山姜素激活孕烷X受体和上调CYP3A4 mRNA转录的研究

目的探讨山姜素能否通过活化孕烷X受体(PXR)诱导CYP3A4的转录表达及对CYP3A4 mRNA的实际诱导作用。方法在人结肠癌LS174T细胞中,用瞬时共转染报告基因实验研究山姜素(1～50μmol.L-1)对PXR介导的CYP3A4基因的转录激活;用荧光定量RT-PCR方法检测其对CYP3A4 mRNA的实际诱导。结果 10μmol.L-1浓度山姜素能够通过活化PXR诱导CYP3A4的转录(1.63倍);10和20μmol.L-1山姜素能够明显上调CYP3A4 mRNA(2.28倍和1.65倍)的表达。结论 PXR途径也许是山姜素诱导CYP3A4基因表达的调控因素之一。     

孕烷X受体对细胞色素P4503A调控机制的研究进展

细胞色素P4503A（CYP3A）是参与临床药物代谢的主要CYP同工酶之一。孕烷X受体（PXR）属于核受体超家族（NR）的NR1 Ⅰ亚家族。该受体作为药物代谢的关键转录调控因子,参与CYP3A的诱导表达。药物可通过多种途径激活PXR受体调控cyp3a基因的表达,其中包括PXR与其他核受体、转录因子及细胞信号转导通路间的相互作用等多种途径。目前,基于PXR的筛选方法已广泛应用于早期新药研发。     

Generation of Caco-2 cells with predictable metabolism by CYP3A4, UGT1A1 and CES using the PITCh system

Caco-2 cells are widely used as an in vitro intestinal model. However, the expression levels of the drug-metabolizing enzymes CYP3A4 and UGT1A1 are lower in these cells than in intestinal cells. Furthermore, the majority of prodrugs in use today are ester-containing, and carboxylesterase (CES) 1 and CES2 are among the enzymes that process the prodrugs into drugs. In the human small intestine, CES1 is hardly expressed while CES2 is highly expressed, but the CES expression pattern in Caco-2 cells is the opposite. In this study, we generated CYP3A4-POR-UGT1A1-CES2 knock-in (KI) and CES1 knock-out (KO) Caco-2 (genome-edited Caco-2) cells using a PITCh system. Genome-edited Caco-2 cells were shown to express functional CYP3A4, POR, UGT1A1 and CES2 while the expression of the CES1 protein was completely knocked out. We performed transport assays using temocapril. The Papp value of temocapril in genome-edited Caco-2 cells was higher than that in WT Caco-2 cells. Interestingly, the amount of temocaprilat on the apical side in genome-edited Caco-2 cells was lower than that in WT Caco-2 cells. These results suggest that genome-edited Caco-2 cells are more suitable than WT Caco-2 cells as a model for predicting intestinal drug absorption and metabolism.     

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。     

Bromuconazole-induced hepatotoxicity is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1 gene expression

Despite widespread use of bromuconazole as a pesticide for food crops and fruits, limited studies have been done to evaluate its toxic effects. Here, we evaluated the hepatotoxic effect of bromuconazole using classical toxicological (biochemical analysis and histopathological examination) and gene-based molecular methods. Male rats were treated either orally or topically with bromuconazole at doses equal to no observed adverse effect level (NOAEL) and 1/10 LD50 for 90?d. Bromuconazole increased activities of liver enzymes (ALT, AST, ALP, and ACP), and levels of bilirubin. It also induced hepatic oxidative stress as evidenced by significant decrease in the activities of superoxide dismutase (SOD), and significant increase in levels of malondialdehyde (MDA) in liver. In addition, bromuconazole caused an increase in liver weights and necrobiotic changes (vacuolation and hepatocellular hypertrophy). It also strongly induced the expression of PXR and its downstream target CYP3A1 gene as well as the activity of CYP3A1. However, it inhibited the expression of CAR and its downstream target CYP2B1 gene without significant changing in CYP2B1 activity. Overall, the oral route showed higher hepatotoxic effect and molecular changes than the dermal route and all changes were dose dependent. This is the first investigation to report that bromuconazole-induced liver oxidative damage is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1.     

