细胞色素P450 2E1在依达拉奉减轻免疫性肝损伤氧化应激中的作用

目的观察细胞色素P450 2E1(CYP 2E1)在依达拉奉作用于刀豆蛋白A(Con A)致小鼠免疫性肝损伤模型中的变化。方法先期给予依达拉奉,之后用Con A腹腔注射致敏小鼠产生急性免疫性肝损伤,观察肝脏病理变化,分光光度法测血清AST和ALT浓度,肝匀浆中SOD、MDA、GSH含量,RT-PCR技术检测肝脏组织CYP 2E1 mRNA水平,Westernblot技术检测肝脏组织CYP 2E1蛋白表达水平。结果与模型组比较,依达拉奉能降低免疫性肝损伤小鼠血清中升高的ALT、AST含量;降低肝匀浆中的MDA水平,升高其降低的SOD、GSH水平;CYP 2E1 mRNA水平明显降低,CYP 2E1蛋白表达水平明显下调。结论依达拉奉对小鼠急性免疫性肝损伤具有一定的保护作用,这可能与它降低CYP 2E1的表达,清除自由基,增强机体抗脂质过氧化能力有关。     

心血管药物速效救心丸和通心络胶囊对大鼠细胞色素P450酶的影响

目的:观察速效救心丸和通心络胶囊对大鼠药物代谢酶细胞色素P450(CYP450)的影响。方法:给予大鼠试验药物后制备肝及小肠微粒体,CO还原差示光谱法测定肝微粒体CYP450含量,蛋白免疫印迹法检测肝微粒体中CYP1A2、CYP2C11、CYP2E1、CYP3A和肠微粒体中CYP3A蛋白的表达量。结果:速效救心丸组和通心络胶囊组与空白对照组比较,大鼠肝脏CYP450含量无显著变化(P>0.05);但是West-ern blotting实验对肝脏和肠道中多种CYP450酶表达量测定的结果显示,速效救心丸可诱导肝脏CYP1A2表达,但抑制肝脏CYP2C11和肠道CYP3A表达(P<0.05);通心络胶囊可诱导大鼠肝脏CYP1A2、CYP2E1蛋白表达(P<0.05)。结论:速效救心丸和通心络胶囊对大鼠肝脏CYP450酶总量无影响,但是,在蛋白质水平对特定亚型蛋白的表达量有影响,由此可能引发的药物相互作用不容忽视。     

参麦注射液和注射用血塞通对大鼠肝脏及肠道药物代谢酶CYP450的影响

目的考察参麦注射液和注射用血塞通对大鼠药物代谢酶细胞色素P450(CYP450)的影响。方法连续给予大鼠试验药物后,制备肝及小肠微粒体;用CO还原差示光谱法,测定肝微粒体CYP450含量;用蛋白免疫印迹法,检测肝微粒体中CYP1A2、CYP2C11、CYP2E1、CYP3A和肠微粒体中CYP3A蛋白的表达量。结果参麦注射液和注射用血塞通组与空白对照组大鼠的肝脏总P450含量无显著性差异(P>0.05)。但参麦注射液可显著诱导肝脏CYP2E1表达,但抑制肠微粒体中CYP3A表达;肝CYP1A2、CYP2C11水平均有所上升。血塞通可显著诱导大鼠肝脏CYP1A2、CYP2E1、CYP2C11、CYP3A蛋白表达,肠CYP3A的表达水平显示出下降趋势。结论参麦注射液和注射用血塞通对大鼠肝脏P450酶总量无影响;但对特定亚型蛋白的表达量有影响,由此可能引发的药物相互作用不容忽视。     

