The Mechanism Of Nuclear Receptor FXR In Rifampicin-induced Liver Injury

BackgroundDrug-induced liver injury(DILI)is a liver injury induced by various prescription,non-prescription chemical drugs,biological agents,Chinese herbal medicines and their metabolites,and even auxiliary materials.Drug-induced liver injury is one of the most common and serious adverse drug reactions,and severe cases can cause acute liver failure or even death.It is also related to the genetic background and immune response of the host.Therefore,exploring the relationship between genetic factors and drug toxicology to formulate individualized medication programs is very important for the accuracy and safety of clinical medication.Bile acids are steroid molecules produced by the oxidation of cholesterol in the liver.In addition to promoting lipid absorption and maintaining cholesterol balance,they also act as signaling molecules to activate bile acid nuclear receptor FXR.Bile acid not only regulates self-synthesis and hepatic-intestinal circulation,but also affects liver lipid homeostasis by activating farnesate X receptor and its downstream signaling pathways.Rifampicin can induce cholestasis,steatosis and liver cell damage.The pathophysiological role and mechanism of bile acid and bile acid nuclear receptor FXR in rifampicin-induced liver injury are still unclear.ObjectiveTo explore the relationship between the nuclear receptor FXR and rifampicin-induced liver injury,and to elucidate the molecular mechanism of FXR in rifampicin-induced liver injury.Methods1.The role of FXR in rifampicin-induced acute liver injuryC57BL/6(Wild type,WT)and FXR knockout(FXR-/-)male mice at 6-8 weeks age were selected and divided into the following 4 groups:mice with DMSO treatment,WT mice with rifampicin(200mg/kg/d)treatment,FXR-/-mice with DMSO treatment,and FXR-/-mice with rifampicin(200mg/kg/d)treatment.All mice were continuously administered for 1 week and anesthetized to collect blood to separate serum after 6 hours of fasting,then the tissue samples were obtained and stored at-80℃.The levels of AST,ALT,ALP,TBIL and DBIL in the serum of mice were detected with a multifunctional microplate reader;liver tissues were taken and paraffin sections were prepared for HE staining2.The role of FXR in rifampicin-induced liver lipid toxicityAs described above,all mice are divided into four groups and anesthetized to collect blood after the sacrifice.Then liver samples were sliced and stained with oil red O after quick freezing,and lipid droplets in liver cells were observed under a microscope.Serum lipoproteins were separated by fast liquid chromatography(FPLC)gel filtration,and the levels of serum VLDL-C,LDL-C,and HDL-C were measured.After 4 hours of fasting,mice were injected with Tyloxapol,and blood was collected under anesthesia to measure the concentration of triglyceride in serum.Total RNA was extracted from liver tissues and the mRNA levels of 17 lipid metabolism related genes,including HMGCR,APOB,APOC Ⅱand APOCⅢ,were detected by QRT-PCR.3.The role of FXR in rifampicin-induced cholestasisAs described above,all mice are divided into four groups and the urine and feces were collected during fasting.The bile acid concentration of serum and urine were measured with a microplate reader.The liver,feces and bile acid pool were homogenized and crushed in 75%ethanol to dissolve the bile acid,and the bile acid level in the supernatant was measured after centrifugation.Total RNA was extracted from liver tissues and QRT-PCR was used to detect the mRNA levels of 20 genes related to bile acid metabolism in mice,such as BSEP and CYP7A1.Western blot was used to detect the expression levels of proteins related to bile acid metabolism in mice,including BSEP and NTCP,etcTo detect the effect of rifampicin on bile acid signaling pathway in cells:HepG2 cells were divided into three groups:DMSO treated group,CDCA treated group,CDCA and rifampicin treated group.Total RNA was extracted from cells and the mRNA levels of genes related to bile acid metabolism were detected by QRT-PCR.The expression of proteins related to bile acid metabolism was detected by Western blot.Results1.The role of FXR in rifampicin-induced acute liver injury(1)Serum AST and ALT levels:The AST and ALT levels in WT and FXR-/-mice increased after rifampicin treatment,and those in FXR-/-mice with rifampicin treatment were higher than those in WT mice with rifampicin treatment.(2)Serum ALP level,TBIL and DBIL concentrations:Serum ALP level,TBIL and DBIL concentration in WT and FXR-/-mice increased after rifampicin treatment,whereas there was no significant difference between the FXR-/-mice with rifampicin treatment and WT mice with rifampicin treatment.