P-糖蛋白及多药耐药相关蛋白1抑制剂促进天麻素跨膜转运作用机制研究

Studies on effects and mechanisms of P-gp and MRP1 inhibitor on transportation of gastrodin

目的体外细胞模型法及分子对接法研究P-糖蛋白（P-gP）及多药耐药相关蛋白1（MRP1）抑制剂促进天麻素跨膜转运的作用机制。方法MTT法考察天麻素对多药耐药基因1（MDR1）基因转染马丁达比犬肾上皮细胞（MDCK-MDR1）细胞的毒性，采用分子对接法预测天麻素与P-gP、MRP1结合方式及作用能力的强弱，MDCK-MDR1细胞模型分析天麻素的跨膜转运，以及P-gp抑制剂维拉帕米、MRP1抑制剂丙磺舒对天麻素跨膜转运的影响。结果天麻素在100~1000μg/mL的质量浓度内对细胞无毒性；天麻素与P-gp通过氢键及疏水作用形成稳定的复合物，与MRP1通过氢键、疏水作用及静电作用形成稳定的复合物，LibDock Score表明P-gp-维拉帕米、MRP1-丙磺舒复合物的稳定性强于P-gp-天麻素和MRP1-天麻素；天麻素基底侧（BL）→顶端侧（AP）的表观渗透系数（P_app BL→AP）大于P_app AP→BL，外排率（ER）为1．5左右，提示天麻素在MDCK-MDR1细胞为被动转运，且转运过程可能存在蛋白外排现象；天麻素配伍维拉帕米P_app显著增大（P〈0．05），ER值显著减小（P〈0．05），天麻素配伍丙磺舒后P。有所增大，ER有所减小，但无显著性差异。结论天麻素为P-gp底物，天麻素是否是MRP1的底物有待进一步考证，但维拉帕米及丙磺舒可通过竞争性结合P-gp和MRP1不同程度促进天麻素跨膜转运。

Objective To study the effects and mechanisms ofp-glycoprotein （P-gp） inhibitor and MRP1 inhibitor on the transportation of gastrodin （GAS）. Methods Cell toxicity of GAS was detected by MTT assay, molecular Docking was employed to predicted binding mode and effect ability of GAS with P-gp and MRP1. MDCK-MDR1 cell model was employed to study the influences of Ver, a P-gp inhibitor, and Probenecid, a MRP l inhibitor, on the transportation of GAS. Results There was no cell cytotoxicity of GAS between the concentration of 100 to 1 000 μg/mL. There was hydrogen-bond and hydrophobic interaction between P-gp and GAS, and hydrogen-bond, hydrophobic interaction and electrostatic-interaction between MRPI and GAS. LibDock Score indicated that both P-gp-verapamil （Vet） and MRP 1-Probenecid were more stable than P-gp-GAS and MRP 1-GAS. The Papp of GAS BL→AP was greater than that of AP→BL, Effiux ratio （ER） of GAS was about 1.5, which indicated the effiux pump protein might involve the transportation of GAS. The Papp of GAS was significant increased but the ER of GAS was significant decreased when co-administrated with Ver （P 〈 0.05）. The Papp of GAS was slightly increased and the ER of GAS was slightly decreased when co-administrated with Probenecid, while there was no significance. Conclusion The results indicate that GAS is the substrate of P-gp. However, whether GAS is the substrate of MRP1 needs further research. Both Ver and Probenecid could enhance the transportation of GAS by competitive binding with P-gp and MRP1.