基于肝脏药物代谢酶研究附子配伍干地黄的减毒机制

Detoxification mechanism of Aconiti Lateralis Radix Praeparata combined with dried Rehmanniae Radix based on metabolic enzymes in liver

测定大鼠肝微粒体中CYP1A2和CYP3A4酶活性,并从蛋白和转录水平上阐明附子配伍干地黄对CYP450酶蛋白和基因表达的影响,探索附子配伍干地黄对其减毒的分子机制。采用"Cocktail"探针药物法进行CYP450酶活性测定;采用CO还原差示法测定肝微粒体中CYP450酶含量并采用RT-PCR测定大鼠肝脏CYP1A2和CYP3A4 mRNA表达量。结果显示,与空白组比较,附子干地黄组CYP1A2和CYP3A4酶活性及mRNA表达显著升高(P<0.05);附子组CYP450含量显著降低(P<0.01);干地黄组、附子干地黄组CYP450含量显著升高(P<0.05)。因此,附子配伍干地黄能够诱导CYP1A2和CYP3A4酶活性,增加CYP450酶含量,加快附子毒性成分的代谢,达到附子减毒作用。

The enzymes CYP1 A2 and CYP3 A4 were measured by building a"Cocktail"probe drug and the incubation system of liver microsomes.The compatibility of Aconiti Lateralis Radix Praeparata combined with dried Rehmanniae Radix on CYP450 enzyme protein and gene expression was explored from the level of protein and molecular biology.It explored the molecular mechanism of compatibility detoxication of Aconiti Lateralis Radix Praeparata to provide scientific support for clinical safe and effective application of Aconiti Lateralis Radix Praeparata.The CYP450 enzyme activity was determined by using"Cocktail"probe drugs.The content of CYP450 enzyme was measured by CO reduction of differential spectrum method.The mRNA expression of CYP1 A2 and CYP3 A4 enzyme was detected by RT-PCR technology.Compared with the blank group,the CYP1 A2 and CYP3 A4 enzyme activity and mRNA expression were increased in the dried Rehmanniae Radix combined with Aconiti Lateralis Radix Praeparata group with significant differences(P<0.05),while the CYP3 A4 enzyme activity and mRNA expression were no influence in the Aconiti Lateralis Radix Praeparata group.The CYP3 A4 enzyme activity and mRNA expression were increased in the dried Rehmanniae Radix and the dried Rehmanniae Radix combined with Aconiti Lateralis Radix Praeparata group,and there were significant differences(P<0.05).The content of CYP450 enzyme was decreased in the Aconiti Lateralis Radix Praeparata group,contributed to extremely significant difference(P<0.01).The content of CYP450 enzyme was increased in the dried Rehmanniae Radix and the dried Rehmanniae Radix combined with Aconiti Lateralis Radix Praeparata group,and there were significant differences(P<0.05).The CYP1 A2 and CYP3 A4 enzyme activity and gene expression were enhanced after dried Rehmanniae Radix combined with Aconiti Lateralis Radix Praeparata.The metabolism of toxic ingredients of Aconiti Lateralis Radix Praeparata was accelerated to reach an effect of detoxication.The detoxication mechanism of compatibility of Aconiti Lateralis Radix Praeparata was verified from the viewpoint of liver metabolic enzymes.