(3)Liver morphology:After treatment with rifampicin in WT and FXR-/-mice,the liver was yellowed and its margins changed,the volume increased,and these phenotypic changes in FXR-/-mice with rifampicin treatment were more obvious than those in WT mice with rifampicin treatment.(4)HE staining:After rifampicin treatment,the hepatocytes of WT mice were slightly edema,the hepatocytes of FXR-/-mice were arranged disorderly,with severe edema,steatosis,and pale cytoplasm.2.The role of FXR in rifampicin-induced liver lipid toxicity(1)Oil red O staining:After rifampicin treatment,lipid droplets in hepatocyte of WT and FXR-/-mice increased,and the increase of lipid droplets in FXR-/-mice with rifampicin treatment was more obvious than that in WT mice with rifampicin treatment.(2)Detection of serum and liver lipids in mice:Serum cholesterol concentration:After rifampicin treatment,the serum cholesterol concentration in WT and FXR-/-mice increased,and the serum cholesterol concentration in FXR-/-mice with rifampicin treatment was higher than that in WT mice with rifampicin treatment.Liver cholesterol level:The liver cholesterol level in the WT mice with rifampicin treatment was higher than that in the WT mice with DMSO treatment,whereas there was no significant difference of liver cholesterol levels between the FXR-/-mice with rifampicin treatment and FXR-/-mice with DMOS treatment.Serum triglyceride concentration:The serum triglyceride concentration in the WT mice with rifampicin treatment was lower than that in WT mice with DMOS treatment,whereas there was no significant difference of serum triglyceride concentration between the FXR-/-mice with rifampicin treatment and FXR-/-mice with DMSO treatment.Liver triglyceride levels:After rifampicin treatment,the liver triglyceride level in WT and FXR-/-mice increased,and that in FXR-/-mice with rifampicin treatment was higher than that in WT mice with rifampicin treatment.(3)Fast protein liquid chromatography analysis:After the rifampicin treatment,serum VLDL-C,LDL-C and HDL-C concentrations in WT and FXR-/-mice were increased,and those in the FXR-/-mice with rifampicin treatment were higher than those in WT mice with rifampicin treatment..(4)Detection of liver lipid secretion:After the rifampicin treatment,there was an increase of liver lipid secretion in WT mice,whereas that in FXR-/-mice remained unchanged.3.The role of FXR in rifampicin-induced cholestasis mouse model(1)Serum total bile acid concentration:The serum total bile acid concentration in WT and FXR-/-mice increased after rifampicin treatment,and that in FXR-/-mice with rifampicin treatment was higher than that in WT mice with rifampicin treatment.(2)Liver bile acid levels:The liver total bile acid levels in WT and FXR-/-mice increased after rifampicin treatment,and that in FXR-/-mice with rifampicin treatment was higher than that in WT mice with rifampicin treatment.(3)Urine and fecal bile acid concentration:The urinary bile acid levels in WT and FXR-/-mice increased after the rifampicin treatment,whereas the fecal bile acid levels in WT and FXR-/-mice decreased after the rifampicin treatment.(4)The expression levels of genes related to bile acid metabolism:After rifampicin treatment,the mRNA levels of BSEP gene in WT mice was increased,while the mRNA levels of CYP7A1 gene was suppressed,and the mRNA levels of these two genes were unchanged after the rifampicin treatment.Moreover,the mRNA levels of NTCP and OATP1b1 gene in WT and FXR-/-mice were suppressed,whereas the mRNA levels of MRP3,CYP2B10 and CYP3A11 genes were enhanced.(5)The expression levels of proteins related to bile acid metabolism:After rifampicin treatment,the expression of BSEP protein in WT mice was enhanced,but there was no change in the expression of BSEP protein in FXR-/-mice after the rifampicin treatment.The expression level of p-JNK protein in WT and FXR-/-mice increased after the rifampicin treatment,and that in FXR-/-mice with rifampicin treatment was higher than that in WT mice with rifampicin treatment.(6)The effect of rifampicin on bile acid signaling pathway in cells:under the condition of hypercholic acid,rifampicin promotes the expression of SHP and BSEP genes,and inhibits the expression of CYP7A1 gene.Conclusion1.Nuclear receptor FXR knockout is more serious in rifampicin-induced liver injury.2.FXR knockout aggravated hepatic lipid toxicity induced by rifampicin.3.The activation of FXR promotes the expression of BSEP,thereby reducing the cholestasis induced by rifampicin.