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OCT1及CYP3A4介导的吡咯里西啶生物碱肝脏转运及毒性研究

发布时间:2018-08-27 08:11
【摘要】:吡咯里西啶生物碱(Pyrrolizidine alkaloids, PAs)广泛分布于紫草科、菊科、豆科植物中。PAs可能是目前对人和动物最重要的天然毒性成分,近50%的PAs为毒性化合物,其毒性表现为肝毒性、遗传毒性和致癌性等,有的还有肺毒性。毒性PAs主要分为retronecine型和otonecine型,其中野百合碱和倒千里光碱为retronecine型肝毒PAs。大多数PAs本身无毒或低毒,经CYP酶代谢激活后产生毒性。CYP主要分布在肝脏,因此,肝脏是PAs首要的毒性靶器官。但CYPs位于肝细胞的内质网膜上,PAs首先需通过一定途径进入肝细胞,才能被CYPs代谢。肝细胞膜表达多种转运体,可以介导药物/化合物的细胞摄取及转运,因此肝细胞膜上的转运体也可能参与PAs的肝脏摄取和转运。 SLC (solute carrier)22家族的有机阳离子转运体(organic cation transporters, OCTs),包括三个成员(OCT1-OCT3),它们对有机阳离子化合物、弱碱性化合物及少量中性化合物的细胞摄取起着关键作用。其中OCT1主要表达在肝脏,定位于肝细胞的基底侧膜上,介导其底物化合物的肝脏摄取。由于部分PAs属于弱碱性化合物,在生理条pH条件下可部分电离,推测它们可能与以OCT1为代表的肝脏阳离子转运体发生相互作用。本论文构建并应用稳定表达hOCT1、CYP3A4的转基因细胞模型,研究四种PAs与肝脏主要阳离子转运体的相互作用,并通过原代肝细胞及共表达CYP3A4与hOCTl的细胞,研究OCT1在MCT及RTS肝脏转运及毒性中的作用。 1.稳定表达hOCT1的MDCK细胞模型的构建 本章旨在建立稳定表达人OCT1(hOCT1)野生型及两个突变体的马丁达比狗肾上皮(Madin-Darby canine kidney, MDCK)细胞模型。首先从人肝组织中提取hOCT1野生型基因,经定点突变获得]hOCT1P341L, hOCT1M420del两个突变型基因,构建表达质粒pcDNA3.1(+)-hOCT1, pcDNA3.1(+)-hOCT1P341L, pcDNA3.1(+)-hOCT1M420del。将质粒转染MDCK细胞,通过G418筛选获得抗性克隆,并通过hOCT1的荧光底物4-(4-(dimethylamino)-styryl)-N-methylpyridinium(ASP+)及抑制剂四乙胺(TEA),筛选得到具有较高活性的MDCK-hOCTl,-hOCT1p341L,-hOCT1M420del单克隆细胞。经反转录聚合酶链式反应(RT-PCR)及经典底物1-methyl-4-phenylpyridinium (MPP+)和二甲双胍的摄取实验,鉴定筛选得到的单克隆细胞株hOCT1mRNA的表达及功能。结果显示,本研究获得的hOCT1野生型及两个突变体细胞模型与mock细胞相比,其hOCT1mRNA表达量显著增高,其对经典底物MPP+及二甲双胍的摄取也显著升高,MDCK-hOCT1,-hOCT1P341L,-hOCT1M420del三种细胞对二甲双胍的积聚动力学参数分别为791.5±24.1,779.1±165.3,537.5±62.8(Vmax, pmol/mg protein/min);409.0±55.1,523.2±36.3,913.4±99.1(Km,μmol/L);1.94,1.49,0.59(Clint, Vmax/Km)。上述结果表明,稳定表达hOCT1的细胞模型构建成功,可以作为研究hOCT1与药物相互作用的细胞模型。 2.PAs与肝脏阳离子转运体的相互作用研究 本章利用MDCK-hOCT1细胞模型,首先考察了野百合碱(MCT)、Isoline、倒千里光碱(RTS)、千里光宁四种PAs,对hOCT1介导的MPP+摄取的抑制作用。结果表明,四种PAs均为hOCT1的抑制剂,IC50值分别为:MCT为5.52μmol/L,Isoline为5.35μmol/L, RTS为2.25μmol/L,千里光宁为3.50μmol/L。进一步对上述PAs在MDCK-hOCT1及mock细胞中的积聚进行研究,发现Isoline及千里光宁在两种细胞的积聚无显著差异,而MCT及RTS在MDCK-hOCTl细胞中的积聚显著高于mock细胞,且两者在MDCK-hOCT1细胞内的积聚可以被OCT1的抑制剂,TEA、ASP+、奎尼丁、右旋延胡索乙素((+)-THP)抑制,提示MCT及RTS均为hOCTl的底物;进一步研究获得MCT和RTS在MDCK-hOCTl中积聚的动力学参数,Km值分别为25.0±6.7μmol/L,23.6±3.0μmol/L; Vmax值分别为266.0±63.9pmol/mg protein/min,209.9±69.3pmol/mg protein/min。 此外,我们还应用稳定表达hOCT2、hOCT3.人多药及毒素外排蛋白转运体1(hMATEl)、或P-糖蛋白(P-gp, MDR1)的MDCK细胞,及稳定表达人乳腺癌耐药蛋白(BCRP)的LLC-PK1细胞模型,考察MCT及RTS是否为上述转运体的底物,以阐明除OCT1外,是否还有其它转运体参与MCT及RTS的肝脏转运。结果显示,MCT及RTS不是或仅是上述转运体的弱底物。 最后,我们应用原代培养的大鼠肝细胞(PRCH),进一步验证OCT1在MCT和RTS肝细胞处置中的作用。