Mechanism Study Of Panaxytriol Upregulating The Expression Of CYP3A4 Based On The Interaction Of PXR,CAR,HSP90 And RXR

Background:CYP3A4 is one of the most important drug-metabolizing enzymes in the body.Panaxytriol,which is one of the critical active ingredients in Red Ginseng extract and Shenmai injection,has inhibitory effect on the activity of many tumor cells.Previous studies have shown that panaxytriol can induce the expression of CYP3A4 enzyme mainly by activating the nuclear receptor PXR(pregnane X receptor,PXR;NR1I2)signaling pathway.CAR(constitutive androstane receptor,CAR;NR1I3)signaling pathway also engaged in the regulation of CYP3A4 enzyme at high concentration of panaxytriol(80 μM).The study also found that the high expression of hCAR can significantly inhibit the upregulation of PXR and CYP3A4 by panaxytriol.After silencing hCAR,the up-regulation was significantly enhanced.Therefore,it can be seen that PXR and CAR not only can independently regulate the expression of metabolic enzymes in their own way,but also have some cross-talk regulatory mechanisms.It has been proved that the transcriptional regulation of drug metabolizing enzymes by PXR and CAR is a complex network structure,and multiple elements such as ligands,receptors,corepressor factors,cofactors,target gene response elements,etc.may be involved in it.Then,it is worthy of further study and discussion that under the intervention of panaxytriol,how PXR and CAR regulate the expression of the target gene CYP3A4 through interaction.Objectives:By using HepG2 cells and Huh-7 cell models,we studied the effects of panaxytriol on the binding of nuclear receptors PXR and CAR with co-repressor factor HSP90αand cofactor RXRα,and the mechanism of panaxytriol regulating CYP3A4 target genes,in order to provide the theoretical and experimental basis for in-depth exploring the mechanism of interaction between panaxytriol and other clinical drugs.Methods:1.RT-qPCR,Western-blot and other experimental techniques were used to explore the effect of panaxytriol on the expression of PXR,CAR,HSP90α,RXRα and target gene CYP3A4 in HepG2 cells.2.hCAR-silencing HepG2 cells,co-immunoprecipitation,immunofluorescence and other experimental techniques were used to investigate the effect of panaxytriol on the binding of PXR,CAR to HSP90α and RXRα proteins in HepG2 cells.3.RT-qPCR,Western-blot and co-immunoprecipitation methods were used to explore the effects of panaxytriol on the expression of PXR,CAR and target gene CYP3A4,as well as the effect on the binding of PXR to HSP90α and RXRα proteins in normal and hCAR-silencing Huh-7 cells.Results:1.Effects of panaxytriol on the expression of PXR,CAR,HSP90α,RXRα and target gene CYP3A4 in HepG2 cells.(1)In HepG2 cells,panaxytriol significantly up-regulated CYP3A4 and PXR m RNA expression,and the up-regulation effect increased with the increase of panaxytriol concentration.The expression of CAR m RNA was only up-regulated at panaxytriol high concentration of 80 μM,meanwhile,there was no significant difference in the expression of HSP90α and RXRα m RNA level.(2)In HepG2 cells,panaxytriol significantly upregulated CYP3A4 total protein expression and PXR total and nuclear protein expression,and the up-regulation effect increased with the increase of panaxytriol concentration.The expression of CAR total and nuclear protein was only up-regulated at panaxytriol high concentration of 80 μM.Meanwhile,there was no significant difference in the expression of HSP90α and RXRαtotal protein level,but it can up-regulate the expression of RXR nucleoprotein,and the up-regulation effect increased with the increase in of panaxytriol concentration.2.Effects of panaxytriol on the binding of PXR,CAR to HSP90α,RXRα protein in HepG2 cells.(1)In normal and hCAR-silencing HepG2 cells,panaxytriol at 10～80 μM concentration can weaken the combination of PXR and HSP90α,and enhance the combination of PXR and RXRα,the effect could be enhanced with the increase of panaxytriol concentration.Compared with the normal cells group,after hCARsilencing,panaxytriol obviously promoted the binding effect of PXR and RXRα,but but has no obvious effect on the combination of PXR and HSP90α.Besides,under panaxytriol high concentration of 80 μM,the combination of CAR and HSP90α was weakened,and the combination of CAR and RXRα was enhanced in the normal cells.(2)In normal and hCAR-silencing HepG2 cells,immunofluorescence results showed that panaxytriol promoted the translocation of PXR and RXRα into the nucleus.3.Effects of panaxytriol on the expression of PXR and CAR and their interaction with HSP90α and RXRα in Huh-7 cells.(1)In Huh-7 cells,panaxytriol significantly up-regulated the m RNA and protein expression of CYP3A4 and PXR.The expression of CAR m RNA and protein can only be up-regulated at high concentration of 80 μM.(2)In hCAR-silencing Huh-7 cells,panaxytriol up-regulated the m RNA and protein expression of CYP3A4 and PXR,and the upregulation effect was enhanced with the increase of concentration.Besides,compared with normal Huh-7 cell group,the up-regulation effect of panaxytriol on CYP3A4 and PXR m RNA and protein expression was significantly enhanced.(3)In normal Huh-7 cells,panaxytriol at a concentration of 10～80 μM can weaken the combination of PXR and HSP90α,and enhance the combination of PXR and RXRα,both effects could be enhanced with the increase of panaxytriol concentration.Conclusions:1.In HepG2 cells and Huh-7 cells,panaxytriol can up-regulate the expression of target gene CYP3A4 by weakening the binding of PXR and HSP90α and promoting the binding of PXR-RXRα.CAR also participates in the regulation of CYP3A4 through a similar mechanism at panaxytriol high concentration.2.In the up-regulation pathway of PXR-CYP3A4 by panaxytriol,CAR can induce interactive inhibition by antagonizing the combination of PXR and RXRα.