【作者】 谭亲友；

【导师】 李焕德；

【作者基本信息】 中南大学 ， 药理学， 2012， 博士

【摘要】 祖国医学很早就认识到甘草具有调和诸药和解毒的作用,认为甘草可以缓解其它药物的毒性和烈性。中药方剂中很多配伍甘草以缓其性。大量临床实践和实验研究均证明了甘草“解百药毒”这一传统认识的科学性。甘草酸(glycyrrhizic acid, GL)是甘草的主要活性成分,其钾、钙盐为甘草甜素(glycyrrhizin),水解后产生二分子葡萄糖醛酸和一分子甘草次酸(glycyrrhetinic acid, GA)由于其母核18位手性碳原子C-H键(18H)的构型不同,因此甘草酸存在两种差向异构体,即18a-甘草酸(18a-GL)和18p-甘草酸(18β-GL),两者水解之后可生成相应的18a-甘草次酸(18a-GA)和18p-甘草次酸(18β-GA)。由于在我国具有自主知识产权的异甘草酸镁“手性肝药”在2006年上市,甘草酸异构体的差异日渐受到国内外学者的关注。本课题组前期研究发现18β-GL能够诱导P-gp的表达,本文从Ⅱ相解毒酶的角度,研究了甘草提取物、甘草酸18H差向异构体及其水解产物对Ⅱ相解毒酶的影响及其机制的研究。目的通过体内外实验研究甘草提取物及甘草酸18位差向异构体及其水解产物对UGT1A、UGT2B及其同工酶UGT1A1和UGT1A6和GSTπ的蛋白表达和转录水平mRNA的影响,探讨其对Ⅱ相解毒酶的作用是否具有立体选择性；拟初步证实甘草提取物及甘草酸18位差向异构体及其水解产物对Ⅱ相解毒酶诱导作用及其机制的研究；通过研究甘草酸制剂对对乙酰氨基酚药动学的影响,初步探讨甘草酸制剂与Ⅱ相解毒酶UGT底物在临床上可能发生的相互作用。方法1.甘草提取物和甘草酸18H差向异构体分别从不同浓度和不同时间对大鼠体内Ⅱ相解毒酶的影响通过从不同浓度甘草提取物(90、180、360mg·kg-1)以及18α-GL和18β-GL(12.5、25、50mg·kg-1)和不同时间(3、6、12天)对大鼠进行灌胃诱导实验,用芯片阵列免疫组化技术手段实验考察试验药物对Ⅱ相解毒酶UGT1A和UGT2B及GSTπ的影响。2.甘草提取物、甘草酸18H差向异构体及其水解产物在大鼠体内对Ⅱ相代谢解毒酶的影响42只大鼠随机分为空白组(生理盐水)、甘草提取物(14.3%甘草酸)组、18a-GL、18β-GL、18α-GA、18β-GA、阳性对照组(苯巴比妥),每组6只,分别诱导7天。于第八天处死,采用肝灌流方法取出肝脏组织,通过肝微粒体法、western blot法和阵列组织芯片免疫组化法等“三位一体”的实验方法,从肝组织的亚细胞水平和组织形态学水平考察甘草提取物及其主要成分18α、β-GL和18α、β-GA对UGT1A、UGT1A1、UGT1A6和UGT2B及GSTπ的影响。3. HepG2细胞培养及MTT毒性实验采用DMEM培养基常规培养HepG2细胞,用于研究甘草提取物、甘草酸18位差向异构体及其水解产物对Ⅱ相解毒酶UGT和GST的影响。采用苯基溴化四氮唑蓝法(MTT),判断各试验药物的体外最大非细胞毒性剂量,保证试验过程中细胞的活性,并为甘草提取物、18α、p-GL和18α、p-GA对UGT1A、UGT2B、UGT1A1、UGT1A6和GSTπ以及转染实验的研究提供与细胞作用的最大浓度。4.甘草提取物和甘草酸18位差向异构体及水解产物在HepG2细胞中对Ⅱ相解毒酶蛋白表达和转录的影响以利福平作为阳性对照,不加药处理的HepG2细胞作为阴性对照,通过Western-Blot和Q-PCR法从蛋白的表达水平和mRNA转录水平来研究甘草提取物及其主要成分甘草酸和甘草次酸差向异构体对Ⅱ相酶UGT1A,UGT2B及同工酶UGT1A1和UGT1A6及GSTπ的影响。5.甘草提取物、甘草酸18H差向异构体及其水解产物通过核受体PXR及CAR对UGT1A1和UGT1A6转录活性的影响构建hPXR、hCAR和RXRa真核表达质粒及UGT1A1和UGT1A6荧光素酶报告基因质粒,瞬时共转染外于肝癌HepG2细胞中,DMSO作为空白对照组,利福平作为PXR阳性对照组,PCN作为是PXR阴性对照组,CITCO作为CAR阳性对照组,TCPOBOP作为CAR阴性对照组。药物处理组：甘草提取物(2、20、200mg·mL-1),18α-GL,18β-GL、18α-GA、18β-GA四个差向异构体低、中、高三个浓度为(1、10、100μM)。用荧光素报告基因法检测甘草提取物及其主要成分18α、β-GL和18α、β-GA对报告基因转录活性的影响。6.甘草酸制剂对对乙酰氨基酚药动学的影响采用随机开放,三周期交叉自身对照的拉丁方设计,12名受试者随机等分为3组,分别为甘草酸二胺胶囊组,即试验组；葡醛内酯组,即对照组；以及安慰剂组。洗脱期为2周。受试者于服药前(空白)和服药后0.16、0.33、0.5、0.75、1.0、1.5、2、3、4、6、8、10、12和24小时点前臂静脉血4mL。分离血浆,立即置-70℃冰箱中保存,并在规定的时间内及时进行测定。尿样收集于给药前及给药后0-2,2-4,4-6,6-8,8-12,12-24h各时段尿液收集于一容器中,记录总量并留取标本,于-70℃保存备用。采用HPLC-MS/MS法测定血浆中对乙酰氨基酚及其结合物的浓度。使用DAS2.1.1软件计算药动学参数,SPSS17.0软件进行统计分析。结果与结论1.肝组织的芯片阵列免疫组化实验结果经统计学分析,发现甘草提取物及18α-GL和18β-GL对Ⅱ相解毒酶UGT1A和UGT2B诱导作用成剂量依赖性和时间依赖性,对谷胱甘肽转移酶GSTπ也有诱导作用,但时间依赖性和剂量依赖性不明显。2研究结果表明,大鼠经实验药物诱导七天后,甘草提取物对UGT1A、 UGT1A1、UGT1A6、UGT2B有明显的诱导作用。对GSTπ有诱导作用,但作用不明显。发现甘草的主要成分18α-GL和18β-GL对UGT及其部分同工酶UGT1A1、UGT1A6有诱导作用,其中18β-GL对Ⅱ相酶的诱导作用表现更加明显。18α-GA对UGT2B有明显的诱导作用,与18α-GA相比,18β-GA对UGT1A及其部分同工酶UGT1A1和UGT1A6的诱导作用更加明显。甘草酸和甘草次酸以及其差向异构体的在很多相互比较的研究中都有一定的差异,两者在代谢方面的区别应受到重视。3.