目的 建立以有机阴离子转运多肽1B1（OATP1B1）和OATP1B3为作用靶点的何首乌肝毒性成分快速筛选方法。方法 使用Discovery Studio 2.5软件将何首乌主要单体成分（48个）与OATP1B1/OATP1B3蛋白进行分子对接，以OATP1B1和OATP1B3主要转运底物胆红素作为阳性对照，对目标化合物进行虚拟筛选；采用CCK-8法考察芦荟大黄素-8-O-β-D-葡萄糖苷（AEG）、大黄素-8-O-β-D-葡萄糖苷（EG）、大黄素甲醚-8-O-β-D-葡萄糖苷（PG）、2，3，5，4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷（TSG）处理24 h后对人源肝永生化肝细胞HepaRG的毒性强弱，并采用实时荧光定量PCR （qRT-PCR）技术测定4种化合物对HepaRG细胞的OATP1B1和OATP1B3 mRNA表达量的影响。结果 与OATP1B1对接结果显示，polygonumnolide B3、cis-emodin-physcionbianthrones、polygonumnolide B2、大黄素甲醚-8-β-D-（6'-O-乙酰基）-葡萄糖苷、trans-emodin-physcionbianthrones、大黄素-3-甲醚-8-O-β-D-葡萄糖苷、polygonumnolide B4、大黄酚-8-O-葡萄糖苷、EG、大黄素-1-O-β-D-葡萄糖苷、AEG、大黄酚-8-O-β-D-葡萄糖苷、大黄酸-8-O-葡萄糖苷高于胆红素打分值的80%，可被初步认定为潜在毒性成分；与OATP1B3对接结果显示，trans-emodin-physcionbianthrones、PG、polygonumnolide A4及虎杖苷高于胆红素打分值的80%，可被初步认定为潜在毒性成分。CCK-8实验进一步证实AEG、EG及PG均具肝细胞毒性作用，半数抑制浓度分别为16.10、49.43、69.44 μg·mL^-1，与分子对接结果一致。与对照组比较，AEG、EG均可显著下调OATP1B1的mRNA表达水平（P<0.05）；PG可显著下调OATP1B3的mRNA表达水平（P<0.05）。结论 以OATP1B1/OATP1B3分子对接技术为切入点，可有效预测何首乌潜在肝毒性成分，实现快速高效的高通量筛选，为中药安全性评价提供新思路。

Objective To establish a rapid screening method for hepatotoxic components in Polygonum multiflorum by taking the organic anion transporting polypeptide 1B1 and 1B3 transporters (OATP1B1,OATP1B3) as the target. Methods At first, computer molecular docking technology was adopted, and Discovery Studio 2.5 software was applied to molecularly dock the target compounds with the OATP1B1/OATP1B3, and the target compounds were screened virtually with bilirubin as the reference objects. Subsequently, the cell counting kit (CCK-8) method was used to investigate the toxicity of aloe-emodin-8-O-glucoside(AEG), emodin-8-O-β-D-glucoside (EG), physcion-8-O-β-D glucoside (PG), and 2,3,5,4 '-tetrahydroxy stilbene-2-O-β-D-glucoside (TSG) on human liver immortalized hepatocytes HepaRG after a 24 h treatment, and real-time PCR (qRT-PCR) was used to determine the effects of the target compounds on the relative expression level of the OATP1B1/OATP1B3 gene in HepaRG cells. Results Docking results with OATP1B1 showed that, polygonumnolide B3, cis-emodin-physcionbianthrones, polygonumnolide B2, emodin methyl ether-8-β-D-(6'-O-acetyl)-glucoside, trans-emodin-physcionbianthrones, emodin-3-methylether-8-O-β-D-glucoside, polygonumnolideB4, rhubarb phenol-8-O-glucoside, EG, emodin-1-O-β-D-glucoside, AEG, rhubarb phenol-8-O-β-D-glycosidase, rhein-8-Oglycosidase higher bilirubin score of 80%, can be initially identified as potential toxic ingredients. The results of docking with OATP1B3 showed that trans-emodin-physcionbianthrones, PG, polygonumnolide A4 and polygonumnolide were higher than 80% of the score of bilirubin, which could be initially identified as potential toxic ingredients. CCK-8 assay further confirmed that AEG, EG and PG had hepatocellular toxicity, and the median inhibitory concentrations (IC₅₀) were 16.10, 49.43 and 69.44 μg·mL^-1, respectively, which were consistent with the molecular docking results. Compared with control group, AEG and EG significantly down-regulated OATP1B1 mRNA expression levels (P < 0.05). PG significantly down-regulated OATP1B3 mRNA expression level (P < 0.05). Conclusion This study used the OATP1B1/OATP1B3 molecular docking technology as a starting point to effectively predict the potential hepatotoxic components of Polygonum multiflorum, and a rapid and efficient high-throughput hepatoxicity screening is achieved. These provide new ideas for the safety evaluation of traditional Chinese medicine.

R285.5

国家自然科学基金资助项目（81973476）