Based On Farnesol X Receptor To Explore The Mechanism Of Liver Injury Caused By Quadruple Anti-tuberculosis Drugs

ObjectiveThe combination of isoniazid（INH）,rifampicin（RIF）,ethambutol（EMB）and pyrazinamide（PZA）is the first choice for clinical tuberculosis treatment,but quadruple anti-tuberculosis drugs（QATD）-induced liver injury often leads to the termination of chemotherapy.In recent years,studies have found that anti-tuberculosis drugs-induced liver injury is related to the disorder of bile acids homeostasis and the expression and function of farnesoid X receptor（FXR）,which regulates the balance of bile acids.However,the relationship between QATD-induced liver injury and FXR has not been fully clarified.Based on the above facts,this article intends to study the role of liver FXR in QATD-induced liver injury through in vivo and in vitro experiments.Methods（1）50 blood samples were from tuberculosis patients who were first diagnosed,had taken QATD about two months,and 50 samples were from healthy people.Aspartate aminotransferase（AST）,alanine aminotransferase（ALT）,total bile acid（TBA）,alkaline phosphatase（ALP）,total bilirubin（TBIL）andγ-glutamyltransferase（GGT）in serum were determined by biochemical kits;the level of taurocholic acid（TCA）in serum was determined by HPLC-MS/MS;the level of fibroblast growth factor19（FGF19）in serum was determined by ELISA kit.（2）Male Wistar rats were randomly divided into control group（n=10）,clinical equivalent dose group（n=10）and high dose group（n=10）.The control group was treated with 0.5%sodium carboxymethyl cellulose solution（drug solvent）,clinical equivalent dose group was treated with INH（30 mg/kg b.w t）+RIF（45 mg/kg b.w t.）+PZA（150 mg/kg b.w t.）+EMB（75 mg/kg b.w t.）and the dose of high dose group was twice the dose of each drug in clinical equivalent dose group.Rats were orally administered once daily for consecutive 15 or 30 days.The feces were collected for 10h on 15th or 30th day after administration.The 2 hours after the last dose,rats were anesthetized with ethyl ether,blood was collected from the abdominal aorta and liver tissues were collected.AST,ALT,TBA,TBIL,direct bilirubin（DBIL）,indirect bilirubin（IBIL）,cholesterol（CHOL）and triglycerides（TG）were determined by automatic biochemical analyzer.Liver tissues and feces were collected and the content of TBA was determined by the kit.Hepatic tissues were fixed with formalin for hematoxylin-eosin（HE）staining and oil red O staining.The 19 individual bile acids in serum and liver were determined by UPLC-MS/MS.The expressions of FXR and its target gene,including sodium-taurocholate cotransporting polypeptide（NTCP）,cholesterol 7αhydroxylase（CYP7A1）and multidrug resistance protein 2（MRP2）,were detected by western blotting.（3）The part of cell experiment:（1）Cytotoxicity evaluation:Hepa RG cells were treated by C_max-1（INH 20μM,RIF 10μM,EMB 20μM and PZA 150μM）,C_max-2（INH 30μM,RIF 20μM,EMB 30μM and PZA 300μM）and C_max-3（INH 40μM,RIF 30μM,EMB 40μM and PZA 450μM）for 24,48 and 72 h,or single drug from above four drugs for 48h,respectively,cytotoxicity was detected by MTT assay.The expression of caspase-3 was investigated after QATD treatment for 48 h.（2）Bile acid-dependent cytotoxicity evaluation:Hepa RG cells were treated with C_max-1 for 24 h in serum-free medium containing mixed bile acids to investigate the bile acid-dependent toxicity.（3）FXR-dependent cytotoxicity evaluation:Hepa RG cells were divided into four groups:control group,50-fold mixed bile acids（50×BAs）group,C_max-2 group and 50×BAs plus C_max-2 group.After drugs and/or bile acids treatment for 3,6,12,24 and48 h,cytotoxicity was detected by MTT assay.The expressions of FXR and its target genes NTCP,CYP7A1 and bile salt export pump（BSEP）were detected by western blotting.The Hepa RG cells were treated with C_max-2 for 3 h or 48 h,respectively,then TCA was added to Hepa RG cells,and the content of intracellular TCA was determined by HPLC-MS/MS.FXR-overexpressed Hepa RG(FXR^+/+-Hepa RG)cells and normal Hepa RG cells were treated by QATD,and then cytotoxicity was detected by CCK8assay.Results（1）Compared with the healthy people group,serum AST,TBA,TCA and FGF19were significantly increased（p<0.05）in the tuberculosis patients group,while the content of ALT,ALP,TBIL and GGT had no significant difference.