Establishment And Application Of Cell Model With Stable Expression Of HPEPT1

The present study established a MDCK cell model with stable expressing human peptide transporter1(hPEPT1), to verify the expression of the transporter, we investigated the mRNA expression of hPEPTl, as well as the transport function of hPEPT1. We applied this MDCK-hPEPT1cell model to screen potential inhibitors/substrates of PEPT1from Chinese herbs, for the first time, some monomers from Chinese herbs were found to have inhibitory effects on hPEPT1Objective:To establish a cell model with stable-expressed hPEPT1in MDCK, then apply this cell model to screen potential inhibitors/substrates of hPEPT1from Chinese herbs, evaluate their kinetic parameters, with the hope of providing some reference for certain drug combination and finding new drug targeted hPEPTl for inflammatory bowel disease treatment.Methods:Transfected MDCK cells with recombinant plasmid pcDNA3.1(+)-hPEPTl, after G418screening, monoclones were obtained by limiting dilution. Functional monoclones were selected and validated by uptake of fluorescent substrate β-Ala-Lys(AMCA) and Glysar, respectively. The reverse transcription-polymerase chain reaction (RT-PCR) were employed to validate the expression of hPEPT1at transcription level. Monoclone with the best function were used to screen potential ligands, which was achieved by their effects on the uptake of Glysar into monoclone. Among those monomers, aristolochic acid I (AA-I), neochamaejasmin B (NCB) and silibinin (INN) were chosen to measure their inhibition kinetics. In addition, Caco-2cell model was applied to further test their effects on Glysar uptake and transport.Results:Monoclones obtained by limiting dilution were verified by β-Ala-Lys(AMCA) and Glysar, two of them possessed high transport function, the mRNA level of hPEPTl in these two monoclones were significantly higher than in cells transfected with pcDNA3.1(+); classic substrates of PEPTl inhibited the uptake of Glysar into monoclones. With this MDCK-hPEPTl cell model, we found some monomers from Chinese herbs could inhibit the uptake of Glysar. Among them, AA-I, NCB and INN inhibited Glysar uptake in a concentration-dependent manner with an estimated IC50of4.1μmol·L-1,22.4μmol-L-1,250.1μmol-L-1; uptake of AA-I, NCB and INN had no difference between MDCK-hPEPT1cells and MDCK-pcDNA3.1cells; the Km and Vmax value of Glysar uptake were0.75mmol·L-1,0.32nmol-mg protein-1·min-1without inhibitors, whereas when AA-I, NCB or INN existed, the Km and Vmax were0.99mmol·L-1,0.94mmol·L-1,0.81mmol·L-1and0.25nmol-mg protein-1·min-1,0.24nmol-mg protein-1·min-1,0.18nmol-mg protein-1·min-1, respecively. AA-I, NCB and INN were found to mainly affected the Vmax value of Glysar transport while Km value was not significantly changed; A A-I, NCB and INN could also inhibit the uptake and transport of Glysar in Caco-2cells.Conclusions:Cell model with stable-expressed hPEPTl is successfully established, this model can be employed to high throughput screen inhibitors/substrates of PEPTl. With this model, some monomers from Chinese herbs with inhibitory effects on hPEPT1were obtained; AA-1, NCB and INN may be the non-competitive inhibitors of hPEPTl. The above results supply some references for clinical rational medication and finding new drug targeted hPEPT1for Inflammatory bowel disease treatment.