地塞米松对大鼠肝脏微粒体细胞色素P-450的诱导效应

目的:观察地塞米松(dexamethasone,DEX)对细胞色素P-450(cytochrome P450,CYP450)的诱导效应,并探讨其诱导机制.方法:雄性Wistar大鼠分别以DEX 0,25,50和100mg·kg-1·d-1诱导处理(ip)4d后,测定大鼠肝脏总CYP450含量,CYP3A1,CYP3A2和CYP2B 1/2的mRNA及蛋白的表达水平,肝脏ERD(CYP3A活性),PROD(CYP2B活性)和BROD(总CYP450活性).结果:CYP450含量、ERD、PROD和BROD活性在DEX多次诱导后都有升高.CYP3A1 mRNA表达水平、蛋白含量和酶活性有明显的升高,剂量效应关系明显;CYP3A2蛋白明显升高,但mRNA表达水平却无明显变化.PROD和CYP2B1/2的mRNA表达水平以及总CYP450含量均在50mg·kg-1·d-1剂量组达到最高值.结论:DEX对雄性大鼠主要诱导CYP3A1和CYP3A2两个CYP450成员表达上调,对CYP2B1/2也有一定的诱导作用,其诱导作用主要表现在酶活性、酶蛋白含量和mRNA表达水平的升高.CYP3A1和CYP3A2的诱导方式可能不同.     

淫羊藿苷对不同月龄大鼠肝微粒体细胞色素P450的影响

目的 揭示淫羊藿苷(Ica)对大鼠肝微粒体细胞色素P450的含量及部分亚型的影响,并比较月龄的差异.方法 ig给予6月龄和18月龄的♂SD大鼠Ica( 60 mg· kg -1),4周后取肝脏,用钙沉淀法提取肝微粒体,BCA法测定微粒体蛋白浓度;用一氧化碳还原差示光谱法测定CYP450的含量;用ELISA法测定CYP1 A1、CYPb5的含量;用比色法测定苯胺羟化酶(反映CYP2E1活性)和红霉素-N-脱甲基酶(反映CYP3A活性)的活性;用real - time RT - PCR检测CYP1 A1、CYP2A3、CYP2E1、CYP3A1、CYP3A2和CYP4B1 mRNA的表达.结果 60 mg· kg-1 Ica明显增加了CYP450的总酶和CYP1 A1的含量、CYP3A的活性及CYP1 A1、CYP3A1、CYP3A2 mRNA的表达,降低了CYP2E1的活性及其mRNA的表达;但Ica对上述各指标的诱导或抑制作用在大鼠月龄方面差异不明显;Ica对CYPb5的含量及CYP2A3、CYP4B1 mRNA的表达未见明显影响.结论 Ica对大鼠肝微粒体CYP450总酶、CYPI A1和CYP3A具有诱导作用,对CYP2E1具有抑制作用,该作用未见明显月龄差异.     

五味子乙素对CYP450药物代谢酶诱导作用的体外研究

目的 研究五味子乙素对细胞色素P450(CYP450)酶活性和mRNA表达是否有诱导作用.方法 采用底物法测定人肝原代细胞CYP1A2、CYP2B6和CYP3A4酶的活性,荧光定量PCR检测mRNA的表达.结果 五味子乙素对CYP1A2和CYP3A4的酶活性均低于阳性对照的40%,高浓度的五味子乙素对CYP2B6的酶活性均高于阳性对照的40%;五味子乙素对CYP1A2的mRNA表达小于阴性对照的4倍,高浓度的五味子乙素对CYP2B6和CYP3A4的mRNA表达大于阴性对照的4倍.结论 五味子乙素对CYP1A2和CYP3A4的酶活性没有诱导作用,对CYP2B6的酶活性有潜在诱导作用;五味子乙素对CYP1A2的mRNA表达没有诱导作用,对CYP2B6和CYP3A4的mRNA表达有潜在诱导作用.     

