The Effect Of Ethanol Uptake On The Enterohepatic Circulation Of Bile Acid And Its Mechanism

ObjectivesBile acid(BA)serve as versatile signaling molecules has important physiological functions that regulated lipid,glucose,energy metabolism.And BA has remarkable characteristics of BA enterohepatic circulation including liver,gallbladder,small intestine,colon,and plasma.BA homeostasis mainly depended on enterohepatic circulation and is well maintained in the healthy.However,the exogenous substances,such as drugs and alcohol,could induced cholestasis and sequently caused to numerous hepatobiliary diseases.Patients with alcoholic liver disease are often accompanied by jaundice and cholestasis,which disrupts BA homeostasis.However,the effect of alcohol uptake on the BA homeostasis of enterohepatic circulation is unclear.In recently years,the study for the relationship of alcohol and abnormal metabolism of BA is very hot.The reported that chronic ethanol consumption could altered the metabolism of BA and altered the expression levels of gene related to BA synthetases,transporters,and nuclear receptor in rat.However,the effect of ethanol uptake on the BA enterohepatic circulation,and the expression levels of protein related to BA synthetases,transporters,and nuclear receptor in mice were rarely reported.To address this,the present study utilized animal model of ethanol treated which male FVB mice were fed 0,30,and 50%ethanol solution(v/v)for 6 weeks.Then a stable and sensitive ultra high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)method was established to detect 24 BAs in the whole enterohepatic circulation system(including liver,gallbladder,small intestine,colon,and plasma)which mainly studies the effect of ethanol uptake on the BA profiles in the whole enterohepatic circulation.In the study of mechanism,the expression levels of protein for BA related synthetases:cholesterol 7α-hydroxylase(Cyp7a1),sterol 12-alpha-hydroxylase(Cyp8b1),sterol 27-hydroxylase(Cyp27a1),and amino acid-N-acyl transferase(Baat);transporters:bile salt export pump(Bsep),multidrug resistance-associated protein 2(Mrp2),Na~+-dependent taurocholate cotransporting polypeptide(Ntcp),and apical sodium dependent BA transporter(Asbt);nuclear receptor:Farnesoid x receptor(Fxr),and fibroblast growth factor 15(Fgf15)in the liver and ileum were measured by western blotting to further clarify the action mechanism of ethanol uptake affected enterohepatic circulation of BA.Additionally,we determined the activity of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum and applied liver Histopathological slice to comprehensively evaluate the effect of liver on mice by ethanol uptake.On this foundation,we investigated that pharmacokinetics of irinotecan(CPT-11)in the control and 50% ethanol-treated mice for 6 weeks.To study,how the ethanol uptake altered the pharmacokinetics profile of the drugs with obvious enterohepatic circulation feature and to provide reference for clinical research.Methods1 The experiment of mice by ethanol uptakeMale FVB mice,the age of 6-7 weeks(n=55)were used as animal experiment.The mice were randomly assigned into control,30%,and 50%ethanol group according to body weights.The ethanol groups were oral administrated 30% and 50%ethanol(v/v),while animals in control group received equivalent deionized water.Dose were 0.1mL/10g/day.The weight of animals were monitored weeks.After continued for 6 weeks,biological samples of the liver,gallbladder,small intestine,colon,and plasma were collected.2 Development of UHPLC-MS/MS methods for BAsThe UHPLC-MS/MS methods of BAs were established by Agilent 6490 Triple Quadrupole mass tandem Agilent 1290 UHPLC System,to measure the concentration of 24 BAs in the liver,gallbladder,small intestine,colon,and plasma,respectively.The BAs including free BAs:CA(Cholic acid),CDCA(Chenodeoxycholic acid),DCA(Deoxycholic acid),LCA(Lithocholic acid),UDCA(Ursodeoxycholic acid),HDCA(Hyodexycholic acid),MCA(Muricholic acid),HCA(Hyocholic acid),αMCA(αMuricholic acid),βMCA(βMuricholic acid),ωMCA(ωMuricholic acid);the