The Role Of CYP3A4/5 And PXR In Drug-induced Liver Injury Caused By Cyclosporine

BACKGROUNDChina is a large organ transplant country.The long-term survival rate and quality of life of organ transplant recipients which related to rejection are important indicators for evaluating organ transplantation.Therefore,in order to prevent the rejection of renal transplant recipients,economical,effective and reasonable immunosuppressive treatment options are important.Cyclosporine A(CsA)is a calcineurin inhibitor.As it have great effects in selective immunosuppression and inhibiton on acute rejection,it is widely used in organ transplantation and autoimmune diseases and blood diseases.CsA has become the first choice for anti-rejection drug.However,CsA has a narrow therapeutic window and is prone to adverse reactions.The adverse reaction of CsA is the main cause of its reduction,withdraw and even transplant failure.The incidence of liver injury is about 20%-40%,and liver injury occurrence is related to the plasma concentration.This occurrence of liver injury is inseparable from the CsA disposition,including metabolism-related P450 enzymes,transport-related proteins,and regulatory genes such as PXR,which regulate the expression of metabolic transport-related proteins.CYP3A4 and CYP3A5 are the most important CsA phase I metabolizing enzymes.CYP3 A accounts for more than 30% of the P450 enzyme system In liver,.The expression and activity of CYP3A4 and CYP3A5 explain the individual differences in CsA metabolism to a certain extent;PXR is a nuclear receptor that regulates the expression of drug disposition proteins,CsA is ligand,and the downstream genes regulated include CYP3A4 and CYP3A5.However,in the CsA-induced liver injury,the regulation mechanism of CsA on CYP3A4,CYP3A5 and PXR is still unclear.In this study,we established a model of HepG2 cell liver injury induced by CsA at the cellular level,and combined with gene silencing technology to study the role of the above genes in liver injury induced by CsA,and provide a theoretical basis for the CsA clinical use.OBJECTIVEThis study aimed to determine the correlation between genetic factors and CsAinduced liver injury by measuring the NR1I2 genotype of kidney transplant recipients taking CsA,and to analyze the correlation between genetic factors and CsA-induced liver injury.HepG2 cells were used as a platform to establish CsA-induced liver injury cell model,the biochemical markers of liver injury and changes of CYP3A4,CYP3A5 and PXR in normal cells was investigated.The pLKO.1-CYP3A4/5-shRNA plasmid was constructed,to stablished the CYP3A4/5 silenced cell strain by lentiviral transfection;Basising on the CsA-induced liver injury cell model,we try to explored the effects of CsA on CYP3A4,CYP3A5,PXR and related biochemical indicators,and to elucidate the role of CYP3A4,CYP3A5,PXR in CsA-induced liver injury at the cellular level by comparing the differences among the HepG2 normal cells,CYP3A4/5 silenced cells and PXR silencing cells,.METHODThe study is divided into five parts:Part 1:Genotyping rs2461823,rs3842689,rs6785049,rs12488820,rs2472682,rs3732357 of 188 CsA-taking kidney transplant recipients,recording gender,age at transplant,height,body mass,BMI at transplantation,and transplantation time,dialysis time,Kidney source,family history of diabetes,history of hepatitis B and hepatitis C infection,and other clinical data,as well as CsA dosage and plasma concentration,clinical examination indicators at various time points,retrospective analysis of each SNP and Correlation of CsA-induced liver injury;Part 2:The effect of different concentrations of CsA on the survival rate of HepG2 cells was Investigated by CCK-8 method after eliminating the toxic effect of solvent DMSO on cells,to determined the IC50 of CsA,and selected the appropriate CsA concentration.through detecting the biochemical index AND observing the changes of the expression of CYP3A4,CYP3A5,PXR and LC3,a suitable CsA-induced liver injury model of HepG2 cells WAS finally