Inhibition Mechanism Of Osthole On The Activities Of CYP3A Of Rats In Vivo

Background:In our previous studies, we found that the osthole(Ost) has weaker inhibitory action to CYP3A4、CYP2C9 and CYP2D6 in human liver microsomal. At the same time we studied the influence of Ost on CYP3 A enzyme activity in rat, ascertaining a strong inhibitory effect of Ost on CYP3 A enzyme activity in vivo. Therefore, Our topic is to expore the inhibition mechanism of Ost on the activities of CYP3 A of rats in vivo through the rat metabolic model.Objectives:The aim of this test is to expore the inhibition mechanism of Ost on the activities of CYP3 A of rats in vivo: Firstly, Corroborating the inhibitory action of Ost on CYP3 A enzyme activity and comparing the differences of single and multiple Ost dosing in the effect of CYP3 A enzyme activity in rat. Secondly, studying the effect of Ost on CYP3A1/2 m RNA expression in rat liver through semi-quantitative RT-PCR technology; Thirdly, Investigating the effect of Ost on CYP3A1/2 Protein expression in rat liver by Western-blot technology.Methods:1. Corroborating the inhibitory action of Ost on CYP3 A enzyme of rats in vivo.Designing solvent control group(including polysorbate 80 and 0.5% DMSO saline solution), Ost experimental group(20 mg/kg Ost solution) and positive control group(50 mg/kg ketone solution), using midazolam(MDZ) as the probe drug,And we begined the experiment by injecting single and multiple dosages of Ost to rat abdominal cavity. Then we determinated the plasma concentrations of MDZ at different time points by HPLC method after injecting of MDZ in rat caudal veins. We calculated the MDZ pharmacokinetic parameters to Corroborate the inhibitory action of Ost on CYP3 A enzyme and compare the differences of single and multiple Ost dosing in the effect of CYP3 A enzyme activity in rat.2. Effect of Ost on the CYP3A1/2 m RNA expression in rat liver.Designing solvent control group(including polysorbate 80 and 0.5% DMSO saline solution), low dose group(10 mg/kg Ost solution), medium dose group(20mg/kg Ost solution) and high dose group(40 mg/kg Ost solution). we injected multiple dosages of drug to rat abdominal cavity for seven consecutive days, then taking out the rat liver under the condition of low temperature aseptic and saving to-80 ℃ refrigerator. Using Trizol reagent extracting total RNA and detecting the expression of CYP3A1/2 m RNA by RT-PCR.3. Effect of Ost on the CYP3A1/2 Protein expression in rat liver.Using RIPA cracking fluid extracting the protein of rat liver tissue samples in method 2 and detecting the expression of CYP3A1/2 Protein in rat liver by Westernblot technology.1. Corroborating the inhibitory action of Ost on CYP3 A enzyme of rats in vivo.Establishing the HPLC analyse method to detect the rat plasma concentrations of MDZ.The pharmacokinetic parameters of MDZ after injecting single dosage of Ost were as follows: solvent control group AUC(0-t)(356.23 mg·L·min-1) 、 AUC(0-∞)(377.298mg·L·min-1)、MRT(0-t)(27.555 min)、MRT(0-∞)(30.665min)、T1/2(19.959min)、CLz(0.053L·min-1·kg-1);Ost experimental group AUC(0-t)(503.53mg·L·min-1)、AUC(0-∞)(518.977 mg·L·min-1)、MRT(0-t)(35.792min)、MRT(0-∞)(40.312min)、T1/2(30.241 min)、CLz(0.01L·min-1·kg-1);positive control group AUC(0-t)(1020.462 mg·L·min-1) 、 AUC(0-∞)(1031.317 mg·L·min-1) 、 MRT(0-t)(77.173min)、MRT(0-∞)(80.07min)、T1/2(44.691min)、CLz(0.005L·min-1·kg-1)。