灯盏花素对张氏肝细胞中CYP3A4和CYP2C19的影响和机制

目的：观察灯盏花素对人张氏肝细胞中孕烷X受体（PXR）及细胞色素P450 3A4（CYP3A4）和细胞色素P450 2C19（CYP2C19） mRNA和蛋白表达的影响,初步探讨灯盏花素对CYP3A4和CYP2C19的影响及其作用机制。方法： MTT法检测不同浓度的灯盏花素对张氏肝细胞活力的影响;采用实时荧光定量PCR（RT-qPCR）检测不同浓度灯盏花素对张氏肝细胞中PXR、CYP3A4及CYP2C19 mRNA的表达;采用Western-blot检测不同浓度的灯盏花素对CYP3A4和CYP2C19蛋白的表达影响。结果： MTT结果表明不同浓度的灯盏花素处理张氏肝细胞24 h、48 h后的IC₅₀分别为9 768.8、1 372.8 μmol·L^-1,IC₁₀分别为2 689.2、154.4 μmol·L^-1;RT-qPCR结果表明灯盏花素对CYP3A4和PXR mRNA的影响呈抑制作用,对CYP2C19 mRNA的影响呈不同程度的促进作用;Western-blot结果表明灯盏花素对CYP3A4蛋白的影响呈抑制作用,对CYP2C19蛋白的影响呈促进作用。结论：灯盏花素对CYP3A4的抑制作用可能通过抑制PXR途径;对CYP2C19的促进作用可能不经PXR通路。     

辣木叶及辣木籽对大鼠CYP450酶3种亚型基因表达影响

目的:研究辣木叶及辣木籽对大鼠肝脏CYP450亚型酶中mRNA及蛋白表达量的影响。方法:将SD大鼠随机分为空白组(0.5%羧甲基纤维素钠混悬溶液)、辣木叶高、中、低剂量组(分别为0.813 8,0.406 9,0.203 5g·kg^-1);辣木籽高、中、低剂量组(分别为1.067 4,0.533 7,0.266 9g·kg^-1)。灌胃给药,给药容量为10mL·kg^-1,2次/天,连续给药14d,取大鼠肝脏,采用实时荧光定量PCR(RT-qPCR)及蛋白免疫印迹(Western-blot)法检测大鼠肝脏中CYP2E1、CYP3A1及CYP1A2的mRNA及蛋白相对表达量。结果:辣木叶高剂量组对CYP2E1的mRNA表达有明显抑制作用(P<0.05);在(0.813 8±0.203 5)g·kg^-1剂量范围内,辣木叶对CYP1A2mRNA表达的抑制作用随给药剂量增加而增加(P<0.05)。本实验剂量范围内的辣木籽对3个酶的mRNA表达都有明显的抑制作用(P<0.05),其抑制效率CYP2E1>CYP3A1>CYP1A2。高剂量组辣木叶及辣木籽对CYP2E1蛋白表达都有明显抑制作用(P<0.01);中、低剂量组的辣木籽对CYP3A1及CYP1A2蛋白表达都有不同程度的抑制作用(P<0.05;P<0.05);中剂量辣木叶对CYP1A2蛋白表达抑制作用显著(P<0.01)。结论:辣木叶及辣木籽对大鼠肝脏中3个亚型酶的mRNA及蛋白表达都有不同程度的抑制作用。     

地非三唑对大鼠肝细胞细胞色素P450 CYP1A1/2的诱导作用

目的试从mRNA表达水平阐明地非三唑对鼠肝微粒体中细胞色素P450 CYP1A1/2的诱导机制。方法给SD大鼠腹腔注射地非三唑,采用Tr-izol法提取大鼠肝脏RNA,用RT-PCR测定经地非三唑处理1,2及4d的鼠肝中细胞色素P450 CYP1A1,CYP1A2 mRNA的表达水平。结果地非三唑处理不同时间的鼠肝细胞中细胞色素P450CYP1A1,CYP1A2 mRNA的表达水平比空白对照组明显增加,空白对照组CYP1A1吸光度比值为0.270±0.040,诱导1,2及4d的吸光度比值分别为0.343±0.055,0.417±0.045及0.603±0.083;空白对照组的CYP1A2吸光度比值为0.613±0.189,而诱导1,2及4d的吸光度比值分别为1.510±0.226,3.057±0.518及4.120±0.458。随着诱导时间的增加,细胞色素P450 CYP1A1及CYP1A2 mRNA的表达也逐步增加,诱导时间与表达水平之间存在一定的线性关系,相关系数分别为0.9984和0.9563。结论地非三唑对细胞色素P450 CYP1A1/2 mRNA表达具有诱导作用。     

