3D肝细胞模型研究大黄素型单蒽酮肝细胞毒性
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摘要
目的:首次研究何首乌中大黄素型单蒽酮的潜在肝细胞毒性及其作用机制。方法:本研究利用悬滴技术构建以人肝细胞HepaRG和人星型细胞HSC为基础的3D多细胞聚球体模型,用该模型评价何首乌中大黄素型单蒽酮成分的重复给药毒性,并与2D模型下单次给药毒性进行比较。进一步研究大黄素型单蒽酮对主要肝药酶和外排转运体表达的影响。结果:肝脏3D多细胞聚球体模型体外培养15 d仍然可以维持较好的肝脏功能。大黄素型单蒽酮2D培养给药48 h,IC50为1. 091μg·mL~(-1)。肝脏3D悬滴模型下连续给药6 d,同浓度大黄素型单蒽酮对肝细胞毒性显著性小于2D培养评价结果。RT-PCR结果显示大黄素型单蒽酮体外多次给药上调主要肝药酶CYP3A4,CYP2B6及外排转运体P-糖蛋白和多药耐药转运蛋白2(MRP2)。结论:何首乌中的新化合物大黄素型单蒽酮具有明显的肝细胞毒性,体外3D模型多次给药引起主要肝药酶和外排转运体表达上调,为肝细胞降低大黄素型单蒽酮毒性提供了潜在机制,同时也提示服用含有大黄素型单蒽酮化合物的中药可能会产生药物相互作用。
Objective: To study the hepatotoxicity of monanthone with emodin type in Polygonummultiflorum and its potential mechanisms for the first time. Methods: We used the hanging drop technique to construct a 3D multicellular polysphere model based on human hepatocyte HepaRG and human stellate cells HSC. The model was used to evaluate the repeated administration toxicity of monanthone with emodin type in Polygonummultiflorum and compared with the toxicity of single administration in 2D model. The effects of monoanthrone with emodin type on the expression of major hepatic enzymes and efflux transporters were further studied. Results: 3D multicellular polysphere model still can maintain preferable hepatic function after in vitro culture for 15 d. The IC50 of monanthonewith emodin type in the 2D model was 1. 091 μg·mL~(-1) after culture for 48 h. In the hanging drop 3D model,cytotoxicity was significantly lower than 2D results after 6 d repeated administration. RT-PCR results showed that CYP3 A4,CYP2 B6,P-gp and MRP RNA expressions were significantly increased after in vitro multiple-dose of monanthone with emodin type. Conclusion: The monanthone with emodin type in Polygonummultiflorum has obvious hepatotoxicity. Multiple-dose in vitro in 3D model of the monanthone with emodin type increases the major CYP enzymes and efflux transporters,provides a potential mechanism for reducing the toxicity of emodin-type monoanthrone by hepatocytes. The results also demonstrated that monanthone with emodin type may potentially induce drug-drug interactions.
Objective: To study the hepatotoxicity of monanthone with emodin type in Polygonummultiflorum and its potential mechanisms for the first time. Methods: We used the hanging drop technique to construct a 3D multicellular polysphere model based on human hepatocyte HepaRG and human stellate cells HSC. The model was used to evaluate the repeated administration toxicity of monanthone with emodin type in Polygonummultiflorum and compared with the toxicity of single administration in 2D model. The effects of monoanthrone with emodin type on the expression of major hepatic enzymes and efflux transporters were further studied. Results: 3D multicellular polysphere model still can maintain preferable hepatic function after in vitro culture for 15 d. The IC50 of monanthonewith emodin type in the 2D model was 1. 091 μg·mL~(-1) after culture for 48 h. In the hanging drop 3D model,cytotoxicity was significantly lower than 2D results after 6 d repeated administration. RT-PCR results showed that CYP3 A4,CYP2 B6,P-gp and MRP RNA expressions were significantly increased after in vitro multiple-dose of monanthone with emodin type. Conclusion: The monanthone with emodin type in Polygonummultiflorum has obvious hepatotoxicity. Multiple-dose in vitro in 3D model of the monanthone with emodin type increases the major CYP enzymes and efflux transporters,provides a potential mechanism for reducing the toxicity of emodin-type monoanthrone by hepatocytes. The results also demonstrated that monanthone with emodin type may potentially induce drug-drug interactions.
