基于分子对接和体外大鼠肝微粒体抑制实验综合考察何首乌中潜在肝毒性成分研究

目的 基于UDP-葡萄糖醛酸转移酶1A1（UGT1A1）介导的胆红素代谢，构建UGT1A1酶蛋白模型，用于研究何首乌中潜在致肝毒性成分的毒性作用，并用体外大鼠肝微粒体抑制实验进行验证。方法 采用同源模建方法构建UGT1A1酶蛋白结构，将UGT1A1底物胆红素及何首乌中主要蒽醌类成分大黄素、大黄酚、大黄酸、羟基大黄素、大黄素-8-O-β-D-葡萄糖苷和大黄素甲醚与UGT1A1蛋白进行分子对接，考察分子结合靶点及结合强弱。采用大鼠肝微粒体孵育体系（RLM），加入系列浓度的底物胆红素对照品溶液及待测单体对照品溶液，检测表观抑制常数（K_i），测定蒽醌类单体成分对UGT1A1酶的抑制作用。结果 分子对接结果显示，UGT1A1酶蛋白结构上共有9个活性口袋区，胆红素的结合口袋确定为位点F；6个单体主要集中在两个活性口袋区：大黄素、羟基大黄素、大黄素-8-O-β-D-葡萄糖苷和大黄酚对接进入位点F；大黄素甲醚和大黄酸对接进入位点C。结合于UGT1A1酶蛋白位点C中的单体大黄酸和大黄素甲醚的结合自由能（IE）值较小；位点F区中，单体大黄素-8-O-β-D-葡萄糖苷、大黄素及羟基大黄素具有较高的IE值，结合能力强。体外抑制实验显示，大黄素-8-O-β-D-葡萄糖苷、大黄素表现为较强的竞争型抑制作用，羟基大黄素为较强的混合型抑制作用，与分子对接结果一致。结论 大黄素-8-O-β-D-葡萄糖苷、羟基大黄素、大黄素对UGT1A1酶介导的胆红素代谢产生较强的抑制作用，构建的UGT1A1酶蛋白模型可有效预测药物的潜在风险。

Investigation of potential hepatotoxic components in Polygonum multiflorum based on molecular docking and rat liver microsome inhibition test

Objective Based on UGT1A1 enzyme-mediated bilirubin metabolism UGT1A1 enzyme protein was constructed by homology modelling to study the toxic effects of potential hepatotoxic components in Polygonum multiflorum.Methods The UGT1A1 enzyme protein structure was constructed by homology modeling method.Then bilirubin and the main anthraquinones (emodin,chrysophanol,rhein,hydroxy emodin,emodin-8-O-beta-D-glucoside and emodin methyl ether) in P.multiflorum were molecularly docked with UGT1A1 protein to investigate the binding target and the action mode.Rat liver microsome incubation system (RLM) was used to determine the inhibitory effect of anthraquinone monomer components on UGT1A1 enzyme by adding a series of concentration of bilirubin reference solution and monomer reference solution to calculate the apparent inhibitory constant (K_i).Results Molecular docking results showed that there were nine active pocket regions in UGT1A1 protein structure,and the binding pocket of bilirubin was identified as site F;Six monomers were mainly concentrated in two active pocket regions:emodin,hydroxy emodin,emodin-8-O-beta-D-glucoside and chrysophanol docking entry site F;emodin methyl ether and emodin acid docking entry site C.The binding free energy (IE) of emodin methyl ether and emodin acid in site C of UGT1A1 enzyme protein was lower.In site F,emodin-8-O-beta-D-glucoside,emodin and hydroxy-emodin had higher IE values and stronger binding ability.in vitro inhibition experiments showed that emodin-8-O-beta-D-glucoside,emodin and hydroxy emodin showed strong competitive inhibition,which was consistent with the results of molecular docking.Conclusion Emodin-8-O-beta-D-glucoside,hydroxy emodin and emodin have strong inhibitory effects on UGT1A1-mediated bilirubin metabolism.The UGT1A1 enzyme protein model can effectively predict the potential risks of drugs.