槲皮素对大鼠肝和空肠P-糖蛋白表达及外排功能的影响

doi:10.11843/j.issn.0366-6964.2018.02.022

摘要/Abstract

摘要：

已证实P-糖蛋白（P-glycoprotein，P-gp，mdr1基因编码）可介导药物间的相互作用，且其表达和功能可受外源化合物的影响。本试验拟探讨黄酮类化合物槲皮素对P-gp表达和功能的影响以及其介导药物间相互作用的机制，为临床合理用药提供指导。随机选取30只健康大鼠，分组后分别采用15、60 mg·kg^-1剂量的槲皮素连续灌服5和15 d，比较处理组和对照组大鼠肝和空肠组织中mdr1a、mdr1b、PXR和CAR的mRNA表达量及P-gp的表达差异，并采用免疫组织化学方法研究槲皮素对P-gp在大鼠肝和空肠组织中定位的影响；然后另选取12只大鼠进一步采用单向在体肠灌流法研究高剂量槲皮素（60 mg·kg^-1连续灌胃15 d）处理后对伊维菌素空肠跨膜转运的影响，并采用体外方法研究槲皮素对Caco-2细胞ATP酶活性影响。试验结果显示： 60 mg·kg^-1槲皮素可显著（P<0.05）或极显著（P<0.01）增加大鼠肝和空肠组织中mdr1a、mdr1b、PXR和CAR的mRNA表达量，虽未影响P-gp在肝和空肠组织中的定位，但可极显著（P<0.01）增加P-gp的蛋白质表达量，该变化与CAR的表达存在显著的相关性（P<0.05）。15 mg·kg^-1的槲皮素处理大鼠15 d后，各基因的mRNA表达和P-gp表达量也会发生较为显著的变化。单向在体肠灌流试验结果显示槲皮素处理组大鼠对伊维菌素的肠渗透性显著（P<0.05）低于对照组；且槲皮素可极显著（P<0.01）提高Caco-2细胞的ATP酶活性。高剂量（60 mg·kg^-1）的槲皮素可从mRNA和蛋白质水平显著增加P-gp的表达，降低伊维菌素在大鼠空肠的渗透性。推测一定剂量的槲皮素可通过调控CAR来诱导健康大鼠P-gp的表达，并通过提高ATP酶活性水解ATP提供能量，进一步增强其对药物的跨膜转运功能。该结果提示临床用药时应考虑槲皮素介导的药物-药物间相互作用。

Abstract:

P-glycoprotein (P-gp), encoded by ABCB1/Mdr1, is a most important member of ABC family efflux transporter dictating the bioavailability of the various xenobiotics. The expression and function of P-gp can be influenced by other xenobiotics. The aim of the study is to explore the effect of quercetin, a promising herbal additive, on the expression and function of P-gp as well as the drug-drug interaction. Randomly selected rats were grouped and treated by quercetin (15 and 60 mg·kg^-1) for 5 and 15 days, respectively. mdr1a, mdr1b, PXR and CAR gene mRNA expression level and P-gp protein expression level and location were detected by real-time RT-PCR, Western bolt and immunohistochemistry, respectively. Single-pass intestinal perfusion was used to explore the effect of quercetin on ivermectin permeability in jejunum for another 12 rats wite quercetin for 15 days. Finally, ATPase activity was detected in quercetin-treated Caco-2 cells by using ATP-kit assay. Results were as follows:Quercetin can upregulate the mRNA expression level of mdr1a, mdr1b, PXR and CAR in rats liver and jejunum (P<0.05, P<0.01) as well as P-gp expression level (P<0.01). There is a correlation between P-gp and CAR expression level (P<0.05). Single pass intestinal perfusion experiment proved that quercetin affected the permeability of ivermectin in jejunum with declining K_a and P_app values (P<0.05). Compared with the control group, quercetin significantly induced the ATPase activity in Caco-2 cells (P<0.01). Quercetin upregulated the P-gp expression in liver and jejunum which was transcriptionally regulated through the activation of nuclear receptors (CAR), accordingly, affected the efflux transport of ivermectin from jejunum in the rats. This may have significant implications for long term use of quercetin causing drug-drug interaction during pharmacotherapy.

中图分类号:

S859.7

何方, Bhutto Zohaib, 郭荔, 王丽平. 槲皮素对大鼠肝和空肠P-糖蛋白表达及外排功能的影响[J]. 畜牧兽医学报, 2018, 49(2): 422-431.

HE Fang, Bhutto Zohaib, GUO Li, WANG Li-ping. Effect of Quercetin on P-glycoprotein Expression and Efflux Function in Liver and Jejunum of Rat[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(2): 422-431.

