【作者】 徐德祥；

【导师】 魏伟；

【作者基本信息】 安徽医科大学 ， 药理学， 2004， 博士

【摘要】 CYP3A是肝脏微粒体细胞色素P450中最重要的代谢酶，约占肝脏微粒体P450总量的25-28％，在药物代谢过程中起重要作用，约有60％临床药物在人体内经肝脏CYP3A代谢。孕烷X受体(PXR)是新发现的孤儿核受体(系统名：NR1I2)，主要在肝细胞表达。PXR是CYP3A基因表达的转录活化因子，在调控肝细胞CYP3A基因表达过程中发挥非常重要的作用。本文研究了脂多糖(LPS)对小鼠肝脏PXR及其靶基因CYP3A的下调作用，并深入探讨了Kupffer细胞、活性氧(ROS)和一氧化氮(NO)在LPS下调小鼠肝脏PXR和CYP3A中的作用。 1．LPS对小鼠肝脏核受体PXR及其靶基因CYP3A的下调作用 为研究LPS对小鼠肝脏PXR和CYP3A基因表达的下调作用，经腹腔注射给予小鼠一定剂量(0.1～5.0mg／kg)的LPS，给药后6～24h取肝脏组织，用Trizol一步法提取总RNA，用RT-PCR技术检测PXR和CYP3A11 mRNA水平。结果显示：经腹腔注射给予小鼠1.0 mg／kg的LPS处理后6h，小鼠肝脏PXR mRNA基础表达水平明显下降；LPS处理后12h，CYP3A11 mRNA基础表达水平也明显下降；LPS对PXR和CYP3A11的下调作用至少持续24h。进一步研究发现：所有剂量(0.1～5.0 mg／kg)的LPS对小鼠肝脏PXR和CYP3A11 mRNA基础表达水平均有一定的下调作用。为探讨LPS对地塞米松(DEX)、利福平(RIF)、米非司酮(RU486)和苯巴比妥(PB)诱导型CYP3A11 mRNA和红霉素N脱甲基酶(ERND)活性的影响，小鼠分别用DEX(40mg／kg i．p)、RIF(50 mg／kg，i．g)、RU486(50 mg／kg，s．c)或PB(75mg／kg，i．p)预处理3天。第4天，小鼠经腹腔注射一次给予1.0mg／kg的LPS，LPS处理后2h给予CYP3A诱导剂，LPS处理后12h和18h分别处死小鼠，用RT-PCR技术检测CYP3A11 mRNA水安徽医科大学博士学位论文平，用Nash法检测ERND活性。结果显示:LPS明显抑制DEX、RIF、RU486和PB对CYJ呀刁11 mRNA的诱导作用。这些研究结果表明:LPS对小鼠肝脏PXR和CYP3A有明显的下调作用。2.Kupffer细胞在LPS下调小鼠肝脏PXR及其靶基因CYP3A中的作用 为探讨Kupffer细胞在LPS下调小鼠肝脏PXR和CYP3An mRNA基础表达水平中的作用，经尾静脉注射1.0m留吨的三氯化札(GdCI3)以抑制小鼠肝脏KuPffer细胞功能，GdC13处理后24h经腹腔注射予1.0 mg/kg的LPS;为探讨Kupffer细胞在LPS下调小鼠肝脏诱导型C钾3An mRNA和ERND活性中的作用，小鼠用DEX(40 mg瓜9 i.p)、RIF(50 mg/kg，1.9)、RU486(50 mg/kg，s.e)或pB(75 mg瓜g，i.p)预处理3天，第3天同时经尾静脉注射1.0 mg/kg的GdC13，第4天经腹腔注射1.0 mg瓜g的LPS，LPs处理后Zh给予cYP3A诱导剂。LPs处理后第12h和第18h分别处死小鼠，用Trisof一步法提取总RNA并制备肝微粒体。用Rf-PCR检测PXR和CYP3An mRNA水平，用Nash法测定肝脏微粒体ERND活性。结果显示:LPS处理组小鼠肝脏PXR和CYP3An mRNA基础表达水平显著下降;GdC13预处理明显减弱LPS对小鼠肝脏PXR和CYP3AnmRNA基础表达水平的下调作用。进一步研究发现:LPS明显下调DEX、RIF、RU486和PB诱导的CYP3An mRNA表达水平和ERND活性;经GdC13预处理后，LPS下调诱导型CYP3An mRNA表达水平和ERND活性的作用明显减弱。这些研究结果提示:Kupffer细胞至少部分参与了LPS对小鼠肝脏PxR及其靶基因CYP3A的下调作用。3.ROS和NO在LPS下调小鼠肝脏PXR及其靶基因CYP3A中的作用 为探讨ROS在LPS下调小鼠肝脏PXR和CYP3An mRNA基础表达水平中的作用，Lps(1.0 mglkg i.p)处理前48h、24h、Zh以及LPs处理后Zh、6h，分别给予一定剂量的别嚓醇(ALL，100m岁kg，1.9.)或氯化二亚苯基碘(DPl，1.0mg/kg，s.c)，以抑制小鼠肝脏产生ROS;为探讨ROS在LPS下调小鼠肝脏诱导型eYP3Ali mRNA和ERND活性中的作用，小鼠用DEX(40 mg/kg i.p)、租F安徽医科大学博士学位论文(50mg/kg，1.9)、RU486(50 mg瓜g，s.e)或pB(75 mg/kg，i.p)预处理3天，第4天小鼠给予LpS(1.0 mg/kg i.p)和CYp3A诱导剂，LpS处理后Zh、6h分别给予ALL(100 mg/kg，1.9)或nPI(1.0 mg/kg，s.e);为探讨一氧化氮(No)在LPS下调小鼠肝脏PXR和CYP3An mRNA基础表达水平中的作用，LPS(l .0mg/kg i.p)处理前 48h、24h、Zh以及LPS处理后Zh、6h，分别经腹腔注射150m岁kg的一氧化氮合酶(iNOS)抑制剂氨基肌(AG);为探讨NO在LPS下调小鼠肝脏诱导型CYp3An mRNA和ERND活性中的作用，小鼠用DEx(40m留kgi.p)、RIF(50 mg/kg，1.9)、RU486(50 mg/kg，s.e)或pB(75 mg/kg，i.p)预处理3天，第4天小鼠给予LPS(l .0 mg瓜g，i.P)和CYP3A诱导剂， LPS处理后Zh、6h分别经腹腔注射150 mg/kg的AG。LPs处理后12h和18h分别处死小鼠，用Tr七oI一步法提取总RNA并制备肝微粒体。用Rf-PCR检测PXR和CYP3An mRNA水平，用Nash测定肝微粒体ERND活性。结果显示:经ALL或DH预处理后，LPS下调小鼠肝脏PXR和CYP3An mRNA基础表达水平以及诱导性CYP3An mRNA表达水平和ERND活性的作用明显减弱。然而，经 AG预处理后，LPS下调小鼠肝脏PXR和CYP3An mRNA基础表达水平、诱导性CYP3All mRNA表达水平以及肝脏微粒体ERND活性的作用未见明显变化。进一步研究发现:抗氧化剂N一乙酞半肤氨酸(NAc)和抗坏血酸(AA)?更多还原

