Study On The Effects Of Magnesium Isoglycyrrhizinate On P-gpand CYP3A And Its Molecular Mechanism

Magnesium isoglycyrrhizinate,as the fourth generation of glycyrrhetinic acid derivatives,is wildly applied to the clinical treatment of chronic viral hepatitis and hepatopathy induced by chemotherapy.Magnesium isoglycyrrhizinate has shown a preferable application prospect as hepatic protector which is of great value for its efficacy research.However,its molecular mechanism of drug-drug interaction is still unclear.Therefore,this experiment is to investigate its impacts on P-gp and CYP3 A both in vivo and in vitro through two basic ways of drug transport and metabolism.The aim of this study is to provide a scientific basis for the clinical application.Objective:Firstly this study investigated the effects of MgIg on the expression of P-gp and CYP3A1 in rats with acute liver injury.Then the impacts of MgIg on the expression and activities of P-gp and CYP3A4 were studied in Hep G2 cells.Finally,Hep G2 cells were used to study the influence of MgIg on the m RNA and protein expression of PXR,and to confirm whether the regulation was through PXR signal as preliminarily discussed.Methods:CCl₄ was applied to establish the acute liver injury models in vivo study,and the animal models were randomly divided into three groups.After the medication in each group,the respective samples were extracted for the following tests.Then the serum biochemical parameters were measured by automatic biochemical analyzer and pathological condition of rat hepatic tissues were determined by HE staining.RT-PCR and Western blot were applied to measure m RNA and protein expression of P-gp and CYP3A1 from each group.In vitro study,MTT experiment was aimed to decide the MgIg concentration for cells.The effects of MgIg on the function of P-gp and the activity of CYP3 A were measured by Rho-123 assay and CYP3A4 test kit in Hep G2 cells.Moreover,the molecular mechanism of MgIg on P-gp and CYP3A4 was finally studied,and RT-PCR and Western blot were used to measure the MgIg on the m RNA and protein expression of P-gp,CYP3A4 and PXR,which aimed to analyze the correlation between CYP3A4/P-gp and PXR.Results:1.Acute Liver injury model caused by CCl₄ was stable and successful and the alleviation of MgIg in the model which were verified by biochemical indicators of ALT,AST and liver haematoxylin and eosin?HE?staining.2.RT-PCR and Western blot were applied to measure the impact of MgIg on expression of P-gp and CYP3 A 1enzyme in CCl₄ induced acute rats liver injury model.Results indicated the m RNA and protein expression of P-gp in CCl₄ group was up-regulated comparing to normal group,and the MgIg medication decreased the m RNA and protein expression of P-gp.Conversely,the m RNA and protein expression of CYP3A1 were induced after medication.3.By conducting cytotoxic test via MTT method,Hep G2 cell growth inhibitory rate less than 20% when MgIg concentration was 0-480?M,which was the maximal non-cytotoxic concentration.Based on previous preliminary experiment and literature reviewing,the medication concentration were 7.5 ?M,15 ?M and 30?M.4.Effects of MgIg on P-gp function by Rho-123 assay,the results from accumulation and excretion indicated MgIg of difference concentrations inhibited the function of P-gp,and the enhanced inhibition was observed in higher concentrations.5.By detecting of CYP3A4 enzyme activity in Hep G2 cells,the results showed that MgIg of 7.5 ?M inhibited the enzymatic activity of CYP3A4,whereas MgIg of 15?M or 30 ?M significantly induced the enzymatic activity of CYP3A4.6.RT-PCR and Western blot were utilized to measure the impact of MgIg in different concentrations on the m RNA and protein expression of P-gp,CYP3A4 and their regulation factor PXR.Results indicated MgIg of 7.5 ?M had inhibitory effect on CYP3A4 expression,and MgIg of 15 ?M or 30 ?M showed obvious induction.By contrast,significant inhibition on function and expression were observed at the highest concentration of MgIg.In the measurement experiment of m RNA and protein expression of up-regulation factor PXR,results showed that MgIg of 15 ?M or 30 ?M all up-regulated the expression of PXR.Conclusion:MgIg up-regulated the levels of transcription and translation in Hep G2 cells and rats with acute liver injury,the metabolism of intracellular toxic substance and medicine mediated via CYP3A4 induced by MgIg were promoted which was possibly related to the drug-drug interaction.However,high concentration of MgIg had inhibited function in P-gp function and expression.As for its regulation factor nuclear receptor PXR,MgIg of moderate and high concentrations induced its expression.The outcome of this study will provide certain theoretical basis for the interaction of some clinical drugs by magnesium isoglycyrrhizinate.