醋甘遂与炙甘草免煎颗粒配伍对CYP2E1、CYP3A4基因和蛋白表达的影响

Compatible Effects of Vinegar-Preparing Kansui and Radix Glycyrrhizae Preparata on CYP2E1 and CYP3A4 Expressions in Rats Liver

目的:通过检测大鼠肝功能指标和肝组织CYP2E1、CYP3A4的基因、蛋白表达,初步观察醋甘遂与炙甘草配伍对肝脏的损伤以及对肝药酶2个亚型的诱导作用。方法:将大鼠按体质量随机分为正常组、炙甘草组(免煎颗粒剂)、醋甘遂组(研末)、炙甘草-醋甘遂组、甘遂半夏汤组。其中正常组给予生理盐水灌胃,其余各组给予相应药液灌胃,连续给药14天。实验结束时,腹主动脉取血分离血清和血浆检测谷草转氨酶(AST)、谷丙转氨酶(ALT)、乳酸脱氢酶(LDH)含量;取肝组织一部分用福尔马林固定观察肝组织病理形态,一部分液氮冻存,采用RT-PCR和western-blot技术检测肝组织中CYP2E1、CYP3A4的基因、蛋白表达情况。结果:与正常组相比,各给药组ALT含量各组变化不明显,无统计学差异(P>0.05),AST含量甘遂组以及醋甘遂-炙甘草配伍组、甘遂半夏汤组较正常组降低,其中甘遂半夏汤组降低显著,有统计学差异(P<0.05),LDH含量各给药组均较正常组显著降低,有统计学差异(P<0.05,P<0.01)。与空白组比较,炙甘草组、醋甘遂组、醋甘遂-炙甘草组和甘遂半夏汤组CYP2E1基金和蛋白表达均有所升高,CYP3A4的基因和蛋白表达则有所下降,二者均具有统计学差异(P<0.01),其中以醋甘遂-炙甘草组升高或降低最为明显;与醋甘遂-炙甘草组比较,甘遂半夏汤组下调CYP2E1基金和蛋白表达,上调CYP3A4的基因和蛋白表达。结论:本实验从分子机制上证实了醋甘遂和炙甘草配伍有增毒效应,以复方的形式使用则能降低毒性,但在生化指标方面未表现出明显增毒作用,可能还存在其他配伍机制有待进一步研究,且需要配合病理状态的研究以确定是否有增效作用。

Objective: To study the influence on the rats liver factor and the mRNA,protein expression of cytochrome P450 2E1( CYP2E1),cytochrome P450 3A4( CYP3A4) by combined use of vinegar-preparing kansui and Radix Glycyrrhizae Preparata. Methods: Sixty male Wistar rats,weighted 140 ~ 160 g,were randomly assigned to normal group,vinegar-preparing kansui group,Radix Glycyrrhizae Preparata group,compatibilities of vinegar-preparing kansui and Radix Glycyrrhizae Preparata group,Gansui Banxia Decoction group,12 rats in each group. The normal group received saline irrigation and the remaining groups were given the corresponding liquid orally,administration volume 1 m L/100 g,continuous administration for 14 days. Weigh every 7 days. Freely eat food and water. 14 days after the anesthesia with3. 5% hydrate of chlorine( 1 m L/100 g),the levels of serum alanine transaminase( ALT),aspartate transaminase( AST),lactate dehydrogenase( LDH) were detected by Automatic Biochemistry Analyzer. The liver tissues in the formalin-fixed were studied by light microscopy. The mRNA expression levels of CYP2E1 and CYP3A4 in rats liver microsomes were determined by semi-quantitative reverse transcription polymerase chain reaction( RT-PCR). The protein expression levels of CYP2E1 and CYP3A4 were detected by Western blotting. The experimental data was processed by SPSS 11. 5 software and the measurement data was compared by using analysis of variance. The homogeneity of variance used LSD and homogeneity of variance used Dunnett T3. The count data used non parametric test and the significant difference was at P < 0. 05. Results: There was no significant difference among every groups for ALT. Compared with normal group,administration groups with vinegar-preparing kansui could decrease plasma AST level which was decreased significantly in Gansui Banxia Decoction group. LDH levels of each drug administration group were lowered significantly than that of the normal group. The expression levels of mRNA and protein of CYP2E1 were significantly increased in co-administration group. The expression levels of mRNA and protein of CYP3A4 were significantly decreased in co-administration group. But,Gansui Banxia Decoction group could decrease the level of CYP2E1,with increasing the level of CYP3A4. Conclusion: This experiment from the molecular mechanism of the compatibility of vinegar-preparing kansui and Radix Glycyrrhizae Preparata increased toxic effects,while this compound can reduce the toxicity. However,it was found that the combined medication of vinegar-preparing kansui and Radix Glycyrrhizae Preparata did not affect the mutual toxicity remarkably in biochemical index. Many other mechanisms need to be further studied because drug interaction is complex.