Study On The Absorption Mechanism And Transport Properties Of Anthocyanins Based On Monolayer Model Of Caco-2 Cells

Anthocyanins have a wide range of biological effects,and the bioavailability of anthocyanins is a major problem in the study of their biological effects.At present,there are a lot of studies on the absorption and bioavailability of anthocyanins,but the results and conclusions are controversial and divergent.In this study,anthocyanins were used as research objects,the absorption of Caco-2 in intestinal epithelial cells and its mechanism were studied.The effect of anthocyanin structure on its transport and absorption was studied.Furthermore,the effects of long-term exposure on the absorption of anthocyanins were explored.The main research results are as follows:1.A monolayer model of Caco-2 cells was established to study the transmembrane absorption of malvidin-o-glucoside?Mv-3-g?,geranium-o-glucoside?Pg-3-g?,cyanidin-o-glucoside?Cy-3-g?and delphindin-o-glucoside?Dp-3-g?.After 21 days of Caco-2 cell culture,a monolayer intestinal cell model was successfully established.The compactness of the single-layer cell model is high,and the resistance value is maintained at about 995?cm².The apparent permeability coefficient?Papp?of fluorescent yellow is 0.26⁰.69×10^-6 cm/s.The concentration of four anthocyanins below 200?g/mL has no toxic effect on Caco-2 cells.The structure of anthocyanin can affect the absorption of transmembrane,at the same concentration,the absorptivity is:Mv-3-G>Pg-3-G>Cy-3-G>Dp-3-G.But there was no significant difference between the four anthocyanins.The absorption of anthocyanins in Caco-2 cells is in the process of efflux.The two-way absorption was time-dependent.The active and passive transport are involved in the absorption.The transport rate of anthocyanins from AP side to BL side was much higher than that of reverse transport.In the absorption process of Caco-2 monolayer cells,the positive absorption effect is greater than the reverse efflux effect.And it has obvious directionality.2.The anthocyanin transporter was analyzed by specific inhibitor technique and the effect of exposure on the expression of transporter was studied.SGLT1 and GLUT2are the main transporters in the transmembrane transport of cyanidin-o-glucoside.When glucose transporter inhibitor was added,the transport capacity of cyanidin-o-glucoside from AP-BL surface of Caco-2 cell monolayer model was significantly reduced,but there was no significant difference in the transport capacity of BL-AP surface.At the same inhibitor concentration,GLUT2 protein inhibitor showed more obvious inhibition on the transport of cyanidin-o-glucoside.After chronic exposure of Caco-2 cells with 200?g/mL cyanidin-o-glucoside for 24 hours,the expression of SGLT1 protein on Caco-2 cell membrane increased by 1.46 times,and there was no significant difference between the expression of GLUT2 protein and the control group.After 96 hours of exposure,the expression of SGLT1 and GLUT2 protein increased 1.3and 1.7 times respectively.After 24 and 96 hours of exposure,the absorption rate of cyanidin-o-glucoside across the membrane did not increase but decreased,the increase of expression of anthocyanin transporter did not increase the absorption rate of anthocyanin.3.The effect of glucose on anthocyanin uptake by Caco-2 cells was studied,and the main transporter of cyanidin-o-glucoside was elucidated.After pretreatment of Caco-2 cells with 200?g/mL cyanidin-o-glucoside for 24 hours and 96 hours,glucose uptake decreased by 47%and 60%,respectively.At the same time,the longer the exposure time,the lower the glucose uptake rate.Glucose concentration in the range of0.5-10 mmol/L significantly inhibited the uptake of cyanidin-o-glucoside in intestinal cells,and showed a certain concentration dependence.Both transporter inhibitors significantly reduced the uptake of glucose and cyanidin-o-glucoside in intestinal cells.At the same molar concentration,the transporters that play the main role in transport are different.Glucose plays an important role in the process of intestinal cell uptake in SGLT1 transporter,while cyanidin-o-glucoside plays an important role in the process of intestinal cell uptake in GLUT2 transporter.4.The response of cell transporters after 96 hours of chronic exposure to 200?g/mL cyanidin-o-glucoside was studied by means of transcriptome technique.All genes in the control group and the treatment group were compared and analyzed,390differential genes were obtained,including 271 genes with up-regulated expression and119 genes with down-regulated expression,accounting for 69.49%and 30.51%of the total differential genes respectively.These differentially expressed genes are involved in many metabolic pathways.The results showed that cyanidin-o-glucoside could significantly affect the metabolic and cellular processes of Caco-2 cells.This result provides a reference for analyzing the molecular mechanism of cyanidin-O-glucoside acting on Caco-2 cells and tapping its target.