五酯片提取物对人CYP3A4及CYP3A5重组酶活性的抑制作用及其机制

五酯片是临床上常用的护肝中药单方制剂,常被用来治疗各种原因引起的肝损伤。前期研究表明五酯片可通过抑制CYP3A从而升高移植患者、大鼠体内他克莫司、环孢素、紫杉醇等药物的血药浓度。CYP3A4和CYP3A5是CYP3A的两种重要亚型,但它们在催化活性及对抑制剂敏感性上表现不同。对CYP3A4和CYP3A5抑制作用的差异可能会导致不同的药物相互作用,这种相互作用的风险在体内可能会被进一步放大。可见,研究五酯片对CYP3A4及CYP3A5活性抑制作用的异同具有较好的临床治疗学及经济学意义。然而,目前尚未见五酯片对CYP3A4及CYP3A5活性的抑制作用及作用机制的相关研究。因此,本研究利用人重组CYP3A4 (recombinant human CYP3A4, rhCYP3A4)、人重组CYP3A5 (recombinant human CYP3A5, rhCYP3A5)考察五酯片提取物对CYP3A4、CYP3A5活性的影响及作用特征、机制。结果表明,五酯片提取物对CYP3A4、CYP3A5活性的抑制作用存在NADPH、预孵育时间及浓度依赖性;其与CYP3A4及CYP3A5的结合比较牢固,不...     

中药莪术激活PXR及对大鼠肝细胞色素P450 3A的影响

目的考察莪术能否通过体外激活PXR调节细胞色素P4503A4(CYP3A4)的转录表达及对大鼠肝脏CYP3A在酶活性及mRNA表达的诱导作用。方法在HepG2细胞中,采用瞬时共转染报告基因实验研究莪术对PXR介导的CYP3A4的转录调节作用;在大鼠体内,采用紫外分光光度法和RT-PCR技术检测莪术对大鼠肝脏CYP450含量及CYP3A同工酶红霉素N-脱甲基酶(ERD)的活性和CYP3A基因mRNA表达的影响。结果在体外报告基因实验研究中,莪术提取物及其有效成分能不同程度的诱导CYP3A4的表达;在大鼠体内实验中,莪术提取物能够增加CYP450蛋白含量和CYP3A酶活性;在mRNA水平上,莪术提取物能够明显诱导CYP3A1及CYP3A2的基因表达。结论莪术提取物及其有效成分能够明显激活PXR并诱导CYP3A4的转录表达;莪术提取物对大鼠体内CYP3A的酶活性及mRNA表达均有明显诱导作用。     

RNA干扰抑制CYP3A4基因表达的实验研究

目的 研究质粒表达型短发夹RNA（small hairpin RNA，shRNA）对CHL-3A4转基因细胞的细胞色素P450 3A4（cytochrome P450 3A4，CYP3A4）基因表达的影响。方法设计并构建3条shRNA真核表达载体（CYP3A4Ⅰ、CYP3A4Ⅱ、CYP3A4Ⅲ）．脂质体转染CHL-3A4转基因细胞。转染48h后．TR-PCR和Western Blotting分别观察3条shRNA对CYP3A4mRNA和蛋白表达的影响；噻唑蓝（MTT）观察shRNA抑制环磷酰胺对CHL-3A4转基因细胞毒性作用。结果CYP3A4Ⅲ表达载体的shRNA分别能抑制CHL-3A4细胞的CYP3A4mRNA表达（70％）及蛋白表达（75％），抑制环磷酰胺对CHL-3A4细胞（75％）细胞毒作用。结论RNA干扰能显著抑制CYP3A4基因的表达，RNA干扰技术为肝细胞色素P450的研究提供了新的方法。     

人细胞色素P450 3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18重组酶的异源表达与活性

目的重组表达人细胞色素P450(CYP)3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18蛋白,为CYP3A4代谢活性的体外研究提供单一酶源。方法应用杆状病毒表达系统构建含有上述各CYP3A4突变体基因序列的重组病毒,将其连同含人源还原型烟酰胺腺嘌呤二核苷磷酸-P450氧化还原酶(POR)和细胞色素b5基因的重组病毒共同感染昆虫草地夜蛾细胞Sf9得到CYP3A4突变体与POR和细胞色素b5共表达的重组蛋白,分别以高效液相色谱法和荧光分析法测定各重组酶对睾酮和7-甲氧基-4-三氟甲基香豆素的代谢活性。结果在mRNA分子水平上验证了CYP3A4突变体CYP3A4*3,CYP3A4*4,CYP3A4*5和CYP3A4*18基因在Sf9细胞中的转录。感染了各重组病毒的Sf9细胞裂解液对睾酮和7-苄氧基-4-三氟甲基香豆素有明显代谢。结论应用杆状病毒-昆虫细胞表达系统在体外成功表达了具有催化活性的人CYP3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18蛋白,其中CYP3A4.5活性显著低于野生型蛋白,CYP3A4.18活性显著高于野生型蛋白,而CYP3A4.3和CYP3A4.4与野生型蛋白活性近似。     