引文
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[16] LI L,YI T,LAM CW. Inhibition of human efflux transporter ABCC2(MRP2)by self-emuLsifying drug delivery system:influences of concentration and combination of excipients[J]. J Pharm Pharm Sci,2014,17(4):447-460.
[17] CHEN CY,KAO CY,LIN PJ,et al. Carbon monoxide may enhance bile secretion by increasing glutathione excretion and Mrp2expression in rats[J]. J Chin Med Assoc,2013,76(5):258-264.
[2] FURUKAWA M,KASAJIMA S,NAKAMURA Y,et al. Toxichepatitis induced by Show-Wu-Pian,a Chinese herbal preparation[J]. Inter Med,2010,49(15):1537-1540.
[3] PATIL SM,SAPKALE GN,SURWASE US,et al. Herbal medicines as aneffective therapy in hair loss-a review[J]. Res J Pharm Biol Chem Sci,2010,1(3):773-781.
[4] LV LS,SHAO X,WANG LY,et al. Stilbene glucoside from Polygonum multiflorum Thunb.:a novel natural inhibitor of advanced glycationend product formation by trapping of methylglyoxal[J]. J Agric Food Chem,2010,58(4):2239-2245.
[5] LONGFEI L,BORAN N,HONGMEI L,et al. Traditional usages,botany,phytochemistry,pharmacology and toxicology of Polygonum multiflorum Thunb.:a review[J]. J Ethnopharmacol,2015,159(15):158-183.
[6] YANG JB,LI L,DAI Z,et al. Polygonumnolides C1-C4:minor dianthrone glycosides from the roots of Polygonum multiflorum Thunb.[J]. Asian Nat Prod Res,2016,18(9):813-822.
[7]汪祺,戴忠,张玉杰,等.何首乌中二蒽酮类成分肝毒性研究[J].药物分析杂志,2018,38(2):268-274.
[8]杨建波.何首乌肝毒性和茶芎活性成分以及质量分析方法研究[D].北京:北京中医药大学,2015:92-93.
[9] LECLUYSE EL. Organotypic liver cuture model:meeting current challenges in toxicity testing[J]. Crit Rev Toxicol,2012,42(6):501-548.
[10] CHANG SY,VOELLINGER JL,VAN KP,et al. Characterization of rat or human hepatocytes cuLtured in microphysiological systems(MPS)to identify hepatotoxicity[J]. Toxicol Vitro,2017,40:170-183.
[11] YANG JB,TIAN JY,DAI Z,et al. a-Glucosidase inhibitors extracted from the roots of PolygonummuLtiflorumThunb[J]. Fitoterapia,2017,117:65-70.
[12] LIN RZ,LIN RZ,CHANG HY. Recent advances in three-dimensional muLticelluLar spheroid cu Lture for biomedical research[J]. Biotechnol,2008,3(9/10):1172-1184.
[13]李艳梅,王刚,黄志,等.细胞色素氧化酶CYP3A4基因多态性研究进展[J].儿科药学杂志,2011,17(6):49-52.
[14] SCHINKEL AH. P-Glycoprotein,a gatekeeper in the blood-brain barrier[J]. Adv Drug Deliv Rev,1999,36(2-3):179-186.
[15] DZIERLENGA AL,CLARKE JD,KLEIN DM,et al. Biliary elimination of pemetrexed is dependent on Mrp2 in rats:potential mechanism of variable response in nonalcoholic steatohepatitis[J]. J Pharmacol Exp Ther,2016,358(2):246-253.
[16] LI L,YI T,LAM CW. Inhibition of human efflux transporter ABCC2(MRP2)by self-emuLsifying drug delivery system:influences of concentration and combination of excipients[J]. J Pharm Pharm Sci,2014,17(4):447-460.
[17] CHEN CY,KAO CY,LIN PJ,et al. Carbon monoxide may enhance bile secretion by increasing glutathione excretion and Mrp2expression in rats[J]. J Chin Med Assoc,2013,76(5):258-264.