Study On Mechanism Of Transport And Metabolism Of Theaflavis In Caco-2 Cell Lines

Theaflavins are generated by the oxidation of epimerized catechins.In black tea,theaflavins are primarily composed of four monomers:theaflavin(TF),theaflavin-3-gallated(TF3G),theaflavin-3'-gallated(TF3'G)and theaflavin-3,3'-digallated(TFDG).Known as major naturally functional components,theaflavins have substantial health benefits,such as reducing the risks of cardiovascular,diabetes and cancer.Our study aimed to figure out the transport mechanism of theaflavins in intestine by establishing Caco-2 monolayer model.This would provide a theoretical basis for the application of theaflavins in pharmaceutical and health care products.The main work and conclusions were listed as follows:1.Stability and cytotoxicity of theaflavinsTo gain the cell morphology,we imaged Caco-2 cells differentiating in culture.At 21days postconfluency,Caco-2 cells were closely arranged and spidle-shaped,forming an integrated cell monolayer.At the same time,the transepithelial electrical resistance(TEER)values of monolayers were more than 500?·cm² and the ratio of alkaline phosphatase activity of AP side to BL side was more than 5.5,indicating that the monolayers were well-prepared for intestinal transport of theaflavins.We found that the solvent and its p H value had important influence on the stability of theaflavins.Theaflavins were extremely unstable at p H 7.4 no matter in HBSS solution or in DMEM medium.While at p H 6.0,theaflavins exhibited a high stability with more than 60%recovery after incubated in HBSS solution for 24h.By comparison,the theaflavins incubated in DMEM medium at p H 6.0 showed a poor stability with 50%recovery of TF and less than 20%recovery of TF3G,TF3'G and TFDG after 2h.The stability of theaflavins followed the order of TF>TFDG>TF3G?TF3'G.It is inferred that the galloyl group on the 3 or 3'position of C ring was the cause of structural instability.Additionally,the cytotoxicity of theaflavins was determined by CCK-8 method.Results showed that the cytotoxicity of theaflavins was in the ascending order of TF3'G>TFDG>TF3G>TF.The safe concentration of theaflavins was choosen at 200?mol/L as the viability of Caco-2 cells was more than 80%.2.Theaflavins transport in Caco-2 cellsThe variation of theaflavins bioavailability was due to the different transport mechanism of theaflavins in Caco-2 cells.The bidirectional transport of TF was essentially in a time-dependent manner with no apparent saturation.With increasing incubation time,the bidirectional transport of TF3G and TFDG increased initially and then decreased with a saturation point at 3h and 2h respectively.The effect of incubation time on bidirectional transport of TF3'G was not remarkable.Similarly,the bidirectional transport of TF was sustainably increased in a concentration-dependent manner with the concentration raising from 100 to 500?mol/L.While the didirectional transport of other three theaflavin monomers were all saturated with concentration.The influx and efflux amounts of theaflavins were in the order of TF>TFDG>TF3G>TF3'G,which was consistent with the order of their stability,suggesting the structural stability of theaflavins was positively correlated with their bioavailability.All the theaflavins could penetrate the Caco-2 cell monolayer but their apparent permeability coefficient(P_app)was less than 1×10^-6cm/s,which means a poor oral bioavailability.Compared to TF,the P_app(BL-AP)/P_app(AP-BL)values of TF3G,TF3'G and TFDG were all larger than 1.5,by which we speculated that theaflavins transferred across Caco-2 monolayers by active transport.Furthermore,we also determined the accumulation of theaflavins in Caco-2 cells after 2h-incubation.Only TF3G was detected with 1.67?g/mg protein.The cellular accumulation of the other three theaflavin monomers was not detected.Results suggested that the cellular accumulation was also considered as one reason of low bioavailability of TF3G.3.Mechanism of efflux transport of theaflavins in Caco-2 cellsIn this study,the efflux transporters were found to be positively related to decreasing the bioavailability of theaflavins.To explore the mechanism of low bioavailability of theaflavins in small intestine,the effect of p-glycoprotein(P-G),multidrug resistance associated proteins(MRP1,MRP2,MRP3)and breast cancer resistance protein(BCRP)on the transport of theaflavins was investigated.Initially,verapamil(inhibitor of P-G),MK-571(inhibitor of MRPs)and Cs A(inhibitor of P-G and BCRP)was used to study whether the efflux transporters influenced the absorption of theaflavins.Results showed that verapamil,MK-571,Cs A and FTC could significantly decrease the IC₅₀ values of theaflavins to Caco-2,suggesting an imporving effect of efflux transporters on cytotoxicity of theaflavins.Moreover,verapamil,MK-571,Cs A and FTC could remarkably increase the influx transport of theaflavins and slightly decreased the efflux transport of theaflavins in varying degrees.Strikingly,an obvious efflux amount of TF and TF3G mediated by P-G indicated that P-G has stronger affinity with TF and TF3G.From the results it is deduced that the reduction of theaflavin absorption is owing to the exocytosis of P-g,MRPs and BCRP.4.Effects of theaflavins on the expression of efflux transporters in Caco-2 cellsTheaflavins not just were the substrates of efflux transporters,but also produced influence on the expression of transporters.Western blot analysis revealed that theaflavins increased the protein expression of P-G,MRP1,MRP2,MRP3 and BCRP with noticeable influence.We also found that TF3G showed the strongest up-regulation of P-G,MRPs and BCRP at protein levels.The localization of efflux transporters in Caco-2 cells was investigated by immunofluorescence.P-G,MRP2 and BCRP were mainly localized to the membrane with limited intracellular staining.MRP1,however,was found intracellular and peri-nuclear with no appearant plasma membrane staining.The location of MRP1 in Caco-2 cell allows it to come into indirect contact with theaflavins that are excreted by intestine.Furthermore,the effect of theaflavins on the gene expression of P-G,MRPs and BCRP was also analyzed.Real-time PCR revealed that the effects of theaflavins on m RNA levels of P-G,MRP1,MRP2,MRP3 and BCRP vaired with incubation time.Within a 2h-incubation period,theaflavins significantly increased m RNA levels of MRP1and BCRP,followed by a slight down-regulation after 4h.With the exception of TF3G,other three theaflavin monomers decreased m RNA level of MRP2 within 24h.As for MRP3 and P-G,theaflavins played an important role in promoting transcriptional level.5.Metabolism of TF3G and TF3'G in Caco-2 cellsFinally,as one reason of poor bioavailability,the metabolism of theaflavins was also discussed.The metabolites of TF3G and TF3'G in Caco-2 cells were identified by LC-TOF-MS.Results found that the major metabolites of TF3G and TF3'G were TF and GA.Other metabolites were in extremely minute quantities possibly including sulfation and glucuronidated metabolites as well as small molecular acids like benzoic acid.