Construction And Application Of PPARγ Agonist High-throughput Screening Assay Model

Peroxisome proliferator-activated receptor y(PPARy), which plays an important role in lipid and glucose metabolism, inflammation and tumors, is a member of the nuclear receptor superfamily of ligand-inducible transcription factors. It is expressed in a broad range of cells including adipocyte, macrophages, liver and lung cells, which can store fat. Activation of PPARy by agonists can promote adipogenesis and adipocyte differentiation, improve insulin resistance and tissue fibrosis, display beneficial effects in controlling inflammation and regulate tumor development. With the development of high-throughput drug screening technology, target or target signaling pathway based drug screening gradually become one of the most effective way for drug development. Therefore, identification of specific agonists of PPARy signaling by high-throughput screening will provide more candidate compounds which might create new ways for the treatment of angiocardiopathy diseases.We developed a PPARy agonist high-throughput screening assay model system, applied it for natural products screening, and made a preliminary exploration to the candidate compounds. Through the optimal selection of PPARy constitutive activation cells and the ratio adjustment of PPARy response element PPRE, we got the drug screening model cell, CLPPD. A ratio of 23 PPARy response element PPRE and entire PPARy expression element were chosen, which made the luciferase reporter genes finally match the Chang Liver cell lines, and the stable cell was found out with well elements stability by DNA sequencing, Western Blot analyses and continuous passages assay. Then we screened more than 2500 natural products, and got five lead compounds with the activation of PPARy, especially the 15# compounds. For further study, by MTT method and fluorescence activity tests, we found that 15# compound had nearly no cytotoxicity and a 200% increase ration of PPARy activation under 15 μM concentration compared with control. Furthermore, 15# compound can improve the expression of PPARy in the CLPPD and THP-1 cells as shown by Western Blot and RT-PCR analyses.In conclusion, we successfully developed a PPARy agonist high-throughput screening assay model for more than 2500 natural products screening, got five lead compounds, and made a preliminary exploration to the 15# lead compound which was confirmed with the significant activation of PPARy. Therefore, it confirmed the effectiveness of this drug screening cell model and provide technological support and theoretical basis for the further development of PPARy agonist for angiocardiopathy diseases.