槲皮素对大鼠肝和空肠P-糖蛋白表达及外排功能的影响

Effect of Quercetin on P-glycoprotein Expression and Efflux Function in Liver and Jejunum of Rat

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、60mg·kg-1剂量的槲皮素连续灌服5和15d,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(60mg·kg-1连续灌胃15d)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:60mg·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)。15mg·kg-1的槲皮素处理大鼠15d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(60mg·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。

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,apromising 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.mdr1 a,mdr1 b,PXRand CARgene mRNA expression level and P-gp protein expression level and location were detected by real-time RTPCR,Western bolt and immunohistochemistry,respectively.Single-pass intestinal perfusion wasused to explore the effect of quercetin on ivermectin permeability in jejunum for another 12 rats with 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 mdr1 a,mdr1 b,PXRand CARin 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 CARexpression level（P〈0.05）.Single pass intestinal perfusion experiment proved that quercetin affected the permeability of ivermectin in jejunum with declining Kaand Pappvalues（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.