The Role And Mechanism Of Abcb11/Bsep In The Development Of Mouse Intrahepatic Cholestasis

ObjectivesBackgroundIntrahepatic cholestasis refers to a series of pathological and clinical manifestations of any cause causing bile secretion disorder,or a series of pathological manifestation caused by bile duct obstruction.Intrahepatic cholestasis can damage liver cells without timely intervention,and gradually evolve into cirrhosis,liver failure and even liver cancer.Despite the current standardized recommendations for the diagnosis and treatment of intrahepatic cholestasis,the treatment of intrahepatic cholestasis has not yet achieved a fundamental breakthrough.The bile salt export pump(BSEP)protein encoded by the ABCB11 gene is a major transporter of bile salts in the liver and is critical for bile acid(BA)-dependent bile flow and enterohepatic cycling.When the ABCB11 gene is mutated,abnormalities in function,localization,and degradation of the BSEP protein are found and underlie progressive familial intrahepatic cholestasis 2(PFIC2).In addition,the occurrence of benign recurrent intrahepatic cholestasis type 2(BRIC2),drug-induced cholestasis,and intrahepatic cholestasis of pregnancy(ICP)are also associated with ABCB11 gene defects.Children with PFIC2 have a manifestation of worsening liver function,which can die of cirrhosis or liver failure during infancy and puberty.The incidence of PFIC2 in Western Europe is estimated to be 1/50,000-1/70,000,and there is no corresponding epidemiological data in China.However,the mechanism of ABCB11 in the pathogenesis of PFIC2 is still unclear and lack of relevant researches.At present,the most effective treatment for PFIC2 is liver transplantation,but the chance of liver transplantation is small due to the lack of liver donors for children.Ursodeoxycholic acid(UDCA)is often used in drug therapy,but the treatment effect is limited.Therfore,exploring the role of ABCB11/BSEP in PFIC2 and finding effective therapeutic drugs are of great significance.Based on the above backgrounds,this paper aims to investigate the role and mechanism of Abcb11 gene in the development of intrahepatic cholestasis of mice from the perspective of BA,and to investigate the protective effects of Farnesoid X receptor(FXR)agonist obeticholic acid(OCA)and Chinese herbal monomer geniposide on the intrahepatic cholestasis of Abcb11(-/-)mice.This study expected to provide a theoretical reference for the treatment of clinical relevant disease and scientific researches.Objectives1.To investigate the changes of cholestatic liver injury and BA profile in Abcb 11(-/-)mice with aging.2.To explore the related mechanisms of cholestatic liver injury of Abcb11(-/-)mice.3.To investigate the effect of FXR agonist OCA on cholestatic liver injury of Abcb11(-/-)mice and its related mechanisms.4.To investigate the effect of Chinese herbal monomer geniposide on cholestatic liver injury of Abcb11(-/-)mice and its related mechanisms.Methods1.C57BL/6J and Abcb11(-/-)mice of different ages were dissected to investigate the cholestatic liver injury of Abcb11(-/-)mice.1.1 Alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),triglyceride(TG)and total cholesterol(TCHO)levels in serum and liver were measured using commercial kits.1.2 Liver histology were evaluated by HE staining,Sirius red staining and Oil red O staining.1.3 Enzyme linked immunosorbent assay(ELISA)was used to determine the level of serum Alpha-fetoprotein(Afp).1.4 Immunohistochemistry(IHC)method was used to detect the expression of Ki67 and PCNA,as well as apoptosis determined by TUNEL labeling in liver tumor tissue of Abcbl 1(/-)mice.1.5 Ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLCMS/MS)was used to detect the BA profile and contents in biological samples such as serum and liver.1.6 MTT assay was used to detect the effect of 5 primary BA accumulated in Abcb11(-/-)mice on the proliferation of mouse primary hepatocytes.2.To explore the related mechanisms of intrahepatic cholestasis of Abcbll(-/-)mice.2.1 Real-time quantitative PCR(RT-PCR)method was used to detect the hepatic and intestinal mRNA expression of BA homeostasis-related genes,as well as lipid metabolismrelated genes in mice.2.2 IHC method was used to detect the expression of Fxr in mice liver.2.3 Western-blot method was used to detect the protein expression levels of BA homeostasis-related proteins in mice liver.2.4 The Fgfl5 level in mice serum were detected by ELISA.2.5 The substrate probe method was used to detect the activity of mouse fecal bile salt hydrolase(BSH).The 16S rRNA gene sequencing was adopted to investigate changes in the gut microbiome.3.To observe the effect of OCA on the cholestatic liver injury of Abcb11(-/-)mice.Abcb11(-/-)mice were dissected after intragastrically administered with 20 mg/kg or 40 mg/kg OCA for 6 weeks.The liver injury related indicators were measured using commercial kits and BA levels were measured by UHPLC-MS/MS.The expression of BA homeostasisrelated genes were detected by RT-PCR method.4.To observe the effect of geniposide on the cholestatic liver injury of Abcb11(-/-)mice.Abcb11(-/-)mice were dissected after intragastrically administered with 100 mg/kg or 200 mg/kg geniposide for 4 weeks.The liver injury related indicators were measured using commercial