Enzyme Expression Of Metabolic Isozyme CYP3A4and1A1by Three Pairs Of Isomerides From Phenylpropanoids In HepG2Cells

Objectives：The structure of compound is the most essential foundation of medicine efficacy.This study was designed to investigate if the different structural changes of three pairs ofisomerides form phenylpropanoids-psoralen, isopsoralen, imperatorin, isoimperatorin, ferulicacid and isoferulic acid could modulate variations of metabolism isoenzymes CYP3A4andCYP1A1in HepG2cells and to illustrate the possible mechanisms for providing a theoryfoundation for clinical safety and rational use of chinese herbal medicine.Methods：1. The growth inhibition ratio of human hepatocellular HepG2cells was measured byMTT colorimetric methods after six kinds of drugs treatment for48h, and calculatedIC50for screening effective concentration.2. The cell cycle of HepG2cells was detected by flow cytometry after treatment by sixkinds of drugs.3. Real-time fluorescent quantitative PCR was used to evaluate mRNA expressionchanges of CYP3A4and CYP1A1in HepG2cells after treatment by six kinds ofdrugs.4. Protein expression of CYP3A4after treatment by six kinds of drugs in HepG2cells waspositioned and quantitated by immune-fluorescence methods (IF) and western blottingassay (WB).Results:1. All of six kinds of drugs inhibited proliferation of HepG2cells after48h under seven concentrations (6.25~200μg/mL) and there was a clear dose-response relationship. Cellcycles were blocked at G2-M phase by psoralen, isopsoralen, ferulic acid and isoferulicacid, meanwhile imperatorin, isoimperatorin restrained cell at G2-M and S phases.2. The mRNA expression of CYP3A4was inhibited by all of six drugs. Inhibition effect ofisopsoralen was stronger than psoralen; but the inhibition effect on CYP3A4mRNAexpression by imperatorin was weak under three concentrations, and the inhibition byisoimperatorin was stronger than imperatorin; compared with ferulic acid, the inhibitionby isoferulic acid on CYP3A4mRNA was slightly stronger.3. All of six kinds of drugs were inhibitors on mRNA expression of CYP1A1. Psoralenshowed slightly stronger inhibition than isoimperatorin on CYP1A1mRNA; theinhibitions of CYP1A1mRNA by isoimperatorin was better than imperatorin at lowconcentrations (25,50μg/mL), while two drugs had not evident discrepancy at highconcentration (100μg/mL); the suppression strength of CYP1A1mRNA at all of threeconcentrations of isoferulic acid was significantly stronger than the ferulic acid.4. IF and Western Blot experiments proved that protein of CYP3A4largely existed incytoplasm and the protein expression of CYP3A4was obviously decreased aftertreatment by six kinds of drugs.Conclusion:1. Six kinds of drugs were inhibitors of CYP3A4and CYP1A1enzymes, and implied thatadverse drug reaction should be paid attention to be caused by drug toxicity of savingsin clinical medicines. Clinician should be paid attention to pharmacological function inclinical drug combination.2. Synthesis of RNA and protein were blocked by six kinds of medications to suppress theproliferation of HepG2cells, meanwhile both of imperatorin and isoimperatorin abatedthe synthesis of DNA to restrain cell growth.3. The four compounds-psoralen, isopsoralen, imperatorin and isoimperatorin belonged to furan coumarin, and we found that the structure change of the furan ring was the mainreason for the differences of RNA and protein expression on enzymes CYP3A4andCYP1A1; the heterogeneous of phenolic hydroxyl on the benzene ring was the actionsite of different inhibitory competence of ferulic acid and isoferulic acid on CYP3A4and CYP1A1enzymes.