结果显示,MCT及RTS在肝细胞内的积聚均可被OCT1的抑制剂,(+)-THP或奎尼丁抑制;MCT、RTS均明显降低肝细胞存活率,升高培养基中乳酸脱氢酶(LDH)活力,(+)-THP、奎尼丁可显著削弱MCT和RTS引起细胞存活降低及LDH活力升高。上述结果说明,MCT及RTS具有肝细胞毒性,OCT1介导MCT及RTS的肝细胞摄取,OCT1抑制剂对抗MCT及RTS的肝细胞毒性。 3.共表达hOCTl及CYP3A4细胞模型的构建 双转染、三转染甚至四重转染转运体或/和代谢酶的细胞模型被认为是研究几个蛋白在肝脏药物处置中共同作用的有效工具。OCT1和CYP3A4均在肝细胞内高表达,且两者的底物和抑制剂谱有一定的重合性,我们推测两者在MCT和RTS致肝毒中共同发挥作用。为了给上述假设的验证提供理想的研究模型,本章建立了稳定单表达CYP3A4及共表达hOCT1、CYP3A4的细胞模型。在已有单转染细胞MDCK-hOCT1, MDCK-pcDNA3.1(+)的基础上,继续向细胞转入CYP3A4的表达质粒(pcDNA3.1(+)-Hygro-CYP3A4)或其相应的空载体(pcDNA3.1(+)-Hygro empty vector)。经G418及潮霉素B双重抗性筛选,获得单克隆细胞,通过Western blot和定量PCR (quantitative Real-time PCR)挑选并鉴定阳性克隆。阳性克隆中hOCTl的功能通过荧光底物ASP+的积聚和抑制剂的抑制实验鉴定,CYP3A4的功能则通过P450-Glo CYP3A4Assay (Luciferin-IPA, CYP3A4的灵敏底物)鉴定。经过筛选,共获得四种细胞MDCK-mock, MDCK-hOCT1, MDCK-CYP3A4, MDCK-hOCT1-CYP3A4。功能鉴定结果显示,MDCK-hOCT1及MDCK-hOCT1-CYP3A4的ASP+积聚量均约为mock细胞的16倍;而MDCK-CYP3A4和MDCK-hOCT1-CYP3A4细胞中CYP3A4的活力均约为mock细胞的66倍。上述结果表明,挑选出的阳性克隆均表达了相应的目的蛋白,细胞模型可用于研究hOCTl及CYP3A4各自及共同作用。 4. hOCTl及CYP3A4在RTS致细胞毒中的作用 由于OCT1对RTS的肝脏摄取起重要作用,同时有报道指出CYP3A4可能介导RTS的代谢激活,鉴于人肝脏同时表达OCT1及CYP3A4,本章应用单表达及共表达hOCT1、CYP3A4的细胞模型,考察RTS的细胞毒性,以阐明hOCT1及CYP3A4在RTS致肝细胞毒性中的作用。将不同浓度的RTS分别与mock, MDCK-hOCT1, MDCK-CYP3A4, MDCK-hOCTl-CYP3A4细胞共孵育,通过细胞形态观察、MTT实验及流式细胞术测定细胞周期,比较RTS对不同细胞的毒性差异。结果显示,RTS对mock及MDCK-hOCT1细胞未显示明显的毒性,而对MDCK-CYP3A4细胞,显示出时间、浓度依赖性的细胞损伤,提示CYP3A4介导的代谢激活是RTS发挥毒性的关键步骤;此外,RTS对MDCK-hOCT1-CYP3A4细胞的毒性明显大于对其它细胞的毒性,提示OCT1介导的摄取及CYP3A4介导的代谢激活,对RTS的细胞毒性均起了重要作用。RTS的毒性表现出以下特点:细胞、细胞核显著增大,细胞生长被显著抑制;细胞周期分析实验则显示RTS诱导细胞G2/M期的阻滞从而抑制有丝分裂。对于RTS毒性机制的了解可以为我们寻找RTS的解毒方法提供新的策略。
[Abstract]:Pyrrolizidine alkaloids (PAs) are widely distributed in Arnebiaceae, Compositae and Legumes. PAs may be the most important natural toxic components in humans and animals. Nearly 50% of PAs are toxic compounds. Their toxicity manifests as hepatotoxicity, genetic toxicity and carcinogenicity, and some are also pulmonary toxicity. Roecine and otonecine, of which monocrotaline and castrarizine are retronecine-type hepatotoxic PAs. Most PAs are non-toxic or low toxic and produce toxicity after metabolic activation by CYP enzymes. CYP mainly distributes in the liver, therefore, the liver is the primary toxic target organ of PAs. However, CYPs are located on the endoplasmic reticulum of hepatocytes, and PAs must first pass a certain route. The hepatocyte membrane expresses a variety of transporters, which can mediate the uptake and transport of drugs and compounds. Therefore, the transporters on the hepatocyte membrane may also participate in the uptake and transport of PAs in the liver.
