霍山石斛对小鼠肝脏细胞色素P450酶表达和活性的影响
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摘要
该文旨在研究口服霍山石斛对小鼠肝脏细胞色素P450酶表达和活性的影响,为评价霍山石斛与临床药物联用时的药物-药物的相互作用提供参考。将C57BL/6小鼠随机分为空白对照组、霍山石斛低剂量组(含生药1.25 g·kg~(-1))、霍山石斛高剂量组(含生药7.5 g·kg~(-1))和苯巴比妥阳性对照组(0.08 g·kg~(-1))连续给药2周。提取小鼠肝脏微粒体,采用Western blot(蛋白质印迹)法检测P450酶主要亚型蛋白表达情况。针对蛋白表达发生变化的P450亚型使用探针药物法对其酶活性进行检测。结果显示,口服霍山石斛对小鼠肝脏CYP1A1,CYP1A2,CYP2B蛋白表达有一定程度的诱导作用。进一步的体外酶活实验结果表明,口服霍山石斛组小鼠CYP1A1,CYP2B对应的探针药物乙氧基异吩噁唑、安非他酮代谢与空白组相比没有显著的差异;口服霍山石斛组小鼠中CYP1A2亚型酶对应的探针药物非那西丁代谢速率V_(max)与空白组相比显著升高约20%,清除率CL_(int)也显著升高约32%。因此,口服霍山石斛可能加快对CYP1A2酶催化的药物代谢,但口服霍山石斛对小鼠肝脏中大多数的P450酶的活性没有影响,理论上对绝大多数临床药物不存在与P450酶系相关的药物-药物相互作用。
This study was carried out to investigate the effect of oral administration of Dendrobium huoshanense on the expressions and activities of hepatic microsomal cytochrome P450 s in mice, and to provide a reference for the evaluation of drug-drug interactions between D. huoshanense and clinical drugs. The C57 BL/6 mice were randomly divided into blank control group, D. huoshanense low dose group(crude drug 1.25 g·kg~(-1)), D. huoshanense high dose group(crude drug 7.5 g·kg~(-1)), and phenobarbital positive control group(0.08 g·kg~(-1)). Each group was intragastrically administered with drugs for 2 weeks. The mice were sacrificed and their liver microsomes were prepared. The expressions of major subtypes of P450 enzyme were determined by Western blot and the probe drugs were used to detect the enzyme activities of P450 subtypes with protein expression changes. Western blot analysis showed that the protein expressions of CYP1 A1, CYP1 A2 and CYP2 B in liver tissues were up-regulated in D. huoshanense-treated group. In vitro enzyme activity tests showed that there were no significant difference in metabolism of 7-ethoxyresorufin(a probe drug for CYP1 A1) and bupropion(a probe drug for CYP2 B) between D. huoshanense group and control group. The metabolism of phenacetin(a probe drug for CYP1 A2) showed a statistical difference in rate V_(max), and it was significantly increased by approximately 20% in D. huoshanense group as compared with the blank control group, and the clearance CL_(int) in treated group was also increased by about 32%. Therefore, oral administration of D. huoshanense had no effects on the activities of most hepatic P450 enzymes in mice, with no drug-drug interaction related to the P450 enzyme system in most clinical drugs theoretically. However, oral administration of D. huoshanense may accelerate the metabolism of CYP1 A2-catalyzed drugs, which needs to be considered in clinical practice.
This study was carried out to investigate the effect of oral administration of Dendrobium huoshanense on the expressions and activities of hepatic microsomal cytochrome P450 s in mice, and to provide a reference for the evaluation of drug-drug interactions between D. huoshanense and clinical drugs. The C57 BL/6 mice were randomly divided into blank control group, D. huoshanense low dose group(crude drug 1.25 g·kg~(-1)), D. huoshanense high dose group(crude drug 7.5 g·kg~(-1)), and phenobarbital positive control group(0.08 g·kg~(-1)). Each group was intragastrically administered with drugs for 2 weeks. The mice were sacrificed and their liver microsomes were prepared. The expressions of major subtypes of P450 enzyme were determined by Western blot and the probe drugs were used to detect the enzyme activities of P450 subtypes with protein expression changes. Western blot analysis showed that the protein expressions of CYP1 A1, CYP1 A2 and CYP2 B in liver tissues were up-regulated in D. huoshanense-treated group. In vitro enzyme activity tests showed that there were no significant difference in metabolism of 7-ethoxyresorufin(a probe drug for CYP1 A1) and bupropion(a probe drug for CYP2 B) between D. huoshanense group and control group. The metabolism of phenacetin(a probe drug for CYP1 A2) showed a statistical difference in rate V_(max), and it was significantly increased by approximately 20% in D. huoshanense group as compared with the blank control group, and the clearance CL_(int) in treated group was also increased by about 32%. Therefore, oral administration of D. huoshanense had no effects on the activities of most hepatic P450 enzymes in mice, with no drug-drug interaction related to the P450 enzyme system in most clinical drugs theoretically. However, oral administration of D. huoshanense may accelerate the metabolism of CYP1 A2-catalyzed drugs, which needs to be considered in clinical practice.
