Effect And Mechanism Research Of PXR-CYP3A Pathway On Cyclosporine-induced Liver Injury

BACKGROUND&OBJECTIVECyclosporine A is a widely used immunosuppressant,which greatly improves the survival rate of patients and grafts after solid organ transplantation.However,the liver and kidney toxicity of CsA severely limits its clinical application.Pregnane X receptor(PXR)is a ligand-activated transcription factor that is involved in the regulation of various drug metabolic enzymes and transporters,including CYP3A4 and CYP3A52,the metabolic enzymes of CsA in vivo.Our previous research suggested that the genetic polymorphisms of some sites of nuclear receptor PXR and metabolic enzymes CYP3A4 and CYP3A5 may be related to the liver damage caused by CsA.In vitro experiments show that CsA can inhibit the protein expression of PXR and its target genes CYP3A4 and CYP3A5.Stable interference of PXR and CYP3A genes aggravates the toxicity of CsA in HepG2 cells.Combined with the results of previous studies,we speculate that the PXR-CYP3A pathway may play a key role in CsA-induced liver injury.At present,no related reports of PXR-CYP3A pathway and CsA-induced liver injury have been reported at home and abroad.Based on previous research,this paper continues to explore the role of PXR-CYP3A pathway in CsA-induced HepG2 cell injury and its molecular mechanism,which is of great significance for reducing the occurrence of liver injury and achieving personalized medicine for CsA.METHODS1.Construct PXR over-expressing cell lines and PXR stably interfering cell lines.2.Use rifampicin(Rif)as PXR activator and ketoconazole(KTZ)as PXR inhibitor.In the presence or absence of Rif and KTZ,different concentrations of CsA were administered to HepG2 cells,PXR overexpression and PXR stably interfered with the cells for a certain period of time.3.CCK8 method to determine cell viability,explore the cytotoxicity of CsA on HepG2 and the role of PXR in CsA-induced cytotoxicity4.Determine the content of lactate dehydrogenase(LDH)and aspartate aminotransferase(AST)in the cell supernatant,explore the cell damage effect of CsA on HepG2,and analyze the effect of PXR on cell damage induced by CsA.5.Western blot and qRT-PCR were used to detect the expression of PXR and its target genes CYP3A4 and CYP3A5,to investigate the regulation of CsA on CYP3A4 and CYP3A5,and to further study the mechanism of CsA-induced HepG2 damage.RESULTS1.Compared with control cells,PXR mRNA and protein expression in PXR stable interference cells decreased significantly(p<0.01),and PXR mRNA and protein expression in PXR stable overexpression cells increased significantly(p<0.01).2.In the range of 1-100 ?M,the cell viability of HepG2 decreased with the increase of CsA concentration and the prolongation of administration time(p<0.01).In HepG2 cells pretreated with KTZ or with low PXR expression,the cell viability decreased at the same CsA concentration(p<0.01).However,in HepG2 cells pretreated with RIF or with high PXR expression,the cell viability increased at the same CSA concentration.3.In the range of 5?50 ?M,LDH and AST increased with the increase of CsA concentration(p<0.01)).In HepG2 cells pretreated with KTZ or with low PXR expression,LDH and AST increased at the same CsA concentration(p<0.01).In HepG2 cells pretreated with RIF or with high PXR expression,LDH and AST decreased at the same CsA concentration(p<0.01).4.In the range of 5?50 ?M,with the increase of CsA concentration,the transcription and protein expression of PXR,CYP3A4 and CYP3A5 decreased in a concentration dependent manner(p<0.01).The inhibitory effect of CsA on CYP3A4 and CYP3A5 in HepG2 cells pretreated with RIF or with high PXR expression was significantly lower than that in the control group.In HepG2 cells pretreated with KTZ or with low PXR expression,the inhibitory effect of CsA on CYP3A4 and CYP3A5 was significantly higher than that of the control group.CONCLUSION1.PXR stable interference cell line and PXR stable overexpression cell line were successfully constructed.2.The cytotoxicity of CsA to HepG2 was concentration dependent and time dependent.3.CsA can cause HepG2 cell injury,which is characterized by the increase of LDH and AST.4.The cytotoxicity and damage degree of CsA were related to the expression level of PXR.The increase of CsA concentration,inhibition or low expression of PXR can enhance the cytotoxicity and cell damage of CsA,whereas the decrease of CsA concentration,activation or high expression of PXR can reduce the cytotoxicity and cell damage of CsA.5.CsA is a PXR ligand and an inhibitor of PXR and CYP3A.CsA mediates the down-regulation of CYP3A4 and CYP3A5 transcription and protein expression through PXR pathway,which may lead to the decrease of CsA metabolism,the increase of CsA accumulation and the aggravation of CsA liver damage.In conclusion,PXR-CYP3A metabolic pathway plays an important role in CsA induced hepatotoxicity and may be one of the targets of CsA induced drug-induced liver injury.PXR and CYP3A activators may be used as prospective drugs to prevent and treat liver injury caused by CsA,while the combination of PXR and CYP3A inhibitors may have potential drug interactions,which should be paid attention to clinically.