淫羊藿苷元的肠道跨膜转运机制研究

Study on transmembrane transport mechanism of icariin in intestine

目的探讨淫羊藿苷元(icaritin,ICT)在肠道内的转运机制,并阐明ICT生物利用度低的主要原因。方法采用体外细胞模型人结肠癌Caco-2细胞及小肠癌LS-180细胞和人药物转运蛋白细胞系[P-糖蛋白(P-glycolprotein,P-gp,又名MDR1)、乳腺癌耐药蛋白(breast cancer resistance protein,BCRP)和多药耐药相关蛋白2(multidrug resistance-associated protein2,MRP2)过表达的狗肾细胞系MDCK-MDR1、MDCK-BCRP、MDCK-MRP2及空白转染的狗肾细胞系MDCK-mock;有机阴离子转运多肽2B1(organic anion transporting polypeptides 2B1,OATP2B1)过表达的人胚胎肾细胞HEK293-OATP2B1及空白转染的人胚胎肾细胞HEK293-mock],应用实时定量荧光PCR、LC-MS/MS联用、放射性同位素示踪等技术,共同研究淫羊藿苷元在肠道的跨膜转运机制。结果Caco-2细胞研究中,ICT在低浓度给药时摄入方向表观渗透系数(Papp)较小,渗透性较弱,存在明显外排,且外排可被BCRP抑制剂明显抑制,不能被P-糖蛋白(P-glycolprotein,P-gp)抑制剂明显抑制,随着给药浓度的增加,外排逐渐减弱,摄入方向Papp逐渐增加,渗透性逐渐增强。通过人药物转运蛋白细胞系研究,ICT可显著抑制BCRP和OATP2B1的转运,对BCRP和OATP2B1转运蛋白的半数抑制浓度(IC50)分别为6.33、31.8μmol/L;同时,ICT是BCRP和OATP2B1的底物。通过LS-180细胞诱导研究表明,ICT不能诱导肠道外排蛋白P-gp、BCRP的表达上调。结论ICT在肠内通过被动扩散和主动转运2种机制吸收,其中被动扩散能力弱,主动转运包括外排转运蛋白BCRP的外排和摄入转运蛋白OATP2B1摄入共同介导,且对肠道外排转运蛋白无诱导表达功能。BCRP的外排对ICT吸收影响有限,这可能与ICT的给药浓度及BCRP的饱和浓度有关。

Objective To explore the transport mechanism of icaritin(ICT) in the intestine and clarify the main reasons for the low bioavailability of ICT.Methods In vitro cell models including Caco-2 and LS-180 cells and human drug transporter-transfected cell models including P-glycolprotein(P-gp,also known as MDR1),breast cancer resistance protein(BCRP),multidrug resistanceassociated protein 2(MRP2) overexpressed in canine kidney cell lines MDCK-MDR1,MDCK-BCRP,MDCK-MRP2,and blank transfected canine kidney cell line MDCK-mock;organic anion transporting polypeptides 2 B1(OATP2 B1) overexpressed human embryonic kidney cells HEK293-OATP2 B1 and blank transfected human embryonic kidney cells HEK293-mock were used.Real-time quantitative fluorescence PCR,LC-MS/MS,radioisotope tracer and other techniques were used to study the transmembrane transport mechanism of ICT in intestinal tract.Results In the study of Caco-2 cell,the apparent permeability coefficient(Papp) of ICT in the direction of intake was small and the permeability was weak at low concentrations,and its efflux could be significantly inhibited by BCRP inhibitors,but not by P-gp inhibitors.With the increase of the concentration of administration,the efflux gradually weakened,the intake direction of Papp gradually increased,and the permeability gradually increased.Through the study of the human drug transporter cell line,ICT could significantly inhibit the transport of BCRP and OATP2 B1,with IC50 values of 6.33 and 31.8 μmol/L,respectively.ICT was the substrate of BCRP and OATP2 B1.LS-180 cell induction study showed that ICT could not induce upregulation of the expression of intestinal efflux protein P-gp and BCRP.Conclusion ICT is absorbed in intestine through passive diffusion and active transport,of which passive diffusion capacity is weak.The active transport is mediated by efflux of BCRP and uptake of uptake transporter OATP2 B1,and has no induction expression function for intestinal efflux transporter.The efflux of BCRP has a limited effect on ICT absorption,which may be related to the dose concentration of ICT and the saturation concentration of BCRP.