地非三唑对大鼠肝细胞细胞色素P450 CYP1A1/2的诱导作用

目的 试从mRNA表达水平阐明地非三唑对鼠肝微粒体中细胞色素P450 CYP1A1/2的诱导机制。方法 给SD大鼠腹腔注射地非三唑，采用Trizol法提取大鼠肝脏RNA,用RT-PCR测定经地非三唑处理1, 2及4 d的鼠肝中细胞色素P450 CYP1A1, CYP1A2 mRNA的表达水平。结果 地非三唑处理不同时间的鼠肝细胞中细胞色素P450 CYP1A1, CYP1A2 mRNA的表达水平比空白对照组明显增加，空白对照组CYP1A1吸光度比值为0.270±0.040, 诱导1, 2及4 d的吸光度比值分别为0.343±0.055, 0.417±0.045及0.603±0.083；空白对照组的CYP1A2吸光度比值为0.613±0.189, 而诱导1，2及4 d的吸光度比值分别为1.510±0.226, 3.057±0.518及4.120±0.458。随着诱导时间的增加，细胞色素P450 CYP1A1及CYP1A2 mRNA的表达也逐步增加，诱导时间与表达水平之间存在一定的线性关系,相关系数分别为0.9984和0.9563。结论 地非三唑对细胞色素P450 CYP1A1/2 mRNA表达具有诱导作用。     

川芎嗪诱导小鼠肝药酶Cyp3a11的作用及其机制研究

目的 考察川芎嗪对小鼠肝药物代谢酶Cyp3a11（CYP3A4同源基因）的作用及相关机制。方法 24只8周龄C57BL/6小鼠随机分为对照组和川芎嗪低、中、高剂量（25,50,100 mg·kg^-1）组,连续灌胃给药15 d。以RT-PCR法检测肝脏Cyp3a11 mRNA表达。采用差速离心法提取肝微粒体,以Cyp3a11经典底物咪达唑仑进行孵育,HPLC-MS/MS检测肝脏Cyp3a11活性。以双荧光报告基因法分析川芎嗪对CYP3A4转录活性的影响。结果 川芎嗪可诱导小鼠肝脏中Cyp3a11的mRNA表达,且诱导作用呈剂量依赖性增加。高剂量组可诱导Cyp3a11的代谢活性升高。川芎嗪可通过核受体PXR途径激活CYP3A4启动子转录活性。结论 川芎嗪对CYP3A4同源基因Cyp3a11的表达和活性具有一定的诱导作用,提示在临床使用过程中可能存在潜在的药物相互作用风险。     

Modulatory actions of o-coumaric acid on carcinogen-activating cytochrome P450 isozymes and the potential for drug interactions in human hepatocarcinoma cells

Abstract

Context: Although humans are exposed to o-coumaric acid (OCA) in their diet, there is no available literature related to drug interaction and the carcinogen-activating potential of OCA in the HepG2 cell line.

Objective: This study was undertaken to determine the effects of OCA on the cytochrome P450 (CYP) 1A2, CYP2E1, CYP2C9, and CYP3A4 enzymes, which are primarily involved in carcinogen and drug metabolism.

Materials and methods: The cytotoxicity of OCA in HepG2 cells was investigated by measuring the cleavage of WST-1. The protein and mRNA levels of CYPs were determined by western blotting and RT-PCR, respectively.

Results: The EC10, EC25, and EC50 values of OCA were calculated to be 1.84, 3.91 and 7.39?mM, respectively. A sublethal dose of 5?mM was used throughout this study. The CYP1A2 protein and mRNA levels were increased by 52 and 40% (p?<?0.05), as were the CYP2E1 levels by 225 and 424%, respectively (p?<?0.05). However, OCA treatment caused 52 and 60% decreases in the levels of CYP3A4 protein and mRNA (p?<?0.05), respectively. In contrast to CYP3A4, the CYP2C9 protein and mRNA levels increased by 110 and 130%, respectively.

Discussion and conclusion: Co-administration of OCA with some drugs may lead to undesirable food–drug interactions due to modulatory effects on CYP isozymes involved in drug metabolism. Moreover, exposure to OCA may cause an increase in carcinogenicity and toxicity due to the induction of the CYP isozymes involved in chemical carcinogenesis. Therefore, serious precautions should be taken when using OCA as a supplement.     

Effect of citral on mouse hepatic cytochrome P450 enzymes

Context: Citral is used as a potential natural treatment for various infectious diseases.