豆腐果苷对人孕烷X受体介导的CYP3A4和MDR1的转录调节作用

目的:建立和验证人孕烷X受体(human pregnant X receptor,hPXR)介导的CYP3A4、MDR1药物诱导剂的体外筛选体系,考察豆腐果苷对hPXR介导的CYP3A4、MDR1的转录调节作用。方法:利用构建的双荧光素酶报告基因系统,将表达载体和报告载体共转染HepG2细胞,以10μmol/L利福平为阳性对照,用不同浓度(0.004、0.04、0.4μmol/L)豆腐果苷处理48h后裂解细胞进行双荧光素酶活性检测。结果:不同浓度的豆腐果苷均不能通过激活hPXR来介导CYP3A4和MDR1表达上调,各浓度处理组的双荧光素酶比活性值与DMSO溶媒组差异无统计学意义(P>0.05)。结论:成功构建了hPXR介导的CYP3A4和MDR1药物诱导剂的体外筛选体系,并发现豆腐果苷不能通过激活hPXR介导CYP3A4和MDR1的表达上调。     

中药止咳橘红颗粒对CYP3A4和CYP1A2抑制作用的研究

目的：在人体内研究止咳橘红对CYP3A4和CYP1A2的抑制作用，以预测止咳橘红与常用临床药物的相互作用。方法：咪哒唑仑和咖啡因分别作为CYP3A4和A2的探针药物，采取交叉设计，10名受试者在服用3d止咳橘红的前后均服用7.5mg咪哒唑仑和100mg咖啡因，服药后采血测定两者及代谢产物的代谢动力学参数，探讨针药物及代谢物的浓度用HPLC-MS法测定，Cmax,tmax从药时曲线中直接读出，AUC用梯形法计算，Ke用3P87程序进行拟合计算，分析服药前后CYP3A4和CYP1A2被抑制的情况，结果 服用止咳橘红后，咪哒唑仑的代谢受到了轻微的抑制，它的血药浓度，达峰时间和药时曲线下面积都有了升高趋势，但无显著差异。而咖啡因的代谢未受到影响。结论 止咳橘红对CYP3A4的活性有较弱的抑制作用，能够导致CYP3A4底物咪哒唑仑代谢的轻微抑制，而对CYP1A2的活性没有影响。止咳橘红长期使用或超过治疗剂量使用时是否会对CYP3A4产生显著性影响。尚需进一步的研究证明。     

Polymorphisms in the cytochrome P450 CYP1A2 gene (CYP1A2) in colorectal cancer patients and controls: allele frequencies,linkage disequilibrium and influence on caffeine metabolism

AIMS: Several single nucleotide polymorphisms (SNPs) of the cytochrome P450 enzyme 1A2 gene (CYP1A2) have been reported. Here, frequencies, linkage disequilibrium and phenotypic consequences of six SNPs are described. METHODS: From genomic DNA, 114 British Caucasians (49 colorectal cancer cases and 65 controls) were genotyped for the CYP1A2 polymorphisms -3858G-->A (allele CYP1A2*1C), -2464T-->delT (CYP1A2*1D), -740T-->G (CYP1A2*1E and *1G), -164A-->C (CYP1A2*1F), 63C-->G (CYP1A2*2), and 1545T-->C (alleles CYP1A2*1B, *1G, *1H and *3), using polymerase chain reaction-restriction fragment length polymorphism assays. All patients and controls were phenotyped for CYP1A2 by h.p.l.c. analysis of urinary caffeine metabolites. RESULTS: In 114 samples, the most frequent CYP1A2 SNPs were 1545T-->C (38.2% of tested chromosomes), -164A-->C (CYP1A2*1F, 33.3%) and -2464T-->delT (CYP1A2*1D, 4.82%). The SNPs were in linkage disequilibrium: the most frequent constellations were found to be -3858G/-2464T/-740T/-164A/63C/1545T (61.8%), -3858G/-2464T/-740T/-164C/63C/1545C (33.3%), and -3858G/-2464delT/-740T/-164A/63C/1545C (3.51%), with no significant frequency differences between cases and controls. In the phenotype analysis, lower caffeine metabolic ratios were detected in cases than in controls. This was significant in smokers (n = 14, P = 0.020), and in a subgroup of 15 matched case-control pairs (P = 0.007), but it was not significant in nonsmokers (n = 100, P = 0.39). There was no detectable association between CYP1A2 genotype and caffeine phenotype. CONCLUSIONS: (i) CYP1A2 polymorphisms are in linkage disequilibrium. Therefore, only -164A-->C (CYP1A2*1F) and -2464T-->delT (CYP1A2*1D) need to be analysed in the routine assessment of CYP1A2 genotype; (ii) in vivo CYP1A2 activity is lower in colorectal cancer patients than in controls, and (iii) CYP1A2 genotype had no effect on phenotype (based on the caffeine metabolite ratio). However, this remains to be confirmed in a larger study.     

