Effect Of Panaxytriol Intervention On PXR-CYP3A4 Regulatory Pathway In HepG2 Cells And Its Mechanism

Background:Panaxytriol(PXT),one of the major effective components of korean ginseng,red ginseng extract and Shenmai Injection,which could inhibit the activity of many tumor cells and is conducive to ease the adverse reaction of certain cancer therapy.Thus,it is worth our effort to study the effect of PXT on the cancer treatment.The results from our previous study showed that PXT down-regulated the expression of CYP3A1/2 mRNA in the primary liver cells of rat.But it remains unclear that whther PXT have any impact on the expression of CYP3A4 in HepG2 cells.This problem is worth us to explore.Objectives:To explore the effect of PXT on the expression of PXR and CYP3A4 in the normal HepG2 cells and the HepG2 cells model with high-expression hPXR and the mechanism of PXR-CYP3A4 regulation pathway which were treated with PXT for different time periods.To provide experimental and theoretical basis for further exploring the possible interaction between PXT and clinical drugs.Methods:1.The normal HepG2 cells and the HepG2 cells model with high-expression hPXR was constructed and cell growth curve of HepG2 cells was plotted.The toxicity on HepG2 cells was investigated by MTS.2.The effect of PXT on the expression of PXR,CYP3A4 mRNA and protein in the HepG2 cells was explored and the HepG2 cells with high-expression hPXR which were treated with PXT for different time periods by Q-PCR and Western blot.3.The mechanism of PXT-CYP3A4 regulatory pathway in the HepG2 cells was explored by constructing plasmid,transient co-transfection and dual-luciferase reporter gene assays.Results:1.Effects of PXT on PXR and CYP3A4 in normal HepG2 cellsPXT showed no impact on the expression PXR and CYP3A4 mRNA treated PXT at 5μM,10μM for 1h(P>0.05),while significantly up-regulated by 20μM,40μM and 80μM(P <0.05).The expression of PXR mRNA was up-regulated by 49.53%,82.05% and 115.25%,respectively,and the expression of CYP3A4 mRNA was up-regulated by 14.22%,51.99%,94.25% and 140.78% respectively,and the two trends are consistent(R2=0.9875).Short-term intervention for 1h,different concentrations of PXT on PXR and CYP3A4 protein expression were no significant effect(P> 0.05).PXT showed no impact on the expression PXR and CYP3A4 mRNA in the HepG2 cells treated PXT at 5μM for 24 h,while significantly up-regulated by 10μM,20μM,40μM and 80μM(P <0.05).The expression of PXR mRNA was up-regulated by 33.37%,88.77%,137.90% and 240.43%,respectively,and the expression of CYP3A4 m RNA was up-regulated by 49.88%,145.06%,246.15% and 370.50%,respectively,and the two trends are consistent(R2=0.9904).PXT showed no impact on the expression PXR and CYP3A4 protein in the HepG2 cells treated PXT at 5μM,10μM,while significantly up-regulated by 20μM,40μM and 80μM(P <0.05).The expression of PXR protein was up-regulated by 51.96%,151.72% and 220.21%respectively and the expression of CYP3A4 protein was up-regulated by 98.51%,167.34% and 261.57% respectively,and the two trends are consistent(R2=0.9755).2.Effects of PXT on PXR and CYP3A4 in HepG2 cells with high-expression hPXRPXT showed no impact on the expression PXR and CYP3A4 mRNA in the HepG2 cells with high-expression hPXR treated PXT at 5μM for 1h(P> 0.05),while the expression of PXR mRNA was up-regulated by 23.86%,69.54%,and 122.89%(P<0.05),respectively,and the expression of CYP3A4 mRNA was upregulated by49.65%,101.35%,144.02% and 188.87%(P <0.05),respectively,and the two trends are consistent(R2=0.9713).Short-term intervention for 1h,different concentrations of PXT on PXR and CYP3A4 protein expression were no significant effect(P> 0.05).The hPXR and CYP3A4 m RNA in the HepG2 cells with high-expression hPXR treated PXT was up-regulated by 5μM,10μM,20μM,40μM and 80μM for 24h(P<0.05).The expression of PXR mRNA was up-regulated by 47.80%,87.62%,131.15%,239.80% and 361.15%,respectively,while the expression of CYP3A4 mRNA was up-regulated by 91.93%,139.84%,212.80%,398.28% and 642.02%,respectively.And the two trends were consistent(R2=0.9959).PXT showed no impact on the expression PXR and CYP3A4 protein in the HepG2 cells with high-expression hPXR treated PXT at 5μM for 24 h,while significantly up-regulated by 10μM,20μM,40μM and 80μM(P <0.05).The expression of PXR protein was up-regulated by 50.72%,104.73%,167.02% and 314.50%,respectively and the expression of CYP3A4 protein was up-regulated by 72.24%,130.27%,193.69% and 344.81%,respectively,and the two trends were consistent(R2=0.9973).3.Effects of PXT on CYP3A4 reporter gene in HepG2 cells by transient co-transfectionThere was no induction of CYP3A4 at 5μM(P>0.05)at 1h after PXT intervention.while the induction fold of PXT on CYP3A4 was 1.44,1.63,1.79,1.93,respectively in the HepG2 cells treated with PXT at 10μM,20μM,40μM and 80μM,statistically significant(P <0.05).When HepG2 cells treated with PXT for 6h,the induction fold was 1.42,1.63,2.18,2.69,3.07(P <0.05),respectively.When HepG2 cells treated with PXT for 24 h,the induction fold was1.90,2.44,2.94,3.61,4.20(P<0.05),respectively.When HepG2 cells treated with PXT for 48 h,the induction fold was 1.56,2.06,2.47,3.08,3.51(P <0.05),respectively.The induction time of CYP3A4 increased with the increase of the concentration of PXT at 1h,6h,24 h and 48 h.The effect of PXT on HepG2 cells was the best at 24 h.The induction effect of PXT and RIF is greater than that of PXT and RIF alone.Conclusions:1.It was shown that PXT could induce the expression of PXR and CYP3A4 mRNA in normal HepG2 cells treated with PXT at different concentrations for 1h,while PXT showed no impact on the expression of PXR and CYP3A4 protein.And PXT showed induction both on the expression of PXR and CYP3A4 mRNA and protein in HepG2 cells treated with PXT at different concentrations for 24 h and the up-regulation the expression PXR and CYP3A4 mRNA was concentration-dependent.And PXT on the PXR and CYP3A4 both the same trend.The results suggest that the effect of PXT on the expression of CYP3A4 may be related to the PXR pathway.2.The effect of PXT on HepG2 cells with high-expression hPXR was similar to that of normal HepG2 cells.PXT could induce the expression of PXR and CYP3A4 mRNA for 1h,while PXT showed no impact on the expression of PXR and CYP3A4 protein.And PXT showed induction both on the expression of PXR and CYP3A4 mRNA and protein for 24 h,and PXT on the PXR and CYP3A4 both the same trend.The results indicate that the effect of PXT on the expression of CYP3A4 in HepG2 cells high-expression hPXR may be related to the PXR pathway.3.By dual-luciferase reporter gene assays further confirmed that PXT on HepG2 cells in the up-regulation of CYP3A4 through the nuclear receptor PXR regulation.