目的 建立MDCK/L02体外肝肠共培养模型,评价小肠吸收对雷公藤甲素(triptolide,TP)肝脏毒性的影响。方法 利用Transwell培养体系,建立MDCK/L02模型。通过测定细胞间隙标记物荧光素钠的通透速率,评价共培养的模型,并进一步观察雷公藤甲素对小肠通透的影响。CCK-8法测定雷公藤甲素对MDCK 的毒性。利用活性氧(ROS)探针结合高内涵,研究小肠吸收对雷公藤甲素诱导肝脏活性氧的影响。结果 MDCK/L02共培养模型形成良好的小肠吸收屏障,荧光素钠测得表观渗透系数为(4.10±0.36)×10-8 cm·s-1。CCK-8法测定雷公藤甲素与MDCK 孵育1、2和4 h的IC50分别为14、8和7 μg·mL-1。雷公藤甲素加入MDCK/L02模型受池孵育1 h后,显著性增强小肠通透。经过小肠吸收后,雷公藤甲素对L02诱导活性氧的水平显著性降低。结论 本实验成功建立MDCK/L02模型,并利用该模型评价显示雷公藤甲素显著性增强了小肠吸收通透,雷公藤甲素经小肠吸收后显著性减低雷公藤甲素的肝脏毒性。
Abstract
OBJECTIVE To establish a MDCK/L02 co-culture model and investigate the effect of intestinal absorption on triptolide (TP) induced liver toxicity. METHODS Transwell was used to establish the MDCK/L02 model. The paracellular permeability was measured using fluorescein sodium (FS). The effect of TP on the paracellular pathway was also studied. The cytotoxicity of TP on MDCK was investigated by CCK-8. TP induced reactive oxygen species (ROS) in L02 cells was studied using high content analysis (HCA). RESULTS The MDCK/L02 co-culture model was established with Papp of FS (4.10±0.36)×10-8 cm·s-1. After 1, 2 and 4 h treatment of TP, the IC50 on MDCK were 14, 8 and 7 μg·mL-1, respectively. After 1 h treatment of TP using the MDCK/L02 model, the paracellular permeability of MDCK was significantly increased. After intestinal absorption, TP toxicity on L02 was significantly decreased measured by ROS induction using HCA. CONCLUSION The MDCK/L02 co-culture model is established. TP significantly increases the MDCK permeability. After intestinal absorption (across the MDCK), TP induced liver toxicity is significantly decreased.
关键词
MDCK/L02共培养模型 /
小肠吸收 /
雷公藤甲素 /
肝脏毒性
{{custom_keyword}} /
Key words
MDCK/L02 co-culture model /
intestinal absorption /
triptolide /
liver toxicity
{{custom_keyword}} /
中图分类号：
R969
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] CHHABRA R S. Intestinal absorption and metabolism of xenobiotics. Environ Health Perspect, 1979, 33(12):61-69.[2] VOLPE D A. Variability in Caco-2 and MDCK cell-based intestinal permeability assays. J Pharm Sci, 2008,97(2):712-725.[3] SCHEERS N M, ALMGREN A B, SANDBERG A S. Proposing a Caco-2/HepG2 cell model for in vitro iron absorption studies. J Nutr Biochem, 2014, 25(7):710-715.[4] LI J, LIU X. The study advancement of side effect and attenuation method of Tripterygium wilfordii . J Henan Univer Chin Med (河南中医学院学报),2008, 4(23):102-104.[5] ZHANG X. Research advance in toxicity of tripterygium preparatum and the attenuation method . Chin Pharm J(中国药学杂志), 2013, 48(22):1897-1901.[6] CHENG Y F, CAI H D. Investigation of liver damage associated with traditional Chinese medicines. Adver Drug React J (药物不良反应杂志), 1999,1(1):27-32.[7] GAO L, BAI B, CHAI Z, et al. A review of toxicological research on Tripterygium wilfordii . Chin J Inf Tradit Chin Med (中国中医药信息杂志), 2012, 4(19):107-111.[8] XUE J,JIA X,TAN X, et al. Determination of apparent oil/water partition coefficient and absorption prediction of triptolide. Chin Pharm J(中国药学杂志), 2009, 44(20):1560-1563. [9] KAMINSKY L S, ZHANG Q Y. The small intestine as a xenobiotic-metabolizingorgan . Drug Metab Dispos, 2003, 31(12):1520-1525.[10] ARTHUR J M. The MDCK cell line is made upof populations of cells with diverse resistive andtransport properties. Tissue Cell, 2000, 32(5):446-450.[11] GLAESER H, DRESCHER S, HOFMANN U, et al. Impact of concentration and rate of intraluminal drug delivery on absorption and gut wall metabolism of verapamil in humans . Clin Pharmacol Ther, 2004, 76(3):230-238.[12] WANG Y H, QI J F, LIN H, et al. Effect of total saikosaponins on the intestinal firs-t pass effect and liver Cyp3a, Cyp2e1 . Chin J Clin Pharm Ther(中国临床药理学与治疗学), 2011,16(7):740-748.[13] IRVINE J D, TAKAHASHI L, LOCKHART K, et al. MDCK (Madin-Darby canine kidney) cells: A tool for membrane permeability screening. J Pharm Sci, 1999,88(1):28-33.[14] D′SOUZA V M, SHERTZER H G, MENON A G, et al. High glucose concentration in isotonic media alters caco-2 cell permeability. AAPS Pharm Sci, 2003,5(3):1-9.[15] DEMARCO V G, LI N, THOMAS J, et al. Glutamine and barrier function in cultured Caco-2 epithelial cell monolayers. J Nutr, 2003,133(7):2176-2179.[16] CHO M J, THOMPSON D P, CRAMER C T, et al. The madin darby canine kidney (MDCK) epithelial cell monolayer as a model cellular transport barrier. Pharm Res, 1989,6(1):71-77.[17] MA J, WU X A, LI W, et al. Effects of triptolide on proliferation and apoptosis of human hepatocytes. Chin J Exper Tradit Med Form (中国试验方剂学杂志), 2012, 24(18):283-287.[18] YAO J C, LIU Y, HU L, et al. Reactive oxygen species in induction of apoptosis of human liver cells by triptolide. Central South Pharma (中南药学), 2012,10(8):561-564.[19] CHU K D, CHEN L D, NI F. Experimental study of alkaloids of Tripteryium wilfordii . Pharmacol Clin Chin Mater Med(中药药理与临床), 2011, 27(1):33-36.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
国家“重大新药创制”科技重大专项“药物安全评价技术平台”资助项目(2012ZX09302001)
{{custom_fund}}
PDF(1217 KB)
