甘草次酸18位差向异构体对P-gp底物罗丹明123在Caco-2细胞上跨膜转运的影响

Effect of Glycyrrhetinic Acid C-18 Epimerides on Membrane Transport of P-gp Substrate Rhodamine 123 in Caco-2 Cell Monolayers

目的研究甘草次酸18位差向异构体即18α-甘草次酸(α-GA)、18β-甘草次酸(β-GA)对P-糖蛋白(P-gp)底物罗丹明123在Caco-2细胞上跨膜转运的影响。方法采用MTT法检测不同摩尔浓度的18α-甘草次酸、18β-甘草次酸及维拉帕米对Caco-2细胞生长抑制作用;建立Caco-2单层细胞模型,以P-糖蛋白底物罗丹明123(Rho-123)为荧光探针,评价甘草次酸18位差向异构体和P-糖蛋白抑制剂维拉帕米对罗丹明123在Caco-2细胞上跨膜转运的影响。罗丹明123浓度采用酶标仪检测。结果高浓度(10μmol.L-1)18α-甘草次酸瞬时作用于Caco-2细胞单层模型,或者干预单层模型72 h后,均使罗丹明123单位面积BL-AP侧累积转运量(transport B-A)增加,外排率(ER)增大,诱导P-糖蛋白底物罗丹明123外向转运;高浓度(10μmol.L-1)18β-甘草次酸瞬时作用于Caco-2细胞单层模型没有表现出诱导作用,但在干预Caco-2单层模型72 h后,表现出对罗丹明123外向转运的诱导;各实验药物均没有对罗丹明123 AP-BL侧转运产生影响。结论跨膜转运实验结果表明,18α-甘草次酸、18β-甘草次酸能诱导P-糖蛋白的外向转运,加速P-糖蛋白底物的外排,可能是甘草酸制剂增强肝脏解毒的机制之一。

OBJECTIVE To study the effects of C-18 epimers of glycyrrhetinic acid, 18α-glycyrrhetinic acid （α-GA） and 18β- glycyrrhetinic acid（β-GA）, on membrane transport of P-gp substrate rhodamine 123 in Caco-2 cell monolayers. METHODS MTF assay was applied to determine the maximum non-cytotoxic dose of α-GA,β-GA and verapamil to ensure the activity of the cells during the test and to consult the maximum test concentration of each drug; appropriate Caco-2 monolayers were established in TranswellTM plates. ELISA Reader was applied to detect the concentration of Rho-123 in transfer fluid. The bi-directional transport of Rho-123 after being treated by instantaneous action and incubation with α-GA and β-GA for 72 h was studied. RESULTS High concentration （ 10μmol· L-1） of α-GA induced the efflux of Rho-123 both in the instantaneous action test and the incubation test. High concentration （ 10β-mol· L-1 ） ofβ-GA induced the efflux of Rho-123 only in the incubation test. The transport from AP to BL was not affected by any test drugs. CONCLUSION The results of the transport experiments of P-gp substrates in Caco-2 monolayers indicated that α-GA and α-GA can induce the efflux function of P-gp and accelerate the excretion of P-gp substrates. It may be one of the mechanisms of that glycyrrhizin preparation enhances liver detoxification.