甘草提取物在HepG2细胞中对UGT1A、UGT2B、UGT1A1、UGT1A6和GSTπ蛋白表达和转录水平mRNA的表达有明显的增强作用,18α、β-GL对UGT1A、UGT2B、UGT1A1、UGT1A6和GSTπ蛋白表达和]mRNA的表达有增强作用,其中18β-GL对UGT1A1和UGT1A6蛋白表达和转录水平有显著的增强作用,这点与大鼠肝组织中报道的18β-GL较18α-GL对Ⅱ相酶诱导作用更明显较为一致；18α-GA和18β-GA在细胞水平对UGT1A、UGT2B、UGT1A1、UGT1A6和GSTπ蛋白表达和nRNA表达比在大鼠肝组织中更加明显,尤其18β-GA在转录水平对UGT1A、UGT1A1、UGT1A6、UGT2B和GSTπ的mRNA表达量都具有明显的增加作用,在蛋白表达水平对UGT1A、UGT1A1也有明显增强作用。18β-GL和18β-GA对Ⅱ相酶的诱导作用可能具有同向性。4.甘草提取物、18α、β-GL和18α、β-GA通过核受体CAR或PXR对UGT1A1和UGT1A6转录活性产生影响,在高剂量时这种影响更加明显。另外18α、β-GL对这种转录活性的影响有一定的差异性,尤其是18α-GL除了高剂量有较强的作用外,低、中剂量通过核受体CAR或PXR对UGT1A1和UGT1A6转录活性产生影响不是很明显,然而18β-GL低、中、高剂量通过核受体CAR或PXR对UGT1A1和UGT1A6转录活性的影响均有显著的增强作用。这也从作用机制方面解释了本课题前面研究报道的18β-GL对Ⅱ相解毒酶UGT有显著诱导作用真正原因。18α、β甘草次酸也通过核受体CAR或PXR对UGT1A1和UGT1A6转录活性产生影响,但仅在高剂量时这种作用较为明显。总之,甘草提取物、18α、β-GL和18α、β-GA通过诱导核受体CAR或PXR,从而进一步诱导下游UGT1A1和UGT1A6等一系列解毒靶基因的表达,这可能是甘草解百毒的重要作用机制之一,下一步可以从PXR/CAR介导解毒酶与外排泵的联合作用机制(如P-gp-UGT或MRP-UGT共转运体系)进行研究,以探明甘草解百毒真正联合机理。5.对乙酰氨基酚药动学实验表明,对乙酰氨基酚的药动学参数(AUCo-24,AUCo-∞,Cmax)在试验组和安慰组之间有较大差异,且AUC0-∞有统计学意义(P<0.05),同时也观察到对乙酰氨基酚的总体消除半衰期有所加快。在我们也比较对乙酰氨基酚葡萄糖醛酸结合物(PG)血药浓度在三个实验组中的变化,发现甘草酸二胺胶囊组中的PG主要药动学参数与安慰组相比有显著变化,尤其是AUCo-24,AUCo-∞都有统计学意义(P<0.05)。与安慰组相比,葡醛内酯试验组在整个过程中对对乙酰氨基酚及其结合物的主要药动学参数有所影响,但变化不大。据此我们推测甘草酸二胺胶囊组可能诱导Ⅱ相酶UGT,从而加快底物对乙酰氨基酚的代谢,使结合物的生成增加,进而促进排泄。葡醛内酯组结果显示,增加葡萄糖醛酸能在一定程度加快UGT底物的代谢和排泄,但起主要作用的可能还是由于诱导了Ⅱ相结合酶UGT所致。本试验中也有些APAP和PG主要药动学参数变化不明显,可能是我们诱导时间太短,也有可能是受试者体内影响底物对乙酰氨基酚代谢UGT同工酶的UGT1A6、UGT1A1和UGT1A9的多态性从而对有些药动学参数产生了影响,因此下步实验可以从扩大样本量,基因分型等角度来进一步研究甘草酸制剂对Ⅱ相解毒酶的诱导作用。本研究对甘草提取物、甘草酸18位差向异构体及其水解产物对UGT和GST的影响进行了探讨,在体内外实验中发现实验药物不同程度地对UGT1A,UGT2B及部分同工酶UGT1A1、UGT1A6, GSTπ有诱导作用；其中甘草提取物和18β-GL对Ⅱ相解毒酶的有显著诱导作用,验证了实验药物通过上调上游因子PXR和CAR信号通路从而增加下游UGT1A1和UGT1A6等一系列解毒基因的表达,可能这个信号通路是甘草解毒的重要机理之一,在比较差向异构体对Ⅱ相结合酶UGT和GST的作用是否存在立体选择性发现由于它们之间存在一定的差异,但没有显著的影响。本实验为临床上甘草应用于解毒治疗提供新的理论支持,也为其它具有解毒作用的中药研究提供新的思路,同时为临床药物相互作用研究提供科学的理论依据.更多还原

【Abstract】 Motherland medicine have long recognized that licorice had a role on reconcile the various drugs and detoxification, which considered licorice to be alleviated the toxicity and strong of other drugs. Compatibility of Chinese Herbs and Licorice Root are used to ease their toxicity. The scientific traditional understanding of "Cabernet drug toxicity" have been demonstrated by a large number of clinical practice and experimental studies. Glycyrrhizic acid (GL) is the main active component of licorice (Radix Glycyrrhiza), one molecule of GL can be transformed to two molecule of glucuronic acid and one molecule of glycyrrhetinic acid (GA) after hydrolyzation. Base on the different configuration of C18-H bond of triterpene saponin mother nucleus, GL has two different epimers:18a-GL and18(3-GL and they can be hydrolyzed to corresponding18a-GA and18β-GA. They were not distinguished at most previous researches. But the difference of C-18epimers of glycyrrhizic acid has attracted more and more attention after magnesium isoglycyrrhetate was marketed in2006. Preliminary studies of our research group had found that licorice can