阿司匹林联合华法林对HepG2细胞中CYP3A4酶的抑制作用

目的研究阿司匹林联用华法林对肝药物代谢酶CYP3A4活性的影响及其机制。方法不同质量浓度的阿司匹林、华法林及联合用药处理Hep G2细胞48 h,采用MTT法检测细胞存活率;通过荧光素酶报告基因技术检测各组对PXR转录酶活性和酶CYP3A4活性的影响;采用荧光定量PCR法和Western blotting法检测Hep G2细胞的酶CYP3A4的m RNA和蛋白表达水平。结果与对照组比较,阿司匹林组、联合用药组细胞的孕烷X受体(PXR)转录酶活性、酶CYP3A4活性均显著降低(P0.01),CYP3A4 m RNA和蛋白表达水平显著降低(P0.05、0.01),华法林组均无显著差异。结论阿司匹林联用华法林能够抑制药物代谢酶CYP3A4活性,其机制可能是通过抑制PXR受体的m RNA和蛋白表达实现的。     

酒精性肝损伤大鼠细胞色素P450 CYP2E1和细胞色素P450 CYP3A的代谢活性

目的观察酒精性肝损伤对大鼠细胞色素P450CYP3A(CYP3A)和细胞色素P450CYP2E1(CYP2E1)代谢活性的影响。方法采用ig给予白酒制备大鼠酒精性肝损伤模型,检测血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性,采用HE染色法光镜下观测酒精对肝脏损伤程度。大鼠ip给予CYP3A探针药物咪达唑仑10mg·kg-1或ig给予CYP2E1探针药物氯唑沙宗50mg·kg-1后,采用高效液相色谱法测定不同时间点大鼠血浆中咪达唑仑和氯唑沙宗的血药浓度,并应用3P87软件计算其药代动力学参数,以考察CYP2E1和CYP3A的代谢活性的变化。大鼠ig给予氯唑沙宗80mg·kg-1后,热板方法测定大鼠添足次数和添足反射潜伏期。结果酒精性肝损伤可致大鼠肝小叶结构不清,肝索排列紊乱,肝细胞体积增大,呈弥漫性中度水变性,肝窦受压,大部分肝细胞胞浆内见大小不等的脂肪空泡;与正常对照组相比,酒精性肝损伤组大鼠GPT和GOT活性分别增加了16.0%和20.0%(P<0.05,P<0.01)。酒精性肝损伤致大鼠CYP2E1对探针药物氯唑沙宗的代谢活性增强,AUC,t1/2和cmax分别降低了38.0%,30.5%和35.0%(P<0.05);酒精肝损伤组大鼠氯唑沙宗镇痛效果明显降低;酒精性肝损伤致大鼠CYP3A对探针药物咪达唑仑的代谢活性增强,AUC,t1/2和cmax分别降低了122.6%,54.9%和56.9%(P<0.01,P<0.05)。结论酒精性肝损伤可使大鼠CYP2E1和CYP3A代谢活性增强。     

注射用丹参总酚酸(冻干)对人CYP450酶和P-糖蛋白体外抑制作用及对大鼠CYP1A2和CYP3A体内诱导作用(英文)