taurine BAs:T-CA(Tauro-cholic acid),T-CDCA(Tauro-chenodeoxycholic acid),T-DCA(Tauro-deoxycholic acid),T-LCA(Tauro-lithocholic acid),T-UDCA(Tauro-ursodeoxycholic acid),T-HDCA(Tauro-hyodexycholic acid),T-αMCA(Tauro-αMuricholic acid),T-βMCA(Tauro-βMuricholic acid);the glycine BAs:G-CA(Glyco-cholic acid),G-CDCA(Glycol-chenodeoxycholic acid),G-DCA(Glyco-deoxycholic acid),G-LCA(Glyco-lithocholic acid),G-UDCA(Glycol-ursodeoxycholic acid).3 Detection of the synthetases,transporters,and nuclear receptor related to BAAfter ethanol-treated mice,the expression levels of proteins on the BA enterohepatic circulation related synthetases(Cyp7a1,Cyp27a1,Cyp8b1,and Baat),transporters(Bsep,Mrp2,and Ntcp),and nuclear receptor(Fxr)in the liver.The expression levels of proteins including transporter(Asbt)and nuclear receptors(Fxr and Fgf15)in the ileum by western blotting.4 Detection of activity alkaline aminotransferases and aspirate aminotransferases in the serumAccording to the protocal of the biochemical analysis kit,the alanine aminotransferase activity(ALT)and aspartate aminotransferase activity(AST)in serum were detected to study the effect of liver function caused by ethanol uptake.5 Histopathological examination of liverAfter ethanol uptake,the fresh liver tissues were carefully taken part in tissue box.The liver tissues were fixed by 4%paraformal dehyde for 48hours,and the 4%paraformal dehyde were replaced 70%ethanol.Then,the investigated that ethanol uptake-induced altered the liver tissues by paraffin section.6 In vivo pharmaceutical study of CPT-11 in ethanol-treated miceThe control and 50%ethanol-treated mice were fasted for 10 to 12 hours with free access to water prior to the pharmacokinetics experiments.CPT-11(5mg/kg)intravenous injection(n=6).After the blood samples(30-50μL)were collected from tail vein and placed in dried heparinized tubes at 0,5,15,30,60,120,360,480,720,and 1440 min,respectively.The samples were stored at-80°C until analysis.Results1 The levels of BA increased in the whole enterohepatic circulation by ethanol uptake1.1 The BA pool(bile acid pool)increased by ethanol uptake in miceThe BA pool were significantly increased 1.9 and 3.7 folds in 30%and 50%ethanol-treated mice compared with control mice,respectively(P<0.05).1.2 The levels of BA elevated in the liver by ethanol uptake in miceIn the liver,the concentrations of individual free-BAs(CA,αMCA,βMCA,ωMCA,MCA,HCA,DCA,CDCA,HDCA,UDCA,and LCA)were significantly increased(2.5-,4.9-,4.4-,3.2-,3.9-,4.5-,2.6-,2.6-,2.4-,3.6-,and 1.8-fold,respectively)in 50%ethanol group compared with control group(P<0.05).The concentrations of taurine-BAs in 50%ethanol group(T-CA,T-αMCA,T-βMCA,T-DCA,and T-CDCA)were markedly elevated(5.0-,5.5-,10.5-,4.5-,and37.4-fold,respectively)than control group(P<0.05).The contents of G-CA and G-CDCA were markedly elevated 6.8-and 1.8-fold,respectively,in 50%ethanol group than control group(P<0.05).We also observed that the total bile acids of three forms,the total of individual free-BAs,taurine-BAs,and glycine-BAs were evidently increased 3.7-,7.2-,and 2.7-fold in the 50%ethanol group and the total of individual taurine-BAs significantly increased3.5-fold in 30%ethanol group(P<0.05)compared with control group,respectively(P<0.05).In addition,the TBAs of liver,total amount of all individual BAs,were markedly elevated 2.0-and 4.7-fold in 30%and 50%ethanol group,respectively(P<0.05).1.3 The levels of BA elevated in the gallbladder by ethanol uptake in miceIn the gallbladder,the concentrations of individual BAs(βMCA,ωMCA,HCA,T-LCA,and G-CA)were increased(3.7-,4.8-,2.7-,2.4-,and 3.0-fold,respectively)in 30%ethanol group(P<0.05),while the concentrations of individual free-BAs(CA,αMCA,βMCA,ωMCA,and HCA)were significantly increased(11.5-,5.0-,5.3-,5.7-,and 3.9-fold,respectively)in 50%ethanol group compared with control group(P<0.05).The level of taurine-BAs(T-αMCA,T-βMCA,T-DCA,T-CDCA,T-HDCA,T-UDCA,T-LCA)were significantly elevated(1.8-,1.8-,2.0-,2.0-,1.8-,2.2-,and 3.5-fold,respectively)in 50%group compared with control group(P<0.05).The concentrations