induce the expression of P-gp, The effects of C-18epimers of Licorice extract, glycyrrhizic acid and their hydrolysis products on II phase detoxification enzymes were introduced in this paper.OBJECTIVEStudy the effects of liquorice extract, C-18epimers of glycyrrhizic acid and their hydrolysis products on protein transcription and expression of UGT1A, UGT2B, and its isozyme UGT1A1and UGT1A6and GSTπ in vitro and in vivo studies and investigate that whether there are stereoselectivity on them; and intended confirmation that the induced effect and mechanism of Liquorice extract, C-18epimers glycyrrhizic acid and their hydrolysis products on the II phase detoxification enzyme; Study the effects of glycyrrhizin preparations on the pharmacokinetics of paracetamol, initially investigate possible drug interaction between glycyrrhizin preparations and substrate of II phase detoxification enzyme UGT that may occur in clinical practice.METHODS 1.Liquorice extract and glycyrrhizic acid18H epimer from different concentra-tions and at different times of the two-phase detoxification enzyme of ratsLicorice extract from different concentrations (90,180,360mg.kg-1) and18a-GL and18β-GL (12.5,25,50mg.kg-1) and at different times (3,6,12consecutive days) in rats by induced Experimental (n=5), By Immunohistochem-ical techniques with chip array experiments investigated the impact of the test drugs on the two-phase detoxification enzyme UGT1A and UGT2B and GSTπ.2. Licorice extract, glycyrrhizic acid18H epimer and their hydrolysis products of Ⅱ-phase detoxification enzymes in ratsThe42rats were randomly divided into control group (saline), licorice extract (concluding14.3%glycyrrhizic acid) group,18a-GL,18β-GL,18a-GA18P-GA,positive control group (phenobarbital)(n=6), respectively, which were induced by7consecutive days. Through the liver microsomal method, the Western blot and the array of tissue microarray methods, and the "trinity"of experimental methods from subcellular level and tissue morphology of the liver tissue level investigated the effect on the licorice extract and its main components18α, β-GL and18α, β-GA on UGT1A, the UGT1A1, UGT1A6,UGT2B and GSTπ.3. HepG2cell culture and MTT toxicity testHepG2cell was cultured with MEM (Modified Eagle’s Medium) and was applied to study the effects of licorice extract, GL and GA epimers on the Ⅱ-phase detixification enzyme of UGT and GST. MTT (Methyl thiazolyl tetrazolium) assay was applied to determine the maximum non-cytotoxic dose of each test drugs the activity of cells during the experiment, and ensure maximum test concentration of each drugs.4. The effect licorice extract, glycyrrhizic acid18H epimer and hydrolysis products on the two phase detoxification enzyme protein expression and transcription in HepG2cellsRifampicin was used to