【Abstract】 The cytochrome P450 3A (CYP3A) is a member of the cytochrome P-450 monooxygenase superfamily, which is responsible for the oxidative metabolism of numerous clinically used drugs. Pregnane X receptor (PXR), a member of the nuclear receptor superfamily, regulates CYP3A gene transcription in a ligand-dependent manner. In present study, we investigated the in vivo effects of lipopolysaccharide (LPS) on PXR and CYP3A expressions in mouse liver. We also explored the role of Kupffer cells and reactive oxygen species (ROS) in LPS-induced down-regulation of PXR and its target gene CYP3A in mouse liver.1. LPS down-regulates nuclear receptor Pregnane X receptor and its target gene CYP3A in mouse liverThe in vivo effects of LPS on PXR and its target gene CYP3A expressions in mouse liver were investigated in this study. Mice were injected intraperitoneally with different doses of LPS (0.1-5.0 mg/kg). PXR and CYP3A11 mRNA levels were measured using RT-PCR. Results indicated that LPS significantly inhibited the expressions of PXR mRNA in a dose-dependent manner, followed by suppression of CYP3A11 mRNA in mouse liver. LPS also repressed the up-regulation of CYP3A11 mRNA levels and erythromycin TV-demethylase (ERND) catalytic activities in mice pre-treated with PXR ligand dexamethasone (DEX), rifampicin (RIF), mifepristone (RU486) and Phenobarbital (PB). These results indicated that LPS down-regulatednuclear receptor pregnane X receptor and its target gene CYP3A in mouse liver.2. Kupffer cells partially mediate LPS-induced down-regulation of PXR and its target gene CYP3A in mouse liverTo investigate whether Kupffer cells mediate LPS-induced down-regulation of PXR and CYP3A in mouse liver, mice were treated with GdCl3, a selective Kupffer cell toxicant, to inactivate Kupffer cells. Mice were then injected intraperitoneally with 1.0 mg/kg LPS. PXR and CYP3A11 mRNA levels were measured using RT-PCR. ERND was used as an indicator of CYP3A catalytic activity. Results showed that LPS-induced down-regulation of PXR and CYP3A11 mRNA in liver was significantly attenuated in mice pretreated with GdCl3. A single dose of GdCl3 (10 mg/kg) pretreatment also significantly attenuated LPS-induced down-regulation on DEX, RIF, RU486 and PB-inducible CYP3A11 mRNA expressions and ERND activities in mouse liver. These results indicated that Kupffer cells contribute to LPS-induced down-regulation of PXR and CYP3A in mouse liver.3. The role of ROS and NO in LPS-induced down-regulation of nuclear receptor pregnane X receptor and its target gene CYP3A in mouse liverIn the present study, we investigated the in vivo role of reactive oxygen species ( ROS ) and nitric oxide (NO ) in LPS-induced down-regulation on CYP3A expressions. Mice were treated with allopurinol, an inhibitor of xanthine oxidase, to inhibite xanthine oxidase in mice. LPS-induced down-regulation of PXR and CYP3A11 mRNA in liver was significantly attenuated in mice pretreated with allopurinol. Allopurinol pretreatment also significantly attenuated LPS-induced down-regulation on DEX, RIF, RU486 and PB-inducible CYP3A11 mRNA expressions and ERND activities in mouse liver. Furthermore, LPS-induced down-regulation of PXR and CYP3A11 mRNA was significantly attenuated in mice pretreated withdiphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase. DPI pretreatments also attenuated the repressive effects of LPS on DEX, RIF, RU486 and PB-inducible CYP3A11 mRNA expressions and ERND catalytic activities in mouse liver. However, aminoguanidine, a selective inhibitor of inducible nitric oxide synthase (iNOS), has no effect on LPS-induced down-regulation of PXR and CYP3A11 mRNA. Finally, LPS-induced down-regulation of PXR and CYP3A11 mRNA was prevented in mice pretreated with either N-acetylcysteine (NAC) or ascorbic acid. These antioxidants also prevented the repressive effects of LPS on DEX, RIF, RU486 and PB-inducible CYP3A11 mRNA expressions and ERND catalytic activities in mouse liver. These results indicated that ROS, possibly produced b更多还原