（2）The part of animal experiment:（1）Rats were treated by QATD for 15 days,compared with the control group,the ratio of liver to weight in the clinical equivalent dose group was significantly decreased（p<0.05）,and there was no statistical difference in high dose group.Serum biochemical results showed that there were no significant changes in AST and ALT,while the levels of TBA,TBIL,DBIL and IBIL significantly increased（p<0.05）,and the level of TG significantly decreased（p<0.01）in the clinical equivalent dose group and high dose group.The content of CHOL was no significant difference in the clinical equivalent dose group,while the content of CHOL was significantly increased（p<0.05）in the high dose group.Compared with the control group,the content of TBA in liver of clinical equivalent dose group and high dose group showed a decreasing trend,but there was no statistical difference.There was no significant difference in the content of TBA in feces.The results of pathological HE staining showed slight liver injury in the high dose group.The results of oil red O staining showed there was no lipid accumulation in the equivalent dose group and high dose group.The results of determination of bile acids levels in serum and liver showed that compared with the control group,the contents of serum total bile acids pool were significantly increased（p<0.001）in the clinical equivalent dose group and high dose group,mainly unconjugated bile acids;the contents of liver total bile acids pool were significantly decreased（p<0.05）,mainly taurine conjugated bile acids.Western blotting results showed that compared with the control group,the expression of MRP2 was significantly increased（p<0.05）in the liver of clinical equivalent dose group,CYP7A1 expression showed a decreasing trend,and there was no significant difference in the expression of FXR and NTCP.The expressions of FXR and MRP2 in liver of high dose group were significantly increased（p<0.05）,the expression of CYP7A1 was significantly decreased（p<0.05）and there was no significant difference in the expression of NTCP.（2）Rats were treated by QATD for 30 days,compared with the control group,the ratio of liver to weight in the clinical equivalent dose group and high dose group significantly increased（p<0.01）.Serum biochemical results showed that compared with the control group,the levels of AST,ALT,CHOL and TG were significantly decreased（p<0.05）in the clinical equivalent dose group and high dose group,and the levels of TBA,TBIL,DBIL and IBIL were significantly increased（p<0.05）.Compared with the control group,the content of TBA in the liver were significantly increased（p<0.05）in the clinical equivalent dose group and high dose group;there was no significant difference in the content of TBA in feces.The results of pathological HE staining showed liver injury occurred in both clinical equivalent dose group and high dose group.The results of oil red O staining showed that the lipid coloration was deepened in the clinical equivalent dose group and high dose group.The results of determination of bile acids levels in serum and liver showed that compared with the control group,the content of serum total bile acids pool were significantly increased（p<0.01）in the clinical equivalent dose group and high dose group,mainly unconjugated and taurine conjugated bile acids;the content of liver total bile acids pool only significantly increased（p<0.05）in the high dose group,mainly taurine conjugated bile acids.Western blotting results showed that compared with the control group,the expression of FXR was significantly decreased（p<0.05）in the clinical equivalent dose group,while the expression of NTCP,CYP7A1 and MRP2 did not change significantly;the expression of FXR and MRP2 was significantly decreased（p<0.05）in the high dose group,the expression of NTCP and CYP7A1 showed an increasing trend,but there was no statistical difference.（3）The part of cell experiment:（1）Hepa RG cells showed time-and dose-dependent cytotoxicity after QATD treatment.In the single drug treatment experiment,PZA has a major contribution to the toxic effects.Compared with the control group,the expression of caspase-3 was significantly increased（p<0.05）after treatment for 24 h and 48 h,suggesting that QATD could promote cell apoptosis.（2）Bile acid-dependent cytotoxicity experiments:compared with the control group,QATD significantly increased the cytotoxic（p<0.01）in the presence of 50-fold and 100-fold mixed bile acids.