引文
[1] 彭华胜, 王德群, 彭代银. 道地药材“皖药”的形成及其界定[J]. 中国中药杂志, 2017, 42(9):1617.
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[3] 陈存武, 戴军, 姚厚军, 等. 霍山石斛资源与鉴定研究进展[J]. 现代中药研究与实践, 2013, 27(3): 84.
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[5] 黄静, 李胜立, 赵宏伟, 等. 霍山石斛多糖对四氯化碳致急性肝损伤小鼠的保护作用[J]. 中国中药杂志, 2013, 38(4): 528.
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[8] 汪刚, 刘莹, 侯雪峰,等. 药物特异质肝损伤因素、机制及损伤病机探析[J]. 中国中药杂志, 2017, 42(16):3036.
[9] 董海燕, 邵敬伟, 陈剑峰, 等. 4 种中药调节细胞色素P450 3A4转录表达的研究[J]. 中国中药杂志, 2008, 33(9): 1014.
[10] 王菊, 陆兔林, 毛春芹, 等. Cocktail探针药物法评价生, 醋莪术对CYP450酶亚型的影响[J]. 中国药理学通报, 2012, 28(11): 1562.
[11] 雷霆雯, 李红梅, 许庆忠, 等. 大青叶对小鼠肝脏CYP2E1酶活性及信使核糖核酸表达的影响[J]. 中国医院药学杂志, 2006, 26(9): 1095.
[12] 何益军, 石苏英, 金科涛, 等. 甘草与大戟、甘遂、芫花配伍对大鼠肝脏细胞色素P4501A2酶活性的影响[J]. 中国药物与临床, 2007, 7(4): 278.
[13] Monostory K, Vereczkey L. Interaction of theophylline and ipriflavone at the cytochrome P450 level[J]. Eur J Drug Metab Ph, 1995, 20(1): 43.
[14] Jurica J, Konecn【math52z】ková L Z, et al. Simultaneous HPLC determination of tolbutamide, phenacetin and their metabolites as markers of cytochromes 1A2 and C6/11 in rat liver perfusate[J]. J Pharmaceut Biomed, 2010, 52(4):557.
[15] 张艳辉, 于超, 郭延垒,等. 高效液相色谱法测定鼠肝微粒体中CYP1A2酶的活性及动力学考察[J]. 药物分析杂志, 2012(2):189.
[16] Wang X, Abdelrahman D R, Fokina V M, et al. Metabolism of bupropion by baboon hepatic and placental microsomes [J].Biochem Pharmacol, 2011, 82(3): 295.
[17] Loboz K K, Gross A S, Ray J, et al. HPLC assay for bupropion and its major metabolites in human plasma[J]. J Chromatogr B, 2005, 823(2): 115.
[18] Meunier B, De Visser S P, Shaik S. Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes[J]. Chem Rev, 2004, 104(9): 3947.
[19] 朱大岭,韩维娜,张荣. 细胞色素P450酶系在药物代谢中的作用[J]. 医药导报, 2004, 23(7): 440.
[20] 王宇光. 基于药物代谢酶的中药相互作用研究[D]. 北京: 中国人民解放军军事医学科学院, 2006.
[21] 张晓燕, 许海淼, 王笃军, 等. 原儿茶酸与原儿茶醛对小鼠肝脏细胞色素P450表达的影响[J]. 中国野生植物资源, 2017, 36(1): 18.
[22] 许海淼, 张晓燕, 王笃军, 等. 苍术苷A对小鼠CYP450主要亚型表达的影响[J]. 中成药, 2017,39(2): 398.
[23] 王笃军, 王硕, 严峰, 等. 欧前胡素和异欧前胡素对小鼠肝CYP450的影响[J]. 中成药, 2017, 39(1): 14.
[24] 年华, 马明华, 徐玲玲, 等. 细胞色素P450酶与药物代谢的研究进展与评价[J]. 中国医院用药评价与分析, 2010 (11): 964.
[25] 胡云珍, 姚彤炜. 细胞色素P4501A的研究进展[J]. 中国药学志,2003,38(4):246.
[26] 成龙, 王岚, 王彦礼,等. 金铃子散不同配比方对大鼠肝药酶CYP1A2活性的影响[J]. 中国中药杂志, 2012, 37(5):648.
[27] 周小艳. 基于CYP1A2的麻黄茶碱药物相互作用研究[D]. 哈尔滨:哈尔滨医科大学, 2010.
[2] 谢晋, 查良平, 彭华胜,等. 历代本草中安徽地产药材的品种与分布[J]. 中国中药杂志, 2017, 42(9):1623.