Objective: To examine the effect of citral on the mRNA expression and activities of cytochrome P450 (CYP450) enzymes and establish the relationship between citral-induced liver injury and oxidative stress.

Materials and methods: ICR mice were randomly divided into citral (20, 200, and 2000?mg/kglow), Tween-80, and control groups (0.9% saline), 10 mice in each group. The citral-treated groups were intragastrically administered citral for 3 d, control groups treated with 0.5% Tween-80 and 0.9% saline in the same way. Liver injury and CYP450 enzymes were analyzed by analyzing the histopathological changes and the changes of related enzymes.

Results: Citral treatment (2000?mg/kg) for 3 d increased serum glutamic pyruvic transaminase and glutamic oxaloacetic transaminase levels, as well as glutathione, gydroxyl radicals, malonaldehyde and total superoxide dismutase contents, but decreased the content of total antioxidant capacity. In doses of 20 and 200?mg/kg groups mice, the contents of NO were decreased significantly and other changes were similar to the 2000?mg/kg group mice, but the liver damage was most severe in the 2000?mg/kg group. Citral induced the mRNA expression and activities of CYP450 1A2, 2D22, and 2E1 in the liver of mice at doses of 20 and 200?mg/kg. There were no changes in testing indexes in Tween-80 treated group mice. Due to its toxic effects, the CYP induction effect of citral negatively correlated with its dose. Although the mRNA expression of CYP450 3A11 was induced by citral, its activity was not affected by low and moderate doses of citral. CYP450 3A11 activity was significantly decreased by high-dose citral.

Conclusions: Citral is hepatotoxic and induced oxidative stress in higher dose, which has a negative effect on CYP450 enzymes. These data suggest caution needs to be taken in order to avoid citral-drug interactions in human beings.     

Selective role for tumor necrosis factor-α, but not interleukin-1 or Kupffer cells, in down-regulation of CYP3A11 and CYP3A25 in livers of mice infected with a noninvasive intestinal pathogen

Hepatic cytochrome P450 (P450) gene and protein expression are modulated during inflammation and infection. Oral infection of C57BL/6 mice with Citrobacter rodentium produces mild clinical symptoms while selectively regulating hepatic P450 expression and elevating levels of proinflammatory cytokines. Here, we explored the role of cytokines in the regulation of hepatic P450 expression by orally infecting tumor necrosis factor-α (TNFα) receptor 1 null mice (TNFR1−/−), interleukin-1 (IL1) receptor null mice (IL1R1−/−), and Kupffer cell depleted mice with C. rodentium. CYP4A mRNA and protein levels and flavin monooxygenase (FMO)3 mRNA expression levels were down-regulated, while CYP2D9 and CYP4F18 mRNAs remained elevated during infection in wild-type, receptor knockout, and Kupffer cell depleted mice. CYPs 3A11 and 3A25 mRNA levels were down-regulated during infection in wild-type mice but not in TNFR1−/− mice. Consistent with this observation, CYPs 3A11 and 3A25 were potently down-regulated in mouse hepatocytes treated with TNFα. Oral infection of IL1R1−/− mice and studies with mouse hepatocytes indicated that IL1 does not directly regulate CYP3A11 or CYP3A25 expression. Uninfected mice injected with clodronate liposomes had a significantly reduced number of Kupffer cells in their livers. Infection increased the Kupffer cell count, which was attenuated by clodronate treatment. The P450 mRNA and cytokine levels in infected Kupffer cell depleted mice were comparable to those in infected mice receiving no clodronate. The results indicate that TNFα is involved in the regulation of CYPs 3A11 and 3A25, but IL1β and Kupffer cells may not be relevant to hepatic P450 regulation in oral C. rodentium infection.     