质粒表达型shRNA对CHL-3A4转基因细胞系细胞色素P450 3A4基因表达的抑制作用

目的研究质粒表达型shRNA对CHL-3A4转基因细胞系细胞色素P450 3A4基因表达的抑制作用.方法设计并构建3条shRNA真核表达载体（CYP 3A4 Ⅰ、CYP 3A4Ⅱ、CYP 3A4Ⅲ）,转染CHL-3A4转基因细胞,以空载体组及无关序列的shRNA表达载体组为阴性对照;转染后48 h,Western blotting观察RNA干扰对CYP 3A4蛋白表达的影响;RNA干扰后,用HPLC法测定CHL-3A4转基因细胞的硝苯地平氧化酶的活性.结果CYP3A4Ⅲ表达载体能显著抑制CYP 3A4蛋白表达（75%）,抑制硝苯地平氧化酶的活性.结论RNA干扰能抑制CYP 3A4基因表达.     

淫羊藿总黄酮对大鼠肝微粒体CYP1A2、CYP3A4和CYP2E1活性的影响

评估淫羊藿总黄酮对大鼠肝细胞色素P450及其主要亚型活性的潜在影响。淫羊藿总黄酮以300mg／kg／d的剂量对SD大鼠进行连续灌胃处理15天，测定肝微粒体中cYP450含量与CYP1A2、CYP3A4和CYP2E1亚型活性，观察淫羊藿总黄酮的效应。CYP1A2的活性用荧光比色法进行测定，CYP3A4和CYP2E1的活性用紫外可见分光光度法测定。淫羊藿总黄酮处理后的大鼠肝脏CYP450含量及CYP1A2、CYP3A4和CYP2E1亚型活性均明显增高，其中CYP1A2和CYP2E1活性升高显著（P〈0．01）。淫羊藿总黄酮对大鼠肝脏CYP450及主要亚型CYP1A2、CYP3A4和CYP2E1活性均有诱导效应。     

CYP3A5~*3和CYP3A4~*18B基因多态性对肾移植患者环孢素药代动力学的影响

目的回顾性研究肾脏移植后1mon,CYP3A5*3和CYP3A4*18B基因多态性对CsA药代动力学参数的影响。方法采用PCR-RFLP方法分析了63名肾脏移植患者CYP3A5*3和CYP3A4*18B基因型;荧光偏正免疫法用于检测肾移植患者静脉全血中的CsA浓度。结果在63名肾移植患者中,CYP3A5*3和CYP3A4*18B突变等位基因发生频率分别为0.770(95CI:0.767～0.773),0.235(95CI:0.235～0.241),而且这些等位基因表现出完全连锁不平衡。在移植术后1mon内,携带CYP3A4*1/*1野生型纯合子患者的C0以及剂量校正谷血浓度(C0/D)均明显高于携带CYP3A4*1/*18B杂合子或CYP3A4*18B/*18B突变型纯合子患者(P<0.05,Mann-WhitneyUtest);CYP3A5*1/*1基因型组的给药剂量明显高于CYP3A5*1/*3或CYP3A5*3/*3基因型组(P=0.004<0.01,Kruakal-Wallistest);CYP34*18B和CYP3A5*3联合考虑,对于CYP3A5表达组,同样发现C0、C0/D在CYP3A4*1/*1组C0以及C0/D均明显高于CYP3A4*1/*18B或CYP3A4*18B/*18B组(P<0.05,Mann-WhitneyUtest);而其他药动学参数在CYP3A5*3及CYP3A4*18B各组间相比差异则没有统计学意义。结论CYP3A5*3和(或)CYP3A4*18B基因多态性对肾移植后1monCsA药代动力学有一定影响,移植前CYP3A5*3基因型的分析仍需进一步研究。     

