目的 建立以有機(jī)陰離子轉(zhuǎn)運(yùn)多肽1B1（OATP1B1）和OATP1B3為作用靶點(diǎn)的何首烏肝毒性成分快速篩選方法。方法 使用Discovery Studio 2.5軟件將何首烏主要單體成分（48個(gè)）與OATP1B1/OATP1B3蛋白進(jìn)行分子對接，以O(shè)ATP1B1和OATP1B3主要轉(zhuǎn)運(yùn)底物膽紅素作為陽性對照，對目標(biāo)化合物進(jìn)行虛擬篩選；采用CCK-8法考察蘆薈大黃素-8-O-β-D-葡萄糖苷（AEG）、大黃素-8-O-β-D-葡萄糖苷（EG）、大黃素甲醚-8-O-β-D-葡萄糖苷（PG）、2，3，5，4'-四羥基二苯乙烯-2-O-β-D-葡萄糖苷（TSG）處理24 h后對人源肝永生化肝細(xì)胞HepaRG的毒性強(qiáng)弱，并采用實(shí)時(shí)熒光定量PCR （qRT-PCR）技術(shù)測定4種化合物對HepaRG細(xì)胞的OATP1B1和OATP1B3 mRNA表達(dá)量的影響。結(jié)果 與OATP1B1對接結(jié)果顯示，polygonumnolide B3、cis-emodin-physcionbianthrones、polygonumnolide B2、大黃素甲醚-8-β-D-（6'-O-乙酰基）-葡萄糖苷、trans-emodin-physcionbianthrones、大黃素-3-甲醚-8-O-β-D-葡萄糖苷、polygonumnolide B4、大黃酚-8-O-葡萄糖苷、EG、大黃素-1-O-β-D-葡萄糖苷、AEG、大黃酚-8-O-β-D-葡萄糖苷、大黃酸-8-O-葡萄糖苷高于膽紅素打分值的80%，可被初步認(rèn)定為潛在毒性成分；與OATP1B3對接結(jié)果顯示，trans-emodin-physcionbianthrones、PG、polygonumnolide A4及虎杖苷高于膽紅素打分值的80%，可被初步認(rèn)定為潛在毒性成分。CCK-8實(shí)驗(yàn)進(jìn)一步證實(shí)AEG、EG及PG均具肝細(xì)胞毒性作用，半數(shù)抑制濃度分別為16.10、49.43、69.44 μg·mL^-1，與分子對接結(jié)果一致。與對照組比較，AEG、EG均可顯著下調(diào)OATP1B1的mRNA表達(dá)水平（P<0.05）；PG可顯著下調(diào)OATP1B3的mRNA表達(dá)水平（P<0.05）。結(jié)論 以O(shè)ATP1B1/OATP1B3分子對接技術(shù)為切入點(diǎn)，可有效預(yù)測何首烏潛在肝毒性成分，實(shí)現(xiàn)快速高效的高通量篩選，為中藥安全性評價(jià)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

國家自然科學(xué)基金資助項(xiàng)目（81973476）