of glycine-BAs(G-CA and G-DCA)significantly elevated(3.6-fold and 5.7-fold,respectively)in50%ethanol group compared with control group(P<0.05).We also observed that the total bile acids of three forms,the total of individual free-BAs and glycine-BAs were significantly increased 3.2-and 2.7-fold in 30%ethanol group(P<0.05),while the total free-BAs,taurine-BAs,and glycine-BAs were evidently increased 4.3-,1.5-,and 2.9-fold in 50%ethanol group compared with control group,respectively(P<0.05).In addition,the TBAs in the gallbladder were markedly elevated 1.5-and 1.8-fold in 30%ethanol and 50%ethanol group compared with control group,respectively(P<0.05).1.4 The levels of BA increased in the small intestine by ethanol uptake in miceIn the small intestine,the concentrations of individual free-BAs(CA,αMCA,βMCA,ωMCA,MCA,HCA,DCA,CDCA,and UDCA)were significantly increased(2.7-,2.5-,6.0-,2.5-,4.1-,1.9-,1.6-,1.5-,and 4.0-fold,respectively)in 50%ethanol treated mice(P<0.05),and the concentrations of individual conjugated-BAs(T-CA,T-βMCA,T-DCA,T-CDCA,T-UDCA,T-HDCA,G-UDCA,and G-DCA)were markedly elevated(2.5-,3.5-,5.7-,2.8-,3.3-3.3-,2.8-,and 2.7-fold respectively)in 50%ethanol treated group than the control group(P<0.05).We also observed that the total bile acids of three forms,the total of individual free-BAs,taurine-BAs,and glycine-BAs were significantly increased 3.0-,2.9-,and 1.8-fold in 50%ethanol treated mice compared with the control mice,respectively(P<0.05),while showed no significantly difference in the 30%ethanol group compared with the control group(P>0.05).In addition,TBAs of small intestine were markedly elevated2.9 folds in 50%ethanol treated mice(P<0.05),while showed no significantly difference in the 30%ethanol group compared with the control group(P>0.05).1.5 The levels of BA increased in the colon by ethanol uptake in miceIn the colon,the concentrations of individual free-BAs,CA,βMCA,MCA,DCA,CDCA,HDCA,UDCA,and LCA were significantly increased 3.1-,2.5-,2.5-,1.7-,1.7-,2.2-,2.2-,and 1.6-fold,respectively in 50%ethanol-treated mice(P<0.05),the concentrations of individual taurine-BAs,T-αMCA,T-βMCA,T-DCA,T-UDCA,and T-HDCA were markedly elevated 2.6-,1.8-,2.6-,2.8-,and2.8-fold,respectively in 50%ethanol-treated mice(P<0.05)compared with the control mice.And the G-CA was markedly elevated 2.0 and 2.9 folds in 30%and50%ethanol-treated mice compared with the control mice,respectively(P<0.05).We also observed that the total bile acids of three forms,the total of individual free-BAs,taurine-BAs,and glycine-BAs were markedly increased about 1.6-,1.7-,and 2.8-fold in 50%ethanol group(P<0.05),while showed no significantly difference in the 30%ethanol group than the control group(P>0.05).Additionally,the TBAs in the colon were significantly increased1.7-fold only in 50%ethanol group(P<0.05),while showed no significantly difference in the 30%ethanol group compared with the control group(P>0.05).1.6 The levels of BA increased in the plasma by ethanol uptake in miceIn the plasma,the concentrations of individual free-BAs,(CA,βMCA,ωMCA,MCA,DCA,CDCA,and LCA)were significantly increased(3.5-,2.3-,3.9-,7.0-,1.8-,1.9-,and 2.4-fold,respectively)in 50%ethanol group compared with the control group(P<0.05).The concentrations of individual taurine-BAs(T-CA,T-αMCA,T-βMCA,T-DCA,T-CDCA,T-HDCA,and T-UDCA)were markedly elevated(3.6-,7.1-,5.1-,3.6-,3.1-,2.7-,and 2.5-fold,respectively)in50%ethanol group(P<0.05),and the concentration of G-CA was markedly elevated8.2-fold in 50%ethanol group than the control group(P<0.05).We also observed that the total bile acids of three forms,the total of individual free-BAs,taurine-BAs,and glycine-BAs were significantly increased 2.6,4.8,and 4.7folds in 50%ethanol group,respectively(P<0.05),while showed no significantly difference in the 30%ethanol group than control group(P>0.05).In addition,the TBAs in the plasma were markedly elevated 5.0-fold in 50%ethanol group(P<0.05),while showed no significantly difference in the 30%ethanol group compared with the control group(P>0.05).2 The protein expression of BA