positive control, the HepG2cells of blank treatment as a negative control. By Western-Blot and Q-PCR method to study the effect Licorice extract and its main components18a,β-GL and18a, β-GA of protein expression levels and mRNA levels on the Ⅱ phase detoxification enzyme UGT1A, UGT2B isoenzyme UGT1A1and UGT1A6and GSTπ.5. The effect of licorice extract, glycyrrhizic acid18H epimer and their hydrolysis products on UGT1A1and UGT1A6transcriptional activity by the nuclear receptors PXR and CARBuild hPXR, hCAR and the RXRa Eukaryotic expression plasmid and UGT1A1and UGT1A6luciferase reporter plasmid, which were transiently cotransfected into HepG2cells, DMSO was used to a blank control group, rifampicin was used to PXR positive control group, PCN was used to a PXR-negative control group, CITCO was used to CAR positive control group, TCPOBOP was used to CAR negative control group. Drug treatment groups:licorice extract (2,20,200mg. mL-1) and18a-glycyrrhizic acid,18β-glycyrrhizic acid,18α-glycyrrhetinic acid,18β-glycyrrhetinic acid (1,10,100μM). Detection of licorice extract and its main component of18α, β-GL and of18a,(3-GA on the transcriptional activity by the reporter gene luciferase reporter gene method.6. Effects of glycyrrhizin preparation on the pharmacokinetics of paracetamolThe study were assigned to open-label, randomized-sequence, latin square design which consisted of three1-day treatment periods and two7-day washout periods.12subjects were randomly divided into three groups, Blood samples (4mL each) were collected at0,10,20,30and45min; then1.0,1.5,2.0,3.0,4.0,6.0,8.0,10.0,12.0and24.0hours after dosing. Urine samples were collected prior to the administration and0-2,2-4,4-6,6-8,8-12,12-24hours after administration. The blood and urine samples obtained were frozen at-75℃until analysis. The HPLC-MS/MS method was used to determinated the plasma concentration of paracetamol and its conjugates. The DAS2.1.1software was used to calculate the pharmacokinetic parameters.The SPSS version13.0(SPSS Inc., Chicago, Illinois) was used for calculation of the pharmacokinetic parameters.RESULTS AND CONCLUSION1. The results of the liver tissue array immunohistochemistry chip by statistical analysis found that the induction effect of licorice extract and180-GL on Ⅱ-phase detoxification enzyme UGT1A and UGT2B in a dose-dependent and time-dependent. The glutathione transferase GSTπ also was induced, but the time-dependent and dose-dependent manner is not obvious.2. The results showed that licorice extract induced UGT1A, UGT1A1,UGT1A6, UGT2B significantly after the rats were induced by the experimental drug for seven consecutive days, but the effect is not obvious for GSTπ. The18a-GL and18β-GL have an effect on the UGT and its isozymes UGTIA1, UGT1A6, meantime,18β-GL have an more