Organic cation transporters (OCTs) of the SLC (solute carrier) 22 family, including three members (OCT1-OCT3), play a key role in cellular uptake of organic cationic compounds, weak alkaline compounds and a small number of neutral compounds. OCT1 is mainly expressed in the liver and localized on the basolateral membrane of hepatocytes. Because some PAs belong to weak alkaline compounds, they can be partially ionized at pH in physiological strips. It is speculated that they may interact with OCT1 as a representative of liver cationic transporters. The interaction of major cation transporters in the viscera and the role of OCT1 in the transport and toxicity of MCT and RTS in the liver were studied by primary hepatocytes and co-expression of CYP3A4 and hOCTl.
1. construction of MDCK cell model with stable expression of hOCT1
The aim of this chapter is to establish a stable expression model of human OCT1 wild-type and two mutants in Madin-Darby canine kidney (MDCK) cells. Firstly, the wild-type genes of hOCT1 were extracted from human liver tissues, and two mutant genes, hOCT1P341L and hOCT1M420del, were obtained by site-directed mutagenesis. MDCK cells were transfected with pcDNA3.1 (+) - hOCT1P341L and pcDNA3.1 (+) - hOCT1M420del. Resistant clones were obtained by G418 screening. Monoclonal MDCK cells with high activity were screened by hOCT1 fluorescent substrate 4 - (dimethylamino) - styryl - N - methylpyridinium (ASP +) and inhibitor tetraethylamine (TEA). Cells. The expression and function of hOCT1 mRNA were identified by reverse transcription polymerase chain reaction (RT-PCR) and the uptake of 1-methyl-4-phenylpyridinium (MPP+) and metformin. The results showed that the wild type and two mutant cell models of hOCT1 obtained in this study were compared with mock cells. The accumulation kinetic parameters of metformin in MDCK-hOCT1, -hOCT1P341L, -hOCT1M420del cells were 791.5 [24.1], 779.1 [165.3], 537.5 [62.8] (Vmax, pmol / mg protein / min), 409.0 [55.1], 523.2 [36.3] and 913.4 [99.1] respectively. These results indicate that the cell model stably expressing hOCT1 has been successfully constructed and can be used as a cell model to study the interaction between hOCT1 and drugs.
Interaction between 2.PAs and hepatic cationic transporters
In this chapter, MDCK-hOCT1 cell model was used to investigate the inhibitory effects of four kinds of PAs, monocrotaline (MCT), Isoline, Rotundine (RTS) and seneclinine, on the uptake of MPP+ mediated by hOCT1. The results showed that all of the four PAs were inhibitors of hOCT1. IC50 values were MCT 5.52 micromol/L, Isoline 5.35 micromol/L, RTS 2.25 micromol/L, seneclinine 5.25 micromol/L, respectively. The accumulation of PAs in MDK-hOCT1 and mock cells was further studied. It was found that there was no significant difference in the accumulation of Isoline and Seneclonin between the two kinds of cells, while the accumulation of MCT and RTS in MDK-hOCTl cells was significantly higher than that in mock cells. The accumulation of PAs in MDK-hOCT1 cells could be inhibited by OCT1 inhibitors, TEA, ASP+, quinine. Nicotine and dextran tetrahydropalmatine (+) - THP inhibition suggested that both MCT and RTS were substrates of hOCTl. The kinetic parameters of accumulation of MCT and RTS in MDCK-hOCTl were obtained by further study. The Km values were 25.0 (6.7) micromol/L, 23.6 (3.0) micromol/L, Vmax values were 266.0 (63.9) pmol/mg protein/min, 209.9 (69.3) pmol/mg protein/min, respectively.