establishedPart 3:The CYP3A4/5-shRNA sequence was synthesized by using pLKO.1 plasmid as a vector,the CYP3A4/5-shRNA sequence was ligated with digested pLKO.1 plasmid,introduced into competent cells,Amp resistance of pLKO.1 plasmid was used,screening of a successful pLKO.1-CYP3A4/5-shRNA plasmid;Part 4:The appropriate concentration of puromycin was screened by pre-experiment;the lentivirus was packaged by 293 T cells based on the three-plasmid encapsulation system,and further transfected into HpG2 cells,using puromycin resistance by pLKO.1 plasmid to screened successfully transfected cells.CYP3A4/5 silenced cell strain,and the silencing efficiency was detected by western blotting;Part 5:The CsA-induced liver injury HepG2 cell model as described above was Used,combining with the CYP3A4/5 silenced cell strain constructed above and the PXR silencing cell line constructed in the previous research.the effect of CsA on CYP3A4,CYP3A5,PXR and related biochemical indicators among these three kinds of cells was Compared,to elucidated the role of CYP3A4,CYP3A5,and PXR in CsA-induced liver injury at the cellular level.RESULTPart 1:The distribution of rs12488820 allele was significantly different between the control group and the liver injury group(p=0.032)and the genotype of this genetype was significantly different between groups(p_add =0.032 and p_rec =0.032).suggested that the rs12488820 was associated with CsA-induced liver injury.In the analysis of CsA plasma concentration,the rs12488820 was significantly different at 7 and 6 months(p = 0.0479 and 0.0102);the rs3842689 recessive model was significantly different at 3 years(p_rec=0.0485);the recessive model of the rs3732357 was significantly different at the 1 year(p_rec = 0.0454).Part 2:The CsA-induced liver injury HepG2 cell model was Constructed.The effect of DMSO on the liver damage of HepG2 cells induced by CsA was excluded.The IC50 of CsA was determined at 20.455 ?M.APAP with clear liver injury effect was used at 7.5 mM as the positive control group.Three CsA concentrations of 2?M,20?M and 50?M were used to detect the biochemical markers of liver injury under the abovementioned concentrations.The results showed that LDH and AST were effective indicators of CsA-induced liver injury in HepG2 cells.With the increase of CsA concentration,LDH and AST also increased.The differences in medium and high concentrations were statistical significantly.The results with western blotting showed that the expressions of PXR,CYP3A4 and CYP3A5 were down-regulated,LC3-II and LC3-II/I were up-regulated at medium and high concentrations,and the difference was statistical significace,indicating that CsA has inhibitory effecs ont PXR,CYP3A4,CYP3A5 expression,and autophagy is occurred in the cells;Part 3:The CYP3A4/5-shRNA sequence was ligation with pLKO.1 plasmid.The sequencing results showed that the ligation was successful and no sequence changes were observed.Part 4:HepG2 cells were transfected with lentivirus to construct a silencing cell strain with stable expression.Western blotting results showed that the CYP3A4?CYP3A5 expression rate of CYP3A-silenced cell line was 45% and 46%,the PXR expression rate of PXR-silenced cell line was 30%;Part 5:CYP3A4,CYP3A5,and PXR are related genes of hepatic injury induced by CsA in HepG2 cells.The low expression of the three genes leads to a significant increase in liver injury marker LDH and AST,aggravating CsA-induced liver injury in HepG2 cells,and CYP3A4,CYP3A5,PXR which is related to the autophagy process of CsA-induced liver injury,the autophagy in low expression group is more obvious.CONCLUSIONThe rs12488820 was associated with CsA-induced liver injury;in the recessive model or additive model,three SNPs were associated with plasma concentrations at four time points;CYP3A4,CYP3A5,and PXR were associated with CsA-induced liver injury in HepG2 cells,the low expression aggravated liver injury;CYP3A4,CYP3A5,PXR were associated with autophagy in liver injury,and autophagy was more obvious in low expression group.