syntheses,transporters,and nuclear receptors in the enterohepatic circulation altered by ethanol uptake2.1 The expression levels of protein BA synthases increased by ethanol uptakeIn the liver,the expression levels of synthases Cyp7a1,Cyp8b1,and Cyp27a1 were significantly increased 3.2,2.9,and 2.8 folds in the 30%ethanol group,and markedly elevated 5.3,3.7,and 3.5 folds in the 50%ethanol group compared with control group,respectively(P<0.05).And the expression level of Baat were significantly increased 1.6 folds only in the 50%ethanol group(P<0.05),while showed no significantly difference in the 30%ethanol group compared with the control group(P>0.05).2.2 The expression levels of protein transporters and nuclear receptors related to BA altered by ethanol uptakeIn the liver,the expression levels of transporters Bsep and Mrp2 were significantly increased 1.8 and 2.4 folds in the 30%ethanol group,and significantly elevated 1.7 and 3.2 folds in the 50%ethanol group compared with control group,respectively(P<0.05).However,the expression level of Ntcp was markedly decreased 1.5 and 4.5 folds in the 30%and 50%ethanol group,respectively(P<0.05).The expression level of Fxr was no statistically different in the 30%and 50%ethanol group compared with control group(P>0.05).Additionally,in the ileum the expression level of transporter Asbt was markedly increased 1.8 and 4.5 folds in the 30%and 50%ethanol group,respectively(P<0.05).The expression level of nuclear receptor Fxr was markedly decreased 1.6 folds only in the 50%ethanol group compared with control group(P<0.05),In addition,the expression level of Fgf15 was no statistically different in the 30%and 50%ethanol group(P>0.05),while showed no significantly difference in the 30%ethanol group compared with the control group(P>0.05).3 Ethanol uptake induced liver injure in miceAfter ethanol uptake,the body weight of mice were significantly lower in the 30%and 50%ethanol-treated mice than control mice at 3 to 6 weeks.The activity of ALT and AST were elevated 2.4-fold and 3.1-fold in 30%ethanol group(P<0.05),and were elevated 2.4-fold and 3.5-fold in 50%ethanol group(P<0.05)compared with control group in the serum,respectively.The liver histopathological in mice result that,hepatocytes were no significantly pathological changes in the 30%ethanol group,while the hepatocytes were marked fatty degeneration,balloon denaturalization,and neutrophils infiltration in the 50%ethanol group compared with control group.The results that ethanol uptake induced alcoholism liver injury in mice..4 In vivo pharmacokinetics of CPT-11 altered by ethanol treated in miceAfter i.v.(5 mg/kg)administrations of CPT-11,the blood concentrations of CPT-11 and SN38 were significantly decreased 53%and 25%in mice of 50%ethanol group compared with control group,respectively(P<0.05).The AUC0-t(t=24h)of CPT-11 and SN38 were significantly decreased 57%and 31%in 50%ethanol treated mice compared with control mice,respectively(P<0.05).The CL of CPT-11 and SN38 were significantly increased 158%and 45%in 50%ethanol group compared with control group,respectively(P<0.05).We observed that pharmacokinetic parameters of SN38-G were no significantly changed in 50%ethanol group(P>0.05),as well.The results indicated that mice of 50%ethanol-treated were effected pharmacokinetics of CPT-11 and its metabolites SN38.ConclusionsThe protein levels of BA syntheses(Cyp7a1,Cyp8b1,Cyp27a1,and Baat)and efflux transporters(Bsep and Mrp2)in the liver,and the protein levels of BA uptake transporter(Asbt)in the ileum were significantly increased.Meanwhile,the protein levels of BA intake transporter(Ntcp)in the liver and the nuclear receptor(Fxr)were significantly decreased by ethanol uptake,which contributed to BA levels increase in the whole enterohepatic circulation system(liver,gallbladder,small intestine,colon,and plasma)and induced cholestasis.Additionally,the AUC level of CPT-11,a significantly profile of enterohepatic circulation,were significantly reduced.The present study provided a theoretical basis for reasonable alcohol consumption of people.