| ObjectiveIsoniazid（INH）-induced liver injury is a common clinical problem,but its mechanism is still unclear.In recent years,it has been found that the expression and activity of hepatic farnesoid X receptor（FXR）are significantly decreased in INH-induced liver injury.However,the relationship between FXR and INH-induced liver injury has not been clarified.Therefore,the purposes of this paper are 1)to clarify the inhibition effect of FXR in the pathogenesis of INH-induced liver injury in vivo and in vitro;2)to determine if INH,the major metabolites of INH,INH and vitamin B6（VB6）adduct pyridoxal isonicotinoyl hydrazine（PIH）,and VB6 can inhibit FXR;3)to investigate the effect of exogenous VB6 supplementation on INH-induced liver injury and INH pharmacokinetics.Findings from this present study are expected to provide valuable cues for the prevention and treatment of INH-induced liver injury.MethodsThere are four parts included in this study:（1）Effect of hepatic FXR inhibition on INH-induced liver injuryIn the first series of experiments,male Wistar rats were orally treated with INH（60,180,300 and 600 mg/kg）or INH（300 mg/kg）plus obeticholic acid（OCA,10 mg/kg）once a day for one week.Serum and liver samples were collected after drugs administration,serum biochemical indexes were measured,and liver pathological changes were also observed.The level of bile acids and content of drug in serum and liver were determined by HPLC-MS/MS,respectively.The mRNA level of hepatic nuclear receptors,metabolic enzymes and transporters involved in the process of bile acid synthesis and transport were measured by RT-qPCR.In the second series of experiments,HepaRG cells or FXR-overexpressed HepaRG cells were exposed to INH in the presence or absence of bile acids for 24 h,and then the cytotoxicity effect was evaluated.Moreover,HepaRG cells were exposed to INH for different time intervals,and TUNEL staining was used to evaluate the effect of INH on apoptosis of HepaRG cells.The intracellular content of bile acids was determined by HPLC-MS/MS.（2）Screening FXR inhibitors based on INH and its major metabolitesIn the first series of experiments,FXR potential inhibitors were selected from INH,Acetylisoniazid（ACINH）,isonicotinic acid（INA）,acetylhydrazide（ACHZ）,hydrazine（HZ）,PIH and VB6 by using the virtual screening approach,and further tested the biological activity by TR-FRET FXR coactivator assay.HepaRG cells were exposed to PIH for 24 h and then collected for RT-qPCR assay,the mRNA levels of FXR and its target genes small heterodimer chaperone receptor（SHP）as well as cholesterol 7α-hydroxylase（CYP7A1）were detected.In the second series of experiments,male Wistar rats were orally treated with INH（60mg/kg）,VB6（60 mg/kg）,INH（60 mg/kg）+VB6（60 mg/kg）and PIH（5 mg/kg）,and serum,liver,ileum and ileum contents samples were collected at 16 h post drug administration.Serum biochemical indexes were measured;the bile acid levels in serum and liver,and drug concentrations of serum,liver,ileum and ileum contents were detected by HPLC-MS/MS,respectively.The mRNA levels of FXR and its target genes SHP and CYP7A1 were measured by RT-qPCR.（3）Role of VB6 in INH-induced liver injuryMale Wistar rats were orally treated with INH（300 mg/kg）or INH（300 mg/kg）+VB6（90,120 and 150 mg/kg）once a day for one week.Serum and liver samples were collected after the final drug administration.Serum biochemical indexes were measured and pathological changes in the liver were observed;the bile acids and drug concentrations in serum and liver were measured by HPLC-MS/MS,respectively.The mRNA levels of liver nuclear receptors,metabolic enzymes and transporters involved in the process of bile acid synthesis and transport were measured by RT-qPCR.（4）Pharmacokinetic interaction between VB6 and INHWistar rats were orally treated with INH（300 mg/kg）or INH（300 mg/kg）+VB6（90,120 and 150 mg/kg）,and blood was collected from the heart at 0,10,30,60,120,180,240,360,480,720 and 1440 min post drug administration,respectively.Serum concentration of INH,the major metabolites of INH and VB6 were determined using HPLC-MS/MS.In another set of study,rats were sacrificed at 2 h post drug administration,the samples of liver,kidney and ileum were collected,and the concentrations of INH,the major metabolites of INH and VB6 were measured.Results（1）Effect of hepatic FXR inhibition on INH-induced liver injuryThe pathological results of liver showed that INH 300 and 600 mg/kg could cause localized hepatocyte necrosis and fatty liver injury.Treatment of INH 60,180,300and 600 mg/kg significantly increased the level of bile acids in the liver when compared to that in the control group（p<0.05）,which may come from the expression or activity decrease of hepatic Fxr.OCA significantly alleviated the INH-induced liver injury,also reversed the INH-induced alteration in mRNA expression of hepatic nuclear receptors,metabolic enzymes and transporters.In addition,INH showed obvious bile acids-dependent toxicity on HepaRG cells,but not in FXR-overexpressed HepaRG cells.Meanwhile,the INH induced increase of bile acids in HepaRG cells occurred earlier than cell apoptosis.（2）Screening FXR inhibitors based on INH and its major metabolitesResults of virtual screening showed that INH,ACINH,INA and PIH may be the potential inhibitors of FXR.Furthermore,TR-FRET FXR coactivator assay and cell experiments confirmed that PIH was the inhibitor of FXR(IC50=52.7 nΜ).In addition,in vivo experiments demonstrated that PIH can inhibit FXR activity and increase the hepatic content of bile acids at the early stage of INH-induced liver injury.（3）Role of VB6 in INH-induced liver injuryCompared with the INH group,VB6 treatment significantly reduced the levels of serum bile acids and hepatic total unconjugated bile acids（p<0.05）,accompanied with the alleviation of hepatic pathological damage,indicating that VB6 may up-regulate the expression and activity of Fxr in the liver,and further reverse the INH caused hepatic mRNA alteration in metabolic enzymes and transporters.In addition,VB6could reduce PIH level in the liver in a dose-dependent manner.（4）Pharmacokinetic interaction between VB6 and INHCompared with the INH group,VB6 treatment significantly reduced the area under the plasma concentration-time curve（AUC）as well as the liver distribution of INH（p<0.05）,but markedly increased the total clearance of INH,ACINH and INA（p<0.05）.However,VB6 treatment significantly increased the plasma AUC and reduce the liver distribution of PIH（p<0.05）.ConclusionIn summary,the experimental results from this present study show that:（1）INH can inhibit hepatic FXR expression and activity,and thus lead to hepatic bile acid accumulation at the early stage,which may contribute to INH-induced liver injury;（2）PIH,the adduct of INH and endogenous VB6,is a powerful inhibitor of FXR.PIH can inhibit hepatic FXR activity and increase hepatic content of bile acids;suggesting that PIH is the main substance that responsible for INH-induced enhancement of hepatic bile acids;（3）Supplementation with exogenous VB6 can significantly reduce the AUC of INH and liver distribution of PIH,but increase hepatic FXR expression and activity,which can alleviate INH-induced liver injury. |
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