pronounced induced effect on Ⅱ-phase enzyme.The18a-GA have a significant induction effect on UGT2B, by compared,the18(3-GA have a more obviously effect on UGT1A and its isozyme UGT1A1and UGT1A6. Glycyrrhizic acid and glycyrrhetinic acid as well as their isomers have some differences in a variety of studies, both the difference in metabolism should be taken attention seriously.3. Licorice extract enhanced significantly the level of protein expression and the transcriptional level of mRNA expression of UGT1A, UGT2B, the UGT1A1, UGT1A6and GSTπ in HepG2cells.18a, β-GL enhanced UGT1A, UGT2B, UGTIA1, UGT1A6and GSTπ protein expression and mRNA expression;18β-GL had a significantly enhanced UGTIA1and UGT1A6protein expression and transcriptional levels, which consistent with reported that18(3-GL had a more induction significantly than18a-GL in rat liver tissue;18a-GA and18β-GA at the cellular level have a more obviously induction impact on protein expression and mRNA expression of UGT1A,UGT2B, UGTIAI, UGT1A6and GSTπ than that in the liver tissue of rats, especially the18β-GA have a significant induction at the transcriptional level of UGT1A, UGT1A1,UGT1A6, UGT2B, and GSTπ mRNA. In the protein expression level,18β-GA had also a significant induction role for UGT1A and UGT1A1.4. Licorice extract,18α,β-GL and of18a, β-GA have an impact on UGTIA1and UGT1A6transcriptional activity through the nuclear receptor CAR or PXR, and this effect is more obvious at high doses. The effect of18a, β-GL on this transcriptional activity shows some differences, especially18a-GL have not significant impact at low dose on UGTIA1and UGT1A6transcriptional activity by the nuclear receptor CAR or PXR except for a obvious role at high doses, however,18β-GL have a significant induction at low, medium and high dose through the nuclear receptor CAR or PXR on UGT1A1and UGT1A6transcriptional activity, which also may be the real reason for the mechanism that18β-GL induce significantly Ⅱ-phase detoxification enzyme UGT.18α, β-GA have an impact on the UGT1A1and UGT1A6transcriptional activity by the nuclear receptor CAR or PXR, but only at high doses, this effect is more obvious. In short, licorice extract,18α, β-GL and18a,(3-GA have an induction of through the nuclear receptor CAR or PXR, and further induce downstream target gene UGT1A1and UGT1A6expression, which may be licorice detoxification one of the mechanisms, the next step study can be carried out from the joint mechanism of PXR/CAR-mediated detoxification enzymes and efflux pump (such as P-gp-UGT pumps or MRP-UGT cotransporter system), to proven licorice detoxification of real joint mechanism.5. The pharmacokinetic experiments of paracematol show that the acetamin-ophen pharmacokinetic parameters (AUC0.24, AUC0-∞, Cmax) between the experiment-tal