A Cell-based High-throughput Screening Assay for Farnesoid X Receptor Agonists

Objective To develop a high-throughput screening assay for Farnesoid X receptor （FXR） agonists based on mammalian one-hybrid system （a chimera receptor gene system） for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. Methods cDNA encoding the human FXR ligand binding domain （LBD） was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain （DBD） of yeast GAL4 of pBIND to construct a GAL4-FXR （LBD） chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD （189-472） chimera expression plasmid. Results After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z＇ factor value of 0.65, Conclusion A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.

Abnormal metabolism of gut microbiota reveals the possible molecular mechanism of nephropathy induced by hyperuricemia

The progression of hyperuricemia disease is often accompanied by damage to renal function.However,there are few studies on hyperuricemia nephropathy,especially its association with intestinal flora.This study combines metabolomics and gut microbiota diversity analysis to explore metabolic changes using a rat model as well as the changes in intestinal flora composition.The results showed that amino acid metabolism was disturbed with serine,glutamate and glutamine being downregulated whilst glycine,hydroxyproline and alanine being upregulated.The combined glycine,serine and glutamate could predict hyperuricemia nephropathy with an area under the curve of 1.00.Imbalanced intestinal flora was also observed.Flavobacterium,Myroides,Corynebacterium,Alcaligenaceae,Oligella and other conditional pathogens increased significantly in the model group,while Blautia and Roseburia,the shortchain fatty acid producing bacteria,declined greatly.At phylum,family and genus levels,disordered nitrogen circulation in gut microbiota was detected.In the model group,the uric acid decomposition pathway was enhanced with reinforced urea liver-intestine circulation.The results implied that the intestinal flora play a vital role in the pathogenesis of hyperuricemia nephropathy.Hence,modulation of gut microbiota or targeting at metabolic enzymes,i.e.,urease,could assist the treatment and prevention of this disease.