[3] 陈存武, 戴军, 姚厚军, 等. 霍山石斛资源与鉴定研究进展[J]. 现代中药研究与实践, 2013, 27(3): 84.
[4] 宋广青, 刘新民, 王琼, 等. 石斛药理作用研究进展[J]. 中草药, 2014, 45(17): 2576.
[5] 黄静, 李胜立, 赵宏伟, 等. 霍山石斛多糖对四氯化碳致急性肝损伤小鼠的保护作用[J]. 中国中药杂志, 2013, 38(4): 528.
[6] 王凯, 眭丹娟, 王长锁, 等. 5 种石斛对四氯化碳致小鼠急性肝损伤的保护作用[J]. 中国中药杂志, 2017, 42(10): 1945.
[7] Nelson D R, Schuler M A, Paquette S M, et al. Comparative genomics of rice and arabidopsis analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a dicot[J]. Plant Physiol, 2004, 135(2): 756.
[8] 汪刚, 刘莹, 侯雪峰,等. 药物特异质肝损伤因素、机制及损伤病机探析[J]. 中国中药杂志, 2017, 42(16):3036.
[9] 董海燕, 邵敬伟, 陈剑峰, 等. 4 种中药调节细胞色素P450 3A4转录表达的研究[J]. 中国中药杂志, 2008, 33(9): 1014.
[10] 王菊, 陆兔林, 毛春芹, 等. Cocktail探针药物法评价生, 醋莪术对CYP450酶亚型的影响[J]. 中国药理学通报, 2012, 28(11): 1562.
[11] 雷霆雯, 李红梅, 许庆忠, 等. 大青叶对小鼠肝脏CYP2E1酶活性及信使核糖核酸表达的影响[J]. 中国医院药学杂志, 2006, 26(9): 1095.
[12] 何益军, 石苏英, 金科涛, 等. 甘草与大戟、甘遂、芫花配伍对大鼠肝脏细胞色素P4501A2酶活性的影响[J]. 中国药物与临床, 2007, 7(4): 278.
[13] Monostory K, Vereczkey L. Interaction of theophylline and ipriflavone at the cytochrome P450 level[J]. Eur J Drug Metab Ph, 1995, 20(1): 43.
[14] Jurica J, Konecn【math52z】ková L Z, et al. Simultaneous HPLC determination of tolbutamide, phenacetin and their metabolites as markers of cytochromes 1A2 and C6/11 in rat liver perfusate[J]. J Pharmaceut Biomed, 2010, 52(4):557.
[15] 张艳辉, 于超, 郭延垒,等. 高效液相色谱法测定鼠肝微粒体中CYP1A2酶的活性及动力学考察[J]. 药物分析杂志, 2012(2):189.
[16] Wang X, Abdelrahman D R, Fokina V M, et al. Metabolism of bupropion by baboon hepatic and placental microsomes [J].Biochem Pharmacol, 2011, 82(3): 295.
[17] Loboz K K, Gross A S, Ray J, et al. HPLC assay for bupropion and its major metabolites in human plasma[J]. J Chromatogr B, 2005, 823(2): 115.
[18] Meunier B, De Visser S P, Shaik S. Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes[J]. Chem Rev, 2004, 104(9): 3947.
[19] 朱大岭,韩维娜,张荣. 细胞色素P450酶系在药物代谢中的作用[J]. 医药导报, 2004, 23(7): 440.
[20] 王宇光. 基于药物代谢酶的中药相互作用研究[D]. 北京: 中国人民解放军军事医学科学院, 2006.
[21] 张晓燕, 许海淼, 王笃军, 等. 原儿茶酸与原儿茶醛对小鼠肝脏细胞色素P450表达的影响[J]. 中国野生植物资源, 2017, 36(1): 18.
[22] 许海淼, 张晓燕, 王笃军, 等. 苍术苷A对小鼠CYP450主要亚型表达的影响[J]. 中成药, 2017,39(2): 398.
[23] 王笃军, 王硕, 严峰, 等. 欧前胡素和异欧前胡素对小鼠肝CYP450的影响[J]. 中成药, 2017, 39(1): 14.
[24] 年华, 马明华, 徐玲玲, 等. 细胞色素P450酶与药物代谢的研究进展与评价[J]. 中国医院用药评价与分析, 2010 (11): 964.
[25] 胡云珍, 姚彤炜. 细胞色素P4501A的研究进展[J]. 中国药学志,2003,38(4):246.
[26] 成龙, 王岚, 王彦礼,等. 金铃子散不同配比方对大鼠肝药酶CYP1A2活性的影响[J]. 中国中药杂志, 2012, 37(5):648.
[27] 周小艳. 基于CYP1A2的麻黄茶碱药物相互作用研究[D]. 哈尔滨:哈尔滨医科大学, 2010.