双环醇对组成型雄甾烷受体和孕烷X受体介导的CYP3A4和CYP286转录的调控

目的：构建pGL4．17-CYP3A4启动子嵌合报告基因表达载体,研究双环醇是否通过活化组成型雄甾烷受体（Constitutive Androstane Receptor,CAR）和孕烷X受体（pregnane Xreceptor．PXR）影响CYP3A4及CYP286的转录。方法：以提取的人肝组织基因组DNA为模板,应用RT-PCR技术,扩增获得CYP3A4启动子的近端启动序列（-362／＋53）和远端调控序列（-7836／-7208）,并连接到pGEM-T载体,测序正确后将目的基因片段先后插入至pGL4．17报告基因表达载体。将hPXR、hCAR表达载体分别与CYP3A4及CYP286报告载体共转染HepG2细胞,利用双荧光素酶报告基因系统检测CYP3A4及CYP286均具有启动活性后,双环醇分别与转染hPXR和CYP3A4、hCAR和CYP3A4、hCAR和CYP286的HepG2细胞温孵24h后,收获细胞并裂解,检测活性以分析双环醇对CYP3A4及CYP286的转录调节途径。结果：获得pGL4．17-CYP3A4启动子嵌合报告基因表达载体,经酶切鉴定及核酸序列测定证实该报告基因表达载体构建成功。双环醇主要通过活化CAR受体途径,调控CYP3A4和CYP286的转录,而PXR受体途径较少参与。     

CYP3A4、CYP3A5和CYP3A7的基因多态性对中国汉族肾移植患者他克莫司药动学的影响

摘 要 目的：探讨中国汉族肾移植患者中,CYP3A5*3（rs776746）、CYP3A4*1G(rs2242480)、CYP3A4 rs4646437、CYP3A7 rs2257401及CYP3A7 rs10211单核苷酸多态性对免疫抑制药他克莫司（TAC）药动学的影响。方法：采用直接测序法,对85例肾移植的患者进行基因分型。采用酶放大免疫分析法测定患者TAC的谷浓度（C0）。比较不同基因型患者间TAC浓度剂量比值的差异。结果：在85例肾移植患者中,CYP3A5*3 A、CYP3A4*1G T、CYP3A4 rs4646437 T、CYP3A7 rs2257401C及CYP3A7 rs10211 G等位基因的频率分别为29.4%,27.6%,13.5%,26.5%及27.6%。肾移植术后1个月时,CYP3A5*3、CYP3A7 rs2257401及CYP3A7 rs10211影响TAC的谷浓度（P<0.01）。CYP3A5*3 A、CYP3A7 rs2257401 C及CYP3A7 rs10211 G等位基因携带者,TAC剂量校正浓度分别比其相应非携带者剂量校正浓度低约50%左右。而本研究未发现CYP3A4*1G和CYP3A4 rs4646437基因多态性与TAC校正谷浓度相关（P>0.05）。结论：在中国汉族肾移植患者中,CYP3A5*3、CYP3A7 rs2257401及CYP3A7 rs10211的单核苷酸多态性与移植术后1个月内TAC的药代动力学有相关性,但把这种相关性应用在临床上,以优化他克莫司的剂量,尚待进一步的研究。     

CYP3A4与CYP3A5基因多态性与中国汉族急性白血病易感性的相关性研究

目的检测中国汉族急性白血病(AL)患者和健康受试者中CYP3A4*18和CYP3A5*3基因型分布特征,探讨CYP3A4和CYP3A5基因多态性与中国汉族AL易感性的相关性。方法病例对照研究,用聚合酶链反应(PCR)后直接测序的方法,检测中国汉族AL患者91例,健康受试者200例的CYP3A4*18B和CYP3A5*3位点的基因型分布及等位基因频率。结果中国汉族AL患者和健康受试者CYP3A4*18B和CYP3A5*3基因型分布均符合Hardy-Weinberg平衡。与健康受试者比较,AL患者CYP3A4*18B基因型及等位基因频率差异均无统计学意义(P>0.05),CYP3A5*3基因型及等位基因分布频率差异均有统计学意义(P<0.05)。结论 CYP3A4*18B多态性与中国汉族AL的易感性可能无关,CYP3A5*3多态性与中国汉族AL的易感性可能相关。     

Systematic screening for polymorphisms in the CYP3A4 gene in the Chinese population