参考文献

[1] HENNESSY M, SPIERS J P. A primer on the mechanics of P-glycoprotein the multidrug transporter[J]. Pharmacol Res, 2007, 55(1):1-15.
[2] XIE J, LI D W, CHEN X W, et al. Expression and significance of hypoxia-inducible factor-1α and MDR1/P-glycoprotein in laryngeal carcinoma tissue and hypoxic Hep-2 cells[J]. Oncol Lett, 2013, 6(1):232-238.
[3] ZHANG Y C, ZHANG Y K, WANG Y J, et al. Esters of the marine-derived triterpene Sipholenol A reverse P-GP-mediated drug resistance[J]. Mar Drugs, 2015, 13(4):2267-2286.
[4] JETTÉ L, MURPHY G F, BÉLIVEAU R. Drug binding to P-glycoprotein is inhibited in normal tissues following SDZ-PSC 833 treatment[J]. Int J Cancer, 1998, 76(5):729-737.
[5] CHEN T, WANG C Y, LIU Q, et al. Dasatinib reverses the multidrug resistance of breast cancer MCF-7 cells to doxorubicin by downregulating P-gp expression via inhibiting the activation of ERK signaling pathway[J]. Cancer Biol Ther, 2015, 16(1):106-114.
[6] 萨础拉, 吕航, 姜艳艳, 等. 三七皂苷在大鼠外翻肠囊中的吸收及与P-糖蛋白相互作用研究[J]. 北京中医药大学学报, 2011, 34(12):836-842.
SA C L, LV H, JIANG Y Y, et al. Absorption of panax notoginseng saponins in rat everted gut sac and interaction between it and P-glycoprotein[J]. Journal of Beijing University of Traditional Chinese Medicine, 2011, 34(12):836-842. (in Chinese)
[7] 杨洋, 王世祥, 房敏峰, 等. 安息香醛、香草醛和β-细辛醚对P-糖蛋白功能的影响[J]. 中成药, 2012, 34(7):1364-1366.
YANG Y, WANG S X, FANG M F, et al. The effects of benzoic aldehyde, vanillin and beta-siocin on P-glycoprotein function[J]. Chinese Traditional Patent Medicine, 2012, 34(7):1364-1366. (in Chinese)
[8] BOOTS A W. Health effects of quercetin:from mechanism to nutraceutical[D]. Maastricht:Universitaire Pers Maastricht, 2006.
[9] VAN HOPPE S, SPARIDANS R W, WAGENAAR E, et al. Breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (P-gp/ABCB1) transport afatinib and restrict its oral availability and brain accumulation[J]. Pharmacol Res, 2017, 120:43-50.
[10] 林蓉, 刘俊田, 李旭, 等. 槲皮素对血管内皮细胞损伤的保护作用[J]. 中国循环杂志, 2000, 15(5):304-305.
LIN R, LIU J T, LI X, et al. Protection of vascular endothelial cells from hydrogen peroxide-induced injury by quercetin[J]. Chinese Circulation Journal, 2000, 15(5):304-305. (in Chinese)
[11] SCAMBIA G, PANICI P B, RANELLETTI F O, et al. Quercetin enhances transforming growth factor β₁, secretion by human ovarian cancer cells[J]. Int J Cancer, 1994, 57(2):211-215.
[12] BOERS F, DENG B L, LEMIÈRE G, et al. An improved synthesis of the anti-picornavirus flavone 3-O-methylquercetin[J]. Arch Pharm (Weinheim), 1997, 330(9-10):313-316.
[13] 钟华, 顾春红, 滕晔, 等. 槲皮素对K562/A02细胞多药耐药基因及糖蛋白表达的影响[J]. 上海医学, 2004, 27(1):20-21.
ZHONG H, GU C H, TENG Y, et al. Study of the effect of quercetin on expression of multidrug resistant gene and protein in K562/A02 cells[J]. Shanghai Medical Journal, 2004, 27(1):20-21. (in Chinese)
[14] 单保恩, 杨俊玲. 槲皮素对人子宫内膜癌耐药细胞株B-MD-C1(ADR^+/+)多药耐药基因mdr1及P-gp蛋白表达的影响[J]. 癌变畸变突变, 2009, 21(5):353-357.
SHAN B E, YANG J L. Mechanisms of multidrug resistance reversal on B-MD-C1(ADR^+/+)by quercetin[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2009, 21(5):353-357. (in Chinese)
[15] MITSUNAGA Y, TAKANAGA H, MATSUO H, et al. Effect of bioflavonoids on vincristine transport across blood-brain barrier[J]. Eur J Pharmacol, 2000, 395(3):193-201.
