槲皮素对肉鸡P-gp表达和功能以及肉鸡口服恩诺沙星药动学的影响
发布时间：2019-07-09 06:25
【摘要】：转运体表达的改变是导致临床上产生药物相互作用的重要原因之一,其会影响合用药物的体内浓度以及代谢过程,从而对药物的有效性及安全性产生不良影响。P-糖蛋白(P-glycoprotein,P-gp)是ABC转运蛋白超家族成员之一,是一种能量依赖型的药物外排泵,参与外源性和内源性物质的吸收和排泄。异源质受体(chicken xenobiotic-sensing orphan nuclear receptor,CXR)是一种存在于禽类并与核受体超家族成员具有相似典型结构与功能的核受体,参与P-gp表达的转录与调控,在机体异源性/内源性物质的代谢及排泄过程中起着重要调节作用。槲皮素(Quercetin,Quer)是一种天然且具有多种生理活性的黄酮类化合物,具有抗氧化、抗炎、抗过敏等多方面的药理作用。近年来研究发现槲皮素对部分药物的体内药动学过程具有调节作用,推测与槲皮素影响药物转运蛋白P-gp及CXR的表达有关,本论文以此为切入点,探究了不同剂量的槲皮素(15和60 mg/kg)对健康肉鸡肝脏、肾脏及肠组织mdr1(P-gp编码基因)及CXRmRNA表达、口服恩诺沙星药动学及肠道P-gp外排功能的影响,为阐明槲皮素是否会介导药物间的相互作用提供参考。首先,本实验通过实时荧光定量PCR反应来探究槲皮素不同剂量(15和60mg/kg)与灌服不同时间(1,5和10天)后对健康肉鸡mdr1及CXRmRNA表达的影响。与空白对照组肉鸡相比,槲皮素(15和60mg/kg)在处理1天、5天、10天后可以极显著诱导肝脏组织中mdr1mRNA的表达(P0.01);槲皮素(15和60 mg/kg)连续处理5、10天也可显著诱导肾脏mdr1 mRNA表达(P0.05),除15 mg/kg槲皮素处理1天可极显著抑制十二指肠组织中mdr1 mRNA的表达外,其余不同剂量的槲皮素(15和60mg/kg)对十二指肠及空肠组织中mdr1 mRNA表达均呈现了不同程度的诱导作用,而在回肠组织中其诱导作用不明显。CXRmRNA的检测结果显示:与对照组相比较,不同剂量的槲皮素(15和60 mg/kg)在连续处理10天后对肝脏、肾脏及肠道组织中CXR mRNA的表达均有较强的抑制作用(P0.05);在空肠组织中,槲皮素(15和60 mg/kg)连续处理1、5、10天均可对空肠组织中CXRmRNA的表达有较强的抑制作用。而在十二指肠及回肠组织中其表达也受到了不同程度的抑制。结果提示槲皮素对mdr1mRNA表达的作用可能主要不是通过CXR途径来调控的。基于槲皮素对mdr1 mRNA表达的影响,本论文进一步探讨了不同剂量的槲皮素对健康肉鸡口服恩诺沙星药动学的影响。实验采用高效液相色谱法(HPLC)检测了恩诺沙星在血浆中的浓度变化。结果显示,空白对照组在肉鸡体内的达峰时间Tmax为2.75±0.25h;血浆中峰浓度Cmax为2.67±0.30μg/mL,药时曲线下面积 AUClast为23.75±2.03 μg·h/mL;清除率 CL 为 413.17±32.80 mL/h/kg;表观分布容积 V 为 2.83 ± 0.60 mL/kg。经槲皮素(15 mg/kg、60 mg/kg)处理一天后其达峰时间与对照组相比均有提前;其中槲皮素高剂量组(60 mg/kg)药时曲线下面积AUClast(17.35± 1.04 μg-h/mL,P0.05)以及峰浓度Cmax(1.79 ±0.13 μg/mL,P0.05)与对照组相比均显著减小;而实验组清除率CL与对照组相比都有增加,且高剂量组差异显著(563.45 ±29.76 mL/h/kg,P0.05);经槲皮素诱导后其表观分布容积与对照组相比也有增加的趋势。本论文最后通过建立在体单向灌流模型来探讨经槲皮素处理后肉鸡肠道P-gp对罗丹明123(Rho23)外排的影响。实验分别以15mg/kg和60mg/kg槲皮素经鸡胃连续诱导3天,以Rho123为探针底物,选取空肠段进行在体灌流实验。实验结果显示,槲皮素以不同浓度(15mg/kg、60mg/kg)连续诱导肉鸡3天后,灌流液中Rho123的浓度有明显升高趋势,药物吸收速率常数(Ka)减小;表观渗透系数(Papp)显著减小,且差异显著(P=0.024,P=0.013),提示一定剂量的槲皮素可诱导健康肉鸡空肠内P-gp的mRNA表达并增强其对底物的外排作用。
文内图片：
图片说明：图２－１邋ＣＸ民与其他核堂体氨基酸序列的比较（Ｃｈｒｉｓｔｏｐｈ邋Ｈａｎｄｓｃｈｉｎ邋ｅｔ邋ａｌ，２０００）逡逑Ｆｉｇ．邋１－１邋Ａｍｉｎｏ邋ａｃｉｄ邋ｃｏｍｐａｒｉｓｏｎ邋ｂｅｔｗｅｅｎ邋ＣＸ民邋ａｎｄ邋ａ邋ｓｅｌｅｃｔｉｏｎ邋ｏｆ邋ｎｕｃｌｅａｒ邋ｒｅｃｅｐｔｏｒｓ．逡逑（Ｃｈｒｉｓｔｏｐｈ邋Ｈａｎｄｓｃｈｉｎ邋ｅｔ邋ａｌ，邋２０００）逡逑
[Abstract]:The change in the expression of the transporter is one of the most important causes of drug interaction in the clinic, which affects the in vivo concentration of the co-administered drug and the metabolic process, thereby adversely affecting the effectiveness and safety of the drug. P-glycoprotein (P-gp) is one of the superfamily members of the ABC transporter, an energy-dependent drug efflux pump, which is involved in the absorption and excretion of exogenous and endogenous substances. The heterogenous receptor (CXR) is a kind of nuclear receptor that is present in the poultry and has a similar structure and function to the superfamily members of the nuclear receptor, and is involved in the transcription and regulation of P-gp expression, and plays an important role in the metabolism and excretion of the heterogenous/ endogenous substances in the body. Quercetin (Quercetin, Quer) is a kind of flavonoid compound which is natural and has a variety of physiological activities, and has many pharmacological effects such as