OBJECTIVES: Cytochrome P450 3A4 (CYP3A4) is a major CYP enzyme in the liver and intestine. It is involved in the metabolism of over 50% of all drugs currently in use. The present study was designed to determine the genetic basis of CYP3A4 variability. METHODS: Single nucleotide polymorphism (SNP) analysis of the CYP3A4 gene was performed on 60 healthy Chinese subjects consisting of 20 Han, 30 She and ten Dong subjects, using direct sequencing. Linkage disequilibrium, haplotype inference and Hardy-Weinberg equilibrium were also determined for these samples. RESULTS: A total of 20 SNPs were found in the CYP3A4 gene, including 11 known SNPs and nine novel SNPs. The known SNPs detected in our study were CYP3A4*1B, CYP3A4*1G, CYP3A4*10, CYP3A4*13, CYP3A4*14, CYP3A4*15, CYP3A4*17, CYP3A4*18, rs3091339, rs3091430 and rs28371761, and the novel SNPs were -658 A-->C, G27A (E10K), T48A, G14284A (G167D), A15623G (N191D), C15635A (L196I), T15656C (F203L), G14199A (intron 5) and C15566T (intron 6). The allelic frequencies found in our sample varied from 1-37%. The novel SNPs detected in the CYP3A4 gene suggest that the Chinese population has different patterns of allele frequency compared with other populations. CONCLUSION: Several SNPs were detected in the CYP3A4 gene. The study of genetic variants in CYP3A4 may have an important significance for the understanding of genotype and phenotype relationships.     

Histone deacetylase inhibitor stimulate<Emphasis Type="Italic">CYP3A4</Emphasis> proximal promoter activity in hepg2 cells

The expression of CYP3A4 gene is induced by a variety of structurally unrelated xenobiotics including the antibiotic rifampicin, pregnenolone 16-carbonitrile (PCN), and endogenous hormones, that might mediate through steroid and xenobiotic receptor (SXR) system. The molecular mechanisms underlying regulation of CYP3A4 gene expression have not been understood. In order to gain the insight of the molecular mechanism of CYP3A4 gene expression, study has been undertaken to investigate if the histone deacetylation is involved in the regulation of CYP3A4 gene expression by proximal promoter in human hepatoma HepG2 cells. Also we have investigated to see if SXR is involved in the regulation of CYP3A4 proximal promoter activity in human hepatoma HepG2 cells. HepG2 cells were transfected with a plasmid pCYP3A4-Luc containing approximately 1 kb of the CYP3A4 proximal promoter region (-863 to +64 bp) in front of a reporter gene, luciferase, in the presence or absence of pSAP-SXR. In HepG2 cells, CYP3A4 inducers, such as rifampicin, PCN and RU486 showed minimal stimulation of CYP3A4 proximal promoter activity in the absence of SXR and histone deacetylase (HDAC) inhibitors. 4-Dimethylamino-N-[4-(2-hydroxycarbamoylvinyl)benzyl]benzamide (IN2001), a new class HDAC inhibitor significantly increased CYP3A4 proximal promoter activity over untreated control cells and rifampicin concomitant treatment with IN2001 increased further CYP3A4 proximal promoter activity that was stimulated by IN2001. The results of this study demonstrated that both HDAC inhibitors and SXR are essential to increase of CYP3A4 proximal promoter activity by CYP3A4 inducers such as PCN, rifampicin, and RU486. Especially SXR seems to be important for the dose dependent response of CYP3A4 inducing chemicals to stimulate CYP3A4 proximal promoter activity. Also this data suggested that HDAC inhibitors seemed to facilitate the CYP3A4 proximal promoter to be activated by chemicals.     

服用卡马西平癫痫患者CYP3A4基因多态性的研究

目的:研究服用卡马西平癫痫患者细胞色素P4503A4(CYP3A4)的基因多态性,为临床制定卡马西平个体化给药方案提供依据。方法:收集临床服用卡马西平癫痫患者的血标本,用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)法检测CYP3A4的突变。结果:141例患者中存在CYP3A4*4、CYP3A4*6的突变,个体突变率均为0.71%;未见CYP3A4*5的突变。结论:2例突变型标本与其他标本的血药浓度与临床疗效比较无显著性差异,不能说明CYP3A4*4、CYP3A4*6的突变对其表达的药物代谢酶的活性有影响。     

