黄诺马苷在MDCK单层细胞模型上的转运机制分析
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摘要
目的:研究黄诺马苷在马丁达比犬肾上皮(MDCK)单层细胞模型上的吸收转运特性。方法:利用噻唑蓝(MTT)比色法考察黄诺马苷对MDCK细胞的毒性,使用Millicell-ERS-2型细胞电阻仪检测MDCK单层细胞模型的电阻值,考察黄诺马苷的质量浓度、给药时间以及钠-葡萄糖协同转运蛋白(SGLTs)抑制剂和葡萄糖转运蛋白2(GLUT2)抑制剂对其跨膜转运的影响,采用UPLC-MS/MS测定黄诺马苷的含量,计算表观渗透系数(P_(app))及外排比(ER)。结果:黄诺马苷质量浓度为5. 625～120 mg·L~(-1)时对MDCK细胞无明显毒性作用,黄诺马苷在MDCK单层细胞模型上的转运具有时间、浓度依赖性,且P_(app)基本处于1×10-6～10×10~(-6)cm·s~(-1)。在60 min和90 min时,与空白组相比,根皮苷组中黄诺马苷在MDCK单层细胞模型上的转运量显著减少。结论:黄诺马苷在肠道中属于中等吸收的药物,其跨膜转运机制以被动转运为主,兼有主动转运存在,且SGLTs转运体可能参与介导了黄诺马苷在MDCK单层细胞模型上的转运。
Objective: To explore the absorption and transport properties of flavanomarein in the MadinDarby canine kidney(MDCK) monolayer cell model. Method: Methyl thiazolyl tetrazolium(MTT) assay was used to investigate the toxicity of flavanomarein in MDCK cells. The resistance value of MDCK monolayer cell model was detected by Millicell-ERS-2 cell resistometer. The effects of mass concentration of flavanomarein,administration time, sodium-glucose cotransporter(SGLTs) inhibitor and glucose transporter 2(GLUT2)inhibitor on the transmembrane transport of flavanomarein were investigated. The concentration of flavanomarein was determined by UPLC-MS/MS,and the apparent permeability coefficient(P_(app)) and the efflux ratio(ER) were calculated. Result: When the concentration of flavanomarein was 5. 625-120 mg·L~(-1),there was no significant toxic effect on MDCK cells. The transport of flavanomarein in MDCK monolayer cell model was time-dependent and concentration-dependent. The P_(app)values of flavanomarein were basically between 1 × 10~(-6) cm·s~(-1) to 10 ×10~(-6) cm·s~(-1). Compared with the blank group, the phlorizin group significantly reduced the transport of flavanomarein on the MDCK monolayer cell model at 60 min and 90 min. Conclusion: Flavanomarein is a moderately absorbed drug in the intestine,its transmembrane transport mechanism is dominated by passive transport along with active transport. SGLTs may be involved in mediating the transport of flavanomarein on the MDCK monolayer cell model.
Objective: To explore the absorption and transport properties of flavanomarein in the MadinDarby canine kidney(MDCK) monolayer cell model. Method: Methyl thiazolyl tetrazolium(MTT) assay was used to investigate the toxicity of flavanomarein in MDCK cells. The resistance value of MDCK monolayer cell model was detected by Millicell-ERS-2 cell resistometer. The effects of mass concentration of flavanomarein,administration time, sodium-glucose cotransporter(SGLTs) inhibitor and glucose transporter 2(GLUT2)inhibitor on the transmembrane transport of flavanomarein were investigated. The concentration of flavanomarein was determined by UPLC-MS/MS,and the apparent permeability coefficient(P_(app)) and the efflux ratio(ER) were calculated. Result: When the concentration of flavanomarein was 5. 625-120 mg·L~(-1),there was no significant toxic effect on MDCK cells. The transport of flavanomarein in MDCK monolayer cell model was time-dependent and concentration-dependent. The P_(app)values of flavanomarein were basically between 1 × 10~(-6) cm·s~(-1) to 10 ×10~(-6) cm·s~(-1). Compared with the blank group, the phlorizin group significantly reduced the transport of flavanomarein on the MDCK monolayer cell model at 60 min and 90 min. Conclusion: Flavanomarein is a moderately absorbed drug in the intestine,its transmembrane transport mechanism is dominated by passive transport along with active transport. SGLTs may be involved in mediating the transport of flavanomarein on the MDCK monolayer cell model.
引文
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[15]慈小燕,夏媛媛,曾勇,等.3-乙酰基-11-羰基-β-乙酰乳香酸在Caco-2和MDCK细胞模型中的吸收研究[J].中草药,2013,44(9):1162-1167.
