cells model with different response intensity at high dose of drugs (P<0.05).Conclusions:This study successfully developed a 293T cell-based drug screening platform targeting specific nuclear receptors of hPPARαand hPPARγ, which could be used for drug-screening for unknown hPPARs ligands or activators.