目的探讨注射用丹参总酚酸(冻干)(SLI)对人CYP450酶和P-糖蛋白体外抑制作用以及对大鼠CYP1A2和CYP3A体内诱导作用。方法①应用P450-GloTMCYP450检测试剂盒,通过化学发光法测定SLI和经典抑制剂对细胞色素P4501A2(CYP1A2),CYP2D6,CYP3A4,CYP2C19和CYP2C9的IC50值,通过比较SLI和经典抑制剂对相应细胞色素P450亚型的IC50值来判断SLI对人CYP450酶的体外抑制作用。②Wistar大鼠分别iv给予SLI 3,10和30 mg·kg-1和诱导剂苯巴比妥钠20 mg·kg-1,采用探针底物法,通过比较代谢产物的生成速率来评价SLI对大鼠CYP1A2和CYP3A的诱导作用。③应用ATP酶检测试剂盒,通过化学发光法测定ATP酶活性来评价SLI是否为P-gp的底物或抑制剂。结果①CYP1A2,CYP2C9,CYP2C19,CYP2D6和CYP3A4抑制剂的IC50与SLI对其的IC50进行比较(CYP1A2:0.12μmol·L-1vs 840μmol·L-1;CYP2C9:3.362μmol·L-1vs 704μmol·L-1;CYP2C19:3.236μmol·L-1vs 306μmol·L-1;CYP2D6:0.117μmol·L-1vs 2660μmol·L-1;CYP3A4:0.078μmol·L-1vs 1780μmol·L-1)。②与空白对照组(86.4±6.3)nmol·g-1.min-1相比,SLI 3,10和30 mg·kg-1组CYP1A2活性分别为83.4±6.6,82.5±4.0和(83.4±6.6)nmol·g-1.min-1。与空白对照组(16.1±0.9)nmol·g-1.min-1比较,SLI 3,10和30 mg·kg-1组CYP3A活性分别为15.7±0.6,15.9±0.7和(15.9±1.0)nmol·g-1.min-1,无显著性差异。③以临床血药浓度为依据设计的一系列浓度的SLI 0.0002,0.0006,0.002,0.006,0.017,0.052,0.156和0.468 g.L-1的ATP酶活性分别与空白对照组进行比较(5.8,5.3,5.8,5.5,5.8,5.2,,5.8,5.3,vs 5.75μmol·g-1.min-1),无显著性差异。结论SLI临床给药剂量既不能体外抑制人CYP1A2,CYP2D6,CYP3A4,CYP2C19和CYP2C9酶活性,也不能诱导大鼠CYP1A2和CYP3A,同时也不是P-gp的体外抑制剂或底物。     

健脾和胃方对大鼠肝微粒体CYP3A1酶活性的影响

目的研究健脾和胃方对大鼠肝微粒体细胞色素P450同工酶3A1（CYP3A1）活性的作用。方法 25只大鼠随机分为5组：空白对照组（生理盐水1 mL·kg-1·d-1,14 d）、地塞米松组（100 mg·kg-1·d-1,3 d）和健脾和胃方高、中、低剂量组（7.146,3.573,1.786 mg·kg-1·d-1,14 d）,每组5只,灌胃给予相应的药物。采用高效液相色谱-紫外检测法,以睾酮为探针,测定睾酮经大鼠肝微粒体温孵后转化的代谢产物6β-羟基睾酮（6β-OHT）的生成速率,以评价各组CYP3A1酶活性。结果空白对照组、地塞米松组和健脾和胃方高、中、低剂量组6β-OHT的生成速率分别为（7.29±0.66）、（27.46±2.35）、（3.51±0.48）、（4.90±1.18）、（6.52±1.23）nmol·mg-1·min-1,健脾和胃方高、中、低剂量组6β-OHT的生成速率与地塞米松组均有显著性差异（P〈0.05）。高、中剂量组与空白对照组有显著性差异（P〈0.05）,低剂量组与空白对照组无显著性差异（P〉0.05）。结论本实验结果表明健脾和胃方对大鼠肝药酶CYP3A1酶活性无诱导作用,健脾和胃方的高、中剂量组能使大鼠肝药酶CYP3A1酶活性下降。     

Involvement of CAR and PXR in the transcriptional regulation of CYP2B6 gene expression by ingredients from herbal medicines

Abstract

1.?Induction of hepatic drug-metabolizing enzymes can affect drug efficacy and cause toxicity. However, so far, limited information is available regarding the molecular mechanism how herbal medicines induce human CYP2B6, which metabolizes many of the clinically used therapeutics and activates several pro-carcinogens or toxicants. Accumulated evidence suggests that the human constitutive androstane receptor (hCAR) and the human pregnane X receptor (hPXR) play important roles in trans-activation of CYP2B6. In this study, we investigated the effects of 68 Chinese herbal ingredients on the receptor specificity of hPXR/hCAR-mediated CYP2B6 induction by luciferase reporter gene assays in transiently transfected HepG2 cells and on the expression of CYP2B6 in LS174T cells.