anti-oxidation, anti-inflammatory, antiallergic and the like. In recent years, it has been found that quercetin has a regulating effect on the pharmacokinetics of some drugs, and it is suggested that quercetin (15 and 60 mg/ kg) of quercetin (15 and 60 mg/ kg) can be used for the liver of healthy broilers. The effects of mdr1 (P-gp and P-gp) on the expression of mdr1 (P-gp) and the expression of CXRmRNA, oral enrofloxacin and the efflux of P-gp in the intestinal tract were investigated. First, the effects of different doses of quercetin (15 and 60 mg/ kg) and different time (1,5 and 10 days) on the expression of mdr1 and CXRmRNA in healthy broilers were investigated by real-time fluorescence quantitative PCR. Compared with blank control group, quercetin (15 and 60 mg/ kg) could significantly induce the expression of mdr1 mRNA in liver tissue after 1 day,5 days and 10 days (P 0.01). Quercetin (15 and 60 mg/ kg) was treated continuously for 5 and 10 days, and the expression of mdr1 mRNA in the kidney was also significantly induced (P0.05). In addition to 15 mg/ kg of quercetin, the expression of mdr1 mRNA in the duodenum and jejunum tissues was significantly inhibited, and the expression of mdr1 mRNA in the duodenum and jejunum tissues was induced by the remaining different doses of quercetin (15 and 60 mg/ kg). The results of the detection of the CXRmRNA showed that the expression of the CXR mRNA in the liver, the kidney and the intestinal tissues was stronger than that of the control group (P0.05). In the jejunal tissue, the expression of the CXR mRNA in the liver, the kidney and the intestinal tissues was stronger (P0.05). Quercetin (15 and 60 mg/ kg) can inhibit the expression of CXRmRNA in jejunal tissue by 1,5 and 10 days. The expression of the tissues in the duodenum and the ileum was also inhibited. The results suggest that the effect of quercetin on mdr1mRNA expression may not be regulated by the CXR pathway. Based on the effect of quercetin on mdr1 mRNA expression, this paper further discusses the effect of different doses of quercetin on the pharmacokinetics of oral enrofloxacin in healthy broilers. The concentration of enrofloxacin in plasma was measured by high performance liquid chromatography (HPLC). The results showed that the peak time Tmax of the blank control group in the broiler was 2.75 to 0.25 h, the peak concentration of the plasma was 2.67 to 0.30. m u.g/ mL, the area AUClast was 23.75 and 2.03. m u.g 路 h/ mL, the clearance CL was 413.17 to 32.80 mL/ h/ kg, and the apparent distribution volume V was 2.83 to 0.60 mL/ kg. The peak time of quercetin (15 mg/ kg,60 mg/ kg) was earlier than that in the control group. The area of AUClast (17.35-1.04. mu.g-h/ mL, P0.05) and the peak-concentration Cmax (1.79-0.13. mu.g/ mL, P0.05) were significantly reduced in the high-dose group (60 mg/ kg). Compared with the control group, the clearance CL of the experimental group increased significantly (563.45, 29.76 mL/ h/ kg, P <0.05), and the apparent distribution volume of the experimental group was higher than that of the control group. In this paper, the effect of P-gp on the efflux of Rhodamine 