Mechanism Of The Inductive Effect Of Zolmitriptan On Rat Hepatic CYP3A1/2

We studied the mechanism of the inductive effect of zolmitriptan(Zol) on rat CYP 3A1/2 with report gene,investigated the contribution of PXR in the progress.Based on the results that our team have studied before,we observed the inductive effect of Zol on the expression and activity of juvenile rat CYP 3A2 to studied the mechanism of the sex difference in the inductive effect of Zol on rat CYP 3A1/2.Objective To study the mechanism of the inductive effect of Zol on rat CYP 3A1/2 and to investigate the changes of the inductive effect with the changes of the concentrations of Zol.Method Extract rat liver total DNA,obtain CYP 3A1/2 promoter sequence using the extracted DNA as templates.Connected the two sequences to the plasmid PMD 18-T respectively and verified by sequencing.Choose the right ones,connected to the plasmid pGL3,obtain the plasmid pGL3-3A1 promoter and pGL3-3A2 promoter.Connected the rPXR sequence and PCDNA3.1 plamid to get the plasmid rPXR-PCDNA3.1.Transfected HepG2 cells with the plasmid of pGL3-3A1/2 promoter,rPXR-PCDNA3.1 and pRL-TK with certain ratio.Treated with different agents for 24h to investigate the inductive effect on CYP 3A1/2 promoter by detecting the fluorescence intensity.We studied the inductive effect of different concentrations of Zol(10,50,100μmol·L^-1) on CYP 3A1/2,using the classic inductor pregnenolone-16α-carbonitrile(PCN) as the positive control,and solvent DMSO as the negative control.Administrator the male and female rats 14 days after born with 1ml different reagents,ig for 3 dys,then prepare the microsome.Study the inductive effect of different concentration of Zol(0.1mg,0.5mg,2.5mg/200g) on juvenile rat CYP 3A2,Using 20mg/200g dexamethasone(Dex) as the positive control, and the solvent CMC-Na as the negative control.Investigate the expression of hepatic CYP 3A2 with Western Blot and the activity of CYP 3A with the metabolism of Testosterone.Administrator the male rats with 1ml different concentrations of Zol (0.01mg,0.1mg,0.5mg,2.5mg,5mg,10mg/200g),ig for 3 days,then prepare the hepatic microsome.Investigate the activity of CYP 3A with the metabolism of Testosterone.Using 20mg/200g Dex as the positive control,and the solvent CMC-Na as the negative control.Results In the report gene experiments,compared to the negative groups,the positive and high level Zol groups exhibited a significant induction effect on hepatic CYP 3A1,the activity increased to 1.93 and 1.96 times respectively,the positive. middle level and low level Zol groups exhibited a significant induction effect on hepatic CYP 3A2,the activity increased to 2.51,2.10 and 1.63 times respectively.Zol can induce the express and the activity of male or female juvenile rats,the middle level (0.5mg/200g) and high level(2.5mg/200g) showed a significant inductive effect,the activity of CYP 3A in male juvenile rats increased to 2.20 and 1.85 times respectively, and increased to 1.71 and 1.55 times in female juvenile rats.Investigate the inductive effect of a series concentrations of Zol on the activity of CYP 3A,when treated with 0.5mg/200g body weight of Zol,the activity of CYP 3A can be induced most efficiently,reached 1.46 times of the activity in negative control rats.Conclution As the PXR pathway showed,Zol with a dose-dependent characteristic can significantly induce CYP 3A2 in low and middle levels and induce CYP 3A1 in high level.In a word,we presume PXR participated in the progress of the inducing of Zol on hepatic CYP 3A1/2.The inductive effect of Zol on adult male rats is more efficiently than the effect in adult female rats,the reason caused this fact is that there are two subtypes of CYP 3A(CYP 3A1 and CYP 3A2) in adult male rats,while there is only one subtype of CYP 3A(CYP 3A1) in adult female rats.When the dosage of Zol increasing,the inductive effect of Zol on CYP 3A enhanced in the first then decreased.