[16] LOZOYA-AGULLO I, ZUR M, BEIG A, et al. Segmental-dependent permeability throughout the small intestine following oral drug administration:single-pass vs. Doluisio approach to in-situ rat perfusion[J]. Int J Pharm, 2016, 515(1-2):201-208.
[17] 张海燕, 邬伟魁, 宋民宪, 等. 《中国药典》2010年版药用辅料标准探讨[J]. 中国实验方剂学杂志, 2011, 17(11):289-291.
ZHANG H Y, WU W K, SONG M X, et al. Standard of pharmaceutical excipients in 《Chinese Pharmacopoeia》 2010 edition[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2011, 17(11):289-291. (in Chinese)
[18] 王旭, 张爽. 槲皮素对卵巢癌细胞HO-8910增殖的抑制作用及机制[J]. 山东医药, 2010, 50(6):12-14.
WANG X, ZHANG S. Inhibitory effect and mechanism of quercetin on ovarian cancer cells Ho-8910 proliferation[J]. Shandong Medical Journal, 2010, 50(6):12-14. (in Chinese)
[19] 张继红, 梁力建, 黄洁夫. bcl-2基因表达变化在槲皮素抑制肝细胞癌生长中的作用[J]. 中国普外基础与临床杂志, 2009, 16(6):455-459.
ZHANG J H, LIANG L J, HUANG J F. Role of bcl-2 gene expression in inhibiting the growth of hepatocellular carcinoma of nude mice by quercetin[J]. Chinese Journal of Bases and Clinics in General Surgery, 2009, 16(6):455-459. (in Chinese)
[20] CHU C C, YANG J S, LU H F, et al. Quercetin-mediated cell cycle arrest and apoptosis involving activation of a caspase cascade through the mitochondrial pathway in human breast cancer MCF-7 cells[J]. Arch Pharm Res, 2010, 33(8):1181-1191.
[21] 陈华, 郑军. 槲皮素对HepG2细胞的抑制作用及对耐药基因和P-糖蛋白的影响[J]. 检验医学与临床, 2010, 7(9):791-794.
CHEN H, ZHENG J. The effect of quercetin on the inhibition of the growth of HepG2 and the expression of MDR₁ and P-gp[J]. Laboratory Medicine and Clinic, 2010, 7(9):791-794. (in Chinese)
[22] 翟莺莺, 周蕾, 赖永洪. 槲皮素抗肿瘤作用的研究[J]. 现代临床医学生物工程学杂志, 2005, 11(1):18-20.
ZHAI Y Y, ZHOU L, LAI Y H. Research of quercetin on Antitumour activit[J]. Journal of Modern Clinical Medical Bioengineering, 2005, 11(1):18-20. (in Chinese)
[23] 刘正清, 姚启盛, 杨勇, 等. 靶向mdr1b/mdr1a基因siRNA逆转T24/ADM多药耐药性研究[J]. 海南医学院学报, 2012, 18(7):865-868.
LIU Z Q, YAO Q S, YANG Y, et al. Effect of mdr1b/mdr1a siRNA on multidrug resistance of bladder carcinoma cell line T24/ADM[J]. Journal of Hainan Medical University, 2012, 18(7):865-868. (in Chinese)
[24] WILLSON T M, KLIEWER S A, WILLSON T M, et al. Pxr, car and drug metabolism[J]. Nat Rev Drug Discov, 2002, 1(4):259-266.
[25] YAN J, XIE W. A brief history of the discovery of PXR and CAR as xenobiotic receptors[J]. Acta Pharm Sin B, 2016, 6(5):450-452.
[26] SONODA J, PEI L M, EVANS R M. Nuclear receptors:decoding metabolic disease[J]. FEBS Lett, 2008, 582(1):2-9.
[27] GONG H B, SINZ M W, FENG Y, et al. Animal models of xenobiotic receptors in drug metabolism and diseases[J]. Methods Enzymol, 2005, 400:598-618.
[28] 王秀梅, 彭文兴, 文晓柯. MDR1调控的信号传导机制研究进展[J]. 中国医药指南, 2012, 10(30):436-440.
WANG X M, PENG W X, WEN X K. Progress in signal transduction mechanisms for transcriptional regulation of the MDR1 gene[J]. Guide of China Medicine, 2012, 10(30):436-440. (in Chinese)
[29] GRIFFIN J, FLETCHER N, CLEMENCE R, et al. Selamectin is a potent substrate and inhibitor of human and canine P-glycoprotein[J]. J Vet Pharmacol Ther, 2010, 28(3):257-265.
[30] MOLENTO M B, LIFSCHITZ A, SALLOVITZ J, et al. Influence of verapamil on the pharmacokinetics of the antiparasitic drugs ivermectin and moxidectin in sheep[J]. Parasitol Res, 2004, 92(2):121-127.