Effects Of Zuoguiyin Decoction On Cytochrome P450 And Expression Of MRNA And Protein In Rats

Zuogui Yin is one of the famous prescriptions in Jingyue Quanshu,a classic medical work in China.It is mainly used in various kinds of Andrology and gynecology diseases and has important clinical value.However,due to the increasing reports of drug interaction in recent years,it also brings challenges to the combination of traditional Chinese medicine and other drugs.Therefore,this paper discusses the effects of Zuogui Yin on the expression of cytochrome P450 enzyme,m RNA and protein in rats from three aspects of enzyme activity,transcription and translation,so as to provide certain scientific basis for the clinical safe and effective application of Zuogui Yin.ObjectiveThe purpose of this study is to explore the effect of Zuogui Yin on the activity of six CYP450 enzymes in rats in vitro and in vivo,in order to provide a theoretical basis for the clinical use of Zuogui Yin,and provide reference for future research and clinical application.MethodsIn vitro experiments,phenacetin,bupropion,amodiaquine,omeprazole,dextromethorphan and midazolam were selected as probe substrates of CYP1A2,CYP2B1,CYP2C7,CYP2C11,CYP2D2 and CYP3A1 to prepare a"cocktail"mixed solution,and a UPLC-MS/MS method for simultaneous determination of six CYP450enzyme substrates in rat liver microsomes was established.The experiment was divided into four groups:experimental group?Zuogui Yin water extract group?,inhibitor control group,blank control group and inactive group.The experimental group was divided into10,50,250,500,1000 and 2500?g/L groups according to the different concentrations of Zuogui Yin water extract,three parts of each group were measured in parallel;the control group was added with CYP450 specific inhibitors of different subtypes;the blank group was added with physiological saline of equal volume;the inactive group was not added with NADPH regeneration system to obtain the maximum substrate concentration.Three samples were measured in parallel in each group.To establish a UPLC-MS/MS method for the determination of phenacetin,bupropion,amodiaquine,omeprazole,dextromethorphan and midazolam,which are the probe substrates of CYP1A2,CYP2B1,CYP2C7,CYP2C11,CYP2D2 and CYP3A1 in rats.Twenty two male SD rats were randomly divided into four groups:normal saline group,Zuogui Yin high,medium and low dose?31,20.67,13.78 g/kg?group,six rats in each group.Rats in each group were given intragastric gavage for 2 weeks continuously,and six probe substrate mixtures were injected into tail vein 30 minutes after the last day of intragastric gavage.Blood was collected from the fundus vein plexus at the preset time point after administration.The concentrations of six probe substrates in plasma of rats in each group were measured by UPLC-MS/MS,the main pharmacokinetic parameters of each probe substrate were measured by DAS2.0 software.The pharmacokinetic parameters of each probe substrate were deduced by analyzing the pharmacokinetic parameters of each probe drugFinally,according to the results in vivo and in vitro,the subtypes with different results are further verified.Twelve SD rats of the same batch were divided into high,medium and low dose groups and blank groups.Similarly,RT-q PCR and Western blot were used to determine the expression of CYP450 isoforms in liver and the effect of protein expression in each group.ResultsIn vitro experiment:the UPLC-MS/MS analysis method established in this study can simultaneously determine the concentrations of phenacetin,bupropion,amodiaquine,omeprazole,dextromethorphan and midazolam in six kinds of probe substrates in rat liver microsomes.The method is simple,has good specificity,good linear relationship,high accuracy and precision,and meets the detection requirements of biological samples.The results showed that the IC₅₀values of Zuogui Yin Decoction to CYP1A2,CYP2B1,CYP2C7,CYP2C11,CYP2D2 and CYP3A1 were 1685,3163,1125,1843,2227 and1843?g/m L respectively.In vivo experiment:the UPLC-MS/MS analysis method established in this study can simultaneously determine the plasma concentrations of six probe substrates in rats,namely,phenacetin,bupropion,amodiaquine,omeprazole,dextromethorphan,midazolam.The method is simple in operation,has good specificity,good linear relationship,high accuracy and precision,and meets the detection requirements of biological samples.Compared with the blank group,the main pharmacokinetic parameters AUC_?0-t?,AUC_?0^??and CL of omeprazole,dextromethorphan and midazolam in the high dose group were significantly different,The main pharmacokinetic parameters of phenacetin,bupropion and amodiquine in other dose groups of Zuogui Yin had no significant difference.For CYP450 subtypes with inconsistent results in vitro and in vivo,further verification was carried out at the gene and protein level.The results showed that the expression of CYP2C11 and CYP3A1 was up-regulated in the middle and high dose Zuogui Yin group,CYP2C11 protein was up-regulated in the high dose Zuogui Yin group,and CYP3A1 protein was up-regulated in the middle and high dose Zuogui Yin group.However,Zuogui Yin had no effect on the gene and protein expression of CYP2D2.ConclusionZuogui Yin had no inhibitory effect on CYP1A2,CYP2B1,CYP2C7,CYP2C11,CYP2D2 and CYP3A1 in vitro.Zuogui Yin can induce CYP2C11,CYP2D2 and CYP3A1 enzyme activity in vivo,but has no effect on CYP1A2,CYP2B1 and CYP2C7enzyme activity.The results of PCR and Western blot showed that the expression of CYP2C11 and CYP3A1 m RNA and protein in Zuogui Yin high dose group was significantly up-regulated.This study provides a theoretical basis for the clinical safe and rational use of Zuogui Yin,and has a reference for the further research and development of Zuogui Yin.