目的 基于UDP-葡萄糖醛酸轉(zhuǎn)移酶1A1（UGT1A1）介導(dǎo)的膽紅素代謝，構(gòu)建UGT1A1酶蛋白模型，用于研究何首烏中潛在致肝毒性成分的毒性作用，并用體外大鼠肝微粒體抑制實驗進行驗證。方法 采用同源模建方法構(gòu)建UGT1A1酶蛋白結(jié)構(gòu)，將UGT1A1底物膽紅素及何首烏中主要蒽醌類成分大黃素、大黃酚、大黃酸、羥基大黃素、大黃素-8-O-β-D-葡萄糖苷和大黃素甲醚與UGT1A1蛋白進行分子對接，考察分子結(jié)合靶點及結(jié)合強弱。采用大鼠肝微粒體孵育體系（RLM），加入系列濃度的底物膽紅素對照品溶液及待測單體對照品溶液，檢測表觀抑制常數(shù)（K_i），測定蒽醌類單體成分對UGT1A1酶的抑制作用。結(jié)果 分子對接結(jié)果顯示，UGT1A1酶蛋白結(jié)構(gòu)上共有9個活性口袋區(qū)，膽紅素的結(jié)合口袋確定為位點F；6個單體主要集中在兩個活性口袋區(qū)：大黃素、羥基大黃素、大黃素-8-O-β-D-葡萄糖苷和大黃酚對接進入位點F；大黃素甲醚和大黃酸對接進入位點C。結(jié)合于UGT1A1酶蛋白位點C中的單體大黃酸和大黃素甲醚的結(jié)合自由能（IE）值較小；位點F區(qū)中，單體大黃素-8-O-β-D-葡萄糖苷、大黃素及羥基大黃素具有較高的IE值，結(jié)合能力強。體外抑制實驗顯示，大黃素-8-O-β-D-葡萄糖苷、大黃素表現(xiàn)為較強的競爭型抑制作用，羥基大黃素為較強的混合型抑制作用，與分子對接結(jié)果一致。結(jié)論 大黃素-8-O-β-D-葡萄糖苷、羥基大黃素、大黃素對UGT1A1酶介導(dǎo)的膽紅素代謝產(chǎn)生較強的抑制作用，構(gòu)建的UGT1A1酶蛋白模型可有效預(yù)測藥物的潛在風(fēng)險。

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.

國家自然基金資助項目（81503347）