以MRP2/MRP3转运体为作用靶点的何首乌中肝毒性成分筛选

Screening of Hepatotoxic Components in Polygoni Multiflori Radix Targeting MRP2/MRP3 Transporters

目的:建立以胆红素外排转运体多药耐药相关蛋白2(MRP2)、MRP3为靶点的何首乌中潜在肝毒性成分的快速筛选方法,并评价相关化合物的肝毒性大小。方法:采用计算机分子对接方法对何首乌中的主要单体成分进行高通量筛选,以MRP2、MRP3转运体底物胆红素为参考对象,将48个目标单体与MRP2、MRP3蛋白进行分子对接,实现虚拟筛选;采用CCK-8细胞计数试剂盒法考察目标单体对肝细胞HepaRG的毒性作用;应用实时荧光定量聚合酶链式反应(qRT-PCR)测定目标单体对HepaRG细胞外排转运体MRP2、MRP3 mRNA相对表达量的影响。结果:MRP2对接结果显示,大黄素-6-O-β-D-葡萄糖苷、大黄素甲醚-8-O-β-D-(6’-O-乙酰基)-葡萄糖苷、反式二苯乙烯苷高于底物胆红素与MRP2对接打分值的80%;MRP3对接结果显示,大黄酚-8-O-β-D-葡萄糖苷、大黄素-6-O-β-D-葡萄糖苷、大黄素-8-O-β-D-葡萄糖苷、大黄素甲醚-8-O-β-D-(6’-O-乙酰基)-葡萄糖苷、大黄酸-8-O-β-D-葡萄糖苷、大黄酚-8-O-β-D-葡萄糖苷及芦荟大黄素-8-O-β-D-葡萄糖苷高于底物胆红素与MRP3对接打分值的80%,因此上述单体可被初步推测为潜在肝毒性成分。体外细胞毒性实验结果表明,大黄素-8-O-β-D-葡萄糖苷[半数抑制浓度(IC_(50))为21.37μg·mL^(-1)]和芦荟大黄素-8-O-β-D-葡萄糖苷(IC_(50)为6.96μg·mL^(-1))表现出较明显的肝细胞毒性,并可显著影响外排转运体MRP2、MRP3 mRNA表达水平(P<0.05)。结论:建立的以MRP2、MRP3为靶点的分子对接法初步筛选得到了何首乌中潜在的肝毒性成分,并对其毒性大小进行了评价,为何首乌的临床安全用药提供了参考。

Objective:This study aims to establish a method for rapid screening of potential hepatotoxic components in Polygoni Multiflori Radix which target bilirubin efflux transporters multidrug resistance-associated protein(MRP)2 and MRP3 and evaluate the potential hepatotoxicity of related components.Methods:The main monomer components in Polygoni Multiflori Radix were screened by computer-aided molecular docking.To be specific,with bilirubin as the reference,48 monomers were docked with MRP2 and MRP3 for virtual screening.Cell counting kit-8(CCK-8)was used to investigate the toxicity of the target monomers to HepaRG cells,and real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR)to determine the influence of target monomers on relative mRNA expression of transporters MRP2 and MRP3 in HepaRG cells.Results:The LibDock scores of emodin-6-O-β-D-glucoside,emodin methyl ether-8-O-β-D-(6’-O-acetyl base)-glucoside,and trans-stilbene glycoside to MRP2 were 80%higher than that of substrate bilirubin.The LibDock scores of chrysophanol-8-O-β-D-glucoside,emodin-6-O-β-D-glucoside,emodin-8-O-β-D-glucoside,emodin methyl ether-8-O-β-D-(6’-O-acetyl base)-glucoside,rhein-8-O-β-D-glucoside,and aloe-emodin-8-O-β-D-glucoside to MRP3 were 80%higher than that of the substrate bilirubin.Therefore,the above monomers might be the potential hepatotoxic components.In addition,in vitro cytotoxicity assay further suggested that both emodin-8-O-β-D-glucoside(IC_(50)=21.37μg·mL^(-1))and aloe-emodin-8-O-β-D-glucoside(IC_(50)=6.96μg·mL^(-1))showed obvious hepatotoxicity and significantly affected the mRNA expression of MRP2 and MRP3(P<0.05).Conclusion:In this study,the potential hepatotoxic components in Polygoni Multiflori Radix targeting MRP2 and MRP3 were screened out with the established molecular docking method and their toxicity was evaluated.The result can serve as a reference for the safe use of Polygoni Multiflori Radix in clinical settings.