基于UDP-葡萄糖醛酸转移酶1A1抑制探讨二蒽酮的潜在肝毒性  

Hepatotoxicity mechanism of dianthrones based on UDP-glucuronosyltransferase 1A1 inhibition

作　　者：汪祺[1] 杨建波[1] 文海若 马双成[1] WANG Qi;YANG Jianbo;WEN Hairuo;MA Shuangcheng(National Institutes for Food and Drug Control,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院,北京100050

出　　处：《药物评价研究》2022年第9期1779-1785,共7页Drug Evaluation Research

基　　金：国家自然科学基金资助项目(81973476)。

摘　　要：目的基于胆红素代谢酶UDP-葡萄糖醛酸转移酶1A1(UGT1A1)靶点评价何首乌中二蒽酮类成分的潜在肝毒性。方法采用Discovery Studio 2.5软件的From Receptor Cavities模块对UGT1A1酶蛋白空腔进行自动识别,将反式-大黄素-大黄素二蒽酮(trans-EMD)、顺式-大黄素-大黄素二蒽酮(cis-EMD)与UGT1A1酶蛋白进行对接,确定待测单体与酶蛋白的作用方式以及连接的紧密程度;应用大鼠肝微粒体孵育体系,加入底物胆红素对照品溶液,评价trans-EMD、cis-EMD(0.037、0.110、0.330、0.990、2.970μg·mL^(-1))对UGT1A1酶的作用,同时启动Ⅰ、Ⅱ相代谢,以表观抑制常数(Ki)为评价指标;CCK-8法检测trans-EMD、cis-EMD(0.04、0.10、0.30、1.00、3.00μg·mL^(-1))作用24 h对HepaRG细胞的毒性作用;实时荧光定量PCR(qRT-PCR)实验检测trans-EMD、cis-EMD(0.04、0.30、3.00μg·mL^(-1))作用24 h对HepaRG细胞UGT1A1 mRNA水平的影响。结果分子对接实验显示trans-EMD、cis-EMD可与UGT1A1结合于site F,2个化合物10或10'位不同氢键构型可引起化合物空间构型的改变,影响其与UGT1A1的结合强弱;体外酶抑制实验表明trans-EMD、cis-EMD对UGT1A1酶均表现出竞争型抑制作用,抑制作用较强;细胞毒性实验表明trans-EMD(IC_(50)为1.333μg·mL^(-1))和cis-EMD(IC_(50)为1.715μg·mL^(-1))均表现出较明显的HepaRG细胞毒性,IC_(50)值较小;与对照组比较,trans-EMD和cis-EMD 0.30、3.00μg·mL^(-1)均可显著下调UGT1A1 mRNA表达水平(P<0.05),且作用存在浓度相关性。结论具有大黄素(10→10')大黄素或大黄素(10→10')大黄素母核结构的二蒽酮化合物是一类具有潜在肝毒性的化合物,其毒性作用可能与抑制胆红素代谢酶UGT1A1相关。Objectives To evaluate the potential hepatotoxicity of dianthrones in Polygonum multiflorum based on the target of bilirubin metabolizing enzyme UDP-glucuronyltransferase 1A1(UGT1A1).Methods The Discovery Studio 2.5 software From Receptor Cavities module to automatic identification of UGT1A1 enzyme protein cavity,Trans-emodin-emodin dianthrone(trans-EMD)and Cis-emodin-emodin dianthrone(cis-EMD)were docked with UGT1A1 enzyme protein to determine the action mode and the closeness of the monomer to be tested and the enzyme protein.To evaluate the effects of trans-EMD and cis-EMD(0.037,0.110,0.330,0.990,2.970μg·mL^(-1))on UGT1A1 enzyme and initiate phaseⅠand phaseⅡmetabolism in the incubation system of rat liver microsomes with substrate bilirubin reference solution.The apparent inhibition constant(Ki)was used as the evaluation index.CCK-8 assay was used to detect the toxicity of trans-EMD and cis-EMD(0.04,0.10,0.30,1.00,3.00μg·mL^(-1))for 24 h on HepaRG cells.The effects of trans-EMD and cis-EMD(0.04,0.30,3.00μg·mL^(-1))on UGT1A1 mRNA levels in HepaRG cells for 24 h were detected by quantitative real-time PCR(qRT-PCR).Results Molecular docking experiments showed that trans-EMD and cis-EMD could bind UGT1A1 to Site F.Different hydrogen bond configurations at 10 or 10'positions of the two compounds could cause changes in the spatial configuration of the compounds and affect their binding strength to UGT1A1.In vitro enzyme inhibition experiments showed that both trans-EMD and cis-EMD exhibited competitive inhibitory effects on UGT1A1 enzyme,with strong inhibitory effects.The cytotoxicity test showed that trans-EMD(IC_(50)=1.333μg·mL^(-1))and cis-EMD(IC_(50)=1.715μg·mL^(-1))showed obvious HepaRG cytotoxicity,and the IC_(50) value was smaller.Compared with control group,trans-EMD and cis-EMD 0.30 and 3.00μg·mL^(-1) could significantly down-regulate UGT1A1 mRNA expression level(P<0.05),and the effect was concentration dependent.Conclusion Dianthrone compounds with emodin(10→10')emodin or emodin(10→10')

关 键 词：分子对接 胆红素代谢酶 UDP-葡萄糖醛酸转移酶1A1(UGT1A1) 何首乌 二蒽酮 肝毒性 

分 类 号：R991[医药卫生—毒理学]