123 (Rho23) in broiler chickens treated with Quercetin was studied by establishing a in vivo one-way perfusion model. The experiment was carried out in vivo with 15 mg/ kg and 60 mg/ kg of quercetin for 3 days, and Rho123 was the probe substrate and the jejunum section was selected for the in-vivo perfusion experiment. The results showed that the concentration of Rho123 in the perfusate was significantly higher after 3 days of continuous induction with different concentration (15 mg/ kg and 60 mg/ kg), and the rate constant of drug absorption (Ka) was decreased, the apparent permeability coefficient (Papp) was significantly reduced, and the difference was significant (P = 0.024, P = 0.013). It is suggested that a dose of quercetin can induce the expression of P-gp in the jejunum of the healthy broiler and enhance the efflux of the substrate.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.7
本文编号：2511924
文内图片：
图片说明：图２－１邋ＣＸ民与其他核堂体氨基酸序列的比较（Ｃｈｒｉｓｔｏｐｈ邋Ｈａｎｄｓｃｈｉｎ邋ｅｔ邋ａｌ，２０００）逡逑Ｆｉｇ．邋１－１邋Ａｍｉｎｏ邋ａｃｉｄ邋ｃｏｍｐａｒｉｓｏｎ邋ｂｅｔｗｅｅｎ邋ＣＸ民邋ａｎｄ邋ａ邋ｓｅｌｅｃｔｉｏｎ邋ｏｆ邋ｎｕｃｌｅａｒ邋ｒｅｃｅｐｔｏｒｓ．逡逑（Ｃｈｒｉｓｔｏｐｈ邋Ｈａｎｄｓｃｈｉｎ邋ｅｔ邋ａｌ，邋２０００）逡逑
[Abstract]:The change in the expression of the transporter is one of the most important causes of drug interaction in the clinic, which affects the in vivo concentration of the co-administered drug and the metabolic process, thereby adversely affecting the effectiveness and safety of the drug. P-glycoprotein (P-gp) is one of the superfamily members of the ABC transporter, an energy-dependent drug efflux pump, which is involved in the absorption and excretion of exogenous and endogenous substances. The heterogenous receptor (CXR) is a kind of nuclear receptor that is present in the poultry and has a similar structure and function to the superfamily members of the nuclear receptor, and is involved in the transcription and regulation of P-gp expression, and plays an important role in the metabolism and excretion of the heterogenous/ endogenous substances in the body. Quercetin (Quercetin, Quer) is a kind of flavonoid compound which is natural and has a variety of physiological activities, and has many pharmacological effects such as anti-oxidation, anti-inflammatory, antiallergic and the like. In recent years, it has been found that quercetin has a regulating effect on the pharmacokinetics of some drugs, and it is suggested that quercetin (15 and 60 mg/ kg) of quercetin (15 and 60 mg/ kg) can be used for the liver of healthy broilers. The effects of mdr1 (P-gp and P-gp) on the expression of mdr1 (P-gp) and the expression of CXRmRNA, oral enrofloxacin and the efflux of P-gp in the intestinal tract were investigated. First, the effects of different doses of quercetin (15 and 60 mg/ kg) and different time (1,5 and 10 days) on the expression of mdr1 and CXRmRNA in healthy broilers were investigated by real-time fluorescence quantitative PCR. Compared with blank control group, quercetin (15 and 60 mg/ kg) could significantly induce the expression of mdr1 mRNA in liver tissue after 1 day,5 days and 10 days (P 0.01). Quercetin (15 and 60 mg/ kg) was treated continuously for 5 and 10 days, and the expression