目的 基于膽紅素代謝酶UDP-葡萄糖醛酸轉(zhuǎn)移酶1A1（UGT1A1）靶點(diǎn)評價何首烏中二蒽酮類成分的潛在肝毒性。方法 采用Discovery Studio 2.5軟件的From Receptor Cavities模塊對UGT1A1酶蛋白空腔進(jìn)行自動識別，將反式-大黃素-大黃素二蒽酮（trans-EMD）、順式-大黃素-大黃素二蒽酮（cis-EMD）與UGT1A1酶蛋白進(jìn)行對接，確定待測單體與酶蛋白的作用方式以及連接的緊密程度；應(yīng)用大鼠肝微粒體孵育體系，加入底物膽紅素對照品溶液，評價trans-EMD、cis-EMD（0.037、0.110、0.330、0.990、2.970 μg·mL^-1）對UGT1A1酶的作用，同時啟動Ⅰ、Ⅱ相代謝，以表觀抑制常數(shù)（K_i）為評價指標(biāo)；CCK-8法檢測trans-EMD、cis-EMD（0.04、0.10、0.30、1.00、3.00 μg·mL^-1）作用24 h對HepaRG細(xì)胞的毒性作用；實(shí)時熒光定量PCR（qRT-PCR）實(shí)驗(yàn)檢測trans-EMD、cis-EMD（0.04、0.30、3.00 μg·mL^-1）作用24 h對HepaRG細(xì)胞UGT1A1 mRNA水平的影響。結(jié)果 分子對接實(shí)驗(yàn)顯示trans-EMD、cis-EMD可與UGT1A1結(jié)合于site F，2個化合物10或10'位不同氫鍵構(gòu)型可引起化合物空間構(gòu)型的改變，影響其與UGT1A1的結(jié)合強(qiáng)弱；體外酶抑制實(shí)驗(yàn)表明trans-EMD、cis-EMD對UGT1A1酶均表現(xiàn)出競爭型抑制作用，抑制作用較強(qiáng)；細(xì)胞毒性實(shí)驗(yàn)表明trans-EMD（IC₅₀為1.333 μg·mL^-1）和cis-EMD（IC₅₀為1.715 μg·mL^-1）均表現(xiàn)出較明顯的HepaRG細(xì)胞毒性，IC₅₀值較小；與對照組比較，trans-EMD和cis-EMD 0.30、3.00 μg·mL^-1均可顯著下調(diào)UGT1A1 mRNA表達(dá)水平（P＜0.05），且作用存在濃度相關(guān)性。結(jié)論 具有大黃素（10→10'）大黃素或大黃素（10→10'）大黃素母核結(jié)構(gòu)的二蒽酮化合物是一類具有潛在肝毒性的化合物，其毒性作用可能與抑制膽紅素代謝酶UGT1A1相關(guān)。

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, 2.970 μg·mL^-1) on UGT1A1 enzyme and initiate phase Ⅰ and phase Ⅱ metabolism in the incubation system of rat liver microsomes with substrate bilirubin reference solution. The apparent inhibition constant (K_i) 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, 3.00 μg·mL^-1) for 24 h on HepaRG cells. The effects of trans-EMD and cis-EMD (0.04, 0.30, 3.00 μg·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 (IC₅₀=1.333 μg·mL^-1) and cis-EMD (IC₅₀=1.715 μg·mL^-1) showed obvious HepaRG cytotoxicity, and the IC₅₀ value was smaller. Compared with control group, trans-EMD and cis-EMD 0.30 and 3.00 μg·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') emodin nucleus structure are a class of compounds with potential hepatotoxicity, and their targets are bilirubin metabolizing enzymes UGT1A enzyme.

R991

國家自然科學(xué)基金資助項(xiàng)目（81973476）