Down-Regulation of Hepatic CYP3A in Chronic Renal Insufficiency

Purpose. The objective of this study was to investigate the mechanisms underlying the decrease in hepatic clearance of some drugs metabolized by CYP450 enzymes in chronic renal insufficiency (CRI). Methods. CRI was induced in male Sprague-Dawley rats (n = 7) by the remnant kidney model (RKM); control animals (C) (n = 12) underwent sham surgery, of which n = 6 rats were pair-fed (CPF) with CRI rats and others (n = 6) had free access to food. Serum creatinine (S_cr) and urea nitrogen (SUN) were monitored every 2 weeks. On day 36, livers were isolated, and microsomes were prepared. Catalytic activities were measured through O-demethylation (CYP2D) and N-demethylation of dextromethorphan (CYP3A) and O-deethylation of 7-ethoxyresorufin (CYP1A2). CYP450 protein and mRNA levels were also measured. Results. Compared with CPF, S_cr and SUN levels in CRI rats were increased twofold (p < 0.01) and 2.5-fold (p < 0.01), respectively. No effect on CYP1A2 and CYP2D activities, mRNA, or protein levels was observed between the groups. There was a reduction (41.8 ± 20%, p < 0.01) in CYP3A activity, mRNA (p < 0.05), and protein levels (p < 0.05) in CRI rats compared to CPF. Conclusions. CRI induced by RKM does not have an effect on hepatic CYP1A2 and CYP2D enzymes but does reduce CYP3A activity, probably through down-regulation of CYP3A2.     

Effect of lipopolysaccharide on the xenobiotic-induced expression and activity of hepatic cytochrome P450 in mice

Infection-associated inflammation can alter the expression levels and functions of cytochrome P450s (CYPs). Cyp gene expression is regulated by the activation of several nuclear receptors, including pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR). These receptors can be activated by xenobiotics, including medicines. Here, to study the xenobiotic-induced fluctuations in CYP during inflammation, we examined the effect of lipopolysaccharide (LPS) treatment on the level of mRNAs encoding hepatic CYPs induced by xenobiotic-activated nuclear receptors, in mice. Both the mRNA induction of Cyp genes and the metabolic activities of CYP proteins were examined. LPS treatment caused a significant decrease in the induced expression of the mRNAs for Cyp3a11, 2c29, 2c55, and 1a2, but not for Cyp2b10. To assess the CYP enzymatic activities, CYP3A-mediated testosterone 6β-hydroxylation and the intrinsic clearance (CL(int)) of nifedipine in liver microsomes were measured in mice treated with the xenobiotic pregnenolone-16alpha-carbonitrile (PCN) with or without LPS administration. Both assays revealed that the CYP3A activity, which was induced by PCN, declined significantly after LPS treatment, and this decline correlated with the Cyp3a11 mRNA level. In addition, we found that the mRNAs for interleukin (IL)-1β and tumor necrosis factor (TNF) α were increased after treatment with LPS plus xenobiotics. Our findings demonstrated that LPS treatment reduces the PXR- and AhR-mediated, and possibly CAR-mediated Cyp gene expression and further suggest that these decreases are dependent on inflammatory cytokines in the liver.     

Modulation of aryl hydrocarbon receptor regulated genes by acute administration of trimethylarsine oxide in the lung,kidney and heart of C57BL/6 mice

Abstract

1.?Arsenite alters the expression of aryl hydrocarbon receptor (AhR)-regulated genes in extrahepatic tissues; yet, the effect of organic arsenicals still unknown. Therefore, C57BL/6 mice received trimethylarsine oxide (TMAO; 13?mg/kg i.p.) with or without 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 15?μg/kg), and euthanized at 6 or 24?h.

2.?Our results demonstrated that TMAO increased Cyp1a1 and Cyp1b1 mRNA, protein and activity in the lung. TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1a2 mRNA, protein and activity in the lung. In the kidney, TMAO increased Cyp1b1 mRNA and protein. TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 mRNA, protein and activity. In the heart, TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 mRNA.

3.?Moreover, TMAO induced Nqo1 mRNA in the lung, kidney and heart, with subsequent increase in Nqo1 protein and activity in the lung. TMAO increased Gsta mRNA in the heart; and increased Gsta protein and activity in the lung and kidney. TMAO increased Nqo1 mRNA as compared to TCDD in the kidney and heart, and potentiated the TCDD-mediated induction of Gsta protein and activity in the kidney.

4.?In conclusion, TMAO modulates AhR-regulated genes in a tissue- and enzyme-specific manner.