of mdr1 mRNA in the kidney was also significantly induced (P0.05). In addition to 15 mg/ kg of quercetin, the expression of mdr1 mRNA in the duodenum and jejunum tissues was significantly inhibited, and the expression of mdr1 mRNA in the duodenum and jejunum tissues was induced by the remaining different doses of quercetin (15 and 60 mg/ kg). The results of the detection of the CXRmRNA showed that the expression of the CXR mRNA in the liver, the kidney and the intestinal tissues was stronger than that of the control group (P0.05). In the jejunal tissue, the expression of the CXR mRNA in the liver, the kidney and the intestinal tissues was stronger (P0.05). Quercetin (15 and 60 mg/ kg) can inhibit the expression of CXRmRNA in jejunal tissue by 1,5 and 10 days. The expression of the tissues in the duodenum and the ileum was also inhibited. The results suggest that the effect of quercetin on mdr1mRNA expression may not be regulated by the CXR pathway. Based on the effect of quercetin on mdr1 mRNA expression, this paper further discusses the effect of different doses of quercetin on the pharmacokinetics of oral enrofloxacin in healthy broilers. The concentration of enrofloxacin in plasma was measured by high performance liquid chromatography (HPLC). The results showed that the peak time Tmax of the blank control group in the broiler was 2.75 to 0.25 h, the peak concentration of the plasma was 2.67 to 0.30. m u.g/ mL, the area AUClast was 23.75 and 2.03. m u.g 路 h/ mL, the clearance CL was 413.17 to 32.80 mL/ h/ kg, and the apparent distribution volume V was 2.83 to 0.60 mL/ kg. The peak time of quercetin (15 mg/ kg,60 mg/ kg) was earlier than that in the control group. The area of AUClast (17.35-1.04. mu.g-h/ mL, P0.05) and the peak-concentration Cmax (1.79-0.13. mu.g/ mL, P0.05) were significantly reduced in the high-dose group (60 mg/ kg). Compared with the control group, the clearance CL of the experimental group increased significantly (563.45, 29.76 mL/ h/ kg, P <0.05), and the apparent distribution volume of the experimental group was higher than that of the control group. In this paper, the effect of P-gp on the efflux of Rhodamine 123 (Rho23) in broiler chickens treated with Quercetin was studied by establishing a in vivo one-way perfusion model. The experiment was carried out in vivo with 15 mg/ kg and 60 mg/ kg of quercetin for 3 days, and Rho123 was the probe substrate and the jejunum section was selected for the in-vivo perfusion experiment. The results showed that the concentration of Rho123 in the perfusate was significantly higher after 3 days of continuous induction with different concentration (15 mg/ kg and 60 mg/ kg), and the rate constant of drug absorption (Ka) was decreased, the apparent permeability coefficient (Papp) was significantly reduced, and the difference was significant (P = 0.024, P = 0.013). It is suggested that a dose of quercetin can induce the expression of P-gp in the jejunum of the healthy broiler and enhance the efflux of the substrate.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.7
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