| Aim To establish a screening platform for ligands of human peroxisome proliferator-activated receptors ( hPPARs ) and to explore novel natural ligands for hPPARs from traditional Chinese medicine monomers with this platform.Methods Total RNA was isolated from human hepatic tissue and cDNAs encoding the ligand binding domain (LBD) of hPPARs were amplified by RT-PCR. Then the products were inserted downstream from the malE gene of the vector pMAL-p2x, which encoded maltose-binding protein (MBP). The recombinant plasmids were transformed into E.coli.TB1 and expressed MBP-PPARsLBP fusion proteins under the optimized induction conditions. The host bacteria were lysed by ultrasonic treatment and centrifuged. The fusion proteins in the supernatant were purified through amylose-resin affinity chromatography, digested by the protease Factor Xa, then separated by amylose-resin affinity chromatography and DEAE-52 anion exchange chromatography for preparation of MBP-PPARsLBD and PPARsLBD with high purity. Biological functions of MBP-PPARsLBD and PPARsLBD were characterized by radioligand binding assays. A screening platform for hPPARs ligands was established by high performance liquid chromatography and ultraviolet detection (HPLC-UV) based on the receptor-ligand binding function. Then, several traditional Chinese medicine monomers were assayed with this platform. The results of the screening studies were verificated by the classical radioligand binding competition assays ( RCBA ). The activation function of ligands was determined through trans-activation reporter gene assays.Results The host bacteria harbouring the recombinant plasmids were induced under the optimized induction conditions ( 0.4 mmol/L IPTG, 6 h inducing time and 30℃incubation temperature ) and the amount of the soluble MBP-hPPARsLBD products harvested was as high as 31% of the total cellular proteins and one liter culture could yield approximately 20 mg soluble recombinant proteins. MBP-PPARsLBD and PPARsLBD showing a single protein band on SDS-PAGE were obtained through the purification procedure. The recovery rates of all three purifed fusion proteins and hPPARsLBD were approximately 70% and 45%, the yields were about 14 mg and 5 mg per liter of culture, respectively. Radioligand binding assays showed that both MBP-PPARsLBD and PPARsLBD had biological functions and MBP did not affect ligand to bind with PPARsLBD in the fusion proteins. The HPLC-UV screening platform was established and the screening assays showed that berberine ( Ber ) and naringenin ( Nar ) could specifically bind to hPPARα, daidzein ( Dai ) to both hPPARαand hPPARγ1, which was consistent with the results from the RCBA. The trans-activation reporter gene assays displayed that hPPARαwas activated potently by Dai ( EC50 = 3.7μmol/L ), Ber ( EC50 = 5.8μmol/L ), and Nar ( EC50 = 5.7μmol/L ), hPPARαby Dai ( EC50 = 2.7μmol/L ), Nar (EC50 = 11.4μmol/L ), and curcumin ( Cur, EC50 = 7.3μmol/L ). Moreover, Dai, Ber, Nar, and polydatin ( Pol ) could activate hPPARδweakly.Conclusions 1) The new strategy using MBP as a tag for preparing and purifying large amount of functional MBP-PPARsLBD and PPARsLBD is a convenient and reliable method. It may be used to easily obtain the other nuclear receptors. 2) The HPLC-UV method is a new, convenient, simple, inexpensive, environmental, effective and rapid method that could be used for rapid screening receptor ligands. 3) It was confirmed that Ber and Nar were natural agonists for hPPARα, Dai was a dual agonist for hPPARαand hPPARγ1. In addition, Cur could activate hPPARγ1, Dai, Ber, Nar, and Pol possessed a certain degree of activation on hPPARδ. The results suggest that Chinese medicine monomer ( such as Dai, Ber, Nar, Pol, and Cur ) could activate hPPARs and highly likely affect the expression of a set of downstream target genes of hPPARs, then display a number of pharmacological effects, such as regulation of lipid and glucose metabolism, anti-tumor, anti-inflammation.
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