kits and BA levels were measured by UHPLC-MS/MS.The expression of BA homeostasis-related genes were detected by RT-PCR method.Results1.Abcb11(-/-)mice showed progressive intrahepatic cholestasis,increased liver index,intrahepatic BA accumulation and lipid decrease.52-week-old female Abcb11(-/-)mice spontaneously developed to liver cancer.1.1 The liver index(liver weight/body weight)of Abcb11(-/-)mice was significantly higher than that of C57BL/6J mice.52-week-old female Abcb11(-/-)mice spontaneously developed to liver cancer.1.2 The ALP level and ALT level were significantly increased in Abcb11(-/-)mice serum and liver(P<0.05).Additionally,the lower TG and TCHO levels were found in Abcb11(-/-)mice serum and liver(P<0.05).1.3 HE staining and Sirius red staining showed that the liver injury and liver fibrosis of Abcb11(-/-)mice gradually increased with aging,and female Abcb11(-/-)mice display a more severe pathology than male mice.Reduced lipid were found in Abcb11(-/-)mice liver indicated by Oil red O staining.1.4 Compared with C57BL/6J mice,the levels of BA in serum and liver of Abcb11(-/-)mice,especially T-?MCA,T-?MCA.T-CA,T-CDCA and ?MCA were significantly increased(P<0.05).BA levels in bile,intestine,intestinal contents,and feces were reduced.In addition,the proportion of BA with Fxr agonism activity and 12a-OH BA were significantly decreased in Abcb 11(-/-)mice liver.1.5 T-?MCA,T-?MCA,T-CDCA and PMCA inhibited the proliferation of mouse primary hepatocytes.Among them,T-?MCA was the most toxic BA(IC50=10.44 ?M).2.The hepatic mRNA and protein expression levels of Fxr and sterol 12?-hydroxylase(Cyp8bl),as well as Lkb1-Ampk signaling associated protein were significantly decreased in Abcb11(-/-)mice.Abcb11(-/-)mice have low serum Fgf15 levels.The fecal BSH activity and Bacteroidetes were increased and Firmicutes was decreased in female Abcb11(-/-)mice.2.1 The mRNA and protein expression of Fxr and Cyp8b1 were significantly decreased in Abcb11(-/-)mice liver.Additionally,the levels of most BA homeostasis-related enzymes,nuclear receptors and transporters in Abcb11(-/-)mice liver were significantly decreased at 52 weeks of age(P<0.05).Also,the mRNA expression levels of intestinal BA homeostasisassociated genes in ileum and lipid metabolism-related genes in Abcb11(-/-)mice liver were also altered.2.2 The expression of Fxr in the liver of Abcb11(-/-)mice was significantly decreased.2.3 The protein expression level of cholesterol 7?-hydroxylase(Cyp7a1)in Abcb11(-/-)mice liver is significantly higher than that of C57BL/6J at 4-week-old,and significantly decreased at adulthood(P<0.05).The expression of sterol 27-hydroxylase(Cyp27a1)and Cyp8b1 were significantly decreased in Abcb11(-/-)mice liver(P<0.05).P-glycoprotein(Pgp)was significantly increased in Abcb11(-/-)mice before the age of 19 weeks.2.4 The phosphorylation level of AMP-activated protein kinase(Ampk)was significantly increased,and the protein expression level of liver kinase 1(Lkb1)was significantly decreased in the liver of Abcb11(-/-)mice(P<0.05).2.5 Compared with C57BL/6J mice,the serum Fgfl5 levels were significantly decreased in Abcb11(-/-)mice(P<0.05).2.6 Fecal BSH activity of female Abcb11(-/-)mice was significantly higher than that of female C57BL/6J mice(P<0.05).Additionally,increased Bacteroidetes and reduced Firmicutes were found in Abcb11(-/-)mice feces.3.OCA improved the cholestatic liver injury of Abcb11(-/-)mice,significantly reduced serum BA levels.HE staining results showed that OCA improved the cholestatic liver injury of Abcb11(-/-)mice.40 mg/kg OCA reduced the liver index of female Abcb11(-/-)mice.20 mg/kg OCA reduced serum ALT and AST levels.Both 20 mg/kg and 40 mg/kg OCA significantly reduced the BA levels in Abcb11(-/-)mice serum(P<0.05).In addition,OCA significantly downregulated the hepatic mRNA expression levels of BA synthase,nuclear receptors,and transporters in Abcb11(-/-)mice afer daily oral administration of OCA(P<0,05).4.Geniposide mildly improved the cholestatic liver injury of Abcb11(-/-)mice,significantly reduced serum BA levels.HE staining results showed that geniposide improved the cholestatic liver injury of Abcb11(-/-)mice,and significantly reduced AST levels and BA levels in Abcb11(-/-)mice serum(P<0.05).Additionally,the hepatic mRNA expression of BA synthase and nuclear receptors in Abcb11(-/-)mice were significantly decreased afer daily oral administration of geniposide(P<0.05).ConclusionIn conclusion,inactivation of Abcb11 gene resulted in the progressive intrahepatic cholestasis in mice.The liver index of Abcb11(-/-)mice was gradually increased with aging.BA were accumulated in the liver,and female Abcb11(-/-)mice were suffered from liver cancer spontaneously at 52-week-old.We speculated that the cholestatic liver injury of Abcb11(-/-)mice is likely to be associated with a decreased proportion of BA with Fxr agonism activity mediated by the declining expression of Cyp8b1 in the liver,which leads to a decreased expression of Fxr and further disordered negative feedback regulation.In addition,Lkb1-Ampk pathway and dysbacteriosis may also be associated with the final progression of liver cancer in female mice.Both FXR agonist OCA and Chinese herbal monomer geniposide improved the cholestatic liver injury of Abcb11(-/-)mice.