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[20]Kwon O,Eck P,CHEN S,et al.Inhibition of the intestinal glucose transporter GLUT2 by flavonoids[J].FASEB J,2007,21(2):366-377.
[21]党珏,李燕,刘美辰,等.大黄有效成分与附子有效成分配伍在Caco-2细胞模型上的转运分析[J].中国实验方剂学杂志,2018,24(7):1-6.
[22]杨海涛,王广基.Caco-2单层细胞模型及其在药学中的应用[J].药学学报,2000,35(10):797-800.
[2]Artursson P,Borchardt R T.Intestinal drug absorption and metabolism in cell cultures:Caco-2 and beyond[J].Pharm Res,1997,14(12):1655-1658.
[3]Cho M J,Thompson D P,Cramer C T,et al.The Madin Darby canine kidney(MDCK)epithelial cell monolayer as a model cellular transport barrier[J].Pharm Res,1989,6(1):71-77.
[4]Fogh J,Fogh J M,Orfeo T.One hundred and twentyseven cultured human tumor cell lines producing tumors in nude mice[J].JNCI,1977,59(1):221-226.
[5]张媛,屠鹏飞.两色金鸡菊头状花序的化学成分研究[J].中国中药杂志,2012,37(23):3581-3584.
[6]毛新民,卢伟,李琳琳,等.两色金鸡菊化学成分和药理作用研究进展[J].中国药物应用与监测,2014,11(4):235-239.
[7]姜保平,许利嘉,贾晓光,等.两色金鸡菊的化学成分和药理作用研究进展[J].现代药物与临床,2014,29(5):567-573.
[8]赵军,孙玉华,徐芳,等.昆仑雪菊黄酮类成分研究[J].天然产物研究与开发,2013,25(1):50-52.
[9]胡鹏翼,刘丹,郑琴,等.川芎活性成分对天麻素在MDCK-MDR1细胞上跨膜转运的影响及机制[J].中国药学杂志,2017,52(14):1234-1240.
[10]Dias T,LIU B,Jones P,et al.Cytoprotective effect of Coreopsis tinctoria extracts and flavonoids on t BHP and cytokine-induced cell injury in pancreatic MIN6 cells[J].J Ethnopharmacol,2012,139(2):485-492.
[11]周乐,赵晓莉,狄留庆,等.黄酮类化合物口服吸收与代谢特征及其规律分析[J].中草药,2013,44(16):2313-2320.
[12]WANG J,Guzhalinuer A,LI X X,et al.Testing the absorption of the extracts of Coreopsis tinctoria Nutt.in the intestinal canal in rats using an Ussing chamber[J].J Ethnopharmacol,2016,186:73-83.
[13]陈瑶,李新霞,李琳琳,等.两色金鸡菊血清药物化学研究[J].新疆医科大学学报,2017,40(4):420-424,432.
[14]王华,胡佳慧,刘翠钗,等.运用细胞代谢组学策略探究薯蓣皂苷元的抗肿瘤作用机制[J].中国实验方剂学杂志,2018,24(20):134-139.
[15]慈小燕,夏媛媛,曾勇,等.3-乙酰基-11-羰基-β-乙酰乳香酸在Caco-2和MDCK细胞模型中的吸收研究[J].中草药,2013,44(9):1162-1167.
[16]刘瑶,曾苏.MDCK-MDR1细胞模型及其在药物透过研究中的应用进展[J].药学学报,2008,43(6):559-564.
[17]Wright E M,Loo D D,Hirayama B A.Biology of human sodium glucose transporters[J].Physiol Rev,2011,91(2):733-794.
[18]Kellett G L,Brotlaroche E.Apical GLUT2:a major pathway of intestinal sugar absorption[J].Diabetes,2005,54(10):3056-3062.
[19]Walgren R A,LIN J T,Kinne R K,et al.Cellular uptake of dietary flavonoid quercetin 4'-β-glucoside by sodiumdependent glucose transporter SGLT1[J].J Pharmacol Exp Ther,2000,294(3):837-843.
[20]Kwon O,Eck P,CHEN S,et al.Inhibition of the intestinal glucose transporter GLUT2 by flavonoids[J].FASEB J,2007,21(2):366-377.
[21]党珏,李燕,刘美辰,等.大黄有效成分与附子有效成分配伍在Caco-2细胞模型上的转运分析[J].中国实验方剂学杂志,2018,24(7):1-6.
[22]杨海涛,王广基.Caco-2单层细胞模型及其在药学中的应用[J].药学学报,2000,35(10):797-800.