基于肝药代谢酶的附子配伍芍药减毒机制研究
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摘要
目的:通过建立"Cocktail"探针药物法及体外肝微粒体孵育体系,测定大鼠肝微粒体中CYP1A2和CYP3A4酶的活性,并从蛋白和转录水平阐释附子芍药配伍对CYP450酶基因表达的影响。方法:采用"Cocktail"探针药物法,建立同时测定CYP1A2和CYP3A4酶两种探针底物的体外肝微粒体孵育体系;采用RP-HPLC法测定两种探针底物在大鼠肝微粒体孵育体系中的浓度;CO还原差示光谱法测定大鼠肝微粒体中CYP450酶含量;RT-PCR技术测定大鼠肝脏CYP1A2和CYP3A4酶基因m RNA表达量。结果:与空白组比较,芍药组CYP1A2酶活性有显著性差异(P<0.05),芍药组CYP1A2酶m RNA表达量升高,有极显著差异(P<0.01);附子芍药组对CYP1A2酶有诱导作用,有极显著差异(P<0.01),附子芍药组CYP1A2酶m RNA表达量升高,有显著性差异(P<0.05)。结论:附子配伍芍药能够诱导CYP1A2酶活性,增加CYP1A2酶基因m RNA表达量,加快附子毒性成分的代谢过程,进而达到附子的减毒作用。
To established the method of "Cocktail"probe drugs and liver microsomal system to determine the activity of CYP1 A2 and CYP3 A4 in liver microsomes. The compatibility of Fuzi with peony on CYP450 enzyme protein and gene expression was explored from the level of protein and molecular biology. Methods: The determination of CYP450 enzyme activity was used with the"Cocktail"probe drugs method, which choose caffeine and midazolam as the probe substrates of CYP1 A2 and CYP3 A4 were prepared into a "Cocktail"solution. The RT-HPLC method was established to determine the concentration of the two probe substrates in the liver microsomal incubation system in order to evaluate the effect of the different Fuzi compatibility on the activity of CYP1 A2 and CYP3 A4 and screen the experimental groups which have induced the enzyme activity. The content of CYP450 was determined with a method which is CO reduction differential spectrum with UV spectrophotometer and fluorescence spectrophotometry, and evaluated the effect of the experimental group on the content of CYP450. The gene expression of CYP450 mRNA were determined using reverse transcription polymerase chain reaction RT-PCR technology after getting out liver of all groups and extracting total RNA which was quality authenticated when the rats were sacrificed by cervical dislocation. Results: Compared with the blank group, the CYP1 A2 enzyme activity was induced in peony group, there were significant differences(p<0.05), and mRNA expression was highly significant difference(p<0.01). The CYP1 A2 enzyme activity was induced in Fuzi with peony group, there was extremely significant difference(p<0.01), and mRNA expression was significant differences(p<0.05).Conclusion: The CYP1 A2 enzyme activity and mRNA expression were enhanced after penoy with Fuzi. The metabolism of toxic ingredients of Fuzi was accelerated to reach an effect of detoxication.
To established the method of "Cocktail"probe drugs and liver microsomal system to determine the activity of CYP1 A2 and CYP3 A4 in liver microsomes. The compatibility of Fuzi with peony on CYP450 enzyme protein and gene expression was explored from the level of protein and molecular biology. Methods: The determination of CYP450 enzyme activity was used with the"Cocktail"probe drugs method, which choose caffeine and midazolam as the probe substrates of CYP1 A2 and CYP3 A4 were prepared into a "Cocktail"solution. The RT-HPLC method was established to determine the concentration of the two probe substrates in the liver microsomal incubation system in order to evaluate the effect of the different Fuzi compatibility on the activity of CYP1 A2 and CYP3 A4 and screen the experimental groups which have induced the enzyme activity. The content of CYP450 was determined with a method which is CO reduction differential spectrum with UV spectrophotometer and fluorescence spectrophotometry, and evaluated the effect of the experimental group on the content of CYP450. The gene expression of CYP450 mRNA were determined using reverse transcription polymerase chain reaction RT-PCR technology after getting out liver of all groups and extracting total RNA which was quality authenticated when the rats were sacrificed by cervical dislocation. Results: Compared with the blank group, the CYP1 A2 enzyme activity was induced in peony group, there were significant differences(p<0.05), and mRNA expression was highly significant difference(p<0.01). The CYP1 A2 enzyme activity was induced in Fuzi with peony group, there was extremely significant difference(p<0.01), and mRNA expression was significant differences(p<0.05).Conclusion: The CYP1 A2 enzyme activity and mRNA expression were enhanced after penoy with Fuzi. The metabolism of toxic ingredients of Fuzi was accelerated to reach an effect of detoxication.
引文
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[3]赵欢,杨巧芳,李梦茜.附子毒性研究进展[J].河北中医,2017,39(5):774-777.
[4]周思思,马增春,谭洪玲,等.人参附子不同比例配伍对小鼠急性毒性的影响[J].天津中医,2013,30(1):43-46.
[5]李建波,张莉,张洁.药对配伍理论及相关研究概述[J].中医杂志, 2013,54(15):1335-1340.
[6]孙燕,王雨,刘京华,等.附子双酯型二萜生物碱对代谢酶的影响及心肌毒性研究进展[J].天津中医药大学学报,2018,37(4):274-279.
[7]王阶,王永炎,杨戈.中药方剂配伍理论研究方法和模式[J].中国中药杂志,2005,30(1):6-11.
[8]冯玉华,李静,闫润红.炮附子配伍芍药对阳虚模型大鼠血清LDH、CK的影响[J].山西中医,2015,31(9):46-47.