and control groups are large different, and AUC0-∞had a statistically significance (P<0.05), compared with the placebo group, paracetamol total elimination half life has been accelerated that also been observed in experiment group. We also compared changes in plasma concentrations of PG in the three experimental groups, which found PG main pharmacokinetic parameters of diammonium glycyrrhininate capsules had a significant changes compared with the placebo group, especially in the AUC0-24and AUC0-∞has a significant difference (P<0.05). Compared with the placebo group, glucuronolactone experimental group have an impact on the main pharmacokinetic parameter of paracetamol and its conjugates, but not significantly.Therefore, we speculated that the diammonium glycyrrhininate capsules group may induce two-phase enzyme UGT,thus speeding up the metabolism and excretion of the substrate acetaminophen, so that the PG increased accordingly. Glucuronolactone group showed that increase of glucuronic acid can speed up the UGT substrate metabolism and excretion to a certain extent, but it play a major role that may be induced two phase conjugating enzyme UGT. In our research, it is also observed that some main pharmacokinetic parameters of APAP and PG did not change significantly, possibly induction time is too short, or there may be the aspolymorphism of UGT isozyme UGT1A6, the UGT1A1and UGT1A9in vivo influence of substrate on the metabolism of acetaminophen, the next step experiment can be developmented from the expansion of the sample size, genotyping and further study of glycyrrhizic acid preparations which affect on the Ⅱ-phase detoxification enzyme.In this study, induction effect of licorice extract and of18α-GL and18β-GL on two-phase detoxification enzyme UGT1A and UGT2B in a dose-dependent and time-dependent. The glutathione transferase GSTπ also was induced, but the time-dependent and dose-dependent manner is not obvious. Licorice extract and18β-GL significantly induced phase Ⅱ detoxifying enzymes, and validation of experimental drugs by up-regulating the upstream factor of PXR and CAR signaling pathway, thereby increasing a series of downstream UGT1A1and UGT1A6expression of detoxification genes, which this signal pathway is one of the important mechanism of detoxification of licorice, Compared with the isomers the effect on phase-conjugating enzyme UGT and GST found that there were some differences between them,but there is no significant impact. In this study, it is the provide a new theoretical support that licorice used in detoxification in clinical application, and also provide new ideas for the detoxification of traditional Chinese medicine research, furthermore, it is provide scientific and theoretical basis for licorice clinical drug interaction studies.更多还原