Increased CYP3A4 copy number in TONG/HCC cells but not in DNA from other humans

Two recent screens for copy-number variations in the entire human genome found 12.4 gene copy number variations per person, including 2.5% of individuals with gains between 7q21.1 and 7q22.1, the chromosomal location of CYP3A4. CYP3A4 is involved in the metabolism of approximately 50% of all drugs, including many cancer chemotherapeutic agents. CYP3A4 gene copy was determined in DNA from 143 individuals: normal human livers, primary and secondary liver tumors, human hepatic cell lines, and immortalized cell lines representing eight ethnically diverse populations. CYP3A4 gene copy was normal in all but one sample, a primary human hepatocellular carcinoma cell line (TONG/HCC). Southern blots of TONG/HCC DNA revealed an approximate 10-fold increase in CYP3A and a corresponding increase in CYP3A mRNA expression and catalytic activity. Fluorescent in situ hybridization of TONG/HCC revealed specific amplification of the CYP3A4 gene on chromosome 7q21 but no amplification of the MDR1 gene that localizes 11.9 Mb upstream of CYP3A4. High resolution analysis of DNA copy number by comparative genomic hybridization confirmed amplification at 7q21.3-7q22. The amplicon spanned 1.7 Mb and contained 30 known genes, including the entire CYP3A locus. To determine whether CYP3A4 expression affected chemotherapeutic toxicity, LLC-PK1 cells were transduced with adenoviruses expressing CYP3A4 and P450 reductase. CYP3A4 conferred resistance to taxol, vinblastine and topotecan. These studies demonstrate that CYP3A4 copy number differences do not contribute to the normal variation in CYP3A4 expression. Tumors with increased CYP3A copy number (via amplification or increased chromosome 7q) would be expected to show reduced cytotoxicity to some chemotherapeutic drugs and potentially an increase in the outgrowth of drug resistant tumors.     

Expression of CYP3A4 and CYP3A7 in Human Foetal Tissues and its Correlation with Nuclear Receptors

Previous reports have suggested that the nuclear receptors vitamin D receptor (VDR), peroxisome proliferator‐activated receptor α (PPARα), pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are involved in the regulation of the drug‐metabolizing enzyme cytochrome P450 (CYP) 3A4 expression in adults. The aim of this study was to investigate the gene expression of CYP3A4 and the foetal CYP3A7 in human foetal tissues and their relation to gene expression and genetic variations in the nuclear receptors VDR, PPARα, PXR and CAR. We determined the relative expression of CYP3A4 and CYP3A7 and these nuclear receptors in foetal livers, intestines and adrenals, using quantitative PCR. In addition, the expression of these enzymes was also analysed in adult liver. There was a high interindividual variability in CYP3A4 and CYP3A7, 49 times and 326 times, respectively. Both CYP3A4 and CYP3A7 had the highest expression in the liver. There were significant correlations (p < 0.001) between the nuclear receptors studied and the expression of CYP3A4 and CYP3A7 in foetal liver, as well as the expression of CYP3A4 in foetal intestine. Polymorphisms in the VDR gene, rs1544410 and rs1523130 (TaqI), in the PXR gene, rs1523130, and in the PPARα gene, rs4253728, were not correlated with CYP3A4 or CYP3A7 expression. However, C‐homozygous individuals of the TaqI VDR polymorphism had 60% lower VDR gene expression (p < 0.05), than individuals carrying one or two T alleles. In conclusion, differences in the expression of nuclear receptors might determine the variability in CYP3A4 and CYP3A7 expression observed in foetal liver.     

Assessment of human pregnane X receptor involvement in pesticide-mediated activation of CYP3A4 gene.

Assessment of foreign chemical inducibility on CYP3A4 is necessary to optimize drug therapies. The properties of chemicals such as pesticides, however, are not well investigated. In the present study, properties of various pesticides on human CYP3A4 induction have been tested using HepG2-derived cells stably expressing the CYP3A4 promoter/enhancer (3-1-10 cells) and the human pregnane X receptor (hPXR)-small interfering RNA (siRNA) system. Among the examined pesticides, 13 pesticides were observed to activate the CYP3A4 gene. Surprisingly, pyributicarb was found to increase the CYP3A4 reporter activity at 0.1 to 1 microM more strongly than typical CYP3A4 inducer rifampicin. Expression of hPXR-siRNA clearly diminished the pyributicarb-stimulated CYP3A4 reporter activity in 3-1-10 cells and decreased the endogenous CYP3A4 mRNA levels in HepG2 cells. Pyributicarb caused enhancement of CYP3A4-derived reporter activity in mouse livers introduced with hPXR by adenovirus. These results indicate pyributicarb as a potent activator of CYP3A4 gene, suggesting the existence of pesticides leading to CYP3A4 induction in our environment.