（3）FXR-dependent cytotoxicity experiment:compared with 50×BAs group,the cell survival rate of the 50×BAs plus C_max-2 group significantly increased（p<0.05）after treatment for 6 h,and the cell survival rate significantly decreased（p<0.01）after treatment for 48 h.Western blotting results showed that compared with the control group,the expression of FXR was significantly increased（p<0.01）after QATD treatment for 3 h,while the expression of FXR was significantly decreased（p<0.01）after QATD treatment for 48 h;the expression of BSEP was consistent with FXR;the expression of NTCP significantly was increased（p<0.05）after treatment for 24 h,and showed an increasing trend to increase after treatment for 48 h,but there was no statistical difference;the expression of CYP7A1 was significantly increased（p<0.05）after treatment for 6,24,and 48 h,and the protein expression of CYP7A1 showed an increasing trend after treatment for 12 h,but there was no statistical difference.The results of TCA uptake experiment showed that compared with the control group,the level of intracellular TCA showed a decreasing trend after 3 h,but there was no significant difference;the level of intracellular TCA showed an increasing trend after48 h,but there was no significant difference.FXR^+/+-Hepa RG cells were treated with different concentrations of QATD,the results showed that compared with normal Hepa RG cells,the cell survival rate of FXR^+/+-Hepa RG cells was significantly increased（p<0.05）after treatment for 48 h.Conclusions（1）Human serum results showed that there were no significant change in biochemical indexes ALT,ALP,TBIL and GGT after taking QATD about two months,only AST and TBA levels were significantly increased.The level of AST was significantly increased,but still within the normal range.Although TBA level was beyond the normal range,it is not used as a common indicator for clinical diagnosis of anti-tuberculosis drugs-induced liver injury.These results suggested that patients had not suffered liver injury after taking drugs about two months.Elevated serum TBA and FGF19 levels suggested that QATD may affect bile acid homeostasis in patients.Through elevated serum TCA levels,we speculated that administration of QATD in the early stage may induce FXR agonism.（2）After Wistar rats were treated by QATD for 15 days in the high dose,the protein expression of liver FXR was increased,the expression of bile acid synthase CYP7A1 was decreased,the expression of efflux transporter MRP2 was increased,the expression of uptake transporter NTCP was not significantly changed,and the content of bile acids increased in serum and decreased in liver.These results suggested that the synthesis of bile acid was decreased and the efflux of bile acid was increased.Meanwhile,the pathological results showed no significant liver injury.However,after rats were treated for 30 days,the expression of liver FXR were decreased in the high dose group,the expression of CYP7A1 and NTCP showed an increasing trend,the expression of MRP2 was decreased,and the contents of bile acid in serum and liver were significantly increased,suggesting that the synthesis of bile acid was increased while the efflux of bile acid was decreased.Pathological results showed significant liver injury.Results from animal experiments suggested that QATD-induced liver injury may related to the accumulation of hepatic bile acids,which caused by the down-regulation of FXR expression and function.（3）Cell experiments showed that QATD have a time-and dose-dependent toxic effects on Hepa RG cells,and PZA has a major contribution to the toxic effects of QATD for Hepa RG cells.The expression and function of FXR were up-regulated in the early stage,while down-regulated in the late stage after QATD treatment,which was consistent with the results of animal experiment.When the FXR gene was overexpressed,the cytotoxicity of Hepa RG induced by QATD was significantly reduced.These results suggested that the inhibition of FXR was related to the cytotoxicity of Hepa RG induced by QATD.（4）Results from in vivo and in vitro experiments suggested that FXR plays an important role in QATD-induced liver injury,and the inhibition of FXR expression and function may be the cause of QATD-induced liver injury.