Results showed that the AUC 0-t), AUC(0-∞) of Ost experimental group relative to the solvent control group were increased(P < 0.05), T1/2, MRT(0- t) and MRT(0-∞) all have extended(P < 0.05), CLz was lower(P < 0.05). Compared with positive control group,the pharmacokinetic parameters of MDZ of Ost experimental group changed less,such as the AUC(0-∞) of positive control group increased by 173% relative to the solvent control group, but the AUC(0-∞) of Ost experimental group only increased by37%. It could be seen that single dosage of Ost have weaker inhibition to the eliminate of MDZ in vivo.The pharmacokinetic parameters of MDZ after injecting multiple dosage of Ost were as follows: Ost experimental group AUC(0-t)(851.785 mg·L·min-1) 、AUC(0-∞)(868.542 mg·L·min-1)、MRT(0-t)(82.253min)、MRT(0-∞)(87.711min)、T1/2(53.487 min)、CLz(0.006L·min-1·kg-1); positive control group AUC(0-t)(1256.687mg·L·min-1)、AUC(0-∞)(1482.351 mg·L·min-1)、MRT(0-t)(84.818min)、MRT(0-∞)(141.738min)、T1/2(118.308min)、CLz(0.003L·min-1·kg-1). Results indicated that the AUC(0-∞) of Ost experimental group increased by 130% relative to the solvent control group and the T1/2 increased by 167%. MRT(0-t) and MRT(0-∞) were significantly prolonged(P < 0.05), CLz also significantly reduced(P < 0.05).According to the result of the above it can be manifested that multiple dosage of Ost have strong inhibitory effect to the eliminate of MDZ in vivo. Comparing the differences of the pharmacokinetic parameters of MDZ between single and multiple dosages group of Ost in vivo, It seemed that the multiple dosages group had stronger inhibitory effect than the single dosages group to the metabolism of MDZ and there were significant differences.2. Effect of Ost on the CYP3A1/2 m RNA expression in rat liver.The expression of CYP3A1 m RNA changed With a concentration-dependence of Ost. Compared with solvent control group, The expression of CYP3A1 m RNA of low dose group increased slightly but no significant difference in liver tissue of rats; In the medium and high dose group the expression of CYP3A1 m RNA appeared a obvious downward trend which were reduced by 24.0% and 47.0%, respectively.Compared with solvent control group, In the low and medium dose group the expression of CYP3A2 m RNA had a slight decline; the expression of CYP3A2 m RNA of high dose group increased slightly, but they were no significant differences.It indicated that Ost didn’t affect CYP3A2 m RNA expression level in the liver of rats.3. Effect of Ost on the CYP3A1/2 Protein expression in rat liver.The expression of CYP3A1 Protein reduced in turn with increasing dosage of Ost and it had a concentration-dependence in rat liver. Compared with solvent control group, it was essentially constant of the expression of CYP3A1 Protein in the low dose group; In the medium and high dose group the expression of CYP3A1 Protein showed a obvious downward trend which were reduced by 34.0% and 57.0%,respectively.The expression of CYP3A2 Protein reduced in turn with increasing dosage ofOst and it had a concentration-dependence in rat liver. Compared with solvent control group, In the low dose group the expression of CYP3A2 Protein reduced slightly but no significant differences; In the medium and high dose group the expression of CYP3A2 Protein appeared a downward trend which were reduced by 29.0% and41.0%, respectively.Conclusions:This test established a reliable sensitive HPLC method to detect the rat plasma concentrations of MDZ and confirmed the the inhibitory action of Ost on CYP3 A enzyme activity. Comparing between single and multiple dosages group of Ost in vivo, It indicated that the multiple dosages group had stronger inhibitory effect than the single dosages group to the activity of CYP3 A enzyme. The RT-PCR experimental results showed that Ost didn’t affect CYP3A2 m RNA expression but had inhibitory effect on CYP3A1 m RNA expression in rat liver. The Western-blot experimental results showed that Ost had inhibitory effect on CYP3A1/2 Protein expression and it had a concentration-dependence, And the inhibitory effect of medium, high dose of Ost to CYP3A1/2 Protein expression had significant differences. At the same time it showed that the different inhibitory effect of Ost to CYP3 A enzyme activity in vivo and in vitro might be due to Ost could inhibit CYP3A1/2 m RNA and protein expression.