A Study On The Mechanism Of Gene Polymorphisms Influencing Tacrolimus Metabolism In The Early Stage After Liver Transplantation

OBJECTIVES Liver transplantation has become a lifesaving therapy for patients with end-stage liver disease.Tacrolimus(TAC,FK506)is widely used as a basic component of immunosuppressive regimens.However,tacrolimus has a narrow therapeutic window,and the pharmacokinetics(PK)of tacrolimus is highly variable between individual patients.In addition,those traits are closely associated with several polymorphisms of cytokine genes,drug-metabolizing enzyme genes.Therefore,the aim of this study was to explore the mechanisms of gene polymorphisms influencing tacrolimus metabolism in the early stage after liver transplantation in order to screen the markers for individualized tacrolimus treatment.METHODS A part of tag Single nucleotide polymorphisms(SNPs)belonging to genes such as cytochrome P450 3A family polypeptide 5(CYP3A5),interleukin-6(IL-6)Toll-like receptor 4(TLR4)were sequenced using a Sequenom Mass ARRAY SNP genotyping platform.Additionally,clinic data of liver transplantation patients was recorded and studied at four weeks after liver transplantation.Then,we investigated the relation between SNPs of donors and recipients and tacrolimus concentration/dose(C/D)ratios using statistical method.Fluorescent quantitative polymerase chain reaction(FQ-PCR)and immunohistochemical methods were applied to detect the IL-6 expression at m RNA and protein levels.Moreover,site-directed mutation technique andstable transfection technology mediated by lentivirus was used to constructed normal hepatic cell lines of different genotypes.Subsequently,effects of IL-6 rs800796 locus SNPs on biological function of human hepatocytes were explored based on LO2 cell H/R model.RESULTS The donor and recipient CYP3A5 rs776746 SNPs,the donor TLR4 rs1927907 SNPs,and the donor IL-6 rs1800796 SNP were closely associated with tacrolimus metabolism(P<0.05).The IL-6 expression of extensive metabolizers group were much higher than that in those poor metabolizers at protein and m RNA level(P<0.05).When compared with LO2 G cells,the expression of IL-6 and STAT3 in LO2 C cells more significantly increased after hypoxia reoxygenation about 6 hours.Aditionally,the cell viability in the LO2 C cells were much higher than LO2 G cells(P<0.05).CONCLUSIONS Collectively,the relevance between the donor and recipient CYP3A5 rs776746 SNPs and tacrolimus metabolism was again proved in this study.We also found that the donor IL-6 rs1800796 SNPs and the donor TLR4 rs1927907 SNPs influenced tacrolimus metabolism in the early stage after liver transplantation.Especially,IL-6 rs1800796 SNPs mainly affected the restoration of liver function after liver transplantation through IL-6/STAT3 signal pathway,which caused the individual differences of TAC metabolism.Combined with gene polymorphisms of both donor and recipient CYP3A5,IL-6 and TLR4 genes might have a greater effect on tacrolimus elimination than each SNP separately.Screening for these gene polymorphisms prior to liver transplantation could more accurately predict tacrolimus metabolic phenotype.Moreover,it provided useful experimental basis for establishing individualized TAC and achieving the desired immunosuppressive effect,which would be of great significance for realizing adequate initial daily TAC doses.