發(fā)布時間：2019-07-09 06:25

【摘要】：轉(zhuǎn)運體表達的改變是導(dǎo)致臨床上產(chǎn)生藥物相互作用的重要原因之一,其會影響合用藥物的體內(nèi)濃度以及代謝過程,從而對藥物的有效性及安全性產(chǎn)生不良影響。P-糖蛋白(P-glycoprotein,P-gp)是ABC轉(zhuǎn)運蛋白超家族成員之一,是一種能量依賴型的藥物外排泵,參與外源性和內(nèi)源性物質(zhì)的吸收和排泄。異源質(zhì)受體(chicken xenobiotic-sensing orphan nuclear receptor,CXR)是一種存在于禽類并與核受體超家族成員具有相似典型結(jié)構(gòu)與功能的核受體,參與P-gp表達的轉(zhuǎn)錄與調(diào)控,在機體異源性/內(nèi)源性物質(zhì)的代謝及排泄過程中起著重要調(diào)節(jié)作用。槲皮素(Quercetin,Quer)是一種天然且具有多種生理活性的黃酮類化合物,具有抗氧化、抗炎、抗過敏等多方面的藥理作用。近年來研究發(fā)現(xiàn)槲皮素對部分藥物的體內(nèi)藥動學(xué)過程具有調(diào)節(jié)作用,推測與槲皮素影響藥物轉(zhuǎn)運蛋白P-gp及CXR的表達有關(guān),本論文以此為切入點,探究了不同劑量的槲皮素(15和60 mg/kg)對健康肉雞肝臟、腎臟及腸組織mdr1(P-gp編碼基因)及CXRmRNA表達、口服恩諾沙星藥動學(xué)及腸道P-gp外排功能的影響,為闡明槲皮素是否會介導(dǎo)藥物間的相互作用提供參考。首先,本實驗通過實時熒光定量PCR反應(yīng)來探究槲皮素不同劑量(15和60mg/kg)與灌服不同時間(1,5和10天)后對健康肉雞mdr1及CXRmRNA表達的影響。與空白對照組肉雞相比,槲皮素(15和60mg/kg)在處理1天、5天、10天后可以極顯著誘導(dǎo)肝臟組織中mdr1mRNA的表達(P0.01);槲皮素(15和60 mg/kg)連續(xù)處理5、10天也可顯著誘導(dǎo)腎臟mdr1 mRNA表達(P0.05),除15 mg/kg槲皮素處理1天可極顯著抑制十二指腸組織中mdr1 mRNA的表達外,其余不同劑量的槲皮素(15和60mg/kg)對十二指腸及空腸組織中mdr1 mRNA表達均呈現(xiàn)了不同程度的誘導(dǎo)作用,而在回腸組織中其誘導(dǎo)作用不明顯。CXRmRNA的檢測結(jié)果顯示:與對照組相比較,不同劑量的槲皮素(15和60 mg/kg)在連續(xù)處理10天后對肝臟、腎臟及腸道組織中CXR mRNA的表達均有較強的抑制作用(P0.05);在空腸組織中,槲皮素(15和60 mg/kg)連續(xù)處理1、5、10天均可對空腸組織中CXRmRNA的表達有較強的抑制作用。而在十二指腸及回腸組織中其表達也受到了不同程度的抑制。結(jié)果提示槲皮素對mdr1mRNA表達的作用可能主要不是通過CXR途徑來調(diào)控的。基于槲皮素對mdr1 mRNA表達的影響,本論文進一步探討了不同劑量的槲皮素對健康肉雞口服恩諾沙星藥動學(xué)的影響。實驗采用高效液相色譜法(HPLC)檢測了恩諾沙星在血漿中的濃度變化。結(jié)果顯示,空白對照組在肉雞體內(nèi)的達峰時間Tmax為2.75±0.25h;血漿中峰濃度Cmax為2.67±0.30μg/mL,藥時曲線下面積 AUClast為23.75±2.03 μg·h/mL;清除率 CL 為 413.17±32.80 mL/h/kg;表觀分布容積 V 為 2.83 ± 0.60 mL/kg。經(jīng)槲皮素(15 mg/kg、60 mg/kg)處理一天后其達峰時間與對照組相比均有提前;其中槲皮素高劑量組(60 mg/kg)藥時曲線下面積AUClast(17.35± 1.04 μg-h/mL,P0.05)以及峰濃度Cmax(1.79 ±0.13 μg/mL,P0.05)與對照組相比均顯著減小;而實驗組清除率CL與對照組相比都有增加,且高劑量組差異顯著(563.45 ±29.76 mL/h/kg,P0.05);經(jīng)槲皮素誘導(dǎo)后其表觀分布容積與對照組相比也有增加的趨勢。本論文最后通過建立在體單向灌流模型來探討經(jīng)槲皮素處理后肉雞腸道P-gp對羅丹明123(Rho23)外排的影響。實驗分別以15mg/kg和60mg/kg槲皮素經(jīng)雞胃連續(xù)誘導(dǎo)3天,以Rho123為探針底物,選取空腸段進行在體灌流實驗。實驗結(jié)果顯示,槲皮素以不同濃度(15mg/kg、60mg/kg)連續(xù)誘導(dǎo)肉雞3天后,灌流液中Rho123的濃度有明顯升高趨勢,藥物吸收速率常數(shù)(Ka)減小;表觀滲透系數(shù)(Papp)顯著減小,且差異顯著(P=0.024,P=0.013),提示一定劑量的槲皮素可誘導(dǎo)健康肉雞空腸內(nèi)P-gp的mRNA表達并增強其對底物的外排作用。
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圖片說明：圖２－１邋ＣＸ民與其他核堂體氨基酸序列的比較（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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S859.7

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