2.?The HepG2 cells were transiently transfected with human CYP2B6 luciferase promoter reporter plasmids along with hPXR or hCAR₃. The results indicated that apigenin (Api), curcumol (Cur) and praeruptorin A (Pra A) were identified as potent activators of hPXR, and Pra A was also a ligand of hCAR.

3.?Furthermore, CYP2B6 mRNA expression in LS174T cells treated with the three herbal ingredients was determined by real-time polymerase chain reaction. By combining western blot and LC–MS/MS, CYP2B6 protein expression and catalytic activity induced by the three herbal ingredients were measured.

4.?Our observation showed Api and Cur up-regulated CYP2B6 expression by transactivation of hPXR, and Pra A acted as the ligand of both hPXR and hCAR to induce CYP2B6 expression.     

Differential expression of CYP3A12 and CYP3A26 mRNAs in canine liver and intestine

The cytochrome P450 (CYP) 3A family is often considered the most important CYP subfamily with regard to drug metabolism in people. Certainly, CYP3A4 contributes to poor oral bioavailability of a number of drugs, and a tremendous amount of effort has been made in attempting to find an appropriate model system to predict the oral bioavailability of candidate drugs. The dog is a species widely used as a preclinical model for the evaluation of drug safety and pharmacokinetics. Compared with other species, little information is available on the tissue distribution of CYP enzymes. The purpose of this study was to determine the level of messenger RNA (mRNA) expression of the canine CYP3A subfamily (CYP3A12 and CYP3A26) in the liver and duodenum. Overall, expression of CYP3A mRNA was greater in the liver than in the duodenum. Hepatic expression of CYP3A26 was greater than CYP3A12 in all dogs, with CYP3A26 comprising 75.2% of the hepatic mRNA CYP3A pool. Conversely, duodenal expression of CYP3A12 was greater than CYP3A26 in all dogs, with CYP3A12 comprising 99.8% of the duodenal mRNA CYP3A pool. In summary, these results represent an important step toward the systematic comparison of human and canine CYP3A enzymes, particularly in relation to oral bioavailability of substrate drugs.     

Regulation of CYP3A4 and CYP2B6 expression by liver X receptor agonists

The liver X receptor (LXR) agonists, 24(S),25-epoxycholesterol and T0901317, were previously shown to be capable of inducing CYP3A expression in primary cultured rodent hepatocytes through activation of the pregnane X receptor (PXR). In this study, the abilities of these two LXR agonists to regulate CYP3A4 and CYP2B6 mRNA expression in primary cultures of human hepatocytes were evaluated. Treatment with 10 or 30 microM of the endogenous oxysterol, 24(S),25-epoxycholesterol, had no effect on CYP3A4 mRNA content in five preparations of primary cultured human hepatocytes, while 30 microM 24(S),25-epoxycholesterol treatment increased CYP2B6 mRNA content by approximately two-fold. By comparison, treatment with the synthetic LXR agonist, T0901317, potently increased CYP3A4 and CYP2B6 mRNA levels in the human hepatocyte cultures, producing multi-fold increases at 10nM. Using a HepG2-based transactivation assay, T0901317 activated human PXR with an EC(50) approximately 20nM, which was more than 10-fold lower than that of the potent PXR ligand, SR-12813, while treatment with 24(S),25-epoxycholesterol failed to induce reporter expression in this assay. Therefore, while 24(S),25-epoxycholesterol-mediated PXR activation and CYP3A induction does not appear to be conserved from rodent to human, T0901317 is among the most potent known activators of human PXR.     

Arsenite and its metabolites, MMA and DMA, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA^III) and dimethylarsinous acid (DMA^III) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA^III induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA^III increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA^III induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.