In addition, we investigated whether MCT and RTS were the substrates of these transporters by stably expressing hOCT2, hOCT3, human multidrug and toxin efflux protein transporter 1 (hMATEl), or P-glycoprotein (P-gp, MDR1) MDK cells, and LC-PK1 cells stably expressing human breast cancer resistance protein (BCRP). The liver transport of MCT and RTS showed that MCT and RTS were not or only the weak substrates of the transporters.
The results showed that the accumulation of MCT and RTS in hepatocytes could be inhibited by OCT1 inhibitors, (+) - THP or quinidine. Both MCT and RTS significantly decreased the survival rate of hepatocytes and increased the activity of lactate dehydrogenase (LDH) in the medium. These results suggest that MCT and RTS have hepatotoxicity, OCT1 mediates hepatocyte uptake of MCT and RTS, and OCT1 inhibitors antagonize hepatotoxicity of MCT and RTS.
3. co expression of hOCTl and CYP3A4 cell models
Cell models of double, triple or even quadruple transfection transporters or/and metabolic enzymes are considered to be effective tools for studying the interaction of several proteins in liver drug disposal. OCT1 and CYP3A4 are highly expressed in hepatocytes, and their substrates and inhibitor profiles have some coincidence. We speculate that OCT1 and CYP3A4 are involved in hepatotoxicity induced by MCT and RTS. In order to provide an ideal research model for validating the above hypothesis, a stable single-expression CYP3A4 and co-expression of hOCT1, CYP3A4 cell model were established. On the basis of existing single-transfected cells MDCK-hOCT1, MDCK-pcDNA3.1 (+), CYP3A4 expression plasmids (pcDNA3.1 (+) - Hygro-CYP3A4) or their corresponding plasmids were transfected into the cells. Vacuum vector pcDNA3.1 (+) - Hygro empty vector. Monoclonal cells were screened by G418 and hygromycin B. Positive clones were selected and identified by Western blot and quantitative Real-time PCR. The function of hOCTl in the positive clones was identified by fluorescent substrate ASP + accumulation and inhibitor inhibition test, and the function of CYP3A4 was identified. P450-Glo CYP3A4Assay (sensitive substrate of Luciferin-IPA, CYP3A4) was used to identify the four cell lines. After screening, MDCK-mock, MDCK-hOCT1, MDCK-CYP3A4, MDCK-hOCT1-CYP3A4, MDCK-hOCT1-CYP3A4, MDCK-hOCT1-CYP3A4, MDCK-hOCT1 and MDCK-hOCT1-CYP3A4 were obtained. The results of functional identification showed that the accumulation of ASP + in MDCK-hOCT1 and MDCK-hOCT1-CYP3A4 was 16 times as much as that in moCK-CYP3A4. CYP3A4 activity in A4 cells was 66 times higher than that in mock cells. These results showed that the selected positive clones expressed the corresponding target proteins. Cell models could be used to study the interaction of hOCTl and CYP3A4.
4. the role of hOCTl and CYP3A4 in cytotoxicity induced by RTS
Because OCT1 plays an important role in the uptake of RTS by liver, it has been reported that CYP3A4 may mediate the activation of RTS metabolism. In view of the simultaneous expression of OCT1 and CYP3A4 in human liver, this chapter investigated the cytotoxicity of RTS by using a single expression and co-expression cell model of hOCT1 and CYP3A4 to clarify the role of hOCT1 and CYP3A4 in RTS-induced hepatotoxicity. RTS at the same concentration were co-incubated with mock, MDCK-hOCT1, MDCK-CYP3A4, MDCK-hOCTl-CYP3A4 cells respectively. Cell cycle was determined by cell morphology, MTT assay and flow cytometry. The results showed that RTS had no significant toxicity to mock and MDCK-hOCT1 cells, but to MDCK-CYP3A4 cells. Time-and concentration-dependent cell damage suggests that CYP3A4-mediated metabolic activation is a key step in the toxicity of RTS; moreover, the toxicity of RTS to MDCK-hOCT1-CYP3A4 cells is significantly greater than that to other cells, suggesting that OCT1-mediated uptake and CYP3A4-mediated metabolic activation play an important role in the cytotoxicity of RTS. Cell cycle analysis showed that RTS could induce G2/M phase arrest and inhibit mitosis. The understanding of the toxic mechanism of RTS could provide a new strategy for the detoxification of RTS.
【学位授予单位】:浙江大学
【学位级别】:博士
【学位授予年份】:2014
【分类号】:R917

【参考文献】

相关期刊论文 前1条

1 汤俊;服部征雄;;《中国药典》含吡咯里西啶生物碱的中药品种与用药安全[J];药学学报;2011年07期



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