Study On Effects And Mechanism Of Luteolin On Intestinal Absorption Of Ceftiofur Based On In Vitro Model

With the continuous expansion of breeding scale,the loss caused by bacterial diseases to duck industry can not be ignored.Ceftiofur,as the first third generation cephalosporin antibiotics for animals,is a commonly used drug for the treatment of duck bacterial diseases due to its broad spectrum and high antibacterial activity.Natural flavonoids have been reported to affect drug transport in the gut and thus promote absorption.However,the effect of luteolin on the intestinal absorption of ceftiofur in ducks has not been reported.Therefore,in this study,the effect and mechanism of luteolin,a flavonoid compound,on the intestinal absorption of ceftiofur and luteolin were investigated by establishing duck evagination capsule model and Caco-2 single-layer cell model,which will provide a theoretical basis for veterinary clinical combined application of ceftiofur and luteolin in the prevention and treatment of bacterial diseases in ducks.In this study,an in vitro model-duck everted gut sac model was established,and the concentration of ceftiofur in intestinal sac fluid of each group was detected by HPLC,and the apparent permeability coefficient(P_app),absorption and absorption rate of ceftiofur were calculated and analyzed to investigate the effects of luteolin on the absorption of ceftiofur intestinal fluid.The results showed that the P_app,absorption and absorption rate of ceftiofur were significantly increased in luteolin group,suggesting that luteolin can promote the absorption of ceftiofur in duck jejunum,and its promoting effect was enhanced with the increase of luteolin concentration.The transport mechanism of ceftiofur and the effect of luteolin on the trans-membrane transport of ceftiofur were further investigated by establishing an isolated cell model,Caco-2monolayer cell model.The results showed that the P_app of ceftiofur was 1.065×10^-6 cm/s,and its efflux ratio（ER）was 1.96.Luteolin could significantly reduce the P_app and ER of ceftiofur,and the MRP2 inhibitor probenecid could significantly reduce the P_app and cumulative transport volume of ceftiofur in Caco-2 cells.This indicated that ceftiofur was absorbed in the intestinal tract in an active transport mode with a moderate absorption degree,which was incomplete.MRP2 transporter was involved in the efflux of ceftiofur,and luteolin could promote the transport of ceftiofur in Caco-2 cells,which was consistent with the test results of ceftiofur in duck intestine.Real-time quantitative PCR and Western Blot were used to detect the m RNA expression and protein expression levels of ceftiofur related transporter MRP2 and tight junction proteins Occludin,Claudin-1 and ZO-1,respectively.The results showed that luteolin significantly down-regulated m RNA and protein expression levels of MRP2,Occludin,Claudin-1 and ZO-1（p<0.05）,suggesting that luteolin can significantly reduce the transcription level and protein expression level of ceftiofur related transporter and tight junction protein in duck jejunum and Caco-2 cells,thus promoting intestinal absorption of ceftiofur and inhibiting its efflux.This study suggests that ceftiofur is absorbed in the gut,in the form of active transport,the degree of absorption medium,as the incomplete absorption material,and exists efflux phenomenon.Luteolin can down-regulate the transcription and expression levels of ceftiofur-related transporters and tight junction proteins,thus inhibiting the efflux of ceftiofur and promoting its transport in the intestinal tract.It will lay the experimental foundation for further study of drug interaction and provide theoretical basis for veterinary clinical combined use of ceftiofur and luteolin in the prevention and treatment of duck bacterial diseases.